Benchmarking computational variant effect predictors by their ability to infer human traits.

BACKGROUND: Computational variant effect predictors offer a scalable and increasingly reliable means of interpreting human genetic variation, but concerns of circularity and bias have limited previous methods for evaluating and comparing predictors. Population-level cohorts of genotyped and phenotyped participants that have not been used in predictor training can facilitate an unbiased benchmarking of available methods. Using a curated set of human gene-trait associations with a reported rare-variant burden association, we evaluate the correlations of 24 computational variant effect predictors with associated human traits in the UK Biobank and All of Us cohorts. RESULTS: AlphaMissense outperformed all other predictors in inferring human traits based on rare missense variants in UK Biobank and All of Us participants. The overall rankings of computational variant effect predictors in these two cohorts showed a significant positive correlation. CONCLUSION: We describe a method to assess computational variant effect predictors that sidesteps the limitations of previous evaluations. This approach is generalizable to future predictors and could continue to inform predictor choice for personal and clinical genetics.

An integrated multi-tool analysis contributes elements to interpreting unclassified factor IX missense variants associated with haemophilia B.

BACKGROUND: Dissection of genotype-phenotype relationships in haemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor IX (FIX) missense variants. Here, the identification of a FIX missense variant associated with mild HB, reported but unclassified, prompted a multiple-level approach to contribute elements to interpret unclassified HB-associated FIX missense variants. METHODS: Molecular modelling of wild-type (WT) and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, Western blotting) and activity (aPTT-based/chromogenic assays) levels after recombinant expression, and multiple prediction tools. RESULTS: The F9(NM_000133.4):c.275T>C (p.V92A) variant was found in a mild HB patient (antigen, 45.4 U/dl; coagulant activity, 23.6 IU/dl; specific activity, 0.52). Newly generated molecular models showed alterations in Gla/EGF1-EGF2 domain conformation impacting on Ca++ affinity and protein-protein interactions with FXIa. Multi-tool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient's and modelling data. Expression studies on the V92A variant showed a specific activity (0.49±0.07; WT, 1.0±0.1) recapitulating that of the natural variant, and pointed toward a moderate activation impairment as the main determinant underlying the p.V92A defect. The validated multi-tool approach, integrated with evidence-based data, was challenged on a panel (n=9) of unclassified FIX missense variants, which resulted in inferred protein (secretion/function) outputs and HB severity. CONCLUSIONS: The rational integration of multi-tool and multi-parameter analyses contributed elements to interpret genotype/phenotype relationships of unclassified FIX missense variants, with implications for diagnosis, management and treatment of HB patients, and potentially translatable into other human disorders.

Monoallelic missense variants in MAB21L1 cause a novel autosomal dominant microphthalmia.

PURPOSE: The biallelic variant of MAB21L1 has previously been documented in conjunction with the autosomal recessive cerebellar, ocular, craniofacial, and genital syndrome (COFG). The purpose of this study was to investigate the gene-disease association of MAB21L1 and the newly discovered autosomal dominant (AD) microphthalmia. METHODS: We report the presence of an exceptionally rare missense variant in a single allele of the Arg51 codon of MAB21L1 among four individuals from a single family diagnosed with microphthalmia, which suggesting an autosomal dominant inheritance pattern. Subsequently, based on comprehensive literature review, we identified another 13 families that have reported cases of autosomal dominant microphthalmos. RESULTS: Genotype-phenotype analysis revealed that patients with a single allele missense variant in MAB21L1 exhibited solely eye abnormalities. This starkly diverged from the clinical presentation of COFG, typified by the concurrent occurrence of ocular and extraocular symptoms stemming from the biallelic variant in MAB21L1. Our findings revealed that the heterozygous pathogenic variant in MAB21L1 resulted in the emergence of autosomal dominant microphthalmia. By combining these genetic and experimental evidence, the clinical validity of MAB21L1 and the emerging autosomal dominant microphthalmia can be regarded as moderate. CONCLUSION: In summary, there is sufficient convincing evidence to prove that MAB21L1 is a novel pathogenic gene responsible for autosomal dominant microphthalmia, thus offering valuable insights for precise diagnosis and targeted therapeutic interventions in cases of microphthalmia.

Genetic constraint at single amino acid resolution in protein domains improves missense variant prioritisation and gene discovery.

BACKGROUND: One of the major hurdles in clinical genetics is interpreting the clinical consequences associated with germline missense variants in humans. Recent significant advances have leveraged natural variation observed in large-scale human populations to uncover genes or genomic regions that show a depletion of natural variation, indicative of selection pressure. We refer to this as "genetic constraint". Although existing genetic constraint metrics have been demonstrated to be successful in prioritising genes or genomic regions associated with diseases, their spatial resolution is limited in distinguishing pathogenic variants from benign variants within genes. METHODS: We aim to identify missense variants that are significantly depleted in the general human population. Given the size of currently available human populations with exome or genome sequencing data, it is not possible to directly detect depletion of individual missense variants, since the average expected number of observations of a variant at most positions is less than one. We instead focus on protein domains, grouping homologous variants with similar functional impacts to examine the depletion of natural variations within these comparable sets. To accomplish this, we develop the Homologous Missense Constraint (HMC) score. We utilise the Genome Aggregation Database (gnomAD) 125 K exome sequencing data and evaluate genetic constraint at quasi amino-acid resolution by combining signals across protein homologues. RESULTS: We identify one million possible missense variants under strong negative selection within protein domains. Though our approach annotates only protein domains, it nonetheless allows us to assess 22% of the exome confidently. It precisely distinguishes pathogenic variants from benign variants for both early-onset and adult-onset disorders. It outperforms existing constraint metrics and pathogenicity meta-predictors in prioritising de novo mutations from probands with developmental disorders (DD). It is also methodologically independent of these, adding power to predict variant pathogenicity when used in combination. We demonstrate utility for gene discovery by identifying seven genes newly significantly associated with DD that could act through an altered-function mechanism. CONCLUSIONS: Grouping variants of comparable functional impacts is effective in evaluating their genetic constraint. HMC is a novel and accurate predictor of missense consequence for improved variant interpretation.

Molecular Origins of the Mendelian Rare Diseases Reviewed by Orpha.net: A Structural Bioinformatics Investigation.

The study of rare diseases is important not only for the individuals affected but also for the advancement of medical knowledge and a deeper understanding of human biology and genetics. The wide repertoire of structural information now available from reliable and accurate prediction methods provides the opportunity to investigate the molecular origins of most of the rare diseases reviewed in the Orpha.net database. Thus, it has been possible to analyze the topology of the pathogenic missense variants found in the 2515 proteins involved in Mendelian rare diseases (MRDs), which form the database for our structural bioinformatics study. The amino acid substitutions responsible for MRDs showed different mutation site distributions at different three-dimensional protein depths. We then highlighted the depth-dependent effects of pathogenic variants for the 20,061 pathogenic variants that are present in our database. The results of this structural bioinformatics investigation are relevant, as they provide additional clues to mitigate the damage caused by MRD.

Compound heterozygous variants in SLC45A1 might cause syndromic intellectual disability by localization failure and activity attenuation in cells.

Intellectual disability (ID) is a kind of nervous developmental disorder and affects more than 1% of people worldwide. SLC45A1 as a transmembrane protein is implicated in the regulation of glucose homoeostasis. Through trio-based exome sequencing, the missense mutations of SLC45A1 c.103G>A (p.V35M) and c.1211T>G (p.F404C) were identified in the proband with syndromic ID. The distribution, expression and activity of SLC45A1 wild-type (WT) and variants were assayed in transfected COS7 cells. In SLC45A1 variants, the hydrogen bonds surrounding the 35th and 404th amino acid were changed, location on the cytomembrane was failed, their activity to transport glucose was also significantly decreased to contrast with SLC45A1-WT. No difference was observed at the mRNA and protein level. In conclusion, the compound heterozygous variants of SLC45A1 might be the genetic etiology for syndromic ID. These novel mutations probably attenuated its activity to transport glucose by the alteration of tertiary structure and failure of intracellular location.

A novel AVPR2 gene mutation in a Chinese pedigree with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare genetic disorder primarily associated with mutations in the arginine vasopressin receptor 2 (AVPR2) gene or the aquaporin 2 (AQP2) gene, resulting in impaired water reabsorption in the renal tubules. This report describes a case of a young male patient with NDI from China with a history of polydipsia and polyuria for over 15 years. Laboratory examinations of the proband indicated low urine-specific gravity and osmolality. Urologic ultrasound revealed severe bilateral hydronephrosis in both kidneys, bilateral dilatation of the ureters, roughness of the bladder wall, and the formation of muscle trabeculae. The diagnosis of diabetes insipidus was confirmed by water deprivation tests. The administration of posterior pituitary hormone did not alter urine-specific gravity, and osmolality remained at a low level (<300 mOsm/kg). Based on these findings, and the genetic tests of the proband and his parents were performed. A missense mutation (c.616 G>C) in exon 3 of the AVPR2 gene of the proband was found, caused by the substitution of amino acid valine to leucine at position 206 [p.Val206Leu], which was a hemizygous mutation and consistent with X-chromosome recessive inheritance. The administration of oral hydrochlorothiazide improves the symptoms of polydipsia and polyuria in the proband. This novel AVPR2 gene mutation may be the main cause of NDI in this family, which induces a functional defect in AVPR2, and leads to reduced tubular reabsorption of water.

Diabetes with GLP-1R polymorphism (rs3765467) accompanied by myotonic dystrophy: A case of myotonic dystrophy with p.R131Q polymorphism at the glucagon-like peptide-1 receptor (rs3765467) resulting in marked effects of its agonist, dulaglutide.

A 47-year-old woman was diagnosed with myotonic dystrophy when admitted for traumatic subarachnoid hemorrhage. Her glycemic control was poor despite administration of pioglitazone, a PPARɤ agonist, and subcutaneous insulin infusion. However, adding a GLP-1 receptor (GLP-1R) agonist markedly improved blood glucose levels, resulting in eventual insulin withdrawal. Genetic testing revealed a heterozygous variant, p.R131Q, in the GLP1R (rs3765467), a common variant in Asia. This variant is known to be associated with increased endogenous insulin from beta cells in response to exogenous GLP-1 infusion. This is the first report and short review of a Japanese case of myotonic dystrophy accompanied by GLP-1R gene polymorphism.

Plasminogen missense variants and their involvement in cardiovascular and inflammatory disease.

Human plasminogen (PLG), the zymogen of the fibrinolytic protease, plasmin, is a polymorphic protein with two widely distributed codominant alleles, PLG/Asp453 and PLG/Asn453. About 15 other missense or non-synonymous single nucleotide polymorphisms (nsSNPs) of PLG show major, yet different, relative abundances in world populations. Although the existence of these relatively abundant allelic variants is generally acknowledged, they are often overlooked or assumed to be non-pathogenic. In fact, at least half of those major variants are classified as having conflicting pathogenicity, and it is unclear if they contribute to different molecular phenotypes. From those, PLG/K19E and PLG/A601T are examples of two relatively abundant PLG variants that have been associated with PLG deficiencies (PD), but their pathogenic mechanisms are unclear. On the other hand, approximately 50 rare and ultra-rare PLG missense variants have been reported to cause PD as homozygous or compound heterozygous variants, often leading to a debilitating disease known as ligneous conjunctivitis. The true abundance of PD-associated nsSNPs is unknown since they can remain undetected in heterozygous carriers. However, PD variants may also contribute to other diseases. Recently, the ultra-rare autosomal dominant PLG/K311E has been found to be causative of hereditary angioedema (HAE) with normal C1 inhibitor. Two other rare pathogenic PLG missense variants, PLG/R153G and PLG/V709E, appear to affect platelet function and lead to HAE, respectively. Herein, PLG missense variants that are abundant and/or clinically relevant due to association with disease are examined along with their world distribution. Proposed molecular mechanisms are discussed when known or can be reasonably assumed.

Analysis of mutational variations in TP53 tumour suppressor gene among Pakistani head and neck cancer patients.

The aim of this study was to determine the frequency of TP53 mutation among Pakistani head and neck cancer (HNC) patients who visited Nishtar Hospital Multan and Nishtar Institute of Dentistry (NID), Multan, Pakistan. While significant research has been conducted on the role of p53 in HNC throughout the world, this study is the first of its kind in Southern Punjab, Pakistan. A total of 242 samples (121 cases and 121 controls) were collected from Nishtar Hospital Multan and NID, Multan, Pakistan. After histopathological analysis to determine the stage type and grade of malignancy, DNA extraction and sequencing were carried out to assess any mutations in the TP53 region (exons 5-8). Genetic screening was performed by the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) technique and Chromas 2.6.6 was used to visualise the sequencing results. The mean age of cases was 50.73 ±16.41 years and controls were 37.55 ± 15.51 years. The frequency of HNC was higher in male patients (65.28%) than in female patients (34.71%). Overall, this cancer was found to be significantly more prevalent in the age group of >35-55 years (p < 0.001). Smoking (51% versus 14%), naswar usage (15.7% versus 6.6%), poor oral hygiene (52.9% versus 29.8%) and anemic status (57.0% versus 4.1%) were significantly associated with cases (p ≤ 0.05) compared to controls. Only 04 samples exon 5 (1 sample), exon 7 (2 samples) and exon 8 (1 sample) with changed mobility patterns were found on the SSCP gel. All mutations were missense and heterozygous. Out of four mutant samples, three mutations were in the hotspot regions (codon 175, 245 and 248) of p53. Based on this study, there may be a weak association between the TP53 exon 5-8 mutation and HNC patients in Southern Punjab, Pakistan.

Spectrum and genotype-phenotype relationship of ALPK3 variants in Chinese patients with hypertrophic cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease, and it has obvious genetic and clinical heterogeneity. Recently, heterozygous ALPK3 truncating variants (ALPK3tv) have been shown to cause HCM. However, the spectrum of ALPK3 variants and their relationships with the clinical characteristics of Chinese patients with HCM remain to be elucidated. Methods and results: Whole-exome sequencing data from 986 patients with HCM and 761 controls without HCM were utilized to analyze ALPK3 variants. Eleven ALPK3tv were detected in 18 patients with HCM (1.8 %), while no such variants were identified in controls. We also detected 21 rare ALPK3 missense variants in 16 patients with HCM (1.6 %) and 8 controls (1.1 %), respectively. ALPK3tv were significantly enriched in patients with HCM (P < 0.001), whereas the prevalence of missense variants was comparable between the HCM and control groups (P = 0.309). Patients with ALPK3tv exhibited a significantly lower left ventricular outflow tract gradient (P = 0.011) and a higher prevalence of apical HCM (27.8 %; P = 0.008). Conclusions: Our study supports that heterozygous ALPK3tv, but not APLK3 missense variants, are a genetic cause of HCM. Patients with HCM carrying ALPK3tv have a greater likelihood of developing apical HCM.

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

Clinical and structural insights into the rare but oncogenic HER2-activating missense mutations in non-small cell lung cancer: a retrospective ATLAS cohort study.

BACKGROUND: Unlike human epidermal growth factor receptor 2 (HER2) amplification or exon 20 insertions, missense mutations in the extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD) of the HER2 protein have been implicated as oncogenic in non-small cell lung cancer (NSCLC). However, their molecular subtypes, structural disparities, and clinical responses to current medical treatments, particularly HER2-targeted tyrosine kinase inhibitors (TKIs), remain unclear in NSCLC and warrant investigation. METHODS: A real-world observational ATLAS study was conducted to gather and analyze therapeutic outcomes of chemotherapy or TKIs for heterogeneous HER2 missense mutations in NSCLC. Computational models of typical ECD, TMD, and ICD mutations were utilized to explore their structural variances. RESULTS: We screened 37 eligible patients with HER2-activating missense mutations, of which 35 patients who had received chemotherapy or HER2-targeted TKIs as first-line therapy were available for response assessment. The median progression-free survival (PFS) for chemotherapy was 4.43 months (95% confidence interval [CI], 3.77-5.10), with an objective response rate (ORR) of 26.1% (6/23) and a disease control rate (DCR) of 17/23 (73.9%). The administration of afatinib, dacomitinib, and pyrotinib, HER2-targeted TKIs, achieved a median PFS of 4.65 months, with an ORR of 33.3% (4/12) and a DCR of 83.3% (10/12). Molecular modeling and computational simulations of ECD, TMD, and ICD mutations revealed their distinct structural characteristics. CONCLUSION: In comparison to chemotherapy, HER2-targeted TKIs demonstrated similar activity and PFS benefits for HER2-activating missense mutations in NSCLC.

MTR3D-AF2: Expanding the coverage of spatially derived missense tolerance scores across the human proteome using AlphaFold2.

The missense tolerance ratio (MTR) was developed as a novel approach to assess the deleteriousness of variants. Its three-dimensional successor, MTR3D, was demonstrated powerful at discriminating pathogenic from benign variants. However, its reliance on experimental structures and homologs limited its coverage of the proteome. We have now utilized AlphaFold2 models to develop MTR3D-AF2, which covers 89.31% of proteins and 85.39% of residues across the human proteome. This work has improved MTR3D's ability to distinguish clinically established pathogenic from benign variants. MTR3D-AF2 is freely available as an interactive web server at https://biosig.lab.uq.edu.au/mtr3daf2/.

Discovering predisposing genes for hereditary breast cancer using deep learning.

Breast cancer (BC) is the most common malignancy affecting Western women today. It is estimated that as many as 10% of BC cases can be attributed to germline variants. However, the genetic basis of the majority of familial BC cases has yet to be identified. Discovering predisposing genes contributing to familial BC is challenging due to their presumed rarity, low penetrance, and complex biological mechanisms. Here, we focused on an analysis of rare missense variants in a cohort of 12 families of Middle Eastern origins characterized by a high incidence of BC cases. We devised a novel, high-throughput, variant analysis pipeline adapted for family studies, which aims to analyze variants at the protein level by employing state-of-the-art machine learning models and three-dimensional protein structural analysis. Using our pipeline, we analyzed 1218 rare missense variants that are shared between affected family members and classified 80 genes as candidate pathogenic. Among these genes, we found significant functional enrichment in peroxisomal and mitochondrial biological pathways which segregated across seven families in the study and covered diverse ethnic groups. We present multiple evidence that peroxisomal and mitochondrial pathways play an important, yet underappreciated, role in both germline BC predisposition and BC survival.

Identifying Oncogenic Missense Single Nucleotide Polymorphisms in Human SAT1 Gene Using Computational Algorithms and Molecular Dynamics Tools.

BACKGROUND: The human SAT1 gene encodes spermidine/spermine N1-acetyltransferase 1 (SSAT1), a regulatory biological catalyst of polyamine catabolism. Numerous essential biological processes, such as cellular proliferation, differentiation, and survival, depend on polyamines like spermidine and spermine. Thus, SSAT1 is involved in key cellular activities such as proliferation and survival of cells and mediates various diseases including cancer. A plethora of studies established the involvement of missense single nucleotide polymorphisms (SNPs) in numerous pathological conditions due to their ability to adversely affect the structure and subsequent function of the protein. AIMS: To date, an in silico study to identify the pathogenic missense SNPs of the human SAT1 gene has not been accomplished yet. This study aimed to filter the missense SNPs that were functionally detrimental and pathogenic. METHODS AND RESULTS: The rs757435207 (I21N) was ascertained to be the most deleterious and pathogenic by all algorithmic tools. Stability and evolutionary conservation analysis tools also stated that I21N variant decreased the stability and was located in the highly conserved residue. Molecular dynamics simulation revealed that I21N caused substantial alterations in the conformational stability and dynamics of the SSAT1 protein. Consequently, the I21N variant could disrupt the native functional roles of the SSAT1 enzyme. CONCLUSION: Therefore, the I21N variant was identified and concluded to be an oncogenic missense variant of the human SAT1 gene. Overall, the findings of this study would be a great directory of future experimental research to develop personalized medicine.

Unravelling benzazepines and aminopyrimidine as multi-target therapeutic repurposing drugs for EGFR V774M mutation in neuroglioma patients.

Introduction: Neuroglioma, a classification encompassing tumors arising from glial cells, exhibits variable aggressiveness and depends on tumor grade and stage. Unraveling the EGFR gene alterations, including amplifications (unaltered), deletions, and missense mutations (altered), is emerging in glioma. However, the precise understanding of emerging EGFR mutations and their role in neuroglioma remains limited. This study aims to identify specific EGFR mutations prevalent in neuroglioma patients and investigate their potential as therapeutic targets using FDA-approved drugs for repurposing approach. Methods: Neuroglioma patient's data were analyzed to identify the various mutations and survival rates. High throughput virtual screening (HTVS) of FDA-approved (1615) drugs using molecular docking and simulation was executed to determine the potential hits. Results: Neuroglioma patient samples (n=4251) analysis reveals 19% EGFR alterations with most missense mutations at V774M in exon 19. The Kaplan-Meier plots show that the overall survival rate was higher in the unaltered group than in the altered group. Docking studies resulted the best hits based on each target's higher docking score, minimum free energy (MMGBSA), minimum kd, ki, and IC50 values. MD simulations and their trajectories show that compounds ZINC000011679756 target unaltered EGFR and ZINC000003978005 targets altered EGFR, whereas ZINC000012503187 (Conivaptan, Benzazepine) and ZINC000068153186 (Dabrafenib, aminopyrimidine) target both the EGFRs. The shortlisted compounds demonstrate favorable residual interactions with their respective targets, forming highly stable complexes. Moreover, these shortlisted compounds have drug- like properties as assessed by ADMET profiling. Conclusion: Therefore, compounds (ZINC000012503187 and ZINC000068153186) can effectively target both the unaltered/altered EGFRs as multi-target therapeutic repurposing drugs towards neuroglioma.

Multiple Endocrine Neoplasia Type 1 Regulates TGFß-Mediated Suppression of Tumor Formation and Metastasis in Melanoma.

Over the past few decades, the worldwide incidence of cutaneous melanoma, a malignant neoplasm arising from melanocytes, has been increasing markedly, leading to the highest rate of skin cancer-related deaths. While localized tumors are easily removed by excision surgery, late-stage metastatic melanomas are refractory to treatment and exhibit a poor prognosis. Consequently, unraveling the molecular mechanisms underlying melanoma tumorigenesis and metastasis is crucial for developing novel targeted therapies. We found that the multiple endocrine neoplasia type 1 (MEN1) gene product Menin is required for the transforming growth factor beta (TGFß) signaling pathway to induce cell growth arrest and apoptosis in vitro and prevent tumorigenesis in vivo in preclinical xenograft models of melanoma. We further identified point mutations in two MEN1 family members affected by melanoma that led to proteasomal degradation of the MEN1 gene product and to a loss of TGFß signaling. Interestingly, blocking the proteasome degradation pathway using an FDA-approved drug and RNAi targeting could efficiently restore MEN1 expression and TGFß transcriptional responses. Together, these results provide new potential therapeutic strategies and patient stratification for the treatment of cutaneous melanoma.