Study Of Gene Diagnosis Of Vitamin D Resistant Rickets

Objectivevitamin D resistant rickets inherited in a dominant manner, represents a group ofdisorders characterized by a defect in renal phosphate transport leading to phosphatewasting and hypophosphatemia. It is also characterized by abnormal regulation ofvitamin D metabolism, resulting in inappropriately normal 1, 25-dihydroxyvitamin Dconcentrations in the face of hypophosphatemia. The manifestations include skeletaldeformities, short stature, osteomalacia, dental abscesses, and bone pain.The human PHEX gene consists of 22 exons that encode a 749-amino acidprotein. PHEX gene expression, as a 6.6-kilobase (kb) transcript, has been reported byNorthern blot analysis in adult ovary and fetal lung, and to a lesser extent in adult lungand fetal liver, indicating that only 35% of the PHEX messenger RNA (mRNA)contains the 2247bp coding sequence with the remaining: 65% representing untranslatedregions (UTRs). PHEX has significant homology to the neutral endopeptidase genefamily which includes neutral endopeptidase, Kell antigen, and endothelin-convertingenzyme 1.Although identifying mutations in PHEX may have limited prognostic value,genetic testing may be useful for the early identification and treatment of affectedindividuals. Therefore, we performed mutational analysis of the PHEX gene in patientswith familial and nonfamilial (sporadic) forms of vitamin D resistant rickets. Toestablish a simple, rapid carrier detection and diagnosis; system for vitamin D resistantrickets. Material and Methods1,Samples10 patients with vitamin D resistant rickets came from the Second AffiliatedHospital, China Medical University. All patients had typical manifestation. Veinousblood of members was kept at -20℃for use after anti-coagulation.2,PCR-DGGEPCR primers designed from intronic sequence flanking 22 exons characterised inthe PHEX gene were used to amplify genomic DNA. DGGE was then carried out todetect possible changes in DNA sequence, which were then confirmed by sequenceanalysis.3,PCR-silver staining methodWe choosed 5 short tandem repeats(STR) in PHEX gene and assayed thepolymorphism of them. After PCR and PAGE, short DNA fragment were detected by asimplified silver staining method. thenAssay the polymorphism of the PCR products.Results1,Result of PCR-DGGEMutational analysis of the 22 PHEX exons in 10 individuals with vitamin Dresistant rickets revealed mutations in 3 patients (30%). Two different mutations weredetected.2,Result of PCR-silver staining methodWe deteceted that the heterozygosity degree of AAC repeats, TTG repeats in intron12, TG repeats in intron 15 and GT repeats in 3'side of gene are low. TTTA repeats inexon 16 included 5 alleles, and the heterozygosity was 69.3%, polymorphisminformation content(PIC) was 0.69. Conclusion1,Two different mutations were detected in PHEX gene among 3 individuals withvitamin D resistant rickets. The two mutations are reported for the first time.2,We finded the heterozygosity and polymorphism information content(PIC) ofTTTA repeats in exon 16 were better through polymorphism analysis in 5 STR inPHEX gene for the first time.