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.
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
National genetic variation registries vastly increase the level of detail for the relevant population, while directly affecting patient management. Herein, we report CanVaS, a Cancer Variation reSource aiming to document the genetic variation of cancer patients in Greece. CanVaS comprises germline genetic data from 7,363 Greek individuals with a personal and/or family history of malignancy. The dataset incorporates ~24,000 functionally annotated rare variants in 97 established or suspected cancer susceptibility genes. For each variant, allele frequency for the Greek population, interpretation for clinical significance, anonymized family and segregation information, as well as phenotypic traits of the carriers, are included. Moreover, information on the geographic distribution of the variants across the country are provided, enabling the study of Greek population isolates. Direct comparisons between Greek (sub)populations with relevant genetic resources is supported, allowing fine-grain localized adjustment of guidelines and clinical decision-making. Most importantly, anonymized data are available for download, while the Leiden Open Variation Database schema is adopted, enabling integration/interconnection with central resources. CanVaS could become a stepping-stone for a countrywide effort to characterize the cancer genetic variation landscape, concurrently supporting national and international cancer research. The database can be accessed at: http://ithaka.rrp.demokritos.gr/CanVaS
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.
Loss of heterozygosity in HLA-I (HLA-I LOH) may facilitate immune evasion. However, the large population study of HLA-I LOH in Chinese pan-cancer patients remains to be explored. In this study, analysis was performed in 1504 advanced pan-cancer patients and 134 early-stage NSCLC patients using a 1021-gene panel. The consistency between the 1021-gene panel and whole-exome sequencing (WES) was evaluated in 45 samples, where concordant results were obtained in 95.6% (43/45) of the samples. Analytical results revealed that the prevalence of HLA-I LOH presents considerable differences across cancer types. HLA-I LOH was relevant to genomic instability, reflected in higher tumor mutation burden (TMB) level. The incidence of HLA-I LOH in MSS samples was significantly higher than that in MSI-H samples. The alteration frequencies of p53 pathway, RTK/RAS pathway, Notch pathway, Hippo pathway, and Nrf2 pathway in HLA-I LOH group were significantly higher than that in HLA-I stable group (p<0.0001, p<0.0001, p=0.032, p=0.013, p=0.003, respectively). In DNA damage response (DDR) pathways, alterations in CPF pathway and FA pathway are enriched in HLA-I LOH group (p<0.0001, p=0.023, respectively). Besides, HLA-I LOH was accompanied by higher mutation rates of several tumor suppressors, including TP53 and LRP1B. These results may shed light on follow-up research.
GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 465 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 231 of these confirmed to be familial and 22 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0-78 years) and myelodysplastic syndrome (MDS) is the first diagnosis in 75% of these cases with acute myeloid leukemia (AML) in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss-of-function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing and expression. In this review, we provide the first expert curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.
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
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.
Background: The genetic causes of atrial fibrillation (AF) with slow conduction are unknown. Methods: Eight kindreds with familial AF and slow conduction, including a family affected by early onset AF, heart block and incompletely penetrant non-ischemic cardiomyopathy (NICM) underwent whole exome sequencing. Results: A known pathogenic mutation in the desmin (DES) gene resulting in S13F substitution at a PKC phosphorylation site was identified in all four members of the kindred with early-onset AF and heart block, while only two developed NICM. Higher penetrance of the mutation for AF and heart block prompted the screening for DES modifier(s). A second deleterious mutation in the phosphodiesterase 4D interacting-protein (PDE4DIP) gene resulting in A123T substitution segregated with early onset AF, heart block and the DES mutation. Three additional novel deleterious PDE4DIP mutations were identified in four other unrelated kindreds. Characterization of PDE4DIPA123T in vitro suggested impaired compartmentalization of PKA and PDE4D characterized by reduced colocalization with PDE4D, increased cAMP activation leading to higher PKA phosphorylation of the β2-adrenergic-receptor, and decreased PKA phosphorylation of Desmin in response to isoproterenol stimulation compared to wildtype PDE4DIP. Conclusion: Our findings identify an epistatic interaction between DES and PDE4DIP variants, increasing the penetrance for conduction disease and arrhythmia.
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/.
Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by corneal thinning and fragility, leading to corneal rupture, the main hallmark of this disorder. Non-ocular symptoms include hearing loss, but also signs of connective tissue fragility, placing it in the Ehlers-Danlos syndrome (EDS) spectrum. It is caused by biallelic pathogenic variants in ZNF469 or PRDM5, which presumably encode transcription factors for extracellular matrix components. We report the clinical and molecular features of nine novel BCS families, four of which harbor variants in ZNF469 and five in PRDM5. We also performed a genotype and phenotype-oriented literature overview of all (N=85) reported patients with ZNF469 (N=53) and PRDM5 (N=32) variants. Musculoskeletal findings may be the mean reason for referral, and often raise suspicion of another heritable connective tissue disorder such as kyphoscoliotic EDS, osteogenesis imperfecta or Marfan syndrome, especially when corneal rupture has not yet occurred. Our findings highlight the multisystemic nature of BCS and validate its inclusion in the EDS classification. Importantly, gene panels for heritable connective tissue disorders should include ZNF469 and PRDM5 to allow for timely diagnosis and appropriate preventive measures for this rare condition.
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.
Mutations in the CLCN5 gene encoding the 2Cl-/1H+ exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low molecular weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the “proton glutamate” that serves as a crucial H+-binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for a second group of mutations in X. laevis oocytes were reduced. Molecular Dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations may alter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.
Von Willebrand disease (VWD), the most prevalent congenital bleeding disorder, arises from a deficiency in von Willebrand factor (VWF), which has crucial roles in hemostasis. The present study investigated functional consequences and underlying pathomolecular mechanisms of several VWF propeptide (VWFpp) variants detected in our cohort of VWD patients for the first time. Transient expression experiments in HEK293T cells demonstrated that four out of the six investigated variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) severely impaired secretion. Their co-transfections with the wt partly corrected VWF secretion, displaying loss of large/intermediate multimers. Immunostaining of the transfected HEK293 cells illustrated the retention of the VWF variants in the endoplasmic reticulum (ER). Docking of the COP I and COP II cargo recruitment proteins, ADP-ribosylation factor 1 and Sec24, onto the N-terminal VWF model (D1D2D´D3) revealed that these variants occur at VWFpp putative interfaces, which can hinder VWF loading at the ER exit quality control. Furthermore, quantitative and automated morphometric exploration of the 3-dimensional immunofluorescence images showed changes in the number/size of the VWF storage organelles, Weibel-Palade body (WPB)-like vesicles. The result of this study highlighted the significance of the VWFpp variants on anterograde ER-Golgi trafficking of VWF as well as the biogenesis of WPB-like vesicles.
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 mild-to-moderate 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 mild-to-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 PCRs. 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 converted alleles of widely different lengths, suggesting that they originated from different mechanisms of gene conversion.
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.