QTL Mapping For Modifier Loci Determinant The Severity Of Symptoms Of Hirschsprung Disease In Ednrb^yzcm Mice

Hirschsprung Disease is also known as colonic aganglionosis, its incidence ranked only second to anorectal malformation among all the congenital malformations of digestive tract, having a tendency of familial incidence. This disease is characterized with the deficiency in ganglion cell in the diseased intestinal segment, due to which, the segment is in a state of spasm and stenosis, and thus deprived of the peristalsis and defecation and other functions, resulting in an accumulation of excrement and gas in the intestinal canal, and then a secondary onset of dilation and pachynsis. Through the method of ENU mutagenesis, this laboratory had obtained a case of mice with recessive hereditary Hirschsprung Disease, by the use of acetylcholine esterase staining and intestinal canal tissue HE staining, a deficiency in ganglion cell in the abnormal intestinal segment had been discovered. It was discovered by the mutant gene positioning and identification that, the 857th basic group presented a transformation from T to C in the coding region of endothelin receptor B gene （Ednrb）, resulting in a replacement of the 286th leucine with pro line (this mice was named as Ednrb^mlyzcm). It was discovered by us during the observation on the B6 homozygote obtained from intercross of C57BL/6J （B6） that, the severity of Hirschsp rung Disease of mice of various homozygotes in the context of B6 was similar. It was discovered by the observation on the F2 mutant homozygote obtained from the intercross of [C57BL/6J （B6）×DBA/2J（D2）]F1 that, the severity of Hirschsprung Disease of mice differed from each other, indicating the existence of effect of modifying genes. This study took the above-mentioned mutant line of mice as an object, and started with the following three parts:1. Microsatellite marker siftingBy the use of MGI polymorphic marker database （http://www. informatics.jax.org/）, on the 19 pairs of autosome of mice,110 microsatellite markers with a mean genetic distance of 15cM （centimorgan） or so and a difference in basic group between B6 and D2 which was over 10bp had been selected for microsatellite conditional grope. It was shown by the result that,82 of these 110 microsatellite markers could be used for QTL positioning.2. QTL preliminary positioning93 cases of young rat with F2-generation homozygote Hirschsprung Disease had been determined by the method of enzyme digestion, within 5-10 days after birth, the whole intestine （from the duodenum to the end of rectum） of mice had been taken for acetylesterase staining, phenotypic value was represented by the length ofN intestinal segments having deficiency/the length of whole intestine, the method of whole-genome scan had been applied,82 pairs of well-sifted microsatellite primer were used to respectively conduct a preliminary QTL analysis on the DNA samples of 93 cases of F2-generation mice, it was shown by the result that, the chromosome No.11 and No.15 had two locus whose threshold was over 0.630, and LOD value over 2.52, indicating the existence of QTL locus influencing the phenotype of Hirschsprung Disease, and it was located near the Dl11Mit41 and D15Mit265 locus of microsatellite.3. Part of QTL precise positioningThe quantity of young mouse with F2-generation homozygote Hirschsprung Disease was increased to 152 cases, the chromosome No.11 and 15 had been precisely positioned, it was shown by the result that, the LOD value near D11Mit41 was 5.36, the one near D11Mit338 was 3.83, the one near D11Mit226 was 1.92, the one near D15M265 was 4.92. It was shown by the result of single-gene QTL linear-regression analytical statistics that, near the D11 Mit226、 Dl11Mit41 and D11Mit338 focus, the mice whose genotype was BB-type had a relatively severe Hirschsprung Disease among the F2-generation homozygote mice, while the ones whose genotype was BD-type had a clearly less severe Hirschsprung Disease, the ones whose genotype was DD-type had the least severe Hirschsprung Disease, near the D15Mit265 locus, the ones whose genotype was BB-type or DD-type had a relatively severe Hirschsprung Disease, while the ones whose genotype was BD-type had a clearly less severe Hirschsprung Disease, indicating that, after the introduction of DBA context to the mice with Hirschsprung Disease in the context of B6, the interaction between genes led to a less severe Hirschsprung Disease.