Mutation Screening For Single Gene Disorders And In Vitro Expression System Identification

Mucopolysaccharidosis type IVA (MPS IVA; Morquio Syndrome type A; OMIM 252300) is one of the autosomal recessive lysosomal storage diseases, caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS). Key clinical features include short stature, skeletal dysplasia, dental anomalies, and corneal clouding. Intelligence is normal. Patients with the severe phenotype usually do not survive past the second or third decade of life.MPS IVA is caused by mutations in GALNS gene, located in 16q24, the coding region of which split into 14 exons, encoding for 120kDa N-acetylgalactosamine-6-sulfatase. This enzyme is involved in the lysosomal degradation of keratin sulfate and chondroitin-6-sulfate.We have screened the GALNS gene mutations by PCR and direct sequencing in 19 patients clinically diagnosed as MPS IVA, and identified 14 mutations that are not reported up to date. To investigate the role of 12 missense mutations, and to test the hypothesis that they might contribute to the disease phenotypes, we established the systematic identification on biological function, including in vitro site-direct mutagenesis, the mutant expression recombinants construction, Human embryonic kidney (HEK) 293FT cells transfection, the activities examination of mutant genes'products and Western blotting analysis. The results showed that all the activities of the mutant GALNSs were lower than that of the wild type.In conclusion, we have found the HEK 293FT cells with low background. The establishment of in vitro GALNS assay is valuable for further study on mucopolysaccharidoses. Coffin-Lowry syndrome is a rare X-linked semi-dominant mental disorder. Typical clinical features are of growth and skeletal abnormalities (delayed bone development, short stature, pectus deformity, kyphosis and/or scoliosis), craniofacial dysmorphism (coarse face, a prominent forehead, orbital hypertelorism, downward-slanting palpebral fissures, prominent ears, thick lips, and irregular or missing teeth), limb abnormalities (large and soft hands with lax skin and distally tapering fingers, flat feet), and cognitive deficit (usually, Males with severe mental retardation and IQ scores from moderate to profound(15-60), and females with a wide range from severe to relatively normal). In addition, the phenotype tends to worsen with age thus suggesting that CLS might be a progressive disorder.CLS is caused by mutations in RPS6KA3 (ribosomal protein S6 kinase,90kDa, polypeptide 3), located in Xp22.2. RPS6KA3 splits into 22 exons encoding for a protein of 740 amino acids, a serine/threonine kinase (90-KDa Ribosomal protein S6 Kinase) acting in the ras-mitogen-activated protein kinase (Ras-MAPK) signaling pathway.Up to date,80% of CLS cases are sporadic and RPS6KA3 mutations can only be found in half of the cases by mutation screening. No Chinese patients have been identified by molecular biology methods although there was a clinical case report.In this study, we screened RPS6KA3 mutation by RT-PCR and MLPA in two Chinese boys, who had very likely clinical characters of CLS. A c.889₈90delAG mutation was detected in one patient, and the mutation was confirmed gDNA sequencing. The nucleotide sequence of the patient's mother was normal, implying that it was a de novo mutation, but gonadal mosaicism could not be role out. No mutation was detected on the other patient.By mutation screecing of RPS6KA3, this was the first time that CLS was diagnosed by molecular method in China, and it provided a genetic basis for gene diagnosis and prenatal diagnosis of CLS.