MOLECULAR GENETICS OF HUMAN LEFT-RIGHT AXIS MALFORMATIONS

1)在inv/inv小鼠品系中，酪氨酸酶微小基因的整合导致内脏器官完全镜像颠倒。对转基因整合位点的分析表明，一种新基因Inversin的功能缺失突变导致了正常左右两极的破坏。基于这些结果，我们假设倒置蛋白基因的突变与人类左-右轴畸形的一些病例有关。我们克隆了人INVERSIN基因，鉴定了其基因组结构，并对112例散发性和14例家族性左-右轴畸形进行了突变筛查。我们证明了人类INVERSIN定位于染色体9q3l，编码的蛋白质与小鼠的同源物有90%的同源性。突变分析发现两个独特的核苷酸替换，S371G和A650P导致错义突变。我们从目前的研究中得出结论，INVERSIN突变很少与人类左-右轴畸形相关。2)对左基因侧翼的人/小鼠基因组序列的生物信息学分析表明，小鼠Lefty 1是人类LEFTY2(也称为LEFTYB)的同源基因。老鼠Lefty 1的右侧消音器元件在人类中也是保守的。然而，对100多名有位置异常的患者的突变分析没有发现任何LEFTY2编码突变的患者。3)在非肥胖型糖尿病小鼠(NOD)和ICR品系雄性小鼠的F1后代中，经常观察到左右轴畸形。ICR株在Hnf3β亚型E的启动子区域插入了0.2kb的片段，并在Hnf3β基因的外显子3(基因频率0.36)发生了G到A的改变，2)在5‘控制元件内有5个单碱基替换和Lefty1的一个脯氨酸到丝氨酸的替换(P2S)(0.77)。这些替换可能会增加母亲患糖尿病的易感性。

1) In the inv/inv mouse strain, integration of the tyrosinase minigene causes complete mirror image reversal of internal organs. Analysis of the transgenic integration site revealed that loss of function mutation of a novel gene, Inversin, is responsible for the disruption of normal left-right polarity. Based on these results, we hypothesized that mutations in the Inversin gene are associated with some cases of left-right axis malformations in humans. We cloned of the human INVERSIN cDNA, characterized its genomic structure, and screened for mutations among 112 sporadic and 14 familial cases of left-right axis malformations. We demonstrated that human INVERSIN, localizes to chromosome 9q3l, and encodes a protein with 9O% homology to the mouse homologue. Mutation analysis revealed 2 unique nucleotide substitutions, S371G and A650P which result in missense mutations. We conclude from the current study that INVERSIN mutations are rarely associated with human left-right axis malformations.2) Bioinformatic analyses of the human/mouse genome sequences of the flanking the lefty gene region revealed that mouse Lefty 1 is the ortholog of human LEFTY2 (also known as LEFTYB). Right sided silencer element of the mouse Lefty 1 is conserved in humans as well. Mutation analysis of more than one hundred patients with situs abnormalities, however, did not reveal any patients with LEFTY2 coding mutations.3) Left-right axis malformations is commonly observed in the F1 offspring of NOD (non-obese diabetic) mouse dams and sires from ICR strains. The ICR strain had 1) a 0.2 kb insertion in the putative promoter region of the isoform E of Hnf3beta together with a G to A change that could create a potential splice acceptor in the exon 3 of Hnf3beta (gene frequency 0.36), 2) five single base substitutions within the 5' controlling element and a proline to serine substitution (P2S) of Lefty1 (0.77). These substitutions may contribute to increased susceptibility to maternal diabetes.