A preliminary study on prenatal diagnosis of congenital malformations using chromosome-specific variable number of tandem repeats (VNTR).

以pYNH24(VNTR)为探针，对30例先天畸形标本进行Southern杂交分析。有2例标本检测到异常条带。由于pYNH24基因定位于人类2号染色体，我们进一步研究了先天性畸形与定位于2q33-36的伽玛晶体Pst I多态之间的关系。在2.1kb和1.7kb的条带中发现了该基因的多态性。正常对照组分别为2.1/1.7(53%)和1.7/1.7(27%)，严重先天性畸形组分别为2.1/1.7(7%)和1.7/1.7(73%)。这一结果提示可能存在一个与先天畸形密切相关的基因座。从基因定位数据中我们发现，人类同源异型盒4基因簇存在于伽马晶体基因附近。因此，我们将研究重点放在HOX4基因上。同源框基因是高度保守的基因，被认为是胚胎发育过程中编码身体规划程序的基因。作为研究Homeobox基因突变的第一步，我们分离了Hox 4A基因组DNA克隆，并测定了约6000bp的核苷酸序列。根据测序数据，设计了聚合酶链式反应(PCR)引物，对23例严重先天畸形的DNA样本进行了聚合酶链式反应-单链构象多态性分析。我们未能在PCR-SSCP中发现异常条带，现在正试图检测人类先天性畸形中的HOX 4B基因异常。

DNA samples from 30 cases of congenital malformations were analyzed by Southern blot hybridization by using pYNH24 (VNTR) as a probe. Abnormal bands were detected in 2 samples. Since the locus of pYNH24 was mapped in human chromosome 2, we further investigated the association of congenital malformations and Pst I-polymorphism of gamma-crystalline which was mapped on 2q33-36. The polymorphism was found in 2.1 and 1.7 kb bands. The frequency of these bands was as follows; 2.1/1.7 (53%) and 1.7/1.7 (27%) in normal controls, while 2.1/1.7 (7%) and 1.7/1.7 (73%) in severe congenital malformations. This result suggests that there may be a locus which is closely associated with congenital malformations. From the gene mapping data, we found that human homeobox 4 gene cluster is present in the proximity of gamma-cristalline gene. Therefore, we focused our investigation on Hox 4 genes.Homeobox gene are highly conserved genes and considered to encode programs for body plan during embryogenesis. As a first step to investigate mutations of homeobox genes, we isolated Hox 4A genomic DNA clone and determined the nucleotide sequence of about 6000 bp. Based on the sequence data, PCR primers were synthesized and PCR-SSCP analysis was carried out in 23DNA samples from severe congenital malformations. We failed to find abnormal bands in PCR-SSCP and now trying to detect Hox 4B gene abnormalities in human congenital malformations.