Splice Site Variant Research Articles

High incidence of malignant arrhythmias and heart failure in patients with RBM20-associated cardiomyopathy: a multicenter cohort study and review of the literature

Abstract Background Patients with RBM20 cardiomyopathy commonly experience malignant arrhythmias, sudden cardiac death (SCD), and progressive heart failure (HF). Objective The aim of this study is to investigate genotype-phenotype correlations, clinical outcomes and causes of death in patients with RBM20-associated cardiomyopathy in a multicenter cohort, combined with an overview of the current literature. Methods This international, multicenter cohort included all patients with cardiomyopathy who had a pathogenic (P) or likely pathogenic (LP) RBM20 variant. For survival and regression analysis, we matched a control group based on sex, age, and presence of left ventricular dysfunction. Additionally, we conducted a literature search on studies investigating RBM20-associated cardiomyopathy. Results Sixty-two patients (45% male) were included in the study, with a mean age of 42 ± 15 years at presentation. Of the LP/P RBM20 variants identified, 9 were novel. Eleven variants were truncating, while the remaining variants consisted of missense and splice-site variants. Patients with a truncating variant who developed HF were on average older at the time of diagnosis compared to patients with missense variants (mean age 62 ± 9 vs. 45 ± 14; p=0.01). After a median follow-up duration of 5.0 [1.0 – 10.5] years, 21 (34%) patients reached the composite endpoint of ventricular arrhythmias (VA), implantable cardioverter-defibrillator therapy, heart transplantation (HTx), left ventricular assist device (LVAD) implantation, or cardiovascular death. Of these, 19 (31%) patients experienced malignant VA (mean age 45 ± 15 years, 63% males). Males were at higher risk for the composite endpoint (log-rank p= 0.02), particularly for VA (log-rank p=0.007). The literature review analyzed 34 studies with a total of 678 patients, of whom 53% were male. In these studies, 123 (24%) patients experienced a VA, 58 (12%) underwent HTx or were treated with LVAD, and 52 (11%) died. Conclusion In this multicenter study the patients carrying a LP/P variant in RBM20 variants had a severe phenotype, with a high incidence of VA, particularly in males. Additionally, this study presents 9 novel DNA variants, of which 8 were truncating, and were mainly observed in older individuals. Finally, we report a lower mortality rate compared to the literature, possibly due to a higher number of heart transplantations performed in our patient cohort.Survival composite endpoint for sex

Familial osteochondrodysplastic and cardiomyopathic syndrome in Chianina cattle.

Skeletal dysplasia encompasses a heterogeneous group of genetic disorders characterized by an abnormal development of bones, joints, and cartilage. Two Chianina half-sibling calves from consanguineous mating with congenital skeletal malformations and cardiac abnormalities were identified. To characterize the disease phenotype, to evaluate its genetic cause, and to determine the prevalence of the deleterious alleles in the Chianina population. Two affected calves, their parents and 332 Chianina bulls. The affected animals underwent clinicopathological investigation. Whole-genome sequencing trio-approach and PCR-based assessment of the frequency of TDP-glucose 4,6-dehydratase (TGDS) and laminin subunit alpha 4 (LAMA4) alleles were performed. The cases presented with retarded growth, poor nutritional status associated with muscular atrophy and angular deformities of the hindlimbs. Radiologic examination identified generalized osteopenia and shortening of the limb long bones. Necropsy showed osteochondrodysplastic limbs and dilatation of the heart right ventricle. On histological examination, the physeal cartilages were characterized by multifocal mild to moderate loss of the normal columnar arrangement of chondrocytes. Osteopenia also was observed. Genetic analysis identified a missense variant in TGDS and a splice-site variant in LAMA4, both of which were homozygous in the 2 cases. Parents were heterozygous and allele frequency in the Chianina population for the TGDS variant was 5% and for the LAMA4 variant was 2%. Genetic findings identified 2 potentially pathogenic alleles in TGDS and LAMA4, but no clear mode of inheritance could be determined.

Genotype-phenotype analysis of hearing function in patients with DFNB1A caused by the c.-23+1G>A splice site variant of the GJB2 gene (Cx26).

The audiological features of hearing loss (HL) in patients with autosomal recessive deafness type 1A (DFNB1A) caused by splice site variants of the GJB2 gene are less studied than those of patients with other variants of this gene. In this study, we present the audiological features of DFNB1A in a large cohort of 134 patients with the homozygous splice site variant c.-23+1G>A and 34 patients with other biallelic GJB2 genotypes (n = 168 patients with DFNB1A). We found that the preservation of hearing thresholds in the speech frequency range (PTA0.5,1.0,2.0,4.0 kHz) in patients with the c.[-23+1G>A];[-23+1G>A] genotype is significantly better than in patients with the "severe" c.[35delG];[35delG] genotype (p = 0.005) and significantly worse than in patients with the "mild" c.[109G>A];[109G>A] genotype (p = 0.041). This finding indicates a "medium" pathological effect of this splice site variant on hearing function. A detailed clinical and audiological analysis showed that in patients with the c.[-23+1G>A];[-23+1G>A] genotype, HL is characterized as congenital or early onset (57.5% onset before 12 months), sensorineural (97.8%), bilateral, symmetrical (82.8%), variable in severity (from mild to profound HL, median hearing threshold in PTA0.5,1.0,2.0,4.0 kHz is 86.73±21.98 dB), with an extremely "flat" audioprofile, and with a tendency toward slow progression (a positive correlation of hearing thresholds with age, r = 0.144, p = 0.041). In addition, we found that the hearing thresholds in PTA0.5,1.0,2.0,4.0 kHz were significantly better preserved in females (82.34 dB) than in males (90.62 dB) (p = 0.001). We can conclude that in patients with DFNB1A caused by the c.-23+1G>A variant, male sex is associated with deteriorating auditory function; in contrast, female sex is a protective factor.

Characterization of Two Novel PNKP Splice-Site Variants in a Proband With Microcephaly, Intellectual Disability, and Multiple Malformations.

Polynucleotide kinase phosphatase (PNKP), encoded by the PNKP gene, is a DNA processing enzyme involved in double-strand break and single-strand break repair pathways, which are essential for genome stability and for the correct development and maintenance of human nervous system. PNKP biallelic loss-of-function variants have been associated with a broad spectrum of neurological anomalies, ranging from congenital microcephaly with intellectual disability and seizures (MCSZ), to later onset forms of ataxia-oculomotor apraxia (AOA4) or peripheral neuropathy (CMT2B2). We report the atypical clinical manifestations of a patient with severe microcephaly, short stature, developmental delay, conductive hearing loss, and tracheoesophageal malformation, in the absence of seizures. Whole exome sequencing analysis identified two novel, compound heterozygous splice-site variants in the PNKP gene (NM_007254.4): c.1448+1G > A and c.199-8_199-5del. To demonstrate the effect of both variants on the splicing process and prove their pathogenicity, we performed a hybrid minigene assay, which successfully highlighted a deleterious impact on the transcript, particularly regarding the c.199-8_199-5del variant. The uncommon clinical features of the proband and the identification of two newly associated pathogenic variants add further evidence to the allelic and phenotypic heterogeneity of the PNKP locus.

Intrinsic cardiomyopathy in pediatric Marfan syndrome: predictive factors and risk assessments.

Marfan syndrome (MFS) is associated with cardiovascular complications, particularly valvulopathies; however, its association with primary cardiomyopathy remains unclear. This retrospective cohort study examined the cardiomyopathy characteristics (CMCs) in pediatric patients with MFS. CMCs were defined as meeting at least one of the following echocardiography or clinical parameters: (1) cardiac index (CI) too low for patient's age, (2) ejection fraction (EF) <50%, and (3) diastolic dysfunction. The predictive factors for CMCs were determined using a multivariable logistic regression model. Among 83 patients with MFS (age, median [range], 12.5 [0.4-22.3] years), 39.8% exhibited CMCs. Only 4 patients (5%) showed heart failure symptoms (NYHA > 1). Independent predictors for CMCs included a systemic score of ≥7 (revised Ghent criteria) and likely pathogenic or pathogenic variants in FBN1, including variants that introduce a premature stop codon, splice site variants, and missense variants involving cysteine. A multivariable score was constructed with an AUC of 0.733. This study offers valuable insights into the prevalence and predictors of CMC in pediatric patients with MFS and presents potential strategies for risk assessment of cardiomyopathy. The objective of this study was to elucidate the contentious issue of intrinsic cardiomyopathy in Marfan syndrome and demonstrate its notable occurrence even in pediatric patients who do not exhibit heart failure symptoms or valvular complications. We highlighted the importance of specific FBN1 variants and higher systemic scores in identifying the potential for intrinsic cardiomyopathy in pediatric patients with Marfan syndrome.

Serological and molecular characterization of novel ABO variants including an interesting B(A) subgroup.

ABO grouping is the most important pretransfusion testing that is directly related to the safety of blood transfusion. A weak ABO subgroup is one of the important causes of an ABO grouping discrepancy. Here, we investigated the characterization of four novel ABO variants including a novel B(A) subgroup. RBCs were phenotyped by standard serology methods. The full coding regions of the ABO gene and the erythroid cell-specific regulatory elements in intron one were sequenced. The effect of the possible splice site variant was predicted by Alamut software. The 3D structural modeling of three relative B(A) enzymes (p.Met214Thr, p.Met214Val, and p.Met214Leu) were performed by PyMOL software. Four novel ABO alleles were identified with weak ABO expression in this study, in which two would lead to premature terminations, and two resulted in amino acid changes. In silico analysis revealed that the splice site variant c.155G>T had the potential to alter splice transcripts. 3D structural view shown that the variant amino acid position 214 was spatially adjacent to the donor recognition pocket residues (266Met and 268Ala) and just next to the 211DVD213 motif. The size of the side chain of Thr and Val is the smallest, Leu is medium, and Met is the largest, and the size changes in the critical position 214 may affect the donor recognition pocket. Four ABO subgroup alleles were newly linked to different kinds of ABO variants and the possible mechanism through which they produce weak ABO subgroups was analyzed in silico.

Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy and a leading cause of sudden death. Genetic testing and familial cascade screening play a pivotal role in the clinical management of HCM patients. However, conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Here, using a non-integrative reprogramming strategy, we generated induced pluripotent stem cell (iPSC) lines from four individuals carrying one of two variants within intronic regions of MYBPC3: c.1224-52G > A and c.1898-23A > G. Upon differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), mis-spliced mRNAs were identified in cells harbouring these variants. Both abnormal mRNAs contained a premature termination codon (PTC), fitting the criteria for activation of nonsense mediated decay (NMD). However, the c.1898-23A > G transcripts escaped this mRNA quality control mechanism, while the c.1224-52G > A transcripts were degraded. The newly generated iPSC lines represent valuable tools for studying the functional consequences of intronic variation and for translational research aimed at reversing splicing abnormalities to prevent disease progression.

A Splice Site Variant in ADAMTS3 Is the Likely Causal Variant for Pulmonary Hypoplasia with Anasarca in Persian/Persian-Cross Sheep.

Pulmonary hypoplasia with anasarca, or hydrops fetalis, is characterized by stillbirth, diffuse oedema, and generalized lymph node hypoplasia. The enlarged fetus frequently causes dystocia. The disease has been reported in cattle and sheep as an inherited condition with a recessive mode of inheritance. This is the first report of the disease in Persian/Persian-cross sheep in Australia. Affected fetuses were reported from three flocks, and a total of eleven affected, eleven obligate carrier, and 188 related Persian/Persian-cross animals were available for analysis, as well as unrelated control animals. SNP genotyping revealed a region of homozygosity in affected animals on ovine chromosome six, which contained the functional candidate gene ADAMTS3. Whole genome sequencing of two affected fetuses and one obligate carrier ewe revealed a single nucleotide deletion, ENSOARG00000013204:g.87124344delC, located 3 bp downstream from a donor splice site region in the ADAMTS3 gene. Sanger sequencing of cDNA containing this variant further revealed that it is likely to introduce an early splice site in exon 14, resulting in a loss of 6 amino acids at the junction of exon 14 and intron 14/15. A genotyping assay was developed, and the ENSOARG00000013204:g.87124344delC segregated with disease in 209 animals, allowing for effective identification of carrier animals.

Identification and functional characteristics of CHD1L gene variants implicated in human Müllerian duct anomalies

BackgroundMüllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations.MethodsOur previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays.ResultsUltimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs.ConclusionsThe current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation

BackgroundHereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST.ObjectiveTo determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism.MethodsA four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted.ResultsA novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration.ConclusionThe adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Sudden Cardiac Death and Channelopathies: What Lies behind the Clinical Significance of Rare Splice-Site Alterations in the Genes Involved?

Background and objectives: Sudden cardiac death (SCD) is a natural and unexpected death of cardiac origin that occurs within 1 h from the onset of acute symptoms. The major leading causes of SCD are cardiomyopathies and channelopathies. In this review, we focus on channelopathies, inherited diseases caused by mutations affecting genes encoding membrane ion channels (sodium, potassium or calcium channels) or cellular structures that affect Ca2+ availability. The diagnosis of diseases such as long QT, Brugada syndrome, short QT and catecholaminergic polymorphic ventricular tachycardia (CPVT) is still challenging. Currently, genetic testing and next-generation sequencing allow us to identify many rare alterations. However, some non-coding variants, e.g., splice-site variants, are usually difficult to interpret and to classify. Methods: In our review, we searched for splice-site variants of genes involved in channelopathies, focusing on variants of unknown significance (VUSs) registered on ClinVar up to now. Results: The research led to a high number of splice-site VUSs of genes involved in channelopathies, suggesting the performance of deeper studies. Conclusions: In order to interpret the correlation between variants and pathologies, we discuss experimental studies, such as RNA sequencing and functional analysis of proteins. Unfortunately, as these in vitro analyses cannot always be performed, we draw attention to in silico studies as future perspectives in genetics. This review has the aim of discussing the potential methods of detection and interpretation of VUSs, bringing out the need for a future reclassification of variants with currently unknown significance.

Expanding the spectrum of progressive familial intrahepatic cholestasis: A report of 3 cases.

Progressive familial intrahepatic cholestasis (PFIC) is a group of autosomal recessive disorders caused by defects in bile secretion or transport usually presenting as cholestasis in pediatric age. Herewith, we describe 3 PFIC cases with diagnostic challenges and highlight the role of genetic analysis. The clinical history, laboratory data, liver biopsy, and molecular analysis for each case were reviewed. Case 1, a Hispanic male from Puerto Rico with hepatomegaly since age 2 months, was eventually diagnosed with PFIC3 following identification of a homozygous splice site variant in ATP binding cassette subfamily B member 4 (ABCB4) (c.2784-12T>C) at age 17 years by whole-exome sequencing (WES). Case 2 was a 37-year-old man with a history of alcoholism, abnormal liver function tests, and ductopenia on biopsy. Molecular testing revealed a pathogenic heterozygous ABCB4 mutation (c.1633C>T) variant leading to a diagnosis of PFIC3. Case 3 was a 2-year-old female initially presenting as a drug-induced liver injury but was diagnosed with PFIC10 following identification of a heterozygous frameshift mutation (p.Asp300Trpfs*19) and a heterozygous missense mutation (c.1357T>C) in myosin VB (MYO5B) by WES. These PFIC cases highlight the heterogenous presentation and diagnostic challenges, and they emphasize the role of next-generation sequencing, particularly the utility of WES.

Heterozygous loss-of-function variants in SPTAN1 cause a novel early childhood onset distal myopathy with chronic neurogenic features.

Neurogenetic disorders caused by pathogenic variants in four genes encoding non-erythrocytic spectrins ( SPTAN1, SPTBN1, SPTBN2, SPTBN4) range from peripheral and central nervous system involvement to complex syndromic presentations. Heterozygous pathogenic variants in SPTAN1 are exemplary for this diversity with phenotypes spanning almost the entire spectrum. Through international collaboration we identified 14 families with genetically unsolved distal weakness and unreported heterozygous SPTAN1 loss-of-function variants including frameshift, nonsense and splice-acceptor variants. Clinical data, electrophysiology, muscle CT or MRI and muscle biopsy findings were collected and standardized. SPTAN1 protein, mRNA expression analysis and cDNA sequencing was performed on muscle tissue from two patients. All 20 patients presented with early childhood onset distal weakness. The severity varied both within families and between different families. Foot abnormalities ranged from hammer toes and pes cavus to distal arthrogryposis. Electrophysiology showed mixed myogenic and neurogenic features. Muscle MRI or CT in 10 patients showed fatty infiltration of the distal lower limb anterior compartment and/or selective involvement of the extensor hallucis longus muscle. Muscle biopsy revealed myopathic changes with mild dystrophic and chronic neurogenic changes in 7 patients. Finally, we provide proof for nonsense mediated decay in tissues derived from two patients. We provide evidence for the association of SPTAN1 loss-of-function variants with childhood onset distal myopathy in 14 families. This finding extends the phenotypic spectrum of SPTAN1 loss-of-function variants ranging from intellectual disability to distal weakness with a predominant myogenic cause. SPTAN1 loss-of-function variants, including frameshift, nonsense and splice site variants cause a novel childhood onset distal weakness syndrome with primarily skeletal muscle involvement. Hereditary motor neuropathies and distal myopathic disorders present a well-known diagnostic challenge as they demonstrate substantial clinical and genetic overlap. The emergence of SPTAN1 loss-of-function variants serves as a noteworthy example, highlighting a growing convergence in the spectrum of genotypes linked to both hereditary motor neuropathies and distal myopathies.

Identification of novel likely pathogenic variant in CDH23 causing non-syndromic hearing loss, and a novel variant in OTOGL in an extended Iranian family

BackgroundSensorineural hearing loss (SNHL) is a clinically and genetically heterogeneous group of disorders of the auditory system. SNHL can occur as a symptom in more than 400 syndromes, and mutations in more than 150 genes can lead to SNHL. Mutations in the GJB2 and GJB6 genes are among the most common causes of SNHL worldwide. Mutations in Cadherin 23 (CDH23) can cause Usher syndrome and/or non-syndromic hearing loss (NSHL).Material and methodsIn this study, the Whole Exome Sequencing (WES) was used to detect the cause of hearing loss in a large consanguineous Iranian family with two patients. All family members underwent a thorough Genotype–phenotype correlation assessment and co-segregation analysis to understand the inheritance pattern within the family. The candidate variants were further confirmed by Sanger sequencing. In addition, in silico analysis was performed to predict the functional impact of the variants; the interpretation of the variants was performed in accordance with the American College of Medical Genetics (ACMG) guidelines.ResultsWES results identified two novel variants, a homozygous missense variant in CDH23 (c.2961T > G) and a heterozygous splice site variant in OTOGL that was compatible with the autosomal recessive pattern of inheritance. Bioinformatics studies confirmed the pathogenic effects of novel variants. The c.2961T > G variant was classified as likely pathogenic.ConclusionsThe novel identified variant in the CDH23 was the cause of congenital profound progressive form of HL. Samples were not available from the second family to distinguish which variant is responsible for the molecular pathology of the disease. Further studies and functional examinations are suggested for investigating the role of OTOGL: c. 1863-1G > T in deafness.

Steatotic liver disease associated with 2,4-dienoyl-CoA reductase 1 deficiency.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is considered multifactorial with a number of predisposing gene polymorphisms known. The occurrence of MASLD in 7 and 10 year old siblings, one without classical risk factors and one with type 2 diabetes suggested a monogenic etiology and prompted next-generation sequencing. Exome sequencing was performed in the proband, both parents and both siblings. The impact of a likely disease-causing DNA variant was assessed on the transcript and protein level. Two siblings have hepatomegaly, elevated serum transaminase activity, and steatosis and harbor a homozygous DECR1 splice-site variant, c.330+3A>T. The variant caused DECR1 transcript decay. Immunostaining demonstrated lack of DECR1 in patient liver. These patients may represent the first individuals with DECR1 deficiency, then defining within MASLD an autosomal-recessive entity, well corresponding to the reported steatotic liver disease in Decr1 knockout mice. DECR1 may need to be considered in the genetic work-up of MASLD.

Revising pathogenesis of AP1S1-related MEDNIK syndrome: a missense variant in the AP1S1 gene as a causal genetic lesion.

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. KEY MESSAGES: A missense AP1S1 c.269T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein.

SLC12A1 variant c.1684+1 G>A causes Bartter syndrome type 1 by promoting exon 13 skipping.

Bartter syndrome type 1, an autosomal recessive genetic disorder, is caused by pathogenic loss-of-function variants in the SLC12A1 gene. It is characterized by metabolic alkalosis and prenatal-onset polyuria leading to polyhydramnios. We identified pathogenic gene in a 12-day-old newborn boy with Bartter syndrome type 1 using whole-exome sequencing. Sanger sequencing validated the identified variants. A minigene assay was performed to investigate the effect of a novel splice site variant on pre-mRNA splicing. We found a compound heterozygous variants in the SLC12A1 gene, consisting of a known pathogenic missense mutation (NM_000338: c.769 G>A; p.Gly257Ser) and a novel splice site variant (c.1684+1 G>A). In silico predictions and an in vitro minigene splicing assay demonstrated that the splicing variant c.1684+1 G>A abolished a consensus splice donor site of SLC12A1 intron 13, resulting in complete exon 13 skipping, translational frameshift, and premature termination codon, ultimately leading to loss of SLC12A1 function. Using a cell-based in vitro assay, we revealed the aberrant effect of the pathogenic splicing variant SLC12A1 c.1684+1 G>A on pre-mRNA splicing. Our findings expand the gene mutation spectrum of Bartter syndrome type 1, providing a basis for genetic diagnosis and the development of genetic medicines.

Splice-altering variant of PJVK gene in a Mauritanian family with non-syndromic hearing impairment.

PJVK gene was recently shown to create hypervulnerability to sound in humans and was the first human gene implicated in non-syndromic hearing impairment due to neural defect. Targeted next-generation sequencing of over 150 known deafness genes was performed in the proband. Sanger sequencing was used to validate the PJVK variant and confirm familial segregation of the disease. A minigene-based assay has been performed to assess the impact of the variant on splicing. We identified a novel c.550-6A > G acceptor splice-site variant in the PJVK gene in the homozygous state in a Mauritanian child with severe to profound congenital deafness. The substitution was located in intron 4. The effect of the variation was demonstrated by a minigene assay which showed that the variation, an insertion of an additional 5 bp, created a new splice site resulting in the appearance of a premature stop codon (p.Phe184Tyrfs*26) and likely a truncated protein. This result constitutes a new splice-site variant report in the PJVK gene leading to DFNB59 type associated with autosomal recessive non-syndromic hearing impairment (ARNSHI).

Clinical Characterization of a National Cohort of Patients With Germline WT1 Variants Including Late-Onset Phenotypes

Background and AimsWT1-disorder is a recently introduced term for phenotypes associated with germline WT1-variants, including glomerulopathy, urogenital anomalies and Wilms tumor. Previous studies showed a bias towards missense variants in the DNA-binding/Zinc-finger domain of WT1 (exon 8/9) and patients with early-onset glomerulopathy. Thorough genotype-phenotype correlations including follow-up data on late-onset glomerulopathy risk are lacking. To characterize the genotypic and phenotypic spectrum of WT1 disorder, we describe a national cohort of individuals with WT1-variants. MethodsWe requested clinical and genetic data of all patients with germline WT1-variants at all Dutch genetic laboratories. ResultsWe identified 43 patients with pathogenic WT1-variants (truncating n=19, missense n=13, splice-site n=7 and deletions n=4). Wilms tumor was the only clinical manifestation in 10 patients, of whom nine females. Wilms tumor occurred in 18/19 patients with truncating variants, in 4/4 patients with deletions and was more rare in patients with missense- or splice-site variants. All patients with missense and 6/7 with splice-site variants developed CKD, versus 5/19 patients with truncating variants (three in adulthood, with kidney failure at 24, 26 and 41 years) and 1/4 with a deletion. Urogenital malformations occurred predominantly in 46,XY individuals. ConclusionAmong patients with WT1-variants, a genotype-phenotype correlation was observed for Wilms tumor risk and age of CKD onset. Although childhood-onset CKD was more common in patients with missense variants in the DNA-binding/Zinc-finger domain, other patients may develop CKD and kidney failure later in life. Therefore, life-long surveillance of kidney function is recommended. Alertness of WT1-variants is especially important for girls with Wilms tumor who often miss additional phenotypes.

Dissecting CASK: Novel splice site variant associated with male MICPCH phenotype.

CASK (MIM#300172), encoding a calcium/calmodulin-dependent serine protein kinase, is crucial for synaptic transmission and gene regulation during neural development. Pathogenic variants of CASK are known to cause several neurodevelopmental disorders, including X-linked intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH). This study introduces a novel, de novo synonymous CASK variant (NM_001367721.1: c.1737G>A, p.(Glu579=)), discovered in a male patient diagnosed with MICPCH, characterized by microcephaly, developmental delay, visual impairment, and myoclonic seizures. The variant disrupts a donor splice-site at the end of exon 18. Transcriptomic analysis of blood identified 12 different CASK transcripts secondary to the synonymous variant. Nearly one third of these transcripts were predicted to result in nonsense mediated decay or protein degradation. Protein modeling revealed structural alterations in the PDZ functional domain of CASK, due to exon 18 deletion. Our findings highlight the utility of transcriptomic analysis in demonstrating the underlying disease mechanism in neurodevelopmental disorders.