Genetic Analysis Of Patients With Hypophosphatemic Rickets

IntroductionHypophosphatemic rickets is a group of disorders of impaired bone miniralization characterized by hypophosphatemia induced by renal phosphate wasting. Bone deformities, growth retardation, oral abnormities and bone pain are the main clinical manifestations. The genes responsible for hypophosphatemic rickets include:1. PHEX (phosphate regal ating gene with homologies to endopeptidases on the X chromosome); 2. FGF23 (fibroblast growth factor 23); 3. DMP1 (dentin matrix protein 1); 4. ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1); 5. FAM20C (family with sequence similarity 20, member C); 6. SLC34A3 (solute carrier family 34, member 3). PHEX is identified as the causative gene for X-linked dominant hypophosphatemic rickets (XLH), wihich is the most common form of heritable rickets.Clinical manifestations vary in severity and therefore it is difficult to diagnose the disease. Early molecular diagnosis to genetic hypophosphatemia is very important for improvement of prognosis and prenatal counseling. However, Sanger sequencing of each gene can be much too expensive and time-consuming to reach a molecular diagnosis. Previous genetic analyses from our group showed relatively low detection rate in patients with hypophosphatemic rickets. Targeted gene capture with next-generation sequencing provides a more sensitive, more sufficient and more economic method to make the molecular diagnosis. To summary the genetic characteristics of PHEX gene in our clinical center is very important to provide the theoretical basis for molecular diagnosis in the Chinese population.Objective1. To analyze and summarize the clinical characteritics of the patients with XLH.2. To detect the disease-causing gene mutations in patients with hypophosphatemic rickets.3. To summary the genetic characteristics of PHEX gene in the Chinese population.4. To analyze and summarize the clinical manifestations of the patients with ARHR2.Subjects and MethodsSubjects(1) 89 patients with hypophosphatemic rickets admitted from 2013.6 to 2015.4 in Department of Endocrinology of PUMCH.(2) 83 patients with hypophosphatemic rickets who did not identified the causative gene by Sanger sequencing.Methods1. Clinical characteristics of XLH patients were analyzed.2. PHEX, FGF23, DMP1, SLC34A3 and ENPP1 gene fragments covering the entire coding region and intron/exon boundaries were amplified by PCR. The amplified products were directly sequenced. Mutations were identified by comparing the sequences against the DNA of reference gene sequences. All the new mutations were confirmed in the 50 ethnically matched controls.3. Targeted gene capture with next-generation sequencing (targeted next-generation sequencing) was conducted to identify the probable causative gene mutations, which were verified by Sanger sequencing and MLPA.4. Genotype-phenotype correlation of the PHEX gene was analyzed between groups with different types of mutation and mutation locations.5. The genetic characteristics of PHEX gene in our clinical center were summarized.6. Clinical characteristics in patients with ARHR2 were analyzed.Results1. Clinical characteristics in XLH patients and genetic analysis(1) After summarizing all the clinical information from 76 patients with XLH, we found that some characteristic features such as early onset of disease (median age of onset is 18 months), skeletal deformities, short stature and dental problems were the most common. Biochemical investigations showed hypophosphatemia, renal phosphate wasting, increased ALP and FGF23 levels. Increased PTH levels were observed in 22 patients (28.9%) before treatment.(2) We performed genetic analysis by Sanger sequencing in 89 patients with hypophosphatemic rickets. PHEX gene mutations were identified in 70 patients and 26 different mutations were novle. Gene mutations were identified in 44 of 47 familial probands (93.6%), and in 26 of 42 sporadic probands (61.9%); that is, in 70 of the 89 total probands (78.7%).(3) Fifty PHEX genes mutations were identified and verified by targeted gene capture with target next-generation sequencing, Sanger and MLPA in 83 patients. The sensitivity of target next-generation sequencing was 98.5% and the specificity was 86.7%. The PHEX mutations were 3 missense,6 nonsense,12 splice site,4 deletions,4 insertions, and 21 copy number variants.(4) No phenotype-genotype correlation was detected in any comparisons.(5) In our group we have identified 212 patients with PHEX mutations. The detection rate of gene mutation in PHEX was 86.9%. Using target next-genetation sequencing, the detection rate of gene mutation in PHEX increased from 66.0% to 86.5%. Nonsense mutation was the most common types of PHEX mutation. The PHEX mutations were distributed with the preponderance of exon 18 region.2. Clinical characteristics in ARHR2 patients and genetic analysis Three patients experienced early onset of disease. Compared with XLH patients, genu valgum, bone deformity and short stature affected to a less extent, and highly and narrow arched palate were maybe the characteristic manifestations. Two ENPP1 compound heterozygous mutations and one homozygous mutation were identified by next-generation sequencing and verified by Sanger sequencing.Conclusion1. The typically clinical characteritics are presented in the Chinese XLH patients.2. Next-generation sequencing increases the detection rate of gene mutations and is likely to be an important supplement of determining genetic causes of hypophosphatemic rickets.3. Nonsense mutation is the most common type of mutation and Exon 18 in PHEX is the most frequent region of mutation in 212 XLH patients from our clinical center.4. In the present study we firstly describe the probable characteristic manifestations in ARHR2 patients.