Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.     

Two Novel Missense Mutations in the Connexin 26 Gene in Turkish Patients with Nonsyndromic Hearing Loss

Most nonsyndromic hearing losses are caused by mutations in the GJB2 gene, and studies have revealed that the forms and frequencies of these mutations are largely dependent on ethnic origin. In the present study, we aimed to characterize the mutation profiles of 151 patients with hearing loss in Turkey. The entire coding region of the GJB2 was directly sequenced in all patients. We found 35 (23.2%) individuals carrying GJB2 mutations. Seven different mutations were identified, five of which were previously known (35delG, delE120, R184P, M163V, L90P), the remaining two being novel variants (M34V, L205V). The most common mutation was 35delG followed by delE120. The 35delG mutation was homozygous in 22 cases (14.5%) and heterozygous in 4 cases (2.6%). Compound heterozygosity for 35delG was also observed. The delE120 mutation was found in three patients in homozygous form. A homozygous L90P and heterozygous mutations M163V and M34V were found in single cases.     

Identification of Novel Mutations in the Human HPRT Gene

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch–Nyhan syndrome (LNS) or Lesch–Nyhan variants (LNVs). We report three novel independent mutations in the coding region of HPRT gene: exon 3: c.141delA, p.D47fs53X; exon 5: c.400G>A, p.E134K; exon 7: c.499A>G, p.R167G from three LNS affected male patients.     

46,XY女性性反转患者SRY基因的两个新突变类型

Y染色体上的性别决定区域——SRY基因作为睾丸决定因子,可以调控男性性别发育过程。SRY基因是一种转录因子,属于带有高迁移率族蛋白家族,该家族成员包含能与DNA结合的HMG盒基序。已知SRY基因的缺失和点突变是造成XY女性性反转的病因之一。通过筛查10位中国46,XY女性性反转病人SRY基因的开放阅读框区域,探寻新的突变类型。用标准方法从外周血中抽提gDNA,通过聚合酶链式反应扩增SRY基因中部的609bp的DNA片段。扩增后的PCR片段被克隆到pUCm-T载体中,在ABI377-3自动测序仪上完成测序。运用限制性内切酶酶切分析的方法验证DNA测序的结果。结果表明,在两个患者的SRY基因中分别发现了新的核苷酸点突变,并都导致氨基酸替代。一个突变发生在SRY基因的5’端HMG盒外的核苷酸第113位腺嘌呤(A)被鸟嘌呤(G)取代,并导致谷氨酸被甘氨酸替换;另一个突变是第387位核苷酸发生T被A替换,该突变引起第129位的酪氨酸变成终止密码,她父亲的SRY序列被证明是正常的野生型。通过查询文献和人类基因突变数据库(HGMD),这两个突变都是以前未见报道过的新型SRY基因突变,并使因核苷酸替换引起SRY基因突变总数增加到45。     

Novel Vitamin D Receptor Mutations in Hereditary Vitamin D Resistant Rickets in Chinese

Hereditary 1, 25-dihydroxyvitamin D-resistant rickets (HVDRR), a rare recessive disease, is caused by mutation in the VDR gene encoding the vitamin D receptor leading to the resistance to vitamin D. We described a female toddler with initial presentation of leg tenderness and clinical features of HVDRR including severe rickets, hypocalcemia and hypophosphatemia without alopecia. Genetic analysis revealed novel compound heterozygous mutations of p.M4I and p.H229Q in patient’s VDR gene. In cis p.M4I with FOKI-F eliminated both translation start sites of the VDR protein. The p.H229Q VDR exhibited significantly reduced VDR transactivation activity with intact dimerization with RXR. Our report expanded the mutation spectrum of HVDRR, and provided the first case of a benign variant p.M4I plus a common p.M1T polymorphism leading to a pathogenic allele.     

Identification of Two Novel Mutations in PKHD1 Gene from Two Families with Polycystic Kidney Disease by Whole Exome Sequencing

Background Polycystic kidney disease (PKD) is an autosomal recessive disorder resulting from mutations in the PKHD1 gene on chromosome 6 (6p12), a large gene spanning 470 kb of genomic DNA.ObjectiveThe aim of the present study was to report newly identified mutations in the PKHD1 gene in two Iranian families with PKD.Materials and Methods Genetic alterations of a 3-month-old boy and a 27-year-old girl with PKD were evaluated using whole-exome sequencing. The PCR direct sequencing was performed to analyse the co-segregation of the variants with the disease in the family. Finally, the molecular function of the identified novel mutations was evaluated by in silico study. ResultsIn the 3 month-old boy, a novel homozygous frameshift mutation was detected in the PKHD1 gene, which can cause PKD. Moreover, we identified three novel heterozygous missense mutations in ATIC, VPS13B, and TP53RK genes. In the 27-year-old woman, with two recurrent abortions history and two infant mortalities at early weeks due to metabolic and/or renal disease, we detected a novel missense mutation on PKHD1 gene and a novel mutation in ETFDH gene.Conclusion In general, we have identified two novel mutations in the PKHD1 gene. These molecular findings can help accurately correlate genotype and phenotype in families with such disease in order to reduce patient births through preoperative genetic diagnosis or better management of disorders.     

Loss-of-Function ENPP1 Mutations Cause Both Generalized Arterial Calcification of Infancy and Autosomal-Recessive Hypophosphatemic Rickets

The analysis of rare genetic disorders affecting phosphate homeostasis led to the identification of several proteins that are essential for the renal regulation of phosphate homeostasis; for example, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D synthesis. Here, we report presumable loss-of-function mutations in the ENPP1 gene (ectonucleotide pyrophosphatase/phosphodiesterase) in members of four families affected with hypophosphatemic rickets. We provide evidence for the conclusion that ENPP1 is the fourth gene—in addition to PHEX, FGF23, and DMP1—that, if mutated, causes hypophosphatemic rickets resulting from elevated FGF23 levels. Surprisingly, ENPP1 loss-of-function mutations have previously been described in generalized arterial calcification of infancy, suggesting an as yet elusive mechanism that balances arterial calcification with bone mineralization.     

MEPE, the Gene Encoding a Tumor-Secreted Protein in Oncogenic Hypophosphatemic Osteomalacia, Is Expressed in Bone

The MEPE (matrix extracellular phosphoglycoprotein) gene is a strong candidate for the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia (OHO). X-linked hypophosphatemia (XLH) is phenotypically similar to OHO and results from mutations in PHEX, a putative metallopeptidase believed to process a factor(s) regulating bone mineralization and renal phosphate reabsorption. Here we report the isolation of the murine homologue of MEPE, from a bone cDNA library, that encodes a protein of 433 amino acids, 92 amino acids shorter than human MEPE. Mepe, like Phex, is expressed by fully differentiated osteoblasts and down-regulated by 1,25-(OH)2D3. In contrast to Phex, Mepe expression is markedly increased during osteoblast-mediated matrix mineralization. Greater than normal Mepe mRNA levels were observed in bone and osteoblasts derived from Hyp mice, the murine homologue of human XLH. Our data provide the first evidence that MEPE/Mepe is expressed by osteoblasts in association with mineralization.     

Novel HRPT2/CDC73 Gene Mutations and Loss of Expression of Parafibromin in Chinese Patients with Clinically Sporadic Parathyroid Carcinomas

Objective

It is widely recognized that the diagnosis of parathyroid carcinoma (PC) is often difficult because of the overlap of characteristics between malignant and benign parathyroid tumors, especially at an early stage. Based on the identification of tumor suppressor gene HRPT2/CDC73 and its association with hereditary and sporadic PC, screening of gene mutations and detection of parafibromin immunoreactivity have been suggested as diagnostic instruments of PC in Whites. There is little information about HRPT2/CDC73 mutations and its corresponding protein expression in patients with sporadic PC in Chinese population, and the long-term follow-up data is scarce.

Methods

Paraffin-embedded tissues were obtained from 13 patients with PC, 13 patients with parathyroid adenoma (PA) and 7 patients with parathyroid hyperplasia(PH), and 6 normal parathyroid (NP) tissues as controls. Peripheral blood from 11 patients with PC was collected. PCR products using Genomic DNA extracted from tumor tissues or blood as template was sequenced for HRPT2/CDC73 gene. Expression of parafibromin in tumor tissues was evaluated by immunohistochemical analysis.

Results

Six mutations in 6 of 13 patients with PC were identified, with three being novel. Four of them were germ-line mutations. Patients with mutations were susceptible to recurrence of the PC. Complete (8/13, 61.5%) or partial (5/13, 38.5%) loss of parafibromin expression was observed in PC tissues. All of tissue samples from normal parathyroid or benign parathyroid tumors displayed positive immunostaining of parafibromin except one adenoma.

Conclusions

The present study supplies information on the mutations and protein expression of HRPT2/CDC73 gene and phenotypes of parathyroid carcinoma in Chinese population. And the expanded mutation database of this gene may benefit patients in the diagnosis and treatment of this disease.     

Novel Mutations Found in Two Genes of Thai Patients with Isolated Methylmalonic Acidemia

Molecular genetic analysis of three patients diagnosed with isolated methylmalonic acidemia (MMA) revealed that one was mut ⁰ MMA, with a mutation in the MUT gene encoding the l-methylmalonyl-CoA mutase (MCM), and two were cblB MMA, with mutations in the MMAB gene required for synthesizing the deoxyadenosylcobalamin cofactor of MCM. The mut ⁰ patient was homozygous for a novel nonsense mutation in MUT, p.R31X (c.167C → T), and heterozygous for three previously described polymorphisms, p.K212K (c.712A → G), p.H532R (c.1671A → G), and p.V671I (c.2087G → A). The new MMAB mutation, p.E152X (c.454G → T), was found to be homozygous in one cblB patient and heterozygous in the other patient, who also had four intron polymorphisms in this gene.     

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.     

Novel Mutations in the Human HPRT Gene

Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT), gives rise to Lesch-Nyhan Syndrome (LNS) or HPRT-related gout. Here, we report five novel independent mutations in the coding region of the HPRT gene from five unrelated male patients manifesting different clinical phenotypes associated with LNS: exon 2: c.133A > G, p.45R > G; c.35A > C, p.12D > A; c.88delG; exon 7: c.530A > T, p.177D > V; and c.318 + 1G > C: IVS3 + 1G > C splice site mutation.     

中国北方人苯丙氨酸羟化酶基因外显子7内新突变的鉴定

应用ＰＣＲ－单链构象多态性分析及ＤＮＡ直接测序,对４５例中国北方苯丙酮尿症（ＰＫＵ）患者苯丙氨酸羟化酶（ＰＡＨ）基因外显子７内突变进行了鉴定。共检出６种错义突变及一种静止突变：Ｒ２４３Ｑ．Ｒ４１Ｈ,Ｇ２４７Ｖ．Ｌ２４９Ｈ．Ｐ２５４Ｉ．Ｇ２５７Ｖ和Ｖ２４５Ｖ。经与国际ＰＡＨ基因突变数据库比较,确认Ｇ２５７Ｖ．Ｐ２５４Ｉ和Ｌ２４９Ｈ为国际上首次发现的突变。结果揭示,中国人与其他种族及中国北方与南方人群ＰＡＨ突变特点不同。明确了中国北方人群中ＰＡＨ基因外显子７基因突变分布,有助于提高ＰＫＵ的基因诊断率,对基因的起源、进化研究有参考价值     

Three Novel Mutations in Porphobilinogen Deaminase Gene Identified in Russian Patients with Acute Intermittent Porphyria

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and +5 (IVS7 2–5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position –1 of intron 5 (IVS5 as –1 G–C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mutant mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.     

Novel Mutations of RPGR in Chinese Retinitis Pigmentosa Patients and the Genotype-Phenotype Correlation

X-linked Retinitis Pigmentosa (XLRP) accounts for 10–20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70–75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.     

Rickets and Osteomalacia in the Glasgow Pakistani Community, 1961-71

The prevalence of vitamin D deficiency was reassessed in April and May 1971, 10 years after the discovery of widespread late rickets and osteomalacia in the Glasgow Pakistani community. Evidence of vitamin D deficiency was found in 28 out of 115 adults and children examined (24%). Children at the age of puberty were most severely affected by rickets, whereas most infants and younger children in the survey were protected by vitamin D supplements. Mild biochemical osteomalacia was common in Pakistani women.A total of 21 Pakistani and Indian children with rickets were admitted to Glasgow hospitals during 1968-70. These comprised 10 children with infantile rickets and 11 with late rickets. Four of the latter group required osteotomy for severe rachitic deformity.Late rickets and osteomalacia in Pakistani and Indian immigrants are not primarily due to nutritional deficiency of vitamin D, though the high phytate content of their diet may be of aetiological importance. A combination of environmental, social, and endogenous factors, the relative importance of which is not at present clear, may also be involved. Advice on the prophylaxis of vitamin D deficiency should be given to all Pakistani and Indian communities in the United Kingdom.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

Identification of BRCA1/2 Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

Background

Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China.

Methodology/Principal Findings

A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated.

Conclusion

In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.