低血磷性佝僂病的分子遺傳學(xué)研究
發(fā)布時間：2018-10-23 06:50
【摘要】：背景低血磷性佝僂病(hypophosphatemic rickets)是一組以腎臟排磷增多引起低磷血癥為特征的骨骼礦化障礙性疾病。該病主要臨床表現(xiàn)為骨骼畸形、身材矮小、牙齒異常(如牙周膿腫等)及骨痛等,是一種致殘、致畸率很高的疾病,給社會和家庭帶來很大的負擔(dān)。目前已經(jīng)明確的低血磷性佝僂病的致病基因有以下幾種：1.PHEX (Phosphate regμl ating gene with homologies to endopeptidases on the X chromosome,X染色體上內(nèi)肽酶同源的磷調(diào)節(jié)基因)突變引起X連鎖顯性遺傳性低血磷性佝僂病(XLH); 2.FGF23 (fibroblast growth factor,成纖維生長因子23)突變引起常染色體顯性遺傳性低血磷性佝僂病(ADHR); 3. DMP1 (dentin matrix protein1,牙基質(zhì)蛋白1)突變引起常染色體隱性遺傳性低血磷性佝僂病1型(ARHR1); 4. ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1,核苷酸外焦磷酸酶/磷酸二脂酶1)突變引起常染色體隱性遺傳性低血磷性佝僂病2型(ARHR2); 5. FAM20C (family with sequence similarity 20, member C,序列相似家族成員20)突變引起常染色體隱性遺傳性低血磷性佝僂病3型(ARHR3); 6. SLC34A3 (solute carrier family 34, member 3,鈉磷共轉(zhuǎn)運蛋白Ⅱ型溶質(zhì)轉(zhuǎn)運家族34)突變引起常染色體隱性遺傳性低血磷高尿鈣性佝僂病(HHRH)。其中XLH是低血磷性佝僂病最常見的類型。由于該病臨床表現(xiàn)多樣,嚴重程度不一,給診斷帶來很大困難。盡早地明確分子診斷對于臨床診治、了解疾病預(yù)后及產(chǎn)前咨詢至關(guān)重要。由于低血磷性佝僂病的致病基因有多種,針對多個基因進行傳統(tǒng)的Sanger測序,往往耗時費力,高成本低效率。本課題組既往研究顯示,低血磷性佝僂病患者突變檢出率為25.8%-43.0%,低于國外報道。由于二代測序技術(shù)具有針對性強、費用低、效率高等特點,采用二代高通量測序?qū)Φ脱仔载䞍E病患者進行遺傳學(xué)研究對于早期明確分子診斷及提高突變的檢出率顯得尤為重要�？偨Y(jié)本課題組XLH患者PHEX基因突變特點,不僅為中國人群XLH患者的分子診斷提供理論依據(jù),同時也豐富了世界低血磷性佝僂病遺傳學(xué)數(shù)據(jù)庫。目的1.分析和總結(jié)XLH患者的臨床特點。2.篩查和檢測低血磷性佝僂病患者的致病基因。3.總結(jié)XLH患者PH EX基因突變特點及分布規(guī)律。4.分析和總結(jié)ARHR2患者的臨床特點。對象和方法1.研究對象：(1)2013.6-2015.4北京協(xié)和醫(yī)院收治的89例低血磷性佝僂病患者及相關(guān)家系成員；(2)傳統(tǒng)Sanger測序未檢出致病基因突變的83例低血磷性佝僂病患者。2.研究方法：(1)總結(jié)基因確診的XLH患者的臨床特點；(2)Sanger測序檢測致病基因突變：針對基因(PHEX、FGF23、DMP1、ENPP1及SLC34A3)進行PCR擴增并測序,未報道的突變位點均在50例健康志愿者中進行驗證；(3)二代測序及可疑致病突變的驗證：對本研究及本課題組以往研究經(jīng)Sanger測序未檢出致病基因突變的低血磷性佝僂病患者進行目標區(qū)域捕獲結(jié)合二代測序；檢出的可疑致病突變,通過Sanger測序及MLPA方法(multiplex ligation-dependent probe amplification,多重連接探針擴增技術(shù))進行驗證；(4)表型和基因型相關(guān)性研究：選取基因確診的XLH患者,根據(jù)不同的突變類型及突變位置,對表型與基因型的相關(guān)性進行探討；(5)PHEX基因突變特點總結(jié)：總結(jié)本研究及本課題組以往研究確診的XLH患者PH EX基因突變類型及分布區(qū)域,探討有無熱點突變；(6)總結(jié)ARHR2患者的臨床特點及ENPP1基因突變分析。結(jié)果1.XLH患者的臨床特點及PHEX基因突變分析(1)對76例基因確診且臨床資料完整的XLH患者進行臨床資料的總結(jié)和分析。所有患者均為幼年發(fā)病,發(fā)病年齡中位數(shù)為18m,開始中性磷治療年齡中位數(shù)為4.75歲。57例(75%)患者存在下肢膝內(nèi)翻畸形。44例(64.7%)患者存在口腔疾患。大多數(shù)患者存在身材矮小,其中成年女性終身高平均值為140.9±8.2cm,成年男性終身高平均值為150.3±9.3cm。生化檢查表現(xiàn)為低磷血癥、高尿磷、TmP/GFR降低、血堿性磷酸酶(ALP)及FGF23水平升高。22例(28.9%)患者治療前全段甲狀旁腺激素(i-PTH)升高。(2)本研究首先采用傳統(tǒng)Sanger測序的方法對89例臨床診斷為低血磷性佝僂病的患者進行了包括PHEX、FGF23、DMP1、ENPP1和SLC34A3致病基因的突變檢測,共檢出PHEX基因突變70例,未發(fā)現(xiàn)其他致病基因突變。PHEX基因總體突變檢出率為78.7%,有家族史的患者檢出率為93.6%,散發(fā)性患者檢出率為61.9%。所發(fā)現(xiàn)的PHEX基因突變中,26種是國內(nèi)外尚未報道的突變。(3)對于本研究及本課題組以往研究經(jīng)傳統(tǒng)Sanger測序未檢出致病基因突變的83例低血磷性佝僂病患者,采用目標區(qū)域捕獲結(jié)合二代測序的方法檢出并驗證了PHEX基因突變50例。二代測序檢出的敏感性為98.5%,特異性為86.7%。50例PHEX基因突變中,其中剪切位點突變12例,無義突變6例,微小缺失突變4例,微小插入突變4例,錯義突變3例及大片段缺失／復(fù)制21例。(4)對本研究確診的76例XLH患者,根據(jù)不同的突變類型及突變位置進行表型與基因型的相關(guān)性分析。選取性別比、有無家族史、發(fā)病年齡、骨骼畸形、牙齒疾患、ALP、PTH.1,25(OH)2D3及FGF23作為研究表型,基因型分為截短突變和非截短突變、以編碼氨基酸一半(375位氨基酸)為界的N端和C端突變兩種。本研究的結(jié)果顯示,76例XLH患者所選取的表型與基因型之間無明顯的統(tǒng)計學(xué)相關(guān)性。(5)本研究匯總了課題組檢出的212例PHEX基因突變,PHEX基因突變檢出率為86.9%。通過采用二代測序,突變檢出率由66.0%升至86.5%。無義突變是最常見的PHEX基因突變類型。Exon 18是PHEX基因發(fā)生突變頻率最高的區(qū)域。2. ARHR2患者的臨床特點及突變分析3例ARHR2患者均為幼年起病。與XLH患者不同的是,下肢膝外翻畸形多見、骨骼畸形及身材矮小程度相對較輕、高腭弓可能是ARHR2患者的特征性表現(xiàn)。二代測序檢出并經(jīng)Sanger測序驗證,2例患者均為ENPPl基因的復(fù)合雜合突變,1例患者為純和錯義突變。結(jié)論1.通過較大樣本量的研究,提出了中國人群XLH患者具有典型的臨床特征；2.二代測序明顯提高了低血磷性佝僂病患者致病基因突變的檢出率,是對Sanger測序的重要補充；3.通過匯總本課題組檢出的PHEX基因突變,發(fā)現(xiàn)無義突變是最常見的突變類型,Exon 18是發(fā)生突變頻率最高的區(qū)域；4.首次在中國人群中發(fā)現(xiàn)了3例ARHR2家系,并提出了特征性表現(xiàn)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R681
本文編號：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R681
【參考文獻】
相關(guān)期刊論文 前4條
1 宋瑩;麻宏偉;黎芳;胡曼;任爽;宇亞芬;趙桂杰;;X-連鎖低磷性佝僂病的基因突變分析[J];中國當(dāng)代兒科雜志;2013年11期
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