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€œThe prospect of another cyclone was scary,” says Monica, who lives in where to get viagra Mbenje in Malawi’s Nsanje District. €œWe lived through the same experience with Cyclone Idai and then Cyclone Kenneth. We had to rebuild from scratch.”Word had spread through the village during the where to get viagra day that a powerful cyclone had hit neighbouring Mozambique, and on that Sunday evening two weeks ago the weather suddenly changed. For almost six hours, torrential rain and strong winds pummelled Mbenje. Tropical Storm Ana had made landfall in Malawi.

“I looked where to get viagra outside and saw the water levels rising. From previous experiences, I knew we had to move to safety,” said Monica, who is six months pregnant. €œI alerted my husband who quickly gathered the kids.” Monica and her family trudged through the rain and mud all night to Nyambese camp, one of 27 temporary disaster sites that have sprung where to get viagra up across Nsanje, and which are now sheltering people affected by the storm.The following day, Monica and her husband made the five-kilometre trek back to their village to see whether they could salvage anything from their home. Their worst fears were confirmed. There was now a deep pool of water filled with rubble where their house had once stood, the food grain was gone and their animals had been washed away.

€œAfter seeing the destruction, I knew Nyambese camp would be our home until the flood water subsided,” where to get viagra Monica said wearily. © UNFPAMonica, who already had to rebuild after 2019 Cyclones Idai and Kenneth and lost everything to Tropical Storm Ana, holds her two children close at her new temporary home at Nyambese camp, Nsanje District.Lives and homes destroyedTropical Storm Ana has left a trail of destruction in its wake in Malawi, particularly in the hardest-hit southern districts of Nsanje, Phalombe, Mulanje and Chikwawa. Flooding has cut off roads, hampering relief efforts, while damage to the country’s electricity infrastructure is causing frequent power outages.In Nsanje District, more than 55,000 people are now living in temporary camps. Among them are Monica, who is expecting her where to get viagra third child in May, and approximately 1,500 pregnant women. Forced to share latrines, and with little privacy, women and girls are at increased risk of physical and sexual violence in a country where one in three women are subjected to gender-based violence.Restricted mobility due to floodwaters and electricity blackouts are affecting the delivery of sexual and reproductive health care.

The vast majority of health facilities in Nsanje district – 21 out of 24 – are struggling to provide services where to get viagra. Three newborns have already died in the district when incubators were left inoperable due to a lack of power. Fuel for the generator at the district hospital, as well as supplies including lifesaving maternal health medicines, are running low. UNFPA/ Joseph ScottUNFPA Malawi Deputy Representative, where to get viagra Masaki Watabe helping out with dignity kits distribution at Sekeni Primary School Camp Restoring sexual and reproductive health services The UN Population Fund (UNFPA) and partners were on the ground within days of the disaster. To date 6,600 dignity kits containing basic hygiene items such as menstrual pads, soap and underwear, have been distributed to women and girls in Nsanje and Chikwawa.

Repairs to the generator at Nsanje District Hospital have been completed, restoring power to the facility. Plans are also where to get viagra underway to deliver reproductive health kits containing medical and non-medical supplies, maternal health medicines and contraceptives to affected communities in the two districts. €œOur immediate priority is to restore quality sexual and reproductive health and protection services in the aftermath of the disaster,” said Young Hong, UNFPA Representative in Malawi. €œAs extreme weather events become more frequent in the region, UNFPA’s support to the recovery must focus on strengthening systems and building the resilience of affected communities, where to get viagra particularly women and girls.” For Monica, the road ahead will be challenging. She faces the prospect of rebuilding both her home and her life again.

But, for now, her most pressing concern is her unborn child. "I lost everything, even my health passport," she says, cupping her face where to get viagra in her shaking hands. "I was supposed to go to an antenatal clinic this week, but travelling to the health centre is not possible. The roads are bad and still flooded.".

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This July 2022 issue of the JME contains several articles addressing how was viagra discovered ethical issues related to erectile dysfunction treatment as well as reproductive ethics—a timely topic, given the leaked U.S. Supreme Court document, anticipating the overturn of Roe v. Wade.On the erectile dysfunction treatment front, original articles in this issue include an analysis of ethical issues related to sharing research samples and data between low/middle-income countries and high-income countries,1 a retrospective analysis of European scientific societies’ triage policies early in the viagra,2 an assessment of the fairness of the allocation framework used by the WHO for their erectile dysfunction treatments Global Access Facility (COVAX),3 balancing physicians’ freedom to express opinions on medical matters with public interest when they run contrary to each other,4 and a survey of Americans’ views on race-based and place-based erectile dysfunction treatment prioritisation.5 The articles include a mix of looking back and looking forward.On the reproductive ethics front, articles include an analysis of when the government is justified in coercing parents and the implications for abortion,6 and a Feature Article on the ethics of assisted gestative how was viagra discovered technologies7 —along with many insightful commentaries on that topic.Some of the main arguments that the reader will find in this issue include:That ethical considerations in biobanking (sample collection, storage, sharing) during public health emergencies like the erectile dysfunction treatment viagra ought to include respect for research subjections, promoting the common good, solidary, benefit sharing, and reciprocity. A review of research ethics guidance and regulatory requirements is required to ensure that they reflect these considerations.1That the European Union (EU) ought to provide criteria for resource allocation within its member states in the management of a viagra.

A review of policies found that Italian and Spanish medical societies both prioritised greater probability of survival and better prognosis/longer life-years, whereas Swiss and German medical societies advocated for preservation of as many lives as possible.2That in principle, fair global allocation of treatments would involve distributing doses to those whose need is greatest (a targeted approach), but this how was viagra discovered approach would fail to account for the self-interested nature of states, making it unlikely that they would participate in COVAX, resulting in its collapse. Thus, an equal distribution approach averts more deaths than a targeted approach.3That when physicians engage in “citizen speech” (speech relating to broad matters in healthcare and public policy), they deserve the greatest level of protection of free speech. €œPhysician speech” (when a physician, acting on the authority of her position, offers specific medical how was viagra discovered guidance to the public) should be subject to a greater degree of regulation, however. €œClinical speech” or “professional speech” ought to be significantly regulated to align with professional standards of care.4That policy makers might consider public acceptability views related to erectile dysfunction treatment resource allocation.

A U.S. Based survey found that a little over 50% of people supported place prioritisation in allocation of erectile dysfunction treatments (prioritising zip codes that have been hit harder by erectile dysfunction treatment) and about 40% supported race-based prioritisation (prioritising Black, Indigenous, and Hispanic populations because they have been hit harder by erectile dysfunction treatment).5That since it is permissible for the government to coerce mothers into feeding their infants (when a transfer is not possible), there is a good reason to think that the state should coerce women into not having an abortion—that abortion should be how was viagra discovered illegal if the fetus is a person.6And finally, that the field ought to use and develop the conceptual category of “assisted gestative technologies” (eg, uterine transplants, artificial wombs) since that these technologies raise distinct ethical, legal and social issues from those related to assisted conception.7There is much to absorb and think about in this issue of JME—readers will appreciate the range of issues discussed. Perennial issues in medical ethics continue to warrant further discussion as well as future issues as science and medical technology develops. And ethicists continue to think critically about how was viagra discovered how to handle the erectile dysfunction treatment viagra as well as future ones.

This issue illustrates the broad and encompassing way that bioethicists engage with the most critical ethical issues of today and tomorrow.Ethics statementsPatient consent for publicationNot applicable.Ethics approvalNot applicable.Conflict over the right of the state and professional bodies to regulate the speech of physicians whose views defy societal norms or consensus standards of care dates back centuries to controversies surrounding complementary remedies, patent medicines and contraceptives.1–4 Since the World War II, the US Food and Drug Administration has targeted purveyors of ineffective treatments, such as orgone and laetrile, while the authorities have largely turned a blind eye to those physicians who advocate against public health measures such as vaccination.5 6 American law has carved out a wide berth for dissenting views, a safe harbour known as the respectable minority doctrine.7 Until recently, it was unheard of for a provider to be disciplined for setting a poor example for patients through personal lifestyle choices, such as by smoking cigarettes in front of a hospital.8 While leading ethicists including Arthur Caplan have called for the revocation of the licenses of physicians who oppose childhood vaccination, in the highest profile American case to date, that of prominent treatment sceptic Jack Wolfson, the Arizona Board of Osteopathic Examiners in Medicine and Surgery refused to take action on the grounds that the controversial cardiologist had a right to ‘express his opinion’.9 10 However, the erectile dysfunction treatment viagra has drawn renewed attention to the rights of physicians to express views and engage in conduct that runs contrary to the expertise of the medical establishment. For example, the Oregon Medical Board suspended the how was viagra discovered license of physician Steven LaTulippe for refusing to wear a mask and warning his patients that doing so was ‘very dangerous’, while an oncology nurse in that state, Ashley Grames, was placed on administrative leave, and later consented to stop practising nursing entirely, for not physically distancing or wearing a mask outside of work.11 12 Yet physicians touting debunked cures for erectile dysfunction treatment (eg, hydroxychloroquine) or claiming that the viagra is a ‘manufactured crisis’ continue to speak out in the USA without sanction—although with increasing professional and public backlash.13 Meanwhile in Italy, the Rome Doctors Guild announced that it had warned or suspended 10 physicians ‘accused of unwarranted criticism of vaccinations’.14 15 The challenging issue of physician dissent has become only more fraught in the high-stakes context of a global viagra that has already claimed millions of lives.Law and ethics surrounding the freedom of expression of dissenting physicians has evolved piecemeal as contentious cases have arisen. Opinion 8.12 of the American Medical Association’s Code of Ethics imposes on physicians an obligation to ensure that all of the information they provide to the public is ‘accurate’ and ‘based on valid scientific evidence and insight gained from professional experience’, while many state regulations remain unclear on the subject.16 Nevertheless, authorities have increasingly turned to a bifurcated model of regulation that distinguishes between speech that occurs in the clinical context and that which occurs outside the medical setting. The ‘professional speech doctrine’ takes the approach that while physicians deserve the same right to expression as other individuals outside their clinical work—such as when writing pamphlets or speaking in the public square—their speech may be curtailed substantially during encounters with patients.17 18 This doctrine essentially classifies direct patient counselling as a form of professional conduct as distinguished from traditionally protected categories of speech.

American courts of appeals have used that distinction in addressing state bans on gay conversion therapy, Florida’s restriction on physician inquiries related to firearms and California’s requirement that anti-abortion crisis pregnancy centres postwarnings that ‘free or low-cost’ abortions are available elsewhere.19–21 Unfortunately, the how was viagra discovered approach affords no middle ground. Yet the erectile dysfunction treatment viagra has shed light on a swath of speech that is not directed at individual patients in the examination room or surgery, yet nonetheless offers specific medical advice to the public that differs substantially from merely expressing general ideas on healthcare, medicine and policy. This paper briefly reviews the competing values at stake with regard to physician expression and then proposes a three-tiered how was viagra discovered approach for analysing and regulating such expression.The debate over restricting physician speechBroad protections for the free speech rights of physicians can be justified on numerous public policy grounds. Free expression increases the likelihood that providers will contribute to the marketplace of ideas, and today’s dissenting views may prove tomorrow’s innovations and future generations’ orthodoxies.

From Mendel and Semmelweis to the work of Barry Marshall and Robin Warren linking Helicobacter pylori to peptic ulcers, scientific breakthroughs have often been met with scepticism how was viagra discovered and hostility. Of course, many wrong ideas will have to be permitted in the proverbial marketplace if the best ideas are to gain traction. Prohibiting physician dissent runs the risk of suppressing such ideas and driving potential innovators from the field entirely. A sceptic of the medical consensus might choose a career in academic science or industry, how was viagra discovered fearing regulation of his/her ideas by overzealous medical boards.

Moreover, the blades of censorship and censure, once unsheathed, can be used to slice haphazardly through public discourse. Advocating for racial equality, female suffrage, gay rights, family planning, sex education, drug legalisation and aid-in-dying have all proven anathema how was viagra discovered to some medical critics in the past—and once speech can be limited, history shows that authorities will use that ability to challenge views they oppose. Beyond the policy considerations, physicians may also assert a fundamental right to expression. Why should one give up one’s right to voice one’s opinions after receiving a medical degree any more how was viagra discovered than when becoming licensed as a barber or a plumber?.

Yet some fundamental differences distinguish restrictions on the speech of medical professionals from those of ordinary citizens. In most Western nations, physicians in essence operate as part of how was viagra discovered a guild. Their numbers are fixed by the government or professional bodies in order to regulate quality and increase reimbursement.22 In the USA, for instance, anyone with the skill and training can pay a fee to become a barber. In contrast, residency positions are limited like taxicab medallions or liquor licenses or radio frequencies, and—with rare exceptions—such training is required for licensure.23 Many European nations are even more restrictive in their access to opportunities to practice medicine.24 As such, once an individual accepts the privilege of becoming a physician, it does not seem unreasonable to ask that individual to accept additional responsibilities.

One of these burdens might arguably be limits on speech how was viagra discovered and conduct that significantly compromises the public’s health. State regulations and professional standards already impose such duties in other areas through ‘role morality’25. Barbers are welcome to have romantic relationships with their clients, but physicians who have sex with patients are usually how was viagra discovered subject to discipline. In many nations, certain other professionals are already limited in their right to express themselves based on the nature of their work.

For example, in many jurisdictions, judges how was viagra discovered are prohibited from making statements that might call the impartiality of the courts into question. In the USA, the Hatch Act prevents many government employees from engaging in a range of political endeavours that implicate free speech.26 In addition, the training and expertise that physicians receive increases the likelihood that laypersons will rely on their statements. As a result, one might argue, they have a higher burden to maintain the accuracy of their statements than do private citizens. Similarly, in practising medicine, physicians inevitably become role models in matters of how was viagra discovered health and safety.

Needless to say, a clear conflict exists here between this responsibility and private liberty. For instance, it is hard to imagine a how was viagra discovered state medical board requiring doctors to eat healthily or forgo tobacco. The question remains whether certain conduct exists that is legal yet so extreme as to justify official sanction.27The American courts have historically distinguished speech from conduct, protecting the former far more extensively than the latter.28 This distinction proves reasonably effective when applied to altercations in bars, or distinguishing between academics discussing the hypothetical overthrow of the government from militants attempting revolution. In the medical context, this division how was viagra discovered proves much less useful.

The regulation of certain forms of conduct, such as the wearing of masks, is certainly reasonable. However, both telling patients not to wear masks and lobbying the legislature against mask mandates are both speech, yet forms of speech fundamentally different in kind. An approach that transcends how was viagra discovered the traditional speech-conduct distinction is necessary.A three-tiered frameworkThis paper proposes a three-tiered framework for assessing and regulating the expression of physicians. Speech/Conduct in the first tier (‘citizen speech’), which relates broadly to matters of healthcare and public policy, deserves the greatest level of protection.

Speech/Conduct in the second tier (‘physician speech’) involves situations in which a physician, how was viagra discovered acting on the authority of his/her position, offers specific medical guidance to the public. This speech, as discussed below, should be subject to a greater degree of regulation. Finally, speech/conduct in the third tier (‘clinical speech’), which is closely related to the concept of how was viagra discovered ‘professional speech’, should be subject to significant regulation with regard to the standards of care of the profession. Each of these three tiers of speech is discussed in more detail below.Tier 1.

Citizen speechThe justification for regulating physician speech is at its weakest when the physician is speaking as an ordinary citizen. Such speech might how was viagra discovered be on a political or social topic entirely unrelated to medicine, or it might be on a medically related matter in which the physician is speaking as an advocate for policy change. Whether the physician agrees with the consensus opinion of the medical establishment or not is irrelevant. Such an approach would protect those physicians who support expanding access how was viagra discovered to healthcare and those who oppose doing so, providers who want antiracism measures imposed and those who do not consider antiracism a priority, those who support lockdowns to control the erectile dysfunction viagra and even those who oppose such measures.

These ideas, whether wise or foolish, are best contested in the marketplace, not before professional regulators. Permitting such debate increases the likelihood of innovation and progress, and suppressing such debate runs the risk of tarnishing the credibility of the profession, especially if one of how was viagra discovered these suppressed ideas proves true. In fact, citizen speech of this sort likely requires more protection in the medical profession than currently exists. The structure of academic medicine in the USA, for instance, sees most clinical faculty holding joint appointments how was viagra discovered at both medical schools and hospitals.

The latter provide a large percentage of their financial support. Even with assurances of academic freedom and tenure from their medical schools, these faculty are constrained in their ability to speak on matters of public policy by fear of termination by their hospitals. Physicians have allegedly faced how was viagra discovered discipline for criticising their home institutions on a wide range of issues from diversity to viagra preparedness.29 30 Political pressure from social activists on both the political right and left (so-called ‘cancel culture’), augmented by the rise of social media, also suppresses citizen speech by physicians. Sometimes universities and medical schools are complicit in this suppression.

For example, the University of Pennsylvania’s Perelman School of Medicine issued a statement disavowing former associate dean of curriculum Stanley Goldfarb’s controversial Wall Street Journal column opposing the rise of social justice-related curricula in medical education.31 Whatever the merits of Goldfarb’s arguments—and this author does not happen to agree with how was viagra discovered them—danger lies in universities distancing themselves publicly from the controversial ideas of their own faculty. In public discourse, the right to be wrong without fear of reprisal is essential for intellectual progress.Tier 2. Physician speechIn some circumstances, doctors will address the how was viagra discovered general public on specific medical matters that implicate care choices. While no individual doctor-patient relationship exists under the circumstances, this situation is fundamentally different from that of a physician speaking broadly on a matter of public concern.

Laypeople are likely to rely on such statements and to act accordingly, often at the expense of their own health. That is not to say that all opinions offered to the how was viagra discovered public by physicians should be more heavily regulated merely because members of the public might rely on them. Rather, citizen speech only becomes physician speech when it is presented as factual. So a physician is well within his/her rights as a citizen to discourage the public from accepting the measles, mumps and rubella (MMR)-II treatment because how was viagra discovered it was originally derived from fetal tissue and he views its use as morally objectionable.32 The public can recognise that the physician is offering an opinion based on his/her own personal values and can weigh the recommendation through their own ethical lens.

In contrast, the state should be permitted to regulate more strictly a physician who urges the public not to accept the MMR-II treatment because it causes autism, which numerous studies have shown to be empirically false, or because it does not work.33An analogy might be drawn to a civil engineer commenting on bridge safety. If the engineer declares on television that suspension bridges are safe, and a particular bridge fails, that is analogous to citizen speech and does not merit sanction how was viagra discovered. In contrast, most people would agree that if an engineer meets a driver on a road during a storm and declares that the bridge up ahead is not washed out, while in fact he knows that it is, his conduct clearly merits punishment. Physician speech is analogous to the engineer going on television and announcing the bridge is not washed out—when, in fact, it is.

Merely because the advice is offered collectively, how was viagra discovered rather than individually, does not absolve the speaker from responsibility. Similarly, offering generally commentary on social media stands well within the rights of physicians. However, offering specific false information on these media—such as stating that the MMR-II treatment causes autism on a public Facebook post or Twitter feed—is just as dangerous as the engineer deceiving drivers about the washed-out bridge how was viagra discovered and should be just as open to regulation. Needless to say, challenges will arise regarding blended or mixed speech that contains both statements of value and false statements of fact.

For example, a physician might criticise how was viagra discovered a particular intervention as both ‘immoral and ineffective’. While some difficulty may arise in parsing out these distinctions, it does not mean that regulators should not attempt to do so.The standard for evaluating physician speech should be the malpractice standard. In other words, if the advice given collectively or publicly were offered to an individual patient in a clinical setting, and he or she acted on it, would that speech justify a malpractice claim?. Such an approach protects recommendations that may not reflect the majority consensus, but nonetheless have enough expert support to qualify for immunity under the respected how was viagra discovered minority doctrine.

While the malpractice standard should apply, civil liability—rather than state regulation—is a poor mechanism for regulating such speech. Potential plaintiffs may receive false medical information from many sources and injured parties how was viagra discovered will have incentive to falsely attribute their own poor choices to dissenting physicians after the fact. At the opposite extreme, followers of dissenting physicians may refuse to take action even after suffering injuries, thereby allowing deleterious physician speech to continue unchecked.While it is clear that the state or licensing bodies should be the regulators of physician speech, how to regulate such speech is a more challenging question. While the state or medical board might how was viagra discovered be justified in prohibiting certain statements, especially during a public health crisis such as the erectile dysfunction treatment viagra, that intervention is not the only available step.

At a minimum, the medical authorities might require anyone advancing such misleading physician speech to issue a concrete disclaimer stating they are not offering clinical advice. Regulators might go one step further and require such physicians to make clear to audiences the absence of medical authority or empirical evidence to justify their position—or even to explain to the public how was viagra discovered the actual standard of care. (This approach actually remedies one of the issues that arises with blended speech, as such an approach may force speakers to distinguish value-based and fact-based statement by appending disclaimers to the latter.) Regulators might also choose to prevent those advocating actionable physician speech from profiting from such speech. Sharing false information with the public is one matter, growing rich off the proceeds of such false information is another matter entirely.

Whatever specific regulatory steps they take, regulators should consider the dangers posed by such physician speech when acting, how was viagra discovered rather than deferring to the speaker as though the speech were no different than ordinary citizen speech.Tier 3. Clinical speechState licensing boards and courts have a long history of regulating speech and expression in the context of the physician-patient relationship. Arguably, one of the key purposes of medical licensure and of tort liability is to ensure that certain clinical how was viagra discovered standards are met during these encounters. Needless to say, physicians are generally afforded considerable flexibility in offering clinical advice and management.

Extensive debate exists surrounding whether the state should be able to regulate clinicians’ speech in these encounters how was viagra discovered on a wide range of political and moral issues including abortion, aid-in-dying, conversion therapy, and gun safety. Those debates are beyond the scope of this paper. Also, the exact boundary between physician speech and clinical speech can prove murky on occasion, such as when potential patients at the office of Florida physician Jack Cassell were greeted with a sign in 2010 that read “If you voted for Obama … seek urologic care elsewhere”.34 However, states are clearly within their authority to penalise or revoke the licenses of providers who provide medical information to patients during clinical encounters that is false and dangerous, such as discouraging vaccination on safety or efficacy grounds. Applying this principle becomes somewhat more how was viagra discovered challenging in the setting of social media.

Is a Facebook account or Twitter feed with numerous followers offering a general opinion or offering medical advice to each of them?. Does it matter if some of these followers are also patients of the provider (which also raises how was viagra discovered other questions regarding professional boundaries that lie beyond the scope of this paper)?. In other words, when does physician speech on social media become clinical speech?. The key question appears to be whether the recipient of the information might reasonably interpret that guidance to be offered how was viagra discovered as part of a clinical encounter or ongoing physician-patient relationship.

If so, it can be regulated like any other direct clinical communication between doctors and their patients.By whose authority?. One question that is bound to arise related to the proposed three-tiered model is what mechanism should be used to operationalise and enforce these rules. The two leading possibilities are professional organisations—such as the American Medical how was viagra discovered Association, the American College of Physicians, etc—or, as suggested above, state licensing authorities. Solid arguments can be advanced for each of these positions.

The benefit of regulation by professional organisations is that such an approach allows physicians, who have both insight into the norms of their profession and incentives to protect its how was viagra discovered reputation and interests, to govern themselves. However, recent history has demonstrated that such an approach often lacks teeth. Even after medical organisations proscribe member participation in such morally fraught endeavours as how was viagra discovered force feeding prisoners, capital punishment and so-called enhanced interrogation, non-member physicians continue to engage in such practices with impunity. The most striking example of this may be the inability of the American Psychiatric Association (APA) to enforce a rule preventing the diagnosis of public figures, as psychiatrists opposed to the ‘Goldwater rule’ merely resign their APA membership and continue to engage in the APA-proscribed practice.

States licensing authorities, in contrast, have the power to act decisively against all licensees, regardless of their standing with professional organisations. Since many dissenting physicians are already outliers alienated from traditional professional organisations and their norms, the regulation of their speech is likely better left to state authorities.ConclusionThe goal of this paper is not to persuade regulators to suppress any particular set of ideas nor to how was viagra discovered penalise doctors who hold any specific viewpoints. Rather, the aim is to reframe the debate surrounding physician expression. The current distinction between speech that occurs in the clinical setting and speech that occurs how was viagra discovered in the marketplace ignores a crucial third category of statements (‘physician speech’) that are expressions of clinical guidance offered outside the individual physician-patient relationship.

In the era prior to the rise of mass media and the internet, the ability of dissenting physicians to offer empirically questionable advice to the public was relatively circumscribed, so the bifurcated dichotomy of citizen speech and professional or clinical speech proved workable. In contrast, the current international health crisis has emphasised how inadequate such an how was viagra discovered approach is for the regulation of dissident medical providers during a viagra. In no other field are experts permitted to offer life-threatening advice under the colour of professional authority and to avoid regulation solely because they offer false information collectively rather than to individual clients or patients. Freedom of expression will suffer little if regulators separate ‘physician speech’ from more traditional forms of citizen speech and then monitor and regulate such utterances more closely.Data availability statementThere are no data in this work.Ethics statementsPatient consent for publicationNot required..

This July 2022 issue of the JME contains several articles addressing ethical issues related to erectile dysfunction treatment as well where to get viagra as reproductive ethics—a timely topic, given the leaked U.S investigate this site. Supreme Court document, anticipating the overturn of Roe v. Wade.On the erectile dysfunction treatment front, original articles in this issue include an analysis of ethical issues related to sharing research samples and data between low/middle-income countries and high-income countries,1 a retrospective analysis of European scientific societies’ triage policies early in the viagra,2 an assessment of the fairness of the allocation framework used by the WHO for their erectile dysfunction treatments Global Access Facility (COVAX),3 balancing physicians’ freedom to express opinions where to get viagra on medical matters with public interest when they run contrary to each other,4 and a survey of Americans’ views on race-based and place-based erectile dysfunction treatment prioritisation.5 The articles include a mix of looking back and looking forward.On the reproductive ethics front, articles include an analysis of when the government is justified in coercing parents and the implications for abortion,6 and a Feature Article on the ethics of assisted gestative technologies7 —along with many insightful commentaries on that topic.Some of the main arguments that the reader will find in this issue include:That ethical considerations in biobanking (sample collection, storage, sharing) during public health emergencies like the erectile dysfunction treatment viagra ought to include respect for research subjections, promoting the common good, solidary, benefit sharing, and reciprocity. A review of research ethics guidance and regulatory requirements is required to ensure that they reflect these considerations.1That the European Union (EU) ought to provide criteria for resource allocation within its member states in the management of a viagra. A review where to get viagra of policies found that Italian and Spanish medical societies both prioritised greater probability of survival and better prognosis/longer life-years, whereas Swiss and German medical societies advocated for preservation of as many lives as possible.2That in principle, fair global allocation of treatments would involve distributing doses to those whose need is greatest (a targeted approach), but this approach would fail to account for the self-interested nature of states, making it unlikely that they would participate in COVAX, resulting in its collapse.

Thus, an equal distribution approach averts more deaths than a targeted approach.3That when physicians engage in “citizen speech” (speech relating to broad matters in healthcare and public policy), they deserve the greatest level of protection of free speech. €œPhysician speech” (when a physician, acting on the authority of her position, offers specific medical guidance to the public) should be subject to a greater degree where to get viagra of regulation, however. €œClinical speech” or “professional speech” ought to be significantly regulated to align with professional standards of care.4That policy makers might consider public acceptability views related to erectile dysfunction treatment resource allocation. A U.S. Based survey found that a little over 50% of people supported place prioritisation in allocation of erectile dysfunction treatments (prioritising zip codes where to get viagra that have been hit harder by erectile dysfunction treatment) and about 40% supported race-based prioritisation (prioritising Black, Indigenous, and Hispanic populations because they have been hit harder by erectile dysfunction treatment).5That since it is permissible for the government to coerce mothers into feeding their infants (when a transfer is not possible), there is a good reason to think that the state should coerce women into not having an abortion—that abortion should be illegal if the fetus is a person.6And finally, that the field ought to use and develop the conceptual category of “assisted gestative technologies” (eg, uterine transplants, artificial wombs) since that these technologies raise distinct ethical, legal and social issues from those related to assisted conception.7There is much to absorb and think about in this issue of JME—readers will appreciate the range of issues discussed.

Perennial issues in medical ethics continue to warrant further discussion as well as future issues as science and medical technology develops. And ethicists continue to think critically about how where to get viagra to handle the erectile dysfunction treatment viagra as well as future ones. This issue illustrates the broad and encompassing way that bioethicists engage with the most critical ethical issues of today and tomorrow.Ethics statementsPatient consent for publicationNot applicable.Ethics approvalNot applicable.Conflict over the right of the state and professional bodies to regulate the speech of physicians whose views defy societal norms or consensus standards of care dates back centuries to controversies surrounding complementary remedies, patent medicines and contraceptives.1–4 Since the World War II, the US Food and Drug Administration has targeted purveyors of ineffective treatments, such as orgone and laetrile, while the authorities have largely turned a blind eye to those physicians who advocate against public health measures such as vaccination.5 6 American law has carved out a wide berth for dissenting views, a safe harbour known as the respectable minority doctrine.7 Until recently, it was unheard of for a provider to be disciplined for setting a poor example for patients through personal lifestyle choices, such as by smoking cigarettes in front of a hospital.8 While leading ethicists including Arthur Caplan have called for the revocation of the licenses of physicians who oppose childhood vaccination, in the highest profile American case to date, that of prominent treatment sceptic Jack Wolfson, the Arizona Board of Osteopathic Examiners in Medicine and Surgery refused to take action on the grounds that the controversial cardiologist had a right to ‘express his opinion’.9 10 However, the erectile dysfunction treatment viagra has drawn renewed attention to the rights of physicians to express views and engage in conduct that runs contrary to the expertise of the medical establishment. For example, the Oregon Medical Board suspended the license of physician Steven LaTulippe for refusing to wear a mask and warning his patients that doing so was ‘very dangerous’, while an oncology nurse in that state, Ashley Grames, was placed on administrative leave, and later consented to stop practising nursing entirely, for not physically distancing or wearing a mask outside of work.11 12 Yet physicians touting debunked cures for erectile dysfunction treatment (eg, hydroxychloroquine) or claiming that the viagra is a ‘manufactured crisis’ continue to speak out in the USA without sanction—although with increasing professional and public backlash.13 Meanwhile in Italy, the Rome Doctors Guild announced that it had warned or suspended 10 physicians ‘accused of unwarranted criticism of vaccinations’.14 15 The challenging issue of physician dissent has become only more fraught in the high-stakes context of a global viagra that has already claimed millions of lives.Law and ethics surrounding the freedom where to get viagra of expression of dissenting physicians has evolved piecemeal as contentious cases have arisen. Opinion 8.12 of the American Medical Association’s Code of Ethics imposes on physicians an obligation to ensure that all of the information they provide to the public is ‘accurate’ and ‘based on valid scientific evidence and insight gained from professional experience’, while many state regulations remain unclear on the subject.16 Nevertheless, authorities have increasingly turned to a bifurcated model of regulation that distinguishes between speech that occurs in the clinical context and that which occurs outside the medical setting.

The ‘professional speech doctrine’ takes the approach that while physicians deserve the same right to expression as other individuals outside their clinical work—such as when writing pamphlets or speaking in the public square—their speech may be curtailed substantially during encounters with patients.17 18 This doctrine essentially classifies direct patient counselling as a form of professional conduct as distinguished from traditionally protected categories of speech. American courts of appeals have used that distinction in addressing state bans on gay conversion therapy, Florida’s restriction on physician inquiries where to get viagra related to firearms and California’s requirement that anti-abortion crisis pregnancy centres postwarnings that ‘free or low-cost’ abortions are available elsewhere.19–21 Unfortunately, the approach affords no middle ground. Yet the erectile dysfunction treatment viagra has shed light on a swath of speech that is not directed at individual patients in the examination room or surgery, yet nonetheless offers specific medical advice to the public that differs substantially from merely expressing general ideas on healthcare, medicine and policy. This paper briefly reviews the where to get viagra competing values at stake with regard to physician expression and then proposes a three-tiered approach for analysing and regulating such expression.The debate over restricting physician speechBroad protections for the free speech rights of physicians can be justified on numerous public policy grounds. Free expression increases the likelihood that providers will contribute to the marketplace of ideas, and today’s dissenting views may prove tomorrow’s innovations and future generations’ orthodoxies.

From Mendel and Semmelweis to the work of Barry Marshall and Robin Warren linking Helicobacter pylori to peptic ulcers, scientific breakthroughs have often where to get viagra been met with scepticism and hostility. Of course, many wrong ideas will have to be permitted in the proverbial marketplace if the best ideas are to gain traction. Prohibiting physician dissent runs the risk of suppressing such ideas and driving potential innovators from the field entirely. A sceptic of the medical consensus might choose a career in academic science or industry, fearing regulation of his/her where to get viagra ideas by overzealous medical boards. Moreover, the blades of censorship and censure, once unsheathed, can be used to slice haphazardly through public discourse.

Advocating for racial equality, female suffrage, gay rights, family planning, sex education, drug legalisation and aid-in-dying have all proven anathema to some medical critics in the past—and once speech can be limited, history shows that where to get viagra authorities will use that ability to challenge views they oppose. Beyond the policy considerations, physicians may also assert a fundamental right to expression. Why should one give up one’s right to voice one’s opinions after receiving a medical degree any more than when becoming licensed as a barber or where to get viagra a plumber?. Yet some fundamental differences distinguish restrictions on the speech of medical professionals from those of ordinary citizens. In most Western nations, physicians in essence operate as part of a guild where to get viagra.

Their numbers are fixed by the government or professional bodies in order to regulate quality and increase reimbursement.22 In the USA, for instance, anyone with the skill and training can pay a fee to become a barber. In contrast, residency positions are limited like taxicab medallions or liquor licenses or radio frequencies, and—with rare exceptions—such training is required for licensure.23 Many European nations are even more restrictive in their access to opportunities to practice medicine.24 As such, once an individual accepts the privilege of becoming a physician, it does not seem unreasonable to ask that individual to accept additional responsibilities. One of these burdens might arguably be limits on speech and conduct that significantly where to get viagra compromises the public’s health. State regulations and professional standards already impose such duties in other areas through ‘role morality’25. Barbers are welcome to where to get viagra have romantic relationships with their clients, but physicians who have sex with patients are usually subject to discipline.

In many nations, certain other professionals are already limited in their right to express themselves based on the nature of their work. For example, where to get viagra in many jurisdictions, judges are prohibited from making statements that might call the impartiality of the courts into question. In the USA, the Hatch Act prevents many government employees from engaging in a range of political endeavours that implicate free speech.26 In addition, the training and expertise that physicians receive increases the likelihood that laypersons will rely on their statements. As a result, one might argue, they have a higher burden to maintain the accuracy of their statements than do private citizens. Similarly, in practising medicine, physicians inevitably become where to get viagra role models in matters of health and safety.

Needless to say, a clear conflict exists here between this responsibility and private liberty. For instance, it is hard to imagine a state medical board requiring doctors to eat healthily or where to get viagra forgo tobacco. The question remains whether certain conduct exists that is legal yet so extreme as to justify official sanction.27The American courts have historically distinguished speech from conduct, protecting the former far more extensively than the latter.28 This distinction proves reasonably effective when applied to altercations in bars, or distinguishing between academics discussing the hypothetical overthrow of the government from militants attempting revolution. In the medical context, this division proves much less useful where to get viagra. The regulation of certain forms of conduct, such as the wearing of masks, is certainly reasonable.

However, both telling patients not to wear masks and lobbying the legislature against mask mandates are both speech, yet forms of speech fundamentally different in kind. An approach that transcends the traditional speech-conduct distinction is necessary.A three-tiered frameworkThis paper proposes a three-tiered framework for assessing where to get viagra and regulating the expression of physicians. Speech/Conduct in the first tier (‘citizen speech’), which relates broadly to matters of healthcare and public policy, deserves the greatest level of protection. Speech/Conduct in where to get viagra the second tier (‘physician speech’) involves situations in which a physician, acting on the authority of his/her position, offers specific medical guidance to the public. This speech, as discussed below, should be subject to a greater degree of regulation.

Finally, speech/conduct in the third tier (‘clinical speech’), which is closely related to the concept of ‘professional speech’, should be subject to significant regulation with where to get viagra regard to the standards of care of the profession. Each of these three tiers of speech is discussed in more detail below.Tier 1. Citizen speechThe justification for regulating physician speech is at its weakest when the physician is speaking as an ordinary citizen. Such speech might be on a political or social topic where to get viagra entirely unrelated to medicine, or it might be on a medically related matter in which the physician is speaking as an advocate for policy change. Whether the physician agrees with the consensus opinion of the medical establishment or not is irrelevant.

Such an approach would protect those physicians who support expanding access to healthcare and those who oppose doing so, providers who want antiracism measures where to get viagra imposed and those who do not consider antiracism a priority, those who support lockdowns to control the erectile dysfunction viagra and even those who oppose such measures. These ideas, whether wise or foolish, are best contested in the marketplace, not before professional regulators. Permitting such debate increases the likelihood of innovation and progress, and suppressing such debate runs the risk where to get viagra of tarnishing the credibility of the profession, especially if one of these suppressed ideas proves true. In fact, citizen speech of this sort likely requires more protection in the medical profession than currently exists. The structure of academic medicine in the USA, for instance, sees most clinical faculty holding joint appointments at both where to get viagra medical schools and hospitals.

The latter provide a large percentage of their financial support. Even with assurances of academic freedom and tenure from their medical schools, these faculty are constrained in their ability to speak on matters of public policy by fear of termination by their hospitals. Physicians have allegedly faced discipline for criticising their home institutions on a wide range of issues from diversity to viagra preparedness.29 30 Political pressure from social activists on both the political right and left (so-called ‘cancel culture’), augmented by the rise of social media, where to get viagra also suppresses citizen speech by physicians. Sometimes universities and medical schools are complicit in this suppression. For example, the University of Pennsylvania’s Perelman School of Medicine issued a statement disavowing former associate dean where to get viagra of curriculum Stanley Goldfarb’s controversial Wall Street Journal column opposing the rise of social justice-related curricula in medical education.31 Whatever the merits of Goldfarb’s arguments—and this author does not happen to agree with them—danger lies in universities distancing themselves publicly from the controversial ideas of their own faculty.

In public discourse, the right to be wrong without fear of reprisal is essential for intellectual progress.Tier 2. Physician speechIn some circumstances, doctors will address the general public on specific medical matters where to get viagra that implicate care choices. While no individual doctor-patient relationship exists under the circumstances, this situation is fundamentally different from that of a physician speaking broadly on a matter of public concern. Laypeople are likely to rely on such statements and to act accordingly, often at the expense of their own health. That is not to say where to get viagra that all opinions offered to the public by physicians should be more heavily regulated merely because members of the public might rely on them.

Rather, citizen speech only becomes physician speech when it is presented as factual. So a physician is well within his/her rights as a citizen where to get viagra to discourage the public from accepting the measles, mumps and rubella (MMR)-II treatment because it was originally derived from fetal tissue and he views its use as morally objectionable.32 The public can recognise that the physician is offering an opinion based on his/her own personal values and can weigh the recommendation through their own ethical lens. In contrast, the state should be permitted to regulate more strictly a physician who urges the public not to accept the MMR-II treatment because it causes autism, which numerous studies have shown to be empirically false, or because it does not work.33An analogy might be drawn to a civil engineer commenting on bridge safety. If the engineer declares on television that suspension bridges are where to get viagra safe, and a particular bridge fails, that is analogous to citizen speech and does not merit sanction. In contrast, most people would agree that if an engineer meets a driver on a road during a storm and declares that the bridge up ahead is not washed out, while in fact he knows that it is, his conduct clearly merits punishment.

Physician speech is analogous to the engineer going on television and announcing the bridge is not washed out—when, in fact, it is. Merely because the advice is offered collectively, rather than individually, does not absolve the speaker where to get viagra from responsibility. Similarly, offering generally commentary on social media stands well within the rights of physicians. However, offering specific false information where to get viagra on these media—such as stating that the MMR-II treatment causes autism on a public Facebook post or Twitter feed—is just as dangerous as the engineer deceiving drivers about the washed-out bridge and should be just as open to regulation. Needless to say, challenges will arise regarding blended or mixed speech that contains both statements of value and false statements of fact.

For example, a physician where to get viagra might criticise a particular intervention as both ‘immoral and ineffective’. While some difficulty may arise in parsing out these distinctions, it does not mean that regulators should not attempt to do so.The standard for evaluating physician speech should be the malpractice standard. In other words, if the advice given collectively or publicly were offered to an individual patient in a clinical setting, and he or she acted on it, would that speech justify a malpractice claim?. Such an approach protects recommendations that may not reflect the majority consensus, but nonetheless have where to get viagra enough expert support to qualify for immunity under the respected minority doctrine. While the malpractice standard should apply, civil liability—rather than state regulation—is a poor mechanism for regulating such speech.

Potential plaintiffs may receive false medical information from many sources and injured parties will have incentive to falsely attribute their own poor choices to dissenting where to get viagra physicians after the fact. At the opposite extreme, followers of dissenting physicians may refuse to take action even after suffering injuries, thereby allowing deleterious physician speech to continue unchecked.While it is clear that the state or licensing bodies should be the regulators of physician speech, how to regulate such speech is a more challenging question. While the state or medical board might be justified in prohibiting certain statements, especially during a public health crisis such as where to get viagra the erectile dysfunction treatment viagra, that intervention is not the only available step. At a minimum, the medical authorities might require anyone advancing such misleading physician speech to issue a concrete disclaimer stating they are not offering clinical advice. Regulators might go one step where to get viagra further and require such physicians to make clear to audiences the absence of medical authority or empirical evidence to justify their position—or even to explain to the public the actual standard of care.

(This approach actually remedies one of the issues that arises with blended speech, as such an approach may force speakers to distinguish value-based and fact-based statement by appending disclaimers to the latter.) Regulators might also choose to prevent those advocating actionable physician speech from profiting from such speech. Sharing false information with the public is one matter, growing rich off the proceeds of such false information is another matter entirely. Whatever specific where to get viagra regulatory steps they take, regulators should consider the dangers posed by such physician speech when acting, rather than deferring to the speaker as though the speech were no different than ordinary citizen speech.Tier 3. Clinical speechState licensing boards and courts have a long history of regulating speech and expression in the context of the physician-patient relationship. Arguably, one of the key purposes of medical licensure and of tort liability is to ensure that where to get viagra certain clinical standards are met during these encounters.

Needless to say, physicians are generally afforded considerable flexibility in offering clinical advice and management. Extensive debate exists surrounding whether the state should be able to regulate clinicians’ speech in these encounters on a wide range of political and moral issues including abortion, aid-in-dying, where to get viagra conversion therapy, and gun safety. Those debates are beyond the scope of this paper. Also, the exact boundary between physician speech and clinical speech can prove murky on occasion, such as when potential patients at the office of Florida physician Jack Cassell were greeted with a sign in 2010 that read “If you voted for Obama … seek urologic care elsewhere”.34 However, states are clearly within their authority to penalise or revoke the licenses of providers who provide medical information to patients during clinical encounters that is false and dangerous, such as discouraging vaccination on safety or efficacy grounds. Applying this principle becomes somewhat more challenging in where to get viagra the setting of social media.

Is a Facebook account or Twitter feed with numerous followers offering a general opinion or offering medical advice to each of them?. Does it where to get viagra matter if some of these followers are also patients of the provider (which also raises other questions regarding professional boundaries that lie beyond the scope of this paper)?. In other words, when does physician speech on social media become clinical speech?. The key question appears to be whether the recipient of the information might reasonably interpret that guidance to be offered as part of a clinical where to get viagra encounter or ongoing physician-patient relationship. If so, it can be regulated like any other direct clinical communication between doctors and their patients.By whose authority?.

One question that is bound to arise related to the proposed three-tiered model is what mechanism should be used to operationalise and enforce these rules. The two leading where to get viagra possibilities are professional organisations—such as the American Medical Association, the American College of Physicians, etc—or, as suggested above, state licensing authorities. Solid arguments can be advanced for each of these positions. The benefit of regulation by professional organisations is that such an approach allows physicians, who have both insight into the norms of their profession and incentives to protect its reputation and interests, to where to get viagra govern themselves. However, recent history has demonstrated that such an approach often lacks teeth.

Even after medical organisations proscribe member participation in such morally fraught endeavours as force feeding prisoners, capital punishment and so-called enhanced interrogation, where to get viagra non-member physicians continue to engage in such practices with impunity. The most striking example of this may be the inability of the American Psychiatric Association (APA) to enforce a rule preventing the diagnosis of public figures, as psychiatrists opposed to the ‘Goldwater rule’ merely resign their APA membership and continue to engage in the APA-proscribed practice. States licensing authorities, in contrast, have the power to act decisively against all licensees, regardless of their standing with professional organisations. Since many dissenting physicians are already outliers alienated from traditional professional organisations and their norms, the regulation of their speech is likely better left to state authorities.ConclusionThe goal of this paper is where to get viagra not to persuade regulators to suppress any particular set of ideas nor to penalise doctors who hold any specific viewpoints. Rather, the aim is to reframe the debate surrounding physician expression.

The current distinction between speech that occurs in the clinical setting and speech that occurs in the marketplace ignores a crucial third category of statements (‘physician speech’) that are expressions of clinical guidance offered where to get viagra outside the individual physician-patient relationship. In the era prior to the rise of mass media and the internet, the ability of dissenting physicians to offer empirically questionable advice to the public was relatively circumscribed, so the bifurcated dichotomy of citizen speech and professional or clinical speech proved workable. In contrast, the current international health crisis has emphasised how inadequate where to get viagra such an approach is for the regulation of dissident medical providers during a viagra. In no other field are experts permitted to offer life-threatening advice under the colour of professional authority and to avoid regulation solely because they offer false information collectively rather than to individual clients or patients. Freedom of expression will suffer little if regulators separate ‘physician speech’ from more traditional forms of citizen speech and then monitor and regulate such utterances more closely.Data availability statementThere are no data in this work.Ethics statementsPatient consent for publicationNot required..

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IntroductionNephrolithiasis (NL) is the second most frequent renal disease after http://kcuei.com/propecia-cost-singapore/ hypertension affecting up to 10% of individuals worldwide, and in nearly all countries, its prevalence what does generic viagra look like and incidence are reported to be rising.1 NL is highly recurrent and is a major cause of patient pain, disability and lost working days. It is also associated with increased risks of cardiovascular disease, bone demineralisation and fractures, chronic kidney disease and end-stage renal disease.2–4The vast majority of kidney stones is composed of calcium oxalate.5 Even a small increase in urinary oxalate markedly promotes calcium oxalate crystal formation,6–8 and hence, hyperoxaluria is a major risk factor for kidney stones.7 The oxalate found in urine is either from exogenous or endogenous origin. Dietary oxalate, which accounts for almost 50% of the oxalate excreted in urine,9 is abundantly present in plant and animal sources, absorbed by the gut and excreted unchanged in the urine.9 In addition, endogenous oxalate production occurs in the liver resulting from the metabolism of glyoxylate.10 11 Primary hyperoxaluria (PH) is an inherited autosomal recessive metabolic disorder that results from increased endogenous oxalate production, whereas secondary hyperoxaluria is acquired and caused by excessive intake of dietary oxalate or of oxalate precursors or by factors that can increase net oxalate absorption what does generic viagra look like by the gastrointestinal tract (eg, low calcium diet, lipid malabsorption and abnormal microbiome).

Therefore, identification of the potential mechanisms leading to hyperoxaluria is an important step to optimise care and treatment of oxalate-dependent NL.In mice, ablation of the oxalate transporter SLC26A6 causes a defect in intestinal oxalate secretion that results in increased net absorption of dietary oxalate,12 leading to hyperoxaluria and calcium oxalate NL.12 13 This observation supports the possibility that an additional type of inherited hyperoxaluria might exist in human patients due to mutations leading to defective intestinal oxalate secretion, thereby resulting in enhanced net intestinal absorption of oxalate. Four studies have assessed the impact of SLC26A6 variants in humans.14–17 However, these studies either reported no association of SLC26A6 variants with hyperoxaluria or with NL,14–16 or reported mutations that disrupt a complex between SLC26A6 and the citrate transporter NaDC-1 thereby causing hypocitraturia as a risk factor for NL.17 Therefore, no rare human SLC26A6 variant contributing to hyperoxaluria has been reported so far.Here, we report a family in which a rare heterozygous missense mutation in the SLC26A6 gene is associated with hyperoxaluria and recurrent bilateral calcium oxalate NL. In vitro characterisation of the mutant demonstrated that the mutation what does generic viagra look like leads to defective membrane expression and oxalate transport activity with evidence of a dominant negative effect on wild-type (WT) transporter.

We conclude that rare, deleterious mutations in SLC26A6 gene are a possible cause of inherited hyperoxaluria and therefore can promote NL in humans.Concise methodsA detailed description of material and methods is available as online supplemental information.Supplemental materialParticipants from UK BiobankWe analysed up to 200 619 samples with available exome sequencing data. This research is part of UK Biobank research application #67 575 what does generic viagra look like. The strategy used in this study to identify patients with kidney stones is reported in online supplemental materials.DNA sequence analysesThe DNA proband was assessed through whole-exome sequencing at the genotyping platform of the European Genomic Institute for Diabetes (Lille, France).

For this purpose, we used the Human Core Exome EF Multiplex Complete kit (Twist Bioscience) in combination with Illumina next-generation sequencing.The SLC26A6 mutation was confirmed in the proband through Sanger sequencing, and then assessed in the family members. Primer sequences and PCR conditions are what does generic viagra look like available on request. Fragments were sequenced in both directions and subsequently analysed using the 3730xl DNA Analyzer (Applied Biosystems).

Electropherogram reads were assembled and examined using the SeqScape software what does generic viagra look like (Applied Biosystems).Detection of variants in UK BiobankWe used exome data from pVCF format (field #23 156). Only variants with a coverage higher than 10 reads and quality Genotype quality (GQ) score higher than 20 were kept for further analyses. Annotation of variants was done using the Ensembl Variant Effect Predictor tool V.103 (RefSeq).In silico analyses of the effects of the p.R507W mutation on Slc26a6 proteinA structural model of human SLC26A6 was developed using the experimental structure of the mouse SLC26A9 homodimer anion transporter as structural template.18 Different structural bioinformatics approaches were used to investigate the model structure and the amino acid replacement including the computations of ΔΔG values between the WT and the variant proteins.

The different approaches used and additional structural analyses are reported in the online supplemental material section.SLC26A6 expression constructsAn HA-tagged wild-type human SLC26A6 cDNA expression construct (HA-WT)19 was used to generate HA-tagged and myc-tagged p.R507W human what does generic viagra look like SLC26A6 mutants (HA-MT and myc-MT, respectively) with a Q5 site-directed mutagenesis kit (New England Biolabs).Cell culture and transfectionsOKP cells (American Type Culture Collection(ATTC)) were used for all transfection experiments. 1×105 OKP cells per well of a 24 wells cell culture dish were reverse transiently transfected with 2 µL of Lipofectamine 2000 (Thermo Scientific) and 0.1–0.25 µg of HA-WT, myc-WT or HA-MT cDNA as indicated in the respective figure legend for each experiment. Cells were assayed 72 hours post-transfection.Cell surface biotinylationOKP cells were what does generic viagra look like surface biotinylated 72 hours post-transfection as described previously.19 Membranes were probed with a rabbit anti-SLC26A6 polyclonal antibody19 (R29.

1:50 000 dilution), a rabbit anti-HA polyclonal antibody (71–5500. 1:250 dilution. Thermo Scientific) or a mouse anti-myc monoclonal antibody (R950-25 what does generic viagra look like.

1:5000 dilution. Thermo Scientific) what does generic viagra look like. Primary antibody labelling was detected with either a donkey antirabbit or a donkey antimouse Horse Radish Peroxydase (HRP-labelled secondary antibody (711-035-152 and 715-035-150 respectively.

1:20 000 dilution. Jackson ImmunoResearch),19 visualised by enhanced chemiluminescence (Clarity Western ECL Substrate what does generic viagra look like. Bio-Rad) and recorded on film.CoimmunoprecipitationTransfected OKP cells were cultured for 72 hours prior to solubilisation with 1% digitonin.

Myc-tagged WT SLC26A6 was immunoprecipitated from OKP cell detergent lysates with the anti-myc antibody R950-25 (Thermo. 5 µg IgG/mL what does generic viagra look like lysate). Immunoprecipitates were captured with Protein G Sepharose Fast Flow (Sigma P3296), and then Sepharose beads were washed extensively with a series of normal and high salt washes to reduce non-specific binding.

Immunoprecipitates were released from sepharose beads by incubation with 2× Sodium Dodecyl what does generic viagra look like Sulfate (SDS)-PAGE sample buffer containing 100 mM dithiothreitol (DTT) for 2 min at 100°C. The immunoprecipitates were subjected to SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF membrane and then analysed by western blot with antibodies directed against the HA-epitope and myc-epitope tags (71–5500 and R950-25 antibodies, respectively).Cl−-dependent 14C-oxalate uptakeSee reference ref 19 for a detailed description of the rationale and methodology behind the assessment of Cl−-gradient dependent 14C-oxalate uptake mediated by the SLC26A6 expression constructs (HA-WT and HA-MT) when transfected into OKP cells. Briefly, uptakes were performed for each transfection condition in the presence and absence of an outwardly directed chloride gradient to provide an estimate of the proportion of the total 14C-oxalate cell uptake that was specifically chloride dependent.

Uptakes were also performed in untransfected cells (exposed to transfection reagent alone) to what does generic viagra look like provide an estimate of background levels of endogenous chloride-dependent 14C-oxalate uptake. The background values were then subtracted from the values in transfected cells to determine the level of chloride-dependent 14C-oxalate uptake that is directly and solely attributable to the activity of the transfected SLC26A6 expression constructs.StatisticsData values are presented as mean±SEM. Statistical significance was evaluated by unpaired two-tailed what does generic viagra look like Student’s t-test (GraphPad Prism).ResultsPrimary clinical data of the probandThe patient is a late adolescent woman from non-consanguineous parents.

She has a personal history of Hashimoto’s thyroiditis diagnosed during her early adolescence and replacement with L-thyroxine. She had her first renal colic during her early adolescence. Since then, what does generic viagra look like she has had recurrent renal colic (>20) and passed multiple kidney stones.

Abdominal CT and uasound detected multiple and bilateral stones without nephrocalcinosis (online supplemental figure S1). A spontaneously evacuated kidney stone was analysed by infrared spectrometry revealing a composition of calcium oxalate monohydrate (~80% whewellite) suggesting that what does generic viagra look like NL is oxalate dependent. Biochemical analyses, performed in the absence of any treatment and 2 months after her last renal colic (table 1), identified the presence of frank hyperoxaluria (733 µmol/day, N.

40–330), which was confirmed on a second independent urine sampling (800 µmol/day, N. 40–330). Adequacy of urine collection was attested by measuring the daily excretion of creatinine (0.16 mmol/kg/day, N.

0.12–0.19). Interview with a dietitian excluded excessive intake in oxalate-rich aliments (~50 mg/day) and revealed very low calcium intake (~400 mg/day). The patient had no history of bowel disease or diarrhoea and showed no sign of malabsorption.

Table 1 also showed no biological evidence of malabsorption. Kidney stone-causing conditions like vitamin D excess or primary systemic disease (eg, primary hyperparathyroidism or renal leak of phosphate) were excluded. The calcium-loading test detected increased digestive absorption of calcium as evidenced by the increased delta between postcalcium load calciuria and fasting calciuria.

However, the patient did not exhibit hypercalciuria. The only other risk factor for NL was very mild hypocitraturia (1.18 mmol/24 hours, N>1.67). The patient exhibited no extrarenal symptoms or biological abnormalities suggesting systemic oxalosis, and renal function appeared normal.Supplemental materialIn silico analysis of the putative effects of the R507W substitution on the SLC26A6 protein structure.

The two subunits are presented with one subunit coloured in green and the other in orange (cartoon representation). The olive spheres represent the boundary of the lipid bilayer as computed with the PPM server. A zone predicted to interact with the Cl− ion as proposed in the mouse SLC26A9 experimental structure is highlighted by a black dashed circle in subunit a for orientation.

R507 residues of both subunits are flagged and coloured in blue. This residue is located on the last helix of the transmembrane domain 22 residues prior to the beginning of the Sulfate Transporter Antagonist of anti-Sigma factor (STAS domain. The insertion loop that could not be built is shown as a dashed line.

The mutation is expected to destabilise the protein and to impede optimal insertion into the lipid bilayer." data-icon-position data-hide-link-title="0">Figure 1 In silico analysis of the putative effects of the R507W substitution on the SLC26A6 protein structure. The two subunits are presented with one subunit coloured in green and the other in orange (cartoon representation). The olive spheres represent the boundary of the lipid bilayer as computed with the PPM server.

A zone predicted to interact with the Cl− ion as proposed in the mouse SLC26A9 experimental structure is highlighted by a black dashed circle in subunit a for orientation. R507 residues of both subunits are flagged and coloured in blue. This residue is located on the last helix of the transmembrane domain 22 residues prior to the beginning of the Sulfate Transporter Antagonist of anti-Sigma factor (STAS domain.

The insertion loop that could not be built is shown as a dashed line. The mutation is expected to destabilise the protein and to impede optimal insertion into the lipid bilayer.View this table:Table 1 Characteristics, blood and urine analyses and calcium-loading test of the patientsIdentification of a rare heterozygous missense mutation in Slc26a6Through whole-exome sequencing of the proband’s DNA, we investigated rare coding variants of potential interest with minor allele frequency below 0.1% in the Genome Aggregation Database (GnomAD V.2.1.1) in a list of genes or candidate genes linked with NL (online supplemental table 1). We did not find any pathogenic or likely pathogenic variant according to the criteria of the American College of Medical Genetics and Genomics.20 No pathogenic or likely pathogenic variant or variant of unknown significance was detected in the three genes involved in PH.

However, we found a rare heterozygous variant of unknown significance located in the 13th coding exon of SLC26A6 (NM_022911.2. C.1519C>T. P.R507W).

SLC26A6 encodes an oxalate transporter involved in hyperoxaluria and NL based on mouse knockout studies.12 13 According to GnomAD (V.2.1.1), SLC26A6 is intolerant to loss-of-function variation with an observed/expected score of 0.65 (0.48–0.91), and the p.R507W mutation is very rare, as it has been found in only 5 out of 280 674 alleles reported in GnomAD (in Europeans and South Asians). According to SIFT, Mutation Taster and PolyPhen-2 (HumDiv), the p.R507W mutation was predicted to be deleterious, disease causing and damaging, respectively.21–23Supplemental materialHuman SLC26A6 residue p.R507 was found fully conserved in an interspecies comparison. The conservation of this residue was also very high in this family of proteins (ie, the score at position 507 was equal to 8, the maximum is 9, via the Consurf server).

We then built a homology model using as experimental template the mouse Slc26a9 homodimer anion transporter tri-dimensional (3D structure (figure 1). The modelling was possible as the overall sequence identity between the two proteins is around 40% (see online supplemental materials).The human SLC26A6 model structure positioned into a membrane is shown in figure 1. P.R507 is located on the C-terminal side of the last transmembrane helix, prior to the STAS domain.

The region of p.R507 is predicted to be essentially rigid (PredyFlexy computation) and thus not very tolerant to the p.R507W substitution, which was predicted to likely perturb correct interaction with the membrane. The details of our in silico analyses are provided as online supplemental materials.Family investigation of the Slc26a6 p.R507W mutationSince the SLC26A6 mutation was potentially pathogenic, both parents of the proband were assessed. Sanger sequencing identified the same heterozygous SLC26A6 mutation in the father, while the mother was WT (online supplemental figure S2).

The mother had no personal or familial history of kidney stones and exhibited normal levels of urinary oxalate (250 µmol/day, N. 40–330).Characterisation of the R507W SLC26A6 mutation in transfected OKP cells. OKP cells were transfected with equivalent amounts (0.1 µg cDNA per well of a 24-well dish) of either wild-type (WT.

HA-WT) or mutant (MT. HA-MT) human SLC26A6 cDNA. Cells were assayed 72 hours post-transfection.

For each transfection (Tx-1 to 3), uptakes and companion biotinylations were performed in triplicate. Utx, untransfected control. Panels A+B show representative images selected from each transfection.

Western blots were probed with the antihuman Slc26a6 polyclonal antibody, R29. Panel A. Western analysis of total cell lysates isolated from each transfection condition.

Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. To facilitate comparisons between transfections (n=3 independent transfections), MT expression levels for each transfection were normalised to mean densitometry values for the WT densitometry values for each transfection.

All WT levels are expressed as 100 (%), and the individual MT values vary accordingly. Panel D. Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT.

HA-WT) and mutant (MT. HA-MT) Slc26a6. The uptake values are presented as pmoles 14C-oxalate uptake per well of a 24-well plate and are not normalised to levels of expressed protein.

WT, wild type. HA-WT, hemagglutinin-tagged wild type. MT, mutant.

HA-MT, hemagglutinin-tagged mutant. Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 2 Characterisation of the R507W SLC26A6 mutation in transfected OKP cells.

OKP cells were transfected with equivalent amounts (0.1 µg cDNA per well of a 24-well dish) of either wild-type (WT. HA-WT) or mutant (MT. HA-MT) human SLC26A6 cDNA.

Cells were assayed 72 hours post-transfection. For each transfection (Tx-1 to 3), uptakes and companion biotinylations were performed in triplicate. Utx, untransfected control.

Panels A+B show representative images selected from each transfection. Western blots were probed with the antihuman Slc26a6 polyclonal antibody, R29. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition.

Panel C. Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry.

To facilitate comparisons between transfections (n=3 independent transfections), MT expression levels for each transfection were normalised to mean densitometry values for the WT densitometry values for each transfection. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly. Panel D.

Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT. HA-MT) Slc26a6.

The uptake values are presented as pmoles 14C-oxalate uptake per well of a 24-well plate and are not normalised to levels of expressed protein. WT, wild type. HA-WT, hemagglutinin-tagged wild type.

MT, mutant. HA-MT, hemagglutinin-tagged mutant. Tx, transfection.

Utx, untransfected.Even though the father did not report any renal colic, biochemical analyses revealed that he also had hyperoxaluria (520 µmol/day, N. 40–330). The calcium loading test was strictly normal, and no other risk factor for NL was identified for the father (table 1).

However, we noticed that he exhibited a urine output of almost 4 L/day reflecting a very high-water intake. This high urine output may explain why he never experienced kidney stones. Furthermore, contrasting with the proband, he did not exhibit any hypocitraturia.

No other relatives from same family were accessible for medical examination and genetic testing.Response to medical treatmentWe hypothesised that the proband presented with inherited enteric hyperoxaluria due to deficient SLC26A6-mediated oxalate secretion resulting in increased net absorption of dietary oxalate. We advised her to increase drinking water intake in order to reach a urinary output of at least 3 L/day and to systematically add a dairy product to each meal in order to increase her calcium intake up to the recommended daily amounts of 900 mg/day. The latter intervention turned out to be very effective, as urinary oxalate excretion markedly dropped from >700 µmol/day down to ~300 µmol/day on repeated control analyses while the mild hypocitraturia persisted (1.49 mmol/24 hours N<1.67).

No other medication was initiated, and NL also dramatically improved, as she has not experienced any recurrence of kidney stones for 2 years of follow-up.Physiological characterisation of the variantIn order to assess the pathogenicity of the SLC26A6 p.R507W mutation, we next assessed its effect on protein expression, protein trafficking and chloride-dependent oxalate transport. Under physiological conditions, apical membrane SLC26A6 in epithelial cells mediates oxalate secretion by operating in the direction of exchanging intracellular oxalate for extracellular chloride. However, SLC26A6 can mediate Cl−-oxalate exchange in either direction depending on the direction of the net driving force.

We measured Cl−-gradient stimulated 14C-oxalate uptake as a measure of oxalate transport activity mediated by human SLC26A6 transfected into OKP epithelial cells, as in previous studies of SLC26A6 glycosylation mutants.19 We specifically evaluated the chloride-dependent component of 14C-oxalate uptake that was mediated by the transfected human SLC26A6 expression constructs by correcting for endogenous oxalate transport (see Methods and online supplemental figure S3 for details).The R507W mutation directly impairs chloride-dependent oxalate transport capability in human SLC26A6. The initial characterisation of the Δ507 mutation suggested that the degree of inhibition of the Slc26a6-mediated chloride-dependent oxalate transport activity was significantly greater than that predicted by decreased protein expression alone. To better address that possibility, we transfected OKP cells with 2.5× more mutant (MT) than wild-type (WT) human SLC26A6 (hA6) in an effort to significantly enhance the apparent transport activity of Δ507-hA6.

Cells were transfected with either 0.25 µg of HA-MT (MT) or 0.1 µg HA-WT (WT) cDNA per well of a 24-well plate. The experiments were performed and data presented identically as in panels A–C of figure 1. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition.

Panel C. Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry.

All WT levels are expressed as 100 (%), and the individual MT values vary accordingly. Panel D. Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT.

HA-WT) and mutant (MT. HA-MT) Slc26a6. The principal objective of this experiment was to directly address the transport capability of mutant relative to wild-type Slc26a6 independent of protein expression.

To that end, the transport data presented in panel D were normalised to cell surface biotinylatable hA6 for each transfection. Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 3 The R507W mutation directly impairs chloride-dependent oxalate transport capability in human SLC26A6.

The initial characterisation of the Δ507 mutation suggested that the degree of inhibition of the Slc26a6-mediated chloride-dependent oxalate transport activity was significantly greater than that predicted by decreased protein expression alone. To better address that possibility, we transfected OKP cells with 2.5× more mutant (MT) than wild-type (WT) human SLC26A6 (hA6) in an effort to significantly enhance the apparent transport activity of Δ507-hA6. Cells were transfected with either 0.25 µg of HA-MT (MT) or 0.1 µg HA-WT (WT) cDNA per well of a 24-well plate.

The experiments were performed and data presented identically as in panels A–C of figure 1. Panel A. Western analysis of total cell lysates isolated from each transfection condition.

Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly.

Panel D. Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT.

HA-MT) Slc26a6. The principal objective of this experiment was to directly address the transport capability of mutant relative to wild-type Slc26a6 independent of protein expression. To that end, the transport data presented in panel D were normalised to cell surface biotinylatable hA6 for each transfection.

Tx, transfection. Utx, untransfected.Characterisation of the effect of the p.R507W mutation on expression and function of human SLC26A6 transfected into OKP cells is shown in figure 2. Results are illustrated for three independent experiments in which OKP cells were transfected with identical amounts of cDNA encoding the WT and mutant (MT) transporters.

Figure 2A shows immunoblots of total cell lysate and compared transfected cells with an untransfected control lane. Transfection of OKP cells results in expression of a minor band just under 75 kDa and multiple prominent bands >100 kDa. As demonstrated in a previous study, the lower band corresponds in size to nascent unglycosylated SLC26A6, whereas the upper bands correspond to complex-glycosylated SLC26A6.19 Surface biotinylated SLC26A6 is shown in figure 2B, corresponding in size to the complex-glycosylated forms of SLC26A6.

As illustrated by the immunoblots in figure 2A and B and summarised by the densitometry in figure 2C, there was a marked reduction in glycosylated and cell surface expression of the mutant transporter compared with WT. Although these findings indicate that the p.R507W mutation leads to decreased processing and trafficking, and/or increased degradation of the transporter, the qualitative similarity of the western blot molecular weight band profiles of the WT and MT constructs strongly suggests that the mutation does not directly affect the glycosylation of SLC26A6 per se. As shown in figure 2D, chloride-dependent 14C-oxalate uptake mediated by mutant SLC26A6 was also markedly impaired.

Of interest, whereas the mutation resulted in approximately a 50% reduction in cell surface expression of SLC26A6 (figure 2C), it led to a disproportionately higher reduction of Cl−-dependent oxalate transport activity of approximately 75%. This suggested that the mutation might affect both the expression and the intrinsic transport capability of SLC26A6.We therefore next designed an experiment to test directly whether the mutation causes a defect in the intrinsic transport function of SLC26A6 in addition to the defect in net surface expression demonstrated in figure 2. Cells were transfected with 2.5-fold more mutant than WT cDNA with the goal of approximately equalising surface expression of the mutant and WT transporters.

As illustrated by the immunoblots in figure 3A and B and summarised by the densitometry in figure 3C, the protocol achieved roughly equivalent amounts of the glycosylated and surface expressed forms of the mutant and WT transporters. Most importantly, assessment of transport activity normalised to surface expression, as shown in figure 3D, indicated that the p.R507W mutation does indeed cause a marked defect (approximately 50%) in intrinsic transport activity of SLC26A6. The findings of figures 2 and 3 taken together suggest that the 75% defect in transport activity of mutant compared with WT SLC26A6 when equal amounts of cDNA are transfected (figure 2D) results from about a 50% defect in cell surface expression (figure 2C) and a roughly 50% defect in intrinsic transporter activity (figure 3D).Given that SLC26 family transporters exist as dimers,18 24 we conducted additional studies to test the possibility of a dominant negative effect of the p.R507W mutation on expression of the WT transporter.

Our approach was to assay expression of myc-tagged WT SLC26A6 as a function of cotransfection with either HA-tagged WT or HA-tagged mutant transporter. We chose transfection conditions to achieve equivalent expression levels of HA-WT and HA-MT SLC26A6 (online supplemental figure S4). As illustrated by the immunoblots in figure 4A and B, and summarised by the densitometry in figure 4C, coexpression of mutant SLC26A6 caused 70% reduction in glycosylated and cell surface expression of myc-tagged WT SLC26A6 compared with coexpression of WT SLC26A6.

These results support the possibility that the p.R507W mutation may have a dominant negative effect on expression of the WT transporter so that the hyperoxaluria phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone.Coexpression of the R507W mutant significantly reduces both total and cell surface expression of wild-type SLC26A6. OKP cells were cotransfected with 0.1 µg myc-tagged human SLC26A6 (myc-WT) cDNA and either 0.1 µg HA-tagged wild-type Slc26a6 (HA-WT) cDNA or 0.25 µg HA-tagged Arg507Trp mutant Slc26a6 (HA-MT) cDNA per well of a 24-well dish. Cotransfection conditions were selected to achieve equivalent levels of expression of each HA-tagged cDNA construct to facilitate a direct comparison of the effects of coexpression of each protein.

Cells were assayed 72 hours postcotransfection. See online supplemental figure S3 for a representative cotransfection experiment illustrating simultaneous expression of each epitope-tagged construct. Panel A.

Western analysis of myc-tagged wild-type Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B. Western analysis of cell surface biotinylatable myc-tagged Slc26a6 from each cotransfection.

Western blots depicted in panels A+B were probed with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 1:5000 dilution). Panel C.

Expression levels of total and cell surface biotinylatable myc-tagged Slc26a6. Expression levels were determined by densitometry. To facilitate comparisons between transfections (n=4 independent transfections) myc-WT:HA-MT cotransfection expression levels were expressed relative to the densitometry values for myc-WT:HA-WT cotransfection expression levels for each transfection and myc-WT:HA-WT cotransfection expression levels were normalised to a mean value of 100 (%).

Co-IP, coimmunoprecipitation. Co-Tx, cotransfection. IP, immunoprecipitation.

Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 4 Coexpression of the R507W mutant significantly reduces both total and cell surface expression of wild-type SLC26A6. OKP cells were cotransfected with 0.1 µg myc-tagged human SLC26A6 (myc-WT) cDNA and either 0.1 µg HA-tagged wild-type Slc26a6 (HA-WT) cDNA or 0.25 µg HA-tagged Arg507Trp mutant Slc26a6 (HA-MT) cDNA per well of a 24-well dish.

Cotransfection conditions were selected to achieve equivalent levels of expression of each HA-tagged cDNA construct to facilitate a direct comparison of the effects of coexpression of each protein. Cells were assayed 72 hours postcotransfection. See online supplemental figure S3 for a representative cotransfection experiment illustrating simultaneous expression of each epitope-tagged construct.

Panel A. Western analysis of myc-tagged wild-type Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B.

Western analysis of cell surface biotinylatable myc-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+B were probed with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 1:5000 dilution).

Panel C. Expression levels of total and cell surface biotinylatable myc-tagged Slc26a6. Expression levels were determined by densitometry.

To facilitate comparisons between transfections (n=4 independent transfections) myc-WT:HA-MT cotransfection expression levels were expressed relative to the densitometry values for myc-WT:HA-WT cotransfection expression levels for each transfection and myc-WT:HA-WT cotransfection expression levels were normalised to a mean value of 100 (%). Co-IP, coimmunoprecipitation. Co-Tx, cotransfection.

IP, immunoprecipitation. Tx, transfection. Utx, untransfected.We next performed a series of WT/MT co-immunoprecipitation studies to verify the association of MT with WT transporter to explain the dominant negative phenotype.

OKP cells were cotransfected with myc-tagged WT SLC26A6 and either HA-tagged WT SLC26A6 or HA-tagged MT SLC26A6 as described for the experiment outlined in figure 4. The cells were solubilised with 1% digitonin, subjected to immunoprecipitation with an anti-myc antibody, and the resulting immunoprecipitates were analysed by SDS-PAGE and western blot with antibodies directed against either the myc-tag or the HA-tag (figure 5).The R507W substitution does not inhibit association of MT-A6 with WT-A6. The principal objective of this experiment was to determine if the p.R507W mutation affected the ability of the resultant mutant to associate with wild-type (WT) Slc26a6 and form the prototypic Slc26a6 multimeric complex.

By using different epitope tags for the potential binding partners, we used anti-myc tag (WT) primary immunoprecipitation to assess association with HA-tagged protomers (WT +MT) by monitoring levels of coimmunoprecipitation. OKP cells were cotransfected (Co-Tx) as described for figure 3, solubilised with 1% digitonin and subjected to immunoprecipitation with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 5 µg IgG/mL lysate).

Panel A. Western analysis of myc-tagged WT Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B.

Western analysis of HA-tagged Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel C. Western analysis of primary immunoprecipitated myc-tagged WT Slc26a6 from each cotransfection.

Panel D. Western analysis of coimmunoprecipitated HA-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+C were probed with anti-myc antibody R950-25 (1:5000 dilution) and those in panels B+D were probed with anti-HA antibody 71–5500 (1:50 dilution).

Panel E. Relative expression of primary immunoprecipitated myc-tagged WT Slc26a6 and coimmunoprecipitated HA-tagged WT or mutant Slc26a6. Expression levels were determined by densitometry.

To facilitate comparisons between cotransfections (n=3 individual cotransfection events. Tx-1 to Tx-3), protein expression levels determined for the myc-WT/HA-MT cotransfection condition were expressed relative to the densitometry values for the myc-WT/HA-WT cotransfection condition for each experiment, and myc-WT:HA-WT cotransfection expression levels were normalised to 100 (%). *Not significantly different at p=0.3566.

Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 5 The R507W substitution does not inhibit association of MT-A6 with WT-A6. The principal objective of this experiment was to determine if the p.R507W mutation affected the ability of the resultant mutant to associate with wild-type (WT) Slc26a6 and form the prototypic Slc26a6 multimeric complex.

By using different epitope tags for the potential binding partners, we used anti-myc tag (WT) primary immunoprecipitation to assess association with HA-tagged protomers (WT +MT) by monitoring levels of coimmunoprecipitation. OKP cells were cotransfected (Co-Tx) as described for figure 3, solubilised with 1% digitonin and subjected to immunoprecipitation with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 5 µg IgG/mL lysate).

Panel A. Western analysis of myc-tagged WT Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B.

Western analysis of HA-tagged Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel C. Western analysis of primary immunoprecipitated myc-tagged WT Slc26a6 from each cotransfection.

Panel D. Western analysis of coimmunoprecipitated HA-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+C were probed with anti-myc antibody R950-25 (1:5000 dilution) and those in panels B+D were probed with anti-HA antibody 71–5500 (1:50 dilution).

Panel E. Relative expression of primary immunoprecipitated myc-tagged WT Slc26a6 and coimmunoprecipitated HA-tagged WT or mutant Slc26a6. Expression levels were determined by densitometry.

To facilitate comparisons between cotransfections (n=3 individual cotransfection events. Tx-1 to Tx-3), protein expression levels determined for the myc-WT/HA-MT cotransfection condition were expressed relative to the densitometry values for the myc-WT/HA-WT cotransfection condition for each experiment, and myc-WT:HA-WT cotransfection expression levels were normalised to 100 (%). *Not significantly different at p=0.3566.

Tx, transfection. Utx, untransfected.As observed in the previous experiment (figure 4), cotransfection of mutant HA-A6 with wild-type myc-A6 led to a dramatic decrease in expression of mature WT myc-A6 in both the solubilised lysate (figure 5A) and the companion primary immunoprecipitation (figure 5C). Western analysis of the solubilised lysate with the anti-HA antibody (figure 5B) confirmed that cotransfection with 2.5× HA-MT relative to HA-WT resulted in roughly similar levels of expression of the two HA-tagged SLC26A6 expression constructs.

We observed significant coimmunoprecipitation of both wild-type and mutant HA-A6 with the myc-tagged WT-A6 (figure 5D). Despite the presence of similar amounts of HA-WT and HA-MT in the initial cell lysates for each experiment (figure 5B), their relative recoveries by coimmunoprecipitation could only reflect, at best, the relative recovery of the primary immunoprecipitated binding partner (myc-WT). If the two constructs multimerise with wild-type SLC26A6 with a similar efficacy, their relative recoveries by coimmunoprecipitation should closely match the relative recovery of myc-WT in the primary immunoprecipitation for each transfection condition.

The apparent decrease in coimmunoprecipitation of HA-MT relative to HA-WT observed in figure 5D and quantified in figure 5E is not significantly different to that observed for the primary immunoprecipitation of myc-WT observed in figure 5B when cotransfected with HA-WT versus HA-MT. This strongly suggests that the p.R507W mutation does not impair the efficacy of formation of the SLC26A6 multimeric complex per se. The demonstration of formation of MT/WT heteromultimers can explain the dominant negative effect of the MT transporter if it is misfolded, has altered stability in the plasma membrane and/or has reduced oxalate transport ability.Identification of additional deleterious Slc26a6 mutations in cases with nl from UK BiobankWe next analysed up to 200 619 samples with available exome sequencing data in UK Biobank.

Unfortunately, NL was not well recorded in the UK Biobank phenotypes as we identified 5267 patients with recorded NL that corresponds to a prevalence of 2% only. This prevalence is very far from the well-known prevalence of NL in the UK (13%).25 Following a case-only design, we found two heterozygous protein-truncating SLC26A6 mutations (NM_022911.3:c. 3G>A/p.Met1?.

and NM_022911.3:c.1132C>T/p.Gln378*) in two males. These two mutations were very rare in GnomAD (with a minor allele frequency <0.00005). Those two additional independent cases support a possible contribution of SLC26A6 loss of function mutations to NL.

Unfortunately, urinary oxalate excretion is not among the urine parameters available in the UK Biobank phenotype.DiscussionWe show complementary genetic and functional evidence for a novel mechanism of inherited enteric hyperoxaluria in humans. Using a candidate gene analysis of whole-exome sequencing data, we identified a rare c.1519C>T mutation in the SLC26A6 gene encoding an oxalate transporter in a family affected by hyperoxaluria. This mutation leads to the substitution of the arginine in position 507 by a tryptophan.

Further extensive in vitro characterisation revealed that the mutation decreases both SLC26A6 transport function and membrane expression. Moreover, the mutant allele also exerts a profound dominant negative effect on the WT allele, suggesting that the mutation might affect SLC26A6-mediated oxalate secretion more than gene haploinsufficiency alone would do.The results of our experiments demonstrating that coexpression of mutant and WT SLC26A6 decreases total and cell-surface expression of the WT protein, that mutant SLC26A6 is capable of forming multimeric structures with WT SLC26A6 and that the presence of a tryptophan in position 507 is predicted to destabilise the interaction of the mutant SLC26A6 monomer with the lipid bilayer suggest that potential mutant–mutant homodimers and mutant–wild type heterodimers are both very likely to be unstable in the cell plasma membrane. The dominant-negative effect of the mutant SLC26A6 on the wild-type protein assembled in the heterodimer (WT/MT SLC26A6) combined with the inhibitory effect of the mutation itself expressed in the homodimer (MT/MT SLC26A6) would be expected to decrease the production of functional SLC26A6 in the plasma membrane by much more than the 50% predicted by the heterozygous mutation.SLC26A6 is an anion exchanger from the solute carrier family 26 expressed at the apical membrane of many types of epithelial cells including enterocytes in the gastrointestinal tract.26 It can exchange intracellular oxalate for external chloride, and hence, performs apical oxalate secretion.27 Oxalate transport across the intestinal epithelium results from the combination of passive paracellular absorption and active transcellular secretion.28 Mouse studies have shown that SLC26A6 plays a critical role in intestinal secretion of oxalate.12 13 Therefore, inactivation of Slc26a6 in mice enhances net oxalate absorption, leading to higher plasma oxalate and ultimately increased urinary oxalate excretion, particularly when dietary intake of oxalate is high.12 Our findings suggest that the c.1519C>T/p.R507W mutation in SLC26A6 may cause similar pathophysiology resulting in enteric hyperoxaluria in humans.

In this regard, it is striking that the patient’s hyperoxaluria was dramatically reduced by adding calcium to her diet, strongly suggesting that hyperoxaluria in her case was mostly from enteric origin and can be controlled by treatments that limit passive paracellular absorption of oxalate.Four previous studies have assessed the possible association of SLC26A6 mutations with hyperoxaluria and calcium oxalate urolithiasis:In the first study, the authors screened a cohort of 94 patients with PH and 96 controls.14 The non-synonymous SLC26A6 variants that were detected in cases were actually frequent (minor allele frequency ≥1%) in one or more populations from the GnomAD browser (v2.1.1). A c.616G>A (p.V206M) mutant was most common (11%) and showed a 30% reduction in oxalate transport activity. However, heterozygosity for this variant did not affect plasma or urine oxalate levels in the study population.

There was no significant effect of any identified variants on oxalate excretion. The p.V206M variant of SLC26A6 was subsequently described in a second study.15 This variant was highly frequent (minor allele frequency (MAF) ~10% in all populations). Again, this variant was not associated with urolithiasis or with hyperoxaluria.

In a third study, Lu et al16 functionally assessed non-synonymous SLC26A6 variants that were listed in the public database dbSNP, according to in silico analyses. The authors found a significant association between the low-frequency (but not rare) rs184187143 variant (with a MAF of 0.4% in the Finnish population) and urolithiasis risk (unadjusted p value=0.007). However, no mechanistic insight potentially explaining this association was provided.

Finally, two additional heterozygous variants of SLC26A6 have recently been reported.17 The first rare p.R621G variant was identified in a non-stone former individual who exhibited a normal (<40 mg/d) urinary excretion of oxalate. According to several in silico programmes (PolyPhen, SIFT, Mutation Taster, Align GVGD), this mutation was totally benign. The second rare p.D674N variant that abolished SLC26A6 expression and Cl−-dependent bicarbonate transport when transfected in HEK cells was identified in a patient presenting with calcium oxalate urolithiasis.

However, the authors did not report whether this variant was cosegregating with lithiasis in the family of the patient. Moreover, in the proband the mutation was not associated with hyperoxaluria but rather with a marked hypocitraturia, in line with previous studies from same group showing that mutation of the STAS domain of SLC26A6 can provoke hypocitraturia, another important risk factor for NL, by altering regulation of the citrate transporter NaDC-1.29 30 A very mild hypocitraturia, along with the marked hyperoxaluria, was also observed in our patient. It is very unlikely that such a mild decrease in urinary citrate excretion could cause such a high-frequency recurrent stone disease course, but it may have contributed along with hyperoxaluria to the increased risk for NL.In summary, our observation that mutation p.R507W is associated with hyperoxaluria in humans supports the hypothesis that like in the mouse SLC26A6 plays a critical role in intestinal secretion of oxalate, thereby limiting net absorption of dietary oxalate.

Consistent with this mechanistic hypothesis, we observed a beneficial effect of increasing calcium in the patient’s diet to reduce enteric oxalate absorption and thereby urinary oxalate excretion. Accordingly, other mutations leading to defects in function of oxalate transporter SLC26A6 could be responsible for inherited form of enteric hyperoxaluria and NL in human patients. Therefore, screening for mutations in SLC26A6 should be performed in patients with hyperoxaluria in whom no mutations in the PH disease genes AGXT, or GRHPR or HOGA are found, or in patients with enteric hyperoxaluria who have no evidence of intestinal disease.Supplemental materialData availability statementAll data relevant to the study are included in the article or uploaded as supplementary information.

Not applicable.Ethics statementsPatient consent for publicationConsent obtained directly from patient(s)Ethics approvalThis study involves human participants and was approved by Institutional review board of the University Hospital of La Réunion (ID :2019/CHU/10). Participants gave informed consent to participate in the study before taking part.AcknowledgmentsWe would like to thank the patient and her family for their kind help and participation in the study. We would also like to thank Frédéric Allegaert and Nicolas Larcher for expert technical assistance.

This research has been conducted using the UK Biobank Application #67 575..

IntroductionNephrolithiasis (NL) is the second most frequent renal disease after hypertension affecting Propecia cost singapore up to 10% of individuals worldwide, and in nearly all countries, its prevalence and incidence are reported to be where to get viagra rising.1 NL is highly recurrent and is a major cause of patient pain, disability and lost working days. It is also associated with increased risks of cardiovascular disease, bone demineralisation and fractures, chronic kidney disease and end-stage renal disease.2–4The vast majority of kidney stones is composed of calcium oxalate.5 Even a small increase in urinary oxalate markedly promotes calcium oxalate crystal formation,6–8 and hence, hyperoxaluria is a major risk factor for kidney stones.7 The oxalate found in urine is either from exogenous or endogenous origin. Dietary oxalate, which accounts for almost 50% of the oxalate excreted in where to get viagra urine,9 is abundantly present in plant and animal sources, absorbed by the gut and excreted unchanged in the urine.9 In addition, endogenous oxalate production occurs in the liver resulting from the metabolism of glyoxylate.10 11 Primary hyperoxaluria (PH) is an inherited autosomal recessive metabolic disorder that results from increased endogenous oxalate production, whereas secondary hyperoxaluria is acquired and caused by excessive intake of dietary oxalate or of oxalate precursors or by factors that can increase net oxalate absorption by the gastrointestinal tract (eg, low calcium diet, lipid malabsorption and abnormal microbiome). Therefore, identification of the potential mechanisms leading to hyperoxaluria is an important step to optimise care and treatment of oxalate-dependent NL.In mice, ablation of the oxalate transporter SLC26A6 causes a defect in intestinal oxalate secretion that results in increased net absorption of dietary oxalate,12 leading to hyperoxaluria and calcium oxalate NL.12 13 This observation supports the possibility that an additional type of inherited hyperoxaluria might exist in human patients due to mutations leading to defective intestinal oxalate secretion, thereby resulting in enhanced net intestinal absorption of oxalate.

Four studies have assessed the impact of SLC26A6 variants in humans.14–17 However, these studies either reported no association of SLC26A6 variants with hyperoxaluria or with NL,14–16 or reported mutations that disrupt a complex between SLC26A6 and the citrate transporter NaDC-1 thereby causing hypocitraturia as a risk factor for NL.17 Therefore, no rare human SLC26A6 variant contributing to hyperoxaluria has been reported so far.Here, we report a family in which a rare heterozygous missense mutation in the SLC26A6 gene is associated with hyperoxaluria and recurrent bilateral calcium oxalate NL. In vitro characterisation of where to get viagra the mutant demonstrated that the mutation leads to defective membrane expression and oxalate transport activity with evidence of a dominant negative effect on wild-type (WT) transporter. We conclude that rare, deleterious mutations in SLC26A6 gene are a possible cause of inherited hyperoxaluria and therefore can promote NL in humans.Concise methodsA detailed description of material and methods is available as online supplemental information.Supplemental materialParticipants from UK BiobankWe analysed up to 200 619 samples with available exome sequencing data. This research is part of UK Biobank research application #67 575 where to get viagra.

The strategy used in this study to identify patients with kidney stones is reported in online supplemental materials.DNA sequence analysesThe DNA proband was assessed through whole-exome sequencing at the genotyping platform of the European Genomic Institute for Diabetes (Lille, France). For this purpose, we used the Human Core Exome EF Multiplex Complete kit (Twist Bioscience) in combination with Illumina next-generation sequencing.The SLC26A6 mutation was confirmed in the proband through Sanger sequencing, and then assessed in the family members. Primer sequences and PCR conditions where to get viagra are available on request. Fragments were sequenced in both directions and subsequently analysed using the 3730xl DNA Analyzer (Applied Biosystems).

Electropherogram reads were assembled and examined using the SeqScape software (Applied Biosystems).Detection of variants in UK BiobankWe used exome data from pVCF format (field #23 156) where to get viagra. Only variants with a coverage higher than 10 reads and quality Genotype quality (GQ) score higher than 20 were kept for further analyses. Annotation of variants was done using the Ensembl Variant Effect Predictor tool V.103 (RefSeq).In silico analyses of the effects of the p.R507W mutation on Slc26a6 proteinA structural model of human SLC26A6 was developed using the experimental structure of the mouse SLC26A9 homodimer anion transporter as structural template.18 Different structural bioinformatics approaches were used to investigate the model structure and the amino acid replacement including the computations of ΔΔG values between the WT and the variant proteins. The different approaches used and additional structural analyses are reported in the online supplemental material section.SLC26A6 expression constructsAn HA-tagged wild-type human SLC26A6 cDNA expression construct (HA-WT)19 was used to generate HA-tagged and myc-tagged p.R507W human SLC26A6 mutants (HA-MT and myc-MT, respectively) with a Q5 site-directed mutagenesis kit (New England Biolabs).Cell culture where to get viagra and transfectionsOKP cells (American Type Culture Collection(ATTC)) were used for all transfection experiments.

1×105 OKP cells per well of a 24 wells cell culture dish were reverse transiently transfected with 2 µL of Lipofectamine 2000 (Thermo Scientific) and 0.1–0.25 µg of HA-WT, myc-WT or HA-MT cDNA as indicated in the respective figure legend for each experiment. Cells were assayed where to get viagra 72 hours post-transfection.Cell surface biotinylationOKP cells were surface biotinylated 72 hours post-transfection as described previously.19 Membranes were probed with a rabbit anti-SLC26A6 polyclonal antibody19 (R29. 1:50 000 dilution), a rabbit anti-HA polyclonal antibody (71–5500. 1:250 dilution.

Thermo Scientific) or a where to get viagra mouse anti-myc monoclonal antibody (R950-25. 1:5000 dilution. Thermo Scientific) where to get viagra. Primary antibody labelling was detected with either a donkey antirabbit or a donkey antimouse Horse Radish Peroxydase (HRP-labelled secondary antibody (711-035-152 and 715-035-150 respectively.

1:20 000 dilution. Jackson ImmunoResearch),19 visualised by enhanced chemiluminescence where to get viagra (Clarity Western ECL Substrate. Bio-Rad) and recorded on film.CoimmunoprecipitationTransfected OKP cells were cultured for 72 hours prior to solubilisation with 1% digitonin. Myc-tagged WT SLC26A6 was immunoprecipitated from OKP cell detergent lysates with the anti-myc antibody R950-25 (Thermo.

5 µg IgG/mL lysate) where to get viagra. Immunoprecipitates were captured with Protein G Sepharose Fast Flow (Sigma P3296), and then Sepharose beads were washed extensively with a series of normal and high salt washes to reduce non-specific binding. Immunoprecipitates were released from sepharose beads by incubation with 2× Sodium Dodecyl Sulfate (SDS)-PAGE sample buffer containing 100 mM dithiothreitol (DTT) for where to get viagra 2 min at 100°C. The immunoprecipitates were subjected to SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF membrane and then analysed by western blot with antibodies directed against the HA-epitope and myc-epitope tags (71–5500 and R950-25 antibodies, respectively).Cl−-dependent 14C-oxalate uptakeSee reference ref 19 for a detailed description of the rationale and methodology behind the assessment of Cl−-gradient dependent 14C-oxalate uptake mediated by the SLC26A6 expression constructs (HA-WT and HA-MT) when transfected into OKP cells.

Briefly, uptakes were performed for each transfection condition in the presence and absence of an outwardly directed chloride gradient to provide an estimate of the proportion of the total 14C-oxalate cell uptake that was specifically chloride dependent. Uptakes were also performed in untransfected cells (exposed to transfection reagent alone) where to get viagra to provide an estimate of background levels of endogenous chloride-dependent 14C-oxalate uptake. The background values were then subtracted from the values in transfected cells to determine the level of chloride-dependent 14C-oxalate uptake that is directly and solely attributable to the activity of the transfected SLC26A6 expression constructs.StatisticsData values are presented as mean±SEM. Statistical significance was evaluated by unpaired two-tailed Student’s t-test (GraphPad Prism).ResultsPrimary clinical data of the where to get viagra probandThe patient is a late adolescent woman from non-consanguineous parents.

She has a personal history of Hashimoto’s thyroiditis diagnosed during her early adolescence and replacement with L-thyroxine. She had her first renal colic during her early adolescence. Since then, she has had recurrent renal where to get viagra colic (>20) and passed multiple kidney stones. Abdominal CT and uasound detected multiple and bilateral stones without nephrocalcinosis (online supplemental figure S1).

A spontaneously evacuated kidney stone was analysed by infrared spectrometry where to get viagra revealing a composition of calcium oxalate monohydrate (~80% whewellite) suggesting that NL is oxalate dependent. Biochemical analyses, performed in the absence of any treatment and 2 months after her last renal colic (table 1), identified the presence of frank hyperoxaluria (733 µmol/day, N. 40–330), which was confirmed on a second independent urine sampling (800 µmol/day, N. 40–330).

Adequacy of urine collection was attested by measuring the daily excretion of creatinine (0.16 mmol/kg/day, N. 0.12–0.19). Interview with a dietitian excluded excessive intake in oxalate-rich aliments (~50 mg/day) and revealed very low calcium intake (~400 mg/day). The patient had no history of bowel disease or diarrhoea and showed no sign of malabsorption.

Table 1 also showed no biological evidence of malabsorption. Kidney stone-causing conditions like vitamin D excess or primary systemic disease (eg, primary hyperparathyroidism or renal leak of phosphate) were excluded. The calcium-loading test detected increased digestive absorption of calcium as evidenced by the increased delta between postcalcium load calciuria and fasting calciuria. However, the patient did not exhibit hypercalciuria.

The only other risk factor for NL was very mild hypocitraturia (1.18 mmol/24 hours, N>1.67). The patient exhibited no extrarenal symptoms or biological abnormalities suggesting systemic oxalosis, and renal function appeared normal.Supplemental materialIn silico analysis of the putative effects of the R507W substitution on the SLC26A6 protein structure. The two subunits are presented with one subunit coloured in green and the other in orange (cartoon representation). The olive spheres represent the boundary of the lipid bilayer as computed with the PPM server.

A zone predicted to interact with the Cl− ion as proposed in the mouse SLC26A9 experimental structure is highlighted by a black dashed circle in subunit a for orientation. R507 residues of both subunits are flagged and coloured in blue. This residue is located on the last helix of the transmembrane domain 22 residues prior to the beginning of the Sulfate Transporter Antagonist of anti-Sigma factor (STAS domain. The insertion loop that could not be built is shown as a dashed line.

The mutation is expected to destabilise the protein and to impede optimal insertion into the lipid bilayer." data-icon-position data-hide-link-title="0">Figure 1 In silico analysis of the putative effects of the R507W substitution on the SLC26A6 protein structure. The two subunits are presented with one subunit coloured in green and the other in orange (cartoon representation). The olive spheres represent the boundary of the lipid bilayer as computed with the PPM server. A zone predicted to interact with the Cl− ion as proposed in the mouse SLC26A9 experimental structure is highlighted by a black dashed circle in subunit a for orientation.

R507 residues of both subunits are flagged and coloured in blue. This residue is located on the last helix of the transmembrane domain 22 residues prior to the beginning of the Sulfate Transporter Antagonist of anti-Sigma factor (STAS domain. The insertion loop that could not be built is shown as a dashed line. The mutation is expected to destabilise the protein and to impede optimal insertion into the lipid bilayer.View this table:Table 1 Characteristics, blood and urine analyses and calcium-loading test of the patientsIdentification of a rare heterozygous missense mutation in Slc26a6Through whole-exome sequencing of the proband’s DNA, we investigated rare coding variants of potential interest with minor allele frequency below 0.1% in the Genome Aggregation Database (GnomAD V.2.1.1) in a list of genes or candidate genes linked with NL (online supplemental table 1).

We did not find any pathogenic or likely pathogenic variant according to the criteria of the American College of Medical Genetics and Genomics.20 No pathogenic or likely pathogenic variant or variant of unknown significance was detected in the three genes involved in PH. However, we found a rare heterozygous variant of unknown significance located in the 13th coding exon of SLC26A6 (NM_022911.2. C.1519C>T. P.R507W).

SLC26A6 encodes an oxalate transporter involved in hyperoxaluria and NL based on mouse knockout studies.12 13 According to GnomAD (V.2.1.1), SLC26A6 is intolerant to loss-of-function variation with an observed/expected score of 0.65 (0.48–0.91), and the p.R507W mutation is very rare, as it has been found in only 5 out of 280 674 alleles reported in GnomAD (in Europeans and South Asians). According to SIFT, Mutation Taster and PolyPhen-2 (HumDiv), the p.R507W mutation was predicted to be deleterious, disease causing and damaging, respectively.21–23Supplemental materialHuman SLC26A6 residue p.R507 was found fully conserved in an interspecies comparison. The conservation of this residue was also very high in this family of proteins (ie, the score at position 507 was equal to 8, the maximum is 9, via the Consurf server). We then built a homology model using as experimental template the mouse Slc26a9 homodimer anion transporter tri-dimensional (3D structure (figure 1).

The modelling was possible as the overall sequence identity between the two proteins is around 40% (see online supplemental materials).The human SLC26A6 model structure positioned into a membrane is shown in figure 1. P.R507 is located on the C-terminal side of the last transmembrane helix, prior to the STAS domain. The region of p.R507 is predicted to be essentially rigid (PredyFlexy computation) and thus not very tolerant to the p.R507W substitution, which was predicted to likely perturb correct interaction with the membrane. The details of our in silico analyses are provided as online supplemental materials.Family investigation of the Slc26a6 p.R507W mutationSince the SLC26A6 mutation was potentially pathogenic, both parents of the proband were assessed.

Sanger sequencing identified the same heterozygous SLC26A6 mutation in the father, while the mother was WT (online supplemental figure S2). The mother had no personal or familial history of kidney stones and exhibited normal levels of urinary oxalate (250 µmol/day, N. 40–330).Characterisation of the R507W SLC26A6 mutation in transfected OKP cells. OKP cells were transfected with equivalent amounts (0.1 µg cDNA per well of a 24-well dish) of either wild-type (WT.

HA-WT) or mutant (MT. HA-MT) human SLC26A6 cDNA. Cells were assayed 72 hours post-transfection. For each transfection (Tx-1 to 3), uptakes and companion biotinylations were performed in triplicate.

Utx, untransfected control. Panels A+B show representative images selected from each transfection. Western blots were probed with the antihuman Slc26a6 polyclonal antibody, R29. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. To facilitate comparisons between transfections (n=3 independent transfections), MT expression levels for each transfection were normalised to mean densitometry values for the WT densitometry values for each transfection. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly.

Panel D. Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT. HA-MT) Slc26a6.

The uptake values are presented as pmoles 14C-oxalate uptake per well of a 24-well plate and are not normalised to levels of expressed protein. WT, wild type. HA-WT, hemagglutinin-tagged wild type. MT, mutant.

HA-MT, hemagglutinin-tagged mutant. Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 2 Characterisation of the R507W SLC26A6 mutation in transfected OKP cells. OKP cells were transfected with equivalent amounts (0.1 µg cDNA per well of a 24-well dish) of either wild-type (WT.

HA-WT) or mutant (MT. HA-MT) human SLC26A6 cDNA. Cells were assayed 72 hours post-transfection. For each transfection (Tx-1 to 3), uptakes and companion biotinylations were performed in triplicate.

Utx, untransfected control. Panels A+B show representative images selected from each transfection. Western blots were probed with the antihuman Slc26a6 polyclonal antibody, R29. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. To facilitate comparisons between transfections (n=3 independent transfections), MT expression levels for each transfection were normalised to mean densitometry values for the WT densitometry values for each transfection. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly.

Panel D. Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT. HA-MT) Slc26a6.

The uptake values are presented as pmoles 14C-oxalate uptake per well of a 24-well plate and are not normalised to levels of expressed protein. WT, wild type. HA-WT, hemagglutinin-tagged wild type. MT, mutant.

HA-MT, hemagglutinin-tagged mutant. Tx, transfection. Utx, untransfected.Even though the father did not report any renal colic, biochemical analyses revealed that he also had hyperoxaluria (520 µmol/day, N. 40–330).

The calcium loading test was strictly normal, and no other risk factor for NL was identified for the father (table 1). However, we noticed that he exhibited a urine output of almost 4 L/day reflecting a very high-water intake. This high urine output may explain why he never experienced kidney stones. Furthermore, contrasting with the proband, he did not exhibit any hypocitraturia.

No other relatives from same family were accessible for medical examination and genetic testing.Response to medical treatmentWe hypothesised that the proband presented with inherited enteric hyperoxaluria due to deficient SLC26A6-mediated oxalate secretion resulting in increased net absorption of dietary oxalate. We advised her to increase drinking water intake in order to reach a urinary output of at least 3 L/day and to systematically add a dairy product to each meal in order to increase her calcium intake up to the recommended daily amounts of 900 mg/day. The latter intervention turned out to be very effective, as urinary oxalate excretion markedly dropped from >700 µmol/day down to ~300 µmol/day on repeated control analyses while the mild hypocitraturia persisted (1.49 mmol/24 hours N<1.67). No other medication was initiated, and NL also dramatically improved, as she has not experienced any recurrence of kidney stones for 2 years of follow-up.Physiological characterisation of the variantIn order to assess the pathogenicity of the SLC26A6 p.R507W mutation, we next assessed its effect on protein expression, protein trafficking and chloride-dependent oxalate transport.

Under physiological conditions, apical membrane SLC26A6 in epithelial cells mediates oxalate secretion by operating in the direction of exchanging intracellular oxalate for extracellular chloride. However, SLC26A6 can mediate Cl−-oxalate exchange in either direction depending on the direction of the net driving force. We measured Cl−-gradient stimulated 14C-oxalate uptake as a measure of oxalate transport activity mediated by human SLC26A6 transfected into OKP epithelial cells, as in previous studies of SLC26A6 glycosylation mutants.19 We specifically evaluated the chloride-dependent component of 14C-oxalate uptake that was mediated by the transfected human SLC26A6 expression constructs by correcting for endogenous oxalate transport (see Methods and online supplemental figure S3 for details).The R507W mutation directly impairs chloride-dependent oxalate transport capability in human SLC26A6. The initial characterisation of the Δ507 mutation suggested that the degree of inhibition of the Slc26a6-mediated chloride-dependent oxalate transport activity was significantly greater than that predicted by decreased protein expression alone.

To better address that possibility, we transfected OKP cells with 2.5× more mutant (MT) than wild-type (WT) human SLC26A6 (hA6) in an effort to significantly enhance the apparent transport activity of Δ507-hA6. Cells were transfected with either 0.25 µg of HA-MT (MT) or 0.1 µg HA-WT (WT) cDNA per well of a 24-well plate. The experiments were performed and data presented identically as in panels A–C of figure 1. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly. Panel D.

Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT. HA-MT) Slc26a6. The principal objective of this experiment was to directly address the transport capability of mutant relative to wild-type Slc26a6 independent of protein expression.

To that end, the transport data presented in panel D were normalised to cell surface biotinylatable hA6 for each transfection. Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 3 The R507W mutation directly impairs chloride-dependent oxalate transport capability in human SLC26A6. The initial characterisation of the Δ507 mutation suggested that the degree of inhibition of the Slc26a6-mediated chloride-dependent oxalate transport activity was significantly greater than that predicted by decreased protein expression alone.

To better address that possibility, we transfected OKP cells with 2.5× more mutant (MT) than wild-type (WT) human SLC26A6 (hA6) in an effort to significantly enhance the apparent transport activity of Δ507-hA6. Cells were transfected with either 0.25 µg of HA-MT (MT) or 0.1 µg HA-WT (WT) cDNA per well of a 24-well plate. The experiments were performed and data presented identically as in panels A–C of figure 1. Panel A.

Western analysis of total cell lysates isolated from each transfection condition. Panel B. Western analysis of cell surface biotinylatable hA6 from each transfection condition. Panel C.

Expression levels of total and cell surface biotinylatable hA6. Expression levels were determined by densitometry. All WT levels are expressed as 100 (%), and the individual MT values vary accordingly. Panel D.

Chloride-dependent 14C-oxalate uptake attributable to transfected wild-type (WT. HA-WT) and mutant (MT. HA-MT) Slc26a6. The principal objective of this experiment was to directly address the transport capability of mutant relative to wild-type Slc26a6 independent of protein expression.

To that end, the transport data presented in panel D were normalised to cell surface biotinylatable hA6 for each transfection. Tx, transfection. Utx, untransfected.Characterisation of the effect of the p.R507W mutation on expression and function of human SLC26A6 transfected into OKP cells is shown in figure 2. Results are illustrated for three independent experiments in which OKP cells were transfected with identical amounts of cDNA encoding the WT and mutant (MT) transporters.

Figure 2A shows immunoblots of total cell lysate and compared transfected cells with an untransfected control lane. Transfection of OKP cells results in expression of a minor band just under 75 kDa and multiple prominent bands >100 kDa. As demonstrated in a previous study, the lower band corresponds in size to nascent unglycosylated SLC26A6, whereas the upper bands correspond to complex-glycosylated SLC26A6.19 Surface biotinylated SLC26A6 is shown in figure 2B, corresponding in size to the complex-glycosylated forms of SLC26A6. As illustrated by the immunoblots in figure 2A and B and summarised by the densitometry in figure 2C, there was a marked reduction in glycosylated and cell surface expression of the mutant transporter compared with WT.

Although these findings indicate that the p.R507W mutation leads to decreased processing and trafficking, and/or increased degradation of the transporter, the qualitative similarity of the western blot molecular weight band profiles of the WT and MT constructs strongly suggests that the mutation does not directly affect the glycosylation of SLC26A6 per se. As shown in figure 2D, chloride-dependent 14C-oxalate uptake mediated by mutant SLC26A6 was also markedly impaired. Of interest, whereas the mutation resulted in approximately a 50% reduction in cell surface expression of SLC26A6 (figure 2C), it led to a disproportionately higher reduction of Cl−-dependent oxalate transport activity of approximately 75%. This suggested that the mutation might affect both the expression and the intrinsic transport capability of SLC26A6.We therefore next designed an experiment to test directly whether the mutation causes a defect in the intrinsic transport function of SLC26A6 in addition to the defect in net surface expression demonstrated in figure 2.

Cells were transfected with 2.5-fold more mutant than WT cDNA with the goal of approximately equalising surface expression of the mutant and WT transporters. As illustrated by the immunoblots in figure 3A and B and summarised by the densitometry in figure 3C, the protocol achieved roughly equivalent amounts of the glycosylated and surface expressed forms of the mutant and WT transporters. Most importantly, assessment of transport activity normalised to surface expression, as shown in figure 3D, indicated that the p.R507W mutation does indeed cause a marked defect (approximately 50%) in intrinsic transport activity of SLC26A6. The findings of figures 2 and 3 taken together suggest that the 75% defect in transport activity of mutant compared with WT SLC26A6 when equal amounts of cDNA are transfected (figure 2D) results from about a 50% defect in cell surface expression (figure 2C) and a roughly 50% defect in intrinsic transporter activity (figure 3D).Given that SLC26 family transporters exist as dimers,18 24 we conducted additional studies to test the possibility of a dominant negative effect of the p.R507W mutation on expression of the WT transporter.

Our approach was to assay expression of myc-tagged WT SLC26A6 as a function of cotransfection with either HA-tagged WT or HA-tagged mutant transporter. We chose transfection conditions to achieve equivalent expression levels of HA-WT and HA-MT SLC26A6 (online supplemental figure S4). As illustrated by the immunoblots in figure 4A and B, and summarised by the densitometry in figure 4C, coexpression of mutant SLC26A6 caused 70% reduction in glycosylated and cell surface expression of myc-tagged WT SLC26A6 compared with coexpression of WT SLC26A6. These results support the possibility that the p.R507W mutation may have a dominant negative effect on expression of the WT transporter so that the hyperoxaluria phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone.Coexpression of the R507W mutant significantly reduces both total and cell surface expression of wild-type SLC26A6.

OKP cells were cotransfected with 0.1 µg myc-tagged human SLC26A6 (myc-WT) cDNA and either 0.1 µg HA-tagged wild-type Slc26a6 (HA-WT) cDNA or 0.25 µg HA-tagged Arg507Trp mutant Slc26a6 (HA-MT) cDNA per well of a 24-well dish. Cotransfection conditions were selected to achieve equivalent levels of expression of each HA-tagged cDNA construct to facilitate a direct comparison of the effects of coexpression of each protein. Cells were assayed 72 hours postcotransfection. See online supplemental figure S3 for a representative cotransfection experiment illustrating simultaneous expression of each epitope-tagged construct.

Panel A. Western analysis of myc-tagged wild-type Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B. Western analysis of cell surface biotinylatable myc-tagged Slc26a6 from each cotransfection.

Western blots depicted in panels A+B were probed with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 1:5000 dilution). Panel C. Expression levels of total and cell surface biotinylatable myc-tagged Slc26a6.

Expression levels were determined by densitometry. To facilitate comparisons between transfections (n=4 independent transfections) myc-WT:HA-MT cotransfection expression levels were expressed relative to the densitometry values for myc-WT:HA-WT cotransfection expression levels for each transfection and myc-WT:HA-WT cotransfection expression levels were normalised to a mean value of 100 (%). Co-IP, coimmunoprecipitation. Co-Tx, cotransfection.

IP, immunoprecipitation. Tx, transfection. Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 4 Coexpression of the R507W mutant significantly reduces both total and cell surface expression of wild-type SLC26A6. OKP cells were cotransfected with 0.1 µg myc-tagged human SLC26A6 (myc-WT) cDNA and either 0.1 µg HA-tagged wild-type Slc26a6 (HA-WT) cDNA or 0.25 µg HA-tagged Arg507Trp mutant Slc26a6 (HA-MT) cDNA per well of a 24-well dish.

Cotransfection conditions were selected to achieve equivalent levels of expression of each HA-tagged cDNA construct to facilitate a direct comparison of the effects of coexpression of each protein. Cells were assayed 72 hours postcotransfection. See online supplemental figure S3 for a representative cotransfection experiment illustrating simultaneous expression of each epitope-tagged construct. Panel A.

Western analysis of myc-tagged wild-type Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B. Western analysis of cell surface biotinylatable myc-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+B were probed with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25.

1:5000 dilution). Panel C. Expression levels of total and cell surface biotinylatable myc-tagged Slc26a6. Expression levels were determined by densitometry.

To facilitate comparisons between transfections (n=4 independent transfections) myc-WT:HA-MT cotransfection expression levels were expressed relative to the densitometry values for myc-WT:HA-WT cotransfection expression levels for each transfection and myc-WT:HA-WT cotransfection expression levels were normalised to a mean value of 100 (%). Co-IP, coimmunoprecipitation. Co-Tx, cotransfection. IP, immunoprecipitation.

Tx, transfection. Utx, untransfected.We next performed a series of WT/MT co-immunoprecipitation studies to verify the association of MT with WT transporter to explain the dominant negative phenotype. OKP cells were cotransfected with myc-tagged WT SLC26A6 and either HA-tagged WT SLC26A6 or HA-tagged MT SLC26A6 as described for the experiment outlined in figure 4. The cells were solubilised with 1% digitonin, subjected to immunoprecipitation with an anti-myc antibody, and the resulting immunoprecipitates were analysed by SDS-PAGE and western blot with antibodies directed against either the myc-tag or the HA-tag (figure 5).The R507W substitution does not inhibit association of MT-A6 with WT-A6.

The principal objective of this experiment was to determine if the p.R507W mutation affected the ability of the resultant mutant to associate with wild-type (WT) Slc26a6 and form the prototypic Slc26a6 multimeric complex. By using different epitope tags for the potential binding partners, we used anti-myc tag (WT) primary immunoprecipitation to assess association with HA-tagged protomers (WT +MT) by monitoring levels of coimmunoprecipitation. OKP cells were cotransfected (Co-Tx) as described for figure 3, solubilised with 1% digitonin and subjected to immunoprecipitation with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25. 5 µg IgG/mL lysate).

Panel A. Western analysis of myc-tagged WT Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B. Western analysis of HA-tagged Slc26a6 expression in total cell lysates isolated from each cotransfection.

Panel C. Western analysis of primary immunoprecipitated myc-tagged WT Slc26a6 from each cotransfection. Panel D. Western analysis of coimmunoprecipitated HA-tagged Slc26a6 from each cotransfection.

Western blots depicted in panels A+C were probed with anti-myc antibody R950-25 (1:5000 dilution) and those in panels B+D were probed with anti-HA antibody 71–5500 (1:50 dilution). Panel E. Relative expression of primary immunoprecipitated myc-tagged WT Slc26a6 and coimmunoprecipitated HA-tagged WT or mutant Slc26a6. Expression levels were determined by densitometry.

To facilitate comparisons between cotransfections (n=3 individual cotransfection events. Tx-1 to Tx-3), protein expression levels determined for the myc-WT/HA-MT cotransfection condition were expressed relative to the densitometry values for the myc-WT/HA-WT cotransfection condition for each experiment, and myc-WT:HA-WT cotransfection expression levels were normalised to 100 (%). *Not significantly different at p=0.3566. Tx, transfection.

Utx, untransfected." data-icon-position data-hide-link-title="0">Figure 5 The R507W substitution does not inhibit association of MT-A6 with WT-A6. The principal objective of this experiment was to determine if the p.R507W mutation affected the ability of the resultant mutant to associate with wild-type (WT) Slc26a6 and form the prototypic Slc26a6 multimeric complex. By using different epitope tags for the potential binding partners, we used anti-myc tag (WT) primary immunoprecipitation to assess association with HA-tagged protomers (WT +MT) by monitoring levels of coimmunoprecipitation. OKP cells were cotransfected (Co-Tx) as described for figure 3, solubilised with 1% digitonin and subjected to immunoprecipitation with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25.

5 µg IgG/mL lysate). Panel A. Western analysis of myc-tagged WT Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B.

Western analysis of HA-tagged Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel C. Western analysis of primary immunoprecipitated myc-tagged WT Slc26a6 from each cotransfection. Panel D.

Western analysis of coimmunoprecipitated HA-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+C were probed with anti-myc antibody R950-25 (1:5000 dilution) and those in panels B+D were probed with anti-HA antibody 71–5500 (1:50 dilution). Panel E. Relative expression of primary immunoprecipitated myc-tagged WT Slc26a6 and coimmunoprecipitated HA-tagged WT or mutant Slc26a6.

Expression levels were determined by densitometry. To facilitate comparisons between cotransfections (n=3 individual cotransfection events. Tx-1 to Tx-3), protein expression levels determined for the myc-WT/HA-MT cotransfection condition were expressed relative to the densitometry values for the myc-WT/HA-WT cotransfection condition for each experiment, and myc-WT:HA-WT cotransfection expression levels were normalised to 100 (%). *Not significantly different at p=0.3566.

Tx, transfection. Utx, untransfected.As observed in the previous experiment (figure 4), cotransfection of mutant HA-A6 with wild-type myc-A6 led to a dramatic decrease in expression of mature WT myc-A6 in both the solubilised lysate (figure 5A) and the companion primary immunoprecipitation (figure 5C). Western analysis of the solubilised lysate with the anti-HA antibody (figure 5B) confirmed that cotransfection with 2.5× HA-MT relative to HA-WT resulted in roughly similar levels of expression of the two HA-tagged SLC26A6 expression constructs. We observed significant coimmunoprecipitation of both wild-type and mutant HA-A6 with the myc-tagged WT-A6 (figure 5D).

Despite the presence of similar amounts of HA-WT and HA-MT in the initial cell lysates for each experiment (figure 5B), their relative recoveries by coimmunoprecipitation could only reflect, at best, the relative recovery of the primary immunoprecipitated binding partner (myc-WT). If the two constructs multimerise with wild-type SLC26A6 with a similar efficacy, their relative recoveries by coimmunoprecipitation should closely match the relative recovery of myc-WT in the primary immunoprecipitation for each transfection condition. The apparent decrease in coimmunoprecipitation of HA-MT relative to HA-WT observed in figure 5D and quantified in figure 5E is not significantly different to that observed for the primary immunoprecipitation of myc-WT observed in figure 5B when cotransfected with HA-WT versus HA-MT. This strongly suggests that the p.R507W mutation does not impair the efficacy of formation of the SLC26A6 multimeric complex per se.

The demonstration of formation of MT/WT heteromultimers can explain the dominant negative effect of the MT transporter if it is misfolded, has altered stability in the plasma membrane and/or has reduced oxalate transport ability.Identification of additional deleterious Slc26a6 mutations in cases with nl from UK BiobankWe next analysed up to 200 619 samples with available exome sequencing data in UK Biobank. Unfortunately, NL was not well recorded in the UK Biobank phenotypes as we identified 5267 patients with recorded NL that corresponds to a prevalence of 2% only. This prevalence is very far from the well-known prevalence of NL in the UK (13%).25 Following a case-only design, we found two heterozygous protein-truncating SLC26A6 mutations (NM_022911.3:c. 3G>A/p.Met1?.

and NM_022911.3:c.1132C>T/p.Gln378*) in two males. These two mutations were very rare in GnomAD (with a minor allele frequency <0.00005). Those two additional independent cases support a possible contribution of SLC26A6 loss of function mutations to NL. Unfortunately, urinary oxalate excretion is not among the urine parameters available in the UK Biobank phenotype.DiscussionWe show complementary genetic and functional evidence for a novel mechanism of inherited enteric hyperoxaluria in humans.

Using a candidate gene analysis of whole-exome sequencing data, we identified a rare c.1519C>T mutation in the SLC26A6 gene encoding an oxalate transporter in a family affected by hyperoxaluria. This mutation leads to the substitution of the arginine in position 507 by a tryptophan. Further extensive in vitro characterisation revealed that the mutation decreases both SLC26A6 transport function and membrane expression. Moreover, the mutant allele also exerts a profound dominant negative effect on the WT allele, suggesting that the mutation might affect SLC26A6-mediated oxalate secretion more than gene haploinsufficiency alone would do.The results of our experiments demonstrating that coexpression of mutant and WT SLC26A6 decreases total and cell-surface expression of the WT protein, that mutant SLC26A6 is capable of forming multimeric structures with WT SLC26A6 and that the presence of a tryptophan in position 507 is predicted to destabilise the interaction of the mutant SLC26A6 monomer with the lipid bilayer suggest that potential mutant–mutant homodimers and mutant–wild type heterodimers are both very likely to be unstable in the cell plasma membrane.

The dominant-negative effect of the mutant SLC26A6 on the wild-type protein assembled in the heterodimer (WT/MT SLC26A6) combined with the inhibitory effect of the mutation itself expressed in the homodimer (MT/MT SLC26A6) would be expected to decrease the production of functional SLC26A6 in the plasma membrane by much more than the 50% predicted by the heterozygous mutation.SLC26A6 is an anion exchanger from the solute carrier family 26 expressed at the apical membrane of many types of epithelial cells including enterocytes in the gastrointestinal tract.26 It can exchange intracellular oxalate for external chloride, and hence, performs apical oxalate secretion.27 Oxalate transport across the intestinal epithelium results from the combination of passive paracellular absorption and active transcellular secretion.28 Mouse studies have shown that SLC26A6 plays a critical role in intestinal secretion of oxalate.12 13 Therefore, inactivation of Slc26a6 in mice enhances net oxalate absorption, leading to higher plasma oxalate and ultimately increased urinary oxalate excretion, particularly when dietary intake of oxalate is high.12 Our findings suggest that the c.1519C>T/p.R507W mutation in SLC26A6 may cause similar pathophysiology resulting in enteric hyperoxaluria in humans. In this regard, it is striking that the patient’s hyperoxaluria was dramatically reduced by adding calcium to her diet, strongly suggesting that hyperoxaluria in her case was mostly from enteric origin and can be controlled by treatments that limit passive paracellular absorption of oxalate.Four previous studies have assessed the possible association of SLC26A6 mutations with hyperoxaluria and calcium oxalate urolithiasis:In the first study, the authors screened a cohort of 94 patients with PH and 96 controls.14 The non-synonymous SLC26A6 variants that were detected in cases were actually frequent (minor allele frequency ≥1%) in one or more populations from the GnomAD browser (v2.1.1). A c.616G>A (p.V206M) mutant was most common (11%) and showed a 30% reduction in oxalate transport activity. However, heterozygosity for this variant did not affect plasma or urine oxalate levels in the study population.

There was no significant effect of any identified variants on oxalate excretion. The p.V206M variant of SLC26A6 was subsequently described in a second study.15 This variant was highly frequent (minor allele frequency (MAF) ~10% in all populations). Again, this variant was not associated with urolithiasis or with hyperoxaluria. In a third study, Lu et al16 functionally assessed non-synonymous SLC26A6 variants that were listed in the public database dbSNP, according to in silico analyses.

The authors found a significant association between the low-frequency (but not rare) rs184187143 variant (with a MAF of 0.4% in the Finnish population) and urolithiasis risk (unadjusted p value=0.007). However, no mechanistic insight potentially explaining this association was provided. Finally, two additional heterozygous variants of SLC26A6 have recently been reported.17 The first rare p.R621G variant was identified in a non-stone former individual who exhibited a normal (<40 mg/d) urinary excretion of oxalate. According to several in silico programmes (PolyPhen, SIFT, Mutation Taster, Align GVGD), this mutation was totally benign.

The second rare p.D674N variant that abolished SLC26A6 expression and Cl−-dependent bicarbonate transport when transfected in HEK cells was identified in a patient presenting with calcium oxalate urolithiasis. However, the authors did not report whether this variant was cosegregating with lithiasis in the family of the patient. Moreover, in the proband the mutation was not associated with hyperoxaluria but rather with a marked hypocitraturia, in line with previous studies from same group showing that mutation of the STAS domain of SLC26A6 can provoke hypocitraturia, another important risk factor for NL, by altering regulation of the citrate transporter NaDC-1.29 30 A very mild hypocitraturia, along with the marked hyperoxaluria, was also observed in our patient. It is very unlikely that such a mild decrease in urinary citrate excretion could cause such a high-frequency recurrent stone disease course, but it may have contributed along with hyperoxaluria to the increased risk for NL.In summary, our observation that mutation p.R507W is associated with hyperoxaluria in humans supports the hypothesis that like in the mouse SLC26A6 plays a critical role in intestinal secretion of oxalate, thereby limiting net absorption of dietary oxalate.

Consistent with this mechanistic hypothesis, we observed a beneficial effect of increasing calcium in the patient’s diet to reduce enteric oxalate absorption and thereby urinary oxalate excretion. Accordingly, other mutations leading to defects in function of oxalate transporter SLC26A6 could be responsible for inherited form of enteric hyperoxaluria and NL in human patients. Therefore, screening for mutations in SLC26A6 should be performed in patients with hyperoxaluria in whom no mutations in the PH disease genes AGXT, or GRHPR or HOGA are found, or in patients with enteric hyperoxaluria who have no evidence of intestinal disease.Supplemental materialData availability statementAll data relevant to the study are included in the article or uploaded as supplementary information. Not applicable.Ethics statementsPatient consent for publicationConsent obtained directly from patient(s)Ethics approvalThis study involves human participants and was approved by Institutional review board of the University Hospital of La Réunion (ID :2019/CHU/10).

Participants gave informed consent to participate in the study before taking part.AcknowledgmentsWe would like to thank the patient and her family for their kind help and participation in the study. We would also like to thank Frédéric Allegaert and Nicolas Larcher for expert technical assistance. This research has been conducted using the UK Biobank Application #67 575..

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Again, in humans, staying mentally active with age has been tied to lower odds of cognitive decline -- though, yet again, the cause-effect question remains.

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