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In cancer, recurrent somatic single-nucleotide variants—which are rare in most paediatric cancers—are confined largely to protein-coding genes 1 – 3 . Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5′ splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5′ cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes ( PTCH1 ) and activates oncogenes ( GLI2 and CCND2 ), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer. Highly recurrent hotspot r.3A>G mutations are identified in U1 splicesomal small nuclear RNAs in about 50% of Sonic hedgehog medulloblastomas, which result in disrupted RNA splicing and the activation of oncogenes.

RNA splicing is the process during which introns are excised and exons are spliced. The precise recognition of splicing signals is critical to this process, and mutations affecting splicing comprise a considerable proportion of genetic disease etiology. Analysis of RNA samples from the patient is the most straightforward and reliable method to detect splicing defects. However, currently, the technical limitation prohibits its use in routine clinical practice. In silico tools that predict potential consequences of splicing mutations may be useful in daily diagnostic activities. In this review, we provide medical geneticists with some basic insights into some of the most popular in silico tools for splicing defect prediction, from the viewpoint of end users. Bioinformaticians in relevant areas who are working on huge data sets may also benefit from this review. Specifically, we focus on those tools whose primary goal is to predict the impact of mutations within the 5′ and 3′ splicing consensus regions: the algorithms used by different tools as well as their major advantages and disadvantages are briefly introduced; the formats of their input and output are summarized; and the interpretation, evaluation, and prospection are also discussed. Genet Med 16 7, 497–503.

PurposeTo determine the genetic cause of infertility in two unrelated families of female patients suffering from oocyte degeneration and fertilization failure.MethodsWhole exome sequencing and Sanger sequencing were performed to identify the disease-causing genes of infertility in two unrelated female patients. Minigene experiments were conducted to confirm the effect of splice site mutations on mRNA splicing.ResultsIn two unrelated female infertility patients, a novel compound heterozygous splicing mutation (c.516-1G > T and c.877-1G > A) in PATL2 gene and a novel homozygous splicing mutation (c.1222-1G > A) in WEE2 gene were identified. Minigene splicing assays revealed that the c.516-1G > T mutation in PATL2 resulted in a deletion of 8 bases in mRNA that causes a frameshift (c.516-523delTCCCCCAG, p.P173Q fs*13). The c.877-1G > A mutation led to the skipping of exons 10 and 11 and retention of introns 8–9 in PATL2 mRNA. The c.1222-1G > A mutation resulted in the deletion of exon 9 in WEE2 mRNA, leading to an in-frame deletion of 57 amino acids in the WEE2 protein (p.408-464del).ConclusionOur study discovered novel splicing mutations in PATL2 and WEE2, further expanding the mutation spectrum of these two genes and providing guidance for genetic counseling and diagnosis of female infertility.

Alport syndrome is a hereditary kidney disease with significant variations in onset and prognosis. While 80–85% of cases are due to pathogenic variants in the COL4A5 gene, there is a notable lack of mouse models with Col4a5 mutations for basic research. Our research presents an 8-year-old child with Alport syndrome, exhibiting facial edema and abnormal urine. Next-generation sequencing revealed a c.1517-1G > T mutation in the intron sequence of the COL4A5 gene. Minigene experiments confirmed that this intronic mutation affects mRNA splicing. Using the CRISPR/Cas9 system, we developed a Col4a5 -c.1517-1G > T mutant mouse model. Col4α5 -deficient mice exhibited growth retardation and reduced lifespan. Renal function analysis indicated progressive deterioration, with high levels of BUN and creatinine. Histological and ultrastructural analyses revealed abnormalities such as mesangial sclerosis, interstitial fibrosis and severe irregularity in membrane thickness. Additionally, significant immune cell infiltration was observed in the renal interstitium. This mouse model provides a valuable tool for studying the role of immune cells in the pathogenesis and treatment of XLAS. It is also the first reported X-linked Alport syndrome mouse model caused by a splicing mutation.

Inflammatory bowel disease (IBD) mainly includes Crohn’s disease and ulcerative colitis. These diseases have a progressive course of chronic relapse and remission and affect a large number of children and adults worldwide. The burden of IBD is rising worldwide, with levels and trends varying greatly in countries and regions. Like most chronic diseases, the costs associated with IBD are high, including hospitalizations, outpatient and emergency visits, surgeries, and pharmacotherapies. However, there is no radical cure for it yet, and its therapeutic targets still need further study. Currently, the pathogenesis of IBD remains unclear. It is generally assumed that the occurrence and development of IBD are related to the environmental factors, gut microbiota, immune imbalance, and genetic susceptibility. Alternative splicing contributes to a various diseases, such as spinal muscular atrophy, liver diseases, and cancers. In the past, it has been reported that alternative splicing events, splicing factors, and splicing mutations were associated with IBD, but there were no reports on the practical application for clinical diagnosis and treatment of IBD using splicing-related methods. Therefore, this article reviews research progress on alternative splicing events, splicing factors, and splicing mutations associated with IBD.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with constitutive activation of the MAPKinase RAS-RAF-MEK-ERK cell signaling pathway. We analyzed 9 LCH cases without BRAF V600 and MAP2K1 mutations by whole exome sequencing. We identified a new somatic BRAF splicing mutation in 2 cases. Both cases were childhood single system (SS) LCH cases, with self-healing outcome of the bone lesions. This mutant consisted in a 9 base pair duplication (c.1511_1517 + 2 duplication), encoding for a predicted mutant protein with insertion of 3 amino acids (p.Arg506_Lys507insLeuLeuArg) in the N-terminal lobe of the kinase domain of BRAF. Transient expression of the c.1511_1517 + 2dup BRAF mutant in HEK293 cells enhanced MAPKinase pathway activation, and was not inhibited by vemurafenib but was inhibited by PLX8394, a second-generation BRAF inhibitor able to inhibit signaling of BRAF monomers and dimers. Future LCH molecular screening panel should include this new mutation to better define its prevalence in LCH and its restriction to autoregressive bone SS LCH.

Deleterious variations in STXBP1 are responsible for early infantile epileptic encephalopathy type 4 (EIEE4, OMIM # 612164) because of its dysfunction in the central nervous system. The clinical spectrum of the neurodevelopmental delays associated with STXBP1 aberrations is collectively defined as STXBP1 encephalopathy (STXBP1-E), the conspicuous features of which are highlighted by early-onset epileptic seizures without structural brain anomalies. A girl was first diagnosed with unexplained disorders of movement and cognition, which later developed into STXBP1-E with unexpected leukoaraiosis and late onset of seizures. Genetic screening and molecular tests alongside neurological examinations were employed to investigate the genetic etiology and establish the diagnosis. A heterozygous mutation of c.37+2dupT at the STXBP1 splice site was identified as the pathogenic cause in the affected girl. The de novo mutation (DNM) did not result in any truncated proteins but immediately triggered mRNA degradation by nonsense-mediated mRNA decay (NMD), which led to the haploinsufficiency of STXBP1. The patient showed atypical phenotypes characterized by hypomyelinating leukodystrophy, and late onset of epileptic seizures, which had never previously been delineated in STXBP1-E. These findings strongly indicated that the haploinsufficiency of STXBP1 could also exhibit divergent clinical phenotypes because of the genetic heterogeneity in the subset of encephalopathies.

Background Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. Methods In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. Results The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5 ; The other is the mutation resulting in the deletion of exon76. Conclusions The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.

Background Kallmann syndrome (KS) is a common type of idiopathic hypogonadotropic hypogonadism. To date, more than 30 genes including ANOS1 and FGFR1 have been identified in different genetic models of KS without affirmatory genotype–phenotype correlation, and novel mutations have been found. Methods A total of 35 unrelated patients with clinical features of disorder of sex development were recruited. Custom-panel sequencing or whole-exome sequencing was performed to detect the pathogenic mutations. Sanger sequencing was performed to verify single-nucleotide variants. Copy number variation-sequencing (CNV-seq) was performed to determine CNVs. The pathogenicity of the identified variant was predicted in silico. mRNA transcript analysis and minigene reporter assay were performed to test the effect of the mutation on splicing. Results ANOS1 gene c.709 T > A and c.711 G > T were evaluated as pathogenic by several commonly used software, and c.1063-2 A > T was verified by transcriptional splicing assay. The c.1063-2 A > T mutation activated a cryptic splice acceptor site downstream of the original splice acceptor site and resulted in an aberrant splicing of the 24-basepair at the 5′ end of exon 8, yielding a new transcript with c.1063–1086 deletion. FRFR1 gene c.1835delA was assessed as pathogenic according to the ACMG guideline. The CNV of del(8)(p12p11.22)chr8:g.36140000_38460000del was judged as pathogenic according to the ACMG & ClinGen technical standards. Conclusions Herein, we identified three novel ANOS1 mutations and two novel FGFR1 variations in Chinese KS families. In silico prediction and functional experiment evaluated the pathogenesis of ANOS1 mutations. FRFR1 c.1835delA mutation and del(8)(p12p11.22)chr8:g.36140000_38460000del were assessed as pathogenic variations. Therefore, our study expands the spectrum of mutations associated with KS and provides diagnostic evidence for patients who carry the same mutation in the future.

Acute intermittent porphyria (AIP) is an autosomal dominant genetic disease caused by a lack or decrease in hydroxymethylbilane synthase (HMBS) activity. It is characterized by acute nerve and visceral attacks caused by factors in the process of heme synthesis. The penetrance rate of this disease is low, and the heterogeneity is strong. Here, we reported two novel HMBS mutations from two unrelated Chinese AIP patients and confirmed the pathogenicity of these two mutations. We found the HMBS c.760–771+2delCTGAGGCACCTGGTinsGCTGCATCGCTGAA and HMBS c.88-1G>C mutations by second-generation sequencing and Sanger sequencing. The in vitro expression analysis showed that these mutations caused abnormal HMBS mRNA splicing and premature termination or partial missing of HMBS protein. Homologous modeling analysis showed that the HMBS mutants lacked the amino acids which are crucial for the enzyme activity or the protein stability. Consistently, enzyme activity analysis confirmed that the HMBS mutants’ overexpression cells exhibited the reduced enzyme activity compared with the HMBS wildtype overexpression cells. Our study identified and confirmed two novel pathogenic HMBS mutations which will expand the molecular heterogeneity of AIP and provide further scientific basis for the clinical diagnosis of AIP.