Plamondon Jenna A, Harris Muriel J, Mager Dixie L, Gagnier Liane, Juriloff Diana M
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Birth Defects Res A Clin Mol Teratol. 2011 Aug;91(8):716-27. doi: 10.1002/bdra.20788. Epub 2011 Mar 7.
The A/WySn mouse strain with 15 to 20% penetrance of cleft lip and palate (CLP) is an animal model for human multifactorial CLP. The CLP is due to two unlinked genes that interact epistatically, Wnt9b(clf1) and clf2, plus a maternal effect. The Wnt9b(clf1) mutation is an IAP transposon insertion. The clf2 gene, with unknown function, was located in a 13.6 Mb region of chromosome 13 containing 145 genes.
To reduce the clf2 candidate region, 1146 mice segregating for A/WySn and C57BL/6J alleles at clf2 were screened for recombinants by simple sequence-length polymorphism haplotypes; recombinants' testcross progeny were typed for CLP and simple-sequence length polymorphisms. To identify the function of clf2, the effect of clf2 genotype on risk of CLP was tested in Wnt9b(null/null) knockouts and in compound mutants (Wnt9b(clf1/null) ), and the methylation of the IAP at Wnt9b was assayed in the Wnt9b(clf1/null) mutants by combined bisulfite restriction analysis.
The location of clf2 was redefined to 3.0 Mb between Cntnap3 and AK029746 containing 48 genes, of which 30 are Zfp genes. The clf2 genotype had no detectable effect on Wnt9b(null/null) embryos, but strongly affected risk of CLP and methylation of the IAP in Wnt9b(clf1/null) embryos. CLP was associated with low levels of methylation of the IAP.
The clf2 gene is the first identified polymorphism that affects the epigenetic methylation and silencing of IAP retrotransposons. This CLP model raises the question of whether parallel epigenetic factors are involved in risk and environmental sensitivity of human CLP.
A/WySn小鼠品系具有15%至20%的唇腭裂（CLP）外显率，是人类多因素CLP的动物模型。CLP是由两个非连锁基因Wnt9b(clf1)和clf2上位性相互作用，再加上母体效应导致的。Wnt9b(clf1)突变是一个IAP转座子插入。clf2基因功能未知，位于13号染色体上一个13.6 Mb的区域，该区域包含145个基因。
为缩小clf2候选区域，通过简单序列长度多态性单倍型对1146只在clf2位点上分离A/WySn和C57BL/6J等位基因的小鼠进行重组筛选；对重组体的测交后代进行CLP分型和简单序列长度多态性分析。为确定clf2的功能，在Wnt9b(null/null)基因敲除小鼠和复合突变体（Wnt9b(clf1/null)）中测试clf2基因型对CLP风险的影响，并通过联合亚硫酸氢盐限制分析检测Wnt9b(clf1/null)突变体中Wnt9b处IAP的甲基化情况。
clf2的位置重新定义为Cntnap3和AK029746之间的3.0 Mb区域，包含48个基因，其中30个是Zfp基因。clf2基因型对Wnt9b(null/null)胚胎没有可检测到的影响，但对Wnt9b(clf1/null)胚胎的CLP风险和IAP甲基化有强烈影响。CLP与IAP的低甲基化水平相关。
clf2基因是第一个被鉴定出影响IAP逆转录转座子表观遗传甲基化和沉默的多态性基因。这个CLP模型提出了一个问题，即平行的表观遗传因素是否参与人类CLP的风险和环境敏感性。
