全基因組外顯子測序發(fā)現(xiàn)點狀掌跖角化病致病基因COL14A1
發(fā)布時間：2018-08-12 16:47
【摘要】：研究背景點狀掌跖角化病(Punctate palmoplantar keratoderma; PPPK; OMIM:148600)是一種不規(guī)則分布于掌跖部位的大量的角化性斑塊為特征的罕見的常染色體顯性遺傳性疾病。Buschke和Fischer在1910年第一次描述此病,Brauer在1913年肯定了它的遺傳性。因此,此病又被命名為點狀掌跖角化病Buschke-Fischer-Brauer型。其典型臨床表現(xiàn)為雙手掌與足跖部位的圓形或卵圓形、較硬的黃色或淡黃色角質(zhì)丘疹,若去除角質(zhì)丘疹后,局部可留有火山口樣的凹坑。可以伴有甲變形。典型的點狀皮損可以融合成塊,可能與遭受連續(xù)性高壓有關(guān),一般足跖部位皮疹比掌部較大。少數(shù)患者也可不僅累及掌跖部位,其他部位如膝部、手足背部、肘部也可受累。PPPK先證者發(fā)病年齡是一般是從12-33歲,有報道PPPK與乳腺癌、前列腺癌、胰腺癌、結(jié)腸惡性腺瘤、轉(zhuǎn)移性肺非小細(xì)胞癌有關(guān)。 Martinez-Mir等在3個PPPK家系中進行全基因組連鎖分析,定位在15q22-15q24.1上D15S534和D15S818之間9.98cM區(qū)域。本實驗室張等在2個PPPK家系中進行全基因組連鎖分析,定位在8q24.13-8q24.21上D8S1804和D8S1720之間9.02cM區(qū)域,此結(jié)果提示PPPK有遺傳異質(zhì)性。本實驗室高等后來在另一個中國大家系中進行精心定位,將PPPK致病基因定位在D15S651和D15S988之間5.06cM區(qū)域。El Amri I等肯定了在染色體15q的可疑基因座的遺傳因素,此區(qū)域在D15S987和D15S153之間。但PPPK的致病基因至今尚未發(fā)現(xiàn)。全基因組外顯子測序(靶向外顯子組捕獲)是一種經(jīng)濟的測序方法,其主要是對人類基因組的編碼區(qū)進行測序從而探測罕見和常見疾病相關(guān)的新基因。2009年來,國內(nèi)外已經(jīng)成功利用外顯子捕獲系統(tǒng)和測序系統(tǒng)發(fā)現(xiàn)或驗證了一些單基因疾病的致病基因,其中有一些是用全基因組外顯子測序與連鎖定位相結(jié)合發(fā)現(xiàn)單基因病的致病基因。 目的(1)全基因組外顯子測序結(jié)合全基因組連鎖分析搜尋PPPK的致病基因,為將來的基因診斷與基因治療奠定基礎(chǔ)。(2)總結(jié)中國漢族PPPK家系的臨床和遺傳特征。 方法(1)鑒于我們原定位到8q24.13-8q24.21上的一個家系患病人數(shù)較多且我們收集的血樣本數(shù)較多,從中挑選了癥狀比較典型的4個病例(Ⅰ1,Ⅱ2,Ⅲ2和Ⅲ5)和2個家系內(nèi)正常對照(Ⅰ2和Ⅲ6)進行全基因組外顯子測序。(2)用Sanger sequencing的方法對我們用全基因組外顯子測序所得到的候選基因的第37號外顯子及外顯子、內(nèi)含子交界區(qū)域在家系內(nèi)另外4個患者和6個對照進行家系內(nèi)驗證測序,對此候選基因所有的47個外顯子和啟動子以及外顯子、內(nèi)含子交界區(qū)域進行家系外突變檢測。(3)通過CBMdisc(中國生物醫(yī)學(xué)文獻(xiàn))和Pubmed進行系統(tǒng)文獻(xiàn)回顧,檢索1991年至今已有文獻(xiàn)報道的9個中國漢族PPPK家系,總結(jié)中國漢族PPPK的遺傳流行病學(xué)及臨床特征。統(tǒng)計學(xué)分析用SPSS13.0。 結(jié)果(1)通過逐步濾過法濾過了dbSNP129、eight HapMap、1000Genomes、家系內(nèi)對照后,4個病例共有的新變異點有21個。其中COL14A1基因的第37號外顯子上有一個錯義突變點(c.4505C-T [p.Pro1502Leu]),此突變點位于原定位的連鎖區(qū)域8q24.13-8q24.21的上游3.4Mb的位置。我們選擇COL14A1基因作為PPPK的最重要的候選致病基因。(2)通過Sanger sequencing的方法在此家系的其他成員進行測序,在其他4個病例中也發(fā)現(xiàn)了同樣的錯義突變,在剩余的5個對照中(Ⅲ18除外)未發(fā)現(xiàn)此突變。(3)在另外的無親緣關(guān)系、種族上、地理位置上匹配的676個正常對照中也篩過了此點。此點在此家系的正常對照和676個正常對照不存在,另外在781個其他疾病患者中對此突變也進行了篩選,此對照中也不存在此點。此變異點被ANNOVAR軟件預(yù)測為"damaging",被PolyPhen軟件預(yù)測為"probably damaging"。COL14A1基因的點突變(p.Pro1502Leu)所對應(yīng)的氨基酸在多種物種中為高度保守的氨基酸。(4)在PPPK712家系的2個患者(先證者與其母親)和1個對照中對COL14A1基因的47個編碼外顯子和內(nèi)含子-外顯子交界部進行Sanger Sequencing,未發(fā)現(xiàn)潛在的致病性變異點。(5)目前已報道的9個家系中,所有患者在掌跖部位表現(xiàn)為典型的掌跖角化,所有的家系顯示了一個連續(xù)性遺傳的特征,未見隔代遺傳現(xiàn)象。男女患病比例無顯著性差異(t=1.280,p=0.219)。受累患者皮膚損害都在童年晚期或青春期出現(xiàn),除1個家系中有患者在40多歲發(fā)病外。在5個家系中觀察到下一代的發(fā)病年齡比上一代早,并且同一家系中年齡大的患者癥狀最嚴(yán)重,年齡輕的患者癥狀較輕。在4個家系中可以發(fā)現(xiàn)并發(fā)癥,2個家系患結(jié)腸癌、肺癌等。2個家系中有2個伴發(fā)銀屑病。 結(jié)論(1)通過全基因組外顯子測序結(jié)合連鎖分析的方法發(fā)現(xiàn)點狀掌跖角化病的致病基因-COL14Al。證實了在常染色體顯性遺傳病的研究中,全基因組外顯子測序結(jié)合全基因組連鎖分析方法有較強的效力。(2)點狀掌跖角化病存在遺傳異質(zhì)性,不同的家系致病基因可能不同。(3)點狀掌跖角化病符合常染色體顯性遺傳規(guī)律,在中國漢族人群同一家系中存在著遺傳早現(xiàn)現(xiàn)象。同一家系或不同家系之間患者的表現(xiàn)型和嚴(yán)重程度存在明顯差異。同一家系內(nèi)患者年齡越大,癥狀越嚴(yán)重�？蓭椭覀兏玫睦斫釶PPK的臨床和遺傳特征,有利于我們進一步了解PPPK。
[Abstract]:Background Punctate palmoplantar keratoderma (PPPK; OMIM: 148600) is a rare autosomal dominant inherited disease characterized by a large number of keratinizing plaques irregularly distributed in the palmoplantar region. Buschke and Fischer first described the disease in 1910, and Brauer confirmed its inheritance in 1913. Therefore, the disease is also known as punctate palmoplantar keratosis Buschke-Fischer-Brauer type. Its typical clinical manifestations are round or oval palm and plantar parts of both hands, hard yellow or yellowish keratinous papules, if removed after the local crater-like pits can be left. Can be accompanied by nail deformation. Typical punctate skin lesions can be melted. A few patients may also involve not only palmoplantar sites, but also other sites such as knees, hands, feet, back, and elbows. The age of onset of PPPK is generally from 12 to 33 years old. It has been reported that PPPK is associated with breast cancer, prostate cancer, pancreatic cancer, and colon malignant glands. Tumor is related to metastatic non-small cell lung cancer.
Genome-wide linkage analysis was carried out in three PPPK families by Martinez-Mir et al, and the 9.98 cM region between D15S534 and D15S818 was located in 15q22-15q24.1. Genome-wide linkage analysis was carried out in two PPPK families by Zhang et al. The results indicated that PPPK had genetic heterogeneity between D8S1804 and D8S1720 in 8q24.13-8q24.21. Our laboratory later located the PPPK gene in a 5.06cM region between D15S651 and D15S988 in another large Chinese family. El Amri I and others confirmed the genetic factors at the suspicious locus of chromosome 15q, which was between D15S987 and D15S153. However, the pathogenic gene of PPPK has not been found so far. Exon Sequencing (Targeted Exome Capture) is an economical method of sequencing. It mainly detects new genes related to rare and common diseases by sequencing the coding regions of the human genome. Since 2009, exon capture systems and sequencing systems have been successfully used to discover or verify the pathogenesis of some single gene diseases at home and abroad. Genes, some of which are found to be pathogenic to monogenic diseases by combining exon-wide sequencing with linkage mapping.
Objective (1) Genome-wide exon sequencing combined with genome-wide linkage analysis was used to search for the pathogenic genes of PPPK and lay a foundation for gene diagnosis and gene therapy in the future. (2) To summarize the clinical and genetic characteristics of Chinese Han PPPK families.
Methods (1) In view of the large number of patients in a family originally located at 8q24.13-8q24.21 and the large number of blood samples we collected, four patients with typical symptoms (I 1, II 2, III 2 and III 5) and two normal controls (I 2 and III 6) were selected for genome-wide exon sequencing. The exon 37 and exon of the candidate gene obtained by genome-wide exon sequencing, the intron boundary region in the other four patients and six controls in the family were validated and sequenced. All 47 exons, promoters and exons of the candidate gene, and the intron boundary region were mutated in the family. (3) The genetic epidemiology and clinical characteristics of PPPK in Chinese Han population were summarized by retrospective study of CBMdisc and Pubmed. SPSS13.0 was used for statistical analysis.
Results (1) Stepwise filtration of dbSNP129, 8 HapMap, 1000 Genomes resulted in 21 new mutations in the four cases, including a missense mutation at exon 37 of the COL14A1 gene (c.4505C-T [p.Pro1502Leu]), which was located upstream of the original linkage region 8q24.13-8q24.21. The COL14A1 gene was selected as the most important candidate gene for PPPK. (2) The same missense mutation was found in other 4 cases by Sanger sequencing in other members of the family. No mutation was found in the remaining 5 controls (except for III 18). (3) In other unrelated species This point was also screened out in 676 geographically matched normal controls in the family. Normal controls and 676 normal controls in the family did not exist. The mutation was also screened in 781 patients with other diseases and did not exist in this control. The mutation was predicted as "damaging" by ANNOVAR software and was predicted by PolyPhen software. The amino acid corresponding to the point mutation of COL14A1 gene (p. Pro1502 Leu) is highly conserved in many species. (4) Sanger Sequencing was performed on 47 coding exons and intron-exon boundaries of the COL14A1 gene in two patients (proband their mother) and one control in the PPPK712 family. There was no significant difference between male and female (t = 1.280, P = 0.219). The skin lesions were all in children. The onset age of the next generation was earlier than that of the previous generation in 5 families, and the older patients in the same family had the most severe symptoms, the younger patients had the lighter symptoms. Complications were found in 4 families, colon cancer in 2 families, lung cancer and so on. 2 of the family members had psoriasis.
Conclusion (1) COL14Al, the pathogenic gene of punctate palmoplantar keratosis, was identified by genome-wide exon sequencing and linkage analysis. It was confirmed that the whole genome exon sequencing combined with genome-wide linkage analysis was effective in the study of autosomal dominant inheritance. (2) There was genetic heterogeneity in punctate palmoplantar keratosis. Pathogenic genes may be different in different families. (3) Punctate palmoplantar keratosis is an autosomal dominant inheritance disorder with early onset in the same family in Chinese Han population. It can help us better understand the clinical and genetic characteristics of PPPK, and help us further understand PPPK.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R758.53
本文編號：2179661
[Abstract]:Background Punctate palmoplantar keratoderma (PPPK; OMIM: 148600) is a rare autosomal dominant inherited disease characterized by a large number of keratinizing plaques irregularly distributed in the palmoplantar region. Buschke and Fischer first described the disease in 1910, and Brauer confirmed its inheritance in 1913. Therefore, the disease is also known as punctate palmoplantar keratosis Buschke-Fischer-Brauer type. Its typical clinical manifestations are round or oval palm and plantar parts of both hands, hard yellow or yellowish keratinous papules, if removed after the local crater-like pits can be left. Can be accompanied by nail deformation. Typical punctate skin lesions can be melted. A few patients may also involve not only palmoplantar sites, but also other sites such as knees, hands, feet, back, and elbows. The age of onset of PPPK is generally from 12 to 33 years old. It has been reported that PPPK is associated with breast cancer, prostate cancer, pancreatic cancer, and colon malignant glands. Tumor is related to metastatic non-small cell lung cancer.
Genome-wide linkage analysis was carried out in three PPPK families by Martinez-Mir et al, and the 9.98 cM region between D15S534 and D15S818 was located in 15q22-15q24.1. Genome-wide linkage analysis was carried out in two PPPK families by Zhang et al. The results indicated that PPPK had genetic heterogeneity between D8S1804 and D8S1720 in 8q24.13-8q24.21. Our laboratory later located the PPPK gene in a 5.06cM region between D15S651 and D15S988 in another large Chinese family. El Amri I and others confirmed the genetic factors at the suspicious locus of chromosome 15q, which was between D15S987 and D15S153. However, the pathogenic gene of PPPK has not been found so far. Exon Sequencing (Targeted Exome Capture) is an economical method of sequencing. It mainly detects new genes related to rare and common diseases by sequencing the coding regions of the human genome. Since 2009, exon capture systems and sequencing systems have been successfully used to discover or verify the pathogenesis of some single gene diseases at home and abroad. Genes, some of which are found to be pathogenic to monogenic diseases by combining exon-wide sequencing with linkage mapping.
Objective (1) Genome-wide exon sequencing combined with genome-wide linkage analysis was used to search for the pathogenic genes of PPPK and lay a foundation for gene diagnosis and gene therapy in the future. (2) To summarize the clinical and genetic characteristics of Chinese Han PPPK families.
Methods (1) In view of the large number of patients in a family originally located at 8q24.13-8q24.21 and the large number of blood samples we collected, four patients with typical symptoms (I 1, II 2, III 2 and III 5) and two normal controls (I 2 and III 6) were selected for genome-wide exon sequencing. The exon 37 and exon of the candidate gene obtained by genome-wide exon sequencing, the intron boundary region in the other four patients and six controls in the family were validated and sequenced. All 47 exons, promoters and exons of the candidate gene, and the intron boundary region were mutated in the family. (3) The genetic epidemiology and clinical characteristics of PPPK in Chinese Han population were summarized by retrospective study of CBMdisc and Pubmed. SPSS13.0 was used for statistical analysis.
Results (1) Stepwise filtration of dbSNP129, 8 HapMap, 1000 Genomes resulted in 21 new mutations in the four cases, including a missense mutation at exon 37 of the COL14A1 gene (c.4505C-T [p.Pro1502Leu]), which was located upstream of the original linkage region 8q24.13-8q24.21. The COL14A1 gene was selected as the most important candidate gene for PPPK. (2) The same missense mutation was found in other 4 cases by Sanger sequencing in other members of the family. No mutation was found in the remaining 5 controls (except for III 18). (3) In other unrelated species This point was also screened out in 676 geographically matched normal controls in the family. Normal controls and 676 normal controls in the family did not exist. The mutation was also screened in 781 patients with other diseases and did not exist in this control. The mutation was predicted as "damaging" by ANNOVAR software and was predicted by PolyPhen software. The amino acid corresponding to the point mutation of COL14A1 gene (p. Pro1502 Leu) is highly conserved in many species. (4) Sanger Sequencing was performed on 47 coding exons and intron-exon boundaries of the COL14A1 gene in two patients (proband their mother) and one control in the PPPK712 family. There was no significant difference between male and female (t = 1.280, P = 0.219). The skin lesions were all in children. The onset age of the next generation was earlier than that of the previous generation in 5 families, and the older patients in the same family had the most severe symptoms, the younger patients had the lighter symptoms. Complications were found in 4 families, colon cancer in 2 families, lung cancer and so on. 2 of the family members had psoriasis.
Conclusion (1) COL14Al, the pathogenic gene of punctate palmoplantar keratosis, was identified by genome-wide exon sequencing and linkage analysis. It was confirmed that the whole genome exon sequencing combined with genome-wide linkage analysis was effective in the study of autosomal dominant inheritance. (2) There was genetic heterogeneity in punctate palmoplantar keratosis. Pathogenic genes may be different in different families. (3) Punctate palmoplantar keratosis is an autosomal dominant inheritance disorder with early onset in the same family in Chinese Han population. It can help us better understand the clinical and genetic characteristics of PPPK, and help us further understand PPPK.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R758.53
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相關(guān)期刊論文 前4條
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