Heterozygous Splice Site Variant Research Articles

Partial penetrance and phenotypic variability of aplasia of lacrimal and salivary glands caused by a novel FGF10 donor splice-site mutation.

Thirteen affected individuals of six generations of a single kindred presented with epiphora evident from infancy. Physical exam and Schirmer test revealed variable expression of tear deficiency, congenital punctal atresia, and dry mouth with multiple caries, without concomitant abnormalities of the ears or digits, commensurate with a diagnosis of aplasia of the lacrimal and salivary glands (ALSG). Reconstruction of the upper lacrimal drainage system was performed in some of the affected individuals. Genetic analysis, testing six affected individuals and three non-affected family members, identified a single novel heterozygous splice-site variant, c.429 + 1, G > T in fibroblast growth factor 10 (FGF10) (NM_004465.1), segregating throughout the family as expected for dominant heredity. RT-PCR assays of HEK-293 cells transfected with wild type or mutant FGF10 demonstrated that the variant causes skipping of Exon 2. Notably, individuals sharing the same variant exhibited phenotypic variability, with unilateral or bilateral epiphora, as well as variable expression of dry mouth and caries. Moreover, one of the variant carriers had no ALSG-related clinical findings, demonstrating incomplete penetrance. While coding mutations in FGF10 are known to cause malformations in the nasolacrimal system, this is the second FGF10 splice-site variant and the first donor-site variant reported to cause ALSG. Thus, our study of a unique large kindred with multiple affected individuals heterozygous for the same FGF10 variant highlights intronic splice-site mutations and phenotypic variability/partial penetrance in ALSG.

A novel splice site variant c.1183 + 1 G > C in DFNA5 causing autosomal dominant nonsyndromic hearing loss in a Chinese family

BackgroundThe most frequent clinical presentation of autosomal dominant nonsyndromic hearing loss (ADNSHL) is bilateral, symmetrical, postlingual progressive sensorineural hearing loss, which begins with impairment at high frequencies and eventually progresses to hearing loss at all frequencies. Autosomal dominant deafness-5 (DFNA5) is a subtype of ADNSHL caused by heterozygous variants in the gasdermin E (GSDME, also known as DFNA5) gene.MethodsDeafness gene NGS panel analysis were performed on the proband of a six-generation Chinese family with hearing loss. The co-segregation analysis between the hearing loss and the novel variant was analyzed by Sanger sequencing and pure-tone audiometry. The minigene splicing assay was performed to evaluate the potential effect of the variant on messenger RNA splicing in vitro.ResultsThe family exhibited autosomal dominant, progressive, postlingual, nonsyndromic sensorineural hearing loss, which was similar to that of the previously reported DFNA5 families. A novel heterozygous splice site variant in GSDME gene intron 8 was identified, which co-segregated with the hearing loss phenotype of the family. The variant caused skipping of exon 8 in the mutant transcript, leading to the direct linking of exons 7 and 9.ConclusionsWe identified a novel GSDME splice site variant c.1183 + 1 G > C in an extended Chinese family, which led to the skipping of exon 8. The results extended the pathogenic variants spectrum of the GSDME gene, provided further support for the 'gain-of-function' mechanism of DFNA5, and afforded a molecular interpretation for these patients with ADNSHL.

Prenatal Detection of Novel Compound Heterozygous Splice Site Variants of the KIAA0825 Gene in a Fetus with Postaxial Polydactyly Type A.

Postaxial polydactyly (PAP) is a common abnormality characterized by extra digits on hands and/or feet. To date, sequence variants in seven genes have been identified in non-syndromic PAP. In the present study, a fetus manifesting non-syndromic postaxial polydactyly type A (PAPA) was found by fetal ultrasonography. To better evaluate fetal prognosis, SNP array analysis and trio whole-exome sequencing (trio-WES) were performed to identify the underlying etiology. Although SNP array analysis revealed no abnormality, trio-WES identified compound heterozygous splice site variants in KIAA0825, c.-1-2A>T and c.2247-2A>G in intron 2 and intron 12, respectively. These two splice site variants were absent in control databases and were predicted to influence splicing by in silico analysis. To confirm the potential pathogenicity of the variants, in vitro splicing assays using minigene and RNA from peripheral leukocytes of the heterozygous parents were conducted. Minigene and RT-PCR assays demonstrated that the c.-1-2A>T variant led to the loss of the initiation codon, and the c.2247-2A>G variant mainly resulted in exon 13 skipping. Prenatal WES and subsequent functional studies are important approaches for defining the genetic etiology of fetuses with PAPA and are also essential for accurate genetic counseling and decision making. Taken together, this study expands the spectrum of KIAA0825 variations in PAPA patients and increases the knowledge of the molecular consequences of KIAA0825 splice site variants.

A BRCA1 Splice Site Variant Responsible for Familial Ovarian Cancer in a Han-Chinese Family.

Ovarian cancer (OC) is one of the most common and most lethal gynecological malignancies. OC has an age-dependent incidence and occurs more commonly in females older than 50 years old. Most OC patients are diagnosed at an advanced stage and have a poor prognosis. Germline mutations in the BRCA1 DNA repair associated gene (BRCA1) and the BRCA2 DNA repair associated gene (BRCA2) account for 20%-25% of epithelial ovarian cancer (EOC). BRCA1 germline mutations are more common in Chinese EOC patients. This study reported a three-generation Han-Chinese family containing four EOC patients and a rectal adenocarcinoma patient. Whole-exome sequencing was performed on two EOC patients and an unaffected individual. Variant validation was also performed in all available members by Sanger sequencing. A heterozygous splice site variant, c.4358-2A>G in the BRCA1 gene, was identified. Bioinformatic analysis showed that the variant may change the splicing machinery. The BRCA1 splice site variant, c.4358-2A>G was identified as the likely genetic cause for EOC, and may also be associated with the increased risk of rectal adenocarcinoma in the family. The findings were beneficial for genetic counseling, helpful for cancer prevention in other family members, and may facilitate therapy decision-making in the future to reduce cancer lethality.

Novel pathogenic mutations and further evidence for clinical relevance of genes and variants causing hearing impairment in Tunisian population

IntroductionHearing impairment (HI) is characterized by complex genetic heterogeneity. The evolution of next generation sequencing, including targeted enrichment panels, has revolutionized HI diagnosis. ObjectivesIn this study, we investigated genetic causes in 22 individuals with non-GJB2 HI. MethodsWe customized a HaloplexHS kit to include 30 genes known to be associated with autosomal recessive nonsyndromic HI (ARNSHI) and Usher syndrome in North Africa. ResultsIn accordance with the ACMG/AMP guidelines, we report 11 pathogenic variants; as follows; five novel variants including three missense (ESRRB-Tyr295Cys, MYO15A-Phe2089Leu and MYO7A-Tyr560Cys) and two nonsense (USH1C-Gln122Ter and CIB2-Arg104Ter) mutations; two previously reported mutations (OTOF-Glu57Ter and PNPT1-Glu475Gly), but first time identified among Tunisian families; and four other identified mutations namely WHRN-Gly808AspfsX11, SLC22A4-Cys113Tyr and two MYO7A compound heterozygous splice site variants that were previously described in Tunisia. Pathogenic variants in WHRN and CIB2 genes, in patients with convincing phenotype ruling out retinitis pigmentosa, provide strong evidence supporting their association with ARNSHI. Moreover, we shed lights on the pathogenic implication of mutations in PNPT1 gene in auditory function providing new evidence for its association with ARNSHI. Lack of segregation of a previously identified causal mutation OTOA-Val603Phe further supports its classification as variant of unknown significance. Our study reports absence of otoacoustic emission in subjects using bilateral hearing aids for several years indicating the importance of screening genetic alteration in OTOF gene for proper management of those patients. ConclusionIn conclusion, our findings do not only expand the spectrum of HI mutations in Tunisian patients, but also improve our knowledge about clinical relevance of HI causing genes and variants.

Genetic cause of epilepsy in a Greek cohort of children and young adults with heterogeneous epilepsy syndromes

We describe a cohort of 10 unrelated Greek patients (4 females, 6 males; median age 6.5 years, range 2-18 years) with heterogeneous epilepsy syndromes with a genetic basis. In these patients, causative genetic variants, including two novel ones, were identified in 9 known epilepsy-related genes through whole exome sequencing. A patient with glycine encephalopathy was a compound heterozygote for the p.Arg222Cys and the p.Ser77Leu AMT variant. A patient affected with Lafora disease carried the homozygous p.Arg171His EPM2A variant. A de novo heterozygous variant in the GABRG2 gene (p.Pro282Thr) was found in one patient and a pathogenic variant in the GRIN2B gene (p.Gly820Val) in another patient. Infantile-onset lactic acidosis with seizures was associated with the p.Arg446Ter PDHX gene variant in one patient. In two additional epilepsy patients, the p.Ala1662Val and the novel non-sense p.Phe1330Ter SCN1A gene variants were found. Finally, in 3 patients we observed a novel heterozygous missense variant in SCN2A (p.Ala1874Thr), a heterozygous splice site variant in SLC2A1 (c.517-2A>G), as a cause of Glut1 deficiency syndrome, and a pathogenic variant in STXBP1 (p.Arg292Leu), respectively. In half of our cases (patients with variants in the GRIN2B, SCN1A, SCN2A and SLC2A1 genes), a genetic cause with potential management implications was identified.

Compound heterozygous PLA2G6 loss-of-function variants in Swaledale sheep with neuroaxonal dystrophy

Sporadic occurrences of neurodegenerative disorders including neuroaxonal dystrophy (NAD) have been previously reported in sheep. However, so far no causative genetic variant has been found for ovine NAD. The aim of this study was to characterize the phenotype and the genetic aetiology of an early-onset neurodegenerative disorder observed in several lambs of purebred Swaledale sheep, a native English breed. Affected lambs showed progressive ataxia and stiff gait and subsequent histopathological analysis revealed the widespread presence of axonal spheroid indicating neuronal degeneration. Thus, the observed clinical phenotype could be explained by a novel form of NAD. After SNP genotyping and subsequent linkage mapping within a paternal half-sib pedigree with a total of five NAD-affected lambs, we identified two loss-of-function variants by whole-genome sequencing in the ovine PLA2G6 gene situated in a NAD-linked genome region on chromosome 3. All cases were carriers of a compound heterozygous splice site variant in intron 2 and a nonsense variant in exon 8. Herein we present evidence for the occurrence of a familial novel form of recessively inherited NAD in sheep due to allelic heterogeneity at PLA2G6. This study reports two pathogenic variants in PLA2G6 causing a novel form of NAD in Swaledale sheep which enables selection against this fatal disorder.

<p>A de novo <em>CTNNB1</em> Novel Splice Variant in an Adult Female with Severe Intellectual Disability</p>

The catenin beta-1 (CTNNB1) gene, encoding a sub-unit of the cadherin/catenin protein complex that is involved in the Wnt signalling pathway important for proper interneuron development, is considered to be causative for the rare autosomal dominant mental retardation syndrome, formerly called MRD19 but later renamed neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV). Its main characteristics are moderate to severe intellectual disability (ID), disruptive autistic behaviours, microcephaly, absent or limited speech, facial dysmorphisms, peripheral hypertonia/spasticity, motor delay and visual defects. So far, 35 patients have been reported with a de novo loss-of-function variant in CTNNB1. In two other patients, a deletion comprising the full gene was found. Four out of the 37 patients were of adult age (range: 27–51 years), while the majority was infant or adolescent (range: 0–20 years). Here, a 32-year-old severely intellectually disabled female patient is described in whom exome sequencing disclosed a de novo heterozygous splice site variant in the CTNNB1 gene [Chr3(GRCh37): g.41267064G>T; NM_001904.3: 23. c.734+1G>T; r. spl?]. Somatic investigation disclosed significant microcephaly and minor facial dysmorphisms. Neurological examination demonstrated severe kyphoscoliosis, distal spastic tetraparesis, especially of the legs with increased tendon reflexes and bilateral Babinski sign, resulting in severely impaired walking capability with a broad-based gait. Apart from strabismus, no ophthalmological abnormalities were found. Here, the reported variant in the CTNNB1 gene was not published earlier nor is included in the international databases. This specific variant is considered to be causative for the severe ID, autism and the somato-neurological phenotype of the patient and corresponds with a diagnosis of NEDSDV.

P0060COMBINED NOVEL PBX1 AND RET VARIANTS IN AN ADULT WITH SYNDROMIC RENAL HYPOPLASIA: A CASE REPORT

Abstract Background and Aims Congenital abnormalities of the kidney and urinary tract (CAKUT) represent the main cause of pediatric end-stage renal disease with vast pheno- and genotypic heterogeneity. However, its contribution to adult chronic kidney disease (CKD) is less well characterized. We here report on an adult who underwent genetic testing for previously unrecognized CAKUT. Method Phenotypic characterization of a family with mild syndromic form of kidney hypoplasia. Panel-based genetic analysis for CAKUT associated genes in DNA from peripheral blood cells, oral mucosa, and urinary cells. Results A 20-year-old male presented with bilateral hypoplastic kidneys, first diagnosed with nocturnal enuresis at the age of ten. For CKD stage 2 (eGFR 72ml/min/1.73m), hypertension, and moderate albuminuria (300mg/d) he was treated with Ramipril. Additionally, facial dysmorphism with low-set microtia, slight micrognathia, and slightly impaired intelligence was apparent. Bilateral audiograms did not reveal any hearing impairment. His father showed slightly reduced kidney size without any laboratory anomalies or extrarenal symptoms. Further family history including his mother and three half siblings presented unremarkable. Genetic analysis revealed the following two variants: First, a novel heterozygous truncating variant, absent from patient and population databases, was found in PBX1 c.992C>A p.Ser331* (NM_002585.3). As this variant was detected in 25% of NGS reads, we tested for potential mosaicism by direct sequencing in DNA obtained from oral mucosa and urinary epithelia. While the latter showed no PBX1 variation, mucosa cells revealed the mutated allele in 50% of reads. Segregation analysis of the father showed PBX1 wild-type. The mother was not available for genetic testing. Additionally, we identified a novel heterozygous splice site variant in RET: c.1760-5C>A (NM_020975.4), absent from both patient and population databases. This substitution is predicted to affect splicing at the acceptor site of exon 10. Sanger sequencing demonstrated paternal inheritance. Conclusion Pathogenic PBX1 variants were recently associated with CAKUTHED (Congenital Abnormalities of the Kidney and Urinary Tract with or without Hearing loss, abnormal Ears, or Developmental delay) syndrome (Heidet 2017), characterized by highly variable phenotypes. PBX1 encodes a homeodomain transcription factor taking part in transcriptional regulation during embryonic development. Functional studies suggest disturbed PBX1-dependent transactivation ability and altered nuclear translocation, resulting in abnormal interactions between PBX1 proteins and cofactors (Slavotinek 2017). Prevailing PBX1 expression in embryonic compared to adult kidneys support an important function in development (Le Tanno 2017). An aberrant splicing effect of the identified RET variant is supported by the fact that the father, who carries the same variant but PBX1 wild-type, has slightly reduced kidneys. Associations between abnormal kidney development and alterations of RET were first described in stillborn fetuses with renal agenesis (Skinner 2008), but also in patients with vesicoureteral reflux (Yang 2008). RET encodes for a receptor tyrosine-protein kinase, which while binding ligands such as GDNF (glial cell-line derived neurotrophic factor) is involved in signaling pathways and plays an important part during organ development. Particularly, the RET/GDNF signaling pathway plays a crucial role during development of the kidneys (Costantini 2010). Functional analyses indicate an altered phosphorylation status of RET as possible causative mechanism. To the best of our knowledge, this case is the first to report on combined PBX1 and RET gene variants associated with mild syndromic renal hypoplasia, likely mitigated by genetic mosaicism.

Deleterious Impact of a Novel CFH Splice Site Variant in Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a heterogeneous disorder characterized by microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and acute kidney injury (AKI). In about 50% of cases, pathogenic variants in genes involved in the innate immune response including complement factors complement factor H (CFH), CFI, CFB, C3, and membrane co-factor protein (MCP/CD46) put patients at risk for uncontrolled activation of the alternative complement pathway. As aHUS is characterized by incomplete penetrance and presence of additional triggers for disease manifestation, genetic variant interpretation is challenging and streamlined functional variant evaluation is urgently needed. Here, we report the case of a 27-year-old female without previous medical and family history who presented with confusion, petechial bleeding, and anuric AKI. Kidney biopsy revealed glomerular thrombotic microangiopathy (TMA). Targeted next generation sequencing identified a paternally transmitted novel heterozygous splice site variant in the CFH gene [c.3134-2A>G; p.Asp1045_Thr1053del] which resulted in a partial in-frame deletion of exon 20 transcript as determined by cDNA analysis. On the protein level, the concomitant loss of 9 amino acids in the short consensus repeat (SCR) domains 17 and 18 of CFH includes a highly conserved cysteine residue, which is assumed to be essential for proper structural folding and protein function. Treatment with steroids, plasmapheresis, and the complement inhibitor eculizumab led to complete hematological and clinical remission after several months and stable renal function up to 6 years later. In conclusion, genetic investigation for pathogenic variants and evaluation of their functional impact, in particular in the case of splice site variants, is clinically relevant and enables not only better molecular understanding but helps to guide therapy with complement inhibitors.

Compound heterozygous splice site variants in the SCLT1 gene highlight an additional candidate locus for Senior-L\xf8ken syndrome

Senior Løken syndrome (SLS) is a heterogeneous disorder characterized by severe retinal degenerations and juvenile-onset nephronophthisis. Genetic variants in ten different genes have been reported as the causes of SLS. Clinical evaluation of a patient with SLS and her unaffected parents revealed that the patient had infantile-onset retinal dystrophy and juvenile-onset nephronophthisis. Other systemic abnormalities included hepatic dysfunction, megacystis, mild learning disability, autism, obesity, and hyperinsulinemia. Whole-exome sequencing identified compound heterozygous SCLT1 variants (c.1218 + 3insT and c.1631A > G) in the patient. The unaffected parents were heterozygous for each variant. Transcript analysis using reverse transcription PCR demonstrated that the c.1218 + 3insT variant leads to exon 14 skipping (p.V383_M406del), while the other variant (c.1631A > G) primarily leads to exon 17 skipping (p.D480EfsX11) as well as minor amounts of two transcripts (6 bps deletion in the last of exon 17 [p.V543_K544del] and exons 17 and 18 skipping [p.D480E, S481_K610del]). Immunohistochemical analysis demonstrated that the Sclt1 protein was localized to the distal appendage of the photoreceptor basal body, indicating a ciliary protein. In conclusion, we identified compound heterozygous splice site variants of SCLT1 in a patient with a new form of ciliopathies that exhibits clinical features of SLS.

First report of Klein-Waardenburg Syndrome in Iran and a novel pathogenic splice site variant in PAX3 gene

ObjectivesWaardenburg Syndrome (WS) as a congenital auditory-pigmentary syndrome is a clinically and genetically heterogeneous disorder. Based upon clinical manifestations, it can be classified into four types. Loss of function mutations in PAX3 gene cause WS1 and WS3 (Klein-Waardenburg syndrome). While WS2 and WS4 have locus heterogeneity with multiple causative genes. Here we report a novel splice site variant in a pedigree with multiple affected members. Based on diagnostic criteria, three of them are associated with WS3. The remained patients classified as type 1. MethodsPCR amplification and Sanger sequencing were performed for all exons and all exon-intron boundaries of PAX3 (NM_181,459) gene of the proband. Then available symptomatic and asymptomatic members were screened for the detected variant. Interpretation and classification of the variant were done based on the current guidelines. ResultsWe identified a novel heterozygous splice site variant (c.586+2T > C) in donor site of intron 4 of PAX3 gene in our proband. Moreover, this variant was co-segregated with the disease in other available five affected members. Also, the detected variant was not detected in any of the investigated asymptomatic members. This variant was classified as a pathogenic variant. ConclusionsThis study shows significant intra-familial clinical heterogeneity and absence of phenotype-genotype correlation in a pedigree with Waardenburg Syndrome. However, severity of phenotypes and additional symptoms in the patients can be related to alternative splicing and different levels of PAX3 gene expression. Detailed evaluation of more cases can shed light on this and case-reports are valuable traffic sign in the road. This article is the first report of Waardenburg syndrome type 3 in Iran.

Whole Exome Sequencing Identifies Truncating Variants in Nuclear Envelope Genes in Patients With Cardiovascular Disease.

The genetic variation underlying many heritable forms of cardiovascular disease is incompletely understood, even in patients with strong family history or early age at onset. We used whole exome sequencing to detect pathogenic variants in 55 patients with suspected monogenic forms of cardiovascular disease. Diagnostic analysis of established disease genes identified pathogenic variants in 21.8% of cases and variants of uncertain significance in 34.5% of cases. Three patients harbored heterozygous nonsense or splice-site variants in the nucleoporin genes NUP37, NUP43, and NUP188, which have not been implicated previously in cardiac disease. We also identified a heterozygous splice site variant in the nuclear envelope gene SYNE1 in a child with severe dilated cardiomyopathy that underwent transplant, as well as in his affected father. To confirm a cardiovascular role for these candidate genes in vivo, we used morpholinos to reduce SYNE1, NUP37, and NUP43 gene expression in zebrafish. Morphant embryos displayed cardiac abnormalities, including pericardial edema and heart failure. Furthermore, lymphoblasts from the patient carrying a SYNE1 splice-site variant displayed changes in nuclear morphology and protein localization that are consistent with disruption of the nuclear envelope. These data expand the repertoire of pathogenic variants associated with cardiovascular disease and validate the diagnostic and research use of whole exome sequencing. We identify NUP37, NUP43, and NUP188 as novel candidate genes for cardiovascular disease, and suggest that dysfunction of the nuclear envelope may be an under-recognized component of inherited cardiac disease in some cases.