Database gnomAD Research Articles

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

Crebbp Mutations Are Associated with Shorter Time to Progression in Limited-Stage Follicular Lymphoma Treated with Radiation

Background: Limited-stage follicular lymphoma (FL), defined as stage I or II disease, is generally treated with radiation therapy (RT) to involved nodes with excellent overall response rates. However, nearly half of all limited-stage FL patients will ultimately experience a relapse. Most prior research on genetic prognostication in FL has focused on patients with advanced disease undergoing systemic therapy, however, there is evidence to suggest that there are genetic differences in limited versus advanced-stage FL, for example, in the frequency of KMT2D and BCL2 mutations as well as gene expression profiles. Objective: We hypothesized that genetic characterization of pre-treatment biopsies can identify those limited-stage FL patients that are at high risk of relapse following radiation therapy. Methods: Pre-treatment formalin-fixed and paraffin-embedded tissue block samples were obtained from adult patients with limited-stage FL, grade 1-3A, treated with curative-intent RT diagnosed between 1995-2018, from 6 international sites in Canada, Europe and Australia. Hybridization-based targeted sequencing of a 71 gene panel was performed, which included genes that are known to be recurrently mutated in FL. Samples with >50X mean on target coverage were retained in the analysis. Variant calling was performed using Mutect2. Germline polymorphisms were filtered out by cross-referencing with dbSNP and gnomAD databases. Only coding, non-silent mutations detected at a variant allele frequency greater than 0.1 were considered. Patient demographics and clinical data were examined retrospectively. Genes mutated at a proportion of 10% or more of the cohort were tested for association with time to progression (TTP) and overall survival (OS). Results: One hundred and seventeen pre-treatment samples from distinct patients were accrued. The median age was 58 years (range: 26-86), and 68 (58%) were female. There were 99 (85%) patients with stage I disease, and 18 (15%) with stage II. Grade was 1 or 2 in 86% of patients. Mean maximum tumour diameter was 3.33cm (range: 0.5-8cm). Of 110 patients with FLIPI scores available, 100 patients had a low FLIPI score (91%), 9 (8%) had an intermediate score, 1 (1%) patient had a high score. Patients were staged with cross-sectional imaging, including a PET scan in 73 (63%) of cases, and bone marrow assessment was completed in 105 (90%) of patients. The most commonly mutated gene in the cohort was CREBBP (n=61; 52.1%), followed by KMT2D (n=45; 38.5%), TNFRSF14 (n=40; 34.2%), STAT6 (n=25; 21.4%), and BCL2 (n=20; 17.1%). BCL2 mutations were more common in patients with stage II (n=8; 44%) compared to stage I disease (n=12; 12%) (p-value=0.003; after FDR adjustment, p-value=0.056). The median number of mutated genes per patient was 4 (range: 0-12, mean 4.0). The median follow-up of living patients was 5.65 years (range: 0.63-18.1 years) and there were 37 progression events in our cohort. CREBBP mutations were associated with shorter TTP (log-rank p-value 0.027), with 5-year TTP estimates of 73.3% vs. 86.0% (Figure 1A). CREBBP mutations were not significantly associated with age, stage, tumor bulk or PET staging. Using Cox univariable analysis for TTP, the hazard ratio (HR) for CREBBP mutations was 2.13 (95% confidence interval (CI) 1.07-4.25; p-value=0.031) and in multivariable analysis adjusting for age and sex the HR was 2.05 (95%CI 1.03-4.10; p-value= 0.041). However, statistical significance was not met after adjusting for the false discovery rate (FDR; p-value 0.621). CREBBP mutations were not significantly associated with OS (log-rank p-value=0.76) (Figure 1B), nor transformation (log-rank p-value=0.9), but the numbers of survival (n=14) and transformation events (n=9) were small. No other genes from the panel were associated with patients' outcomes. Conclusions: In conclusion, we found an association between CREBBP mutations and shorter time to progression in limited-stage FL treated with RT. To our knowledge, this represents a novel genetic prognostic finding in limited-stage FL. Further studies with larger sample sizes are required to confirm the association. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

De Novo Germline DHX38 Variant Associated with Alternative Splicing of Multiple Transcripts in Iron-Related Pathways in a Patient with Atypical Congenital Dyserythropoietic Anemia with Ring Sideroblasts

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare hereditary anemias caused by ineffective and dyspoietic erythropoiesis, with the pathognomonic feature of binuclear and/or multinuclear erythroid precursors in the bone marrow. The clinical diagnosis of CDA is based on identifying the characteristic morphology of erythroblasts in the bone marrow of patients presenting with chronic anemia, frequently with evidence of hemolysis but suboptimal reticulocytosis, and iron overload. CDAR (ClinicalTrials.gov identifier: NCT02964494), a registry for patients with CDA in North America, has been established with the goal of providing a longitudinal database and associated biorepository to facilitate natural history studies and research on the pathogenetic mechanisms in this heterogenous disease. A 2-year-old female, enrolled in CDAR, had transfusion-dependent anemia since birth. She demonstrated accelerated iron overload and had failure to thrive despite regular transfusions maintaining trough hemoglobin higher than 9.5 g/dL. Bone marrow evaluation revealed dyserythropoiesis along with ring sideroblasts (RS), resembling acquired myelodysplastic syndrome (MDS) with the characteristics of Refractory Anemia with RS (RARS). Whole-exome sequencing for the proband and her parents in trio analysis identified a de novo, heterozygous germline DHX38 missense variant c.3310A>C, p.Thr1104Pro in the proband. In silico prediction programs suggest this rare variant is deleterious. DEAH box RNA helicase DHX38 is an RNA helicase, homolog to the yeast Prp16 that has been shown to play a dual role in the spliceosome complex: an ATP-independent role in the first catalytic step mediating proofreading of the branchpoint sequence, and an ATP-dependent role in the second step, mediating removal of the lariat and joining the exons to complete the splicing (Tseng et al, RNA 2011). DHX38 is highly expressed during terminal erythropoiesis similarly to SF3B1, the splicing factor gene most frequently mutated in MDS-RARS. RNA-seq analysis of CD71+ reticulocytes isolated from the proband at three different time points compared to three different normal control samples, using AltAnalyze software, revealed approximately 3,000 differentially expressed genes. More than 1,500 differentially spliced events were identified, affecting over 200 genes, including SLC25A37, coding for an iron importer localized in the mitochondrial inner membrane and PPOX, coding for protoporphyrinogen oxidase (Figure 1A), both essential for the synthesis of mitochondrial heme. Mis-splicing of the same genes is a characteristic finding in MDS-RARS associated with SF3B1 pathogenic variants, most commonly p.Lys700Glu, and may contribute to RS formation. Moreover, DHX38 is known to interact with centromeric noncoding RNA, regulating chromosome segregation during mitosis, and its depletion in vitro causes abnormal grape-shaped nuclear morphology (Nishimura et al, Genes Cells 2019). Review of the patient's bone marrow studies revealed several erythroblasts with grape-shaped nuclei (Figure 1B). Erythropoiesis cultures from patient-derived iPSCs were utilized to phenocopy the disease in vitro. Cytospins of the generated erythroblasts revealed dyserythropoiesis that was verified quantitatively by increased frequency of nuclear abnormalities in erythroblasts produced in vitro from patient-derived iPSCs analyzed by imaging flow cytometry. Extra-hematopoietic abnormalities of the nucleus and mitotic defects were explored in fibroblasts generated from the patient's skin biopsy, which also tested positive for DHX38 p.Thr1104Pro. Grape-shaped nuclei were detected in cultures of patient's fibroblasts indicating improper mitotic exit due to aberrant chromosome segregation. Our findings indicate that DHX38 is likely the causative gene for this CDA with a RARS phenotype, causing mis-splicing of a multitude of genes critical for iron metabolism and erythropoiesis as well as defects in chromosome segregation and cell division. Out of approximately 250,000 alleles in the gnomAD database, the DHX38 p.Thr1104Pro variant has been detected in 4 alleles; however, SF3B1 p.Lys700Glu is also detected in 22. Therefore, the rare presence of this DHX38 variant in "healthy" population may indicate that acquired DHX38 pathogenic variants exist and could be responsible for SF3B1-negative MDS-RARS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel homozygous mutation in TRAPPC9 gene causing autosomal recessive non-syndromic intellectual disability

BackgroundThe etiology of intellectual disabilities is diverse and includes both genetic and environmental factors. The genetic causes of intellectual disabilities range from chromosomal aberrations to single gene disorders. The TRAPPC9 gene has been reported to cause autosomal recessive forms of intellectual disabilities in 56 patients from consanguineous and non-consanguineous families around the world.MethodsWe analyzed two siblings with intellectual disability, microcephaly and delayed motor and speech development from a consanguineous Sudanese family. Genomic DNA was screened for mutations using NGS panel (NextSeq500 Illumina) testing 173 microcephaly associated genes in the Molecular Genetics service in Robert Debre hospital in Paris, France.ResultsA novel homozygous mutation (NM_031466.7 (TRAPPC9):c.2288dup, p. (Val764Glyfs*7) in exon 14 of TRAPPC9 gene was found in the two patients. The mutation was predicted to cause nonsense mediated decay (NSMD) using SIFT prediction tool. The variant has not been found in either gnomAD or Exac databases. Both parents were heterozygous (carriers) to the mutation.ConclusionThis is the first study to report patients with TRAPPC9-related disorder from Sub-Saharan Africa.

Abstract 11984: A Novel Pathogenic Gata6 Variant Identified in a Family With Persistent Truncus Arteriosus, Childhood-Onset Diabetes Mellitus and Spontaneous Intestinal Perforation

GATA6 is a member of the GATA family of transcription factors, among which GATA4, 5 and 6 are known to play critical roles during heart development. Pathogenic variants in GATA6 have been primarily associated with congenital heart disease (CHD) and pancreatic agenesis in humans. Although the phenotypic spectrum associated with pathologic variation in GATA6 has expanded to include developmental delay along with congenital malformations affecting the biliary system and diaphragm, the full disease spectrum of GATA6 pathogenic variation and genotype-phenotype correlations are unclear. Here, we report a family where the father had persistent truncus arteriosus (PTA) and three children had CHD, 2 with PTA and 1 with an atrial septal defect. Notably, the father had childhood-onset diabetes mellitus and one child had spontaneous intestinal perforation (SIP). Using exome sequencing, we identified a novel, heterozygous missense variant in GATA6 (c.1403G&gt;A; p.Cys468Tyr) in affected members. This variant is absent from the gnomAD and ExAC databases and causes a missense change predicted to be damaging by all in silico bioinformatic tools. Sanger sequencing confirmed the exome sequencing results and segregation of this variant. GATA6 p.Cys468Tyr variant affects the highly conserved zinc-finger domain of GATA6 and impedes its ability to bind DNA. Using wild-type and mutant GATA6 constructs driving atrial natriuretic peptide promoter reporter, in vitro luciferase assays showed that p.Cys468Tyr mutant protein exhibited significantly decreased luciferase transcriptional activity. Furthermore, immunofluorescence revealed that p.Cys468Tyr mutant demonstrated an impaired localization pattern in the nucleus and induced protein aggregation. Our findings expand the variant and phenotypic spectrum for GATA6. Our study highlights that GATA6 variants can cause not only cardiac and pancreatic malformations but also gastrointestinal abnormalities, including neonatal SIP. Further investigation is needed to define the mechanisms underlying the full phenotypic spectrum associated with pathogenic variation in GATA6 .

Whole exome sequencing identified a novel POT1 variant as a candidate pathogenic allele underlying a Li-Fraumeni-like family.

Li-Fraumeni syndrome (LFS) and Li-Fraumeni-like (LFL) syndrome are rare hereditary diseases characterized by predisposition to a diverse spectrum of cancer types, primarily sarcoma. The pathogenic variants underlying the majority of LFL cases remain to be explored. We performed whole-exome sequencing (WES) on 13 core members of a large LFL family with highly aggregated incidences of cancers, including cases with sarcoma, non-small cell lung cancer and cardiac angiosarcoma, and conducted a comprehensive literature review of candidate gene associations in LFS/LFL syndromes or sarcoma to identify potential pathogenic germline variants. No germline variants in the best-known LFL/LFS-associated gene TP53 were detected. Of all the genes associated with LFS/LFL or sarcoma that we have surveyed, we identified a novel p.P35L germline variant in POT1 (protection of telomeres 1). Germline and somatic alterations in POT1 have been implicated in a series of familial cancers, including angiosarcoma, glioma, melanoma and colorectal cancer. This particular variant is located in the telomere-binding OB1 domain, which is important in maintaining the proper telomere length, and showed high conservation across different POT1 orthologues. No record of the variant was found in any of the 1000 genomes, ExAC, gnomAD, dpSNP and COSMIC databases. Prediction algorithms and in silico structural analysis suggested completely disrupted protein structure and function of POT1 in the presence of this mutation. Leveraging WES, we identified a novel germline risk allele, p.P35L in POT1, that likely predisposes to LFL syndrome. Our results support the routine testing of POT1 and other LFL/LFS-associated genes in the risk populations to enable early cancer diagnosis, prevention and intervention.

Novel rare mutation in a conserved site of PTPRB causes human hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a rare but fatal birth defect in which the left side of the heart is underdeveloped. HLHS accounts for 2% to 4% of congenital heart anomalies. Whole genome sequencing (WGS) was conducted for a family trio consisting of a proband and his parents. A homozygous rare variant was detected in the PTPRB (Protein Tyrosine Phosphatase Receptor Type B) gene of the proband by functional annotation and co-segregation analysis. Sanger sequencing was used to confirm genotypes of the variant. The in silico prediction tools, including Mutation Taster, SpliceAI, and CADD, were used to predict the impact of the mutation. The allele frequencies across populations were compared based on multiple databases, including "1000 genomes" and "gnomAD". We used two vectors (pcMINI and pcDNA3.1) to generate a minigene construct to validate the mutational effect at the transcriptional level. Family-based WGS analyses showed that only a homozygous splice acceptor variant (NC_000012.12: g.70636068T>G, NM_001109754.4: c.56-2A>C, NG_029940.2: g.6373A>C) at the exon-intron border of PTPRB gene associates with HLHS. This variant is also within the region with the enhancer activity based on UCSC genome annotation. Genotyping and Sanger sequencing revealed that the proband's parents are heterozygous for this variant. Evolutionary conservation analysis revealed that the site (NC_000012.12: g.70636068) is extremely conserved across species, supporting the evolutionary functional constraints of the ancestral wild type (T). In silico tools universally predicted a deleterious or disease-causing impact of the mutation from T to G. The mutation was not found in the 1000 genomes and gnomAD databases, which indicates that this mutation is very rare in most human populations. A splicing assay indicated that the mutated minigene caused aberrant splicing of mRNA, in which a 3bp missing in the second exon resulted in the deletion of one amino acid (NP_001103224.1:p.Glu19del) compared to the normal protein of PRPTB (also the VE-PTP). Structure prediction revealed that the deletion occurred within the C-region of the signal peptide of VE-PTP, suggesting signal peptide-related defects as a potential mechanism for the HLHS cellular pathogeny. We report a rare homozygous variant with splicing error in PTPRB associated with HLHS. Previous model species studies revealed conserved functions of PTPRB in cardiovascular and heart development in mice and zebrafish. Our study is the first report to show the association between PTPRB and HLHS in humans.

P.10 Compound CACNA1S heterozygosity resulting in a novel phenotype of congenital myopathy and early onset periodic paralysis: report of two probands

Skeletal muscle contraction is mediated by excitation-contraction coupling (ECC), a process involving the rapid elevation of cytosolic calcium. L-type voltage gated calcium channels located on T-tubule membranes are the voltage sensors of ECC. The dihydropyridine receptor (DHPR) contains the pore forming basic functional elements of these calcium channels and is encoded by the <i>CACNA1S</i> gene. Recessive mutations in the <i>CACNA1S</i> gene have been recently reported with a severe congenital myopathy and facial involvement. We describe two sisters with compound heterozygous mutations involving the <i>CACNA1S</i> gene. Both had neonatal hypotonia, muscle weakness, and delayed motor milestones. Prior to one year of age, periodic paralysis was evident as episodic flaccidity and loss of postural control. Periodic paralysis episodes continued to occur most notably with cold exposure, lasting around 10 minutes each. Lab evaluation revealed normal CPK. Electrophysiological evaluation revealed normal motor and sensory nerve conduction studies, and normal long exercise testing. Limb cooling and cold provocation led to clinical weakness and a drop in motor amplitude. Magnetic resonance imaging of the older sister showed symmetrical tigroid pattern fat replacement and atrophy of bilateral gluteus maximus muscles. Targeted next generation sequencing revealed two variants in the <i>CACNA1S</i> gene: both c.3526-2A>G and c.2831G>A (p.Cys944Tyr) are not described in the gnomAD database. Both variants co-segregated into affected sisters and Sanger sequencing revealed that c.3526-2A>G was inherited from the unaffected mother and c.2831G>A from the unaffected father, thus revealing a recessive pattern of inheritance. The c.3526-2A>G is expected to disrupt the acceptor splice site of intron 27 by <i>in silico</i> analysis, whereas the c.2831G>A variant replaces a highly conserved cysteine residue with tyrosine at codon 944 in the DIII-S5 of the Cav1.1 protein. This novel phenotype of a milder congenital recessive myopathy and early onset periodic paralysis expands the phenotypic spectrum of <i>CACNA1S</i> gene mutations.

Determining the Likelihood of Disease Pathogenicity Among Incidentally Identified Genetic Variants in Rare Dilated Cardiomyopathy-Associated Genes.

Background As utilization of clinical exome sequencing (ES) has expanded, criteria for evaluating the diagnostic weight of incidentally identified variants are critical to guide clinicians and researchers. This is particularly important in genes associated with dilated cardiomyopathy (DCM), which can cause heart failure and sudden death. We sought to compare the frequency and distribution of incidentally identified variants in DCM-associated genes between a clinical referral cohort with those in control and known case cohorts to determine the likelihood of pathogenicity among those undergoing genetic testing for non-DCM indications. Methods and Results A total of 39 rare, non-TTN DCM-associated genes were identified and evaluated from a clinical ES testing referral cohort (n=14 005, Baylor Genetic Laboratories) and compared with a DCM case cohort (n=9442) as well as a control cohort of population variants (n=141 456) derived from the gnomAD database. Variant frequencies in each cohort were compared. Signal-to-noise ratios were calculated comparing the DCM and ES cohort with the gnomAD cohort. The likely pathogenic/pathogenic variant yield in the DCM cohort (8.2%) was significantly higher than in the ES cohort (1.9%). Based on signal-to-noise and correlation analysis, incidental variants found in FLNC, RBM20, MYH6, DSP, ABCC9, JPH2, and NEXN had the greatest chance of being DCM-associated. Conclusions The distribution of pathogenic variants between the ES cohort and the DCM case cohort was gene specific, and variants found in the ES cohort were similar to variants found in the control cohort. Incidentally identified variants in specific genes are more associated with DCM than others.

Whole-genome sequencing identifies new candidate genes for nonobstructive azoospermia.

Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established. To identify low-frequency NOA-associated single nucleotide variants (SNVs) using whole-genome sequencing (WGS). Men with various types of NOA (n = 39), including samples that had been previously tested with whole-exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed. Using WGS, we revealed potential NOA-associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis. In this study, we identified novel potential candidate NOA-associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA-associated gene findings.

When LUCA met gnomAD: genetic constraints on universal genes in humans.

LUCA, the last universal common ancestor, is the hypothetical most recent common ancestor of the three domains of life which share the universal genes (UG). It seems interesting to evaluate whether the UG phylogeny has had an impact on current Human gene constraints. A list of human homologs of UG was retrieved from the eggNOG database. We analyzed this LUCA gene (LG) group, and a random sample of 500 genes from the gnomAD database (RG group). Gene constraint metrics were retrieved from gnomAD and associations with Mendelian diseases and modes of inheritance were retrieved from OMIM. The LG group consisted of 277 genes and the RG group, 492 (8 genes were in LG group). 38.6% of the genes in the LG group and 25.2% of the genes in the RG group were associated with a Mendelian disease (p < 0.0001). The mode of inheritance was more often autosomal recessive (69.0 vs. 50.5%), and less often autosomal dominant (19.0 vs. 31.3%), or mixed (6.0 vs. 12.1%) for those associated with the LG group (p = 0.048). The LG group was significantly more constrained for missense variants (MOEUF, 0.919 vs. 0.997, p < 0.0001) and was borderline significantly more constrained for loss-of-function variants (LOEUF, 0.872 vs. 0.947, p = 0.051). These results suggest that the UG in humans differs from the rest of the genome in terms of constraints and associated Mendelian diseases. It suggests that phylogenic data can explain some of the characteristics of human genes and could help in interpreting variants.

Variants in HNRNPH1 are associated with high myopia in humans and ocular coloboma in zebrafish.

High myopia is one of the most common causes for blindness due to its associated complications. Genetic factor has been considered as the major cause for early-onset high myopia (eoHM), but exact genetic defects for most eoHM are yet to be identified. Through multi-step bioinformatics analysis of our in-house whole exome sequencing dataset from 6397 individuals, variants from 928 probands with eoHM were further compared with those from in-house controls as well as gnomAD database. The results showed that loss-of-function (LoF) variants in a novel gene HNRNPH1 were identified in two of 928 probands with eoHM but in none of 5469probands with other eye conditions (p=0.02). LoF variants in HNRNPH1 were extremely rare and intolerant, while two LoF variants in 928 eoHM were statistically higher than their frequency in gnomAD (p=5.98 × 10-4 ). These two LoF variants, c.2dup/p.(M1?) and c.121dup/p.(Q41Pfs*20), were absent from existing database. Variants in HNRNPH1 have not been associated with any inherited eye disease before. Expression of HNRNPH1 was enriched in ganglion cell layer and inner nuclear layer in humans. Knockdown of hnrnph1 in zebrafish resulted in ocular coloboma. All these suggest that HNRNPH1 is potential contribution to eoHM when mutated.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion.In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations.The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5.In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77.In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

CELSR1 variants are associated with partial epilepsy of childhood.

CELSR1 gene, encoding cadherin EGF LAG seven-pass G-type receptor 1, is mainly expressed in neural stem cells during the embryonic period. It plays an important role in neurodevelopment. However, the relationship between CELSR1 and disease of the central nervous system has not been defined. In this study, we performed trios-based whole-exome sequencing in a cohort of 356 unrelated cases with partial epilepsy without acquired causes and identified CELSR1 variants in six unrelated cases. The variants included one de novo heterozygous nonsense variant, one de novo heterozygous missense variant, and four compound heterozygous missense variants that had one variant was located in the extracellular region and the other in the cytoplasm. The patients with biallelic variants presented severe epileptic phenotypes, whereas those with heterozygous variants were associated with a mild epileptic phenotype of benign epilepsy with centrotemporal spikes (BECTS). These variants had no or low allele frequency in the gnomAD database. The frequencies of the CELSR1 variants in this cohort were significantly higher than those in the control populations. The evidence from ClinGen Clinical-Validity Framework suggested a strong association between CELSR1 variants and epilepsy. These findings provide evidence that CELSR1 is potentially a candidate pathogenic gene of partial epilepsy of childhood.

Abstract EC101: Premature Stop Mutations In Mybphl Share The Common Pathomechanism Of Preventing Protein Incorporation Into The Cardiac Myofilament

Cardiomyopathy and arrhythmia are diseases with significant genetic linkage. Cardiac myosin binding protein-C (cMyBP-C) is a myofilament associated protein with a C-terminal composed of repeating immunoglobulin (Ig) and fibronectin III (FnIII) domains that bind to the myosin thick filaments and titin. Truncating mutations in the C-terminal of MyBP-C result in improper myofilament incorporation and degradation of the truncated protein and is a major cause of hypertrophic cardiomyopathy. Myosin-binding protein-H like ( MyBP-HL ) is a protein that was recently identified that is structurally related to MyBP-C. Both cMyBP-C and MyBP-HL contain repeated C-terminal Ig and FnIII domains that bind to myosin. MyBP-HL is different in that it is primarily expressed in the atria where it interacts with the myofilament. MyBP-HL premature stop mutations are associated with improper myofilament incorporation, and has been linked to ventricular conduction system abnormalities, atria enlargement, and dilated cardiomyopathy (DCM) in humans. The gnomAD database reports 9 stop-gain mutations for MYBPHL in humans including: Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255 and Tyr307. Prior investigation modeled the MyBP-HL Arg255X mutation and showed no MyBP-HL localization into the myofilament. A null mouse model showed that loss of MyBP-HL resulted in arrhythmia and cardiomyopathy, similar to that observed in humans. We hypothesize that all MyBP-HL truncation mutations will share a common mechanism by failing to produce MyBP-HL that integrates into the sarcomere. We test this hypothesis by transfecting neonatal rat cardiomyocytes with MyBP-HL constructs containing these mutations. We tested Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250 and Arg255 MyBP-HL mutations. We measured co-localization events between MyBP-HL mutants and MyBP-C in atrial and ventricular overexpressed cells and observed that stop gained mutations disrupt normal localization as we expected. Moreover, we tested wild-type MyBP-HL overexpression, and both showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. These truncating mutations in MYBP-HL may increase risk for developing arrhythmias, atria enlargement and DCM.

Whole exome sequencing identifies novel inherited genetic variants in tetralogy of Fallot

BackgroundTetralogy of Fallot (TOF) is the most common neonatal cyanotic heart defect, and genetic variation is an important risk factor for the etiology of TOF. Identifying TOF-associated genetic variants is critical to understanding susceptibility and outcome in patients with TOF and may help delineate pathological mechanisms.MethodsWhole exome sequencing (WES) was performed 19 patients with sporadic TOF and 3 healthy controls. The dbSNP, GnomAD, Denovo-db, and ClinVar databases were used to annotate the mutations. PolyPhen, SIFT, MutationTaster, and FATHMM softwares were used for mutation pathogenicity analysis. Sanger sequencing was used to validate candidate variants.ResultsWe identified 21 genetic variants involving 16 genes were found in 12 patients with sporadic TOF. The types of mutations were missense and splicing variants. None of these genes were detected in samples from the 3 healthy controls. These variants include 9 pathogenic variants, 6 suspected pathogenic variants, and 6 variants of unknown significance (VUS). Further analysis showed that the patients with apolipoprotein B (APOB) and ring finger protein 135 (RNF135) variants had more serious clinical symptoms. Sanger sequencing confirmed that the two variants were heterozygous in TOF patients.ConclusionsWe identified several genetic variants associated with TOF and confirmed that RNF135 and ABOB variants were associated with TOF severity. These findings provide new evidence for exploring the genetic mechanism of TOF.

Characterization of ADME Gene Variation in Colombian Population by Exome Sequencing.

In genes related to drug pharmacokinetics, molecular variations determine interindividual variability in the therapeutic efficacy and adverse drug reactions. The assessment of single-nucleotide variants (SNVs) is used with growing frequency in pharmacogenetic practice, and recently, high-throughput genomic analyses obtained through next-generation sequencing (NGS) have been recognized as powerful tools to identify common, rare and novel variants. These genetic profiles remain underexplored in Latin-American populations, including Colombia. In this study, we investigated the variability of 35 genes included in the ADME core panel (absorption, distribution, metabolism, and excretion) by whole-exome sequencing (WES) of 509 unrelated Colombian individuals with no previous reports of adverse drug reactions. Rare variants were filtered according to the minor allele frequencies (MAF) <1% and potential deleterious consequences. The functional impact of novel and rare missense variants was assessed using an optimized framework for pharmacogenetic variants. Bioinformatic analyses included the identification of clinically validated variants described in PharmGKB and ClinVar databases. Ancestry from WES data was inferred using the R package EthSEQ v2.1.4. Allelic frequencies were compared to other populations reported in the public gnomAD database. Our analysis revealed that rare missense pharmacogenetic variants were 2.1 times more frequent than common variants with 121 variants predicted as potentially deleterious. Rare loss of function (LoF) variants were identified in 65.7% of evaluated genes. Regarding variants with clinical pharmacogenetic effect, our study revealed 89 sequence variations in 28 genes represented by missense (62%), synonymous (22.5%), splice site (11.2%), and indels (3.4%). In this group, ABCB1, ABCC2, CY2B6, CYP2D6, DPYD, NAT2, SLC22A1, and UGTB2B7, are the most polymorphic genes. NAT2, CYP2B6 and DPYD metabolizer phenotypes demonstrated the highest variability. Ancestry analysis indicated admixture in 73% of the population. Allelic frequencies exhibit significant differences with other Latin-American populations, highlighting the importance of pharmacogenomic studies in populations of different ethnicities. Altogether, our data revealed that rare variants are an important source of variability in pharmacogenes involved in the pharmacokinetics of drugs and likely account for the unexplained interindividual variability in drug response. These findings provide evidence of the utility of WES for pharmacogenomic testing and into clinical practice.

Whole Exome Sequencing Enhanced Imputation Identifies 85 Metabolite Associations in the Alpine CHRIS Cohort.

Metabolites are intermediates or end products of biochemical processes involved in both health and disease. Here, we take advantage of the well-characterized Cooperative Health Research in South Tyrol (CHRIS) study to perform an exome-wide association study (ExWAS) on absolute concentrations of 175 metabolites in 3294 individuals. To increase power, we imputed the identified variants into an additional 2211 genotyped individuals of CHRIS. In the resulting dataset of 5505 individuals, we identified 85 single-variant genetic associations, of which 39 have not been reported previously. Fifteen associations emerged at ten variants with >5-fold enrichment in CHRIS compared to non-Finnish Europeans reported in the gnomAD database. For example, the CHRIS-enriched ETFDH stop gain variant p.Trp286Ter (rs1235904433-hexanoylcarnitine) and the MCCC2 stop lost variant p.Ter564GlnextTer3 (rs751970792-carnitine) have been found in patients with glutaric acidemia type II and 3-methylcrotonylglycinuria, respectively, but the loci have not been associated with the respective metabolites in a genome-wide association study (GWAS) previously. We further identified three gene-trait associations, where multiple rare variants contribute to the signal. These results not only provide further evidence for previously described associations, but also describe novel genes and mechanisms for diseases and disease-related traits.

Novel compound heterozygous cadherin 3 mutations in hypotrichosis and juvenile macular dystrophy.

Novel compound heterozygous cadherin 3 mutations in hypotrichosis and juvenile macular dystrophy.

Complex Inheritance of Rare Missense Variants in PAK2, TAP2, and PLCL1 Genes in a Consanguineous Arab Family With Multiple Autoimmune Diseases Including Celiac Disease

BackgroundAutoimmune diseases (AIDs) share a common molecular etiology and often present overlapping clinical presentations. Thus, this study aims to explore the complex molecular basis of AID by whole exome sequencing and computational biology analysis.MethodsMolecular screening of the consanguineous AID family and the computational biology characterization of the potential variants were performed. The potential variants were searched against the exome data of 100 healthy individuals and 30 celiac disease patients.ResultA complex inheritance pattern of PAK2 (V43A), TAP2 (F468Y), and PLCL1 (V473I) genetic variants was observed in the three probands of the AID family. The PAK2 variant (V43A) is a novel one, but TAP2 (F468Y) and PLCL1 (V473I) variants are extremely rare in local Arab (SGHP and GME) and global (gnomAD) databases. All these variants were localized in functional domains, except for the PAK2 variant (V43A) and were predicted to alter the structural (secondary structure elements, folding, active site confirmation, stability, and solvent accessibility) and functional (gene expression) features. Therefore, it is reasonable to postulate that the dysregulation of PAK2, TAP2, and PLCL1 genes is likely to elicit autoimmune reactions by altering antigen processing and presentation, T cell receptor signaling, and immunodeficiency pathways.ConclusionOur findings highlight the importance of exploring the alternate inheritance patterns in families presenting complex autoimmune diseases, where classical genetic models often fail to explain their molecular basis. These findings may have potential implications for developing personalized therapies for complex disease patients.