Research On Clinical And Molecular Etiology Of Monogenic Hereditary Bone Disease

Part I Three Novel PHEX Gene Mutations in Four Chinese Familieswith X-linked Dominant Hypophosphatemic RicketsObjective: Hypophosphatemic rickets (HR) is a group of disorders accompanied by adefect in bone mineralization due to renal phosphate wasting, leading to hypophosphatemia.X-linked hypophosphatemia (XLH) is the most prevalent genetic form of HR, which is adominant disorder, characterized by rickets with short stature, bone pain, dental anomalies,lower extremity deformities, and radiographic evidence of rickets in children. Laboratoryindexes of the desease include low serum phosphorus levels, normal serum calcium levels,increased activity of serum alkaline phosphatases. The related gene with inactivatingmutations associated with XLH has been identified to be PHEX, which is aphosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inpresent study, a variety of PHEX mutations were identified in four Chinese families withXLH.Methods: We reported four unrelated Chinese families who exhibited typical features ofXLH. We used PCR to analyze mutations and performed gene sequencing among7patients, other normal members of four families and the control group which included250volunteers (male:125, female:125) without XLH. Laboratory and radiologicalinvestigations were carried out simultaneously.Results: Three novel mutations were found in these four families. In family2, we found aframeshift mutation c.2033dupT in exon20, which inserted a thymidine and changedcodon ACA to CAT and resulted in a subsequent change of the threonine codon to thehistidine codon at p.679and truncating at p.717. In family3, we found a nonsensemutation c.1294A>T in exon11, which changed codon AAG to TAG and transformed thelysine codon to stop codon and truncated at p.432. In family4, a missense mutationc.2192T>C in exon22was found, which caused a subsequent change of the phenylalaninecodon (TTT) to serine codon (TCT) at p.731. In addition, by detecting the DNA sequencefor the PHEX gene among13different biological species, we found that the two gene sites: p.534in family1and p.731in family4, were conserved. We did not detect above PHEXgene mutations in other normal family members and250healthy volunteers.Conclusions: We found that the PHEX gene mutations: p.T679H, p.K432X, p.F731S werenovel mutations, which were responsible for XLH in these Chinese patients. Our findingswere useful to understand the genetic basis of Chinese patients with XLH and maycontribute to the early molecular diagnosis and treatment of XLH. Part II Mutation Screening for theEXT1and EXT2Genes in ChinesePatients with Multiple OsteochondromasObjective: Multiple osteochondromas (MO), an autosomal dominant skeletal disease, ischaracterized by the presence of multiple cartilage-capped bone tumors (exostoses). Theincidence of MO has been estimated to be1-2/100000. Exostoses develop after birth andprogress gradually in size and number till skeletal maturity is achieved. In MO patients, theexostoses can appear in several joints and at multiple sites in the same joint. The two geneswith mutations that are most commonly associated with MO have been identified as EXT1and EXT2, which are Exostosin-1and Exostosin-2. In this study, a variety of EXT1andEXT2gene mutations were identified in ten Chinese families with MO.Methods: We investigated ten unrelated Chinese families involving a total of46patientswho exhibited the typical features of MO. We used PCR to analyze mutations andperformed gene sequencing among10probands, other normal members of four familiesand the control group which included250volunteers (male:125, female:125) without MO.The radiological investigation was conducted simultaneously.Results: Nine mutations were identified, five in EXT1and four in EXT2. In Family1, anonsense mutation in exon1of EXT1, p.Gln27X (c.79C>T), was present in the proband. InFamily2, a frameshift mutation in exon1of EXT1, p.Asn173LysfsX16(c.518dupA) andtwo missense mutations in exon9of EXT2, p.Gln452Lys (c.1354C>A) and p.Ile473Asn(c.1418T>A), were found in the proband. The missense mutation, p.Ile473Asn(c.1418T>A) in exon9of EXT2was present in the proband’s mother. In Family4, aframeshift mutation in exon1in EXT1, p.Leu272ArgfsX4(c.815delT), was identified inthe proband. In Family5, a nonsense mutation in exon2in EXT2, p.Gln44X (c.130C>T),was identified in the proband. In Family7, the proband harbored a nonsense mutation in exon8, p.Trp396X (c.1187G>A), in EXT2. In Family8, the proband had a nonsensemutation in exon9, p.Tyr592X (c.1776C>A), in EXT1. In Family10, the proband and hismother had the same missense mutation in exon2, p.Arg340His (c.1019G>A), in EXT1.No gene mutations were discovered in Family3, Family6, or Family9. We did not detectthe above EXT1or EXT2gene mutations in the unaffected family members or in the250healthy volunteers.Conclusions: We found that the EXT1gene mutations: p.Gln27X, p.Asn173LysfsX16,p.Leu272ArgfsX4, and the EXT2gene mutations: p.Ile473Asn, p.Gln452Lys, p.Gln44Xwere novel mutations. The EXT1gene mutations: p.Asn173LysfsX16in family2andp.Tyr592X in family8, the EXT2gene mutation: p.Gln452Lys in family2, were de novomutations. Our findings are useful for extending the mutational spectrum in EXT1andEXT2and understanding the genetic basis of MO in Chinese patients. Part III FGFR3Gene G380R Mutation in Five Chinese Families withAchondroplasiaObjective: Achondroplasia (ACH) is the most common form of short-limbed dwarfism inhuman beings, with an estimated prevalence between1/15000and1/77000. It is inheritedas an autosomal dominant trait with essentially complete penetrance, and is characterizedby short stature caused by rhizomelic shortening of the limbs, characteristic facies withfrontal bossing and midface hypoplasia, exaggerated lumbar lordosis, limitation of elbowextension, genu varum, and trident hand. The gene for ACH has been identified to be thefibroblast growth factor-3(FGFR3), which has been mapped on human chromosome4p16.3, contains19exons spanning16.5kb and encodes a membrane-spanning tyrosinekinase receptor of806amino acids. In present study, we analyzed five Chinese familieswith autosomal dominant ACH, and identified a common mutation of FGFR3gene.Methods: We investigated five unrelated Chinese families who exhibited typical featuresof ACH. We used PCR to analyze mutation and performed gene sequencing among thepatients, other normal family members and the control group which included250volunteers (male:125, female:125) without ACH. Radiological investigations were carriedout simultaneously. Results: A common missense mutation of exon9was found. It was a heterozygous G-to-Atransition at c.1138, which resulted in a glycine-to-arginine substitution at p.380. We didnot detect FGFR3gene mutations among other family members or250healthy volunteers.This missense mutation was a de novo mutation in four families.Conclusions: We found that the FGFR3gene G380R mutation was responsible for ACHin our patients. Our results supported the argument that the G380R mutation of FGFR3isthe most frequent mutation causing ACH. Our finding was useful to understand the geneticbasis of Chinese patients with ACH and may contribute to the early molecular diagnosisand treatment of ACH.