常染色体显性遗传“珊瑚状”核性白内障的分子遗传学研究
摘要
先天性白内障是一种较常见的儿童眼病，在全球范围内，每万人中就有1-6人因此而失明。大约25％的先天性白内障与遗传缺陷有关。遗传性先天性白内障有三种不同的遗传方式：常染色体显性遗传(AD)、常染色体隐性遗传(AR)和X连锁隐性遗传(XR)。在三种遗传型中，以AD型最多见，AR型较少，XR型罕见。
遗传性白内障具有复杂的临床表现，常以白内障浑浊所累及的位置来分类：如，核性白内障、绕核性白内障、缝状白内障、前极白内障、后极白内障和全白内障等。但还有许多难以归类的白内障表型被不断发现，其浑浊可以同时累及晶状体的不同部位，如：皮刺状白内障(aculeiform cataract)，其皮刺状浑浊从核向外周分布，累及了晶状体的胚胎核、胎儿核和外周皮质；中心袋样伴缝状白内障的浑浊在晶状体核处形成一六边形小袋，且浑浊还累及前后“Y”字缝；Nettleship首次报道的罕见的珊瑚状白内障(coralliform cataract)表型是以指状浑浊从核向外周分布，状似海中珊瑚为特点的，其浑浊累及了整个晶状体。
分子生物学技术的迅猛发展，使得越来越多的遗传性疾病基因被定位，到目前已发现了30多个不同的白内障疾病基因分布在人类十条染色体上的定位区域，有20多个与白内障相关的突变被确认，有10多个基因的突变被证实导致了常染色体
浙江大攀协士研究生毕业论文
显性遗传性白内障的形成，这些基因包括:晶状体蛋白基因(cRYAA[71，cRYAB[81，
eRYBA〔，，，eRYBB〔，。，，eRY“〔，”，CRYGD〔，，，)，homeobox基因PITx3〔，，，，主要内源性蛋
白基因(the major intrinsi。protein，MIP)【，‘，，珠状丝蛋白基因(beaded filament
pr。tein，BFsPZ)〔“〕，。 onnexins基因(exso〔，‘，，ex46〔，，，)和热休克转录因子4基
因(HSF4)〔，日，。
由于遗传性白内障的表型复杂多样，且遗传性白内障有着极为明显的遗传异质
性，即同一种基因的不同或同一突变可有不同的临床表现，甚至出现在同一家系
中;而同一临床表现又可源于不同的致病基因的突变。目前研究工作主要从两方
面着手:一方面，从与白内障形成有关的重要功能蛋白出发，通过各种杂交技术
找到其染色体编码位点，作为突变研究中的重要候选基因;另一方面，对典型的
大样本家系资料，进行连锁分析、全基因组扫描确定染色体定位，然后对定位区
域内候选基因筛选测序发现突变位点，来明确与白内障形成相关的基因。
本研究在临床中发现一个延续四代带有罕见表型的白内障家系，其表型非常特
殊:双侧晶状体核性混浊，伴后极向前方呈放射状点片状白色混浊，象大海中的
珊瑚，其间还可见点状金属样反光。其表型与Nettleship首次报道的罕见的珊瑚
状白内障表型部分类似。目前国内外尚未有与珊瑚状白内障形成相关的基因缺陷
报道，对此家系进行分子遗传学研究，希望能明确该病的致病基因，为明确白内
障的发生机制，致病途径和进一步的基因诊断提供基础。
第一部分珊瑚状核性白内障家系
通过系列眼病检查和全身检查发现，在38个家系成员中调查到的所有23例
患者中，晶体混浊表现除了程度上略有差别外，形态完全一致:双侧晶状体核性
混浊，点片状白色混浊从核向四周放射状分布在晶体前后极之间的中轴部及其附
近，其间还可见点状金属样反光，为较少见的珊瑚状核性白内障。本家系的白内
障生来就有，其四代中每代都有患者，且该病未见延伸至无亲缘关系的个体，疾
病的临床特点符合遗传病的诊断，为先天遗传性白内障。另外，该家系中未发现
其他全身疾病，可排除各类综合症，即不是染色体病。从眼部观察和检查来看，
除晶状体混浊外均未发现其他明显的异常，不存在一般先天性白内障较常见的小
浙东〔大学俘士研究六毕典论文
眼球、虹膜缺损、眼球震颤等症状，这说明病变局限于晶状体。
第二部分家系遗传模式的判断
通过家系调查、家系图谱的绘制了解该珊瑚状核性白内障这一性状在家系中
传递的情况。家系的发病率明显高于一般人群，且每代之间无明显差异，不存在
多基因疾病的微效累加基因效应，可以用自由组合律和连锁互换律来分析，其遗
传传递方式明显符合孟德尔分离律，故可认为此家系疾病为单基因遗传病。从系
谱中，我们可以看到，此白内障在家系中代代相传且无性别分布比例上的差异，
符合常染色体显性遗传的典型特征。为了进一步明确和证明这一遗传方式，本研
究进行了数理统计学分析。首先对家系各代发病率进行卡方检验，发现各代发病
率之间无统计学差异。课题又进行了分离分析(segregation analysis)。由于通过详
细的眼部检查，本家系中患者性状一白内障是完全确认。故选用单病例法(Singles
method)。经过公式计算该家系估计分离比P值为0.52，95%可信区间为
30.938%一73.862%。常染色体隐性遗传的理论分离比为0.25，不包括在此区域内，
故不是常染色体隐性遗传，95%可信区间包含了0.5，故认为本病的遗传方式为常
染色体显性遗传。
第三部分疾病相关基因的定位
本研究选用覆盖了人类整个基因组(22条常染
Congenital or infantile cataract is a sight-threatening lens defect that presents with an estimated incidence of 1-6 cases per 10 000 live births and cause 10-30 % of all blindness in children . Approximately 25 % of all cases are probably due to genetic defects, the majority of these inherited non-syndromic cataracts show autosomal dominant inheritance (AD); autosomal recessive (AR) and X linked (XR) forms are seldom observed.
Classification of human congenital cataract is difficult because so many cataract phenotypes have been reported. Cataract affecting the nucleus is common, such as central pulverulent cataract,. Lamellar cataract (e.g. zonular pulverulent cataract) have been also called zonular, perinuclear or Marner's cataract; the accurate localization of the opacity is to the zonular area surrounding the embryonal nucleus. Cataract limited to the cortex and not affecting the nucleus is rare, blue dot (cerulean) cataract was the first described cataract only affecting the cortex. Cataract limited to the anterior or posterior pole of lens are called anterior or posterior cataract respectively. Opacities affecting the sutures of lens are called sutural cataract. But many cataract phenotypes were reported that the opacities affected more than one location in lens, such as "aceuliform" cataract ,"central pouchlike" cataract, and "coralliform" cataract.
Development of advanced molecular biological techniques has facilitated the identification of more than 30 independent cataract loci located in ten different human chromosomes, from which 20 mutations have been identified. Mutations in more than 10 genes have been identified as genetic causes for autosomal dominant cataract (ADC); these genes include: crystalline gene (CRYAA, CRYAB, CRYBA, CRYBB, CRYGC, CRYGD), homeobox gene PITX3, the major intrinsic protein gene (MIP), beaded filament protein gene BFSP2, connexins gene (CX50, CX46). More recently, Len Bu et al. reported that mutations in the DNA-binding domain of heat-shock transcription factor 4 gene (HSF4) were associated with autosomal dominant lamellar and Marner cataract.
Genetic heterogeneity of cataract is evident, more than one gene has been reported to cause the same phenotype. On the other hand, an identical mutation can result in different phenotypes. There are two major methods used in cataract research: on one hand, basing on major functional protein associated with cataractgenesis , mapping the disease gene location by hybridization; on the other hand, mapping the locus for hereditary cataract in typical family using candidate gene linkage analysis, genomic genescan , and candidate gene screening, then identify the cataract interrelated gene.
In this study, we identified a four-generation family with hereditary cataracts transmitted as an autosomal dominant mode. The cataract phenotype is characterized by a large number of shiny slice-like and punctiform opacities extending radially from the nucleus, which resemble sea coral . this phenotype is similar to the coralliform cataract phenotype which first described by Nettleship. The pathogenesis of coralliform cataract is far from clear and the correlative reports are lacking, so studying the cataract in this family could help us understand better the cataract pathogenetic mechanisms in human.
Part I the hereditary central coralliform cataract family
23 from 38 family members received careful examination, including slit-lamp
examination and photography of lens to record the cataract type. All affected members have identical phenotype: a large number of shiny slice-like and punctiform opacities extending radially from the nucleus, which resemble sea coral. Ophthalmic examination of the unaffected siblings in family did not reveal any ocular abnormalities. No other diseases aside from age-related disorders were identified by medical examination.
Part II Estimation of the family inherited model
On the basis of the pedigree and generation-to-generation transmission, autosomal
dominant inheritance was supported : the equa
遗传性白内障具有复杂的临床表现，常以白内障浑浊所累及的位置来分类：如，核性白内障、绕核性白内障、缝状白内障、前极白内障、后极白内障和全白内障等。但还有许多难以归类的白内障表型被不断发现，其浑浊可以同时累及晶状体的不同部位，如：皮刺状白内障(aculeiform cataract)，其皮刺状浑浊从核向外周分布，累及了晶状体的胚胎核、胎儿核和外周皮质；中心袋样伴缝状白内障的浑浊在晶状体核处形成一六边形小袋，且浑浊还累及前后“Y”字缝；Nettleship首次报道的罕见的珊瑚状白内障(coralliform cataract)表型是以指状浑浊从核向外周分布，状似海中珊瑚为特点的，其浑浊累及了整个晶状体。
分子生物学技术的迅猛发展，使得越来越多的遗传性疾病基因被定位，到目前已发现了30多个不同的白内障疾病基因分布在人类十条染色体上的定位区域，有20多个与白内障相关的突变被确认，有10多个基因的突变被证实导致了常染色体
浙江大攀协士研究生毕业论文
显性遗传性白内障的形成，这些基因包括:晶状体蛋白基因(cRYAA[71，cRYAB[81，
eRYBA〔，，，eRYBB〔，。，，eRY“〔，”，CRYGD〔，，，)，homeobox基因PITx3〔，，，，主要内源性蛋
白基因(the major intrinsi。protein，MIP)【，‘，，珠状丝蛋白基因(beaded filament
pr。tein，BFsPZ)〔“〕，。 onnexins基因(exso〔，‘，，ex46〔，，，)和热休克转录因子4基
因(HSF4)〔，日，。
由于遗传性白内障的表型复杂多样，且遗传性白内障有着极为明显的遗传异质
性，即同一种基因的不同或同一突变可有不同的临床表现，甚至出现在同一家系
中;而同一临床表现又可源于不同的致病基因的突变。目前研究工作主要从两方
面着手:一方面，从与白内障形成有关的重要功能蛋白出发，通过各种杂交技术
找到其染色体编码位点，作为突变研究中的重要候选基因;另一方面，对典型的
大样本家系资料，进行连锁分析、全基因组扫描确定染色体定位，然后对定位区
域内候选基因筛选测序发现突变位点，来明确与白内障形成相关的基因。
本研究在临床中发现一个延续四代带有罕见表型的白内障家系，其表型非常特
殊:双侧晶状体核性混浊，伴后极向前方呈放射状点片状白色混浊，象大海中的
珊瑚，其间还可见点状金属样反光。其表型与Nettleship首次报道的罕见的珊瑚
状白内障表型部分类似。目前国内外尚未有与珊瑚状白内障形成相关的基因缺陷
报道，对此家系进行分子遗传学研究，希望能明确该病的致病基因，为明确白内
障的发生机制，致病途径和进一步的基因诊断提供基础。
第一部分珊瑚状核性白内障家系
通过系列眼病检查和全身检查发现，在38个家系成员中调查到的所有23例
患者中，晶体混浊表现除了程度上略有差别外，形态完全一致:双侧晶状体核性
混浊，点片状白色混浊从核向四周放射状分布在晶体前后极之间的中轴部及其附
近，其间还可见点状金属样反光，为较少见的珊瑚状核性白内障。本家系的白内
障生来就有，其四代中每代都有患者，且该病未见延伸至无亲缘关系的个体，疾
病的临床特点符合遗传病的诊断，为先天遗传性白内障。另外，该家系中未发现
其他全身疾病，可排除各类综合症，即不是染色体病。从眼部观察和检查来看，
除晶状体混浊外均未发现其他明显的异常，不存在一般先天性白内障较常见的小
浙东〔大学俘士研究六毕典论文
眼球、虹膜缺损、眼球震颤等症状，这说明病变局限于晶状体。
第二部分家系遗传模式的判断
通过家系调查、家系图谱的绘制了解该珊瑚状核性白内障这一性状在家系中
传递的情况。家系的发病率明显高于一般人群，且每代之间无明显差异，不存在
多基因疾病的微效累加基因效应，可以用自由组合律和连锁互换律来分析，其遗
传传递方式明显符合孟德尔分离律，故可认为此家系疾病为单基因遗传病。从系
谱中，我们可以看到，此白内障在家系中代代相传且无性别分布比例上的差异，
符合常染色体显性遗传的典型特征。为了进一步明确和证明这一遗传方式，本研
究进行了数理统计学分析。首先对家系各代发病率进行卡方检验，发现各代发病
率之间无统计学差异。课题又进行了分离分析(segregation analysis)。由于通过详
细的眼部检查，本家系中患者性状一白内障是完全确认。故选用单病例法(Singles
method)。经过公式计算该家系估计分离比P值为0.52，95%可信区间为
30.938%一73.862%。常染色体隐性遗传的理论分离比为0.25，不包括在此区域内，
故不是常染色体隐性遗传，95%可信区间包含了0.5，故认为本病的遗传方式为常
染色体显性遗传。
第三部分疾病相关基因的定位
本研究选用覆盖了人类整个基因组(22条常染
Congenital or infantile cataract is a sight-threatening lens defect that presents with an estimated incidence of 1-6 cases per 10 000 live births and cause 10-30 % of all blindness in children . Approximately 25 % of all cases are probably due to genetic defects, the majority of these inherited non-syndromic cataracts show autosomal dominant inheritance (AD); autosomal recessive (AR) and X linked (XR) forms are seldom observed.
Classification of human congenital cataract is difficult because so many cataract phenotypes have been reported. Cataract affecting the nucleus is common, such as central pulverulent cataract,. Lamellar cataract (e.g. zonular pulverulent cataract) have been also called zonular, perinuclear or Marner's cataract; the accurate localization of the opacity is to the zonular area surrounding the embryonal nucleus. Cataract limited to the cortex and not affecting the nucleus is rare, blue dot (cerulean) cataract was the first described cataract only affecting the cortex. Cataract limited to the anterior or posterior pole of lens are called anterior or posterior cataract respectively. Opacities affecting the sutures of lens are called sutural cataract. But many cataract phenotypes were reported that the opacities affected more than one location in lens, such as "aceuliform" cataract ,"central pouchlike" cataract, and "coralliform" cataract.
Development of advanced molecular biological techniques has facilitated the identification of more than 30 independent cataract loci located in ten different human chromosomes, from which 20 mutations have been identified. Mutations in more than 10 genes have been identified as genetic causes for autosomal dominant cataract (ADC); these genes include: crystalline gene (CRYAA, CRYAB, CRYBA, CRYBB, CRYGC, CRYGD), homeobox gene PITX3, the major intrinsic protein gene (MIP), beaded filament protein gene BFSP2, connexins gene (CX50, CX46). More recently, Len Bu et al. reported that mutations in the DNA-binding domain of heat-shock transcription factor 4 gene (HSF4) were associated with autosomal dominant lamellar and Marner cataract.
Genetic heterogeneity of cataract is evident, more than one gene has been reported to cause the same phenotype. On the other hand, an identical mutation can result in different phenotypes. There are two major methods used in cataract research: on one hand, basing on major functional protein associated with cataractgenesis , mapping the disease gene location by hybridization; on the other hand, mapping the locus for hereditary cataract in typical family using candidate gene linkage analysis, genomic genescan , and candidate gene screening, then identify the cataract interrelated gene.
In this study, we identified a four-generation family with hereditary cataracts transmitted as an autosomal dominant mode. The cataract phenotype is characterized by a large number of shiny slice-like and punctiform opacities extending radially from the nucleus, which resemble sea coral . this phenotype is similar to the coralliform cataract phenotype which first described by Nettleship. The pathogenesis of coralliform cataract is far from clear and the correlative reports are lacking, so studying the cataract in this family could help us understand better the cataract pathogenetic mechanisms in human.
Part I the hereditary central coralliform cataract family
23 from 38 family members received careful examination, including slit-lamp
examination and photography of lens to record the cataract type. All affected members have identical phenotype: a large number of shiny slice-like and punctiform opacities extending radially from the nucleus, which resemble sea coral. Ophthalmic examination of the unaffected siblings in family did not reveal any ocular abnormalities. No other diseases aside from age-related disorders were identified by medical examination.
Part II Estimation of the family inherited model
On the basis of the pedigree and generation-to-generation transmission, autosomal
dominant inheritance was supported : the equa
引文
1. Francois J. Genetics of cataracts. Surv Ophthalmol 1996;40:427-58.
2．李凤鸣 眼科全书 北京：人民出版社，1996：1600-1605
3．李凤鸣 眼科全书 北京：人民出版社，1996：607-615
4. Vogt A. Weitere ergebnisse der spaltlampenmikroskopie des vorderen Bulbsabschnittes.Ⅲ.angeborene und fruth erworbene linsenveranderungen. Von Greafes Arch Ophthalmol. 1922; 107:237-240
5. Vanita, Jai RS, Virinder K, et al. A novel form of "central pouchlike" cataract, with sutural opacities, maps to chromosome 15q21-22.
6. Nettleship E. A peculiar form of congenital cataract. Trans OPhthal Soc UK 1906;26: 191-206.
7. Litt M, Kramer P, LaMorticella DM, et al. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystalline gene CRYA. Hum Mol Genet, 1998,7:471-474.
8. Berry V, Francis P, Reddy MA, et al. Alpha-B crystalline gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Am J Hum Genet, 2001,69:1141-1145.
9. Kannabiran C, Rogan PK, Olmos L, et al. Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the beta A3/A1-crystallin gene. Mol Vis, 1998,4:18-18.
10. Litt M, Carrero-Valenzuela R, LaMorticella DM, et al. Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human
beta-crystallin gene CRYBB2. Hum Mol Genet, 1997,6:665-668.
11. Heon E, Priston M, Schorderet DF, et al. The gamma-crystallins and human cataracts: a puzzle made clearer. Am J Hum Genet, 1999,65:1261-1267.
12. Kmoch S, Brynda J, Asfaw B, et al. Link between a novel human γ D-crystallin allele and a unique cataract phenotype explained by protein crystallography. Hum Mol Genet, 2000,9:1779-1786.
13. Semina EV, Ferrell RE, Mintz-Hittner HA, et al. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet, 1998,19:167-170.
14. Berry V, Francis P, Aushal S, et al. Missense mutations in MIP underlie autosomal dominant"polymorphic" and lamellar cataracts linked to 12q. Nat Genet, 2000,25:15-17.
15. akobs PM, Hess JF, FitzGerald PG, et al. Autosomal dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. Am J Hum Genet, 2000,66:1432-1436.
16. Shiels A, Mackay D, Ionides A,et al. Amissense mutation in the human Connexin 50 gene (GJA8) underlins autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hun Genet, 1998,62:526-532.
17. Mackay D, Ionides A, Kibiar Z, et al. Connexin 46 mutations in autosomal dominant congenital cataract. Am J Hum, 1999,64:1357-1364.
18. Bu L, Jin YP, Shi YF, et al. Mutant DNA-binding domain of HSF4 is associated
with autosomal dominant lamellar and Marner cataract. Nat Genet, 2002,31:276-278.
2．李凤鸣 眼科全书 北京：人民出版社，1996：1600-1605
3．李凤鸣 眼科全书 北京：人民出版社，1996：607-615
4. Vogt A. Weitere ergebnisse der spaltlampenmikroskopie des vorderen Bulbsabschnittes.Ⅲ.angeborene und fruth erworbene linsenveranderungen. Von Greafes Arch Ophthalmol. 1922; 107:237-240
5. Vanita, Jai RS, Virinder K, et al. A novel form of "central pouchlike" cataract, with sutural opacities, maps to chromosome 15q21-22.
6. Nettleship E. A peculiar form of congenital cataract. Trans OPhthal Soc UK 1906;26: 191-206.
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