The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch Syndrome (LS, OMIM 120435), represent variants of unknown significance (VUS). The inability to assess pathogenicity of such VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based tool suited for VUS classification. To derive a formula that translates CIMRA assay results for PMS2 VUS into the odds of pathogenicity (OddsPath), we used a set of clinically classified PMS2 variants, supplemented by inactivating variants generated by an in cellulo genetic screen, as proxies for pathogenic variants. Validation of this OddsPath revealed very high predictive values for PMS2 VUS. We conclude that this OddsPath provides an integral metric that, similar to the other, higher penetrance, MMR proteins MSH2, MLH1 and MSH6, can be incorporated into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). This will represent a seminal step forward in enabling personalized healthcare for individuals suspected of LS and their relatives.

A major challenge in validating genetic causes for patients with rare diseases (RDs) is the difficulty in identifying other RD patients with overlapping phenotypes and variants in the same candidate gene. This process, known as matchmaking, requires robust data sharing solutions in order to be effective. In 2014 we launched PhenomeCentral, a RD data repository capable of collecting computer-readable genotypic and phenotypic data for the purposes of RD matchmaking. Over the past 7 years PhenomeCentral’s features have been expanded and its dataset has consistently grown. There are currently 1,615 users registered on PhenomeCentral, which have contributed over 12,000 patient cases. Most of these cases contain detailed phenotypic terms, with a significant portion also providing genomic sequence data or other forms of clinical information. Matchmaking within PhenomeCentral, and with connections to other data repositories in the Matchmaker Exchange, have collectively resulted in over 60,000 matches, which have facilitated multiple gene discoveries. The collection of deep phenotypic and genotypic data has also positioned PhenomeCentral well to support next generation of matchmaking initiatives that utilize genome sequencing data, ensuring that PhenomeCentral will remain a useful tool in solving undiagnosed RD cases in the years to come.

In hemophilia A and B, analysis of the F8 and F9 variants has become standard over recent decades, giving information on the severity of hemophilia, inhibitor formation and allowing counseling for the families. The PedNet Registry collects data on hemophilia in children and has more than 2000 children listed. Genetic reports are collected uniformly and re-evaluated following international guidelines. We report 90 novel variants in the F8 and F9 gene, respectively, causing hemophilia with detailed information on severity, factor level and inhibitor formation. This will lead to further guidance for genetic laboratories and the treating physician. These findings can be implemented in hemophilia variant databases. The study highlights the need to re-evaluate and update earlier genetic reports in hemophilia both locally but also in variant databases in the light of changed nomenclature, the use of in silico prediction and new sequencing techniques.

TAR syndrome is a rare congenital disorder whose genetic bases have remained unclear for many years. It has now been understood that the disease is caused by the compound inheritance of a rare null allele (usually the 1q21.1 deletion) and a low-frequency hypomorphic noncoding single nucleotide polymorphism (SNP) in RBM8A gene. Nevertheless, only a limited set of variants has been identified so far. A recent report of Boussion et al. described four novel RBM8A noncoding SNPs (i.e., 1) c.205 + 3_205 + 6del, 2) c.206 − 13C>A, 3) c. − 19G>T, and 4) c.*6C>G) increasing the mutational spectrum of TAR syndrome. Here, based on the recently published manuscript by Boussion et al., we report data regarding an additional African TAR patient carrying the 1q21.1q21.2 deletion in trans with the 3’UTR (c.*6C>G) variant. Present data further confirm the pathogenic role of this hypomorphic SNP and highlights its relevance in the African population, leading to advice geneticists to directly search for the c.*6C>G variant in African patients affected by TAR syndrome and carrying the 1q21.1 deletion, shortening the diagnostic time window.

The vast volume of data that has been generated as a result of the next-generation sequencing revolution is overwhelming to sift through and interpret. Parsing functional vs. non-functional and benign vs. pathogenic variants continues to be a challenge. Out of three billion bases, the genomes of two given individuals will only differ by about 3 million variants (0.1%). Furthermore, only a small fraction of these are biologically-relevant and, of those that are functional, only a handful actually drive disease pathology. While whole genome and exome sequencing have transformed our collective understanding of the role that genetics plays in disease pathogenesis, there are certain conditions and populations for whom DNA-level data has failed to produce a molecular diagnosis. Patients of non-White race/non-European ancestry are disproportionately affected by “variants of unknown/uncertain significance” (VUS). This limits the scope of precision medicine for minority patients and perpetuates health disparities. VUS often include deep intronic and splicing variants which are difficult to interpret in DNA alone. RNA analysis is capable of illuminating the consequences of VUS thereby allowing for their reclassification as pathogenic vs. benign. Here we review the critical role, going forward, of transcriptome analysis for clarifying VUS in both neoplastic and non-neoplastic diseases.

The genes MECP2, CDKL5, FOXG1, UBE3A, SLC9A6, and TCF4 present unique challenges for current ACMG/AMP variant interpretation guidelines. To address those challenges, the Rett and Angelman-like Disorders Variant Curation Expert Panel (Rett/AS VCEP) drafted gene-specific modifications. A pilot study was conducted to test the clarity and accuracy of using the customized variant interpretation criteria. Multiple curators obtained the same interpretation for 78 out of the 87 variants (~90%), indicating appropriate usage of the modified guidelines the majority of times by all the curators. The classification of 13 variants changed using these criteria specifications compared to when the variants were originally curated and as present in ClinVar. Many of these changes were due to internal data shared from laboratory members however some changes were because of changes in strength of criteria. There were no two step classification changes and only 1 clinically relevant change (Likely pathogenic to VUS). The Rett/AS VCEP hopes that these gene-specific variant curation rules and the assertions provided help clinicians, clinical laboratories, and others interpret variants in these genes but also other fully penetrant, early-onset genes associated with rare disorders.

Skeletal dysplasias are a heterogeneous group of disorders ranging from mild to lethal skeletal defects. We investigated two unrelated families with individuals presenting with a severe skeletal disorder. In family NMD02, affected individuals had a dysostosis multiplex-like skeletal dysplasia and severe short stature (<-8.5 SD). They manifested increasing coarse facial features, protruding abdomens and progressive skeletal changes, reminiscent of mucopolysaccharidosis. The patients gradually lost mobility and the two oldest affected individuals died in their twenties. The affected child in family ID01 had coarse facial features and severe skeletal dysplasia with clinical features similar to mucopolysaccharidosis. She had short stature, craniosynostosis, kyphoscoliosis and hip-joint subluxation. She died at the age of 5 years. Whole-exome sequencing identified two homozygous variants c.133C>T; p.(Arg45Trp) and c.215dupA; p.(Tyr72Ter) respectively, in the two families, affecting an evolutionary conserved gene TMEM251. Immunofluorescence and confocal studies on Human Osteosarcoma cells indicated that TMEM251 localized to the Golgi complex and plasma membrane. However, p.Arg45Trp mutant TMEM251 protein was targeted less efficiently to the membranes and the localization was punctate. Tmem251 knockdown by siRNA induced dedifferentiation of rat primary chondrocytes. Our work implicates TMEM251 in the pathogenesis of a novel disorder and suggests its potential function in chondrocyte differentiation.

The growing use of new generation sequencing technologies on genetic diagnosis has produced an exponential increase in the number of Variants of Uncertain Significance (VUS). In this manuscript we compare three machine learning methods to classify VUS as Pathogenic or No pathogenic, implementing a Random Forest (RF), a Support Vector Machine (SVM), and a Multilayer Perceptron (MLP). To train the models, we extracted 82,463 high quality variants from ClinVar, using 9 conservation scores, the loss of function tool and allele frequencies. For the RF and SVM models, hyperparameters were tuned using cross validation with a grid search. The three models were tested on a set of 5,537 variants that had been classified as VUS any time along the last three years but had been reclassified in august 2020. The three models yielded superior accuracy on this set compared to the benchmarked tools. The RF based model yielded the best performance across different variant types and was used to create VusPrize, an open source software tool for prioritization of variants of uncertain significance. We believe that our model can improve the process of genetic diagnosis on research and clinical settings.

γ-Glutamyl carboxylase (GGCX) catalyses γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-haemorrhagic phenotypes remain elusive. Therefore, we analyzed the effect of 22 GGCX pathogenic variants on γ-carboxylation of six non-haemostatic VKD proteins (UCMA/GRP, MGP, BGLAP, GAS6, PRGP1, TMG4) in a GGCX-/- HEK293T cell line by a functional ELISA. We observed that biallelic deficiency to γ-carboxylate Gla-rich protein lead to the development of skin laxity. Markedly reduced level of γ-carboxylated MGP is crucial but not exclusive for causing facial dysmorphologies. Moreover, we identified the vitamin K hydroquinone binding site in GGCX in an in silico model by docking studies, which was further validated by functional assays. Variants affecting this site result into loss-of-function or severely diminished ability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to develop new treatment options for VKCFD1 patients, where individualized therapy with γ-carboxylated VKD proteins may represent a promising strategy.

This letter is a response to the commentary by Jonson & Do (Johnson and Do 2020) on our paper, entitled “A Vietnamese human genetic variation database” (Vinh et al. 2019). The commentators concerned about two issues: Firstly, the relation of Southeast Asian (SEA) and East Asian (EA) groups to African and European groups; Secondly, the history of migration and settlement in Southeast Asia. Our responses will clarify both concerns from the commentators.

It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect mRNA processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 81 exonic variants that impact splicing regulatory elements in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2. We utilized a pre-existing large-scale BRCA1 functional dataset to map functional splicing regulatory elements, assess the relative performance of different tools to predict effects of 283 variants on such elements, and develop a generic workflow to prioritize variants that may impact splicing regulatory elements. We also describe rare examples of intronic variants that impact branchpoint sites and create pseudoexons. We discuss the challenges in predicting variant effect on branchpoint site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review highlights the importance of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.

Xq28 (involving MECP2) duplication syndrome is a severe neurodevelopmental disorder in males, most females are asymptomatic carriers, but there are phenotypic heterogeneities in the females. Skewed X-chromosome inactivation (XCI) seems to prevent duplicated region activation in asymptomatic females, but it remains controversial. Herein we reported two asymptomatic females (daughter and mother) with interstitial Xq28 duplication. HUMARA and RP2 assays showed that both had complete skewed XCI, the Xq28 duplicated chromosome was inactivated in the daughter, but surprisingly, it was activated in her mother. Interestingly, by combining RNA sequencing and whole-exome sequencing, we confirmed that XIST only expressed in the Xq28 duplication chromosomes of the two females, indicating that the Xq28 duplication chromosomes were inactive. Meanwhile, MECP2 and most XCI genes in the duplicated X-chromosomes were not transcriptionally expressed or upregulated, precluding major clinical phenotypes in the two females, especially the mother. We showed that XCI status detected by RNA sequencing was more relevant for establishing the clinical phenotype of MECP2 duplication females. It suggested there were other factors maintaining the XCI status in addition to DNA methylation, a possible additional inhibition mechanism occured at the transcriptional level in the unmethylated X-chromosome, counter balancing the MECP2 duplication’s detrimental phenotype effects

Defects in DNA repair genes have been extensively associated to cancer susceptibility. Germline pathogenic variants (GPV) in genes involved in homologous recombination repair pathway predispose to cancers arising mainly in breast and ovary, but also other tissues. The RAD51 paralogs RAD51C and RAD51D were included in this group 10 years ago, when germline variants were associated to non-BRCA1/2 familial ovarian cancer. However, whether GPVs in these genes are associated with other cancers remains unknown. Here, we have reviewed the landscape of RAD51C and RAD51D germline variants in cancer reported in the literature during the last decade, curating a total of 341 variants and the phenotypes found in families with RAD51C/D variant carriers. A comprehensive catalogue has been generated pinpointing to the existence of recurrent variants in both genes. Investigation of pedigrees found fourteen other cancer types reported more than five times in families with carriers of RAD51C/D pathogenic variants. Among those, colorectal (3.72% and 4.43%) (RAD51C/D respectively), pancreatic (1.19% and 0.86%), lung (1.27% and 2.58%), prostate (1.56% and 1.48%), and leukemia (1.56% and 1.11%) cancer were the most prevalent types. This work highlights how both genes might confer susceptibility to a broader spectrum of cancer types than ovary and breast.

Pathogenic variants in JAG1 are known to cause Alagille syndrome (ALGS), a disorder that primarily affects the liver, lung, kidney and skeleton. Whereas cardiac symptoms are also frequently observed in ALGS, thoracic aortic aneurysms have only been reported sporadically in post-mortem autopsies. We here report two families with segregating JAG1 variants that present with isolated aneurysmal disease, as well as the first histological evaluation of aortic aneurysm tissue of a JAG1 variant carrier. Our observations shed more light on the pathomechanisms behind aneurysm formation in JAG1 variant carriers and underline the importance of cardiovascular imaging in the clinical follow-up of JAG1 variant carrying individuals.

Recently, we demonstrated that the qualitative American College of Medical Genetics and Genomics/ Association for Medical Pathology (ACMG/AMP) guidelines for evaluation of Mendelian disease gene variants are fundamentally compatible with a quantitative Bayesian formulation. Here, we show that the underlying ACMG/AMP “strength of evidence categories” can be abstracted into a point system. These points are proportional to Log(odds), are additive, and produce a system that recapitulates the Bayesian formulation of the ACMG/AMP guidelines. Strengths of this system are its simplicity and that the connection between point values and odds of pathogenicity allows empirical calibration of strength of evidence for individual data types. Weaknesses include that a narrow range of prior probabilities is locked in, and that the Bayesian nature of the system is inapparent. We conclude that a points-based system has useful attributes of user friendliness and can be useful so long as the underlying Bayesian principles are acknowledged.

Neural Tube Defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to cerebral folate deficiency by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole genome sequencing (WGS) data of 140 isolated spina bifida cases and identified 8 missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants down regulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

Male infertility has become a serious health and social problem troubling approximately 15% of couples worldwide; however, the genetic and phenotypic heterogeneity of human infertility poses a substantial obstacle to effective diagnosis and therapy. A previous study reported that heterozygous mutations in solute carrier family 26 member 8 (SLC26A8, NG_033897.1) were causatively linked to asthenozoospermia. Interestingly, in our research, three deleterious heterozygous mutations of SLC26A8 were separately detected in three unrelated patients who were suffered from teratozoospermia. These three heterozygous mutations resulted in the reduce of SLC26A8 expression in transfected cells, while no disrupt expression of SLC26A8 was observed in sperm from the affected individuals. Noticeably, two of the three SLC26A8 heterozygous mutations detected in the patients were inherited from their fertile fathers. Thus, we suggested that male infertility associated with SLC26A8 mutations should be involved in a recessive-inherited pattern, considering the infertile homozygous Slc26a8 KO male mice. Given that SLC26A8 heterozygous mutations were detected in the infertile patients, and SLC26A8 is predominantly expressed in the various germ cells during spermatogenesis, the heterozygous mutations in SLC26A8 may not be the direct genetic cause but contribute to male infertility to a certain degree.

The American College of Medical Genetics and Genomics, and the Association for Molecular Pathology (ACMG/AMP) have proposed a set of evidence-based guidelines to support sequence variant interpretation. The ClinGen hearing loss expert panel (HL-EP) introduced further specifications into the ACMG/AMP framework for genetic hearing loss. This study developed a tool named VIP-HL, aiming to semi-automate the HL ACMG/AMP rules. VIP-HL aggregates information from external databases to automate 13 out of 24 ACMG/AMP rules specified by HL-EP, namely PVS1, PS1, PM1, PM2, PM4, PM5, PP3, BA1, BS1, BS2, BP3, BP4, and BP7. We benchmarked VIP-HL using 50 variants where 83 rules were activated by the ClinGen HL-EP. VIP-HL concordantly activated 96% (80/83) rules, significantly higher than that of by InterVar (47%; 39/83). Of 4948 ClinVar star 2+ variants from 142 deafness-related genes, VIP-HL achieved an overall variant interpretation concordance in 88.0% (4353/4948). VIP-HL is an integrated online tool for reliable automated variant classification in hearing loss genes. It assists curators in variant interpretation and provides a platform for users to share classifications with each other. VIP-HL is available with a user-friendly web interface at http://hearing.genetics.bgi.com/.