CADD Scores Research Articles

A structural genomics approach to investigate Dystrophin mutations and their impact on the molecular pathways of Duchenne muscular dystrophy

BackgroundDystrophin is a key protein encoded by the DMD gene, serves as a scaffold linking the cytoskeleton to the extracellular matrix that plays a critical role in muscle contraction, relaxation, and structural integrity. Mutations, particularly single-point amino acid substitutions, can lead to dysfunctional Dystrophin, causing muscular dystrophies, with Duchenne muscular dystrophy (DMD) being the most severe form.ObjectiveThis study aimed to evaluate the effects of 184 single-point amino acid substitutions on the structure and function of Dystrophin using computational approaches.MethodsMany computational tools were used to predict the impact of amino acid substitutions on protein stability, solubility, and function. Pathogenic potential was assessed using disease phenotype predictors and CADD scores, while allele frequency data from gnomAD contextualized mutation prevalence. Additionally, aggregation propensity, frustration analysis, and post-translational modification sites were analyzed for functional disruptions.ResultsOf the 184 substitutions analyzed, 50 were identified as deleterious, with 41 predicted to be pathogenic. Seventeen mutations were localized in the Calponin-homology (CH) 1 domain, a critical functional region of Dystrophin. Six substitutions (N26H, N26K, G47W, D98G, G109A, and G109R) were predicted to decrease protein solubility and were located in minimally frustrated regions, potentially compromising Dystrophin functionality and contributing to DMD pathogenesis.ConclusionThis study provides novel insights into the molecular mechanisms of DMD, highlighting specific mutations that disrupt Dystrophin’s solubility and function. These findings could inform future therapeutic strategies targeting Dystrophin mutations to address DMD pathogenesis.

Whole-genome sequencing reveals rare variants associated with gout in Taiwanese males.

To identify rare variants (RVs) of gout, we sequenced the whole genomes of 321 male gout patients and combined these with those of 64 male gout patients and 682 normal controls at Taiwan Biobank. We performed ACAT-O to identify 682 significant RVs (p < 3.8 × 10-8) clustered on chromosomes 1, 7, 10, 16, and 18. To prioritize causal variants effectively, we sifted them by Combined Annotation-Dependent Depletion score >10 or |effect size| ≥ 1.5 for those without CADD scores. In particular, to the best of our knowledge, we identified the rare variants rs559954634, rs186763678, and 13-85340782-G-A for the first time to be associated with gout in Taiwanese males. Importantly, the RV rs559954634 positively affects gout, and its neighboring gene NPHS2 is involved in serum urate and expressed in kidney tissues. The kidneys play a major role in regulating uric acid levels. This suggests that rs559954634 may be involved in gout. Furthermore, rs186763678 is in the intron of NFIA that interacts with SLC2A9, which has the most significant effect on serum urate. Note that gene-gene interaction NFIA-SLC2A9 is significantly associated with serum urate in the Italian MICROS population and a Croatian population. Moreover, 13-85340782-G-A significantly affects gout susceptibility (odds ratio 6.0; P = 0.038). The >1% carrier frequencies of these potentially pathogenic (protective) RVs in cases (controls) suggest the revealed associations may be true; these RVs deserve further studies for the mechanism. Finally, multivariate logistic regression analysis shows that the rare variants rs559954634 and 13-85340782-G-A jointly are significantly associated with gout susceptibility.

Genetic profile of progressive myoclonic epilepsy in Mali reveals novel findings.

Progressive myoclonic epilepsy (PME) is a group of neurological disorders characterized by recurrent myoclonic seizures with progressive neurological deterioration. We investigated the genetics of three unrelated patients with PME from Mali, a country in sub-Saharan Africa highly underrepresented in genetic and genomic research. Participants were carefully examined and phenotyped. DNA was obtained for genetic analysis including whole exome sequencing (WES). In silico prediction tools and ACMG criteria were used to assess the deleteriousness of putative candidate variants. Pedigree analysis suggests autosomal recessive inheritance patterns for one family and sporadic forms of PME for the two other cases. WES identified novel homozygous missense variants in all the three patients, one each for NHLRC1, EPM2A, and NEU1. The sequence variants segregated with PME in each family and in silico studies including protein 3D structures, CADD scores and ACMG criteria suggested that they were damaging. PME is a group of clinically heterogeneous neurological disorders. Most reported cases in the literature are from European background with only a few cases described in North Africa. We report here novel pathogenic variants in three different genes causing PME phenotypes in three unrelated Malian patients, suggesting that genetic studies of underrepresented populations may expand the genetic epidemiology of PME. These findings also emphasize the need for inclusive genetic research to ensure a more targeted diagnostic and therapeutic approaches for diverse patient populations.

Meta-analysis of 46,000 germline de novo mutations linked to human inherited disease

BackgroundDe novo mutations (DNMs) are variants that occur anew in the offspring of noncarrier parents. They are not inherited from either parent but rather result from endogenous mutational processes involving errors of DNA repair/replication. These spontaneous errors play a significant role in the causation of genetic disorders, and their importance in the context of molecular diagnostic medicine has become steadily more apparent as more DNMs have been reported in the literature. In this study, we examined 46,489 disease-associated DNMs annotated by the Human Gene Mutation Database (HGMD) to ascertain their distribution across gene and disease categories.ResultsMost disease-associated DNMs reported to date are found to be associated with developmental and psychiatric disorders, a reflection of the focus of sequencing efforts over the last decade. Of the 13,277 human genes in which DNMs have so far been found, the top-10 genes with the highest proportions of DNM relative to gene size were H3-3 A, DDX3X, CSNK2B, PURA, ZC4H2, STXBP1, SCN1A, SATB2, H3-3B and TUBA1A. The distribution of CADD and REVEL scores for both disease-associated DNMs and those mutations not reported to be de novo revealed a trend towards higher deleteriousness for DNMs, consistent with the likely lower selection pressure impacting them. This contrasts with the non-DNMs, which are presumed to have been subject to continuous negative selection over multiple generations.ConclusionThis meta-analysis provides important information on the occurrence and distribution of disease-associated DNMs in association with heritable disease and should make a significant contribution to our understanding of this major type of mutation.

Evaluating the Utility of REVEL and CADD for Interpreting Variants in Amyotrophic Lateral Sclerosis Genes

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease affecting approximately two per 100,000 individuals globally. While there are many benefits to offering early genetic testing to people with ALS, this has also led to an increase in the yield of novel variants of uncertain significance in ALS-associated genes. Computational (in silico) predictors, including REVEL and CADD, are widely employed to provide supporting evidence of pathogenicity for variants in conjunction with clinical, molecular, and other genetic evidence. However, in silico predictors are developed to be broadly applied across the human genome; thus, their ability to evaluate the consequences of variation in ALS-associated genes remains unclear. To resolve this ambiguity, we surveyed 20 definitive and moderate ClinGen-defined ALS-associated genes from two large, open-access ALS sequencing datasets (total people with ALS = 8,230 ; controls = 9,671 ) to investigate REVEL and CADD’s ability to predict which variants are most likely to be disease-causing in ALS. While our results indicate a predetermined pathogenicity threshold for REVEL that could be of clinical value for classifying variants in ALS-associated genes, an accurate threshold was not evident for CADD, and both in silico predictors were of limited value for resolving which variants of uncertain significance (VUS) may be likely pathogenic in ALS. Our findings allow us to provide important recommendations for the use of REVEL and CADD scores for variants and indicate that both tools should be used with caution when attempting to evaluate the pathogenicity of VUSs in ALS genetic testing.

Exome sequencing findings in children with annular pancreas.

Annular pancreas (AP) is a congenital defect of unknown cause in which the pancreas encircles the duodenum. Theories include abnormal migration and rotation of the ventral bud, persistence of ectopic pancreatic tissue, and inappropriate fusion of the ventral and dorsal buds before rotation. The few reported familial cases suggest a genetic contribution. We conducted exome sequencing in 115 affected infants from the California birth defects registry. Seven cases had a single heterozygous missense variant in IQGAP1, five of them with CADD scores >20; seven other infants had a single heterozygous missense variant in NRCAM, five of them with CADD scores >20. We also looked at genes previously associated with AP and found two rare heterozygous missense variants, one each in PDX1 and FOXF1. IQGAP1 and NRCAM are crucial in cell polarization and migration. Mutations result in decreased motility which could possibly cause the ventral bud to not migrate normally. To our knowledge, this is the first study reporting a possible association for IQGAP1 and NRCAM with AP. Our findings of rare genetic variants involved in cell migration in 15% of our population raise the possibility that AP may be related to abnormal cell migration.

Genome-wide identification of dominant polyadenylation hexamers for use in variant classification.

Polyadenylation is an essential process for the stabilization and export of mRNAs to the cytoplasm and the polyadenylation signal hexamer (herein referred to as hexamer) plays a key role in this process. Yet, only 14 Mendelian disorders have been associated with hexamer variants. This is likely an under-ascertainment as hexamers are not well defined and not routinely examined in molecular analysis. To facilitate the interrogation of putatively pathogenic hexamer variants, we set out to define functionally important hexamers genome-wide as a resource for research and clinical testing interrogation. We identified predominant polyA sites (herein referred to as pPAS) and putative predominant hexamers across protein coding genes (PAS usage >50% per gene). As a measure of the validity of these sites, the population constraint of 4532 predominant hexamers were measured. The predominant hexamers had fewer observed variants compared to non-predominant hexamers and trimer controls, and CADD scores for variants in these hexamers were significantly higher than controls. Exome data for 1477 individuals were interrogated for hexamer variants and transcriptome data were generated for 76 individuals with 65 variants in predominant hexamers. 3' RNA-seq data showed these variants resulted in alternate polyadenylation events (38%) and in elongated mRNA transcripts (12%). Our list of pPAS and predominant hexamers are available in the UCSC genome browser and on GitHub. We suggest this list of predominant hexamers can be used to interrogate exome and genome data. Variants in these predominant hexamers should be considered candidates for pathogenic variation in human disease, and to that end we suggest pathogenicity criteria for classifying hexamer variants.

DNA damage response (DDR) variants and effects on the tumor transcriptome and microenvironment in east Asian (EA) prostate cancers (PCa).

75 Background: Aberrations of the DDR pathway have been shown to be prognostic of survival and predictive of PARP inhibition in PCa. We have previously reported on the prevalence of germline DDR mutations based on a 32-gene set in localized PCa in EA men, and observed comparable rates with that of White men. Here, we performed additional analyses investigating for associations between DDR variants to the somatic transcriptome of localized PCa. Methods: We utilized a prospectively recruited cohort of 172 EA patients, with centrally assessed Gleason’s score (GS) by an expert pathologist. Germline profiling was performed by whole exome sequencing (100X) using the Illumina Novaseq (CA). Joint genotyping was performed to annotate variants that were deemed to be pathogenic (P)/likely pathogenic (LP)/conflict of interest pathogenic (CIP) based on the CADD and REVEL scores. Tumor transcriptome was profiled using the Decipher Genomic Classifier (GC, Veracyte, CA) based on the Affymetrix ST array (ThermoFisher, CA). Results: Among the subset of 172 EA men, we observed DDR variants in 26 (15%) patients, which was comparable to the larger cohort of 890 men from the same institution. Among the DDR mutations, 2 (7.7%) were classified as P/LP, while 24 (92.3%) were CIP. Decipher GC scores were comparable between patients with and without DDR mutations (median GC score 0.87 vs 0.53, Mann-Whitney U P=0.10). For luminal-basal subtyping by either the PAM50 and Prostate specific subtyping models, we did not observe a difference for the basal (P=0.48) and luminal subtypes (P=0.21). Interestingly, DNA repair gene-set expression were comparable between patients with DDR mutations versus those without (median score -0.2 vs -0.17). However, we observed microenvironment differences for immune-related genes, where patients harboring DDR mutations manifested a less immune-suppressive environment versus those without (PDL2: -0.15 vs 0.2; Treg: 0.09 vs 0.13; MDSC: 0.03 vs 0.07, respectively). Conclusions: Herein, we report an extension of our previous work (Lua, et al. ASCO GU, 2023), where we show potential linkages between the germline DDR variant status and the tumor transcriptome. Our findings suggest the influence of germline DDR variants that extends beyond tumorigenesis.

Gene panel analysis of literature-based infertility genes in Sertoli cell-only syndrome patients

Sertoli cell-only syndrome (SCOS) is a condition of male infertility characterized by a total absence of spermatogenic cells in nearly all seminiferous tubules. Apart from well-established genetic changes such as Klinefelter syndrome, CFTR variants, and Y-chromosome microdeletions, several hundred candidate genes were reported as associated with male infertility. We selected 92 evidence-based genes associated with infertility and investigated data from whole-exome sequencing in 6 individuals with clinically diagnosed SCOS. Eight heterozygous variants passed our filtering criteria, including population frequency ≤ 0.1% and high functional impact indicated by Sift, Polyphen, and CADD scores. Out of them, we considered only variants with putative autosomal dominant effects on infertility that were subsequently validated by Sanger sequencing. This filtering pipeline has led to the final likely causative variants detected in CHD7 and SCYP3 genes that potentially explain SCOS in two of our patients. Our discoveries suggest that gene panel testing of patients with SCOS could improve the diagnostic outcome; however, assembling a gene panel consisting of only genuine causative genes is crucial.

Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are “variants of uncertain significance” (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Quantitative determination of SLC2A1 variant functional effects in GLUT1 deficiency syndrome.

The goal of this study is to demonstrate the utility of a growth assay to quantify the functional impact of single nucleotide variants (SNVs) in SLC2A1, the gene responsible for Glut1DS. The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio. Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores. Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.

De novo variants in MAST4 related to neurodevelopmental disorders with developmental delay and infantile spasms: Genotype-phenotype association.

This study aims to prove that the de novo variants in MAST4 gene are associated with neurodevelopmental disorders (NDD) with developmental delay (DD) and infantile spasm (IS) and to determine the genotype-phenotype correlations. Trio-based exome sequencing (ES) was performed on the four families enrolled in this study. We collected and systematically reviewed the four probands' clinical data, magnetic resonance images (MRI), and electroencephalography (EEG). We also carried out bioinformatics analysis by integrating published exome/genome sequencing data and human brain transcriptomic data. We described four patients whose median age of seizure onset was 5 months. The primary manifestation was infantile spasms with typical hypsarrhythmia on EEG. Developmental delays or intellectual disabilities varied among the four individuals. Three de novo missense variants in MAST4 gene were identified from four families, including chr5:66438324 (c.2693T > C: p.Ile898Thr) z, chr5:66459419 (c.4412C > T: p.Thr1471Ile), and chr5:66462662 (c.7655C > G:p.Ser2552Trp). The missense variant p.Ile898Thr is mapped to the AGC-kinase C-terminal with phosphatase activity. The other variant p.Ser2552Trp is located in a phosphoserine-modified residue which may affect cell membrane stability and signal transduction. Besides, the variant p.Thr1471Ile is a recurrent site screened out in two unrelated patients. Compared to private mutations (found only in a single family or a small population) of MAST4 in the gnomAD non-neuro subset, all de novo variants were predicted to be damaging or probably damaging through different bioinformatic analyses. Significantly higher CADD scores of the variant p.Thr1471Ile indicate more deleteriousness of the recurrent site. And the affected amino acids are highly conserved across multiple species. According to the Brainspan Atlas database, MAST4 is expressed primarily in the mediodorsal nucleus of the thalamus and medial prefrontal cortex during the prenatal period, potentially contributing to embryonic brain development. Our results revealed that the variants of MAST4 gene might lead to neurodevelopmental disorders with developmental delay and infantile spasm. Thus, MAST4 variants should be considered the potential candidate gene in patients with neurodevelopmental disorders clinically marked by infantile spasms.

In Silico Analysis of BMAL1 and CLOCK SNPs in the Ensembl Database

Objective: A circadian rhythm in mammals controls the sleep-wake cycle, blood pressure, hormone secretion, metabolism and many other physiological processes. The circadian clock mechanism is regulated by four genes: Bmal1, Clock, Cry, and Per. Mutations in these regulatory genes are associated with sleep and mood disorders, obesity, and cancer. Several PER2 and CRY2 SNPs are associated with advanced sleep phase syndrome. It is, therefore, critical to understand the effect of clock genes’ SNPs on the circadian clock. In this study, we determined “pathogenic” BMAL1 and CLOCK SNPs in the Ensembl database for biochemical characterization. Materials and Methods: BMAL1 and CLOCK SNPs in the Ensemble database were filtered out for only missense mutations. Among the missense mutations, pathogenic ones were determined according to SIFT, PolyPhen, and CADD scores, REVEL, MetalR, Mutation Assessor, I-Mutant, PROVEAN, and FireDock programs. BMAL1 and CLOCK SNPs were visualized by using PyMol. Results: Thousands of BMAL1 and CLOCK missense SNP mutations were reported in the Ensembl database. After the classification of those SNPs according to their SIFT, PolyPhen, and CADD pathogenicity, twelve SNPs for each protein remained as pathogenic. A further analysis with all in silico tools revealed that BMAL1 SNPs causing Ala154Val, Arg166Gln, and Val440Gly mutations; and CLOCK SNPs causing Gly120Val, Asp119Val, Gly120Ser, Ala117Val, and Cys371Gly mutations were predicted as the most “pathogenic” ones. Conclusion: Overall, by using in silico tools, we provided a starting point for experimental studies for determining the effect of pathogenic BMAL1 and CLOCK SNPs on the circadian clock mechanism.

Molecular dynamics study of tropical calcific pancreatitis (TCP) associated calcium-sensing receptor single nucleotide variation.

Tropical Calcific Pancreatitis (TCP) is a chronic non-alcoholic pancreatitis characterised by extensive calcification. The disease usually appears at a younger age and is more common in tropical regions. This disease’s progression can lead to pancreatic diabetes, which can subsequently lead to pancreatic cancer. The CASR gene encodes a calcium-sensing receptor (CaSR), which is a GPCR protein of class C. It is expressed in the islets of Langerhans, the parathyroid gland, and other tissues. It primarily detects small gradients in circulating calcium concentrations and couples this information to intracellular signalling, which helps to regulate PTH (parathyroid hormone) secretion and mineral ion homeostasis. From co-leading insulin release, CaSR modulates ductal HCO3− secretion, Ca2+ concentration, cell-cell communication, β-cell proliferation, and intracellular Ca2+ release. In pancreatic cancer, the CaSR limits cell proliferation. TCP-related four novel missense mutations P163R, I427S, D433H and V477A, found in CaSR extracellular domain (ECD) protein, which were reported in the mutTCPdb Database (https://lms.snu.edu.in/mutTCPDB/index.php). P163R mutation occurs in ligand-binding domain 1 (LBD-1) of the CaSR ECD. To investigate the influence of these variations on protein function and structural activity multiple in-silico prediction techniques such as SIFT, PolyPhen, CADD scores, and other methods have been utilized. A 500 ns molecular dynamic simulation was performed on the CaSR ECD crystal structure and the corresponding mutated models. Furthermore, Principal Component Analysis (PCA) and Essential Dynamics analysis were used to forecast collective motions, thermodynamic stabilities, and the critical subspace crucial to CaSR functions. The results of molecular dynamic simulations showed that the mutations P163R, I427S, D433H, and V477A caused conformational changes and decreased the stability of protein structures. This study also demonstrates the significance of TCP associated mutations. As a result of our findings, we hypothesised that the investigated mutations may have an effect on the protein’s structure and ability to interact with other molecules, which may be related to the protein’s functional impairment.

Novel Missense CNTNAP2 Variant Identified in Two Consanguineous Pakistani Families With Developmental Delay, Epilepsy, Intellectual Disability, and Aggressive Behavior.

We report the genetic analysis of two consanguineous pedigrees of Pakistani ancestry in which two siblings in each family exhibited developmental delay, epilepsy, intellectual disability and aggressive behavior. Whole-genome sequencing was performed in Family 1, and we identified ~80,000 variants located in regions of homozygosity. Of these, 615 variants had a minor allele frequency ≤ 0.001, and 21 variants had CADD scores ≥ 15. Four homozygous exonic variants were identified in both affected siblings: PDZD7 (c.1348_1350delGAG, p.Glu450del), ALG6 (c.1033G>C, p.Glu345Gln), RBM20 (c.1587C>G, p.Ser529Arg), and CNTNAP2 (c.785G>A, p.Gly228Arg). Sanger sequencing revealed co-segregation of the PDZD7, RBM20, and CNTNAP2 variants with disease in Family 1. Pathogenic variants in PDZD7 and RBM20 are associated with autosomal recessive non-syndromic hearing loss and autosomal dominant dilated cardiomyopathy, respectively, suggesting that these variants are unlikely likely to contribute to the clinical presentation. Gene panel analysis was performed on the two affected siblings in Family 2, and they were found to also be homozygous for the p.Gly228Arg CNTNAP2 variant. Together these families provide a LOD score 2.9 toward p.Gly228Arg CNTNAP2 being a completely penetrant recessive cause of this disease. The clinical presentation of the affected siblings in both families is also consistent with previous reports from individuals with homozygous CNTNAP2 variants where at least one allele was a nonsense variant, frameshift or small deletion. Our data suggests that homozygous CNTNAP2 missense variants can also contribute to disease, thereby expanding the genetic landscape of CNTNAP2 dysfunction.

Gene-based whole genome sequencing meta-analysis of 250 circulating proteins in three isolated European populations

ObjectiveDeep sequencing offers unparalleled access to rare variants in human populations. Understanding their role in disease is a priority, yet prohibitive sequencing costs mean that many cohorts lack the sample size to discover these effects on their own. Meta-analysis of individual variant scores allows the combination of rare variants across cohorts and study of their aggregated effect at the gene level, boosting discovery power. However, the methods involved have largely not been field-tested. In this study, we aim to perform the first meta-analysis of gene-based rare variant aggregation optimal tests, applied to the human cardiometabolic proteome. MethodsHere, we carry out this analysis across MANOLIS, Pomak and ORCADES, three isolated European cohorts with whole-genome sequencing (total N = 4,422). We examine the genetic architecture of 250 proteomic traits of cardiometabolic relevance. We use a containerised pipeline to harmonise variant lists across cohorts and define four sets of qualifying variants. For every gene, we interrogate protein-damaging variants, exonic variants, exonic and regulatory variants, and regulatory only variants, using the CADD and Eigen scores to weigh variants according to their predicted functional consequence. We perform single-cohort rare variant analysis and meta-analyse variant scores using the SMMAT package. ResultsWe describe 5 rare variant pQTLs (RV-pQTL) which pass our stringent significance threshold (7.45 × 10−11) and quality control procedure. These were split between four cis signals for MARCO, TEK, MMP2 and MPO, and one trans association for GDF2 in the SERPINA11 gene. We show that the cis-MPO association, which was not detectable using the single-point data alone, is driven by 5 missense and frameshift variants. These include rs140636390 and rs119468010, which are specific to MANOLIS and ORCADES, respectively. We show how this kind of signal could improve the predictive accuracy of genetic factors in common complex disease such as stroke and cardiovascular disease. ConclusionsOur proof-of-concept study demonstrates the power of gene-based meta-analyses for discovering disease-relevant associations complementing common-variant signals by incorporating population-specific rare variation.

Exome sequencing identifies variants in infants with sacral agenesis.

Sacral agenesis (SA) consists of partial or complete absence of the caudal end of the spine and often presents with additional birth defects. Several studies have examined gene variants for syndromic forms of SA, but only one has examined exomes of children with non-syndromic SA. Using buccal cell specimens from families of children with non-syndromic SA, exomes of 28 child-parent trios (eight with and 20 without a maternal diagnosis of pregestational diabetes) and two child-father duos (neither with diagnosis of maternal pregestational diabetes) were exome sequenced. Three children had heterozygous missense variants in ID1 (Inhibitor of DNA Binding 1), with CADD scores >20 (top 1% of deleterious variants in the genome); two children inherited the variant from their fathers and one from the child's mother. Rare missense variants were also detected in PDZD2 (PDZ Domain Containing 2; N=1) and SPTBN5 (Spectrin Beta, Non-erythrocytic 5; N=2), two genes previously suggested to be associated with SA etiology. Examination of variants with autosomal recessive and X-linked recessive inheritance identified five and two missense variants, respectively. Compound heterozygous variants were identified in several genes. In addition, 12 de novo variants were identified, all in different genes in different children. To our knowledge, this is the first study reporting a possible association between ID1 and non-syndromic SA. Although maternal pregestational diabetes has been strongly associated with SA, the missense variants in ID1 identified in two of three children were paternally inherited. These findings add to the knowledge of gene variants associated with non-syndromic SA and provide data for future studies.

The Role of Microtubule Associated Serine/Threonine Kinase 3 Variants in Neurodevelopmental Diseases: Genotype-Phenotype Association.

Objective: To prove microtubule associated serine/threonine kinase 3 (MAST3) gene is associated with neurodevelopmental diseases (NDD) and the genotype-phenotype correlation.Methods: Trio exome sequencing (trio ES) was performed on four NDD trios. Bioinformatic analysis was conducted based on large-scale genome sequencing data and human brain transcriptomic data. Further in vivo zebrafish studies were performed.Results: In our study, we identified four de novo MAST3 variants (NM_015016.1: c.302C > T:p.Ser101Phe; c.311C > T:p.Ser104Leu; c.1543G > A:p.Gly515Ser; and c.1547T > C:p.Leu516Pro) in four patients with developmental and epileptic encephalopathy (DEE) separately. Clinical heterogeneities were observed in patients carrying variants in domain of unknown function (DUF) and serine-threonine kinase (STK) domain separately. Using the published large-scale exome sequencing data, higher CADD scores of missense variants in DUF domain were found in NDD cohort compared with gnomAD database. In addition, we obtained an excess of missense variants in DUF domain when compared autistic spectrum disorder (ASD) cohort with gnomAD database, similarly an excess of missense variants in STK domain when compared DEE cohort with gnomAD database. Based on Brainspan datasets, we showed that MAST3 expression was significantly upregulated in ASD and DEE-related brain regions and was functionally linked with DEE genes. In zebrafish model, abnormal morphology of central nervous system was observed in mast3a/b crispants.Conclusion: Our results support the possibility that MAST3 is a novel gene associated with NDD which could expand the genetic spectrum for NDD. The genotype-phenotype correlation may contribute to future genetic counseling.

Discriminating head trauma outcomes using machine learning and genomics.

A percentage of the population suffers prolonged and persistent post-concussion symptoms (PCS) following average head injuries or develops severe neurological dysfunction following minor head trauma. Genetic variants that may contribute to individual response to head trauma have been investigated in some studies, but to date none have explored the use of machine learning (ML) methods with genomic data to specifically explore outcomes of head trauma. Whole exome sequencing (WES) was completed for three groups of individuals (N = 60): (a) 16 individuals with severe neurological responses to minor head trauma, (b) 26 individuals with persistent PCS and (c) 18 individuals with normal recovery from concussion or mTBI. Gradient boosted tree algorithms were applied to the data using XGBoost. By using variants with CADD scores above 15 in the training set (randomly sampled 70%), we identified signatures that accurately distinguish to accurately distinguish the test groups with an average area under the curve (AUC) of 0.8 (SE = 0.019). Metrics including positive and negative prediction values, as well as kappa were all within acceptable range to support the prediction accuracy. This study illustrates how ML methods in combination with WES data have the potential to predict severe or prolonged responses to head trauma from healthy recovery. KEY MESSAGES: Linear association analysis has been inconclusive in concussion genetics. Non-linear methods as boosted trees can offer better insights in small samples. Strong discrimination trends can be achieved from exome data of cases and controls.

The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA.We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project.To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells.To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors.To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair.Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1).In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. DisclosuresSridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.