Genetic Studies Of Low-phosphorus Rickets / Osteomalacia Dysplasia Of Bone Resistance Of Fibrous Tissue Gene Mutation Detection

Part I Genetic analysis of patients with hypophosphatemic rickets/OsteomalaciaIntroductionX-linked dominant hypophosphatemia, the most prevalent form of inherited rickets in humans, is a dominant disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal phosphate wasting. The gene responsible for XLH was identified as PHEX (formerly PEX) to depict a phosphate regulating gene with homology to endopeptidases on the X chromosome. Some previous studies of genotype-phenotype relationship analysis showed that there was no correlation between severity of disease and type and location of PHEX mutation. However, the conclusion is controversial. Moreover, functional study about PHEX gene will reveal the precise mechanism of this kind of disease, which provides more advices of follow-up and treatment.Objective1. To analyze the clinical manifestations of the patients diagnosed with XLH.2. To detect the disease-causing gene mutations in total 128 patients with hypophosphatemic rickets/osteomalacia in Department of Endocrinology of PUMCH.3. To find the phenotype-genotype association of the patients with XLH.4. To find the functional change of PHEX protein via in vitro study.Subjects and MethodsSubjects:128 patients with hypophosphatemic rickets/osteomalacia in Department of Endocrinology of PUMCH.Methods1. All the clinical information was analyzed.2. After obtaining appropriate consent, blood samples were taken from the patients and their family members. Genomic DNA was extracted from peripheral blood of the patient and 50 ethnically matched controls.3. PHEX, FGF23, DMP1 and SLC34A3 gene fragments covering the entire coding region and intron/exon boundaries were amplified by PCR. The amplified products were directly purified and sequenced. Mutations were identified by comparing the sequences against the DNA of reference gene sequences. All the mutations were confirmed in the 50 ethnically matched controls.4. We constructed the PHEX expression vectors (with or without mutation) and transfected the vectors into HEK293 cells. After 36 hours expression, we extracted the total RNA from cells and performed RT-PCR to detect PHEX mRNA. We performed Western-Blot to detect the expressed PHEX protein. The substrates of PHEX we used in the proteolysis study include human recombined i-FGF23 and ASARM polypeptide and we used ELISA kit and LC-MS/MS to detect the product.5. Statistical analysis:we performed Fisher's exact test in the phenotype-genotype association study.Results1. We performed gene mutation analysis in 128 patients with hypophosphatemic rickets/osteomalacia and found 33 PHEX gene mutations and one FGF23 gene mutation.2. After summarizing all the clinical information from 33 patients with XLH (including 8 familial cases), we found that some characteristic features such as early onset of disease (mean age of onset=18 months), severe hypophosphatemia, skeletal deformities and dental problems are most common. Many patients had increased serum ALP concentration. Several patients were found to have nephrolithiasis after long-time treatment of neutral phosphorus and active vitamin D. The reason is not clear.3. PHEX gene analysis of the 33 Chinese pedigrees revealed 29 different mutations, including 20 novel mutations and nine mutations that have been previously described in the literature or entered in PHEXdb as personal submission. It consists of 7 missense (24.1%),7 nonsense (24.1%),3 deletions (10.3%),7 insertions (24.1%) and 5 splice site mutations (17.2%). The 29 mutations are scattered widely throughout the whole gene and 9 of them (31%) located in conserved domains. Of the 20 novel mutations,3 were missense,3 were nonsense,3 were deletions,6 were insertions and 5 were splice site mutations. And 6 of them (30%) were found in familial cases. 4. The phenotype-genotype association study showed no significant relationship between the age of onset or dental problems and gene mutations.5. We reported a family of Chinese ethnic with ADHR. The diagnosis was confirmed based on the clinical and molecular genetic analysis. A single heterozygous c.527G>A (p.R176Q) mutation in the FGF23 gene was detected in three of the family members including the proband, her brother and their mother.6. A functional study was performed to elucidate the effect of three missense mutations including c.254G>C (p.C85S),c.1216 T>A (p.C406S) and c.1843A>C (p.T615P) on proteolysis of PHEX protein. We successfully established the HEK293 expression cell culture model, and the expression of wild type and the mutant PHEX gene were proved similar. More studies are necessary for functional analysis and the mechanism remains to elucidate.ConclusionIn the present study we concluded the clinical manifestations and analyzed the PHEX gene mutations in 33 Chinese pedigrees with XLH, including familial and sporadic cases. The phenotype-genotype association study showed no significant relationship between the age of onset or dental problems and gene mutations. We reported a family of Chinese ethnic with ADHR carrying a heterozygous c.527G>A (p.R176Q) mutation in the FGF23 gene. A functional study was performed to elucidate the effect of three missense mutations including c.254G>C (p.C85S),c.1216 T>A (p.C406S) and c.1843A>C (p.T615P) on proteolysis of PHEX protein. We successfully established wildtype and mutanted expression plasmid. However, more investigations should be performed to elucidate the precise mechanism of how PHEX involved in the pathology of those phosphate-wasting diseases. Part II Mutation analysis of ACVR1 gene in two Chinese patients with fibrodysplasia ossificans progressivaIntroductionFibrodysplasia ossificans progressiva (FOP, OMIM 135100) is a rare heritable disorder of connective tissue characterized by congenital malformations of the great toes and recurrent episodes of painful soft-tissue swelling, leading to heterotopic ossifications. First described in 1692, FOP has been documented in all races. In 1982, the incidence of FOP was estimated to be approximately 1:1640000. Autosomal dominant transmission with variable expressivity has been established. Recently, Shore et al. successfully mapped FOP to chromosome 2q23-24 by genome-wide linkage analysis. They identified an identical heterozygous mutation (c.617G> A; p.R206H) in the glycine-serine (GS) activation domain of ACVR1 (OMIM 102576; also known as Alk2 or ActRIA), a BMP type I receptor, in all affected individuals examined. The c.617G> A mutation in the ACVR1 gene is also recurrent in Asian patients with FOP. In this study, we investigated the ACVR1 gene mutation in two Chinese patients diagnosed as FOP.ObjectiveOur study is amied to investigate the ACVR1 gene mutation in two Chinese patients diagnosed with FOP.Subjects and methods1. Subjects:We observed two Chinese patients suffered from progressive pain and ankylosis of major joints with congenital bilateral hallus valgo deformation, neck stiffness and several post-traumatic ossified lesions on the head and dorsum. Both of them were diagnosed with FOP. Patient 1 is an 18 year old boy, suffered from multiple progressive painful swellings in synovial joints including hips and knee joints over 13 years. He also had limitation of the spinal movement and progressive ankylosis of lumbar spine. He was found to have bilateral hallus valgo deformation, neck stiffness and several post-traumatic ossified lesions on the head (temporal), dorsum and left hip since he was 3 years old. Patient 2 was a 16 year old boy who suffered from multiple progressive painful swellings in synovial joints, including hips and knee joints, over 13 years. From the age of 3 years, the patient had several post-traumatic ossified lesions on the head, dorsum and right hip. The patient had scoliosis and cervical stiffness since the age of 4. He had bilateral hallus valgus malformation. The family history of both patients was negative.2. After obtaining appropriate consent, blood samples were taken from the patient2, the parents of the two families and 50 ethnically-matched unrelated controls. Genomic DNA was isolated from extracted from peripheral blood of the patient and his parents by standard Phenol-chloroform extraction procedure. The ACVR1 gene fragments covering the entire coding region and intron/exon boundaries were amplified by PCR. The amplified products were directly purified and sequenced. Mutations were identified by comparing the sequences against the DNA of the ACVR1 gene.3. Mutation confirmed by restriction enzyme digestion. Samples from parents of the patient's family and 50 healthy controls were amplified by PCR. The production were digested by Cac8I and the digested samples were underwent electrophoreses.ResultsWe detected a heterozygote missense mutation 617G>A (R206H) in the ACVR1 gene in two Chinese patients with FOP. The 617G>A mutation was de novo in their families. It was a recurrent mutation of ACVR1 gene in two Chinese patients.ConclusionsWe investigated the ACVR1 gene mutation in two Chinese patients diagnosed with FOP. As we expected, we found the 617G> A (R206H) mutation in two Chinese FOP patients. It was a recurrent mutation of ACVR1 gene in Chinese patients.