0521定位克隆-路李(课外)

基因型和连锁分析
DNA样本采集和纯化 基因组DNA的PCR扩增 380个不同形态的微卫星标记 基于标记的Two-point Linkage分析

– Disease－gene frequency – Two-point Linkage LOD scores
LOD值

LOD scores 连锁分数：lg（连锁概率/不连 锁的概率）
ห้องสมุดไป่ตู้
单体型分析
Haplotype Analyis

对保守序列的判定
2号常染色体的长臂2q35－2q36 标记D2S2248－D2S360之间
分析结果
两个家系的疾病可能由同一个基因的突变 引起 两个家系非同源：不共享同一个单体型（ haplotype）

进一步分析
家系中22，23，35号个体将定位间隔缩小 到介于D2S2249 和D2S1242 (2q35-q36)间 的8.1cM 。 区段候选基因:
Figure 4 Figure and toe features of family 2. The pictures provide the numbers of the individuals. All the affected members had phenotypes similar to those of family 1. In most affected individuals examined by radiography, the middle phalanges were missing (e.g., individuals 5, 7, and 11). Individual 19 had shortened middle phalanges in digits 2, 3, and 4. The proximal phalanges of the thumbs and digit 5 were markedly shortened and broadened in most of the affected members (e.g., individuals 5, 7, and 11). The affected family members also have thin shafts and broad epiphyses of metacarpals and proximal phalanges. The clinodactyly of fingers 5 and 4 was present in some affected individuals (e.g., individual 18 had clinodactyly in finger 5, and individuals 5 and 19 had clinodactyly in finger 4). Most of the affected had the proximal phalanges of digit 5 shortened, broadened, or both (e.g., individuals 5, 7, 11, and 18). Distal phalanges and metacarpals were also shortened in two affected members of this family (e.g., in individual 5, the distal phalanges, metacarpal 3, and metacarpal 5 were shortened; in individual 7, the distal phalanges were severely shortened).
A-1类的短指畸形

主要特征： 指(趾)中关节，与末端关节几近融合，关节 消失。
存在指（趾）的外观变形


NOTE.—A plus sign ( ) indicates presence, a minus sign ( ) indicates absence, and a question mark (?) indicates that presence or absence of the anomaly could not be determined because the individual was too young. Anomalies are numbered, as follows: 1 = all middle phalanges severely shortened; some were fused or missing; 2 = thin shafts and broad epiphyses of metacarpals and proximal phalanges; 3 = clinodactyly; 4 = proximal phalanges of the thumbs shortened and broad-ened; 5 = third metacarpal, fifth metacarpal, or both shortened; 6 = shortened distal phalanges; 7=proximal phalange of digit 5 shortened and broadened; A = middle phalange of digit 5 missing; B= middle phalange of digits 2 and 5 missing; C = middle phalange of digits 2, 4, and 5 missing; D= all middle phalanges missing; E = radial clinodactyly of fingers 4 and 5; and F = ulnar clinodactyly of fingers 2 and 3. A Individual not examined by radiography.
– 两个家系一个来自湖南省，另一个来自贵州省。
病例分型
1951年科学家根据指（趾）的畸形情况将 可遗传的短指畸形分成了5类－A、B、C、 D、E. 指（趾）的中间关节变短是短指（趾）畸 形（BDA）的主要特征。 BDA又有几个亚型。研究对象是BDA-1。

Figure 3 Finger and toe features of family 1. The pictures provide the numbers of the individuals. Anomalies were mainly confined to the middle phalanges. In some affected individuals, all the middle phalanges were missing or fused to the distal ones (e.g., individuals 6 and 20). In some affected family members, one to three fingers had middle phalanges missing or fused (e.g., individual 30 had the middle phalanges of digit 5 missing; individual 23 had the middle phalanges of digits 2 and 5 missing; and individuals 8 and 16 had the middle phalanges of digits 2, 4, and 5 missing).The proximal phalanges of the thumbs were markedly shortened and broadened in most of the affected members (e.g., individuals 16, 20, 23, 30, and 31). All affected adults in this family had the feature of thin shafts and broad epiphyses of metacarpals and proximal phalanges, but affected children did not have such a feature (e.g., individual 31, 11 years old; and individual 36, 5 years old).The clinodactyly of the second, fourth, or third fingers was present in most of the affected family members (e.g., individuals 8, 16, 23, 30, 31, and 36). All the manifestations described above are common to other reports reviewed by Fitch (1979). The distal phalanges and metacarpals were also shortened in two of the affected family members: individual 6 and individual 16. Individual 6 had very short distal phalanges in digits 2, 3, 4, and 5 and had a shortened metacarpal 3 of the right hand. Individual 16 had short distal phalanges in digits 1, 3, and 5 and a short third metacarpal of the left hand.
定位克隆的案例
－－家族性短指（趾）基因的定位 克隆
关于短指畸形

看看这手有什么不同？
贺林研究室
1903年的发现－－百年之谜 找到三个大的隔离的家系 对各种表形作了归类 精确定位在第二号染色体的长臂2q35－q36 区域 找到了致病基因IHH

家谱分析

对联各家系的辐射照片进行了鉴定
Markers 和疾病 位点的连锁性 分析 家系一两点间的 连锁分析数据 从数据上可以看 出： 在D2S2249－ D2S1242之间 的区域连锁可 能性大
家系二两点间的 连锁分析数据 从数据上可以看 出： 在D2S2248－ D2S2308之间 的区域连锁性 强
单体型分析
单体型（Haplotype） 基因组的DNA序列可以划分为若干“单体型 ”的基本结构单元所排列组合而成。

由此推断PAX3上的某种突变引可能是发BDA1的原因。
IHH基因的研究
使用老鼠IHH基因mRNA 设计引物 扩增出编码基因、启动子基因、连结区域 三个家系分别发现

– 外显子1的283位点存在G-->A的转变，意味着 Glu95Lys的转变 – 外显子2的391位点存在G A的转变，意味着Glu131 Lys的转变 – 外显子1的300位点存在C A的转变，意味着Asp100 Glu的转变

– PAX3 (paired box homeotic gene 3) – IHH（ Indian hedgehog ）
进一步分析

PAX3 基因在标定的位点内部 不同的PAX3 缺陷引起不同的表型
– WS3 上肢骨骼发育不全 – craniofacial-deafness-hand syn-drome 鼻骨和上颌骨发育不全。 – 老鼠的PAX3 在不同组织结构层面上表达包括在老鼠胚胎时期的肢 体发育过程中