Clinical Significance Of Genetic Variants Research Articles

POI-associated EIF4ENIF1 mutations exhibit impaired translation regulation abilities

Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.

Sex-specific molecular differences in glioblastoma: assessing the clinical significance of genetic variants.

Glioblastoma multiforme (GBM) is one of the most aggressive types of brain cancer, and despite rigorous research, patient prognosis remains poor. The characterization of sex-specific differences in incidence and overall survival (OS) of these patients has led to an investigation of the molecular mechanisms that may underlie this dimorphism. We reviewed the published literature describing the gender specific differences in GBM Biology reported in the last ten years and summarized the available information that may point towards a patient-tailored GBM therapy. Radiomics analyses have revealed that imaging parameters predict OS and treatment response of GBM patients in a sex-specific manner. Moreover, gender-based analysis of the transcriptome GBM tumors has found differential expression of various genes, potentially impacting the OS survival of patients in a sex-dependent manner. In addition to gene expression differences, the timing (subclonal or clonal) of the acquisition of common GBM-driver mutations, metabolism requirements, and immune landscape of these tumors has also been shown to be sex-specific, leading to a differential therapeutic response by sex. In male patients, transformed astrocytes are more sensitive to glutaminase 1 (GLS1) inhibition due to increased requirements for glutamine uptake. In female patients, GBM is more sensitive to anti-IL1β due to an increased population of circulating granulocytic myeloid-derived suppressor cells (gMDSC). Moving forward, continued elucidation of GBM sexual dimorphism will be critical in improving the OS of GBM patients by ensuring that treatment plans are structured to exploit these sex-specific, molecular vulnerabilities in GBM tumors.

Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

Abstract P3054: Further Delineation And Exploration Of The Cardiac MicroRNA-Target Interactome

MicroRNAs (miRs) have emerged as key players in cardiac biology and disease. These non-coding RNAs are loaded into Argonaute (Ago) proteins to direct suppression of target mRNAs via base-pairing. Despite a mass of research on cardiac miRs in model systems and humans, the translational impacts have been limited since miR-mRNA target interactions in heart tissues have not been adequately defined at the “-omics” level. Researchers heavily rely on predicted miR targets and are often misled by false predictions, since imperfect base-pairing is sufficient for miR binding. Our goal is to comprehensively define miR targeting events and their biological relevance in the heart and to understand the clinical significance of genetic variants (e.g. SNPs) that modulate cardiac miR functions. We previously used high-throughput biochemical means (Ago2 CLIP-seq) to generate the first transcriptome-wide map of miR binding sites in non-failing human hearts, yielding ~4000 sites across &gt;2000 genes. While exploring this dataset, we discovered translationally relevant miR-SNP interactions that associate with heart failure patient outcomes, most notably in SCN5A . We have since identified multiple common missense variants in TTN that disrupt miR binding. These associate with heart structure/function measures in the general population and could have significant implications for cardiomyopathy patients with TTN mutations. Beyond SNPs, we made the surprising discovery that TTN mRNA harbors &gt;20 miR-133 binding sites and has the potential to bind up to 20% of the miR-133 pool in cardiomyocytes, perhaps serving as an endogenous miR “sponge”. We experimentally validated the potential for this using a TTN mini-gene harboring 18 of the most strongly bound miR-133 target sites; we found that this construct was capable of inhibiting miR-133 activity in cells. Finally, to further expand these studies, we optimized our protocol by adapting state-of-the-art CLIP methods, which recently yielded our 2 nd generation map of human cardiac miR binding sites (now in failing hearts), leading to the identification of &gt;12,000 bindings sites across &gt;6000 genes. We are now interrogating these data and processing additional samples to investigate how miR binding profiles rewire in failing hearts.

Significance of α-Myosin Heavy Chain (MYH6) Variants in Hypoplastic Left Heart Syndrome and Related Cardiovascular Diseases.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with complex genetic inheritance. HLHS segregates with other left ventricular outflow tract (LVOT) malformations in families, and can present as either an isolated phenotype or as a feature of a larger genetic disorder. The multifactorial etiology of HLHS makes it difficult to interpret the clinical significance of genetic variants. Specific genes have been implicated in HLHS, including rare, predicted damaging MYH6 variants that are present in >10% of HLHS patients, and which have been shown to be associated with decreased transplant-free survival in our previous studies. MYH6 (α-myosin heavy chain, α-MHC) variants have been reported in HLHS and numerous other CHDs, including LVOT malformations, and may provide a genetic link to these disorders. In this paper, we outline the MYH6 variants that have been identified, discuss how bioinformatic and functional studies can inform clinical decision making, and highlight the importance of genetic testing in HLHS.

Clinical and molecular relevance of genetic variants in the non-coding transcriptome of patients with cytogenetically normal acute myeloid leukemia

Expression levels of long non-coding RNA (lncRNA) have been shown to associate with clinical outcome of patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, the frequency and clinical significance of genetic variants in the nucleotide sequences of lncRNA in AML patients is unknown. Herein, we analyzed total RNA sequencing data of 377 younger adults (aged <60 years) with CN-AML, who were comprehensively characterized with regard to clinical outcome. We used available genomic databases and stringent filters to annotate genetic variants unequivocally located in the non-coding transcriptome of AML patients. We detected 981 variants, which are recurrently present in lncRNA that are expressed in leukemic blasts. Among these variants, we identified a cytosine-to-thymidine variant in the lncRNA RP5-1074L1.4 and a cytosine-to-thymidine variant in the lncRNA SNHG15, which independently associated with longer survival of CN-AML patients. The presence of the SNHG15 cytosine-to-thymidine variant was also found to associate with better outcome in an independent dataset of CN-AML patients, despite differences in treatment protocols and RNA sequencing techniques. In order to gain biological insights, we cloned and overexpressed both wild-type and variant versions of the SNHG15 lncRNA. In keeping with its negative prognostic impact, overexpression of the wild-type SNHG15 associated with higher proliferation rate of leukemic blasts when compared with the cytosine-to-thymidine variant. We conclude that recurrent genetic variants of lncRNA that are expressed in the leukemic blasts of CN-AML patients have prognostic and potential biological significance.

Clinical and molecular relevance of genetic variants in the non-coding transcriptome of patients with cytogenetically normal acute myeloid leukemia.

Expression levels of long non-coding RNA (lncRNA) have been shown to associate with clinical outcome of patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, the frequency and clinical significance of genetic variants in the nucleotide sequences of lncRNA in AML patients is unknown. Herein, we analyzed total RNA sequencing data of 377 younger adults (aged <60 years) with CN-AML, who were comprehensively characterized with regard to clinical outcome. We used available genomic databases and stringent filters to annotate genetic variants unequivocally located in the non-coding transcriptome of AML patients. We detected 981 variants, which are recurrently present in lncRNA that are expressed in leukemic blasts. Among these variants, we identified a cytosine-to-thymidine variant in the lncRNA RP5-1074L1.4 and a cytosine-to-thymidine variant in the lncRNA SNHG15, which independently associated with longer survival of CN-AML patients. The presence of the SNHG15 cytosine-to-thymidine variant was also found to associate with better outcome in an independent dataset of CN-AML patients, despite differences in treatment protocols and RNA sequencing techniques. In order to gain biological insights, we cloned and overexpressed both wild-type and variant versions of the SNHG15 lncRNA. In keeping with its negative prognostic impact, overexpression of the wild-type SNHG15 associated with higher proliferation rate of leukemic blasts when compared with the cytosine-to-thymidine variant. We conclude that recurrent genetic variants of lncRNA that are expressed in the leukemic blasts of CN-AML patients have prognostic and potential biological significance.

The Clinical Utility of Determining the Allelic Background of Mutations Causing Alpha-1 Antitrypsin Deficiency: The Case with the Null Variant Q0(Mattawa)/Q0(Ourém).

Alpha-1 antitrypsin deficiency (AATD) is caused by genetic variants in the SERPINA1 gene conferring risk of developing emphysema. The clinical expression of AATD-related emphysema mostly occurs in carriers of 2 deficient alleles. By DNA sequencing of SERPINA1, numerous rare variants have been identified. Clarifying whether 2 mutations observed in 1 patient are on the same or distinct alleles has obvious clinical implications. We studied 7 carriers of a rare variant, Leu353Phe_fsTer24, known to lead to undetectable serum levels of AAT. Two of them were also carriers of the S or Z allele. We developed an allele-specific DNA sequencing method to characterize the allelic background of the Leu353Phe_fsTer24 variant. The Leu353Phe_fsTer24 variant was transmitted on the same allele as the M3 variant (E376D) in all patients. This mutation is thus named Q0Ourém on the conventional PI system. We demonstrated that individuals harboring the E264V (S) and E342K (Z) mutations had them on distinct alleles from Q0Ourém and are, thus, compound heterozygotes. The 7 Q0Ourém carriers had AAT levels ranging from 0.18g/l to 0.82g/l. The lowest AAT serum levels were observed in compound heterozygotes (S/Q0Ourém and Z/Q0Ourém) suggesting higher risk of developing emphysema. For the 7 patients, Leu353Phe_fsTer24 is transmitted on the M3 background and they are, thus, carriers of the Q0Ourém allele. Allele-specific DNA sequencing was useful to distinguish 1 or 2 deficient alleles in carriers of 2 mutations. In rare cases, this method is important to understand the clinical significance of genetic variants found in SERPINA1.

Estimation of allele-specific fitness effects across human protein-coding sequences and implications for disease.

A central challenge in human genomics is to understand the cellular, evolutionary, and clinical significance of genetic variants. Here, we introduce a unified population-genetic and machine-learning model, called Linear Allele-Specific Selection InferencE (LASSIE), for estimating the fitness effects of all observed and potential single-nucleotide variants, based on polymorphism data and predictive genomic features. We applied LASSIE to 51 high-coverage genome sequences annotated with 33 genomic features and constructed a map of allele-specific selection coefficients across all protein-coding sequences in the human genome. This map is generally consistent with previous inferences of the bulk distribution of fitness effects but reveals pervasive weak negative selection against synonymous mutations. In addition, the estimated selection coefficients are highly predictive of inherited pathogenic variants and cancer driver mutations, outperforming state-of-the-art variant prioritization methods. By contrasting our estimated model with ultrahigh coverage ExAC exome-sequencing data, we identified 1118 genes under unusually strong negative selection, which tend to be exclusively expressed in the central nervous system or associated with autism spectrum disorder, as well as 773 genes under unusually weak selection, which tend to be associated with metabolism. This combination of classical population genetic theory with modern machine-learning and large-scale genomic data is a powerful paradigm for the study of both human evolution and disease.

Germline variants in Hamartomatous Polyposis Syndrome-associated genes from patients with one or few hamartomatous polyps

Objective: A subgroup of patients with hamartomatous polyps in the GI tract has a hereditary Hamartomatous Polyposis Syndrome with an increased risk of cancer. The distinction between patients with one or few polyps and patients with a syndrome can be difficult. A pathogenic germline mutation can be detected in a majority of HPS patients. This study investigates whether patients with one or few hamartomatous polyps could have a syndrome based on genetic screening of relevant genes.Methods: We designed a gene panel including 26 hamartomatous polyposis-associated genes. Using targeted Next Generation Sequencing, DNA samples from 77 patients with 84 hamartomatous polyps were sequenced. The detected germline variants were classified into pathogenicity classes.Results: We detected several germline variants, among them three in ENG, two in BMPR1A, one in PTEN, and one in SMAD4. Although some of the detected variants have been reported previously none could be definitely pathogenic or likely pathogenic.Conclusions: Our study points towards that genetic testing for the Hamartomatous Polyposis Syndromes in patients with one or few polyps does not improve diagnostics, however we illustrate that the clinical significance of genetic variants can be difficult to interpret. A family history of polyps, cancer, or extraintestinal findings or a minimum of 3–5 polyps seems to be relevant information to include before genetic testing.

A systematic approach to assessing the clinical significance of genetic variants.

Molecular genetic testing informs diagnosis, prognosis, and risk assessment for patients and their family members. Recent advances in low-cost, high-throughput DNA sequencing and computing technologies have enabled the rapid expansion of genetic test content, resulting in dramatically increased numbers of DNA variants identified per test. To address this challenge, our laboratory has developed a systematic approach to thorough and efficient assessments of variants for pathogenicity determination. We first search for existing data in publications and databases including internal, collaborative and public resources. We then perform full evidence-based assessments through statistical analyses of observations in the general population and disease cohorts, evaluation of experimental data from in vivo or in vitro studies, and computational predictions of potential impacts of each variant. Finally, we weigh all evidence to reach an overall conclusion on the potential for each variant to be disease causing. In this report, we highlight the principles of variant assessment, address the caveats and pitfalls, and provide examples to illustrate the process. By sharing our experience and providing a framework for variant assessment, including access to a freely available customizable tool, we hope to help move towards standardized and consistent approaches to variant assessment.

Free the Data: The End of Genetic Data as Trade Secrets

Genetic Testing and Molecular BiomarkersVol. 17, No. 8 PerspectiveFree AccessFree the Data: The End of Genetic Data as Trade SecretsStephanie Nguyen and Sharon F. TerryStephanie NguyenUniversity of Virginia School of Law, Charlottesville, Virginia.Genetic Alliance, Washington, District of Columbia.Search for more papers by this author and Sharon F. TerryGenetic Alliance, Washington, District of Columbia.Search for more papers by this authorPublished Online:1 Aug 2013https://doi.org/10.1089/gtmb.2013.1547AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail For patients and their clinicians, broad access to information about DNA sequence variation is critical. The information is essential to interpreting the clinical significance of genetic variants, but it is not always readily available. At the present time, genetic test providers have the ability to create private databases containing information they have collected through testing and to restrict access to this vital information.A case study of BRCA1/2 testing by Myriad Genetics reveals the implications of creating a private database of clinical information and the importance of open access of this information. Until the June 2013 U.S. Supreme Court ruling that invalidated patents on isolated, naturally occurring human DNA, Myriad Genetics provided the only commercially available test for BRCA1/2 diagnostic testing in countries where patents were filed (for example, the United States). Myriad has tested over 1 million people and has gained valuable experience and information.A particularly significant consequence of being the sole provider of the BRCA1/2 diagnostic tests is that Myriad has collected a vast number of the genetic variants associated with the BRCA1 and BRCA2 genes, along with the accompanying interpretation of each variant. If this testing becomes available elsewhere, this information will not be available to researchers, clinicians, and patients.Some BRCA1/2 test results have a clear-cut interpretation. Perhaps the most clear-cut is a negative result upon testing a relative of someone with a known mutation. A negative result could be uncertain unless the negative result occurs in a person with a known mutation in the family and that mutation was not detected. A positive result, or detection of a known deleterious mutation, is also clear-cut when that mutation has been disclosed. However, some variants are particularly difficult to interpret because the effect of the variation is not yet known. These BRCA variations are aptly named “variants of unknown significance” (VUS).Myriad's patent exclusivity on the BRCA1/2 genes allowed the company to be the sole distributors of the genetic test, and thus it has been the largest collector of any clinically discovered VUS. Through the information gained from this collection, Myriad reports it was able to reduce the frequency of reporting a VUS. Furthermore, when a patient receives a test result with a VUS, Myriad offers free testing to the family members in an effort to gain more information and help determine the clinical significance of the variant. The ability to include additional data on the VUS and expand the proprietary database is a clear incentive for Myriad to offer free testing to the family members of the patient with the VUS. Patients also benefit from this practice.However, other providers of the BRCA1/2 tests will face unnecessary challenges due to the lack of transparency of data. The practice of privatizing clinical data obstructs the scientific community from independently verifying the data's clinical significance and accuracy.The ideal course of action to mitigate this problem would be to require every laboratory that tests for BRCA1/2 to register each test in the National Center for Biotechnology Information (NCBI), National Institutes of Health, Genetic Testing Registry, and deposit all BRCA1/2 variants in the NCBI ClinVar database.Another powerful way of liberating the BRCA1/2 variants is for the very people who have been tested to “free the data” and independently submit their BRCA1/2 report to directly contribute to an open-access database. Current projects that have undertaken this effort include the Sharing Clinical Reports Project (www.sharingclinicalreports.org), where physicians are encouraged to submit their patients' de-identified report to be uploaded to the ClinVar database, and the complementary Free the Data! Campaign (www.free-the-data.org), where the individuals are empowered to share the information themselves. The Sharing Clinical Reports Project avoids violating the Health Insurance Portability and Accountability Act by the de-identification of the patient's personal information, but the Free the Data! Campaign goes one step further in individual autonomy by enabling tested individuals to share their variant in ClinVar and to set their own sharing, privacy, and data access preferences. They can also share phenotypic information, thus providing researchers with information to help advance an understanding of these variants.BRCA1/2 is just an example of the clinical trade secret issue. Allowing public access to variants and associated phenotypes on all genes is essential for the accelerated understanding of health and disease that everyone desires. Private databases thwart progress and slow understanding of the contribution of genetics to health and disease. Laboratories need to share clinical variants for the public good; it cannot be held as a proprietary interest. Individuals need to exert their influence and “free the data” to let the data speak.FiguresReferencesRelatedDetailsCited byEnabling openness of valuable information resources: Curbing data subtractability and exclusion12 March 2018 | Information Systems Journal, Vol. 29, No. 4Bringing Genomics to MedicineIf you build it, they will come: unintended future uses of organised health data collections6 September 2016 | BMC Medical Ethics, Vol. 17, No. 1Premises for Clinical Genetics Data Governance: Grappling with Diverse Value Logics4 August 2016Challenges of web-based personal genomic data sharing27 March 2015 | Life Sciences, Society and Policy, Vol. 11, No. 1 Volume 17Issue 8Aug 2013 InformationCopyright 2013, Mary Ann Liebert, Inc.To cite this article:Stephanie Nguyen and Sharon F. Terry.Free the Data: The End of Genetic Data as Trade Secrets.Genetic Testing and Molecular Biomarkers.Aug 2013.579-580.http://doi.org/10.1089/gtmb.2013.1547Published in Volume: 17 Issue 8: August 1, 2013PDF download

Butyrylcholinesterase gene mutations in patients with prolonged apnea after succinylcholine for electroconvulsive therapy

patients undergoing electroconvulsive therapy (ECT) often receive succinylcholine as part of the anesthetic procedure. The duration of action may be prolonged in patients with genetic variants of the butyrylcholinesterase enzyme (BChE), the most common being the K- and the A-variants. The aim of the study was to assess the clinical significance of genetic variants in butyrylcholinesterase gene (BCHE) in patients with a suspected prolonged duration of action of succinylcholine after ECT. a total of 13 patients were referred to the Danish Cholinesterase Research Unit after ECT during 38 months. We determined the BChE activity and the BCHE genotype using molecular genetic methods, the duration of apnea, time to sufficient spontaneous ventilation and whether neuromuscular monitoring was used. The duration of apnea was compared with published data on normal subjects. in 11 patients, mutations were found in the BCHE gene, the K-variant being the most frequent. The duration of apnea was 5-15 min compared with 3-5.3 min from the literature. Severe distress was noted in the recovery phase in two patients. Neuromuscular monitoring was used in two patients. eleven of 13 patients with a prolonged duration of action of succinylcholine had mutations in BCHE, indicating that this is the possible reason for a prolonged period of apnea. We recommend objective neuromuscular monitoring during the first ECT.

Application of Embryonic Lethal or Other Obvious Phenotypes to Characterize the Clinical Significance of Genetic Variants Found in Trans with Known Deleterious Mutations

This work describes an approach to characterize the clinical significance of genetic variants detected during the genetic testing of BRCA1 in patients from hereditary breast/ovarian cancer families. Results from transgenic mice and extensive clinical testing support the hypothesis that biallelic BRCA1 mutations result in embryonic lethality. Therefore, it is reasonable to conclude that variants of uncertain clinical significance found to reside in trans with known deleterious mutations impart reduced risk for cancer. This approach was applied to a large data set of 55,630 patients who underwent clinical BRCA1 screening by whole gene direct DNA sequencing. Fourteen common single nucleotide polymorphisms (SNPs) were used to assign 10 previously defined common, recurrent, or canonical haplotypes in 99% of these cases. From a total of 1,477 genetic variants detected in these patients, excluding haplotype-tagging SNPs, 877 (59%) could be unambiguously assigned to one or more haplotypes. In 41 instances, variants previously classified as being of uncertain clinical significance, mostly missense variants, were excluded as fully penetrant mutations due to their coincidence in trans with known deleterious mutations. From a total of 1,150 patients that harbored these 41 variants, 956 carried one as the sole variant of uncertain clinical significance reported. This approach could have widespread application to other disease genes where compound heterozygous mutations are incompatible with life or result in obvious phenotypes. This largely computational technique is advantageous because it relies upon existing clinical data and is likely to prove informative for prevalent genetic variants in large data sets.