【文献阅读】TBX6 Null Variants and a Common Hypomorphic Allele in Congenital Scoliosis

【文献阅读】TBX6 Null Variants and a Common Hypomorphic Allele in Congenital Scoliosis (TBX6基因无效变异联合常见亚效等位基因导致先天性脊柱侧凸)

摘要

背景：

先天性脊柱侧凸（Congenital scoliosis, CS）具有遗传易感性

方法：

在161 例中国汉族散发性CS病例，166个对照，和2个包含16p11.2染色体片段缺失的家系中进行比较基因组杂交（CGH）、定量聚合酶链反应分析（qPCR）和DNA测序。
验证集中使用了76莉中国汉族CS病例和42个来自多个中心的具有16p11.2片段缺失的个体。

结果：

161例中国汉族散发性CS病例中发现了17个TBX6杂合无效变异（11%）；166个对照个体中没有发现TBX6无效变异。这些无效变异包含拷贝数变异（12例16p11.2染色体片段缺失影响TBX6基因）和单核苷酸变异（1例无义突变和4例移码突变）。
但TBX6无效杂合突变还不足够引起先天性脊柱侧凸。我们在17个病例中均发现了一个常见的TBX6单倍体作为第二风险等位基因。
验证集也证实了这个复杂遗传模型。
体外功能实验表明该风险单倍型是一个亚效等位基因。
TBX6相关的先天性脊柱侧凸的特征是半椎体畸形。

结论：

在我们的病例中，罕见TBX6无效突变和亚效等位基因组成的复杂遗传模型占据了11%。

正文内容

方法：

脊柱畸形可以分为hemivertebra or hypoplasia （半椎体或发育不全）, segmentation defect（分割缺损）, or butterfly vertebra（蝶形椎体 ）

主要结果

1. 先天性脊柱侧弯患者16p11.2片段缺失流行
   20组家庭（1位患者+2个健康父母）参与拷贝数变异的全基因组分析（CNV GWAS），用到了1×1, CGH芯片。
   结果发现20个患者中有2例具有16p11.2染色体片段缺失。剩余的141个患者中有10个具有杂合缺失，相比较于166个对照个体中0个杂合缺失，差异显著。
   
   Figure 1. Effect of the 16p11.2 Deletion on TBX6
   Panel A shows the region affected by the proximal 16p11.2 deletion based on the human genome assembly hg19. A pair of long direct genomic repeats (shown in orange) can mediate 0.6-Mb recurrent deletions. The genes affected by this deletion, including TBX6 (circled), are shown.
   Panel B shows three common single-nucleotide polymorphisms (SNPs) in TBX6. Two SNPs, rs3809624 and rs3809627, are located in the 5’ noncoding region, whereas rs2289292 is a synonymous SNP in the last exon.
   Panels C and D show two pedigrees with 16p11.2 deletions (SE1 and SE2). Squares denote male pedigree members, circles female pedigree members, solid symbols members with congenital scoliosis, and open symbols unaffected members; the probands are indicated by black arrows. The red bracket represents the deletion allele, and the nonreference alleles of rs2289292, rs3809624, and rs3809627 on the nondeletion chromosomes are shown in blue. The slash denotes a deceased family member. NA denotes not available.
2. TBX6功能缺失突变与CS关系
   161 CS患者中有5例TBX6无义突变，166例对照中没有任何人有该基因的无义突变，证明TBX6无义突变与CS有关。
3. 无义突变携带者中的常见风险单倍体
   作者观察在两个家系中都有一个常见TBX6单倍型（T-C-A），随后在17个包含无义突变的患者中都观察到此单倍型存在于第二条染色体上。这里作者提出TBX6复杂遗传模型包含一个罕见无义突变和一个常见风险等位基因（T-C-A）共同导致CS。
4. 验证集
   76例患者中6个含有无义突变，且均包含T-C-A单倍型。
   42例包含16p11.2片段缺失的来自多国家的志愿者中，共有6例CS患者，其中5人含有T-C-A半合子。唯一的特例是一个非洲样本，非洲群体中T-C-A单倍型很罕见（<1%）。无脊柱侧凸的组别中，30人中5人包含此单倍型。证明T-C-A在CS中是一个高风险因素。
5. 亚效等位基因体外实验
   LD分析中发现TBX6的LD区域很短，并且在该区域中没有发现罕见突变。最简单的解释是此常见单倍型具有功能。
   后采用体外荧光素酶实验研究rs3809624和rs3809627对基因表达的影响，发现一致支持rs3809624和rs3809627的alternative allele组合对TBX6表达的亚形效应。
6. TBX6相关的先天性脊柱侧弯患者具有半椎体畸形。
   
   Figure 3. Results of In Vitro Functional Assays of Common TBX6 Variants and the Mechanistic Model of TBX6-Associated Congenital Scoliosis
   Panel A shows the results of luciferase reporter assays. Two cell types (P19CL6 cells and human induced pluripotent stem cells [hiPSCs]) with high levels of expression of TBX6 during induced differentiation were used. The nonreference alleles of rs3809624 and rs3809627 are shown in blue, and the risk haplotype is underlined. At least three independent experiments of normalized luciferase activity were quantified, with the graph showing mean values and their standard errors (I bars). Panel B shows a simplified genetic model of TBX6-associated congenital scoliosis. TBX6 expression is critical for normal vertebral formation (the reference allele of TBX6 is indicated by a black line). Heterozygous hypomorphic variants (shown in blue) cause only a moderate reduction in TBX6 expression. Even homozygous hypomorphic variants do not reduce TBX6 expression dramatically. Heterozygous TBX6 null mutations (indicated by red brackets) may reduce gene expression by one half. Independently, these mutations hardly reach the gene-dosage threshold for congenital scoliosis. In combination, however, a null allele and a hypomorphic allele of TBX6 cause further reductions in gene expression that may confer a high risk of congenital scoliosis.

笔记

1. 亚效等位基因： 亚效等位基因有基因功能，但功能比正常的要少。这种等位基因通常由于基因损害，更常见的是损害调控这个基因的功能区。通常有蛋白产物，但少于正常。也就是说该基因或者该基因的调控区域发生突变，但没有完全损害基因的功能，是基因产物的功能降低，或者基因的产物表达量降低。大多数错义突变都产生亚效等位基因，而不会完全丧失蛋白功能。
2. 无义突变（nonsense mutation ）是指由于某个碱基的改变使代表某种氨基酸的密码子突变为终止密码子，从而使肽链合成提前终止。
3. 半合子（hemizygous）具有二组相同的染色体组，但有一个或多个基因是单价的，没有与之相对应的等位基因，这种合子称为半合子。

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https://www.medsci.cn/article/show_article.asp?id=f05845312a6