全基因组外显子测序发现点状掌跖角化病致病基因COL14A1
发布时间：2018-08-12 16:47
【摘要】：研究背景点状掌跖角化病(Punctate palmoplantar keratoderma; PPPK; OMIM:148600)是一种不规则分布于掌跖部位的大量的角化性斑块为特征的罕见的常染色体显性遗传性疾病。Buschke和Fischer在1910年第一次描述此病,Brauer在1913年肯定了它的遗传性。因此,此病又被命名为点状掌跖角化病Buschke-Fischer-Brauer型。其典型临床表现为双手掌与足跖部位的圆形或卵圆形、较硬的黄色或淡黄色角质丘疹,若去除角质丘疹后,局部可留有火山口样的凹坑。可以伴有甲变形。典型的点状皮损可以融合成块,可能与遭受连续性高压有关,一般足跖部位皮疹比掌部较大。少数患者也可不仅累及掌跖部位,其他部位如膝部、手足背部、肘部也可受累。PPPK先证者发病年龄是一般是从12-33岁,有报道PPPK与乳腺癌、前列腺癌、胰腺癌、结肠恶性腺瘤、转移性肺非小细胞癌有关。 Martinez-Mir等在3个PPPK家系中进行全基因组连锁分析,定位在15q22-15q24.1上D15S534和D15S818之间9.98cM区域。本实验室张等在2个PPPK家系中进行全基因组连锁分析,定位在8q24.13-8q24.21上D8S1804和D8S1720之间9.02cM区域,此结果提示PPPK有遗传异质性。本实验室高等后来在另一个中国大家系中进行精心定位,将PPPK致病基因定位在D15S651和D15S988之间5.06cM区域。El Amri I等肯定了在染色体15q的可疑基因座的遗传因素,此区域在D15S987和D15S153之间。但PPPK的致病基因至今尚未发现。全基因组外显子测序(靶向外显子组捕获)是一种经济的测序方法,其主要是对人类基因组的编码区进行测序从而探测罕见和常见疾病相关的新基因。2009年来,国内外已经成功利用外显子捕获系统和测序系统发现或验证了一些单基因疾病的致病基因,其中有一些是用全基因组外显子测序与连锁定位相结合发现单基因病的致病基因。 目的(1)全基因组外显子测序结合全基因组连锁分析搜寻PPPK的致病基因,为将来的基因诊断与基因治疗奠定基础。(2)总结中国汉族PPPK家系的临床和遗传特征。 方法(1)鉴于我们原定位到8q24.13-8q24.21上的一个家系患病人数较多且我们收集的血样本数较多,从中挑选了症状比较典型的4个病例(Ⅰ1,Ⅱ2,Ⅲ2和Ⅲ5)和2个家系内正常对照(Ⅰ2和Ⅲ6)进行全基因组外显子测序。(2)用Sanger sequencing的方法对我们用全基因组外显子测序所得到的候选基因的第37号外显子及外显子、内含子交界区域在家系内另外4个患者和6个对照进行家系内验证测序,对此候选基因所有的47个外显子和启动子以及外显子、内含子交界区域进行家系外突变检测。(3)通过CBMdisc(中国生物医学文献)和Pubmed进行系统文献回顾,检索1991年至今已有文献报道的9个中国汉族PPPK家系,总结中国汉族PPPK的遗传流行病学及临床特征。统计学分析用SPSS13.0。 结果(1)通过逐步滤过法滤过了dbSNP129、eight HapMap、1000Genomes、家系内对照后,4个病例共有的新变异点有21个。其中COL14A1基因的第37号外显子上有一个错义突变点(c.4505C-T [p.Pro1502Leu]),此突变点位于原定位的连锁区域8q24.13-8q24.21的上游3.4Mb的位置。我们选择COL14A1基因作为PPPK的最重要的候选致病基因。(2)通过Sanger sequencing的方法在此家系的其他成员进行测序,在其他4个病例中也发现了同样的错义突变,在剩余的5个对照中(Ⅲ18除外)未发现此突变。(3)在另外的无亲缘关系、种族上、地理位置上匹配的676个正常对照中也筛过了此点。此点在此家系的正常对照和676个正常对照不存在,另外在781个其他疾病患者中对此突变也进行了筛选,此对照中也不存在此点。此变异点被ANNOVAR软件预测为"damaging",被PolyPhen软件预测为"probably damaging"。COL14A1基因的点突变(p.Pro1502Leu)所对应的氨基酸在多种物种中为高度保守的氨基酸。(4)在PPPK712家系的2个患者(先证者与其母亲)和1个对照中对COL14A1基因的47个编码外显子和内含子-外显子交界部进行Sanger Sequencing,未发现潜在的致病性变异点。(5)目前已报道的9个家系中,所有患者在掌跖部位表现为典型的掌跖角化,所有的家系显示了一个连续性遗传的特征,未见隔代遗传现象。男女患病比例无显著性差异(t=1.280,p=0.219)。受累患者皮肤损害都在童年晚期或青春期出现,除1个家系中有患者在40多岁发病外。在5个家系中观察到下一代的发病年龄比上一代早,并且同一家系中年龄大的患者症状最严重,年龄轻的患者症状较轻。在4个家系中可以发现并发症,2个家系患结肠癌、肺癌等。2个家系中有2个伴发银屑病。 结论(1)通过全基因组外显子测序结合连锁分析的方法发现点状掌跖角化病的致病基因-COL14Al。证实了在常染色体显性遗传病的研究中,全基因组外显子测序结合全基因组连锁分析方法有较强的效力。(2)点状掌跖角化病存在遗传异质性,不同的家系致病基因可能不同。(3)点状掌跖角化病符合常染色体显性遗传规律,在中国汉族人群同一家系中存在着遗传早现现象。同一家系或不同家系之间患者的表现型和严重程度存在明显差异。同一家系内患者年龄越大,症状越严重。可帮助我们更好的理解PPPK的临床和遗传特征,有利于我们进一步了解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.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.53
本文编号：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.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.53
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相关期刊论文 前4条
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