Category Archives: Transhuman News

15 Aug 2017

Genetic forces drive a sizable portion of Alzheimers disease, yet only a fraction of cases thus far are explained by known mutations. A handful of recent papers used genomic sequencing to fish out new variants that, while exceedingly rare, pack a wallop in those who carry them. In the July 24 JAMA Neurology, researchers led by Margaret Pericak-Vance at the University of Miami in Florida reported that mutations in four endolysosomal transport genes boosted risk of early onset AD (EOAD). A few weeks earlier, a large collaboration of French researchers reported rare new TREM2, ABCA7, and SORL1 variants in Neurobiology of Aging, while scientists led by Henne Holstege at VU University Medical Center in Amsterdam characterized the pathogenicity of SORL1 variants and even proposed classifying this endosomal sorting protein as the fourth autosomal-dominant AD gene. A team led by Dominique Campion at University of Rouen, France, dug deep into the well-trodden territory of the three autosomal-dominant genesAPP, PS1, and PS2and uncovered de novo pathogenic variants that cropped up in people with no family history of AD. Last but not least, Anne Rovelet-Lecrux, also at Rouen, linked a duplication in the tau gene to people with an AD diagnosis who lacked Aplaques.

Together, the studies move the field a step closer to filling in the missing genetic influence on AD, and could provide new targets for therapeutic strategies, commented Liana Apostolova at the University of Indiana in Bloomington. There are more genetic risk factors in hiding that have yet to be discovered, and these studies suggest were on the right track, she toldAlzforum.

In the JAMA Neurology study, first author Brian Kunkle and colleagues report on their search for rare variants with large effects on AD risk. Reasoning that people with EOAD are likely carriers of damaging genetic mutations, they conducted whole-exome sequencing in 51 non-Hispanic white EOAD patients, plus 53 people from 19 Caribbean Hispanic families with EOAD; all had tested negative for known causal mutations in APP, PS1, and PS2. The scientists combed the exomes for variants predicted to have damaging effects, then attempted to validate each variants association with AD using exome genotyping data from a separate cohort of 1,500 EOAD patients, 7,000 LOAD patients, and 7,000 controls. Developed by the Alzheimers Disease Genetics Consortium (ADGC), the exome chip used to genotype this separate cohort contains more than 200,000 variants, most of which are functional, rare, single nucleotidemutations.

In their original sequencing cohort, the researchers identified mutations in known or suspected EOAD genes, including SORL1, PS1, PS2, TREM2, and MAPT. Some were known; others were new variants in genes previously tied to LOAD, including HLA-DRB1, ABCA7, and RIN3. Suspicious mutations also cropped up in genes without an AD record. A missense mutation in TCIRG1, present in a non-Hispanic white person with EOAD and segregating with EOAD in three Hispanic families, was twice as frequent in EOAD than in controls in the validation cohort. Deleterious mutations in PSD2 appeared in multiple unrelated cases in the sequencing cohort, and associated with EOAD in the validation cohort, at least when considered in the aggregate. Mutations in RIN3 and RUFY1 appeared in the discovery cohort, but their EOAD association in the validation group was nominal. Importantly, all four genes function in different parts of the endolysosomal transport pathway, which is essential for clearing cellular debris and unwanted proteins, includingA.

The researchers found additional rare mutations in EOAD patients, but these were not on the exome chip used for the validation cohort. For example, of 151 potentially damaging variants that appeared in the original exome sequencing cohort, only 43 were included on the exomechip.

While this filtering process allows researchers to test whether a variant is truly linked to disease, it also precludes consideration of totally new, potentially damaging mutations, said Holstege. The mutations that are most damaging are also the most rare, Holstege told Alzforum. If you filter them out in this way, you quench yoursignal.

Holstege took a different tack to find and classify rare SORL1 variants. Rather than filtering out undocumented variants, Holstege and colleagues made them their bread and butter. In the May 24 European Journal of Human Genetics, they reported 115 SORL1 variants from the exomes of a Dutch cohort of 640 AD cases and 1,268 controls. Fifteen of these variants were common, and not associated with AD. The other 100 were rare, occurring in less than 0.01 percent of the population. Of those, 19 were predicted to be strongly damaging, based on high scores on CADD, an algorithm that considers more than 100 variant characteristics to predict how likely a given mutation is to alter protein function orexpression.

Strikingly, 16 of these suspicious variants only appeared in a single person within the entire cohort, and 14 of those had AD. None of the variants were included in prior exome genotyping studies, so the researchers could not draw upon past data to validate whether they truly associated with the disease. Instead, the researchers developed a pathogenicity scale. Weaving in data from more than 3,000 exomes sequenced separately, the researchers classified a total 181 SORL1 variants based on their combined CADD scores and rarity. They categorized those that had high CADD scores and were very rare as pathogenic. Estimated pathogenicity decreased from likely pathogenic to uncertain to likely benign to benign as variants became less damaging and morecommon.

The scientists found that a combination of high CADD scores and extremely low allele frequency selected out those SORL1 variants that occurred much more often in cases than in controls. The 13 variants with the highest pathogenicity resulted in truncations of SORL1, and occurred only in AD cases. The researchers predicted they would cause dominantly inherited AD, though none have yet been traced in familypedigrees.

Holstege and colleagues proposed that SORL1 take a spot alongside PS1, PS2, and APP as an autosomal-dominant AD gene. Pathogenic SORL1 mutations occurred in 2 percent of the AD cases in this study, placing them at a higher frequency than other ADAD genes. Like PS1, PS2, and APP, SORL1 protein strongly influences A, as it protects APP from amyloidogenic processing and ushers A to lysosomes for disposal (see Sep 2007 news; Feb 2014 news).

Classifying SORL1 as an ADAD gene would raise new questions. How to provide genetic counseling to affected families? Should mutation carriers be eligible to join the Dominantly Inherited Alzheimers Network (DIAN)? Clinical-grade genetic tests for SORL1 variants would be needed, a challenge developers may postpone until further data has confirmed the mutations are pathogenic, commented Apostolova. She added that while Holsteges pathogenicity scale is an exciting tool that should be used in future studies, validation of each mutation in other cohorts, as well as functional evidence in animal and cell culture studies, should be required to elevate SORL1 to ADAD status. Rovelet-Lecrux agreed that designating SORL1 an ADAD gene will have to await discovery of multigenerational families in which SORL1 segregates with disease in an autosomal-dominant pattern. Until we accumulate more genetic evidence, we cannot tell SORL1 mutation carriers how likely they are to develop disease, shesaid.

A new study led by Rouens Campion and co-authored by Rovelet-Lecrux further supports pathogenicity of SORL1 variants, even if it does not provide evidence of multigenerational segregation. As reported July 13 in the Neurobiology of Aging, the researchers detected SORL1 missense and protein-truncating variants that associated strongly with early onset disease by doing whole-exome and genome sequencing of a French cohort of 852 EOAD, 927 LOAD, and 1,273 control cases. All but one of 13 protein-truncating variants occurred only in EOAD cases, and eight of 10 cases with available family information had a history of the disease. Besides SORL1, TREM2 and ABCA7 also harbored potentially damaging EOAD-associated variants in this sample. The researchers estimated that variants in these three genes accounted for 1.42, 1.17, and 1.33 percent of EOAD heritability, respectively. By comparison, ApoE4 accounted for 9.12percent.

New Finds in Old Genes While many pathogenic mutations in PS1, PS2, and APP have been traced in family pedigrees, additional rare variants in these established ADAD genes may yet be discovered. In search of them, researchers led by Rouens Campion sequenced these genes in 129 sporadic cases of early onset AD, as well as in 53 affected families. Published March 28 in PLOS Medicine, the findings included data from participants who joined the ongoing French study after 2012, when the researchers had published a similar analysis (Wallon et al., 2012).In all, first author Hlne-Marie Lanoisele and colleagues identified 44 PS1, two PS2, and 20 APP mutations, as well as five APP duplications; 12 of the PS1 and one PS2 mutation had not been reportedpreviously.

The most striking finding was the existence of de novo mutations in PS1. Indeed, seven of 12 new mutations occurred in sporadic cases of EOAD. In three of these mutations, the researchers were able to confirm that the carriers parents did not carry the new mutation. Rovelet-Lecrux, a co-author on the paper, said that the prevalence of de novo mutations in ADAD genes is likely underestimated, because routine genetic screening for these mutations is done only in familial AD cases. The de novo find underscores the importance of checking for pathogenic mutations even in patients without a family history of AD, especially people with an early age at onset, Holstege commented. Similar to the situation with rare SORL1 variants, researchers will need to decide how to categorize carriers of new and de novo mutations in established ADAD genes, shesaid.

Finally, results from a slightly older study led by Rovelet-Lecrux pose a different kind of classification conundrum. The authors deployed whole-exome sequencing to hunt specifically for copy number variations (CNVs) such as duplications and deletions in 335 genes predicted to influence A processing, clearance, or aggregation. The researchers found CNVs in 30 out of 522 people with EOAD, but only 18 out of 584 controls. Most of these CNVs occurred in a single person in the cohort, and they included novel deletions in the PS1, ABCA7, and SLC30A3 genes previously tied toAD.

A surprising finding reared its head in four AD cases, who all had a duplicationin a region of chromosome 17 including MAPT, the gene encoding none other than tau. The duplication appeared in two sporadic cases of EOAD and two with a family history. DNA available from one of those families confirmed that the duplication segregated with EOAD. Even though these four carriers had symptoms consistent with AD, the three who underwent amyloid-PET imaging had negative scans, to Rovelet-Lecruxs surprise. All four duplication carriers had abnormal levels of CSF p-tau and tau, and three of them also had abnormal concentrations of A42. The researchers also found nearly double the amount of tau mRNA in the blood of carriers than incontrols.

Together, the findings suggest that despite the lack of A plaques visible on PET, carriers of a tau duplication have a clinical disorder markedly similar to AD. The abnormality of CSF A42 in three of the duplication carriers suggests that they could have accumulated A just below the level of PET detection, a sub-threshold aggregation the researchers speculated could even be somehow caused by elevatedtau.

Do these tau duplication carriers have AD? Not if you consider A accumulation as a defining feature of the disease, said Apostolova. Indeed, in the paper, the researchers defined their disease as a tau-related dementia, proposing that it could account for a significant proportion of early onset dementia cases with no genetic explanation. While some researchers view A as a mere forerunner to the more destructive tau pathology, which they consider the main event in AD, Rovelet-Lecrux shied away from separating A from AD, saying that AD is ultimately diagnosed via its neuropathological hallmarks of A plaques and tau tangles. She believes it will be important to screen EOAD patients without A plaques for tau pathology, especially in the future once both A- or tau-targeted therapies exist.JessicaShugart

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The Search for the Missing AD Heritability Turns Up New Rare Variants - Alzforum

In an article published in American Journal of Human Genetics on 3 August 2017, an international group of 11 organisations with genetics expertise has issued a joint position statement, setting out 3 key positions on the question of human germline genome editing:

(1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy.

(2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research.

(3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.

This serendipitously timed statement comes on the heels of Shoukhrat Mitalipov and colleagues at Oregon Health and Science Universitys publication of an article in Nature reporting the successful use of CRISPR/Cas9 in human embryos to correct a mutation in a gene called MYBPC3 that causes a potentially fatal heart condition known as hypertrophic cardiomyopathy. The publication of this article has drawn the attention of the wider mainstream media and reignited the public debate as to the desirability, feasibility and ethics of editing the human genome in an inheritable way.

Gene editing - putting the paper in context

Whilst debates about the ethics of gene editing (both somatic and germline) go back decades, human germline genome editing has never before been realistically possible from a technical standpoint. That has changed with the advent of the CRISPR/Cas9 system, whose efficiency and ease of use has not only opened up the field of gene editing to a far larger number of companies and laboratories than previously, but has brought the editing of specific genes in a human embryo out of the realms of fantasy into reality. The potential for such technology to improve quality of life and prevent suffering caused by debilitating genetically inherited diseases has captured the imagination of many, particularly people living with currently intractable genetic conditions, their friends and family. However, the power of the technology has also conjured up the familiar spectres of playing God, the uncertainty of long term effects on individuals (and what it means to be human itself), marginalisation of the disabled or genetically inferior and the potential for inequality to manifest itself genetically as well as socioeconomically.

Germline cell editing poses significantly more concerning ethical and regulatory issues than somatic cell editing. The latter will only result in uninheritable changes to the genome of a population of cells in the particular individual treated, whilst the former involves genetic changes that will be passed down, for better or worse, to the individuals offspring.

In early 2015, the first study demonstrating that CRISPR/Cas9 could be used to modify genes in early-stage human embryos was published. Although the embryos employed for those experiments were not capable of developing to term, the work clearly demonstrated that genome editing with CRISPR/Cas9 in human embryos can readily be performed. That report stimulated many scientists and organisations to clarify their stance on the use of genome-editing methods. The United Kingdom and Sweden have both approved experiments for editing DNA of a human embryo but not those that involve implanting embryos. In the UK, Human Fertilisation and Embryology Authority (HFEA) has approved an application by developmental biologist Kathy Niakan, at the Francis Crick Institute in London, to use CRISPR/Cas9 in healthy human embryos. Currently, such experiments cannot be done with federal funding in the United States given a congressional prohibition on using taxpayer funds for research that destroys human embryos. Congress has also banned the U.S. Food and Drug Administration from considering a clinical trial of embryo editing. As would be expected, the safety requirements for any human clinical genome-editing application are extremely stringent.

However, earlier this year, US-based National Academy of Sciences (NAS) and the National Academy of Medicine (NAM), published a report that concluded using genome-editing technology, such as CRISPR/Cas9, to make alterations to the germline would be acceptable if the intention was to treat or prevent serious genetic disease or disorders, and the procedure was proven to be safe ( significant and, to an extent, subjective hurdles to be overcome).

The ASHG position paper

The position paper was the product of a working group established by the American Society of Human Genetics (ASHG), including representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counsellors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counsellors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The group, composed of a combination of research and clinical scientists, bioethicists, health services researchers, lawyers and genetic counsellors, worked together to integrate the scientific status of and socio-ethical views towards human germline genome editing.

As part of this process, the working group reviewed and summarised nine existing policy statements on gene editing and embryo research and interventions from national and international bodies, including The International Society for Stem Cell Research (2015) Statement on Human Germline Genome Modification, The Hinxton Group (2015) Statement on Genome Editing Technologies and the statement released following the International Summit on Human Gene Editing (2015) co-hosted by the National Academy of Sciences, National Academy of Medicine, Chinese Academy of Sciences and The Royal Society (UK). It was observed that differences in these policies include the very definition of what constitutes a human embryo or a reproductive cell, the nature of the policy tool adopted to promote the positions outlined, and the oversight/enforcement mechanisms for the policy. However, by and large, the majority of available statements and recommendations restrict applications from attempting to initiate a pregnancy with an embryo or reproductive cell whose germline has been altered. At the same time, many advocate for the continuation of basic research (and even preclinical research in some cases) in the area. One notable exception is the US National Institutes of Health, which refuses to fund the use of any gene-editing technologies in human embryos. Accordingly, due to the lack of public funding in the US, work such as that done by Mitalipovs group must be privately funded.

The working group considered that ethical issues around germline genome editing largely fall into two broad categories those arising from its potential failure and those arising from its success. Failure exposes individuals to a variety of health consequences, both known and unknown, while success could lead to societal concerns about eugenics, social justice and equal access to medical technologies.

The 11 organisations acknowledged numerous ethical issues arising from human germline genome editing, including:

Having touched on each of these issues, the group then outlined its consensus positions:

1. At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy.

It was noted that there is not yet a high quality evidence base to support the use of germline genome editing, with unknown risk of health consequences and ethical issues still to be explored and resolved by society.

The group observed that two major categories of safety concerns are (i) the effect of unwanted or off-target mutations, and (ii) the potential unintended effects of the desired on-target base changes (edits) being made. It noted that it is reasonable to presume that any human genome-editing therapeutic application will require stringent monitoring of off-target mutation rates, but there remains no consensus on which methods would be optimal for this, or what a desirable maximum off-target mutation rate would be when these techniques are translated clinically. The working-group thus outlined its views on the minimum necessary developments that would be required (at least from a safety perspective) before germline genome editing could be used clinically:

2. Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research.

The group agreed that conducting basic scientific [techniques?] involving editing of human embryos and gametes can be performed ethically via compliance with applicable laws and policies, and that any study involving in vitro genome editing on human embryos and gametes should be conducted under rigorous and independent governance mechanisms, including approval by ethics review boards and meeting any other policy or regulatory requirements. Public funding for such research was seen as important in ensuring that such research is not driven overseas or underground, where it would be subject to less regulation, oversight and transparency.

3. Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.

Even if the technical data from preclinical research reaches a stage where it supports clinical translation of human germline genome editing, the working group stresses that many more things need to happen before translational research in human germline genome editing is considered. The criteria identified by the group in this position cut across medical, ethical, economic and public participation issues and represent the setting of an appropriately high and comprehensive standard to be met before human germline genome editing may be applied clinically. The group acknowledges the challenges of public engagement with such technical subject matter but encourages new approaches to public engagement and engagement of broader stakeholder groups in the public discussion.

The ethical implications of altering the human germline has been the subject of intense discussion in recent years, with calls for such work to be put on hold until the process of genome editing is better understood. ASHG supports somatic genome editing and preclinical (in vitro human and animal) germline genome research but feels strongly that it is premature to consider human germline genome editing in any translational manner at this time.

The working group concludes that Many scientific, medical, and ethical questions remain around the potential for human germline genome editing. ASHG supports somatic genome editing and preclinical (in vitro human and animal) germline genome research but feels strongly that it is premature to consider human germline genome editing in any translational manner at this time. We encourage ethical and social consideration in tandem with basic science research in the upcoming years.

This appears a reasonable position largely in line with the recommendations from the major national and international groups surveyed by the working group. It balances the need to encourage further basic research and validation with strong awareness of the ethical and societal implications of human germline genome editing, setting a high bar before such technology should be translated to the clinic. No doubt, however, the debate will continue, particularly in respect of public funding for such work. Whether the US will maintain their stance against public funding, in the face of international competition, and potential loss of talent and investment, remains to be seen.

For more information about the science of CRISPR, its wide range of applications in life sciences and beyond, and latest developments in the field, please see Allen & Overys dedicated CRISPR microsite.

Original post:
Human Germline Genome Editing - Genetics bodies weigh in on debate with position paper - Lexology (registration)