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Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics (ACMG) and Association of Molecular Pathology (AMP) standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel based consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well specifications for utilization of mtDNA genomic databases and computational algorithms.

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Purpose Hearing loss (HL) is the most common sensory disorder in children. Prompt molecular diagnosis may guide screening and management; especially in syndromic cases when HL is the single presenting feature. Exome sequencing (ES)is an appealing diagnostic tool for HL as the genetic causes are highly heterogeneous. Methods ES was performed on a prospective cohort of 43 probands with HL. Sequence data were analyzed for primary and secondary findings. Capture and coverage analysis was performed for genes and variants associated with HL. Results The diagnostic rate using ES was 37.2% compared to 15.8% with clinical HL panel. Secondary findings were discovered in three patients. For 247 genes associated with HL, 94.7% of the exons were targeted for capture and 81.7% of these exons were covered at 20× or greater. Further analysis of 454 randomly selected HL-associated variants showed 89% were targeted for capture and 75% were covered at a read depth of at least 20×. Conclusion ES has an improved yield to clinical testing and may capture diagnoses not initially considered due to subtle clinical phenotypes. Technical challenges were identified, including inadequate capture and coverage of HL genes. Additional considerations of ES include secondary findings, cost, and turnaround time.

PurposeThis study aimed to establish variants inCBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder.MethodsPatients withCBX1variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performedin vitrocellular assays, neurobehavioral and cytological analyses of neuronal cells obtained from newly generatedCbx1mutant mouse lines.ResultsIn three unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygousde novovariants inCBX1. The identified variants were in the chromodomain, the functional domain of HP1 β, which mediates interactions with chromatin.Cbx1chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, while HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β.ConclusionThese collective findings confirm the role ofCBX1in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. As HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects.

Powerful, recent advances in technologies to analyze the genome have had a profound impact on the practice of medical genetics, both in the laboratory and in the clinic. Increasing utilization of genome-wide testing such as chromosomal microarray analysis and exome sequencing have lead a shift toward a "genotype-first" approach. Numerous techniques are now available to diagnose a particular syndrome or phenotype, and while traditional techniques remain efficient tools in certain situations, higher-throughput technologies have become the de facto laboratory tool for diagnosis of most conditions. However, selecting the right assay or technology is challenging, and the wrong choice may lead to prolonged time to diagnosis, or even a missed diagnosis. In this review, we will discuss current core technologies for the diagnosis of classic genetic disorders to shed light on the benefits and disadvantages of these strategies, including diagnostic efficiency, variant interpretation, and secondary findings. Finally, we review upcoming technologies posed to impart further changes in the field of genetic diagnostics as we move toward "genome-first" practice.

Background Clinical genome and exome sequencing (CGES) is primarily used to address specific clinical concerns by detecting risk of future disease, clarifying diagnosis, or directing treatment. Additionally, CGES makes possible the disclosure of autosomal recessive and X‐linked carrier results as additional secondary findings, and research about the impact of carrier results disclosure in this context is needed. Methods Representatives from 11 projects in the clinical sequencing exploratory research (CSER) consortium collected data from their projects using a structured survey. The survey focused on project characteristics, which variants were offered and/or disclosed to participants as carrier results, methods for carrier results disclosure, and project‐specific outcomes. We recorded quantitative responses and report descriptive statistics with the aim of describing the variability in approaches to disclosing carrier results in translational genomics research projects. Results The proportion of participants with carrier results was related to the number of genes included, ranging from 3% (three genes) to 92% (4,600 genes). Between one and seven results were disclosed to those participants who received any positive result. Most projects offered participants choices about whether to receive some or all of the carrier results. There were a range of approaches to communicate results, and many projects used separate approaches for disclosing positive and negative results. Conclusion Future translational genomics research projects will need to make decisions regarding whether and how to disclose carrier results. The CSER consortium experience identifies approaches that balance potential participant interest while limiting impact on project resources.

Inherited platelet disorders (IPD) are a heterogeneous group of rare disorders that affect platelet number and function and often predispose to other significant medical complications. In spite of the identification of over 50 IPD disease-associated genes, a molecular diagnosis is only identified in a minority (10%) of affected patients without a clinically suspected etiology. We studied a cohort of 21 pediatric patients with suspected IPDs by exome sequencing (ES) to: (1) examine the performance of the exome test for IPD genes, (2) determine if this exome-wide diagnostic test provided a higher diagnostic yield than has been previously reported, (3) to evaluate the frequency of variants of uncertain significance identified, and (4) to identify candidate variants for functional evaluation in patients with an uncertain or negative diagnosis. We established a high priority gene list of 53 genes, evaluated exome capture kit performance, and determined the coverage for these genes and disease-related variants. We identified likely disease causing variants in 5 of the 21 probands (23.8%) and variants of uncertain significance in 52% of patients studied. In conclusion, ES has the potential to molecularly diagnose causes of IPD, and to identify candidate genes for functional evaluation. Robust exome sequencing also requires that coverage of genes known to be associated with clinical findings of interest need to be carefully examined and supplemented if necessary. Clinicians who undertake ES should understand the limitations of the test and the full significance of results that may be returned.

© 2018, American College of Medical Genetics and Genomics. Purpose: Clinical sequencing emerging in health care may result in secondary findings (SFs). Methods: Seventy-four of 6240 (1.2%) participants who underwent genome or exome sequencing through the Clinical Sequencing Exploratory Research (CSER) Consortium received one or more SFs from the original American College of Medical Genetics and Genomics (ACMG) recommended 56 gene–condition pair list; we assessed clinical and psychosocial actions. Results: The overall adjusted prevalence of SFs in the ACMG 56 genes across the CSER consortium was 1.7%. Initially 32% of the family histories were positive, and post disclosure, this increased to 48%. The average cost of follow-up medical actions per finding up to a 1-year period was $128 (observed, range: $0–$678) and $421 (recommended, range: $141–$1114). Case reports revealed variability in the frequency of and follow-up on medical recommendations patients received associated with each SF gene–condition pair. Participants did not report adverse psychosocial impact associated with receiving SFs; this was corroborated by 18 participant (or parent) interviews. All interviewed participants shared findings with relatives and reported that relatives did not pursue additional testing or care. Conclusion: Our results suggest that disclosure of SFs shows little to no adverse impact on participants and adds only modestly to near-term health-care costs; additional studies are needed to confirm these findings.

Multiple questions relating to contributions of cultural and demographical factors in the process of human geographical dispersal remain largely unanswered. India, a land of early human settlement and the resulting diversity is a good place to look for some of the answers. In this study, we explored the genetic structure of India using a diverse panel of 78 males genotyped using the GenoChip. Their genome-wide single-nucleotide polymorphism (SNP) diversity was examined in the context of various covariates that influence Indian gene pool. Admixture analysis of genome-wide SNP data showed high proportion of the Southwest Asian component in all of the Indian samples. Hierarchical clustering based on admixture proportions revealed seven distinct clusters correlating to geographical and linguistic affiliations. Convex hull overlay of Y-chromosomal haplogroups on the genome-wide SNP principal component analysis brought out distinct non-overlapping polygons of F*-M89, H*-M69, L1-M27, O2a-M95 and O3a3c1-M117, suggesting a male-mediated migration and expansion of the Indian gene pool. Lack of similar correlation with mitochondrial DNA clades indicated a shared genetic ancestry of females. We suggest that ancient male-mediated migratory events and settlement in various regional niches led to the present day scenario and peopling of India.

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC in three unrelated probands with a novel syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS), that we have named CHOPS syndrome (C for Cognitive impairment and Coarse facies, H for Heart defects, O for Obesity, P for Pulmonary involvement and S for Short stature and Skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 between CdLS and CHOPS syndrome. Direct molecular interaction between SEC, cohesin and RNAP2 was demonstrated. This data supports a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine.

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

The aim of this study is to describe genetic variation in the TNF promoter in the ethnically diverse population of Misiones, north-eastern Argentina. We analysed 210 women including 66 Amerindians of the Mbya-Guarani ethnic group and 144 white-admixed individuals from urban and rural areas of Misiones. Their DNA samples were surveyed for TNF polymorphisms -376 A/G, -308 A/G -244 A/G and -238 A/G by PCR amplification and direct sequencing and for the Amerindian marker -857 C/T by real-time PCR. Our main findings are as follows:(i) a distinctive pattern of Single Nucleotide Polymorphism (SNP) distribution among these groups, (ii) genetic differentiation between the Mbya-Guarani and the white-admixed populations (P < 0.05), (iii) lower gene diversity (~0.05) in Mbya-Guarani compared with the whiteadmixed group (~0.21); and (iv) linkage disequilibrium between the -376A and -238A SNPs in white-admixed populations. These data highlight the principal role of population history in establishing present-day genetic variation at the TNF locus and provide a framework for undertaking ethnographic and disease association studies in Misiones. Fil: Badano, Ines. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina; Fil: Schurr, T. G.. University of Pennsylvania; Estados Unidos de América; Fil: Stietz, S. M.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina; Fil: Dulik, M. C.. University of Pennsylvania; Estados Unidos de América; Fil: Mampaey, M.. Provincia de Misiones. Ministerio de Salud Pública; Argentina; Fil: Quintero, I. M.. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina; Fil: Zinovich, J. B.. Provincia de Misiones. Ministerio de Salud Pública; Argentina; Fil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina; Fil: Liotta, Domingo Javier. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

The linguistically distinctive Haida and Tlingit tribes of Southeast Alaska are known for their rich material culture, complex social organization, and elaborate ritual practices. However, much less is known about these tribes from a population genetic perspective. For this reason, we analyzed mtDNA and Y-chromosome variation in Haida and Tlingit populations to elucidate several key issues pertaining to the history of this region. These included the genetic relationships of Haida and Tlingit to other indigenous groups in Alaska and Canada; the relationship between linguistic and genetic data for populations assigned to the Na-Dene linguistic family, specifically, the inclusion of Haida with Athapaskan, Eyak, and Tlingit in the language family; the possible influence of matrilineal clan structure on patterns of genetic variation in Haida and Tlingit populations; and the impact of European entry into the region on the genetic diversity of these indigenous communities. Our analysis indicates that, while sharing a “northern” genetic profile, the Haida and the Tlingit are genetically distinctive from each other. In addition, Tlingit groups themselves differ across their geographic range, in part due to interactions of Tlingit tribes with Athapaskan and Eyak groups to the north. The data also reveal a strong influence of maternal clan identity on mtDNA variation in these groups, as well as the significant influence of non-native males on Y-chromosome diversity. These results yield new details about the histories of the Haida and Tlingit tribes in this region.

This report describes a pilot study examining aDNA from a skeletal population excavated in the 1990s at the late Early Bronze Age (EBA, c. 2300-2100 BC) urban settlement of Titris Hoyuk in southeastern Turkey. Typically, late EBA burials at Titris Hoyuk consisted of periodically reused underground family crypts contained within houses. However, one unique set of remains dated to the latest phase of the late EBA occupation at the site departed from this burial pattern entirely. It consisted of an above ground mortuary installation (B98.87) displaying the skulls and post-cranial bones of 19 individuals, most exhibiting a variety of fatal traumas. In this article, we compare the mtDNA sequences of these individuals with those buried in contemporary traditional late EBA intramural crypts. After successful extraction and amplification of ancient DNA molecules during a double blind study of 13 skeletons selected for the pilot study, our team was able to compare the genetic relatedness of individuals displayed in B98.87 to individuals buried elsewhere on the site. Based on archaeological evidence alone, earlier we had suggested that the occupants of B98.87 were perhaps outsiders, possibly soldiers vanquished in hostilities taking place shortly before the abandonment of the city at the end of the late EBA. However, our pilot study showed no clear genetic difference between the population of B98.87 and the broader population of the late EBA city, contrary to our expectations. Copyright © 2010 John Wiley & Sons, Ltd.

The Altaian Kazakhs, a Turkic speaking group, now reside in the southern part of the Altai Republic in south-central Russia. According to historical accounts, they are one of several ethnic and geographical subdivisions of the Kazakh nomadic group that migrated from China and Western Mongolia into the Altai region during the 19th Century. However, their population history of the Altaian Kazakhs and the genetic relationships with other Kazakh groups and neighboring Turkic-speaking populations is not well understood. To begin elucidating their genetic history, we analyzed the mtDNAs from 237 Altaian Kazakhs through a combination of SNP analysis and HVS1 sequencing. This analysis revealed that their mtDNA gene pool was comprised of roughly equal proportions of East (A-G, M7, M13, Y and Z) and West (H, HV, pre-HV, R, IK, JT, X, U) Eurasian haplogroups, with the haplotypic diversity within haplogroups C, D, H, and U being particularly high. This pattern of diversity likely reflects the complex interactions of the Kazakhs with other Turkic groups, Mongolians, and indigenous Altaians. Overall, these data have important implications for Kazakh population history, the genetic prehistory of the Altai-Sayan region, and the phylogeography of major mitochondrial lineages in Eurasia.

In 1653, the Patriarch Nikon modified liturgical practices to bring the Russian Orthodox Church in line with those of the Eastern (Greek) Orthodox Church, from which it had split 200 years earlier. The Old Believers (staroveri) rejected these changes and continued to worship using the earlier practices. These actions resulted in their persecution by the Russian Orthodox Church, which forced them into exile across Siberia. Given their history, we investigate whether populations of Old Believers have diverged genetically from other Slavic populations as a result of their isolation. We also examine whether the three Old Believer populations analyzed in this study are part of a single gene pool (founder population) or are instead derived from heterogeneous sources. As part of this analysis, we survey the mitochondrial DNAs (mtDNAs) of 189 Russian Old Believer individuals from three populations in Siberia and 201 ethnic Russians from different parts of Siberia for phylogenetically informative mutations in the coding and noncoding regions. Our results indicate that the Old Believers have not significantly diverged genetically from other Slavic populations over the 200-300 years of their isolation in Siberia. However, they do show some unique patterns of mtDNA variation relative to other Slavic groups, such as a high frequency of subhaplogroup U4, a surprisingly low frequency of haplogroup H, and low frequencies of the rare East Eurasian subhaplogroup D5.

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

Background: Inherited Platelet Disorders (IPD) are individually rare disorders that have many different molecular causes. Diagnosis of IPD is often complicated by the need for complex testing that is not readily available at many centers and the lack of available testing to define the molecular cause of some disorders. While some platelet disorders are sufficiently defined by functional characterization, recent data suggests that some platelet disorders may predispose to significant other complications including cancer predisposition, myelofibrosis or hearing loss. Therefore, it may be important to establish a molecular diagnosis to better counsel families about necessary follow up and possible risks. The goal of this study was to determine the diagnostic yield of whole exome sequencing in a cohort of 22 pediatric patients with clinical presentation suggesting an underlying genetic cause (or positive family history of platelet disorder with no prior genetic diagnosis). Methods: Peripheral blood was collected from patients identified as likely to have an inherited platelet disorder after informed consent. Samples were also obtained from parents and siblings for co-segregation and variant calling. Genomic DNA was extracted manually using the Gentra Puregene Blood Kit. Exome capture was performed using the Agilent SureSelect v4 and 100 base paired end sequencing was done on an IlluminaHiSeq 2000 with 100X average coverage. Sequencing reads were generated in FASTQ format and mapped to human genome GRCh37 (hg19) and Novoalign v2.08 was used for optimal alignment. Disease-related variants were extracted from HGMD to identify variants that might be missed. Variant filtering and pathogenicity classification was performed using a customized pipeline and manual curation. We identified 53 genes of interest and on average across all exomes with an indication for a platelet disorder, bases were sequenced at a minimum depth of 15X to be considered covered within an exon. 80.4% of exons were 100% covered with this technology completely, while 10.4% of exons were partially covered (>40 to Results: 22 patients were enrolled over a 12-month period. Overall, 82% of patients had variants identified in platelet related genes on whole exome sequencing with 64% of patients returning at least one variant of uncertain significance (14) and 23% (5) patients returning definite positive results. One patient referred for further work up carried the initial diagnosis of ITP, but had macrothrombocytopenia since early childhood and bleeding out of proportion to the platelet count. Flow cytometry and functional studies performed on referral suggested possible Bernard Soulier Syndrome and sequencing confirmed homozygous pathogenic mutation in GP9. One patient with congenital thrombocytopenia and history of intracranial hemorrhage with a similarly affected sibling had confirmed pathogenic MYH9 mutation, allowing clinicians to offer prenatal diagnosis during a third pregnancy. One patient with a significant bleeding phenotype was a compound heterozygote for two novel RASGRP2 variants, but the functional significance of those variants is uncertain and further studies are underway to determine whether these variants are causative. 18% of patients (4) had negative sequencing results (no reportable variants in platelet related genes identified). Conclusions: Whole exome sequencing can be a powerful diagnostic tool in identifying the molecular cause of disease in a cohort of patients with suspected inherited platelet disorders. The majority of patients, however, will receive results of uncertain significance and centers that undertake this testing will require an infrastructure to allow for further functional evaluation, which will help in reclassification of these variants, and ensure that results are correctly interpreted. Clinicians who undertake ES to diagnose IPD need to understand limitations of the test as well as the full significance of results that may be returned. Disclosures Lambert: Novartis: Consultancy.

As genomic and exomic testing expands in both the research and clinical arenas, determining whether, how, and which incidental findings to return to the ordering clinician and patient becomes increasingly important. Although opinion is varied on what should be returned to consenting patients or research participants, most experts agree that return of medically actionable results should be considered. There is insufficient evidence to fully inform evidence-based clinical practice guidelines regarding return of results from genome-scale sequencing, and thus generation of such evidence is imperative, given the rapidity with which genome-scale diagnostic tests are being incorporated into clinical care. We present an overview of the approaches to incidental findings by members of the Clinical Sequencing Exploratory Research network, funded by the National Human Genome Research Institute, to generate discussion of these approaches by the clinical genomics community. We also report specific lists of "medically actionable" genes that have been generated by a subset of investigators in order to explore what types of findings have been included or excluded in various contexts. A discussion of the general principles regarding reporting of novel variants, challenging cases (genes for which consensus was difficult to achieve across Clinical Sequencing Exploratory Research network sites), solicitation of preferences from participants regarding return of incidental findings, and the timing and context of return of incidental findings are provided.Genet Med 15 11, 860-867.Genetics in Medicine (2013); 15 11, 860-867. doi:10.1038/gim.2013.133.

Purpose To determine whether the incidence of and susceptibility to climatic droplet keratopathy (CDK), an acquired, often bilateral degenerative corneal disease, is influenced by the genetic background of the individuals who exhibit the disorder. Methods To determine whether the disease expression was influenced by the genetic ancestry of CDK cases in native Mapuche of the northwest area of Patagonia in Argentina, we examined mitochondrial DNA and Y-chromosome variation in 53 unrelated individuals. Twenty-nine of them were part of the CDK (patient) population, while 24 were part of the control group. The analysis revealed the maternal and paternal lineages that were present in the two study groups. Results This analysis demonstrated that nearly all persons had a Native American mtDNA background, whereas 50% of the CDK group and 37% of the control group had Native American paternal ancestry, respectively. There was no significant difference in the frequencies of mtDNA haplogroups between the CDK patient and control groups. Although the Y-chromosome data revealed differences in specific haplogroup frequencies between these two groups, there was no statistically significant relationship between individual paternal genetic backgrounds and the incidence or stage of disease. Conclusions These results indicate a lack of correlation between genetic ancestry as represented by haploid genetic systems and the incidence of CDK in Mapuche populations. In addition, the mtDNA appears to play less of a role in CDK expression than for other complex diseases linked to bioenergetic processes. However, further analysis of the mtDNA genome sequence and other genes involved in corneal function may reveal the more precise role that mitochondria play in the expression of CDK. Fil: Schurr, Theodore G.. University Of Pennsylvania. Penn Museum Of Archaeology And Anthropology; United State of America; Fil: Dulik, Matthew C.. University Of Pennsylvania. Penn Museum Of Archaeology And Anthropology; United State of America; Fil: Cafaro, Thamara Analia. Consejo Nacional de Investigaciones Científicas y Técnicas.Centro Científico Tecnológico - CONICET - Cordoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; Fil: Suarez, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas.Centro Científico Tecnológico - CONICET - Cordoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; Fil: Urrets-Zavalia, Julio A.. Universidad Catolica de Cordoba. Facultad de Medicina. Clinica Universitaria Reina Fabiola. Dto de Oftalmologia; Fil: Serra, Horacio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas.Centro Científico Tecnológico - CONICET - Cordoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

For decades, the peopling of the Americas has been explored through the analysis of uniparentally inherited genetic systems in Native American populations and the comparison of these genetic data with current linguistic groupings. In northern North America, two language families predominate: Eskimo-Aleut and Na-Dene. Although the genetic evidence from nuclear and mtDNA loci suggest that speakers of these language families share a distinct biological origin, this model has not been examined using data from paternally inherited Y chromosomes. To test this hypothesis and elucidate the migration histories of Eskimoan- and Athapaskan-speaking populations, we analyzed Y-chromosomal data from Inuvialuit, Gwich’in, and Tłįchǫ populations living in the Northwest Territories of Canada. Over 100 biallelic markers and 19 chromosome short tandem repeats (STRs) were genotyped to produce a high-resolution dataset of Y chromosomes from these groups. Among these markers is an SNP discovered in the Inuvialuit that differentiates them from other Aboriginal and Native American populations. The data suggest that Canadian Eskimoan- and Athapaskan-speaking populations are genetically distinct from one another and that the formation of these groups was the result of two population expansions that occurred after the initial movement of people into the Americas. In addition, the population history of Athapaskan speakers is complex, with the Tłįchǫ being distinct from other Athapaskan groups. The high-resolution biallelic data also make clear that Y-chromosomal diversity among the first Native Americans was greater than previously recognized.

Y-chromosome Haplogroup O is the dominant lineage of East Asians, comprising more than a quarter of all males on the world; however, its internal phylogeny remains insufficiently investigated. In this study, we determined the phylogenetic position of recently defined markers (L127, KL1, KL2, P164, and PK4) in the background of Haplogroup O. In the revised tree, subgroup O3a-M324 is divided into two main subclades, O3a1-L127 and O3a2-P201, covering about 20 and 35% of Han Chinese people, respectively. The marker P164 is corrected from a downstream site of M7 to upstream of M134 and parallel to M7 and M159. The marker PK4 is also relocated from downstream of M88 to upstream of M95, separating the former O2(*) into two parts. This revision evidently improved the resolving power of Y-chromosome phylogeny in East Asia.

Discovered in the early 16th century by European colonists, Bermuda is an isolated set of islands located in the mid-Atlantic. Shortly after its discovery, Bermuda became the first English colony to forcibly import its labor by trafficking in enslaved Africans, white ethnic minorities, and indigenous Americans. Oral traditions circulating today among contemporary tribes from the northeastern United States recount these same events, while, in Bermuda, St. David's Islanders consider their histories to be linked to a complex Native American, European, and African past. To investigate the influence of historical events on biological ancestry and native cultural identity, we analyzed genetic variation in 111 members of Bermuda's self-proclaimed St. David's Island Native Community. Our results reveal that the majority of mitochondrial DNA (mtDNA) and Y-chromosome haplotypes are of African and West Eurasian origin. However, unlike other English-speaking New World colonies, most African mtDNA haplotypes appear to derive from central and southeast Africa, reflecting the extent of maritime activities in the region. In light of genealogical and oral historical data from the St. David's community, the low frequency of Native American mtDNA and NRY lineages may reflect the influence of genetic drift, the demographic impact of European colonization, and historical admixture with persons of non-native backgrounds, which began with the settlement of the islands. By comparing the genetic data with genealogical and historical information, we are able to reconstruct the complex history of this Bermudian community, which is unique among New World populations.

Kazakh populations have traditionally lived as nomadic pastoralists that seasonally migrate across the steppe and surrounding mountain ranges in Kazakhstan and southern Siberia. To clarify their population history from a paternal perspective, we analyzed the non-recombining portion of the Y-chromosome from Kazakh populations living in southern Altai Republic, Russia, using a high-resolution analysis of 60 biallelic markers and 17 STRs. We noted distinct differences in the patterns of genetic variation between maternal and paternal genetic systems in the Altaian Kazakhs. While they possess a variety of East and West Eurasian mtDNA haplogroups, only three East Eurasian paternal haplogroups appear at significant frequencies (C3*, C3c and O3a3c*). In addition, the Y-STR data revealed low genetic diversity within these lineages. Analysis of the combined biallelic and STR data also demonstrated genetic differences among Kazakh populations from across Central Asia. The observed differences between Altaian Kazakhs and indigenous Kazakhs were not the result of admixture between Altaian Kazakhs and indigenous Altaians. Overall, the shared paternal ancestry of Kazakhs differentiates them from other Central Asian populations. In addition, all of them showed evidence of genetic influence by the 13(th) century CE Mongol Empire. Ultimately, the social and cultural traditions of the Kazakhs shaped their current pattern of genetic variation.

Background The ancestry of African-descended Americans is known to be drawn from three distinct populations: African, European, and Native American. While many studies consider this continental admixture, few account for the genetically distinct sources of ancestry within Africa – the continent with the highest genetic variation. Here, we dissect the within-Africa genetic ancestry of various populations of the Americas self-identified as having primarily African ancestry using uniparentally inherited mitochondrial DNA. Methods and Principal Findings We first confirmed that our results obtained using uniparentally-derived group admixture estimates are correlated with the average autosomal-derived individual admixture estimates (hence are relevant to genomic ancestry) by assessing continental admixture using both types of markers (mtDNA and Y-chromosome vs. ancestry informative markers). We then focused on the within-Africa maternal ancestry, mining our comprehensive database of published mtDNA variation (∼5800 individuals from 143 African populations) that helped us thoroughly dissect the African mtDNA pool. Using this well-defined African mtDNA variation, we quantified the relative contributions of maternal genetic ancestry from multiple W/WC/SW/SE (West to South East) African populations to the different pools of today's African-descended Americans of North and South America and the Caribbean. Conclusions Our analysis revealed that both continental admixture and within-Africa admixture may be critical to achieving an adequate understanding of the ancestry of African-descended Americans. While continental ancestry reflects gender-specific admixture processes influenced by different socio-historical practices in the Americas, the within-Africa maternal ancestry reflects the diverse colonial histories of the slave trade. We have confirmed that there is a genetic thread connecting Africa and the Americas, where each colonial system supplied their colonies in the Americas with slaves from African colonies they controlled or that were available for them at the time. This historical connection is reflected in different relative contributions from populations of W/WC/SW/SE Africa to geographically distinct Africa-derived populations of the Americas, adding to the complexity of genomic ancestry in groups ostensibly united by the same demographic label.

The name "Wampanoag" means "Eastern People" or "People of the First Light" in the local dialect of the Algonquian language. Once extensively populating the coastal lands and neighboring islands of the eastern United States, the Wampanoag people now consist of two federally recognized tribes, the Aquinnah and Mashpee, the state-recognized Seaconke Wampanoag tribe, and a number of bands and clans in present-day southern Massachusetts. Because of repeated epidemics and conflicts with English colonists, including King Philip's War of 1675-76, and subsequent colonial laws forbidding tribal identification, the Wampanoag population was largely decimated, decreasing in size from as many as 12,000 individuals in the 16th century to less than 400, as recorded in 1677. To investigate the influence of the historical past on its biological ancestry and native cultural identity, we analyzed genetic variation in the Seaconke Wampanoag tribe. Our results indicate that the majority of their mtDNA haplotypes belongs to West Eurasian and African lineages, thus reflecting the extent of their contacts and interactions with people of European and African descent. On the paternal side, Y-chromosome analysis identified a range of Native American, West Eurasian, and African haplogroups in the population, and also surprisingly revealed the presence of a paternal lineage that appears at its highest frequencies in New Guinea and Melanesia. Comparison of the genetic data with genealogical and historical information allows us to reconstruct the tribal history of the Seaconke Wampanoag back to at least the early 18th century.

BACKGROUND Population history can be reflected in group genetic ancestry, where genomic variation captured by the mitochondrial DNA (mtDNA) and non-recombining portion of the Y chromosome (NRY) can separate female- and male-specific admixture processes. Genetic ancestry may influence genetic association studies due to differences in individual admixture within recently admixed populations like African Americans. PRINCIPAL FINDINGS We evaluated the genetic ancestry of Senegalese as well as European Americans and African Americans from Philadelphia. Senegalese mtDNA consisted of approximately 12% U haplotypes (U6 and U5b1b haplotypes, common in North Africa) while the NRY haplotypes belonged solely to haplogroup E. In Philadelphia, we observed varying degrees of admixture. While African Americans have 9-10% mtDNAs and approximately 31% NRYs of European origin, these results are not mirrored in the mtDNA/NRY pools of European Americans: they have less than 7% mtDNAs and less than 2% NRYs from non-European sources. Additionally, there is

The Altai region of southern Siberia has played a critical role in the peopling of northern Asia as an entry point into Siberia and a possible homeland for ancestral Native Americans. It has an old and rich history because humans have inhabited this area since the Paleolithic. Today, the Altai region is home to numerous Turkic-speaking ethnic groups, which have been divided into northern and southern clusters based on linguistic, cultural, and anthropological traits. To untangle Altaian genetic histories, we analyzed mtDNA and Y chromosome variation in northern and southern Altaian populations. All mtDNAs were assayed by PCR-RFLP analysis and control region sequencing, and the nonrecombining portion of the Y chromosome was scored for more than 100 biallelic markers and 17 Y-STRs. Based on these data, we noted differences in the origin and population history of Altaian ethnic groups, with northern Altaians appearing more like Yeniseian, Ugric, and Samoyedic speakers to the north, and southern Altaians having greater affinities to other Turkic speaking populations of southern Siberia and Central Asia. Moreover, high-resolution analysis of Y chromosome haplogroup Q has allowed us to reshape the phylogeny of this branch, making connections between populations of the New World and Old World more apparent and demonstrating that southern Altaians and Native Americans share a recent common ancestor. These results greatly enhance our understanding of the peopling of Siberia and the Americas.

Background Conditions associated with sudden cardiac arrest/death (SCA/D) in youth often have a genetic etiology. While SCA/D is uncommon, a pro-active family screening approach may identify these inherited structural and electrical abnormalities prior to symptomatic events and allow appropriate surveillance and treatment. This study investigated the diagnostic utility of exome sequencing (ES) by evaluating the capture and coverage of genes related to SCA/D. Methods Samples from 102 individuals (13 with known molecular etiologies for SCA/D, 30 individuals without known molecular etiologies for SCA/D and 59 with other conditions) were analyzed following exome capture and sequencing at an average read depth of 100X. Reads were mapped to human genome GRCh37 using Novoalign, and post-processing and analysis was done using Picard and GATK. A total of 103 genes (2,190 exons) related to SCA/D were used as a primary filter. An additional 100 random variants within the targeted genes associated with SCA/D were also selected and evaluated for depth of sequencing and coverage. Although the primary objective was to evaluate the adequacy of depth of sequencing and coverage of targeted SCA/D genes and not for primary diagnosis, all patients who had SCA/D (known or unknown molecular etiologies) were evaluated with the project’s variant analysis pipeline to determine if the molecular etiologies could be successfully identified. Results The majority of exons (97.6 %) were captured and fully covered on average at minimum of 20x sequencing depth. The proportion of unique genomic positions reported within poorly covered exons remained small (4 %). Exonic regions with less coverage reflect the need to enrich these areas to improve coverage. Despite limitations in coverage, we identified 100 % of cases with a prior known molecular etiology for SCA/D, and analysis of an additional 30 individuals with SCA/D but no known molecular etiology revealed a diagnostic answer in 5/30 (17 %). We also demonstrated 95 % of 100 randomly selected reported variants within our targeted genes would have been picked up on ES based on our coverage analysis. Conclusions ES is a helpful clinical diagnostic tool for SCA/D given its potential to successfully identify a molecular diagnosis, but clinicians should be aware of limitations of available platforms from technical and diagnostic perspectives. Electronic supplementary material The online version of this article (doi:10.1186/s40246-015-0038-y) contains supplementary material, which is available to authorized users.

Background Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate and accuracy for the Affymetrix MitoChip v2.0 platform. We used this custom pipeline to analyze MitoChip v2.0 data from 24 DNA samples representing a broad range of tissue types (18 whole blood, 3 skeletal muscle, 3 cell lines), mutations (a 5.8 kilobase pair deletion and 6 known heteroplasmic mutations), and haplogroup origins. All results were compared to those obtained by at least one other mitochondrial DNA sequence analysis method, including Sanger sequencing, denaturing HPLC-based heteroduplex analysis, and/or the Illumina Genome Analyzer II next generation sequencing platform. Results An average call rate of 99.75% was achieved across all samples with our custom pipeline. Comparison of calls for 15 samples characterized previously by Sanger sequencing revealed a total of 29 discordant calls, which translates to an estimated 0.012% for the base call error rate. We successfully identified 4 known heteroplasmic mutations and 24 other potential heteroplasmic mutations across 20 samples that passed quality control. Conclusions Affymetrix MitoChip v2.0 analysis using our optimized MitoChip Filtering Protocol (MFP) bioinformatics pipeline now offers the high sensitivity and accuracy needed for reliable, high-throughput and cost-efficient whole mitochondrial genome sequencing. This approach provides a viable alternative of potential utility for both clinical diagnostic and research applications to traditional Sanger and other emerging sequencing technologies for whole mitochondrial genome analysis.

Background Exome sequencing is a promising method for diagnosing patients with a complex phenotype. However, variant interpretation relative to patient phenotype can be challenging in some scenarios, particularly clinical assessment of rare complex phenotypes. Each patient’s sequence reveals many possibly damaging variants that must be individually assessed to establish clear association with patient phenotype. To assist interpretation, we implemented an algorithm that ranks a given set of genes relative to patient phenotype. The algorithm orders genes by the semantic similarity computed between phenotypic descriptors associated with each gene and those describing the patient. Phenotypic descriptor terms are taken from the Human Phenotype Ontology (HPO) and semantic similarity is derived from each term’s information content. Results Model validation was performed via simulation and with clinical data. We simulated 33 Mendelian diseases with 100 patients per disease. We modeled clinical conditions by adding noise and imprecision, i.e. phenotypic terms unrelated to the disease and terms less specific than the actual disease terms. We ranked the causative gene against all 2488 HPO annotated genes. The median causative gene rank was 1 for the optimal and noise cases, 12 for the imprecision case, and 60 for the imprecision with noise case. Additionally, we examined a clinical cohort of subjects with hearing impairment. The disease gene median rank was 22. However, when also considering the patient’s exome data and filtering non-exomic and common variants, the median rank improved to 3. Conclusions Semantic similarity can rank a causative gene highly within a gene list relative to patient phenotype characteristics, provided that imprecision is mitigated. The clinical case results suggest that phenotype rank combined with variant analysis provides significant improvement over the individual approaches. We expect that this combined prioritization approach may increase accuracy and decrease effort for clinical genetic diagnosis. Electronic supplementary material The online version of this article (doi:10.1186/1471-2105-15-248) contains supplementary material, which is available to authorized users.

Behar, Doron M. et al.-- The Genographic Consortium, Different lines of evidence point to the resettlement of much of western and central Europe by populations from the Franco-Cantabrian region during the Late Glacial and Postglacial periods. In this context, the study of the genetic diversity of contemporary Basques, a population located at the epicenter of the Franco-Cantabrian region, is particularly useful because they speak a non-Indo-European language that is considered to be a linguistic isolate. In contrast with genome-wide analysis and Y chromosome data, where the problem of poor time estimates remains, a new timescale has been established for the human mtDNA and makes this genome the most informative marker for studying European prehistory. Here, we aim to increase knowledge of the origins of the Basque people and, more generally, of the role of the Franco-Cantabrian refuge in the postglacial repopulation of Europe. We thus characterize the maternal ancestry of 908 Basque and non-Basque individuals from the Basque Country and immediate adjacent regions and, by sequencing 420 complete mtDNA genomes, we focused on haplogroup H. We identified six mtDNA haplogroups, H1j1, H1t1, H2a5a1, H1av1, H3c2a, and H1e1a1, which are autochthonous to the Franco-Cantabrian region and, more specifically, to Basque-speaking populations. We detected signals of the expansion of these haplogroups at ∼4,000 years before present (YBP) and estimated their separation from the pan-European gene pool at ∼8,000 YBP, antedating the Indo-European arrival to the region. Our results clearly support the hypothesis of a partial genetic continuity of contemporary Basques with the preceding Paleolithic/Mesolithic settlers of their homeland., This work was supported by the Institut Pasteur, National Geographic, and the Histoire des populations et variation linguistique dans les Pyrénées de l'Ouest project, which received funding from the Conseil Régional d'Aquitaine, the Conseil Général des Pyrénées-Atlantiques, the Conseil des Elus du Pays-Basque, and the Centre National de la Recherche Scientifique interdisciplinary program Origine de l'Homme, des Langues et du Langage. This study also benefited from the support of Department of Hematology, Centre Hospitalier de la Côte Basque, in Bayonne, and Association Sang 64.