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Y‐chromosomal haplogroups and the Y‐SNPs defining them are relevant for the exploration of male lineages, inference of paternal ancestry, and reconstruction of migration pathways, to name a few. Currently, over 300,000 Y‐SNPs have been reported, defining 20 main haplogroups. However, ascertainment bias in the investigations has led to some haplogroups being overlooked, which hinders a representative depiction of certain populations and their migration events. For migration pattern analyses of the first settlers of the Americas, the Native American main founding lineage Q‐M3 needs to be further investigated to allow clear genetic differentiation of individuals of different ethnogeographic origins. To increase the resolution within this haplogroup, a total of 7.45 Mb of the Y chromosome of 59 admixed South Americans of haplogroup Q was targeted for sequencing using hybridization capture enrichment. Data were combined with 218 publicly available sequences of Central and South Americans of haplogroup Q. After rigorous data processing, variants not meeting the quality criteria were excluded and 4128 reliable Y‐SNPs were reported. A total of 2224 Y‐SNPs had previously unknown positions in the phylogenetic tree, and 1291 of these are novel. The phylogenetic relationships between the Y‐SNPs were established using the software SNPtotree in order to report a redesigned phylogenetic tree containing 300 branches, defined by 3400 Y‐SNPs. The new tree introduces 117 previously undescribed branches and is the most comprehensive phylogenetic tree of the Native American haplogroup Q lineages to date. The 214 sequences were assigned to 135 different low‐ to high‐resolution branches, while in the previous phylogenetic tree, only 195 sequences could be sorted into 14 low‐resolution branches with the same quality criteria. The improved genetic differentiation of subhaplogroup Q‐M3 has a great potential to resolve migration patterns of Native Americans. [ABSTRACT FROM AUTHOR]

The Y-chromosome is a valuable kinship indicator in family history and forensic research. To reconstruct genealogies, the time to the most recent common ancestor (tMRCA) between paternal relatives can be estimated through Y-STR analysis. Existing models are the stepwise mutation model (SMM, only one-step Y-STR changes) and the infinite allele model (IAM, new allele per Y-STR change). In this study, these mutation models and all existing tMRCA calculators were validated through a genetic-genealogy database containing 1,120 biologically related genealogical pairs confirmed by 46 Y-STRs with known tMRCA (18,109 generations). Consistent under- and overestimation and broad confidence intervals were observed, leading to dubious tMRCA estimates. This is because they do not include individual mutation rates or multi-step changes and ignore hidden multiple, back, or parallel modifications. To improve tMRCA estimation, we developed a user-friendly calculator, the "YMrCA", including all previously mentioned mutation characteristics. After extensive validation, we observed that the YMrCA calculator demonstrated a promising performance. The YMrCA yields a significantly higher tMRCA success rate (96%; +20%) and a lower tMRCA error (7; -3) compared to the mutation models and all online tMRCA calculators. Therefore, YMrCA offers the next step towards more objective tMRCA estimation for DNA kinship research., (© 2021 Wiley Periodicals LLC.)

Null variants are prevalent within the human genome, and their accurate interpretation is critical for clinical management. In 2018, the ClinGen Sequence Variant Interpretation (SVI) Working Group refined the only criterion with a very strong pathogenicity rating (PVS1). To streamline PVS1 interpretation, we have developed an automatic classification tool with a graphical user interface called AutoPVS1. The performance of AutoPVS1 was assessed using 56 variants manually curated by the ClinGen's SVI Working Group; it achieved an interpretation concordance of 93% (52/56). A further analysis of 28,586 putative loss-of-function variants by AutoPVS1 demonstrated that at least 27.7% of them do not reach a very strong strength level, 17.5% because of variant-specific issues and 10.2% due to disease mechanism considerations. Notably, 41.0% (1,936/4,717) of splicing variants were assigned a decreased preliminary PVS1 strength level, a significantly greater fraction than in frameshift variants (13.2%) and nonsense variants (10.8%). Our results reinforce the necessity of considering variant-specific issues and disease mechanisms in variant interpretation and demonstrate that AutoPVS1 meets an urgent need by enabling biocurators to easily assign accurate, reliable and reproducible PVS1 strength levels in the process of variant interpretation. AutoPVS1 is publicly available at http://autopvs1.genetics.bgi.com/., (© 2020 Wiley Periodicals LLC.)

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10 -3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10 -2 ), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications., (© 2020 Wiley Periodicals LLC.)

Routine exome sequencing (ES) in individuals with neurodevelopmental disorders (NDD) remains inconclusive in >50% of the cases. Research analysis of unsolved cases can identify novel candidate genes but is time‐consuming, subjective, and hard to compare between labs. The field, therefore, requires automated and standardized assessment methods to prioritize candidates for matchmaking. We developed AutoCaSc (https://autocasc.uni-leipzig.de) based on our candidate scoring scheme. We validated our approach using synthetic trios and real in‐house trio ES data. AutoCaSc consistently (94.5% of all cases) scored the relevant variants in valid novel NDD genes in the top three ranks. In 93 real trio exomes, AutoCaSc identified most (97.5%) previously manually scored variants while evaluating additional high‐scoring variants missed in manual evaluation. It identified candidate variants in previously undescribed NDD candidate genes (CNTN2, DLGAP1, SMURF1, NRXN3, and PRICKLE1). AutoCaSc enables anybody to quickly screen a variant for its plausibility in NDD. After contributing >40 descriptions of NDD‐associated genes, we provide usage recommendations based on our extensive experience. Our implementation is capable of pipeline integration and therefore allows the screening of large cohorts for candidate genes. AutoCaSc empowers even small labs to a standardized matchmaking collaboration and to contribute to the ongoing identification of novel NDD entities. [ABSTRACT FROM AUTHOR]

Polymorphic inversions are ubiquitous in humans and they have been linked to both adaptation and disease. Following their discovery in Drosophila more than a century ago, inversions have proved to be more elusive than other structural variants. A wide variety of methods for the detection and genotyping of inversions have recently been developed: multiple techniques based on selective amplification by PCR, short‐ and long‐read sequencing approaches, principal component analysis of small variant haplotypes, template strand sequencing, optical mapping, and various genome assembly methods. Many methods apply complex wet lab protocols or increasingly refined bioinformatic analyses. This review is an attempt to provide a practical summary and comparison of the methods that are in current use, with a focus on metrics such as the maximum size of segmental duplications at inversion breakpoints that each method can tolerate, the size range of inversions that they recover, their throughput, and whether the locations of putative inversions must be known beforehand. [ABSTRACT FROM AUTHOR]

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes. [ABSTRACT FROM AUTHOR]

ABSTRACT During the last few decades, a wealth of studies dedicated to the human Y chromosome and its DNA variation, in particular Y-chromosome single-nucleotide polymorphisms ( Y- SNPs), has led to the construction of a well-established Y-chromosome phylogeny. Since the recent advent of new sequencing technologies, the discovery of additional Y- SNPs is exploding and their continuous incorporation in the phylogenetic tree is leading to an ever higher resolution. However, the large and increasing amount of information included in the 'complete' Y-chromosome phylogeny, which now already includes many thousands of identified Y- SNPs, can be overwhelming and complicates its understanding as well as the task of selecting suitable markers for genotyping purposes in evolutionary, demographic, anthropological, genealogical, medical, and forensic studies. As a solution, we introduce a concise reference phylogeny whereby we do not aim to provide an exhaustive tree that includes all known Y- SNPs but, rather, a quite stable reference tree aiming for optimal global discrimination capacity based on a strongly reduced set that includes only the most resolving Y- SNPs. Furthermore, with this reference tree, we wish to propose a common standard for Y-marker as well as Y-haplogroup nomenclature. The current version of our tree is based on a core set of 417 branch-defining Y- SNPs and is available online at http://www.phylotree.org/Y. [ABSTRACT FROM AUTHOR]

Despite recent biomedical breakthroughs and large genomic studies growing momentum, the Middle Eastern population, home to over 400 million people, is underrepresented in the human genome variation databases. Here we describe insights from Phase 1 of the Qatar Genome Program with whole genome sequenced 6047 individuals from Qatar. We identified more than 88 million variants of which 24 million are novel and 23 million are singletons. Consistent with the high consanguinity and founder effects in the region, we found that several rare deleterious variants were more common in the Qatari population while others seem to provide protection against diseases and have shaped the genetic architecture of adaptive phenotypes. These results highlight the value of our data as a resource to advance genetic studies in the Arab and neighboring Middle Eastern populations and will significantly boost the current efforts to improve our understanding of global patterns of human variations, human history, and genetic contributions to health and diseases in diverse populations. [ABSTRACT FROM AUTHOR]

Runs of long homozygous (ROH) stretches are considered to be the result of consanguinity and usually contain recessive deleterious disease‐causing mutations. Several algorithms have been developed to detect ROHs. Here, we developed a simple alternative strategy by examining X chromosome non‐pseudoautosomal region to detect the ROHs from next‐generation sequencing data utilizing the genotype probabilities and the hidden Markov model algorithm as a tool, namely ROHMM. It is implemented purely in java and contains both a command line and a graphical user interface. We tested ROHMM on simulated data as well as real population data from the 1000G Project and a clinical sample. Our results have shown that ROHMM can perform robustly producing highly accurate homozygosity estimations under all conditions thereby meeting and even exceeding the performance of its natural competitors. [ABSTRACT FROM AUTHOR]

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co‐occur with a disease‐causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild‐type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild‐type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis‐interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function. [ABSTRACT FROM AUTHOR]

The pioneering discovery research of X‐linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide; however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene‐regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single‐nucleotide, and repeat expansion analyses of genome sequencing. RNA‐Seq from patient‐derived cell lines, reverse‐transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery. [ABSTRACT FROM AUTHOR]

KATK is a fast and accurate software tool for calling variants directly from raw next‐generation sequencing reads. It uses predefined k‐mers to retrieve only the reads of interest from the FASTQ file and calls genotypes by aligning retrieved reads locally. KATK does not use data about known polymorphisms and has NC (no call) as the default genotype. The reference or variant allele is called only if there is sufficient evidence for their presence in data. Thus it is not biased against rare variants or de‐novo mutations. With simulated datasets, we achieved a false‐negative rate of 0.23% (sensitivity 99.77%) and a false discovery rate of 0.19%. Calling all human exonic regions with KATK requires 1–2 h, depending on sequencing coverage. [ABSTRACT FROM AUTHOR]

Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often‐overlooked splicing effect of missense variants, we developed the functional assay ("minigene") for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon–intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning‐based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the "minigene" functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics. [ABSTRACT FROM AUTHOR]

Genome sequencing is positioned as a routine clinical work‐up for diverse clinical conditions. A commonly used approach to highlight candidate variants with potential clinical implication is to search over locus‐ and gene‐centric knowledge databases. Most web‐based applications allow a federated query across diverse databases for a single variant; however, sifting through a large number of genomic variants with combination of filtering criteria is a substantial challenge. Here we describe the Clinical Genome and Ancestry Report (CGAR), an interactive web application developed to follow clinical interpretation workflows by organizing variants into seven categories: (1) reported disease‐associated variants, (2) rare‐ and high‐impact variants in putative disease‐associated genes, (3) secondary findings that the American College of Medical Genetics and Genomics recommends reporting back to patients, (4) actionable pharmacogenomic variants, (5) focused reports for candidate genes, (6) de novo variant candidates for trio analysis, and (7) germline and somatic variants implicated in cancer risk, diagnosis, treatment and prognosis. For each variant, a comprehensive list of external links to variant‐centric and phenotype databases are provided. Furthermore, genotype‐derived ancestral composition is used to highlight allele frequencies from a matched population since some disease‐associated variants show a wide variation between populations. CGAR is an open‐source software and is available at https://tom.tch.harvard.edu/apps/cgar/. [ABSTRACT FROM AUTHOR]

Postaxial polydactyly (PAP) is a frequent limb malformation consisting in the duplication of the fifth digit of the hand or foot. Morphologically, this condition is divided into type A and B, with PAP‐B corresponding to a more rudimentary extra‐digit. Recently, biallelic truncating variants in the transcription factor GLI1 were reported to be associated with a recessive disorder, which in addition to PAP‐A, may include syndromic features. Moreover, two heterozygous subjects carrying only one inactive copy of GLI1 were also identified with PAP. Herein, we aimed to determine the level of involvement of GLI1 in isolated PAP, a condition previously established to be autosomal dominantly inherited with incomplete penetrance. We analyzed the coding region of GLI1 in 95 independent probands with nonsyndromic PAP and found 11.57% of these subjects with single heterozygous pathogenic variants in this gene. The detected variants lead to premature termination codons or result in amino acid changes in the DNA‐binding domain of GLI1 that diminish its transactivation activity. Family segregation analysis of these variants was consistent with dominant inheritance with incomplete penetrance. We conclude that heterozygous changes in GLI1 underlie a significant proportion of sporadic or familial cases of isolated PAP‐A/B. [ABSTRACT FROM AUTHOR]

Lysosomal acid lipase (LAL) deficiency is an autosomal recessive disorder caused by LIPA gene mutations that disrupt LAL activity. We performed in vitro functional testing of 149 LIPA variants to increase the understanding of the variant effects on LAL deficiency and to improve disease prevalence estimates. Chosen variants had been reported in literature or population databases. Functional testing was done by plasmid transient transfection and LAL activity assessment. We assembled a set of 165 published LAL deficient patient genotypes to evaluate this assay's effectiveness to recapitulate genotype/phenotype relationships. Rapidly progressive LAL deficient patients showed negligible enzymatic activity (<1%), whereas patients with childhood/adult LAL deficiency typically have 1–7% average activity. We benchmarked six in silico variant effect prediction algorithms with these functional data. PolyPhen‐2 was shown to have a superior area under the receiver operating curve performance. We used functional data along with Genome Aggregation Database (gnomAD) allele frequencies to estimate LAL deficiency birth prevalence, yielding a range of 3.45–5.97 cases per million births in European‐ancestry populations. The low estimate only considers functionally assayed variants in gnomAD. The high estimate computes allele frequencies for variants absent in gnomAD, and uses in silico scores for unassayed variants. Prevalence estimates are lower than previously published, underscoring LAL deficiency's rarity. [ABSTRACT FROM AUTHOR]

Large scale human genome projects have created tremendous human genome databases for some well‐studied populations. Vietnam has about 95 million people (the 14th largest country by population in the world) of which more than 86% are Kinh people. To date, genetic studies for Vietnamese people mostly rely on genetic information from other populations. Building a Vietnamese human genetic variation database is a must for properly interpreting Vietnamese genetic variants. To this end, we sequenced 105 whole genomes and 200 whole exomes of 305 unrelated Kinh Vietnamese (KHV) people. We also included 101 other previously published KHV genomes to build a Vietnamese human genetic variation database of 406 KHV people. The KHV database contains 24.81 million variants (22.47 million single nucleotide polymorphisms (SNPs) and 2.34 million indels) of which 0.71 million variants are novel. It includes more than 99.3% of variants with a frequency of >1% in the KHV population. Noticeably, the KHV database revealed 107 variants reported in the human genome mutation database as pathological mutations with a frequency above 1% in the KHV population. The KHV database (available at https://genomes.vn) would be beneficial for genetic studies and medical applications not only for the Vietnamese population but also for other closely related populations. [ABSTRACT FROM AUTHOR]

Precision medicine and sequence‐based clinical diagnostics seek to predict disease risk or to identify causative variants from sequencing data. The Critical Assessment of Genome Interpretation (CAGI) is a community experiment consisting of genotype‐phenotype prediction challenges; participants build models, undergo assessment, and share key findings. In the past, few CAGI challenges have addressed the impact of sequence variants on splicing. In CAGI5, two challenges (Vex‐seq and MaPSY) involved prediction of the effect of variants, primarily single‐nucleotide changes, on splicing. Although there are significant differences between these two challenges, both involved prediction of results from high‐throughput exon inclusion assays. Here, we discuss the methods used to predict the impact of these variants on splicing, their performance, strengths, and weaknesses, and prospects for predicting the impact of sequence variation on splicing and disease phenotypes. [ABSTRACT FROM AUTHOR]

Precise estimates of mutation rates at Y-chromosomal microsatellite STR (short tandem repeat) loci make an important basis for paternity diagnostics and dating of Y chromosome lineage origins. There are indications of considerable locus mutation rate variability between (inter-) and within (intra-) loci. We have studied nine Y-STR loci-DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS385, and DYS388-in 1,766 father-son pairs of confirmed paternity (a total of 15,894 meioses). Five biallelic markers were also analyzed in the fathers-Tat, YAP, 12f2, SRY1532, and 92R7-defining haplogroups 1, 2, 3, 4, 9, and 16, respectively. A total of 36 fragment length mutations were observed: 24 gains (22 single-step, two double-step) and 12 single-step losses. Thus, there was a significant surplus of gains (p=0.045). Overall, the mutation rate was positively correlated to STR repeat length and there was a significant relative excess of losses in long alleles and gains in short alleles (p=0.043). In contrast to the situation in autosomal STR loci and in MSY-1, no noteworthy correlation between mutation rate and the father's age at the child's birth was observed. We observed significant interlocus differences in Y-STR mutation rates (p<0.01). The number of observed mutations ranged from zero in DYS392 to eight in DYS391 and DYS390. We have also demonstrated obvious differences in mutation rates between the haplogroups studied (p=0.024), a phenomenon that is a reflection of the dependence of mutation rate on allele size. Our study has thus demonstrated the necessity of not only locus-specific, but even allele-specific, mutation rate estimates for forensic and population genetic purposes, and provides a considerable basis for such estimates., (Copyright 2003 Wiley-Liss, Inc.)

Accurate and detailed understanding of the effects of variants in the coding and noncoding regions of the genome is the next big challenge in the new genomic era of personalized medicine, especially to tackle newer findings of genetic and phenotypic heterogeneity of diseases. This is necessary to resolve the gene-variant--disease relationship, the pathogenic variant spectrum of genes, pathogenic variants with variable clinical consequences, and multiloci diseases. In turn, this will facilitate patient recruitment for relevant clinical trials. In this review, we describe the trends in research at the intersection of basic and clinical genomics aiming to (a) overcome molecular diagnostic challenges and increase the clinical utility of next-generation sequencing (NGS) platforms, (b) elucidate variants associated with disease, (c) determine overall genomic complexity including epistasis, complex inheritance patterns such as "synergistic heterozygosity," digenic/multigenic inheritance, modifier effect, and rare variant load.We describe the newly emerging field of integrated functional genomics, in vivo or in vitro large-scale functional approaches, statistical bioinformatics algorithms that support NGS genomics data to interpret variants for timely clinical diagnostics and disease management. Thus, facilitating the discovery of new therapeutic or biomarker options, and their roles in the future of personalized medicine. [ABSTRACT FROM AUTHOR]

Hereditary fructose intolerance (HFI) is an inborn error of fructose metabolism of autosomal recessive inheritance caused by pathogenic variants in the ALDOB gene that lead to aldolase B deficiency in the liver, kidneys, and intestine. Patients manifest symptoms, such as ketotic hypoglycemia, vomiting, nausea, in addition to hepatomegaly and other liver and kidney dysfunctions. The treatment consists of a fructose-restricted diet, which results in a good prognosis. To analyze the distribution of ALDOB variants described in patients and to estimate the prevalence of HFI based on carrier frequency in the gnomAD database, a systematic review was conducted to assess ALDOB gene variants among patients with HFI. The prevalence of HFI was estimated from the carrier frequency of variants described in patients, as well as rare variants predicted as pathogenic by in silico tools. The p.(Ala150Pro) and p.(Ala175Asp) variants are the most frequent and are distributed worldwide. However, these variants have particular distribution patterns in Europe. The analysis of the prevalence of HFI showed that the inclusion of rare alleles predicted as pathogenic is a more informative approach for populations with few patients. The data show that HFI has a wide distribution and an estimated prevalence of ~1:10,000., (© 2021 Wiley Periodicals LLC.)

Whole exomes of patients with a genetic disorder are nowadays routinely sequenced but interpretation of the identified genetic variants remains a major challenge. The increased availability of population-based human genetic variation has given rise to measures of genetic tolerance that have been used, for example, to predict disease-causing genes in neurodevelopmental disorders. Here, we investigated whether combining variant information from homologous protein domains can improve variant interpretation. For this purpose, we developed a framework that maps population variation and known pathogenic mutations onto 2,750 'meta-domains.' These meta-domains consist of 30,853 homologous Pfam protein domain instances that cover 36% of all human protein coding sequences. We find that genetic tolerance is consistent across protein domain homologues, and that patterns of genetic tolerance faithfully mimic patterns of evolutionary conservation. Furthermore, for a significant fraction (68%) of the meta-domains high-frequency population variation re-occurs at the same positions across domain homologues more often than expected. In addition, we observe that the presence of pathogenic missense variants at an aligned homologous domain position is often paired with the absence of population variation and vice versa. The use of these meta-domains can improve the interpretation of genetic variation. [ABSTRACT FROM AUTHOR]

Brazilians are highly admixed with ancestry from Europe, Africa, America, and Asia and yet still underrepresented in genomic databanks. We hereby present a collection of exomic variants from 609 elderly Brazilians in a census-based cohort (SABE609) with comprehensive phenotyping. Variants were deposited in ABraOM (Online Archive of Brazilian Mutations), a Web-based public database. Population representative phenotype and genotype repositories are essential for variant interpretation through allele frequency filtering; since elderly individuals are less likely to harbor pathogenic mutations for early- and adult-onset diseases, such variant databases are of great interest. Among the over 2.3 million variants from the present cohort, 1,282,008 were high-confidence calls. Importantly, 207,621 variants were absent from major public databases. We found 9,791 potential loss-of-function variants with about 300 mutations per individual. Pathogenic variants on clinically relevant genes (ACMG) were observed in 1.15% of the individuals and were correlated with clinical phenotype. We conducted incidence estimation for prevalent recessive disorders based upon heterozygous frequency and concluded that it relies on appropriate pathogenicity assertion. These observations illustrate the relevance of collecting demographic data from diverse, poorly characterized populations. Census-based datasets of aged individuals with comprehensive phenotyping are an invaluable resource toward the improved understanding of variant pathogenicity. [ABSTRACT FROM AUTHOR]

Detailed characterization of chromosomal abnormalities, a common cause for congenital abnormalities and pregnancy loss, is critical for elucidating genes for human fetal development. Here, 2,186 product-of-conception samples were tested for copy-number variations (CNVs) at two clinical diagnostic centers using whole-genome sequencing and high-resolution chromosomal microarray analysis. We developed a new gene discovery approach to predict potential developmental genes and identified 275 candidate genes from CNVs detected from both datasets. Based on Mouse Genome Informatics (MGI) and Zebrafish model organism database (ZFIN), 75% of identified genes could lead to developmental defects when mutated. Genes involved in embryonic development, gene transcription, and regulation of biological processes were significantly enriched. Especially, transcription factors and gene families sharing specific protein domains predominated, which included known developmental genes such as HOX, NKX homeodomain genes, and helix-loop-helix containing HAND2, NEUROG2, and NEUROD1 as well as potential novel developmental genes. We observed that developmental genes were denser in certain chromosomal regions, enabling identification of 31 potential genomic loci with clustered genes associated with development. [ABSTRACT FROM AUTHOR]

ABSTRACT Scientific and clinical communities produce ever increasing amounts of data and details about health and disease. Our ability to understand and utilize this information is limited because of imprecise language and lack of well-defined concepts. This problem involves also the principal concepts of health, disease, and pathogenicity. Here, a systematic model is presented for pathogenicity, as well as for health and disease. It has three components: extent, modulation, and severity, which jointly define the continuum of pathogenicity. The model is population based, and once implemented, it can be used for numerous purposes such as diagnosis, patient stratification, prognosis, finding phenotype-genotype correlations, or explaining adverse drug reactions. The new model has several benefits including health economy by allowing evidence-based personalized/precision medicine. [ABSTRACT FROM AUTHOR]

ABSTRACT The aryl hydrocarbon receptor interacting protein ( AIP) founder mutation R304* (or p.R304*; NM_003977.3:c.910C>T, p.Arg304Ter) identified in Northern Ireland (NI) predisposes to acromegaly/gigantism; its population health impact remains unexplored. We measured R304* carrier frequency in 936 Mid Ulster, 1,000 Greater Belfast (both in NI) and 2,094 Republic of Ireland (ROI) volunteers and in 116 NI or ROI acromegaly/gigantism patients. Carrier frequencies were 0.0064 in Mid Ulster (95%CI = 0.0027-0.013; P = 0.0005 vs. ROI), 0.001 in Greater Belfast (0.00011-0.0047) and zero in ROI (0-0.0014). R304* prevalence was elevated in acromegaly/gigantism patients in NI (11/87, 12.6%, P < 0.05), but not in ROI (2/29, 6.8%) versus non-Irish patients (0-2.41%). Haploblock conservation supported a common ancestor for all the 18 identified Irish pedigrees (81 carriers, 30 affected). Time to most recent common ancestor (tMRCA) was 2550 (1,275-5,000) years. tMRCA-based simulations predicted 432 (90-5,175) current carriers, including 86 affected (18-1,035) for 20% penetrance. In conclusion, R304* is frequent in Mid Ulster, resulting in numerous acromegaly/gigantism cases. tMRCA is consistent with historical/folklore accounts of Irish giants. Forward simulations predict many undetected carriers; geographically targeted population screening improves asymptomatic carrier identification, complementing clinical testing of patients/relatives. We generated disease awareness locally, necessary for early diagnosis and improved outcomes of AIP-related disease. [ABSTRACT FROM AUTHOR]

ABSTRACT Adoption of next-generation sequencing (NGS) in a diagnostic context raises numerous questions with regard to identification and reports of secondary variants (SVs) in actionable genes. To better understand the whys and wherefores of these questioning, it is necessary to understand how they are selected during the filtering process and how their proportion can be estimated. It is likely that SVs are underestimated and that our capacity to label all true SVs can be improved. In this context, Locus-specific databases (LSDBs) can be key by providing a wealth of information and enabling classifying variants. We illustrate this issue by analyzing 318 SVs in 23 actionable genes involved in cancer susceptibility syndromes identified through sequencing of 572 participants selected for a range of atherosclerosis phenotypes. Among these 318 SVs, only 43.4% are reported in Human Gene Mutation Database (HGMD) Professional versus 71.4% in LSDB. In addition, 23.9% of HGMD Professional variants are reported as pathogenic versus 4.8% for LSDB. These data underline the benefits of LSDBs to annotate SVs and minimize overinterpretation of mutations thanks to their efficient curation process and collection of unpublished data. [ABSTRACT FROM AUTHOR]

ABSTRACT Acute intermittent porphyria results from hydroxymethylbilane synthase ( HMBS) mutations that markedly decrease HMBS enzymatic activity. This dominant disease is diagnosed when heterozygotes have life-threatening acute attacks, while most heterozygotes remain asymptomatic and undiagnosed. Although >400 HMBS mutations have been reported, the prevalence of pathogenic HMBS mutations in genomic/exomic databases, and the actual disease penetrance are unknown. Thus, we interrogated genomic/exomic databases, identified non-synonymous variants (NSVs) and consensus splice-site variants (CSSVs) in various demographic/racial groups, and determined the NSV's pathogenicity by prediction algorithms and in vitro expression assays. Caucasians had the most: 58 NSVs and two CSSVs among ∼92,000 alleles, a 0.00575 combined allele frequency. In silico algorithms predicted 14 out of 58 NSVs as 'likely-pathogenic.' In vitro expression identified 10 out of 58 NSVs as likely-pathogenic (seven predicted in silico), which together with two CSSVs had a combined allele frequency of 0.00056. Notably, six presumably pathogenic mutations/NSVs in the Human Gene Mutation Database were benign. Compared with the recent prevalence estimate of symptomatic European heterozygotes (∼0.000005), the prevalence of likely-pathogenic HMBS mutations among Caucasians was >100 times more frequent. Thus, the estimated penetrance of acute attacks was ∼1% of heterozygotes with likely-pathogenic mutations, highlighting the importance of predisposing/protective genes and environmental modifiers that precipitate/prevent the attacks. [ABSTRACT FROM AUTHOR]

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomaldominant connective tissue disorder. [ABSTRACT FROM AUTHOR]

Insertion/deletion variants (indels) alter protein sequence and length, yet are highly prevalent in healthy populations, presenting a challenge to bioinformatics classifiers. Commonly used features--DNA and protein sequence conservation, indel length, and occurrence in repeat regions--are useful for inference of protein damage. However, these features can cause false positives when predicting the impact of indels on disease. Existing methods for indel classification suffer from low specificities, severely limiting clinical utility. Here, we further develop our variant effect scoring tool (VEST) to include the classification of in-frame and frameshift indels (VEST-indel) as pathogenic or benign. We apply 24 features, including a new "PubMed" feature, to estimate a gene's importance in human disease. When compared with four existing indel classifiers, our method achieves a drastically reduced false-positive rate, improving specificity by as much as 90%. This approach of estimating gene importance might be generally applicable to missense and other bioinformatics pathogenicity predictors, which often fail to achieve high specificity. Finally, we tested all possible meta-predictors that can be obtained from combining the four different indel classifiers using Boolean conjunctions and disjunctions, and derived a meta-predictor with improved performance over any individual method. [ABSTRACT FROM AUTHOR]

ABSTRACT Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan® Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD ( P = 1.05 × 10−3) and VKH syndrome ( P = 2.56 × 10−3). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD ( P = 3.35 × 10−8) and VKH syndrome ( P = 9.77 × 10−8). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4+ T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers ( P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls ( P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome. [ABSTRACT FROM AUTHOR]

ABSTRACT Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach. [ABSTRACT FROM AUTHOR]

ABSTRACT SNPs from the non-recombining part of the human Y chromosome (Y-SNPs) are informative to classify paternal lineages in forensic, genealogical, anthropological, and evolutionary studies. Although thousands of Y-SNPs were identified thus far, previous Y-SNP multiplex tools target only dozens of markers simultaneously, thereby restricting the provided Y-haplogroup resolution and limiting their applications. Here, we overcome this shortcoming by introducing a high-resolution multiplex tool for parallel genotyping-by-sequencing of 530 Y-SNPs using the Ion Torrent PGM platform, which allows classification of 432 worldwide Y haplogroups. Contrary to previous Y-SNP multiplex tools, our approach covers branches of the entire Y tree, thereby maximizing the paternal lineage classification obtainable. We used a default DNA input amount of 10 ng per reaction but preliminary sensitivity testing revealed positive results from as little as 100 pg input DNA. Furthermore, we demonstrate that sample pooling using barcodes is feasible, allowing increased throughput for lower per-sample costs. In addition to the wetlab protocol, we provide a software tool for automated data quality control and haplogroup classification. The unique combination of ultra-high marker density and high sensitivity achievable from low amounts of potentially degraded DNA makes this new multiplex tool suitable for a wide range of Y-chromosome applications. [ABSTRACT FROM AUTHOR]

ABSTRACT Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes ( EMGs) to determine the RNA and protein products generated by splice site variants ( n = 10) implicated in cystic fibrosis ( CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney ( HEK293) and CF bronchial epithelial ( CFBE41o-) cells. Quantification of variant induced aberrant m RNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585-1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants ( MES), 8/10 variants ( NNSplice), and 7/10 variants ( SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 ( HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. [ABSTRACT FROM AUTHOR]

ABSTRACT Recurrent miscarriage (RM) is a multifactorial disorder with acknowledged genetic heritability that affects ∼3% of couples aiming at childbirth. As copy number variants (CNVs) have been shown to contribute to reproductive disease susceptibility, we aimed to describe genome-wide profile of CNVs and identify common rearrangements modulating risk to RM. Genome-wide screening of Estonian RM patients and fertile controls identified excessive cumulative burden of CNVs (5.4 and 6.1 Mb per genome) in two RM cases possibly increasing their individual disease risk. Functional profiling of all rearranged genes within RM study group revealed significant enrichment of loci related to innate immunity and immunoregulatory pathways essential for immune tolerance at fetomaternal interface. As a major finding, we report a multicopy duplication (61.6 kb) at 5p13.3 conferring increased maternal risk to RM in Estonia and Denmark (meta-analysis, n = 309/205, odds ratio = 4.82, P = 0.012). Comparison to Estonian population-based cohort (total, n = 1000) confirmed the risk for Estonian female cases ( P = 7.9 × 10−4). Datasets of four cohorts from the Database of Genomic Variants (total, n = 5,846 subjects) exhibited similar low duplication prevalence worldwide (0.7%-1.2%) compared to RM cases of this study (6.6%-7.5%). The CNV disrupts PDZD2 and GOLPH3 genes predominantly expressed in placenta and it may represent a novel risk factor for pregnancy complications. [ABSTRACT FROM AUTHOR]

ABSTRACT Targeted sequencing using next-generation sequencing technologies is currently being rapidly adopted for clinical sequencing and cancer marker tests. However, no existing bioinformatics tool is available for the analysis and visualization of multiple targeted sequencing datasets. In the present study, we use cancer panel targeted sequencing datasets generated by the Life Technologies Ion Personal Genome Machine Sequencer as an example to illustrate how to develop an automated pipeline for the comparative analyses of multiple datasets. Cancer Panel Analysis Pipeline ( CPAP) uses standard output files from variant calling software to generate a distribution map of SNPs among all of the samples in a circular diagram generated by Circos. The diagram is hyperlinked to a dynamic HTML table that allows the users to identify target SNPs by using different filters. CPAP also integrates additional information about the identified SNPs by linking to an integrated SQL database compiled from SNP-related databases, including db SNP, 1000 Genomes Project, COSMIC, and db NSFP. CPAP only takes 17 min to complete a comparative analysis of 500 datasets. CPAP not only provides an automated platform for the analysis of multiple cancer panel datasets but can also serve as a model for any customized targeted sequencing project. [ABSTRACT FROM AUTHOR]

ABSTRACT We performed whole-exome sequencing of a family with autosomal dominant Dandy- Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix ( ECM) protein-encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1-binding partner. Structural modeling of the NID1- LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy- Walker spectrum disorders. [ABSTRACT FROM AUTHOR]

ABSTRACT Heterozygous mutations in DNA mismatch repair ( MMR) genes result in predisposition to colorectal cancer (hereditary nonpolyposis colorectal cancer or Lynch syndrome). Patients with biallelic mutations in these genes, however, present earlier, with constitutional mismatch repair deficiency cancer syndrome ( CMMRD), which is characterized by a spectrum of rare childhood malignancies and café-au-lait skin patches. The hallmark of MMR deficiency, microsatellite instability ( MSI), is readily detectable in tumor DNA in Lynch syndrome, but is also present in constitutional DNA of CMMRD patients. However, detection of constitutional or germline MSI (g MSI) has hitherto relied on technically difficult assays that are not routinely applicable for clinical diagnosis. Consequently, we have developed a simple high-throughput screening methodology to detect g MSI in CMMRD patients based on the presence of stutter peaks flanking a dinucleotide repeat allele when amplified from patient blood DNA samples. Using the three different microsatellite markers, the g MSI ratio was determined in a cohort of normal individuals and 10 CMMRD patients, with biallelic germline mutations in PMS2 (seven patients), MSH2 (one patient), or MSH6 (two patients). Subjects with either PMS2 or MSH2 mutations were easily identified; however, this measure was not altered in patients with CMMRD due to MSH6 mutation. [ABSTRACT FROM AUTHOR]

ABSTRACT Polymorphisms in regulatory DNA regions are believed to play an important role in determining phenotype, including disease, and in providing raw material for evolution. We devised a new pipeline for the systematic identification of functional variation in human regulatory sequences. The algorithm is based on the identification of SNPs leading to significant changes in both the affinity of a regulatory region for transcription factors ( TFs) and the expression in vivo of the regulated gene. We tested the algorithm by identifying SNPs leading to altered regulation by STAT3 in human promoters and introns, and experimentally validated the top-scoring ones, showing that most of the SNPs identified by the algorithm indeed correspond to differential binding of STAT3 and differential induction of the target gene upon stimulation with IL6. Using the same computational approach, we compiled a database of thousands of predicted functional regulatory SNPs for hundreds of human TFs, which we provide as online Supporting Information. We discuss possible applications to the interpretation of noncoding SNPs associated with human diseases. The method we propose and the database of predicted functional cis-regulatory polymorphisms will be useful in future studies of regulatory variation and in particular to interpret the results of past and future genome-wide association studies. [ABSTRACT FROM AUTHOR]