Mutation Screening And Molecular Genetics Of Single Gene Disorders

Limb deformity is regarded as the most frequently occurring congenital defect in newborns. Preaxial polydactyly typeⅡ(PPD2) and non-syndromic postaxial polydactyly are two types of limb deformities, both of which inherited in autosomal dominant mode. PPD2 patients have triphalangeal thumb and may have duplication of big toes. Expressivity difference can be observed among individuals from the same pedigree, or between limbs of the same patient. Patients with non-syndromic postaxial polydactyly have one or more extra finger on the side of the fifth finger without any other physical abnormality.We performed mutation screening in a PPD2 pedigree and a non-syndromic postaxial polydactyly pedigree, in order to locate mutation in each pedigree.Numeric abnormality of fingers is related to the interruption of the pattern of anteroposterior axis during limb development. Experimental evidences proved that PPD2, along with other triphalangeal-thumb-featured diseases, is caused by ectopic expression of SHH in the anterior region in limb bud. Mutations in ZRS (ZPA (zone of polarizing activity) regulatory sequence), a SHH limb-specific cis-acting element lies 1Mb upstream, are responsible for the mis-expression.Linkage analysis was performed in the PPD2 pedigree with microsatellite markers around ZRS and the haplotype was constructed. Linkage was established between the phenotype and ZRS with two markers (LOD score>3,θ=0). A G>C substitution was identified at ZRS 404. The mutation was screened on all the family members and normal unrelated individuals by allele-specific PCR. This single nucleotide substitution (ZRS:404 G>C) is present in all patients and absent in all non-patients (except an obligate carrier) and 64 unrelated normal control individuals. This mutation occurred at the same nucleotide where a G>A substitution has been reported previously in a Cuban pedigree.Four locus were proved to be responsible for non-syndromic postaxial polydactyly by linkage analysis. But mutation was detected in only one of those so far. According to bioinformatics features and reported functions, we selected 37 potential target genes, and performed mutation screening in each exon of those genes. But no pathological mutation has been detected. Mucopolysaccharidosis typeⅣA (MPSIVA; Morquio Syndrome type A) can be defined by its classical symptoms including bone dysplasia, short trunk dwarf ism and corneal clouding. MPS IVA is an autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase, which is encoded by GALNS on 16q24.3. The cDNA of GALNS is 1.5kb, harboring 14 exons. Over 150 mutations of this gene are reported, most of which are missense/nonsense mutations.All the 19 patients were clinically diagnosed in the Genetic Counseling Clinic, Department of Pediatrics, PUMC Hospital, and confirmed by enzymology method. We use PCR to amplify each GALNS exon with necessary flanking sequences, or RT-PCR to amplify the coding region and UTRs of GALNS cDNA, and then screen mutations by direct sequencing.Up to date, thirty-four mutant alleles were identified, sorted to twenty-three different mutations. Nineteen missense/nonsense mutations, 1 small deletion qnd 1 splicing mutation were detected by PCR-sequencing method. They are G155R, G290S, M318R, G340D, G21R, H142R, P163H, G167L, H236D, V257A, D288E, N289S, T312A, G316V, A324E, L366P, Q422X, Q422K, F452L, L36_L37del and c.634-1G>A. The first four missense mutations were reported in Japanese and Bulgarian patients previously, the others were not reported up to date. Two splicing mutations were detected with RT-PCR-sequencing method. One is c.567-1G>T causing 11bp deletion between cDNA 568 to 578; the other causes skipping of exon 5. All mutations identified in this study located in exons 1,5,7,8,9,10 and 12.Compare to previously released database, the result suggests there is difference of the mutation spectrum between the Chinese patients and the Caucasian and Japanese patients. These crucial areas can be-regard as hot spot during screening in Chinese patients. Moreover, it also demonstrated that RT-PCR sequencing procedure facilitates the identification of small deletion/insertions and splicing mutations.