Résumé
Objective:To identify the genetic variation in a mucopolysaccharidosis type Ⅱ(MPS Ⅱ)family, and conduct a functional study of iduronate-2-sulfatase(IDS): c.323A>C.Methods:A five-generation MPS Ⅱ family of 83 individuals including 4 patients from northern China was collected. Urine mucopolysaccharide and Alder-Reilly body were tested to assist the clinical diagnosis of MPS Ⅱ. IDS enzyme activity was detected on core family members. By the whole exome sequencing of a MPS Ⅱ patient in this family and bioinformatics analysis, the variant was screened and further identified by PCR-Sanger sequencing. Finally, to validate the function of the variant in vitro, the wild-type IDS overexpression plasmid(pCMV-hIDS-WT)and the IDS overexpression plasmid carrying the mutation site(pCMV-hIDS-c.323A>C)were transfected into COS-7 cells and the IDS activity was detected. Results:The proband(Ⅳ3)and Ⅳ4 were diagnosed as MPS Ⅱ by urine mucopolysaccharide, Alder-Reilly body, and IDS enzyme activity tests. Ⅳ3, Ⅳ4, Ⅲ19, and Ⅲ32 were determined to carry IDS: c.323A>C missense variant through the whole-exome sequencing, and diagnosed as MPS Ⅱ. Meanwhile, Ⅱ2, Ⅱ4, Ⅱ8, Ⅱ12, Ⅱ14, Ⅲ5, Ⅲ7, Ⅳ14 in the MPS Ⅱ family carried IDS: c.323A>C missense variant, and were excluded as MPS Ⅱ. The in vitro experiment in COS-7 cells showed that the missense mutation led to a significant decrease in IDS enzyme activity. Conclusion:The variant IDS: c.323A>C: p.Y108S significantly decreases the activity of IDS enzyme in vivo and in vitro, and it is identified as a pathogenic variant for MPS Ⅱ.
Résumé
Objective:To explore the underlying genetic cause in two patients with mucopolysaccharidosis(MPS)using the whole-exome sequencing.Methods:Genomic DNA was extracted from the peripheral blood of two patients with MPS and their family members. Sanger sequencing and pedigree verification were performed on the pathogenic variants filtered by whole-exome sequencing. The function of the mutation sites was analyzed by bioinformatics software. The effect of the splice mutation on mRNA was further determined by reverse transcription-PCR(RT-PCR).Results:The proband 1 was a 25-year-old male, who carried compound heterozygous mutations of α-L-iduronidas(IDUA) gene: p. T179R and p. S633L, and was diagnosed as MPSⅠ. His mother and sister carried heterozygous p. T179R, while his father carried heterozygous p. S633L, consistent with the autosomal recessive inheritance pattern. The proband 2 was a 3-year-old male, who was hemizygous for IVS 6-8A>G of iduronate-2-sulfatase(IDS) gene. His mother and grandmother were heterozygous for this mutation, consistent with the X-linked recessive inheritance. The proband 2 was diagnosed as MPSⅡ. Sequencing of RT-PCR products showed that the IVS 6-8A>G mutation activated an upstream cryptic splice-site in intron 6, leading to 7 nucleotide insertion in exon 7, frameshift, and shorter peptide chain.Conclusion:In this study, IDUA p. T179R and p. S633L, and IDS IVS 6-8A>G mutations were found in two patients with MPS by whole exome sequencing, which further expanded the genotypic and phenotype spectrum of MPS.