Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1 or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the pre-mRNA splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 8 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1( c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108*; c.1571C>T, p.Pro524Leu and c.1572G>A, p.Pro524Pro) were identified to result in whole or part of exon skipping by either disruption of ESEs and generation of ESSs, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro

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

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the reoccurrence of several alleles in different populations.

Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the in total 252 PRPH2 variants, more than half (n=137) were missense variants. All variants were uploaded into the Leiden Open source Variation Database. Our study underscores the need of experimental assays for variants of unknown significance to improve pathogenicity classification, which is needed to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.

N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.-3065A>C and NM_153006.2:c-3098C>T reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Sequence variants NM_153006.2:c.426+326G>A and NM_153006.2:c.427-218A>C reside in the first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression of NAGS. These findings show that analyzing non-coding regions of NAGS and other urea cycle genes can reveal molecular causes of disease and identify novel regulators of ureagenesis.

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.

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previously, 94.21% of variants were identified using Sanger sequencing and multiplex ligation-dependent probe amplification. To investigate the remaining variants, whole-genome sequencing (WGS) was performed in four patients with PKU with unknown genotype to identify deep intronic or structural variants. Three novel heterozygous variants (c.706+368T>C; c.1065+241C>A; and c.1199+502A>T) were identified in a deep PAH gene intron. The c.1199+502A>T variant was detected in 60% (6/10) PKU patients. In silico prediction showed that the three deep variants may impact splice site selection and result in inclusion of a pseudo-exon. The c.1199+502A>T PAH minigene and reverse transcription PCR of blood RNA in a patient with PKU and compound heterozygous variants (c.1199+502A>T/ c.1199G>A) confirmed that the c.1199+502A>T variant creates a novel branch point and leads to the inclusion of a 25 bp in PAH mRNA (r.1199_2000ins1199+538_1199+562). Furthermore, the c.1199G>A mutation leads to the retention of an additional 17 nt in the PAH mRNA transcript (r.1199_2000ins1199+1_1199+17). These results expand the PAH genotypic spectrum and highlight that deep intronic analysis of PAH can improve genetic diagnosis in undiagnostic patients.

The Ensembl Variant Effect Predictor (VEP) is a freely available, open source tool for the annotation and filtering of genomic variants. It predicts variant molecular consequence using the Ensembl/GENCODE or RefSeq gene sets. It also reports phenotype associations from databases such as ClinVar, allele frequencies from studies including gnomAD, and predictions of deleteriousness from tools such as SIFT and CADD. Ensembl VEP includes filtering options to customise variant prioritisation. It is well supported and updated roughly quarterly to incorporate the latest gene, variant and phenotype association information. Ensembl VEP analysis can be performed using a highly configurable, extensible command-line tool, a Representational State Transfer (REST) application programming interface (API) and a user-friendly web interface. These access methods are designed to suit different levels of bioinformatics experience and meet different needs in terms of data size, visualisation and flexibility. In this tutorial, we will describe performing variant annotation using the Ensembl VEP web tool, which enables sophisticated analysis through a simple interface.

Pathogenic variants of the nuclear receptor subfamily 2 group F member 1 gene (NR2F1) are responsible for Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by optic atrophy associated with developmental delay and intellectual disability, but with a clinical presentation which appears to be multifaceted. We created the first public locus-specific database (LSDB) dedicated to NR2F1. All variants and clinical cases reported in the literature, as well as new unpublished cases, were integrated into the database using standard nomenclature to describe both molecular and phenotypic anomalies. We subsequently pursued a comprehensive approach based on computed representation and analysis suggesting a refinement of the BBSOAS clinical description with respect to neurological features and the inclusion of musculoskeletal hypotonia and intestinal signs with feeding difficulties. This database is fully accessible for both clinician and molecular biologists and should prove useful in further refining the clinical synopsis of NR2F1 as new data is recorded.

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.

Hereditary papillary renal cell carcinoma (HPRCC) is a rare inherited renal cancer syndrome characterized by bilateral and multifocal papillary type 1 renal tumors (PRCC1). Activating germline pathogenic variants of MET gene were identified in HPRCC families. We reviewed the medical and molecular records of a large French series of 158 patients screened for MET oncogenic variants (153 index-cases and five relatives). MET pathogenic variant rate was 10.4% (16/153) with 37.5% among patients with familial PRCC1 and 3.3% among patients with sporadic PRCC1 presentation. The phenotype in MET mutated cases was characteristic as PRCC1 tumors were mainly bilateral (82.3%) and multifocal (85.8%). Histologically, six out of seven patients with MET germline pathogenic variant harboured biphasic squamoid alveolar PRCC. Genetic screening identified in four index-cases a novel missense pathogenic variant within the tyrosine kinase domain: MET c.3389T>C, p.(Leu1130Ser). Functional assay confirmed its oncogenic effect with a constitutive phosphorylation of ERK protein and an abnormal focus formation induced. The genotype-phenotype correlation between MET pathogenic variants and PRCC1 presentation should encourage to widen the screening, especially toward non-familial PRCC1. This precise phenotype also constitutes a strong argument for the classification of novel missense variants within the tyrosine kinase domain when functional assays aren’t accessible.

Microdeletions and gross deletions are important causes (~20%) of human inherited disease. Their genomic locations are strongly influenced by the local DNA sequence environment. Yet no systematic study has examined the generative mechanisms. Here, we obtained 42,098 pathogenic microdeletions and gross deletions from the Human Gene Mutation Database (HGMD) that together form a continuum of germline deletions ranging in size from 1 bp to 28,394,429 bp. We analyzed the sequence within 1-kb of the breakpoint junctions and found the frequencies of non-B DNA-forming repeats, GC content, and the presence of seven of 78 specific sequence motifs in the vicinity of pathogenic deletions correlated with deletion length for deletions of length ≤30 bp. Furthermore, we found the repeats of DR, GQ, and STR appear to be important for the formation of longer deletions (>30 bp) but not for the formation of shorter deletions (≤30 bp) and significantly (Chi-square test P-value < 2E-16) more microhomologies were identified in flanking short deletions than long deletions (length >30 bp). We provide evidence to support a functional distinction between microdeletions and gross deletions. A deletion length cut-off of 25-30 bp may serve as an objective means to functionally distinguish microdeletions from gross deletions.

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.

Juvenile open angle glaucoma (JOAG) is a severe type of glaucoma with childhood onset and dominant inheritance. Using exome sequencing we identified 3 independent families from the Philippines with rare EFEMP1 variants (c.238A>T, p.Asn80Tyr; c.1480T>C p.Ter494Glnext*29; and c.1429C>T, p.Arg477Cysteine ) co-segregating with disease. Affected variant carriers (N= 34) exhibited severe disease with average age of onset of 16 years and with 76% developing blindness. To investigate functional effects, we transfected COS7 cells with vectors expressing the three novel EFEMP1 variants and showed that all three variants found in JOAG patients caused significant intracellular protein aggregation and retention compared to wild type and also compared to EFEMP1 variants associated with other ocular phenotypes including an early-onset form of macular degeneration, Malattia Leventinese/Doyne’s Honeycomb retinal dystrophy. These results suggest that rare EFEMP1 coding variants can cause JOAG through a mechanism involving protein aggregation and retention, and that the extent of intracellular retention correlates with disease phenotype. This is the first report of EFEMP1 variants causing JOAG, expanding the EFEMP1 disease spectrum. Our results suggest that EFEMP1 mutations appear to be a relatively common cause of JOAG in Filipino families, an ethnically diverse population.

Mucopolysaccharidoses (MPSs) are rare, heterogeneous inborn errors of metabolism (IEM) diagnosed through a combination of clinical, biochemical, and genetic investigations. The aim of this study was molecular characterization of the largest cohort of Iranian MPS patients (302 patients from 289 unrelated families), along with tracking their ethnicity and geographical origins. 185/289 patients were studied using an IEM-targeted NGS panel followed by complementary Sanger sequencing, which led to the diagnosis of 154 MPS patients and 5 non-MPS IEMs (diagnostic yield: 85.9%). Furthermore, 106/289 patients who were referred with positive findings went through re-analysis and confirmatory tests which confirmed MPS diagnosis in 104. Among the total of 258 MPS patients, 225 were homozygous, and 9 had copy number variations, and 90 had novel variants. MPS IV was the most common type (34.8%) followed by MPS I (22.7%) and MPS VI (22.5%). Geographical origin analysis unveiled a pattern of distribution for frequent variants in ARSB (c.430G>A, c.962C>T, c.281C>A), GALNS (c.319G>A, c.860C>T, c.1042A>G), and IDUA (c.1A>C, c.1598C>G). Our extensive patient cohort reveals the genetic and geographic landscape of MPS in Iran, which provides insight into the genetic epidemiology of MPS and can facilitate a more cost-effective, time-efficient diagnostic approach based on the region-specific variants.

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.

With great sadness, we report that Haig Kazazian, Founding Co-Editor of Human Mutation, died on January 19, 2022 of complications related to congestive heart failure.

Over 7,000 rare genetic diseases have been identified, and millions of newborns are affected by severe rare genetic diseases each year. A variety of Human Phenotype Ontology (HPO)-based clinical decision support systems (CDSS) and patient repositories have been developed to support clinicians in diagnosing patients with suspected rare genetic diseases. In September 2017, we released PubCaseFinder (https://pubcasefinder.dbcls.jp), a web-based CDSS that provides ranked lists of genetic and rare diseases using HPO-based phenotypic similarities, where top-listed diseases represent the most likely differential diagnosis. We also developed a Matchmaker Exchange (MME) application programming interface (API) to query PubCaseFinder, which has been adopted by several patient repositories. In this paper, we describe notable updates regarding PubCaseFinder, the GeneYenta matching algorithm implemented in PubCaseFinder, and the PubCaseFinder API. The updated GeneYenta matching algorithm improves the performance of the CDSS automated differential diagnosis function. Moreover, the updated PubCaseFinder and new API empower patient repositories participating in MME and medical professionals to actively use HPO-based resources.