A novel pathogenic splicing mutation of RPGR in a Chinese family with X-linked retinitis pigmentosa verified by minigene splicing assay

AIM:To report a novel splicing mutation in the RPGR gene(encoding retinitis pigmentosa GTPase regulator)in a three-generation Chinese family with X-linked retinitis pigmentosa(XLRP).METHODS:Comprehensive ophthalmic examinations including best corrected visual acuity,fundus photography,vision field,and pattern-visual evoked potential were performed to identify the disease phenotype of a six-yearold boy from the family(proband).Genomic DNA was extracted from peripheral blood of five available members of the pedigree.Whole-exome sequencing(WES),Sanger sequencing,and pSPL3-based exon trapping were used to investigate the aberrant splicing of RPGR.Human Splice Finder v3.1 and NNSPLICE v0.9 were used for in silico prediction of splice site variants.RESULTS:The proband was diagnosed as having retinitis pigmentosa(RP).He had severe symptoms with early onset.A novel splicing mutation,c.619+1G>C in RPGR was identified in the proband by WES and in four family members by Sanger sequencing.Minigene splicing assays verified that c.619+1G>C in RPGR would result in the formation of a damaging alternative transcript in which the last 91 bp of exon 6 were skipped,leading to the subsequent deletion of 623 correct amino acids(c.529_619del p.Val177Glnfs*16).CONCLUSION:We identify a novel splice donor site mutation causing aberrant splicing of RPGR.Our findings add to the catalog of pathological mutations of RPGR and further emphasize the functional importance of RPGR in RP pathogenesis and its complex clinical phenotypes.

A novel splice site mutation of CRYBA3/A 1 gene associated with congenital cataract in a Chinese family

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, KEYWORDS： splice site mutation; congenital cataract; CRYBA3/A1 gene

Novel mutation c.1210-3C>G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis

Cystic fibrosis(CF)is a rare autosomal recessive disease with only one pathogenic gene cystic fibrosis transmembrane conductance regulator(CFTR).To identify the potential pathogenic mutations in a Chinese patient with CF,we conducted Sanger sequencing on the genomic DNA of the patient and his parents and detected all 27 coding exons of CFTR and their flanking intronic regions.The patient is a compound heterozygote of c.2909G>A,p.Gly970Asp in exon 18 and c.1210-3C>G in cis with a poly-T of 5T(T5)sequence,3 bp upstream in intron 9.The splicing effect of c.1210-3C>G was verified via minigene assay in vitro,indicating that wild-type plasmid containing c.1210-3C together with T7 sequence produced a normal transcript and partial exon 10-skipping-transcript,whereas mutant plasmid containing c.1210-3G in cis with T5 sequence caused almost all mRNA to skip exon 10.Overall,c.1210-3C>G,the newly identified pathogenic mutation in our patient,in combination with T5 sequence in cis,affects the CFTR gene splicing and produces nearly no normal transcript in vitro.Moreover,this patient carries a p.Gly970Asp mutation,thus confirming the high-frequency of this mutation in Chinese patients with CF.

A novel PNPLA2 mutation causing total loss of RNA and protein expression in two NLSDM siblings with early onset but slowly progressive severe myopathy

Neutral lipid storage disease with myopathy(NLSDM)is a rare autosomal recessive disorder,due to an enzymatic error of lipid metabolism.Patients present always with skeletal muscle myopathy and variable cardiac and hepatic involvement.NLSDM is caused by mutations in the PNPLA2 gene,which encodes the adipose triglyceride lipase(ATGL).Here we report the molecular characterization and clinical findings of two NLSDM siblings carrying the novel c.187t1G>C homozygous PNPLA2 mutation,localized in the splice site of intron 2.Molecular analyses revealed that neither aberrant PNPLA2 mRNA isoforms,nor ATGL mutated protein were detectable in patient’s cells.Clinically,both patients presented early onset muscle weakness,in particular of proximal upper limb muscles.In almost 15 years,muscle damage affected also distal upper limbs.This is a NLSDM family,displaying a severe PNPLA2 mutation in two siblings with clinical presentation characterized by an early onset,but a slowly evolution of severe myopathy.