Case report: A novel splice-site mutation of MTX2 gene caused mandibuloacral dysplasia progeroid syndrome: the first report from China and literature review.

Background: Mandibuloacral dysplasia (MAD) syndrome is a rare genetic disease. Several progeroid syndromes including mandibuloacral dysplasia type A (MADA), mandibuloacral dysplasia type B(MADB), Hutchinson-Gilford progeria (HGPS) and mandibular hypoplasia, deafness, and lipodystrophy syndrome (MDPL) have been reported previously. A novel MAD progeroid syndrome (MADaM) has recently been reported. So far, 7 cases of MADaM diagnosed with molecular diagnostics have been reported in worldwide. In the Chinese population, cases of MAD associated with the MTX2 variant have never been reported. Methods: The clinical symptoms and the genetic analysis were identified and investigated in patients presented with the disease. In addition, we analyzed and compared 7 MADaM cases reported worldwide and summarized the progeroid syndromes reported in the Chinese population to date. Results: The present study reports a case of a novel homozygous mutation c.378 + 1G > A in the MTX2 gene, which has not been previously reported in the literature. Patients present with early onset and severe symptoms and soon after birth are found to have growth retardation. In addition to the progeroid features, skeletal deformities, generalized lipodystrophy reported previously, and other multisystem involvement, e.g. hepatosplenic, renal, and cardiovascular system, this case was also reported to have combined hypogammaglobulinemia. She has since been admitted to the hospital several times for infections. Among 22 previously reported progeroid syndromes, 16/22 were MADA or HGPS caused by LMNA gene mutations, and the homozygous c.1579C > T (p.R527C) mutation may be a hot spot mutation for MAD in the Chinese population. MAD and HGPS mostly present in infancy with skin abnormalities or alopecia, MDPL mostly presents in school age with growth retardation as the first manifestation, and is often combined with an endocrine metabolism disorder after several decades. Conclusion: This is the first case of MAD syndrome caused by mutations in MTX2 gene reported in the Chinese population. MTX2 gene c.378 + 1G > A homozygous mutation has not been previously reported and the report of this patient expands the spectrum of MTX2 mutations. In addition, we summarized the genotypes and clinical characteristics of patients with progeroid syndromes in China.

Identification and functional characterization of a novel heterozygous splice­site mutation in the calpain 3 gene causes rare autosomal dominant limb­girdle muscular dystrophy.

Limb-girdle muscular dystrophies are a group of extremely heterogenous neuromuscular disorders that manifest with gradual and progressive weakness of both proximal and distal muscles. Autosomal dominant limb-girdle muscular dystrophy (LGMDD4) or calpainopathy is a very rare form of myopathy characterized by weakness and atrophy of both proximal and distal muscles with a variable age of onset. LGMDD4 is caused by germline heterozygous mutations of the calpain 3 (CAPN3) gene. Patients with LGMDD4 often show extreme phenotypic heterogeneity; however, most patients present with gait difficulties, increased levels of serum creatine kinase, myalgia and back pain. In the present study, a 16-year-old male patient, clinically diagnosed with LGMDD4, was investigated. The proband had been suffering from weakness and atrophy of both of their proximal and distal muscles, and had difficulty walking and standing independently. The serum creatine kinase levels (4,754 IU/l; normal, 35-232 IU/l) of the patient were markedly elevated. The younger sister and mother of the proband were also clinically diagnosed with LGMDD4, while the father was phenotypically normal. Whole exome sequencing identified a heterozygous novel splice-site (c.2440-1G>A) mutation in intron 23 of the CAPN3 gene in the proband. Sanger sequencing confirmed that this mutation was also present in both the younger sister and mother of the proband, but the father was not a carrier of this mutation. This splice-site (c.2440-1G>A) mutation causes aberrant splicing of CAPN3 mRNA, leading to the skipping of the last exon (exon 24) of CAPN3 mRNA and resulting in the removal of eight amino acids from the C-terminal of domain IV of the CAPN3 protein. Hence, this splice site mutation causes the formation of a truncated CAPN3 protein (p.Trp814*) of 813 amino acids instead of the wild-type CAPN3 protein that consists of 821 amino acids. This mutation causes partial loss of domain IV (PEF domain) in the CAPN3 protein, which is involved in calcium binding and homodimerization; therefore, this is a loss-of-function mutation. Relative expression of the mutated CAPN3 mRNA was reduced in comparison with the wild-type CAPN3 mRNA in the proband, and their younger sister and mother. This mutation was also not present in 100 normal healthy control individuals of the same ethnicity. The present study reported the first case of CAPN3 gene-associated LGMDD4 in the Chinese population.

A hypomorphic variant of choroideremia is associated with a novel intronic mutation that leads to exon skipping.

INTRODUCTION: 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. METHODS AND MATERIALS: 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. RESULTS: 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. DISCUSSION: 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.

X-linked hypophosphatemia due to a de novo novel splice-site variant in a 7-year-old girl with scaphocephaly, Chiari syndrome type I and syringomyelia.

X-linked hypophosphatemia (XLH) is a rare X-linked dominant inherited disorder caused by loss-of-function variants in the PHEX gene and characterized by renal phosphate wasting, hypophosphatemia, abnormal vitamin D metabolism, growth retardation and lower limb deformities. We describe a case of XLH-rickets in a 7-year-old girl with scaphocephaly, Chiari syndrome type I and syringomyelia, with a de novo non-canonical splice variant (c.1080-3C > G) in intron 9 of the PHEX gene, that has not been previously described.

Prenatal to preimplantation genetic diagnosis of a novel compound heterozygous mutation in HSPA9 associated with Even-Plus syndrome.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Whole-Exome sequencing identifies GYS2 biallelic variants in individuals with suspected epilepsy.

BACKGROUND: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2. METHODS: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families. RESULTS: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency. CONCLUSIONS: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene.

Compound Heterozygosity for Late-Onset Cardiomyopathy-Causative ALPK3 Coding Variant and Novel Intronic Variant Cause Infantile Hypertrophic Cardiomyopathy.

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.

Identification of a Novel de novo Splicing Mutation in Duchenne Muscular Dystrophy Gene in an Iranian Family.

Introduction: Duchenne muscular dystrophy (DMD) (NM_004006.3) is one of the most notable neuromuscular disorders of early years. The majority of DMD cases are caused by deletions or duplications in dystrophin, while point mutations are less prevalent in dystrophin abnormalities. It is a common knowledge that the severity of the disease depends on the effect of the mutation on the translational reading frame of the dystrophin mRNA. Case Report: We studied an 8-year-old boy with relevant clinical presentations for DMD. Deletion/duplication screening was performed by using multiplex ligation-dependent probe amplification, and whole-exome sequencing was conducted in order to identify potential variants. A novel de novo splice site variant was identified in the DMD gene (DMD: c.8548-2A>G). To explore the effect of a novel variant in DMD, various in silico analyses were carried out to investigate the pathogenicity of the causative variant. To study the structure of a DMD protein and information on how the genetic variant impacts splicing site in models of wild-type and mutated DMD, we carried out different computational studies. Sanger sequencing was performed for the purpose of variant confirmation and familial segregation analysis. Discussion: This novel de novo variant was predicted to have an effect on splicing, which leads to DMD due to its significant impacts on dystrophin functionality. The novel mutation would be expected to disrupt the protein structure.

Novel homozygous leptin receptor mutation in an infant with monogenic obesity.

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.

Hypoparathyroidism, deafness and renal dysplasia syndrome caused by a GATA3 splice site mutation leading to the activation of a cryptic splice site.

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.

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.

Loss-of-function mutations in MYO15A and OTOF cause non-syndromic hearing loss in two Yemeni families.

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.

Case report: Functional characterization of a novel CHD7 intronic variant in patients with CHARGE syndrome.

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.

New splice site mutations in MYO7A causing Usher syndrome type 1: a study on a Chinese consanguineous family.

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.

Alternative splicing in CEP290 mutant cats results in a milder phenotype than LCACEP290 patients.

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.

Compound heterozygous loss-of-function variants in BRAT1 cause lethal neonatal rigidity and multifocal seizure syndrome.

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.

Identification of a novel CACNA1F mutation in a Chinese family with CORDX3.

BACKGROUND: X-linked cone-rod dystrophy (CORDX) is one form of inherited retinal disorders (IRDs) characterized by progressive dysfunction of photoreceptor. Three types of CORDX were reported and CACNA1F gene defect can cause CORDX3. The aim of this study was to investigate the pathogenic variant in a Chinese family with IRD. METHODS: The two affected subjects including the proband and his elder sister underwent ophthalmic examinations. Whole exome sequencing (WES) was performed in the proband at first, then co-segregation analysis was performed in the family by Sanger sequencing. Minigene approach was used to verify the effect of the mutation on the splicing of CACNA1F. X-chromosomal inactivation assay was performed to evaluate the inactivation patterns of the female carriers. RESULTS: The ophthalmic examination results of the proband fit the clinical description of CORDX3, and the female patient presented with only mild symptoms due to mildly skewed X-chromosomal inactivation (ratio 67: 33). Molecular genetic testing identified a novel splice-site mutation c.3847-2A > G in CACNA1F (NM_005183.4) gene in the patients, which inherited from their asymptomatic mother. Minigene approach confirmed that c.3847-2A > G could affect the splicing of CACNA1F. CONCLUSION: Our study identified a novel splice-site mutation in the CACNA1F gene, which expanded the mutational spectrum of CACNA1F-releated diseases and demonstrated the importance of combining clinical and genetic testing in the diagnosis of IRDs.

A novel UBE2A splice site variant causing intellectual disability type Nascimento.

X-linked intellectual disability type Nascimento (XLID) is a rare disease caused by variants in the ubiquitin-conjugating enzyme E2A gene (UBE2A). Patients with XLID have similar phenotypes, including speech impairments, severe intellectual disability, hearing loss, wide facies, synophrys, generalized hirsutism, and urogenital abnormalities. Till date, only two splice-site variants of the UBE2A gene have been observed in patients with X-linked ID type Nascimento. Here, we report the case of a Chinese boy with a syndrome clinically similar to XLID with speech impairment, severe intellectual disability, and moderate hearing loss. However, different characteristics were also present in the patient, including an inability to maintain his head in an upright posture. Both of the patient's palms have a single transverse palmar crease. Subsequent whole-exome sequencing revealed a novel splice site variant in UBE2A (c.241 + 1 G > A). Our study not only expands the variant spectrum and clinical characteristics of UBE2A deficiency syndrome but also provides clinical evidence for genetic diagnoses.

Case Report: A Novel Splice-Site Mutation in DNAJB6 Associated With Juvenile-Onset Proximal-Distal Myopathy in a Chinese Patient.

DNAJB6 was identified as the causative gene of limb-girdle muscular dystrophy type 1D. In recent years, the phenotypic and molecular spectrum of DNAJB6-myopathy has been expanded, and several mutations of DNAJB6 have been identified in Europe, North America, and Asia. Interestingly, almost all identified mutations in previous reports were point mutations, and most of them were clustered in exon 5, which encodes the G/F domain of DNAJB6. The so-far unique splice site mutation eliminating the entire G/F domain was reported to cause a severe, early-onset phenotype. Here, we report a juvenile-onset Chinese patient who presented with proximal-distal myopathy as well as esotropia and facial weakness. Muscle pathology showed rimmed vacuolation and myofibrillar disarrangement. A novel splice-site mutation NM_058246:c.236-1_240delGGTGGA of the DNAJB6 gene was identified by targeted exome sequencing, which results in a severe defect of the G/F domain. This rare mutation type expands the molecular spectrum of DNAJB6-myopathy and further underlines the importance of the G/F region.

Predicting functional riboSNitches in the context of alternative splicing.

RNAs are the major regulators of gene expression, and their secondary structures play crucial roles at different levels. RiboSNitches are disease-associated SNPs that cause changes in the pre-mRNA secondary structural ensemble. Several riboSNitches have been detected in the 5' and 3' untranslated regions and lncRNA. Although cases of secondary structural elements playing a regulatory role in alternative splicing are known, regions specific to splicing events, such as splice junctions have not received much attention. We tested splice-site mutations for their efficiency in disrupting the secondary structure and hypothesized that these could play a crucial role in alternative splicing. Multiple riboSNitch prediction methods were applied to obtain overlapping results that are potentially more reliable. Putative riboSNitches were identified from aberrant 5' and 3' splice site mutations, cancer-causing somatic mutations, and genes that harbor the regulatory RNA secondary structural elements. Our workflow for predicting riboSNitches associated with alternative splicing is novel and paves the way for subsequent experimental validation.