Exome Data Research Articles

Germline and somatic albinism variants in amelanotic/hypomelanotic melanoma: Increased carriage of TYR and OCA2 variants.

Amelanotic/hypomelanotic melanoma is a clinicopathologic subtype with absent or minimal melanin. This study assessed previously reported coding variants in albinism genes (TYR, OCA2, TYRP1, SLC45A2, SLC24A5, LRMDA) and common intronic, regulatory variants of OCA2 in individuals with amelanotic/hypomelanotic melanoma, pigmented melanoma cases and controls. Exome sequencing was available for 28 individuals with amelanotic/hypomelanotic melanoma and 303 individuals with pigmented melanoma, which were compared to whole exome data from 1144 Australian controls. Microarray genotyping was available for a further 17 amelanotic/hypomelanotic melanoma, 86 pigmented melanoma, 147 melanoma cases (pigmentation unknown) and 652 unaffected controls. Rare deleterious variants in TYR/OCA1 were more common in amelanotic/hypomelanotic melanoma cases than pigmented melanoma cases (set mixed model association tests P = 0.0088). The OCA2 hypomorphic allele p.V443I was more common in melanoma cases (1.8%) than controls (1.0%, X2 P = 0.02), and more so in amelanotic/hypomelanotic melanoma (4.4%, X2 P = 0.007). No amelanotic/hypomelanotic melanoma cases carried an eye and skin darkening haplotype of OCA2 (including rs7174027), present in 7.1% of pigmented melanoma cases (P = 0.0005) and 9.4% controls. Variants in TYR and OCA2 may play a role in amelanotic/hypomelanotic melanoma susceptibility. We suggest that somatic loss of function at these loci could contribute to the loss of tumor pigmentation, consistent with this we found a higher rate of somatic mutation in TYR/OCA2 in amelanotic/hypomelanotic melanoma vs pigmented melanoma samples (28.6% vs 3.0%; P = 0.021) from The Cancer Genome Atlas Skin Cutaneous Melanoma collection.

Novel mutation identification and copy number variant detection via exome sequencing in congenital muscular dystrophy.

BackgroundCongenital muscular dystrophy type 1A (MDC1A), also termed merosin‐deficient congenital muscular dystrophy (CMD), is a severe form of CMD caused by mutations in the laminin α2 gene (LAMA2). Of the more than 300 likely pathogenic variants found in the Leiden Open Variant Database, the majority are truncating mutations leading to complete LAMA2 loss of function, but multiple copy number variants (CNVs) have also been reported with variable frequency.MethodsWe collected a cohort of individuals diagnosed with likely MDC1A and sought to identify both single nucleotide variants and small and larger CNVs via exome sequencing by extending the analysis of sequencing data to detect splicing changes and CNVs.ResultsStandard exome analysis identified multiple novel LAMA2 variants in our cohort, but only four cases carried biallelic variants. Since likely truncating LAMA2 variants are often found in heterozygosity without a second allele, we performed additional splicing and CNV analysis on exome data and identified one splice change outside of the canonical sequences and three CNVs, in the remaining four cases.ConclusionsOur findings support the expectation that a portion of MDC1A cases may be caused by at least one CNV allele and show how these changes can be effectively identified by additional analysis of existing exome data.

Genome-Wide Association for HbA1c in Malay Identified Deletion on SLC4A1 that Influences HbA1c Independent of Glycemia

Glycated hemoglobin A1c (HbA1c) level is used to screen and diagnose diabetes. Genetic determinants of HbA1c can vary across populations and many of the genetic variants influencing HbA1c level were specific to populations. To discover genetic variants associated with HbA1c level in nondiabetic Malay individuals. We conducted a genome-wide association study (GWAS) analysis for HbA1c using 2 Malay studies, the Singapore Malay Eye Study (SiMES, N = 1721 on GWAS array) and the Living Biobank study (N = 983 on GWAS array and whole-exome sequenced). We built a Malay-specific reference panel to impute ethnic-specific variants and validate the associations with HbA1c at ethnic-specific variants. Meta-analysis of the 1000 Genomes imputed array data identified 4 loci at genome-wide significance (P < 5 × 10-8). Of the 4 loci, 3 (ADAM15, LINC02226, JUP) were novel for HbA1c associations. At the previously reported HbA1c locus ATXN7L3-G6PC3, association analysis using the exome data fine-mapped the HbA1c associations to a 27-bp deletion (rs769664228) at SLC4A1 that reduced HbA1c by 0.38 ± 0.06% (P = 3.5 × 10-10). Further imputation of this variant in SiMES confirmed the association with HbA1c at SLC4A1. We also showed that these genetic variants influence HbA1c level independent of glucose level. We identified a deletion at SLC4A1 associated with HbA1c in Malay. The nonglycemic lowering of HbA1c at rs769664228 might cause individuals carrying this variant to be underdiagnosed for diabetes or prediabetes when HbA1c is used as the only diagnostic test for diabetes.

Deep geno- and phenotyping in two consanguineous families with CMT2 reveals HADHA as an unusual disease-causing gene and an intronic variant in GDAP1 as an unusual mutation.

Charcot-Marie-Tooth (CMT) disease is a prevalent and heterogeneous peripheral neuropathy. Most patients affected with the axonal form of CMT (CMT2) do not harbor mutations in the approximately 90 known CMT-associated genes. We aimed to identify causative genes in two CMT2 pedigrees. Neurologic examination, laboratory tests and brain MRIs were performed. Genetic analysis included exome sequencing of four patients from the two pedigrees. The predicted effect of a deep intronic mutation on splicing was tested by regular and real-time PCR and sequencing. Clinical data were consistent with CMT2 diagnosis. Inheritance patterns were autosomal recessive. Exome data of CMT2-101 did not include mutations in known CMT-associated genes. Sequence data, segregation analysis, bioinformatics analysis, evolutionary conservation, and information in the literature strongly implicated HADHA as the causative gene. An intronic variation positioned 23 nucleotides away from following intron/exon border in GDAP1 was ultimately identified as cause of CMT in CMT2-102. It was shown to affect splicing. The finding of a HADHA mutation as a cause of CMT is of interest because its encoded protein is a subunit of the mitochondrial trifunctional protein (MTP) complex, a mitochondrial enzyme involved in long chain fatty acid oxidation. Long chain fatty acid oxidation is an important source of energy for skeletal muscles. The mutation found in CMT2-102 is only the second intronic mutation reported in GDAP1. The mutation in the CMT2-102 pedigree was outside the canonical splice site sequences, emphasizing the importance of careful examination of available intronic sequences in exome sequence data.

Infectivity and Progression of COVID-19 Based on Selected Host Candidate Gene Variants.

Introduction: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has spread around the globe. Susceptibility has been associated with age, biological sex, and other prior existing health conditions. However, host genes are involved in viral infectivity and pathogenicity, and polymorphisms in these could be responsible for the interethnic/interindividual variability observed in infection and progression of COVID-19.Materials and Methods: Clinical exome data of 103 individuals was analyzed to identify sequence variants in five selected candidate genes: ACE2, TMPRSS2, CD209, IFITM3, and MUC5B to assess their prevalence and role to understand the COVID-19 infectivity and progression in our population.Results: A total of 497 polymorphisms were identified in the five selected genes in the exomes analyzed. Thirty-eight polymorphisms identified in our cohort have been reported earlier in literature and have functional significance or association with health conditions. These variants were classified into three groups: protective, susceptible, and responsible for comorbidities.Discussion and Conclusion: The two polymorphisms described in literature as risk inducing are rs35705950 in MUC5B gene and TMPRSS2 haplotype (rs463727, rs34624090, rs55964536, rs734056, rs4290734, rs34783969, rs11702475, rs35899679, and rs35041537) were absent in our cohort explaining the slower infectivity of the disease in this part of India. The 38 functional variants identified can be used as a predisposition panel for the COVID-19 infectivity and progression and stratify individuals as “high or low risk,” which would help in planning appropriate surveillance and management protocols. A larger study from different regions of India is warranted to validate these results.

Integrated analysis of metabolomic profiling and exome data supplements sequence variant interpretation, classification, and diagnosis

PurposeA primary barrier to improving exome sequencing diagnostic rates is the interpretation of variants of uncertain clinical significance. We aimed to determine the contribution of integrated untargeted metabolomics in the analysis of exome sequencing data by retrospective analysis of patients evaluated by both exome sequencing and untargeted metabolomics within the same clinical laboratory. MethodsExome sequencing and untargeted metabolomic data were collected and analyzed for 170 patients. Pathogenic variants, likely pathogenic variants, and variants of uncertain significance in genes associated with a biochemical phenotype were extracted. Metabolomic data were evaluated to determine if these variants resulted in biochemical abnormalities that could be used to support their interpretation using current American College of Genetics and Genomics (ACMG) guidelines. ResultsMetabolomic data contributed to the interpretation of variants in 74 individuals (43.5%) over 73 different genes. The data allowed for the reclassification of 9 variants as likely benign, 15 variants as likely pathogenic, and 3 variants as pathogenic. Metabolomic data confirmed a clinical diagnosis in 21 cases, for a diagnostic rate of 12.3% in this population. ConclusionUntargeted metabolomics can serve as a useful adjunct to exome sequencing by providing valuable functional data that may not otherwise be clinically available, resulting in improved variant classification.

Abstract 1790: PDJ amplicon heterogeneity in triple negative breast cancers

Abstract Background: Amplification of chromosome 9p24.1 targeting PD-L1, PD-L2, and JAK2 (PDJ amplicon) is present in ~15% of TNBCs and is associated with activation of the JAK/STAT pathway and poor clinical outcomes. In preclinical studies, PD-L1 expression is markedly and rapidly inducible by low-dose IFN-γ in a PDJ amplicon copy-number dependent manner, mimicking an in situ inflammatory response. This suggests PDJ amplification could identify those TNBCs that would respond to ICIs and/or JAK2 targeted-therapies. The goals in this study were to determine the PDJ status of a well annotated cohort of TNBC PDXs with a 3 color FISH assay and to further define the genomes and transcriptomes of PDJ+ and PDJ- TNBCs. Methods: Tissue microarrays (TMAs) of 49 cores from 27 breast cancer PDX samples, including 21 TNBCs were obtained from the Baylor College of Medicine. The TMAs were interrogated with a 3 color FISH assay consisting of three probes that simultaneously target the PDJ amplicon, 9q22, and 9q34.1. The ratios of PDJ to 9q22 and PDJ to 9q34.1 were calculated to distinguish PDJ amplification from polyploidy. In each case, 50 cells were scored. Samples with PDJ signal &amp;gt;4.0, PDJ/9q22 ≥2 and/or PDJ/9q34.1 ≥2 were called positive by FISH. A subset of PDJ+ PDXs was then flow sorted by DNA content and interrogated with 400k whole genome CNV arrays. These results were integrated with existing clinical, exome, and transcriptome data for the TMAs (pdxportal.research.bcm.edu). Results: PDJ amplification was identified in 6/27 PDX samples including 3/21 (14%) TNBCs. Notably the number of PDJ+ cells in each sample ranged from 2-18. Single cells with ≥10 copies were observed in each PDJ+ sample as well as two PDJ- TNBCs. PDJ+ samples had increased expression of PD-L1 and JAK2 in their RNAseq data. Whole genome CNV analysis of flow sorted PDJ+ samples confirmed the presence of the PDJ amplicon and identified co-occurring amplifications and deletions. A similar pattern of PDJ amplicon heterogeneity was seen in a Mayo Clinic Arizona cohort of 70 surgically resected TNBCs. Previous whole genome CNV analysis of flow sorted samples identified 13/70 (18%) PDJ+ cases. FISH analysis of 14 TNBCs from this cohort (PDJ+ and PDJ- cases) validated these results and identified PDJ+ tumors with cells containing &amp;gt;20 copies of the amplicon. Conclusion: Our multicolor FISH assay of PDX and primary resected samples confirms a prevalence of ~15% PDJ+ TNBCs. Previous studies have reported an enrichment of JAK2 amplification in neoadjuvant treated TNBCs. Given the heterogeneous FISH patterns in both PDJ+ and PDJ- samples in our cohorts, we hypothesize that neoadjuvant therapy could enrich the presence of PDJ+ cells and thereby sensitize TNBCs to ICIs and JAK2 targeted therapies. Future work will explore the role of PDJ amplification and the presence of PDJ+ cells in TNBCs in response to neoadjuvant therapy. Citation Format: Alexander S. Roesler, Smriti Malasi, Elizabeth Lenkiewicz, Barbara A. Pockaj, Michael T. Lewis, Lacey E. Dobrolecki, Lori Koslosky, Peter Hartmayer, Karen S. Anderson, Michael T. Barrett. PDJ amplicon heterogeneity in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1790.

D190Y mutation in C-terminal tail region of TNNI3 gene causing severe form of restrictive cardiomyopathy with mild hypertrophy in an Indian patient

BackgroundRestrictive cardiomyopathy (RCM) is a rare condition with stiffening of the heart. Cardiac Troponin I gene is the most common causation of RCM constituting 8–10% of RCM cases and known responsible for either RCM with hypertrophy or hypertrophy with restrictive physiology. MethodsThirty RCM patients, 62 family members with 100 age, sex and ethnic matched subjects as controls were recruited from cardiology department of the All India Institute of Medical Sciences (AIIMS), New Delhi with written informed consent. 5 ml of blood sample was subjected to DNA extraction and all eight exons of the TNN13 gene were PCR amplified by 5 primer sets for Sanger sequencing. ResultsA rare heterozygous, de-novo missense mutation c.G568T p.D190Y is identified in a RCM patient having mild hypertrophy with restrictive physiology as it is absent in all unaffected family members, controls, other RCM patients of the study and several genome and exome data bases. This mutation is highly conserved and in-silico functional and protein structure analyses found it likely pathogenic. ConclusionIn the present study, out of 30 RCM Indian patients screened for all 8 exons of the TNNI3 gene, only one patient (3.33%) was found with the mutation. Variations and the substitution of different amino acids on same codons of TNNI3 gene confer different range phenotypes. In summary, this study contributes to the mutation spectrum in exon 8 as hot spot region of the TNNI3 gene causing RCM.

Identification of New Potential LncRNA Biomarkers in Hirschsprung Disease.

Hirschsprung disease (HSCR) is a neurocristopathy defined by intestinal aganglionosis due to alterations during the development of the Enteric Nervous System (ENS). A wide spectrum of molecules involved in different signaling pathways and mechanisms have been described in HSCR onset. Among them, epigenetic mechanisms are gaining increasing relevance. In an effort to better understand the epigenetic basis of HSCR, we have performed an analysis for the identification of long non-coding RNAs (lncRNAs) by qRT-PCR in enteric precursor cells (EPCs) from controls and HSCR patients. We aimed to test the presence of a set lncRNAs among 84 lncRNAs in human EPCs, which were previously related with crucial cellular processes for ENS development, as well as to identify the possible differences between HSCR patients and controls. As a result, we have determined a set of lncRNAs with positive expression in human EPCs that were screened for mutations using the exome data from our cohort of HSCR patients to identify possible variants related to this pathology. Interestingly, we identified three lncRNAs with different levels of their transcripts (SOCS2-AS, MEG3 and NEAT1) between HSCR patients and controls. We propose such lncRNAs as possible regulatory elements implicated in the onset of HSCR as well as potential biomarkers of this pathology.

Identification of Neuropsychiatric Copy Number Variants in a Health Care System Population

Population screening for medically relevant genomic variants that cause diseases such as hereditary cancer and cardiovascular disorders is increasing to facilitate early disease detection or prevention. Neuropsychiatric disorders (NPDs) are common, complex disorders with clear genetic causes; yet, access to genetic diagnosis is limited. We explored whether inclusion of NPD in population-based genomic screening programs is warranted by assessing 3 key factors: prevalence, penetrance, and personal utility. To evaluate the suitability of including pathogenic copy number variants (CNVs) associated with NPD in population screening by determining their prevalence and penetrance and exploring the personal utility of disclosing results. In this cohort study, the frequency of 31 NPD CNVs was determined in patient-participants via exome data. Associated clinical phenotypes were assessed using linked electronic health records. Nine CNVs were selected for disclosure by licensed genetic counselors, and participants' psychosocial reactions were evaluated using a mixed-methods approach. A primarily adult population receiving medical care at Geisinger, a large integrated health care system in the United States with the only population-based genomic screening program approved for medically relevant results disclosure, was included. The cohort was identified from the Geisinger MyCode Community Health Initiative. Exome and linked electronic health record data were available for this cohort, which was recruited from February 2007 to April 2017. Data were collected for the qualitative analysis April 2017 through February 2018. Analysis began February 2018 and ended December 2019. The planned outcomes of this study include (1) prevalence estimate of NPD-associated CNVs in an unselected health care system population; (2) penetrance estimate of NPD diagnoses in CNV-positive individuals; and (3) qualitative themes that describe participants' responses to receiving NPD-associated genomic results. Of 90 595 participants with CNV data, a pathogenic CNV was identified in 708 (0.8%; 436 women [61.6%]; mean [SD] age, 50.04 [18.74] years). Seventy percent (n = 494) had at least 1 associated clinical symptom. Of these, 28.8% (204) of CNV-positive individuals had an NPD code in their electronic health record, compared with 13.3% (11 835 of 89 887) of CNV-negative individuals (odds ratio, 2.21; 95% CI, 1.86-2.61; P < .001); 66.4% (470) of CNV-positive individuals had a history of depression and anxiety compared with 54.6% (49 118 of 89 887) of CNV-negative individuals (odds ratio, 1.53; 95% CI, 1.31-1.80; P < .001). 16p13.11 (71 [0.078%]) and 22q11.2 (108 [0.119%]) were the most prevalent deletions and duplications, respectively. Only 5.8% of individuals (41 of 708) had a previously known genetic diagnosis. Results disclosure was completed for 141 individuals. Positive participant responses included poignant reactions to learning a medical reason for lifelong cognitive and psychiatric disabilities. This study informs critical factors central to the development of population-based genomic screening programs and supports the inclusion of NPD in future designs to promote equitable access to clinically useful genomic information.

Genetic architecture of complex traits and disease risk predictors

Genomic prediction of complex human traits (e.g., height, cognitive ability, bone density) and disease risks (e.g., breast cancer, diabetes, heart disease, atrial fibrillation) has advanced considerably in recent years. Using data from the UK Biobank, predictors have been constructed using penalized algorithms that favor sparsity: i.e., which use as few genetic variants as possible. We analyze the specific genetic variants (SNPs) utilized in these predictors, which can vary from dozens to as many as thirty thousand. We find that the fraction of SNPs in or near genic regions varies widely by phenotype. For the majority of disease conditions studied, a large amount of the variance is accounted for by SNPs outside of coding regions. The state of these SNPs cannot be determined from exome-sequencing data. This suggests that exome data alone will miss much of the heritability for these traits—i.e., existing PRS cannot be computed from exome data alone. We also study the fraction of SNPs and of variance that is in common between pairs of predictors. The DNA regions used in disease risk predictors so far constructed seem to be largely disjoint (with a few interesting exceptions), suggesting that individual genetic disease risks are largely uncorrelated. It seems possible in theory for an individual to be a low-risk outlier in all conditions simultaneously.

Comprehensive Genomic Analysis of Translocation Renal Cell Carcinoma Reveals Copy-Number Variations as Drivers of Disease Progression.

Translocation renal cell carcinoma (tRCC) is a rare, aggressive renal cell carcinoma (RCC) subtype. There is currently limited understanding on the role of molecular alterations in the pathogenesis and progression of these tumors. We investigated the association between somatic alterations and clinical outcomes in two independent cohorts profiled using DNA sequencing. Twenty-two tRCCs underwent targeted sequencing [Memorial Sloan Kettering Cancer Center (MSK)-IMPACT]; a subset was profiled using exome-sequencing and combined with exome data from The Cancer Genome Atlas (TCGA) for analysis. The prognostic value of specific somatic aberrations, tumor mutation burden (TMB), and fraction of copy-number-altered genome (FCNAg) was explored. In TCGA cases, neoantigen prediction and immune cell deconvolution were performed using RNA-sequencing and exome data. Overall survival estimates were computed using the Kaplan-Meier method; time-on-treatment was calculated for 14 MSK-IMPACT patients who underwent systemic therapy. Associations between molecular features and outcomes were evaluated using nonparametric testing. Copy-number aberrant tRCCs were associated with poor overall survival (P = 0.03). Pediatric patients had tumors with lower FCNAg (P = 0.01). In one adult case with two chronologically distinct tumor samples sequenced, we confirmed that copy-number events occurred early during evolution. TERT promoter mutations were found exclusively in high-stage tumors. We found that tRCCs displayed distinct angiogenesis and PD-L1 gene expression profiles compared with other RCC subtypes. Tumors molecularly defined by increased copy-number variations were associated with aggressive disease in tRCC. A higher burden of genomic events in adults compared with pediatric cases likely reflects a more aggressive clinical course. The unique immunophenotypic characteristics of tRCC merit further exploration.

New Insights on Genetic Diagnostics in Cardiomyopathy and Arrhythmia Patients Gained by Stepwise Exome Data Analysis.

Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype–genotype correlations, as well as the identification of novel candidate genes.

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

Loqusdb: added value of an observations database of local genomic variation

BackgroundExome and genome sequencing is becoming the method of choice for rare disease diagnostics. One of the key challenges remaining is distinguishing the disease causing variants from the benign background variation. After analysis and annotation of the sequencing data there are typically thousands of candidate variants requiring further investigation. One of the most effective and least biased ways to reduce this number is to assess the rarity of a variant in any population. Currently, there are a number of reliable sources of information for major population frequencies when considering single nucleotide variants (SNVs) and small insertion and deletions (INDELs), with gnomAD as the most prominent public resource available. However, local variation or frequencies in sub-populations may be underrepresented in these public resources. In contrast, for structural variation (SV), the background frequency in the general population is more or less unknown mostly due to challenges in calling SVs in a consistent way. Keeping track of local variation is one way to overcome these problems and significantly reduce the number of potential disease causing variants retained for manual inspection, both for SNVs and SVs.ResultsHere, we present loqusdb, a tool to solve the challenge of keeping track of any type of variant observations from genome sequencing data. Loqusdb was designed to handle a large flow of samples and unlike other solutions, samples can be added continuously to the database without rebuilding it, facilitating improvements and additions. We assessed the added value of a local observations database using 98 samples annotated with information from a background of 888 unrelated individuals.ConclusionsWe show both how powerful SV analysis can be when filtering for population frequencies and how the number of apparently rare SNVs/INDELs can be reduced by adding local population information even after annotating the data with other large frequency databases, such as gnomAD. In conclusion, we show that a local frequency database is an attractive, and a necessary addition to the publicly available databases that facilitate the analysis of exome and genome data in a clinical setting.

Workflow for the Implementation of Precision Genomics in Healthcare.

To enable the implementation of precise genomics in a local healthcare system, we devised a pipeline for filtering and reporting of relevant genetic information to healthy individuals based on exome or genome data. In our analytical pipeline, the first tier of filtering is variant-centric, and it is based on the selection of annotated pathogenic, protective, risk factor, and drug response variants, and their one-by-one detailed evaluation. This is followed by a second-tier gene-centric deconstruction and filtering of virtual gene lists associated with diseases, and VUS-centric filtering according to ACMG pathogenicity criteria and pre-defined deleteriousness criteria. By applying this filtering protocol, we were able to provide valuable insights regarding the carrier status, pharmacogenetic profile, actionable cardiovascular and cancer predispositions, and potentially pathogenic variants of unknown significance to our patients. Our experience demonstrates that genomic profiling can be implemented into routine healthcare and provide information of medical significance.

Identification of novel genetic variants associated with short stature in a Baka Pygmies population

Human growth is a complex trait determined by genetic factors in combination with external stimuli, including environment, nutrition and hormonal status. In the past, several genome-wide association studies (GWAS) have collectively identified hundreds of genetic variants having a putative effect on determining adult height in different worldwide populations. Theoretically, a valuable approach to better understand the mechanisms of complex traits as adult height is to study a population exhibiting extreme stature phenotypes, such as African Baka Pygmies. After phenotypic characterization, we sequenced the whole exomes of a cohort of Baka Pygmies and their non-Pygmies Bantu neighbors to highlight genetic variants associated with the reduced stature. Whole exome data analysis revealed 29 single nucleotide polymorphisms (SNPs) significantly associated with the reduced height in the Baka group. Among these variants, we focused on SNP rs7629425, located in the 5′-UTR of the Hyaluronidase-2 (HYAL2) gene. The frequency of the alternative allele was significantly increased compared to African and non-African populations. In vitro luciferase assay showed significant differences in transcription modulation by rs7629425 C/T alleles. In conclusion, our results suggested that the HYAL2 gene variants may play a role in the etiology of short stature in Baka Pygmies population.

50 Years Ago in TheJournal ofPediatrics: Familial Hyperphosphatasia with Mental Retardation, Seizures, and Neurologic Deficits

Mabry C, Bautista A, Kirk RFH, Dublilier LD, Braunstein H, Koepke JA. J Pediatr 1970;77:74-85. Mabry et al reported the first 4 patients with hyperphosphatasia with mental retardation syndrome (HPMRS) to present with generalized seizures and facial dysmorphism. The syndrome can be distinguished from other developmental disabilities by the stable elevation of alkaline phosphatase without bone disease.1Thompson M.D. Nezarati M.M. Gillessen-Kaesbach G. Meinecke P. Mendoza-Londono R. Mornet E. et al.Hyperphosphatasia with seizures, neurologic deficit, and characteristic facial features: five new patients with Mabry syndrome.Am J Med Genet A. 2010; 152A: 1661-1669Crossref PubMed Scopus (36) Google Scholar Few published reports of the syndrome appeared before we identified a patient whose seizures responded to pyridoxine.2Cole D.E. Thompson M.D. Neurogenic aspects of Mabry syndrome.Subcell Biochem. 2015; 76: 343-361Crossref PubMed Scopus (12) Google Scholar Improved syndromology1Thompson M.D. Nezarati M.M. Gillessen-Kaesbach G. Meinecke P. Mendoza-Londono R. Mornet E. et al.Hyperphosphatasia with seizures, neurologic deficit, and characteristic facial features: five new patients with Mabry syndrome.Am J Med Genet A. 2010; 152A: 1661-1669Crossref PubMed Scopus (36) Google Scholar made it possible to use next-generation sequencing to identify the recessive mutations that cause what became known as Mabry syndrome (OMIM 239300).2Cole D.E. Thompson M.D. Neurogenic aspects of Mabry syndrome.Subcell Biochem. 2015; 76: 343-361Crossref PubMed Scopus (12) Google Scholar There are at least 6 phenotypes. HPMRS 1, 2, 5, and 6 result from disruption of 4 genes encoding phosphatidylinositol glycan (PIG) anchor biosynthesis enzymes that act in the endoplasmic reticulum: type V (PIGV) type O (PIGO), type W (PIGW), and type Y (PIGY). HPMRS 4 and 3 result from disruption of 2 genes encoding postattachment to proteins (PGAP) enzymes that stabilize glycosylphosphatidylinositol attachment to proteins in the golgi: PGAP3 and PGAP2.3Carmody L. Blau H. Danis D. Gourdine J.-P. Vasilevsky N. Krawitz P. et al.Significantly different clinical phenotypes associated with mutations in synthesis and transamidase+remodeling glycosylphosphatidylinositol (GPI)-anchor biosynthesis genes.Orphanet J Rare Dis. 2020; 15: 40Crossref PubMed Scopus (13) Google Scholar The report by Mabry et al describes the first of at least 21 inherited glycosylphosphatidylinositol biosynthesis defects (GPIBDs) that together compose approximately 0.15% of all developmental disabilities.4Pagnamenta A.T. Murakami Y. Taylor J.M. Anzilotti C. Howard M.F. Miller V. et al.Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders.Eur J Hum Genet. 2017; 25: 669-679Crossref PubMed Scopus (48) Google Scholar The HPMRS3 (GPIBD8 [MIM: 614207]) phenotype presented by Mabry et al, resulting from biallelic inheritance of PGAP2 mutations, is the prototypical HPMRS phenotype among GPIBDs.5Thompson M.D. Knaus A.A. Barshop B.A. Caliebe A. Muhle H. Nguyen T.T.M. et al.A post glycosylphosphatidylinositol (GPI) attachment to proteins, type 2 (PGAP2) variant identified in Mabry syndrome index cases: Molecular genetics of the prototypical inherited GPI disorder.Eur J Med Genet. 2019; (https://www.ncbi.nlm.nih.gov/pubmed/31805394. Accessed May 15, 2020)PubMed Google Scholar This work demonstrates the value of case studies to basic science, clinical innovation, and patient follow-up.

Distinct immune evasion in APOBEC-enriched, HPV-negative HNSCC.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Decoding whole-genome mutational signatures in 37 human pan-cancers by denoising sparse autoencoder neural network.

Millions of somatic mutations have recently been discovered in cancer genomes. These mutations in cancer genomes occur due to internal and external mutagenesis forces. Decoding the mutational processes by examining their unique patterns has successfully revealed many known and novel signatures from whole exome data, but many still remain undiscovered. Here, we developed a deep learning approach, DeepMS, to decompose mutational signatures using 52,671,908 somatic mutations from 2780 highly curated cancer genomes with whole genome sequencing (WGS) in 37 cancer types/subtypes. With rigorous model training and comparison, we characterized 54 signatures for single base substitutions (SBSs), 11 for doublet base substitutions (DBSs) and 16 for small insertions and deletions (Indels). Compared to the previous methods, DeepMS could discover 37 SBS, 5 DBS and 9 Indel new signatures, many of which represent associations with DNA mismatch or base excision repair and cisplatin therapy mechanisms. We further developed a regression-based model to estimate the correlation between signatures and clinical and demographical phenotypes. The first deep learning model DeepMS on WGS somatic mutational profiles enable us identify more comprehensive context-based mutational signatures than traditional NMF approaches. Our work substantially expands the landscape of the naturally occurring mutational signatures in cancer genomes, and provides new insights into cancer biology.