刘万清
论文题目：家族性恒齿缺失基因的定位、克隆以及精神疾病相关基因分析
作者简介：刘万清，男，1974年01月出生，1996年09月师从于中国科学院上海生命科学研究院贺林教授，于2000年08月获博士学位。
摘 要
自19世纪60年代以来，遗传学成为一门真正独立的学科。在整个20世纪中，遗传学得到了日新月异的发展。但这些发展从一开始的较长时间里都停留在理论阶段。随着核酸的发现、认识到核酸是遗传物质的载体再到揭示DNA的双螺旋结构到细胞遗传学的兴起和新发现，遗传学的理论才真正与实实在在的实验基础相结合。20世纪70年代以来，分子遗传学的手段得到了巨大的发展，人们第一次能够操纵遗传物质。80年代PCR等技术的发明更使人们对遗传物质的研究的到了大众化的普及。遗传学研究手段的不断进步使人们在疾病研究的道路上大大前进了一步。众多单基因遗传病的研究过程就是最为明显的例子。
20世纪40－60年代，随着核酸和蛋白质研究的深入，生物化学得到了较大的发展。许多研究生物化学、生物大分子的手段如电子显微镜、生物分子分离、提纯、蛋白质电泳相继出现，使人们在分子水平上研究疾病病因成为可能。许多酶缺陷症就是利用这些方法将其基因确定的。如苯丙酮尿症等。镰刀形红细胞贫血病致病突变的发现使人们第一次有了“分子病“的概念。所有上述的方法都是基于疾病与正常之间明显可见的或直接与生化功能相关的线索确定与疾病相关的基因。这些方法的线索是基于基因的功能产物，因此又称为功能克隆。70－80年代，细胞遗传学的发展使人们认识到，染色体上的许多缺陷是导致疾病发生的根本原因。利用染色体畸变，人们确定了包括杜氏肌营养不良（DMD）以及多种白血病的相关基因。基于疾病－染色体位置－基因的反向遗传学策略的提出引导人们能够对那些没有明显表型线索或生化功能的疾病进行定位克隆。细胞遗传学的发展以及分子遗传学手段的进步尤其是核酸杂交技术和PCR技术的出现，催生了许多新的确定疾病相关基因的技术手段。如差异显示PCR
(Differential-Display PCR, DDPCR)，基因组错配扫描 (Genome Mismatch Scanning,
GMS)，代表性差异分析 (Representative
Difference Analysis, RDA)等。这些技术的特点是既不考虑基因的功能线索，也不考虑基因在染色体上的位置信息，而是直接在疾病与正常样本之间寻找分子水平上的差异。这种方法直接联系疾病的表型与基因，故又称为表型克隆。近年出现的二维蛋白质电泳、DNA芯片、DNA阵列等也是这种表型克隆意义上的技术。
以上是利用遗传学手段确定疾病基因的主要策略。在实际的应用中，每种策略都各具优缺点：如功能克隆依赖于明显的功能线索，但可以较快地找到与疾病有关的基因；而定位克隆则依赖于家系和疾病的群体样本，即使对基因功能和生化产物的了解较少时也可以找到与疾病相关的基因，但周期较长。表型克隆可以直接对疾病和正常样本进行快速比较，但有时对样本的依赖性太强，多适用于肿瘤等相关基因的研究。由于目前对孟德尔遗传的疾病多用定位克隆的方式，复杂疾病的多种研究方法也是从该策略延伸出的方法。
定位克隆的核心是连锁分析技术。连锁分析是基于家系研究的一种方法，是单基因遗传病定位克隆方法的核心。它是利用遗传标记在家系中进行分型(Genotyping)，再利用数学手段计算遗传标记在家系中是否与疾病产生共分离。连锁分析是利用连锁的原理研究致病基因与参考位点(遗传标记)的关系。根据孟德尔分离率，如果同一染色体上的位点不连锁，那么遗传标记标将独立于致病基因而分离，与致病基因位于同一单倍体或不同单倍体的机会各占一半，否则表明连锁的存在。
对多基因遗传病的研究目前主要有两种方法：基于家系的连锁分析与基于群体的连锁不平衡分析。其中，患病同胞对法，关联分析，核心家系的关联分析以及连锁不平衡分析等是常用的方法。 这些方法都是从单基因遗传病的研究延伸出的方法。
本论文利用定位克隆的策略对一个单基因遗传疾病”家族型恒齿缺失(贺-赵缺陷症)”进行了基因定位与克隆。该疾病的特点是患者乳牙相对正常，而恒牙发生缺失。首先采用DNA Pooling的方法对遗传标记进行了快速扫描, 得到候选的区域后再结合连锁分析的方法将该疾病相关基因定位于人染色体10q11.2区域5.5cM的范围内。在D10S196标记上取得最大两点间LOD值为13.29(重组率为0)。 构建单倍型后发现连锁区域位于D10S604与D10S568之间。接下来对该区域中的YAC和BAC文库进行了Contig 搭建、部分测序和候选基因的克隆以及突变检测，迄今尚未发现与疾病相关的基因突变。
在关于精神性疾病的基因分析中检测了多个基因位点，得到了两个阳性位点，其中一项较大规模的关联研究(579例精神分裂症患者，1528例正常对照)证实，载脂蛋白E基因的E4型等位基因与精神分裂症之间存在极强的关联关系(p<10-6)。对该基因位点的等位基因在精神分裂症患者和正常对照中的年龄分布作了详细研究和分析后，发现该基因与精神疾病之间的关联主要来自于1948年以前和1957~1967年之间出生的群体。 由于这两个时期的人群均处于不同程度的环境应激条件下，如营养不良。推测可能该基因与饥饿等应激或其它环境因素之间存在的相互作用使疾病易发。 另一项在中国人群中5－羟色胺转运酶与精神疾病之间的关联研究表明该基因在上海人群中与精神分裂症及单相抑郁症存在一定的关联关系，否定了该位点与双相情感障碍之间的关系。同时，在11个群体包括高加索人群、中国汉族人群以及9个中国少数民族群体中对5－羟色胺转运酶基因单核苷酸多态性 (SNP) 的研究中，发现了22个新的多态位点。对该基因内SNP的进一步分析表明该基因在进化上存在较低的核苷酸多样性、弱阳性选择、较弱的连锁不平衡水平以及较高的重组率。利用新发现的SNP与精神分裂症之间的进一步研究重复了以前的研究结果，为揭示该基因与精神疾病之间的关系提供了更多的证据。
ABSTRACT
From 1860s, genetics became
a separate branch of sciences. In the whole 20th century, genetics
got dramatic development. However, in a long period, genetics was sticked in
the observations and assumptions. With the discovery of nucleic acid and the
double helix structure of DNA, scientists were aware of the inherited basis of
phenotyps. In 1970s, genetics met the turning point to molecular level. for the
first time, genetists could operate a DNA fragment. The invention of PCR
technology in 1980s promoted the genetics into common population. The
increasing development of theories and technologies in genetics made it become
a crutial tool to disclose hundreds and thousands of inherited diseases.
During the period from 1940s
to 1960s, the rising of biochemistry based on the exploding inventions on
electronic microscope, separating techniques for biomolecules, purification and
electrophoresis technologies for proteins facilitated the identification of
mutated genes for inherited diseases. The strategy targeting directly on the
functional products of genes, so called the Functional Cloning made many
diseases caused by enzymy deficiency clear. In 1970s and the middle of 1980s,
the development of cytogenetics indicated that many chromosomal rearrangments
were essential for many iherited diseases. Molecular genetics techniques on
chromosomal rearrangments discovered dozens of mutant including the DMD and
many leukemia-causative mutations. However, for more inherited diseases, it was
very difficult to connect any clinical clue to the etiology of these diseases
either because of the limitation of technologies or the limited depth on the
experimental attempts based on the living human body. A strategy called
Positional Cloning trying to link the disease, causative chromosomal region and
mutated gene led genetists to identify the genes for diseases--theoretically
only based on the disease status itself regardless of other informations.
Nowadays, genetic technologies like molecular hybridization and PCR hatched out
plenty of new strategies on identification of disease related gene. These
strategies include the Differential-Display PCR, Genome Mismatch Scanning, Representative
Difference Analysis, 2-D gel, Microarry and DNA chip, etc. Most of them try to
link the phenotype and the genotype directly, therefore, a name of Phenotypic
Cloning was given.
In the practices, each of
these trategies has advantages and disadvatages. For example, the Functional
Cloning depends much on the clinical informations, but the cloning would be
relatively rapid, while Positional Cloning does not require many clinical clues
but depends much on the collection of pedigrees and pupolation samples, a long
time would be expected if a gene get cloned before the human genome project
were launched. The Phenotypic Cloning is capable to comparing the samples
between disease and normal control, but the disadvantages are also
obvious—samples, tissue or disease related tissue, would be difficult to get.
With the straightforward identifications of most genes with obvious clinical
information, Postional Cloning was increasingly used in the most Medelian
inherited diseases.
The essential core of
Positional Cloning is linkage analysis strategy, a method based on the
pedigree, in which genetic markers distributed evenly in the genome were
genotyped and calculated in the pedigree to test the co-segregation with the
disease trait. Using this method, hundreds of monogenic disease genes have been
cloned till now.
Currently, the most
difficult and unsolved problem is the lacking of powerful strategies on complex
disease such as schizophrenia, hypertension, asthma, etc. Most of these
diseases are polygenic causes with strong interaction between genes and
environment. Almost all the method and techniques used now are based on the
strategies practised in the Medelian-iherited disease, for example, sib pair
analysis, association study and linkage disequilibrium study. These methods can
be summized by two mechanisms: pedigree based and population based method. In
my Ph. D works, Positional Cloning strategies were used in a Medelian-inherited
disease, He-Zhao Deficiency and complex diseases, schizophrenia and depression.
The thesis contains two main pieces of work. In the first one, we mapped the locus for He-Zhao deficiency (HZD) in a large kindred to chromosome 10q11.2. He-Zhao deficiency was a novel distinct form of agenesis of permanent teeth observed in a large Chinese kindred; affected individuals lacked the permanent teeth while the primary teeth were relatively normal. DNA pooling method combined with linkage analysis was employed in this study. The maximum two-points LOD [Zmax] scores at marker D10S196 was 13.29 at recombination fraction of 0.00. Haplotype analysis refined the locus within an interval of 5.5 cM flanked by markers D10S604 and D10S568. Our discovery has identifed a locus for a novel gene associated with tooth development and shed new light to understanding of mechanism underlying the development of permanent teeth. Subsequently, we tried to positionally clone the gene responsible for the HZD. YAC and BAC Contigs were constructed and candidate genes were cloned and sequenced. However, no disease-causing mutation was found so far.
In another piece of work, association studies were performed for psychiatric disorders and several candidate genes, from which positive results were found between both the Apolipoprotein E (ApoE)gene and the Serotonin transporter(SERT) gene and schizophrenia. For the ApoE study, we conducted a large-scale case-control investigation using 579 schizophrenics and 1528 healthy controls. A very significant association was observed between ApoE E4 allele and schizophrenia. Further analysis found that the association mainly resulted from two age periods (before 1948 and during 1957~1967). We speculated that strong stress such as starvation might be involved in the interaction with ApoE gene, which could increase the susceptibility to schizophrenia.
In three surveys of association
between psychiatric disorders and SERT gene, we found a VNTR
polymorphism in the SERT gene showing association with schizophrenia and
unipolar depression, but not with bipolar disorder in Chinese population. We
also investigated the SNP profile of the SERT gene by re-sequencing the SERT
gene in 204 individuals consisted of Chinese Han, Scottish Caucasian and other
9 minorities in China. A low degree of nucleotide diversity and a low level of
linkage disequilibrium were observed from the SNP profile. The diversity of LD
level and haplotypes distribution were demonstrated among different
populations. An independent case-control study using several identified SNPs
confirmed the association between SERT gene and schizophrenia, which
added more evidence supporting the role of this gene played in psychiatric
disorders.
