Congenital Dislocation Of The Hip: Study Of Genetic Epidemiology And Associations With HOXB9 And COL1A1 Genes

ObjectiveCongenital dislocation of the hip (CDH) , also called developmental dys-plasia of the hip ( DDH) , is one of the most common congenital malformations in the Department of Pediatric Orthopaedics, which does severe harm to children s health. The prevalence of CDH varies according to racial and geographic parameters throughout the world. It has been reported that the prevalence in China ranges between 0.91%‰ and 3. 80‰. Up to now, the genetic model and patho-genesis of CDH remains elusive. More and more clinical data support such a hypothesis that genetic factor and some related environmental factors involved in utero, perinatally, or subsequently with development may play a crucial role in the etiology of CDH. However, no comprehensive studies on epidemiology and molecular genetics of CDH have been reported so far. Therefore, the studies on genetic and environmental risk factors, as well as susceptibility genes would be of great help not only to shed light on the pathogenesis of CDH, but also to identify the susceptibility genes and genetic markers for at - risk individuals or population.It is considered that CDH is a kind of congenital malformation that results from the abnormal development of the hip joint during the embryonic development. The growth and development of the hip joint is a complicated process involved in the sequential regulation and expression of multiple genes in different temporal and spacial development of embryogenesis. Recently, progress has been made in the research into regulatory genes of embryonic development, ofwhich homeobox gene family is the most important. The family comprises a large number of genes that play a fundamental role in temporal and spacial regulation of growth and development of organisms. Nearly thousands of homeobox genes distribute in different regions of genome, of which HOX genes are present in a clustered way, while others are scattered throughout the genome. All the homeobox genes contain a common sequence element of 18Obp, the homeobox, which has been highly conserved throughout evolution. The homeobox encodes a 60 - amino acid homeodomain that is responsible for sequence - specific DNA binding of much larger heomodomain proteins. These proteins are transcription transregulators that regulate the expression of target genes and control various aspects of embryogenesis and cell differentiation.Genetic analyses in animals have revealed that the HOX genes are the major regulatory genes of the morphogenesis of the vertebrate embryo and limb, and the alterations of their function result in abnormalities of limb development. Moreover, It has been reported that some of human congenital malformations are caused by mutaions of HOX genes. There is a possibility that HOX genes play a regulatory role in pathogenesis of CDH. According to the rule of temporal and spacial colinearity of HOX gene expression, Davis et al. suggested that the expression of the ninth paralogous HOX genes lie within the region of the hip joint. Therefore, of the HOX family, H0XA9 , H0XB9 , H0XC9 and H0XD9 are most likely associated with pathogenesis of CDH.Type I collagen, a major extracellular matrix component in tissues such as skin, bone, cartilage, tendon, ligament, and capsule, et al. , plays a significant role influencing cell behavior and maintaining tissue structure. Collagen type I is composed of three polypeptide chains encoded by collagen type I alpha 1 gene ( COLlAl ) and collagen type I alpha 2 gene ( COL1A2 ). As a result, mutations in COLlAl and COLlAl genes may result in laxity of tendon, ligament and capsule, as well as abnormal differentiation of cartilage cell. The laxity of tendon, ligament and capsule, as well as acetabular dysplasia resulted from abnormal differentiation of cartilage cell are thought to be the crucial causes of CDH. In 1999, Giunta et al. reported that 20 patients with Ehlers - Danlos syndrome type VII all presented with bilateral CDH, suggesting that COLlAl andCOLlAl be candidate genes of CDH.According to the forementioned characteristics of pathogenesis of CDH, we chose the candidate cloning strategy to focus the study on the regulatory gene involved in the embryonic development and Type I collagen gene. For the first time, our study mapped the susceptibility region of CDH to 17q21 where exists H0XB9 gene and COLlAl gene, so H0XB9 gene and COLlAl gene are considered to be candidate genes of CDH. Further studies on mutation screening of the homeobox of H0XB9 gene in CDH patients and association analysis between CDH and PCOL2 and Spl polymorphisms of the COLlAl gene would be of great value to elucidate the pathogenesis of CDH.Methods1. Genetic epidemiology of CDHA case - control study including 444 pairs of CDH proband and normal control pedigrees was performed to analyze the effect of genetic factor and other risk factors on the liability of CDH. Logistic regression model was used for multifac-tor analysis, and Falconer Threshold Model was utilized to estimate the heritabil-ity.2. Mapping of susceptibility gene of CDHA microsatellite DNA marker D17S1820 in the region of chromosome 17q21 where exists H0XB9 gene which regulates the embryonic limb development and COLlAl gene was chosen. Genotypes of 303 members in 101 CDH nuclear family trios were analyzed by the techniques of polymerase chain reaction ( PCR) and denaturing polyacrylamide gel electrophoresis. Then transmission disequilibrium test (TDT) was used to test the data of genotypes.3. Mutation analysis of the homeoboxPolymerase chain reaction - single strand conformation polymorphism (PCR -SSCP) was used to detect the mutation of exon 2 of #0X49 , H0XB9 and H0XD9 genes in 130 CDH patients.4. Association between polymorphisms of PC0L2 and Spl and CDHThe PC0L2 polymorphism ( - 1997 G/T) in the promoter and Spl poly-morphism (1546 G/T) in the intron 1 of COLlAl gene were genotyped in 81 CDH nuclear families by the technique of polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP) , and then TDT was used to analyze the data of genotypes.5. Distribution of PCOL2 polymorphism in various populations 156 Chinese North populations were chosen to analyze the genotype and al-lele frequency distributions, and then to perform Hardy - Weinberg Equilibrium test. The significant differences of genotype and allele frequency distributions were detected between the Chinese North population and Spanish Caucasian population, and the Chinese North population and American Caucasian population by x2 test.Results1. Genetic epidemiology of CDH42 CDH probands in 444(9. 46% ) had the CDH history, compared with only 7 controls in 444( 1. 58% ). The odd ratio ( OR) and 95% confidence interval (95% CI) of the CDH family history were slightly lower in multifactor a-nalysis (5.209, 95%CI = 12.284 ~2.209) than those in monofactorial analysis (6. 522, 95% CI = 14. 684 ~2. 897) , but still suggesting that it was significantly related to CDH. Additionally, OR value and 95% CI of breech position and swaddling position in extension and adduction were 3. 504 (5. 626 ~2.182) and 4.842 (6. 507 ~3. 604) , respectively. No association was identified between Cesarean section and CDH(P =0. 579). The prevalence rate of the first - degree and the second - degree relatives in proband group was significantly higher than that in control group, and their OR value and 95%CI were 25.441 (188. 150 -3.440) and 3.453 (10.502-1. 135) , respectively. The order of prevalence rate was as follows: first - degree relatives > second - degree relatives > third - degree relatives > control relatives, the heritability of liability to CDH was 67. 88% ±5. 12% in the first - degree relatives, 48. 53% ± 11.06% in the second - degree relatives, 31.82% ±42.89% in the third -degree relatives, and their weighted mean heritability was 41. 74% ±4. 62%.2. Mapping of susceptibility gene of CDH12 alleles and 42 genotypes were observed at D17S1820 polymorphysm. The heterozygosity was 0. 7970. Transmission disequilibrium was identified between CDH and the fourth allele of D17S1820 (X2 = 6.025, P = 0.022 ) , suggesting that CDH is significantly associated with the fourth allele of D17S1820.3. Mutation analysis of the homeoboxNo mutation in exon 2 oiH0XA9 , H0XB9 andH0XD9 genes was identified in 130 CDH patients.4. Association between polymorphisms of PCOL2 and Spl and CDHNo significant association was observed between CDH and PCOL2 polymorphism (x2=0.381,P=0. 537 ). For the 120 individuals tested, no polymorphic restriction enzyme site of Spl polymorphism was identified in our sample, and the allele s was absent.5. Distribution of PCOL2 polymorphism in various populationsThe genotype distribution of PCOL2 polymorphism in 156 Chinese North populations is as follows; GG 35.9% , GT 53. 8% , TT 10. 3%. No significant difference between the observed and expected numbers of different genotypes was found, suggesting that the population was under Hardy - Weinberg Equilibrium. Genotype and allele frequency distributions in Chinese North population were significantly different from those in Spanish Caucasian population, and American Caucasian population.Conclusion1. CDH conforms to the characteristics of polygenic disorder. Genetic factor plays an important role in the liability of CDH. The breech position and swaddling position in extension and adduction are two crucial environmental risk factors of CDH. Cesarean section is not the risk factor of CDH.2. CDH is significantly associated with the region of chromosome 17q21. H0XB9 gene and COLIA1 gene are candidate genes of CDH.3. No pathogenic mutation of CDH may exist in the homeobox of H0XA9 , H0XB9 and H0XD9 genes.