Hereditary disorders are frequently caused by genetic variants that affect pre-mRNA splicing. Whilst genetic variants in the canonical splice motifs are almost always disrupting splicing, the pathogenicity of variants in the non-canonical splice sites (NCSS) and deep intronic (DI) regions are difficult to predict. Multiple splice prediction tools have been developed for this purpose, with the latest tools employing deep learning algorithms. We benchmarked established and deep learning splice prediction tools on gold standard sets of variants in the ABCA4 and MYBPC3 genes associated with Stargardt disease (STGD1) and cardiomyopathy, respectively, with functional assessment in midigene and minigene splice assays. The best performing splice prediction tool for both NCSS and DI variants in ABCA4 was SpliceAI, whilst SpliceSiteFinder-like performed best for NCSS variants in MYBPC3. Overall, the performance in a real time clinical setting is much more modest than reported by the developers of the tools.

Bi-allelic loss-of-function variants of OTOA are a well-known cause of moderate-to-severe hearing loss. Whereas non-allelic homologous recombination-mediated deletions of the gene are well known, gene conversions to pseudogene OTOAP1 have been reported in the literature but never fully described nor their pathogenicity assessed. Here, we report two unrelated patients with moderate hearing-loss, who were compound heterozygotes for a converted allele and a deletion of OTOA. The conversions were initially detected through sequencing depths anomalies at the OTOA locus after exome sequencing, then confirmed with long range polymerase chain reactions. Both conversions lead to loss-of-function by introducing a premature stop codon in exon 22 (p.Glu787*). Using genomic alignments and long read nanopore sequencing, we found that the two probands carry stretches of converted DNA of widely different lengths (at least 9 kbp and around 900 bp, respectively).

To the Editor, This letter is a response to the commentary by Dr. Finsterer (Finsterer, 2020) on our paper entitled “Homozygous mutations in C1QBP as cause of progressive external ophthalmoplegia (PEO) and mitochondrial myopathy with multiple mtDNA deletions” (Marchet et al. 2020). Here we try to address the key concerns raised by him.We did not make any distinction between pure PEO and PEO plus, but just considered PEO as a clinical sign which is extremely useful to suggest the presence of a mitochondrial disorder, in particular of primary mitochondrial myopathy (PMM). We stressed the use of the term PEO because it is the one used in OMIM to describe these genetic diseases, with 10 entry genes classified as “Progressive external ophthalmoplegia with mtDNA deletions” (Phenotypic Series - PS157640). Moreover, we stated that PEO usually (and not necessarily) starts with ptosis; indeed, the two patients with C1QBP mutations we described (P1 and P2) presented with ptosis at disease onset. Accordingly, the consortium on Trial Readiness in Mitochondrial Myopathies confirmed that “the most common presentation of PMM is chronic PEO” and that “PEO is usually accompanied by bilateral eyelid ptosis, which is often the presenting symptom” (Mancuso et al. 2017).In the first paper about C1QBP mutations (Feichtinger et al. 2017), all the four reported patients presented with cardiac involvement leading the authors to sustain in the title that biallelic C1QBPmutations cause severe neonatal-, childhood-, or later-onset cardiomyopathy. Although we cannot exclude subtle cardiac dysfunction in our patients (since they did not undergo long-term ElectroCardioGram recordings, trans-esophageal echocardiography, or cardiac MRI), their standard ECGs and echoCGs were normal and thus we still consider valid the main message of our paper: subjects with C1QBP mutations may present with adult-onset PEO/PMM phenotype, without overt cardiomyopathy.We obviously agree that mtDNA genetics is peculiar, and that different level of heteroplasmy may explain the variable phenotypic expression of mtDNA mutations, but we are talking here about mutations in a nuclear gene with an autosomal recessive inheritance. No DNA from any family members was available for segregation studies but we expect that the parents of P1 and P2 (reported to be second-grade and third-grade cousins, respectively) would have tested as heterozygous carrier. Both patients have no siblings. Anyway, detailed clinical investigations of first-degree relatives, not harboring the homozygous C1QBPmutation, would be not informative and hence, in our opinion, useless.The presence of multiple mtDNA deletions is a secondary effect of the mutations in C1QBP , although the exact mechanism linking C1QBP with mtDNA maintenance and stability is not known. The assertion that residual protein amount and different localization of the C1QBPmutations could explain the variable observed phenotypes, including both clinical symptoms and molecular/biochemical defects (mtDNA deletions, mitochondrial respiratory chain - MRC - complex activities, histochemical staining) remains plausible. As suggested by Dr. Finsterer, it is possible that the phenotypic variability ofC1QBP variants is fairly attributable to variable heteroplasmy of secondary mtDNA deletions and/or mtDNA copy number, but it is not possible to test this hypothesis in detail (e.g. throughout assessment of heteroplasmy in different muscle types, including extraocular muscle, and at different time points during disease progression). All the experiments reported in our paper (Marchet et al. 2020) were performed on a single muscle biopsy from quadriceps of the two patients. Densitometry analysis of the Southern blot (Fig. 1C reported in Marchet et al. 2020) revealed 58% and 48% mtDNA deleted species in P1 and P2, respectively. Nevertheless, an exponential accumulation of multiple mtDNA deletions has been reported in post-mitotic tissues during aging (Cortopassi et al. 1992), and thus we cannot exclude an influence of the age at biopsy and of duration from onset disease on this result.Another important issue is related to the presence of mosaic of cells in the same tissue, which is expected also in our patients based on histological analyses showing fibers with different features likely related to different levels of mtDNA deletions. Accordingly, previous single-cell analysis has revealed that mtDNA deletions are distributed as a mosaic of affected and non-affected cells (He et al. 2002). While the link between heteroplasmy level and biochemical/clinical phenotype is well established in patients with single large‐scale mtDNA deletion, it is more complex in patients with multiple mtDNA deletions, where each muscle fiber may contain different, and more than one, mtDNA deleted species (Lehman et al. 2019).Regarding mtDNA copy number, we did not assess it directly but we expect the same limitations reported above, because of experimental data from a single specimen characterized by intercellular heterogeneity. Nevertheless, some indirect indications can be obtained by already reported histological and biochemical findings. Muscle cells with high levels of mtDNA deletions typically show mitochondrial proliferation as compensatory mechanism, which is reflected by the presence of Ragged Red Fibers (RRF). Moreover, the activity of citrate synthase (CS) is often used as a quantitative marker for mitochondrial mass. In both P1 and P2, we observed the presence of many RRF but the CS activity in total muscle homogenate was in the normal range (118% and 100% of the controls’ mean for P1 and P2, respectively) again confirming variable heteroplasmy in different fibers but indicating an overall normal amount of mitochondria and, roughly, of mtDNA copy number.All the above considerations are useful to explain also the last concern by Dr. Finsterer, i.e. why biochemical investigations of P2 were normal. Notably, the histochemical staining for cytochrome c oxidase (i.e. complex IV) was decreased in scattered fibers from P2, despite biochemical assay showed normal values for MRC complexes. It has already been reported that the activities of respiratory complexes in muscle from PEO patients range from normal to about 50% of the controls’ mean (Viscomi & Zeviani, 2017). Likewise, normal MRC activity has been observed in several patients presenting with mtDNA deletions caused by mutations in nuclear genes (e.g. POLG, POLG2, RNASEH1 …). More recently, by single cell studies some authors demonstrated that genetic defects do not strictly correlate with MRC deficiency in fibers with multiple mtDNA deletions (Lehman et al. 2019).In conclusion, the very limited number of C1QBP cases reported up to now and their allelic heterogeneity hamper to define any genotype–phenotype correlations, but nevertheless indicate a huge clinical spectrum associated with C1QBP mutations, ranging from early-onset severe cardiomyopathy to adult-onset PEO/PMM.CONFLICT OF INTERESTSThe authors declare that there are no conflicts of interests.

A document by josef finsterer. Click on the document to view its contents.

Osteoporotic fractures cause major morbidity and mortality in the aging population. Genome-wide association studies (GWAS) have identified USF3 as the novel susceptibility gene of osteoporosis. However, the functional role in bone metabolism and the target gene of the bHLH transcription factor USF3 are unclear. Here we show that USF3 enhances osteoblast differentiation and suppresses osteoclastogenesis in cultured human osteoblast-like U-2OS cells. Mechanistic studies revealed that transcription factor USF3 antagonistically interacts with anti-osteogenic TWIST1/TCF12 heterodimer in the WNT16 and RUNX2 promoter, and counteracts CREB1 and JUN/FOS in the RANKL promoter. Importantly, the osteoporosis GWAS lead SNP rs2908007 risk A allele abolishes USF3 binding in the WNT16 promoter, conferring allele-specific downregulation of the osteoclastogenesis suppressor WNT16. While the risk G allele of osteoporosis GWAS lead SNP rs4531631 facilitates binding of CREB1 and JUN/FOS in the RANKL promoter, resulting in enhanced transactivation of RANKL, the principal contributor to osteoclastogenesis. Our findings uncovered functional mechanisms of osteoporosis novel GWAS associated gene USF3 and lead SNPs rs2908007 and rs4531631 in the regulation of bone formation and resorption.

In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

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.

Background: CHEK2 variants are associated with intermediate breast cancer risk among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. Methods: First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1,848 prospective patients with HC suspicion. We refined ACMGAMP criteria and applied different combinatorial rules to classify CHEK2 variants and define risk alleles. Results: We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; 2 can also be considered “established risk-alleles” and one as “likely risk-allele”. The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary non-polyposis colorectal cancer patients). Conclusions: Here we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this work would be useful for variant classification of other genes with low effect variants

Full genome analysis of a young girl with deafness, dystonia, central hypomyelination, refractory seizure, and fluctuating liver function impairment revealed a heterozygous, de novo variant in the BCAP31 gene on chromosome X28q (NC_000023.11(BCAP31_v001):c.92G>A), mutations of which caused the X-linked recessive severe neurologic disorder DDCH (Deafness, Dystonia, and Cerebral Hypomyelination, OMIM#300475). Reverse transcription-PCR (RT-PCR) of the patient’s white blood cells showed the absence of wild-type BCAP31 mRNA but the presence of two novel BCAP31 mRNAs. The major alternatively-spliced mRNA is due to exon 2 skipping and the utilization of a new initiation site in exon 3 that leads to a frameshift and truncated transcript while the minor novel mRNA has a 110 nucleotide insertion to exon 2. Phasing studies showed that the de novo variant arose in the paternal X chromosome. X chromosome inactivation assay was done and confirmed that the patient’s maternal X chromosome was preferentially inactivated, providing evidence that the mutated BCAP31 gene was the predominantly expressed. According to the ACMG guideline, this variant is deemed “pathogenic” (PS2, PS3, PM2, PP3, PP4) and deleterious. This is the first reported female patient in BCAP31-related syndrome resulted from skewed X-inactivation and a de novo mutation in the active X chromosome.

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.

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.

Purpose:There have been concerted efforts towards cataloging rare and deleterious variants in different world population using high throughput genotyping and sequencing based methods. The Indian populations are underrepresented or its information w.r.t. clinically relevant variants are sparse in public datasets. The aim of this study was to estimate the burden of monogenic disease causing variants in Indian populations. Towards this, we have assessed the frequency profile of monogenic phenotype associated ClinVar variants. Methods: The study utilized genotype dataset (global-screening-array, Illumina) from 2795 individuals (multiple in-house genomics cohorts) representing diverse ethnic and geographically distinct Indian populations. Results: Of the analyzed variants from GSA, ~12% were found to be informative and were either not known earlier or underrepresented in public databases in terms of their frequencies. These variants were linked to disorders, viz. Inborn-errors of Metabolism, Monogenic-diabetes, hereditary cancers and various other hereditary conditions. We have also shown that our study cohort is genetically better representatives of Indian populations than its representation in1000 genome project (South-Asians). Conclusion: We have created a database, ClinIndb [(http://clinindb.igib.res.in) and (https://databases.lovd.nl/shared/variants?search_owned_by_=%3D%22Mohamed%20Faruq%22)], to help clinicians and researchers in diagnosis, counseling and development of appropriate genetic screening tools relevant to the Indian populations and Indians living abroad.

Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic hedgehog (SHH) is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-Protein Coupled Receptor (GPCR) SMOOTHENED (SMO), whose signalling activity is tightly regulated, is the sole obligate transducer of hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct non-synonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the hedgehog gene regulatory network (GRN) in overall medical interpretations.

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.