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There is currently no effective treatment strategy for recurrent/metastatic adenoid cystic carcinoma (R/M ACC). Furthermore, recent single-agent and combination immunotherapy trials have failed in unselected ACC cohorts, unlike non-ACC salivary gland cancers. Genomic profiling revealed no actionable targets but NOTCH1 and KDM6A frameshift and CTCF splice site mutations (no MYB/L fusion) with a low tumor mutational burden (TMB), microsatellite stable (MSS) and negative programmed death ligand 1 (PD-L1) were observed. We recommended an anti-programmed cell death protein 1 (anti-PD-1) plus anti-Cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) combination based on TMB 2-fold greater-than-median TMB in ACC, tumor harboring multiple immunogenic frameshift or splice site mutations, and PD-L1 negativity. Accordingly, we achieved a complete response in a radiotherapy (RT) and chemotherapy (CT)-refractory patient with locally recurrent lacrimal gland (LG) ACC and lung metastasis following personalized immunotherapy in combination with integrative therapeutics. Therefore, it is crucial to assess not only conventional immune biomarkers but also patient-specific parameters, especially in "immune-cold" cancer types. [ABSTRACT FROM AUTHOR]

Molecular confirmation of pathogenic sequence variants in the CHM gene is required prior to enrolment in retinal gene therapy clinical trials for choroideremia. Individuals with mild choroideremia have been reported. The molecular basis of genotype–phenotype associations is of clinical relevance since it may impact on selection for retinal gene therapy. Genetic testing and RNA analysis were undertaken in a patient with mild choroideremia to confirm the pathogenicity of a novel intronic variant in CHM and to explore the mechanism underlying the mild clinical phenotype. A 42-year-old male presented with visual field loss. Fundoscopy and autofluorescence imaging demonstrated mild choroideremia for his age. Genetic analysis revealed a variant at a splice acceptor site in the CHM gene (c.1350-3C > G). RNA analysis demonstrated two out-of-frame transcripts, suggesting pathogenicity, without any detectable wildtype transcripts. One of the two out-of-frame transcripts is present in very low levels in healthy controls. Mild choroideremia may result from +3 or −3 splice site variants in CHM. It is presumed that the resulting mRNA transcripts may be partly functional, thereby preventing the development of the null phenotype. Choroideremia patients with such variants may present challenges for gene therapy since there may be residual transcript activity which could result in long-lasting visual function which is atypical for this disease. [ABSTRACT FROM AUTHOR]

Hypertrophic and dilated cardiomyopathy (HCM, DCM) are leading causes of cardiovascular morbidity and mortality in children. The pseudokinase alpha-protein kinase 3 (ALPK3) plays an essential role in sarcomere organization and cardiomyocyte differentiation. ALPK3 coding mutations are causative of recessively inherited pediatric-onset DCM and HCM with variable expression of facial dysmorphism and skeletal abnormalities and implicated in dominantly inherited adult-onset cardiomyopathy. We now report two variants in ALPK3—a coding variant and a novel intronic variant affecting splicing. We demonstrate that compound heterozygosity for both variants is highly suggestive to be causative of infantile-onset HCM with webbed neck, and heterozygosity for the coding variant presents with adult-onset HCM. Our data validate partial penetrance of heterozygous loss-of-function ALPK3 mutations in late-onset hypertrophic cardiomyopathy and expand the genotypic spectrum of autosomal recessive ALPK3-related cardiac disease with Noonan-like features. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Hearing loss is a rare hereditary deficit that is rather common among consanguineous populations. Autosomal recessive non-syndromic hearing loss is the predominant form of hearing loss worldwide. Although prevalent, hearing loss is extremely heterogeneous and poses a pitfall in terms of diagnosis and screening. Using next-generation sequencing has enabled a rapid increase in the identification rate of genes and variants in heterogeneous conditions, including hearing loss. We aimed to identify the causative variants in two consanguineous Yemeni families affected with hearing loss using targeted next-generation sequencing (clinical exome sequencing). The proband of each family was presented with sensorineural hearing loss as indicated by pure-tone audiometry results. Results We explored variants obtained from both families, and our analyses collectively revealed the presence and segregation of two novel loss-of-function variants: a frameshift variant, c.6347delA in MYO15A in Family I, and a splice site variant, c.5292-2A > C, in OTOF in Family II. Sanger sequencing and PCR–RFLP of DNA samples from 130 deaf and 50 control individuals confirmed that neither variant was present in our in-house database. In silico analyses predicted that each variant has a pathogenic effect on the corresponding protein. Conclusions We describe two novel loss-of-function variants in MYO15A and OTOF that cause autosomal recessive non-syndromic hearing loss in Yemeni families. Our findings are consistent with previously reported pathogenic variants in the MYO15A and OTOF genes in Middle Eastern individuals and suggest their implication in hearing loss.

The HDR syndrome is a rare autosomal dominant disorder characterised by Hypoparathyroidism, Deafness, and Renal dysplasia, and is caused by inactivating heterozygous germline mutations in the GATA3 gene. We report an 11-year-old girl with HDR syndrome caused by a heterozygous mutation located at the splice acceptor site of exon 5 of the GATA3 gene (NM_001002295.2: c.925-1G>T). Functional studies using a minigene assay showed that this splice site mutation abolished the normal splicing of the GATA3 pre-mRNA and led to the use of a cryptic splice acceptor site, resulting in the loss of the first seven nucleotides (TCTGCAG) of exon 5 in the GATA3 mRNA. These findings increase the understanding of the mechanisms by which GATA3 splicing mutations can cause HDR syndrome. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the new splice site mutations of Myosin VIIA (MYO7A) in patients with Usher syndrome type 1 (USH1) from a three-generation Chinese consanguineous family. Methods: All subjects underwent comprehensive ophthalmic examinations and an audiometric test. Demographic data, family history, and peripheral blood leukocytes were collected. We performed whole exome sequencing (WES) to analyze the genomic DNA of the family. DNA sequence and restriction fragment length polymorphism (RFLP) analyses were also done. The identified genetic variants were validated by conducting polymerase chain reaction (PCR) in 100 healthy control subjects and comparing with the NCBI VARIANT database and the 1000 Genomes Project. The functional consequences were further analyzed. Results: WES identified two new splice site mutations (c.5648G > A(rs111033215) and c.6238-1G > C) in MYO7A in two patients with USH1, i.e., the proband and her elder brother. DNA sequence and RFLP analyses showed that other members without USH1 carried only one of the two mutations. In the analysis of healthy controls, neither mutation existed. Both mutations were predicted to be damaging and were most likely associated with USH1. Conclusion: In the three-generation Chinese consanguineous family with USH1, c.5648G > A(rs111033215) and c.6238-1G > C mutations in MYO7A are most likely associated with the disease. Our findings expand the mutational spectrum of MYO7A, which will enhance the understanding of the genetic abnormalities in USH1 and provide more evidence for future investigations on therapeutic strategies such as precise gene replacement or gene editing. [ABSTRACT FROM AUTHOR]

Background: Hearing loss is a rare hereditary deficit that is rather common among consanguineous populations. Autosomal recessive non-syndromic hearing loss is the predominant form of hearing loss worldwide. Although prevalent, hearing loss is extremely heterogeneous and poses a pitfall in terms of diagnosis and screening. Using next-generation sequencing has enabled a rapid increase in the identification rate of genes and variants in heterogeneous conditions, including hearing loss. We aimed to identify the causative variants in two consanguineous Yemeni families affected with hearing loss using targeted next-generation sequencing (clinical exome sequencing). The proband of each family was presented with sensorineural hearing loss as indicated by pure-tone audiometry results. Results: We explored variants obtained from both families, and our analyses collectively revealed the presence and segregation of two novel loss-of-function variants: a frameshift variant, c.6347delA in MYO15A in Family I, and a splice site variant, c.5292-2A > C, in OTOF in Family II. Sanger sequencing and PCR–RFLP of DNA samples from 130 deaf and 50 control individuals confirmed that neither variant was present in our in-house database. In silico analyses predicted that each variant has a pathogenic effect on the corresponding protein. Conclusions: We describe two novel loss-of-function variants in MYO15A and OTOF that cause autosomal recessive non-syndromic hearing loss in Yemeni families. Our findings are consistent with previously reported pathogenic variants in the MYO15A and OTOF genes in Middle Eastern individuals and suggest their implication in hearing loss. [ABSTRACT FROM AUTHOR]

Monogenic obesity can be caused by a mutation in one of the single genes involved in hunger and satiety. The most common mutations affect melanocortin 4 (MC4) followed by the leptin gene and its receptor. Leptin receptor (LEPR) gene mutation is an extremely rare endocrine disease characterized by early-onset obesity, hyperphagia in addition to pituitary hormone deficiency, and metabolic abnormalities. We report the case of a 12-month-old male infant born of a non-consanguineous marriage. He presented to us with rapid weight gain from 2 months of age along with hyperphagia. Biochemistry revealed a deranged lipid profile, elevated transaminases, and markedly raised serum leptin levels. On genetic analysis, a novel mutation was detected, which was a homozygous variation In exon 12 of the LEPR gene (chr1:g.65608901G>A) that resulted in the synonymous amino acid change of lysine at codon 584 proximal to donor splice site (p.Lys584). The in silico prediction of the variant was 'damaging' by MutationTaster2. The mutation was classified as a 'variant of uncertain significance' due to a lack of published literature and had to be correlated carefully with the clinical symptoms. It was recommended to do Sanger sequencing of the parents and other family members. However, due to financial constraints, the family could not afford the same. At the time of writing, funds were being arranged for procuring setmelanotide, which is a novel and effective therapy for monogenic obesity due to LepR mutation. [ABSTRACT FROM AUTHOR]

The HDR syndrome is a rare autosomal dominant disorder characterised by Hypoparathyroidism, Deafness, and Renal dysplasia, and is caused by inactivating heterozygous germline mutations in the GATA3 gene. We report an 11-year-old girl with HDR syndrome caused by a heterozygous mutation located at the splice acceptor site of exon 5 of the GATA3 gene (NM_001002295.2: c.925-1G>T). Functional studies using a minigene assay showed that this splice site mutation abolished the normal splicing of the GATA3 pre-mRNA and led to the use of a cryptic splice acceptor site, resulting in the loss of the first seven nucleotides (TCTGCAG) of exon 5 in the GATA3 mRNA. These findings increase the understanding of the mechanisms by which GATA3 splicing mutations can cause HDR syndrome.

Purpose: The rdAc cat has an intronic mutation in the centrosomal 290 kDa (CEP290) gene resulting in a frameshift and a premature stop codon (c.6960 + 9 T > G, p.Ile2321AlafsTer3) predicted to truncate the protein by 157 amino acids. CEP290 mutations in human patients cause a range or phenotypes including syndromic conditions and severe childhood loss of vision while the rdAc cat has a milder phenotype. We sought to further characterize the effect of rdAc mutation on CEP290 expression. Methods: TaqMan quantitative real‐time polymerase chain reaction assays were used to compare wildtype and truncated transcript levels. Relative protein abundance was analyzed by Western blot. Immunohistochemistry (IHC) was performed to detect CEP290 protein. Results: CEP290 mutant cats show low‐level (17.4% of wildtype cats) use of the wildtype splice site and usage of the mutant splice site. Western analysis shows retina from cats homozygous for the mutation has CEP290 protein that likely comprises a combination of both wildtype and truncated protein. IHC detects CEP290 in affected and control retina labeling the region of the interconnecting cilium. Conclusions: The comparably milder phenotype of CEP290 mutant cats is likely due to the retained production of some full‐length CEP290 protein with possible functional contributions from presence of truncated protein. [ABSTRACT FROM AUTHOR]

Background: Lethal neonatal rigidity and multifocal seizure syndrome (RMFSL, OMIM 614498) is a rare autosomal recessive disease characterized by the onset of rigidity and intractable seizures at or soon after birth. The BRAT1 has been identified to be the disease‐causing gene for RMFSL. This study aimed to determine the underlying pathogenic mutations of a Chinese family with RMFSL and to confirm the effect of the splice‐site mutation by reverse transcription analysis. Methods: Detailed family history and clinical data were recorded, and peripheral blood samples were collected from all available family members. Whole exome sequencing (WES), Sanger sequencing, and bioinformatics analysis were performed to investigate the causative variants. The impact of the intronic variant on splicing was subsequently analyzed by RT‐PCR analysis. Results: We identified two compound heterozygous variants in the BRAT1, c.431‐2A>G in intron 3 and c.1359_1361del(p.Leu454del) in exon 9 in the proband, one inherited from each parent. Furthermore, the 3′‐splice site acceptor (c.431‐2A>G) variant was found to activate a cryptic acceptor splice site, which resulted in the loss of 29 nucleotides and generation of a premature stop codon at code 180, producing a truncated BRAT1 (c.432_460del; p.Ala145Argfs*36). Conclusions: This research identified two mutations in the BRAT1 of one Chinese family with RMFSL. These data can aid in developing clinical diagnoses as well as providing genetic counseling and prenatal interventions to the family. These findings also expand our knowledge of the spectrum of BRAT1 pathogenic variants in RMFSL syndrome. [ABSTRACT FROM AUTHOR]

Lung cancer is one of the major oncological problems in Poland. Pembrolizumab monotherapy can be applied as first-line treatment in patients with advanced non-small-cell lung cancer (NSCLC) with the expression of programmed death ligand 1 (PD-L1) in = 50% of tumor cells. The article presents a case report of a female patient with advanced lung adenocarcinoma and high PD-L1 expression and an additional MET exon 14 skipping mutation. Despite the advanced stage of the disease, the patient benefited spectacularly from pembrolizumab administered following stereotactic radiotherapy for central nervous system (CNS) metastases. Partial remission followed by long-term stabilization of the disease was achieved. Unfortunately, the therapy was discontinued due to grade-3 pulmonary toxicity observed after 3 years of treatment. Despite the discontinuation of the pembrolizumab therapy, the disease has currently been stabilized and inflammatory changes have slowly resolved upon administration of corticosteroid. [ABSTRACT FROM AUTHOR]

Congenital CD59 deficiency is a recently described rare autosomal recessive disease associated with CD59 gene mutations that lead to deficient or dysfunctional CD59 protein on the cell surface. The disease is characterized by the early onset of chronic hemolysis, relapsing peripheral demyelinating neuropathy, and recurrent ischemic strokes. To date, there are 14 patients with 4 exon mutations reported globally. A young boy with early onset peripheral neuropathy and atypical hemolytic uremic syndrome is presented. Next-generation sequencing (NGS) identified a homozygous splice site variant in intron 1 of the CD59 gene (c.67 + 1G > T). This variant alters a consensus donor splicing site. Quantitative reverse transcription PCR showed that CD59 mRNA expression in the patient is significantly reduced to 0.017-fold compared to the controls. Flow cytometry showed the lack of CD59 protein on the surface of the patient’s red blood cells. This variant is the first splice site mutation reported to be associated with congenital CD59 deficiency.

Abstract Background Left ventricular non-compaction cardiomyopathy (LVNC) is a rare congenital heart defect. Gene defections have been found in patients with LVNC and their family members; and MYH7 is the most frequent gene associated with LVNC. Methods We performed a complete prenatal ultrasound and echocardiographic examination on a fetus with cardiac abnormality and a parent–child trio whole-exome sequencing to identify the potential genetic causes. When the genetic abnormality in MYH7 was identified in the fetus, we performed echocardiography and genetic screening on its high-risk relatives. Results Second trimester ultrasound and echocardiography showed several malformations in the fetus: Ebstein’s anomaly (EA), heart dilatation, perimembranous ventricle septal defects, mild seroperitoneum, and single umbilical artery. Heterozygous genotyping of a splicing variant allele (NM_00025.3: c.732+G>A) was identified in this fetus and her mother, not her father, indicating a maternal inheritance. Subsequently, direct sequencing confirmed the presence of this splicing variant among her grandmother (mother of mother), mother, older sister, and herself in a heterozygous manner. No PCR products were amplified by qRT-PCR for the RNA samples extracted from peripheral blood cells. In addition to this proband who was diagnosed with EA, her older sister and grandmother (mother of mother) were diagnosed with isolated asymptomatic LVCN, but her mother was just a carrier with no marked clinical manifestations after family screening. Conclusion The presence of MYH7 splicing variant c.732+G>A can be inherited maternally, and its cardiac phenotypes are different from one carrier to another.

Background: Because CHARGE syndrome is characterized by high clinical variability, molecular confirmation of the clinical diagnosis is of pivotal importance. Most patients have a pathogenic variant in the CHD7 gene; however, variants are distributed throughout the gene and most cases are due to de novo mutations. Often, assessing the pathogenetic effect of a variant can be challenging, requiring the design of a unique assay for each specific case.Method: Here we describe a new CHD7 intronic variant, c.5607+17A>G, identified in two unrelated patients. In order to characterize the molecular effect of the variant, minigenes were constructed using exon trapping vectors.Results: The experimental approach pinpoints the pathogenetic effect of the variant on CHD7 gene splicing, subsequently confirmed using cDNA synthetized from RNA extracted from patient lymphocytes. Our results were further corroborated by the introduction of other substitutions at the same nucleotide position, showing that c.5607+17A>G specifically alters splicing possibly due to the generation of a recognition motif for the recruitment of a splicing effector.Conclusion: Here we identify a novel pathogenetic variant affecting splicing, and we provide a detailed molecular characterization and possible functional explanation.

Abstract Background Lethal neonatal rigidity and multifocal seizure syndrome (RMFSL, OMIM 614498) is a rare autosomal recessive disease characterized by the onset of rigidity and intractable seizures at or soon after birth. The BRAT1 has been identified to be the disease‐causing gene for RMFSL. This study aimed to determine the underlying pathogenic mutations of a Chinese family with RMFSL and to confirm the effect of the splice‐site mutation by reverse transcription analysis. Methods Detailed family history and clinical data were recorded, and peripheral blood samples were collected from all available family members. Whole exome sequencing (WES), Sanger sequencing, and bioinformatics analysis were performed to investigate the causative variants. The impact of the intronic variant on splicing was subsequently analyzed by RT‐PCR analysis. Results We identified two compound heterozygous variants in the BRAT1, c.431‐2A>G in intron 3 and c.1359_1361del(p.Leu454del) in exon 9 in the proband, one inherited from each parent. Furthermore, the 3′‐splice site acceptor (c.431‐2A>G) variant was found to activate a cryptic acceptor splice site, which resulted in the loss of 29 nucleotides and generation of a premature stop codon at code 180, producing a truncated BRAT1 (c.432_460del; p.Ala145Argfs*36). Conclusions This research identified two mutations in the BRAT1 of one Chinese family with RMFSL. These data can aid in developing clinical diagnoses as well as providing genetic counseling and prenatal interventions to the family. These findings also expand our knowledge of the spectrum of BRAT1 pathogenic variants in RMFSL syndrome.

BackgroundX-linked Alport syndrome (XLAS) is caused by pathogenic variants in COL4A5 and is characterized by progressive kidney disease, hearing loss, and ocular abnormalities.The aim of this study was to identify gene mutations in a Chinese family with XLAS, confirm a diagnosis, and provide an accurate genetic counseling.MethodsThe proband was a 5-year-old male with microscopic hematuria and a family history of renal disease in 5 relatives.His relatives had microhematuria with or without proteinuria. His maternal uncle developed renal failure at the age of 35 years. He was evaluated by renal biopsy,whole-exome sequencing (WES) and whole-genome sequencing (WGS) for Alport syndrome. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from the skin of the proband. Then, a splicing reporter minigene assay was used to examine the effect of the variation on the splicing of the primary transcript in transfected cells.ResultsPathological examination of the kidney of the proband revealed diffuse thinning of the glomerular basement membrane, and immunofluorescence analysis indicated normal expression of the α5 chain in the basement membrane. No phenotype-associated candidate variant was detected in the proband via WES. A novel deep intronic COL4A5 variant (c.385–716G > A), which is segregated with disease in this family, was identified using WGS. In-vitro minigene assay and in-vivo RT-PCR analysis demonstrated that the variant could produce both normal and abnormal transcripts. The abnormal transcripts showed that the variant activated a cryptic splice site, introducing a 147 bp pseudoexon into the mRNA sequence and consequently generating a premature termination codon (p.G129Afs*38) and leading to frameshifting and truncation of the α5 (collagen IV) protein.ConclusionThis is the first report of the novel c.385–716G > A splicing mutation in the COL4A5 gene, which illustrates the importance of performing WGS to find additional mutations in WES-negative patients with highly suspected forms of genetic diseases. The same results obtained from the in-vitro and in-vivo splicing experiments confirm the consistency between the minigene assay and RT-PCR analysis. In addition, this study highlights the importance of functional analysis in diagnosis and genetic counseling in AS.

Congenital CD59 deficiency is a recently described rare autosomal recessive disease associated with CD59 gene mutations that lead to deficient or dysfunctional CD59 protein on the cell surface. The disease is characterized by the early onset of chronic hemolysis, relapsing peripheral demyelinating neuropathy, and recurrent ischemic strokes. To date, there are 14 patients with 4 exon mutations reported globally. A young boy with early onset peripheral neuropathy and atypical hemolytic uremic syndrome is presented. Next-generation sequencing (NGS) identified a homozygous splice site variant in intron 1 of the CD59 gene (c.67 + 1G > T). This variant alters a consensus donor splicing site. Quantitative reverse transcription PCR showed that CD59 mRNA expression in the patient is significantly reduced to 0.017-fold compared to the controls. Flow cytometry showed the lack of CD59 protein on the surface of the patient's red blood cells. This variant is the first splice site mutation reported to be associated with congenital CD59 deficiency. [ABSTRACT FROM AUTHOR]

A 12-year-old boy with a history of decreased vision and photophobia since he was 1 year old. Comprehensive clinical and molecular approaches were applied to evaluate his condition by which a detailed ophthalmological examination revealed bilateral isolated foveal hypoplasia with the absence of the avascular zone. Novel homozygous aryl hydrocarbon receptor (AHR) splice site mutation NM_001621.4: c.899_908 + 15del (p.?) was identified and segregated within the family members. This case represents the first report of autosomal recessive isolated foveal hypoplasia without infantile nystagmus in the literature and the second reported AHR mutation with autosomal recessive isolated foveal hypoplasia post the original cloning paper. Our identified novel splice site AHR mutation supports the pathogenicity of the AHR gene and expands its phenotypic spectrum. [ABSTRACT FROM AUTHOR]

Background: Left ventricular non-compaction cardiomyopathy (LVNC) is a rare congenital heart defect. Gene defections have been found in patients with LVNC and their family members; and MYH7 is the most frequent gene associated with LVNC. Methods: We performed a complete prenatal ultrasound and echocardiographic examination on a fetus with cardiac abnormality and a parent–child trio whole-exome sequencing to identify the potential genetic causes. When the genetic abnormality in MYH7 was identified in the fetus, we performed echocardiography and genetic screening on its high-risk relatives. Results: Second trimester ultrasound and echocardiography showed several malformations in the fetus: Ebstein's anomaly (EA), heart dilatation, perimembranous ventricle septal defects, mild seroperitoneum, and single umbilical artery. Heterozygous genotyping of a splicing variant allele (NM_00025.3: c.732+G>A) was identified in this fetus and her mother, not her father, indicating a maternal inheritance. Subsequently, direct sequencing confirmed the presence of this splicing variant among her grandmother (mother of mother), mother, older sister, and herself in a heterozygous manner. No PCR products were amplified by qRT-PCR for the RNA samples extracted from peripheral blood cells. In addition to this proband who was diagnosed with EA, her older sister and grandmother (mother of mother) were diagnosed with isolated asymptomatic LVCN, but her mother was just a carrier with no marked clinical manifestations after family screening. Conclusion: The presence of MYH7 splicing variant c.732+G>A can be inherited maternally, and its cardiac phenotypes are different from one carrier to another. [ABSTRACT FROM AUTHOR]

Objective: To investigate the impact of a novel heterozygous FUS mutation in the acceptor splice site of intron 14 (c.1542 − 1 g > t) on protein expression in Peripheral Blood Mononuclear Cells (PBMC) from a familial ALS patient. Methods: PBMC were isolated for mRNA analysis (cDNA synthesis, sequencing and one-step RT-PCR), Western Immunoblot (WI), and Immunofluorescence (IF). Results: cDNA analysis revealed the skipping of exon 15 and a premature stop codon at c.228. RT-PCR showed reduced FUS mRNA by more than half compared to a healthy control (HC) and an ALS patient without genetic mutations (wtALS). In WI FUS band intensity in the proband was 30–50% compared to HC and wtALS. An antibody expected to detect only the wild-type protein did not reveal any reduction of FUS band intensity compared to the other antibodies. IF showed no difference among HC, wtALS, and the proband. Discussion: The reduction of FUS mRNA and protein in PBMC suggests the absence of the truncated protein, probably due to nonsense-mediated decay, leading to loss of function. [ABSTRACT FROM AUTHOR]

The manifestation of intermediate uveitis (IU) in patients with retinitis pigmentosa (RP) is uncommon and poses diagnostic and management challenges. In this case, we describe the clinical features and management outcomes in an RP patient with a novel homozygous splice site mutation in PRPF8. A 21-year-old male presented with unilateral decrease of vision in the right eye for 1 week. Retinal dystrophy features were present in the left eye. After 2 weeks of topical steroid therapy, near-total resolution of IU was achieved and vision improved to 20/30. Signs of (RP) were present bilaterally, with the right eye more affected than the left. Genetic testing indicated a novel homozygous c. 3061-6_3061-3del mutation in the PRPF8 gene. IU in young patients with RP can be effectively treated with a short course of topical steroids, sparing the need for systemic immunosuppressives. After the improvement in IU, the right eye showed more advanced RP changes. [ABSTRACT FROM AUTHOR]

Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4 , c.322-57_322-8del in PAX3 , c.991-15_991-13del in DFNA5 , c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4 , c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis. [ABSTRACT FROM AUTHOR]

Patterns of p53 immunostaining are used as a surrogate marker for tumor protein 53 (TP53) mutations in the diagnosis of ovarian high-grade serous carcinoma (HGSC). We present a rare case of ovarian HGSC that metastasized to the diaphragm and cardiophrenic lymph nodes and showed the immunostaining pattern of wild-type p53 and aberrant neural cell adhesion molecule (CD56) expression. A 63-year-old woman developed multifocal metastases in the diaphragmatic pleura and cardiophrenic lymph nodes. Because she had a history of ovarian HGSC and pulmonary adenocarcinoma, we considered the possibility that the metastatic carcinoma was of either ovarian or pulmonary origin. Immunostaining revealed that the tumor cells were negative for thyroid transcription factor 1 but positive for Wilms tumor 1. The tumor additionally exhibited strong membranous CD56 expression and patchy p53 expression, both of which were inconsistent with the characteristics of ovarian HGSC. However, targeted sequencing analysis revealed that the tumor harbored a pathogenic mutation at the splice acceptor site of TP53 intron 9 (c.994-1G>C). [ABSTRACT FROM AUTHOR]

Autosomal recessive congenital ichthyosis (ARCI) is a rare form of keratinization disorder of the skin, which can be caused by mutations in 14 ARCI genes. We present a rare case of ARCI that carried a novel null mutation and a novel splice site mutation in the CYP4F22 gene. [ABSTRACT FROM AUTHOR]

Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.

Abstract Autosomal recessive congenital ichthyosis (ARCI) is a rare form of keratinization disorder of the skin, which can be caused by mutations in 14 ARCI genes. We present a rare case of ARCI that carried a novel null mutation and a novel splice site mutation in the CYP4F22 gene.

• Novel complex heterozygous variations in HSPA9 causing Even-Plus syndrome were found. • The mutation spectrum leading to Even-Plus syndrome was expanded upon. • Genotype–phenotype relationships in early pregnancy were characterized. • A basis for future embryonic studies was provided. • Assisted reproductive technology was used to avoid transmission of the same mutation. Heat shock protein family A member 9 (HSPA9) prevents unfolded and dysfunctional protein accumulation, with genetic variants known to be pathogenic. Here, we determined the genetic cause of Even-Plus syndrome (OMIM: 616854) in a Chinese family. We collected samples from two affected and two normal individuals. Whole-exome sequencing was performed to identify their genetic profiles. Potential variants were validated using Sanger sequencing. Assisted reproduction with mutation-free embryos successfully blocked the transmission of mutations. We identified novel inherited pathogenic complex heterozygous variations in the HSPA9 gene in the two affected fetuses. Three-dimensional spatial simulation of the HSPA9 protein after prediction of the mutated RNA splicing pattern abolished part of the substrate-binding domain of the protein. According to ACMG guidelines, c. 1822-1G>A and c. 1411-3T>G were classified as pathogenic and likely pathogenic, respectively. Mutation-free embryos were selected for transplantation and reconfirmed to possess no mutations. A healthy daughter was successfully born into the family. This study is the first to report complex heterozygous variations in the HSPA9 gene that influence alternative splicing in early pregnancy. Our findings expand on the mutational spectrum leading to Even-Plus syndrome and provide a basis for genetic counseling and future embryonic studies. [ABSTRACT FROM AUTHOR]

Background: Alström syndrome (AS) is a very rare childhood disorder characterized by cardiomyopathy, progressive hearing loss and blindness. Inherited genetic variants of ALMS1 gene are the known molecular cause of this disease. The objective of this study was to characterize the genetic basis and understand the genotype–phenotype relationship in Saudi AS patients.Methods: Clinical phenotyping and whole-exome sequencing (WES) analysis were performed on six AS patients belonging to two unrelated consanguineous Saudi families. Sanger sequencing was performed to determine the mode of inheritance of ALMS1 variant in first-degree family relatives and also to ensure its rare prevalence in 100 healthy population controls.Results: We identified that Alström patients from both the families were sharing a very rare ALMS1, 3′-splice site acceptor (c.11873−2 A>T) variant, which skips entire exon-19 and shortens the protein by 80 amino acids. This disease variant was inherited by AS patients in autosomal recessive mode and is not yet reported in any population-specific genetic databases. AS patients carrying this mutation showed heterogeneity in clinical presentations. Computational analysis of the mutant centroid structure of ALMS1 mRNA revealed that exon-19 skipping enlarges the hairpin loop and decreases the free energy, eventually affecting its folding pattern, stability, and function. Hence, we propose c.11873–2A as an AS causative potential founder mutation in Saudi Arabia because it is found in two families lacking a common lineage.Conclusions: We conclude that WES analysis potentially helps in clinical phenotyping, early diagnosis, and better clinical management of Alström patients showing variable clinical expressivity.

Summary: Pre‐mRNA splicing is an essential step for the regulation of gene expression. In order to specifically capture splicing variants in plants for genome‐wide association studies (GWAS), we developed a software tool to quantify and visualise Variations of Splicing in Population (VaSP).VaSP can quantify splicing variants from short‐read RNA‐seq datasets and discover genotype‐specific splicing (GSS) events, which can be used to prioritise causal pre‐mRNA splicing events in GWAS. We applied our method to an RNA‐seq dataset with 328 samples from 82 genotypes from a rice diversity panel exposed to optimal and saline growing conditions.In total, 764 significant GSS events were identified in salt stress conditions. GSS events were used as markers for a GWAS with the shoot Na+ accumulation, which identified six GSS events in five genes significantly associated with the shoot Na+ content. Two of these genes, OsNUC1 and OsRAD23 emerged as top candidate genes with splice variants that exhibited significant divergence between the variants for shoot growth under salt stress conditions.VaSP is a versatile tool for alternative splicing analysis in plants and a powerful tool for prioritising candidate causal pre‐mRNA splicing and corresponding genomic variations in GWAS. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is a rare X-linked glycosphingolipidosis caused by mutations in GLA, a gene responsible for encoding α-galactosidase A, an enzyme required for degradation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in all cells of the body. FD patients present a broad spectrum of clinical phenotype and many symptoms are shared with other diseases, making diagnosis challenging. Here we describe a novel GLA variant located in the 5′ splice site of the intron 3, in four members of a family with neuropsychiatric symptoms. Analysis of the RNA showed the variant promotes alteration of the wild type donor site, affecting splicing and producing two aberrant transcripts. The functional characterization showed absence of enzymatic activity in cells expressing both transcripts, confirming their pathogenicity. The family presents mild signs of FD, as angiokeratoma, cornea verticillata, acroparesthesia, tinnitus, vertigo, as well as accumulation of plasma lyso-Gb3 and urinary Gb3. Interestingly, the man and two women present psychiatric symptoms, as depression or schizophrenia. Although psychiatric illnesses, especially depression, are frequently reported in patients with FD and studies have shown that the hippocampus is an affected brain structure in these patients, it is not clear whether the Gb3 accumulation in the brain is responsible for these symptoms or they are secondary. Therefore, new studies are needed to understand whether the accumulation of Gb3 could produce neuronal alterations leading to psychiatric symptoms. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegias (HSPs) is a rare heterogeneous group of neurodegenerative diseases, with upper and lower limb spasticity motor neuron disintegration leading to paraplegias. NT5C2 gene (OMIM: 600417) encode a hydrolase enzyme 5'-nucleotidase, cytosolic II play an important role in maintaining the balance of purine nucleotides and free nucleobases in the spinal cord and brain. In this study we have identified a large consanguineous Saudi family segregating a novel homozygous splice site donor alteration in NT5C2 gene leading to spastic diplegia cerebral palsy, developmental delay and microcephaly. Whole exome sequencing (WES) was performed for the affected members of the family to study the novel mutation. WES data analysis, confirmed by Sanger sequencing analysis, identifies a homozygous splice site donor alteration of possible interest in NT5C2 (ENST00000343289: c.539+1G > T) at the sixth exon/intron boundaries. The mutation was further ruled out in 100 healthy control from normal population. The novel homozygous mutation observed in this study has not been reported in the literature or variant databases. The identified splicing alteration broadens the mutation spectrum of NT5C2 gene in neurodevelopmental disorders. To the best of our knowledge this is the first report from Saudi Arabia.

Abstract Background Wiskott-Aldrich syndrome is an X-linked recessive immunodeficiency due to mutations in Wiskott-Aldrich syndrome (WAS) gene. WAS gene is encoded for a multifunctional protein with key roles in actin polymerization, signaling pathways, and cytoskeletal rearrangement. Therefore, the impaired protein or its absence cause phenotypic spectrum of the disease. Since identification of novel mutations in WAS gene can help uncover the exact pathogenesis of Wiskott-Aldrich syndrome, the purpose of this study was to investigate disease causing-mutation in an Iranian male infant suspicious of this disorder. Case presentation The patient had persistent thrombocytopenia from birth, sepsis, and recurrent gastrointestinal bleeding suggestive of both Wiskott-Aldrich syndrome and chronic colitis in favor of inflammatory bowel disease (IBD). To find mutated gene in the proband, whole exome sequencing was performed for the patient and its data showed a novel, private, hemizygous splice site mutation in WAS gene (c.360 + 1G > C). Conclusions Our study found a novel, splice-site mutation in WAS gene and help consider the genetic counselling more precisely for families with clinical phenotypes of both Wiskott-Aldrich syndrome and inflammatory bowel disease and may suggest linked pathways between these two diseases.

Abstract Background The mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease resulting from the defective activity of the enzyme α-L-iduronidase (IDUA). The disease has three major clinical subtypes (severe Hurler syndrome, intermediate Hurler–Scheie syndrome and attenuated Scheie syndrome). We aim to identify the genetic variants in MPS I patients and to investigate the effect of the novel splice site mutation on splicing of IDUA- mRNA variability using bioinformatics tools. Methods The IDUA mutations were determined in four MPS I patients from four families from Northern Tunisia, by amplifying and sequencing each of the IDUA exons and intron–exon junctions. Results One novel splice site IDUA mutation, c.1650 + 1G > T in intron 11 and two previously reported mutations, p.A75T and p.R555H, were detected. The patients in families 1 and 2 who have the Hurler phenotype were homozygotes for the novel splice site mutation c.1650 + 1G > T. The patient in family 3, who also had the Hurler phenotype, was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.A75T. The patient in family 4 who had the Hurler–Scheie phenotype was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.R555H. In addition, four known IDUA polymorphisms were identified. Bioinformatics tools allowed us to associate the variant c.1650 + 1G > T with the severe clinical phenotype of MPS I. This variant affects the essential nucleotide + 1 (G to T) of the donor splice site of IDUA intron 11. The G > T in intron 11 leads to wild type donor site broken with minus 19.97% value compared to normal value with 0%, hence the new splice site acceptor has plus 5.59%. Conclusions The present findings indicate that the identified mutations facilitate the accurate carrier detection (genetic counseling of at-risk relatives) and the molecular prenatal diagnosis in Tunisia.

Background: Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder with no effective treatment, which underscores the importance of avoiding the birth of children with DMD by identifying pathogenic mutations and obtaining an accurate prenatal diagnosis. Objective: The objective of this study was to analyze the genetic defect of a Chinese family where all male patients have died of DMD. Methods: Multiplex ligation dependent probe analysis (MLPA) and next-generation sequencing (NGS) were employed to detect DMD mutations. The candidate mutations were then validated by Sanger sequencing. In vitro splicing assay was further conducted to examine the potential effect of the novel DMD splice site mutation on splicing. Results: We found that two rare DMD mutations c.1318G>A and c.6438+2T>G passed from generation to generation among female carriers and they may be used as genetic markers in the Chinese DMD family. In vitro splicing assay further revealed that the novel classical splice site mutation c.6438+2T>G gave rise to a new donor splice site, which resulted in a frame shift of the transcripts and a premature termination at position 2159 in exon 45 (p.Y2144Nfs*16). Conclusion: We found that two co-inherited mutations passed from generation to generation in female carriers and they may be used as genetic markers in the Chinese DMD family. Our findings not only expanded the DMD mutation spectrum, but also provided an important basis for identifying of female carriers and avoiding the birth of affected male children in this DMD family. [ABSTRACT FROM AUTHOR]

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome. [ABSTRACT FROM AUTHOR]

Hereditary spastic paraplegias (HSPs) is a rare heterogeneous group of neurodegenerative diseases, with upper and lower limb spasticity motor neuron disintegration leading to paraplegias. NT5C2 gene (OMIM: 600417) encode a hydrolase enzyme 5'-nucleotidase, cytosolic II play an important role in maintaining the balance of purine nucleotides and free nucleobases in the spinal cord and brain. In this study we have identified a large consanguineous Saudi family segregating a novel homozygous splice site donor alteration in NT5C2 gene leading to spastic diplegia cerebral palsy, developmental delay and microcephaly. Whole exome sequencing (WES) was performed for the affected members of the family to study the novel mutation. WES data analysis, confirmed by Sanger sequencing analysis, identifies a homozygous splice site donor alteration of possible interest in NT5C2 (ENST00000343289: c.539+1G > T) at the sixth exon/intron boundaries. The mutation was further ruled out in 100 healthy control from normal population. The novel homozygous mutation observed in this study has not been reported in the literature or variant databases. The identified splicing alteration broadens the mutation spectrum of NT5C2 gene in neurodevelopmental disorders. To the best of our knowledge this is the first report from Saudi Arabia. [ABSTRACT FROM AUTHOR]

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

AIM: To identify the disease-causing mutation responsible for the presence of congenital cataract in a Chinese family. METHODS: The study recruited a four-generation Chinese pedigree affected by autosomal dominant congenital cataract (ADCC). Family history and the history of cataract extraction were recorded. Blood samples were collected from individuals for DNA extraction. Direct sequencing of congenital cataract-associated genes was performed. Single-strand conformational polymorphism and bioinformatic analysis were conducted to further study the mutation. RESULTS: Direct sequencing revealed a novel splice site mutation of c.30-2 A>G in the CRYBA3/A1 gene. The mutation co-segregated within all affected individuals in the family and was not found in unaffected members or 100 unrelated normal controls. These results were further confirmed by single-strand conformational polymorphism and bioinformatic analysis using the Human Splicing Finder and MaxEnt online software and Annovar computer software. CONCLUSION: c.30-2 A>G mutation of CRYBA3/A1 gene is a novel mutation and broadens the genetic spectrum of ADCC.

AIM:To analyze the clinical features of a Usher syndrome family and explore the pathogenic gene of the disease. METHODS: A Chinese family with Usher syndrome was involved in our study. After informed consent, careful clinical examinations were taken and 4mL blood were obtained. The whole genome DNA was extracted and target-captured next generation sequencing of the proband was performed to identify suspected mutations. We used Sanger sequencing to verify the detected mutations in all the members of the family, as well as in 100 normal controls. RESULTS: In addition to typical retinitis pigmentosa, the patients suffered from mild to moderate sensorineural deafness. Sequencing results revealed compound heterozygous mutations(c.2310_2311insA and c.8559-2A>G)of USH2A gene in the patients, and either of the mutations was found in normal relatives that had consanguinity with the patients. Both of the mutations were not found in other members of the family and normal individuals. CONCLUSION: USH2A is the pathogenic gene of the disease in this family. The mutation c.8559-2A>G(IVS42)is a previously reported mutation, while the mutation c.2310_2311 insA(p.E771Rfs*8)is a novel mutation. The study has expanded the mutation spectrum of USH2A gene resulting in Usher syndrome.

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET, most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling.

Fabry disease (FD) is a rare and underdiagnosed X-linked disorder resulting from the deficient activity of the lysosomal hydrolase α-galactosidase A, which leads to storage of complex glycosphingolipids inside of lysosomes in critical organs and tissues, impairing their functions and consequently resulting in a progressive multisystem disease. FD is caused by mutations in the GLA gene, and only 4.6% of described mutations are located in the splice site regions. RNA splicing is an essential step to the formation of functional proteins, and mutations in splice site regions can cause formation of aberrant transcripts leading to disease. Here we report a novel GLA insertion at position c.801+3 in intron 5 (c.801+2_801+3insT) in a Brazilian family with suspicion of FD. The index case, a 46-year-old male, presented undetectable α-galactosidase A activity. Analysis of blood cDNA found two aberrant GLA transcripts. In the first transcript, a novel donor splice site was created promoting formation of an intron inclusion with 37 bp. The splice site was not recognized in the second transcript and the intron 5 was not excised. The wild-type transcript was not formed and both aberrant transcripts lead to a premature stop codon. Despite not being in the canonical site, this new mutation disrupts existing 5’ splice site and produces two aberrant transcripts leading to FD.

Abstract Background Primary carnitine deficiency (PCD) is an autosomal recessive disorder of carnitine transportation caused by mutations in the SLC22A5 that lead to low serum carnitine levels and decreased intracellular carnitine accumulation. Characteristic clinical findings are hypoketotic hypoglycemia and skeletal and cardiac myopathy. Objective To genetically diagnose 24 unrelated Chinese patients with PCD, including 18 infants and six adults. Methods The entire coding region and the intron–exon boundaries of SLC22A5 were amplified by polymerase chain reaction (PCR). In silico analyses and reverse transcription‐polymerase chain reaction (RT‐PCR) were used to predict variants’ impact on protein structure and function. Results Disease‐causing variants in the SLC22A5 were identified in all 24 subjects, and c.288delG, c.495C>A, c.774_775insTCG, c.824+1G>A, and c.1418G>T were novel. The novel variant c.824+1G>A caused a truncated protein p.Phe276Tyrfs*8. Conclusions We identified 13 variants in the SLC22A5 in 24 PCD patients, and five of these variants are novel mutations. c.824+1G>A was confirmed to alter mRNA splicing by reverse transcription PCR. Furthermore, our findings broaden the mutation spectrum of SLC22A5 and the understanding of the diverse and variable effects of PCD variants.

• MSH2 c.2634+1G>C mutation was not reported previously as LS associated. • We confirmed that MSH2 c.2634+1G>C mutation has damaging effect on splicing, causing exon 15 excision. • in silico tools predict loss of domains important for its function, implicating disrupted MMR, predisposing to cancer. • MSH2 c.2634+1GC and other substitutions on the same position should be (re)classified as Class 5 - Pathogenic. • Our results support previous suggestion of inclusion of bladder cancer in LS cancer spectrum. Lynch syndrome (LS) is predisposing mainly to colorectal and endometrial carcinomas, but also to urinary tract cancers. LS association with upper urinary tract carcinomas is known, but its association with bladder cancer is not so clear. Confirmation of pathogenicity of detected mutations in LS-associated genes is required for adequate counseling. Tested young female has family history of two early colorectal and two bladder carcinomas. NGS sequencing revealed MSH2 splice site mutation c.2634+1G>C, which was confirmed by Sanger sequencing. MSH2 cDNA part containing potential splicing change was sequenced. in silico softwares were used to predict the effect of detected mutation on splicing and protein structure. ACMG Guidelines were used for mutation classification. in silico softwares predict damaging effect of detected mutation on splicing and loss of protein-binding domains. cDNA sequencing confirmed this mutation causes exon 15 excision. ACMG Guidelines classify this mutation as Pathogenic. MSH2 c.2634+1G>C mutation was not reported previously as LS associated. We confirmed its damaging effect on splicing. in silico tools predict consequent loss of protein domains implicating disrupted protein function. Our results suggest that this mutation should be classified as Pathogenic, and indicate inclusion of bladder cancer in LS cancer spectrum. [ABSTRACT FROM AUTHOR]

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET , most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling. [ABSTRACT FROM AUTHOR]