This research analyzes the clinical data, whole-exome sequencing results, and in vitro minigene functional experiments of a child with developmental delay and intellectual disability. The male patient, aged 4, began experiencing epileptic seizures at 3 months post-birth and has shown developmental delay. Rehabilitation training was administered between the ages of one and two. There were no other significant family medical histories. Through comprehensive family exome genetic testing, a hemizygous variant in the 11th exon of the OPHN1 gene was identified in the affected child: c.1025 + 1G > A. Family segregation analysis confirmed the presence of this variant in the patient\'s mother, which had not been previously reported. According to the ACMG guidelines, this variant was classified as a likely pathogenic variant. In response to this variant, an in vitro minigene functional experiment was designed and conducted, confirming that the mutation affects the normal splicing of the gene\'s mRNA, resulting in a 56 bp retention on the left side of Intron 11. It was confirmed that OPHN1: c.1025 + 1G > A is the pathogenic cause of X-linked intellectual disabilities in the child, with clinical phenotypes including developmental delay and seizures.

Background: Dysferlinopathy is an autosomal recessive disorder caused by mutations in the DYSF gene. This study reported two homozygous adjacent missense mutations in the DYSF gene, presenting clinically with bilateral lower limb weakness and calf swelling. Two homozygous adjacent missense mutations in the DYSF gene may be associated with the development of dysferlinopathy, but the exact mechanism needs further investigation. Methods: A retrospective analysis of clinical data from a dysferlinopathy-affected family was conducted. Peripheral blood samples were collected from members of this family for whole-exome sequencing (WES) and copy number variation analysis. Sanger sequencing was employed to confirm potential pathogenic variants. The Human Splicing Finder, SpliceAI, and varSEAK database were used to predict the effect of mutations on splicing function. The pathogenic mechanism of aberrant splicing in dysferlinopathy due to two homozygous adjacent missense mutations in the DYSF gene was determined by an in vivo splicing assay and an in vitro minigene assay. Results: The proband was a 42-year-old woman who presented with weakness of the lower limbs for 2 years and edema of the lower leg. Two homozygous DYSF variants, c.5628C>A p. D1876E and c.5633A>T p. Y1878F, were identified in the proband. Bioinformatics databases suggested that the mutation c.5628C>A of DYSF had no significant impact on splicing signals. Human Splicing Finder Version 2.4.1 suggested that the c.5633A>T of DYSF mutation caused alteration of auxiliary sequences and significant alteration of the ESE/ESS motif ratio. VarSEAK and SpliceAI suggested that the c.5633A>T of DYSF mutation had no splicing effect. Both an in vivo splicing assay and an in vitro minigene assay showed two adjacent mutations: c.5628C>A p. D1876E and c.5633A>T p. Y1878F in the DYSF gene leading to an Exon50 jump that resulted in a 32-aa amino acid deletion within the protein. Point mutation c.5628C>A p. D1876E in the DYSF gene affected splicing in vitro, while point mutation c.5633A>T p. Y1878F in the DYSF gene did not affect splicing function. Conclusion: This study confirmed for the first time that two homozygous mutations of DYSF were associated with the occurrence of dysferlinopathy. The c.5628C>A p. D1876E mutation in DYSF affected the splicing function and may be one of the contributing factors to the pathogenicity.

Autosomal Recurrent Primary Microscopic (MCPH, OMIM: 251200) is a neurodevelopmental disorder that is characterized by a noticeable decrease in brain size, particularly in the cerebral cortex, but with a normal brain structure and a non-progressive intellectual disability. MCPH1 has been identified as the gene that triggers primary microcephaly (MCPH1，OMIM: 607117). Here we report a case of autosomal recessive primary microcephaly as caused by a novel variant in the MCPH1 gene. Head circumference was measured by Magnetic Resonance Imaging (MRI), while the Wechsler Intelligence Scale was used to evaluate the intelligence of the individual being tested. B-ultrasound was used to assess gonadal development, and semen routine was used to assess sperm status. The whole-exome sequencing (WES) was performed on the proband. Sanger sequencing was conducted on the parents of the proband to determine if the novel variant in the MCPH1 gene was present. The effect of the mutation on the splicing of MCPH1 was verified by minigene approach. It was observed that the proband had autosomal recessive primary microcephaly and azoospermatism. A novel splice-site homozygous mutation (c.233+2T > G) of the MCPH1 gene was identified, which inherited from his parents. Minigene approach confirmed that c.233+2T > G could affect the splicing of MCPH1. Therefore, our findings contributed to the mutation spectrum of the MCPH1 gene and may be useful in the diagnosis and gene therapy of MCPH.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Germline MICAL1 defects have been rarely reported in patients with epilepsy and the genotype-phenotype association remains unclear. In this study, the patient was a 4.6 years old girl who presented with onset of recurrent focal seizures with onset at age 3.4 years. EEG showed abnormal δ-wave activity in the right central and middle temporal lobe. Trio WES showed a novel heterozygous variant c.-43-1G > A in the MICAL1 gene in the patient and her normal mother. Minigene verified two abnormal transcripts due to the mutation, which was predicted to interrupt 5\'UTR structures of MICAL1. The patient was clinically diagnosed with benign childhood epilepsy with centrotemporal spike (BECTS). As far as we know, this is the first BECTS case with documented MICAL1 mutation. Novel MICAL1 variant c.-43-1G > A putatively interrupted MICAL1 translation by changing 5\'UTR structures and, however, further functioning study is needed.

Polycystic kidney disease (PKD) is common genetic renal disorder. In present study, we performed WES to identify pathogenic variant in nine families including 26 patients with PKD and 19 unaffected members. The eight pathogenic variants were identified in known PKD associated genes including PKD1 (n = 6), PKD2 (n = 1), and OFD1 (n = 1) in eight families. There is one missense, one stopgain, two non-frameshifts, two canonical splicing variants, three frameshift variants and one potential non-canonical splicing variant (NCSV) in 8 families. The six variants were novel variants and not reported in ClinVar database. In addition, the compound heterozygous variants in PKHD1 were identified including one frameshift variants (PKHD1: NM_138694.4, c.9841del, p.S3281Lfs*4) and one non-canonical splicing variant (PKHD1: NM_138694.4, c.6332 + 40A > G) which were defined as deleterious variant by four splicing prediction tools (CADD-splice, SpliceAI, Spliceogen, Squirl). We used the minigene method to validate whether the prioritized potential NSCVs disrupt the typical mRNA splicing process and found abnormally larger PCR production of minigene carrying potential NCSV comparing to wild-type minigene. Sanger sequencing confirmed the 39-bp insertion of intron 38 between exon 38 and exon 39, which results in non-frameshift and 13 amino acid insertions. In conclusion, our study expands the variant spectrum and highlight the important role of non-canonical splicing variant in PKD.

BACKGROUND: Primary periodic paralysis (PPP) is an inherited disorders of ion channel dysfunction characterized by recurrent episodes of flaccid muscle weakness, which can classified as hypokalemic (HypoPP), normokalemic (NormoPP), or hyperkalemic (HyperPP) according to the potassium level during the paralytic attacks. However, PPP is charactered by remarkable clinical and genetic heterogeneity, and the diagnosis of suspected patients is based on the characteristic clinical presentation then confirmed by genetic testing. At present, there are only limited cohort studies on PPP in the Chinese population.
RESULTS: We included 37 patients with a clinical diagnosis of PPP. Eleven (29.7%) patients were tested using a specific gene panel and 26 (70.3%) by the whole-exome sequencing (WES). Twenty-two cases had a genetic variant identified, representing a diagnostic rate of 59.5% (22/37). All the identified mutations were either in the SCN4A or the CACNA1S gene. The overall detection rate was comparable between the panel (54.5%: 6/11) and WES (61.5%: 16/26). The remaining patients unresolved through panel sequencing were further analyzed by WES, without the detection of any mutation. The novel atypical splicing variant c.2020-5G > A affects the normal splicing of the SCN4A mRNA, which was confirmed by minigene splicing assay. Among 21 patients with HypoPP, 15 patients were classified as HypoPP-2 with SCN4A variants, and 6 HypoPP-1 patients had CACNA1S variants.
CONCLUSIONS: Our results suggest that SCN4A alleles are the main cause in our cohort, with the remainder caused by CACNA1S alleles, which are the predominant cause in Europe and the United States. Additionally, this study identified 3 novel SCN4A and 2 novel CACNA1S variants, broadening the mutation spectrum of genes associated with PPP.

UNASSIGNED: Combined Oxidative Phosphorylation Deficiency 23 (COXPD23) is a rare mitochondrial disease caused by mutations in the GTPBP3 gene. The rare incidence of the disease and the high clinical heterogeneity pose challenges in making a precise diagnosis. Investigations into the rare COXPD23 patients are of pathophysiological and etiological value. In this study, we investigated the genotype-phenotype relationship in a COXPD23 patient from a Manchu family, with GTPBP3 mutations.
UNASSIGNED: Routine physical examinations, laboratory assays and imaging analyses were performed. The metabolic profiles of amino acids in blood, acylcarnitine in blood and organic acids in urine were used to determine the presence of inherited metabolic diseases. Genetic variations in the family were investigated using whole-exome sequencing and Sanger sequencing. Splicing disruption by a mutation was predicted and verified using a minigene assay.
UNASSIGNED: The patient presented with severe lactic acidosis, neurological symptoms, multiple symmetrical lesions in the brain and serious mitochondrial energy metabolism disturbances. The c.689A > C (p.Q230P) and c.809-1_809delinsA compound heterozygous mutations were detected in GTPBP3. The novel c.809-1_809delinsA mutation was located at the splicing site of exon 7 and intron 6 and multiple tools predicted that it would disrupt the normal splicing. The minigene assay proved that the novel mutation resulted in two aberrant transcripts that created premature termination codons.
UNASSIGNED: The clinical manifestations, brain imaging change, mitochondrial metabolism disturbances and the detection and validation of the GTPBP3 mutations expand the profile of COXPD23 and the pathogenic mutation spectrum. Our study improves the understanding of the pathophysiology and etiology of COXPD23.

BACKGROUND: Treacher Collins syndrome (TCS; OMIM 154500) is a craniofacial developmental disorder.
METHODS: To investigate the genetic features of a four-generation Chinese family with TCS, clinical examinations, hearing tests, computed tomography, whole-exome sequencing (WES), Sanger sequencing, reverse transcription (RT)-PCR, and the Minigene assay were performed.
RESULTS: The probands, an 11-year-old male and his cousin exhibited typical clinical manifestations of TCS including conductive hearing loss, downward slanting palpebral fissures, and mandibular hypoplasia. Computed tomography revealed bilateral fusion of the anterior and posterior stapedial crura and malformation of the long crura of the incus. WES of both patients revealed a novel heterozygous intronic variant, i.e., c.4342 + 5_4342 + 8delGTGA (NM_001371623.1) in TCOF1. Minigene expression analysis revealed that the c.4342 + 5_4342 + 8delGTGA variant in TCOF1 caused a partial deletion of exon 24 (c.4115_4342del: p.Gly1373_Arg1448del), which was predicted to yield a truncated protein. The deletion was further confirmed via RT-PCR and sequencing of DNA from proband blood cells. A heterozygous variant in the POLR1C gene (NM_203290; exon6; c.525delG) was found almost co-segregated with the TCOF1 pathogenic variant.
CONCLUSIONS: In conclusion, we identified a heterozygous TCOF1 splicing variant c.4342 + 5_4342 + 8delGTGA (splicing) in a Chinese TSC family with ossicular chain malformations and facial anomalies. Our findings broadened the spectrum of TCS variants and will facilitate diagnostics and prognostic predictions.

Dysferlin protein deficiency can cause neuromuscular dysfunction, resulting in autosomal recessive dysferlinopathy, which is caused by DYSF gene mutation. Dysferlin proteins belongs to the Ferlin1-like protein family and are associated with muscle membrane repair and regeneration. In China, pathogenic mutations of the protein often result in two clinical phenotypes of Miyoshi muscular or limb band muscular dystrophy type 2B. It is clinically characterized by progressive muscle weakness and elevated serum creatine kinase. The data of the child were collected, blood samples of the child and his family members were collected, and whole exome sequencing (WES) was performed. The recombinant expression vector was constructed, the function of the mutation was verified by minigene, and the pathogenicity of the mutation was further analyzed by combining with biological information analysis. The patient initially presented with asymptomatic elevation of serum creatine kinase（CK）. Then progressive lower limb weakness, mainly distal limb weakness. Large amounts of scattered necrosis, myogenic lesions, and complete deletion of dysferlin protein were observed under muscle biopsy, which further improved genetic detection. Whole exome sequencing showed compound mutations (c.1397 + 1_1397 + 3del and c.1375dup p.M459Nfs*15) in DYSF gene. c.1375dup p.M459Nfs*15 have been reported. The other mutation is the deletion of c.1397 + 1_1397 + 3 in Intron15, which is an intron mutation that may affect splicing and the pathogenesis is still unknown. Minigene splicing assay verified that c.1397 + 1_1397 + 3del resulted in exon15 skipping and produced a premature termination codon. We report a novel pathogenic mutation in DYSF gene with Miyoshi myopathy and demonstrate this variant causes skipping of exon15 by minigene splicing assay. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation. The finding enriches the mutation spectrum of DYSF gene and laid a foundation for future studies on the correlation between genotype and phenotype.