低血磷性佝偻病的分子遗传学研究
发布时间：2018-10-23 06:50
【摘要】：背景低血磷性佝偻病(hypophosphatemic rickets)是一组以肾脏排磷增多引起低磷血症为特征的骨骼矿化障碍性疾病。该病主要临床表现为骨骼畸形、身材矮小、牙齿异常(如牙周脓肿等)及骨痛等,是一种致残、致畸率很高的疾病,给社会和家庭带来很大的负担。目前已经明确的低血磷性佝偻病的致病基因有以下几种：1.PHEX (Phosphate regμl ating gene with homologies to endopeptidases on the X chromosome,X染色体上内肽酶同源的磷调节基因)突变引起X连锁显性遗传性低血磷性佝偻病(XLH); 2.FGF23 (fibroblast growth factor,成纤维生长因子23)突变引起常染色体显性遗传性低血磷性佝偻病(ADHR); 3. DMP1 (dentin matrix protein1,牙基质蛋白1)突变引起常染色体隐性遗传性低血磷性佝偻病1型(ARHR1); 4. ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1,核苷酸外焦磷酸酶/磷酸二脂酶1)突变引起常染色体隐性遗传性低血磷性佝偻病2型(ARHR2); 5. FAM20C (family with sequence similarity 20, member C,序列相似家族成员20)突变引起常染色体隐性遗传性低血磷性佝偻病3型(ARHR3); 6. SLC34A3 (solute carrier family 34, member 3,钠磷共转运蛋白Ⅱ型溶质转运家族34)突变引起常染色体隐性遗传性低血磷高尿钙性佝偻病(HHRH)。其中XLH是低血磷性佝偻病最常见的类型。由于该病临床表现多样,严重程度不一,给诊断带来很大困难。尽早地明确分子诊断对于临床诊治、了解疾病预后及产前咨询至关重要。由于低血磷性佝偻病的致病基因有多种,针对多个基因进行传统的Sanger测序,往往耗时费力,高成本低效率。本课题组既往研究显示,低血磷性佝偻病患者突变检出率为25.8%-43.0%,低于国外报道。由于二代测序技术具有针对性强、费用低、效率高等特点,采用二代高通量测序对低血磷性佝偻病患者进行遗传学研究对于早期明确分子诊断及提高突变的检出率显得尤为重要。总结本课题组XLH患者PHEX基因突变特点,不仅为中国人群XLH患者的分子诊断提供理论依据,同时也丰富了世界低血磷性佝偻病遗传学数据库。目的1.分析和总结XLH患者的临床特点。2.筛查和检测低血磷性佝偻病患者的致病基因。3.总结XLH患者PH EX基因突变特点及分布规律。4.分析和总结ARHR2患者的临床特点。对象和方法1.研究对象：(1)2013.6-2015.4北京协和医院收治的89例低血磷性佝偻病患者及相关家系成员；(2)传统Sanger测序未检出致病基因突变的83例低血磷性佝偻病患者。2.研究方法：(1)总结基因确诊的XLH患者的临床特点；(2)Sanger测序检测致病基因突变：针对基因(PHEX、FGF23、DMP1、ENPP1及SLC34A3)进行PCR扩增并测序,未报道的突变位点均在50例健康志愿者中进行验证；(3)二代测序及可疑致病突变的验证：对本研究及本课题组以往研究经Sanger测序未检出致病基因突变的低血磷性佝偻病患者进行目标区域捕获结合二代测序；检出的可疑致病突变,通过Sanger测序及MLPA方法(multiplex ligation-dependent probe amplification,多重连接探针扩增技术)进行验证；(4)表型和基因型相关性研究：选取基因确诊的XLH患者,根据不同的突变类型及突变位置,对表型与基因型的相关性进行探讨；(5)PHEX基因突变特点总结：总结本研究及本课题组以往研究确诊的XLH患者PH EX基因突变类型及分布区域,探讨有无热点突变；(6)总结ARHR2患者的临床特点及ENPP1基因突变分析。结果1.XLH患者的临床特点及PHEX基因突变分析(1)对76例基因确诊且临床资料完整的XLH患者进行临床资料的总结和分析。所有患者均为幼年发病,发病年龄中位数为18m,开始中性磷治疗年龄中位数为4.75岁。57例(75%)患者存在下肢膝内翻畸形。44例(64.7%)患者存在口腔疾患。大多数患者存在身材矮小,其中成年女性终身高平均值为140.9±8.2cm,成年男性终身高平均值为150.3±9.3cm。生化检查表现为低磷血症、高尿磷、TmP/GFR降低、血碱性磷酸酶(ALP)及FGF23水平升高。22例(28.9%)患者治疗前全段甲状旁腺激素(i-PTH)升高。(2)本研究首先采用传统Sanger测序的方法对89例临床诊断为低血磷性佝偻病的患者进行了包括PHEX、FGF23、DMP1、ENPP1和SLC34A3致病基因的突变检测,共检出PHEX基因突变70例,未发现其他致病基因突变。PHEX基因总体突变检出率为78.7%,有家族史的患者检出率为93.6%,散发性患者检出率为61.9%。所发现的PHEX基因突变中,26种是国内外尚未报道的突变。(3)对于本研究及本课题组以往研究经传统Sanger测序未检出致病基因突变的83例低血磷性佝偻病患者,采用目标区域捕获结合二代测序的方法检出并验证了PHEX基因突变50例。二代测序检出的敏感性为98.5%,特异性为86.7%。50例PHEX基因突变中,其中剪切位点突变12例,无义突变6例,微小缺失突变4例,微小插入突变4例,错义突变3例及大片段缺失／复制21例。(4)对本研究确诊的76例XLH患者,根据不同的突变类型及突变位置进行表型与基因型的相关性分析。选取性别比、有无家族史、发病年龄、骨骼畸形、牙齿疾患、ALP、PTH.1,25(OH)2D3及FGF23作为研究表型,基因型分为截短突变和非截短突变、以编码氨基酸一半(375位氨基酸)为界的N端和C端突变两种。本研究的结果显示,76例XLH患者所选取的表型与基因型之间无明显的统计学相关性。(5)本研究汇总了课题组检出的212例PHEX基因突变,PHEX基因突变检出率为86.9%。通过采用二代测序,突变检出率由66.0%升至86.5%。无义突变是最常见的PHEX基因突变类型。Exon 18是PHEX基因发生突变频率最高的区域。2. ARHR2患者的临床特点及突变分析3例ARHR2患者均为幼年起病。与XLH患者不同的是,下肢膝外翻畸形多见、骨骼畸形及身材矮小程度相对较轻、高腭弓可能是ARHR2患者的特征性表现。二代测序检出并经Sanger测序验证,2例患者均为ENPPl基因的复合杂合突变,1例患者为纯和错义突变。结论1.通过较大样本量的研究,提出了中国人群XLH患者具有典型的临床特征；2.二代测序明显提高了低血磷性佝偻病患者致病基因突变的检出率,是对Sanger测序的重要补充；3.通过汇总本课题组检出的PHEX基因突变,发现无义突变是最常见的突变类型,Exon 18是发生突变频率最高的区域；4.首次在中国人群中发现了3例ARHR2家系,并提出了特征性表现。
[Abstract]:Background Hypophosphatemia is a group of bone mineral disorders characterized by hypophosphatemia caused by increased excretion of phosphorus in the kidneys. The main clinical manifestation of this disease is bone deformity, short stature, abnormal tooth (such as periodontal abscess, etc.) and bone pain. It is a kind of disease with high disability and high incidence rate, which brings great burden to society and family. There are several pathogenic genes that have been identified at present: 1. PHEX (Physalis reg. l ating gene with Immunologies to endopeptidases on the X chromomome, X chromosome) mutation causes X-linked dominant low blood phosphorus resistance (XLH); 2. FGF23 (fibroblast growth factor, The mutation of fibroblast growth factor (23) causes autosomal dominant hypophosphatemia (ADHR); 3. DMP1 (dentinmatrix protein1, tooth-based protein 1) mutation causes autosomal recessive hypophosphatemia type 1 (ARHR1); 4. The mutations of ENPP1 (econovaco pyrovatase/ Lipodista1, extracellular pyrophosphatase/ dilipoxygenase 1) resulted in autosomal recessive hypophosphatemia type 2 (ARHR2); 5. FAM20C (family with sequence similarity 20, member C, sequence similarity family member 20) mutation causes autosomal recessive hypophosphatemia type 3 (ARHR3); 6. SLC34A3 (solute carrier family 34, member 3, sodium-phosphorus co-transporter type II solute transport family 34) mutations cause autosomal recessive hypophosphatemia hypercalciuria (HSLA). where XLH is the most common type of hypophosphatemia. Because the clinical manifestation of the disease is diverse, the severity is not one, it brings great difficulty to the diagnosis. Early identification of molecular diagnosis is critical to clinical diagnosis and treatment, understanding of disease prognosis and pre-production counseling. Due to the multiple pathogenic genes of the low blood phosphorus resistance gene, the traditional SLAMP sequencing for a plurality of genes is often time consuming and labor intensive and high in cost and low in efficiency. The previous study showed that the rate of mutation in patients with hypophosphatemia was 25.8%-43.0%, which was lower than that of foreign reports. Because the second-generation sequencing technology has the characteristics of strong pertinence, low cost, high efficiency and the like, the second-generation high-throughput sequencing is adopted to carry out genetic research on low-blood-phosphorus-resistant patients, and the detection rate of early definite molecular diagnosis and mutation detection is particularly important. To sum up the characteristics of PHX gene mutation in XLH patients of our research group, it not only provides theoretical basis for molecular diagnosis of XLH patients in Chinese population, but also enriches the low-blood-phosphorus cytogenetic database in the world. Purpose 1. To analyze and summarize the clinical features of XLH patients. Screening and testing of pathogenic genes in patients with hypophosphatemia. To summarize the characteristics and distribution of PH EX gene mutation in XLH patients. To analyze and summarize the clinical characteristics of ARHR2 patients. Object and Method 1. Study subjects: (1) In 2013. 6-2015, 89 patients with hypophosphatemia and related family members were admitted to Peking Union Medical College Hospital in 2013. (2) 83 patients with low blood phosphorus resistance were not detected by traditional SMR sequencing. Methods: (1) To summarize the clinical characteristics of XLH patients diagnosed by gene; (2) sequencing and detection of pathogenic gene mutation: target genes (PHEX, FGF23, DMP1, ENPP1 and SLC34A3) were amplified and sequenced. The unreported mutation sites were verified in 50 healthy volunteers. (3) verification of the second generation sequencing and the suspicious pathogenic mutation: the present study and the previous research of the current research group have carried out target area capture and second generation sequencing of the low blood-phosphorus hepatitis B patient with no pathogenic gene mutation detected by Sanger sequencing; the detected suspicious pathogenic mutation, (4) Phenotypic and genotypic correlation: The correlation between phenotype and genotype was discussed based on different mutation types and mutation sites. (5) Summary of the characteristics of PHX gene mutation: To sum up the mutation types and distribution regions of PH EX gene mutation in XLH patients diagnosed by this study and the previous research group, explore whether there is hot spot mutation, and (6) summarize the clinical features of ARHR2 patients and the analysis of ENPP1 gene mutation. Results The clinical features of XLH patients and the analysis of PHX gene mutation (1) summarize and analyze the clinical data of 76 cases of XLH patients diagnosed with gene diagnosis and complete clinical data. All patients were young, the median age was 18m, the median age of onset of neutropenia was 4. 75 years. 57 (75%) patients had lower limb knee varus. 44 patients (64. 7%) had oral disorders. In most patients, short stature was found, among which adult female lifelong high average was 140. 9/ 8. 2cm, and adult male lifelong high average was 150. 3/ 9. 3cm. Biochemical examination showed hypophosphatemia, high urinary phosphorus, TmP/ GFR decreased, serum alkaline phosphatase (ALP) and FGF23 levels increased. 22 (28. 9%) patients had increased total parathyroid hormone (i-PTH) before treatment. (2) In this study, 89 patients with hypophosphatemia were detected by traditional Sanger sequencing. The mutations of PHEX, FGF23, DMP1, ENPP1 and SLC34A3 genes were detected in 89 patients with hypophosphatemia, and 70 cases of PHEX gene mutation were detected, and no other pathogenic gene mutation was found. The overall mutation rate of PHEX gene was 72.7%, the positive rate of patients with family history was 93.6% and 61.9% respectively. Of the identified PHEX gene mutations, 26 were not reported at home and abroad. (3) In this study and the previous research group, 83 cases with low blood phosphorus resistance were studied by using the target region capture and second-generation sequencing, and 50 cases of PHEX gene mutation were detected by using the target region capture and the second-generation sequencing method. The sensitivity of the second-generation sequencing was 96.5%, the specificity was 86.7%. In the 50 cases of PHEX gene mutation, there were 12 cases with mutation site mutation, 6 cases without sense mutation, 4 cases of microdeletion mutation, 4 cases of microinsertion mutation, 3 cases of missense mutation and 21 cases of deletion/ duplication of large segment. (4) The correlation between phenotype and genotype was analyzed according to different mutation types and mutation sites in 76 patients with XLH confirmed by this study. There are two types of N-terminal and C-terminal mutations which encode amino acid half (375-bit amino acid) as the research phenotype, including family history, age of onset, bone deformity, tooth disease, ALP, PTH. 1, 25 (OH) 2D3 and FGF23 as the study phenotype. The results of this study showed no significant statistical correlation between the phenotype selected by 76 patients with XLH and the genotype. (5) In this study, 212 PHEX gene mutations were detected by our group, and the positive rate of PHX gene mutation was 86.9%. By using the second-generation sequencing, the mutation rate was increased from 66. 0% to 86.5%. Undefined mutations are the most common types of PHX gene mutations. Exon 18 is the highest frequency region of the PHEX gene. The clinical features and mutations of ARHR2 patients were juvenile onset in 3 ARHR2 patients. In contrast to XLH patients, the degree of valgus deformity of the lower extremities is seen, and the degree of bone deformity and short stature is relatively light, and the high aortic arch may be the characteristic expression of ARHR2 patients. The second-generation sequencing showed that 2 patients had complex heterozygous mutation of ENPPl gene, and 1 patient had pure and missense mutation. Conclusion 1. According to the study of large sample size, the typical clinical features of XLH patients in Chinese population are presented. Second-generation sequencing significantly increased the detection rate of pathogenic gene mutation in patients with hypophosphatemia, and was an important supplement to SMDV sequencing. By summarizing the PHEX gene mutation detected by our research group, it was found that no-sense mutation was the most common mutation type, and Exon 18 was the highest frequency region; 4. Three ARHR2 families were found in Chinese population for the first time, and characteristic expression was put forward.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R681
本文编号：2288378
[Abstract]:Background Hypophosphatemia is a group of bone mineral disorders characterized by hypophosphatemia caused by increased excretion of phosphorus in the kidneys. The main clinical manifestation of this disease is bone deformity, short stature, abnormal tooth (such as periodontal abscess, etc.) and bone pain. It is a kind of disease with high disability and high incidence rate, which brings great burden to society and family. There are several pathogenic genes that have been identified at present: 1. PHEX (Physalis reg. l ating gene with Immunologies to endopeptidases on the X chromomome, X chromosome) mutation causes X-linked dominant low blood phosphorus resistance (XLH); 2. FGF23 (fibroblast growth factor, The mutation of fibroblast growth factor (23) causes autosomal dominant hypophosphatemia (ADHR); 3. DMP1 (dentinmatrix protein1, tooth-based protein 1) mutation causes autosomal recessive hypophosphatemia type 1 (ARHR1); 4. The mutations of ENPP1 (econovaco pyrovatase/ Lipodista1, extracellular pyrophosphatase/ dilipoxygenase 1) resulted in autosomal recessive hypophosphatemia type 2 (ARHR2); 5. FAM20C (family with sequence similarity 20, member C, sequence similarity family member 20) mutation causes autosomal recessive hypophosphatemia type 3 (ARHR3); 6. SLC34A3 (solute carrier family 34, member 3, sodium-phosphorus co-transporter type II solute transport family 34) mutations cause autosomal recessive hypophosphatemia hypercalciuria (HSLA). where XLH is the most common type of hypophosphatemia. Because the clinical manifestation of the disease is diverse, the severity is not one, it brings great difficulty to the diagnosis. Early identification of molecular diagnosis is critical to clinical diagnosis and treatment, understanding of disease prognosis and pre-production counseling. Due to the multiple pathogenic genes of the low blood phosphorus resistance gene, the traditional SLAMP sequencing for a plurality of genes is often time consuming and labor intensive and high in cost and low in efficiency. The previous study showed that the rate of mutation in patients with hypophosphatemia was 25.8%-43.0%, which was lower than that of foreign reports. Because the second-generation sequencing technology has the characteristics of strong pertinence, low cost, high efficiency and the like, the second-generation high-throughput sequencing is adopted to carry out genetic research on low-blood-phosphorus-resistant patients, and the detection rate of early definite molecular diagnosis and mutation detection is particularly important. To sum up the characteristics of PHX gene mutation in XLH patients of our research group, it not only provides theoretical basis for molecular diagnosis of XLH patients in Chinese population, but also enriches the low-blood-phosphorus cytogenetic database in the world. Purpose 1. To analyze and summarize the clinical features of XLH patients. Screening and testing of pathogenic genes in patients with hypophosphatemia. To summarize the characteristics and distribution of PH EX gene mutation in XLH patients. To analyze and summarize the clinical characteristics of ARHR2 patients. Object and Method 1. Study subjects: (1) In 2013. 6-2015, 89 patients with hypophosphatemia and related family members were admitted to Peking Union Medical College Hospital in 2013. (2) 83 patients with low blood phosphorus resistance were not detected by traditional SMR sequencing. Methods: (1) To summarize the clinical characteristics of XLH patients diagnosed by gene; (2) sequencing and detection of pathogenic gene mutation: target genes (PHEX, FGF23, DMP1, ENPP1 and SLC34A3) were amplified and sequenced. The unreported mutation sites were verified in 50 healthy volunteers. (3) verification of the second generation sequencing and the suspicious pathogenic mutation: the present study and the previous research of the current research group have carried out target area capture and second generation sequencing of the low blood-phosphorus hepatitis B patient with no pathogenic gene mutation detected by Sanger sequencing; the detected suspicious pathogenic mutation, (4) Phenotypic and genotypic correlation: The correlation between phenotype and genotype was discussed based on different mutation types and mutation sites. (5) Summary of the characteristics of PHX gene mutation: To sum up the mutation types and distribution regions of PH EX gene mutation in XLH patients diagnosed by this study and the previous research group, explore whether there is hot spot mutation, and (6) summarize the clinical features of ARHR2 patients and the analysis of ENPP1 gene mutation. Results The clinical features of XLH patients and the analysis of PHX gene mutation (1) summarize and analyze the clinical data of 76 cases of XLH patients diagnosed with gene diagnosis and complete clinical data. All patients were young, the median age was 18m, the median age of onset of neutropenia was 4. 75 years. 57 (75%) patients had lower limb knee varus. 44 patients (64. 7%) had oral disorders. In most patients, short stature was found, among which adult female lifelong high average was 140. 9/ 8. 2cm, and adult male lifelong high average was 150. 3/ 9. 3cm. Biochemical examination showed hypophosphatemia, high urinary phosphorus, TmP/ GFR decreased, serum alkaline phosphatase (ALP) and FGF23 levels increased. 22 (28. 9%) patients had increased total parathyroid hormone (i-PTH) before treatment. (2) In this study, 89 patients with hypophosphatemia were detected by traditional Sanger sequencing. The mutations of PHEX, FGF23, DMP1, ENPP1 and SLC34A3 genes were detected in 89 patients with hypophosphatemia, and 70 cases of PHEX gene mutation were detected, and no other pathogenic gene mutation was found. The overall mutation rate of PHEX gene was 72.7%, the positive rate of patients with family history was 93.6% and 61.9% respectively. Of the identified PHEX gene mutations, 26 were not reported at home and abroad. (3) In this study and the previous research group, 83 cases with low blood phosphorus resistance were studied by using the target region capture and second-generation sequencing, and 50 cases of PHEX gene mutation were detected by using the target region capture and the second-generation sequencing method. The sensitivity of the second-generation sequencing was 96.5%, the specificity was 86.7%. In the 50 cases of PHEX gene mutation, there were 12 cases with mutation site mutation, 6 cases without sense mutation, 4 cases of microdeletion mutation, 4 cases of microinsertion mutation, 3 cases of missense mutation and 21 cases of deletion/ duplication of large segment. (4) The correlation between phenotype and genotype was analyzed according to different mutation types and mutation sites in 76 patients with XLH confirmed by this study. There are two types of N-terminal and C-terminal mutations which encode amino acid half (375-bit amino acid) as the research phenotype, including family history, age of onset, bone deformity, tooth disease, ALP, PTH. 1, 25 (OH) 2D3 and FGF23 as the study phenotype. The results of this study showed no significant statistical correlation between the phenotype selected by 76 patients with XLH and the genotype. (5) In this study, 212 PHEX gene mutations were detected by our group, and the positive rate of PHX gene mutation was 86.9%. By using the second-generation sequencing, the mutation rate was increased from 66. 0% to 86.5%. Undefined mutations are the most common types of PHX gene mutations. Exon 18 is the highest frequency region of the PHEX gene. The clinical features and mutations of ARHR2 patients were juvenile onset in 3 ARHR2 patients. In contrast to XLH patients, the degree of valgus deformity of the lower extremities is seen, and the degree of bone deformity and short stature is relatively light, and the high aortic arch may be the characteristic expression of ARHR2 patients. The second-generation sequencing showed that 2 patients had complex heterozygous mutation of ENPPl gene, and 1 patient had pure and missense mutation. Conclusion 1. According to the study of large sample size, the typical clinical features of XLH patients in Chinese population are presented. Second-generation sequencing significantly increased the detection rate of pathogenic gene mutation in patients with hypophosphatemia, and was an important supplement to SMDV sequencing. By summarizing the PHEX gene mutation detected by our research group, it was found that no-sense mutation was the most common mutation type, and Exon 18 was the highest frequency region; 4. Three ARHR2 families were found in Chinese population for the first time, and characteristic expression was put forward.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R681
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