Genetic Linkage Analysis by Mitotic Recombination

描述（由申请人提供）：在开发方面取得的巨大进步 遗传性疾病的治疗和治愈将来自于识别 导致这些疾病的基因突变。对于罕见的遗传疾病， 家庭谱系是稀缺的，遗传连锁和定位克隆， 困难、低效和/或不可能。 进行性骨化性纤维发育不良（FOP），是最致残的形式， 异位骨化为人类所知，是一种非常罕见的常染色体显性遗传病 低生殖适合度遗传病及其亲本遗传 家庭中的孩子很少见到。FOP的特点是骨骼 大脚趾畸形和骨形成的渐进诱导 在异位部位。BMP 4 mRNA和蛋白在淋巴细胞中过表达， 来自FOP和BMP 4过表达患者的病变细胞作为一种 可靠的分子标记BMP 4基因没有突变， FOP和BMP 4基因座已被排除在连锁的条件。 虽然FOP与染色体4 q27 -31位点存在遗传连锁， 在四个小家庭的分析中，这些数据在预测中的可靠性 遗传位点和连锁中突变基因的鉴定 轨迹不确定。多代同堂家庭的缺乏也使得 怀疑的保真度的定位，因为遗传异质性可能 存在. 我们的FOP研究小组协调一个整合的全球医生网络 负责全球FOP患者的护理。即使有这种 但是，不太可能会有更多的多代家庭。 鉴定用于进一步的连锁分析（减数分裂重组）。我们 因此，他们设计了一种新的和创新的替代方法， 体细胞中的重组。通过增强有丝分裂的产生 基因产生的重组后代细胞，这种方法将产生大量 来自单个BMP 4过表达“亲本”FOP细胞的细胞假家族 线体细胞重组会造成突变染色体的丢失 在细胞“后代”的子集中的基因座，其将通过免疫组织化学鉴定。 BMP 4过表达（FOP表型的标志物）的丧失。的 染色体重组位点将通过缺失 这些微卫星标记的杂合性（洛） “后代”细胞，并将促进高度集中和靶向的定位， 克隆方法来鉴定FOP患者中的突变基因位点。

DESCRIPTION (provided by applicant): Great advancements in developing therapeutics and cures for inherited disorders will result from identification of the gene mutations that cause these disorders. For rare genetic disorders, family pedigrees are scarce and genetic linkage and positional cloning is difficult, inefficient and/or impossible. Fibrodysplasia ossificans progressiva (FOP), the most disabling form of heterotopic ossification known to mankind, is a very rare autosomal dominant genetic disorder with low reproductive fitness and its inheritance from parent to child in families is rarely seen. FOP is characterized by skeletal malformations of the great toes and by progressive induction of bone formation at ectopic sites. BMP4 mRNA and protein are over-expressed in lymphocytes and lesional cells from patients who have FOP and BMP4 over-expression serves as a reliable molecular marker for the condition. The BMP4 gene is not mutated in FOP, and the BMP4 locus has been excluded from linkage to the condition. Although FOP has been linked to chromosome locus 4q27-31 by genetic linkage analysis in four small families, the reliability of these data in predicting the genetic locus and the identification of the mutated gene within the linked locus is uncertain. The paucity of multi -generational families also renders doubt about the fidelity of the localization, as genetic heterogeneity may exist. Our FOP research group coordinates an integrated global network of physicians who are responsible for the care of FOP patients worldwide. Even with this resource, it is unlikely that additional multigenerational families will be identified for use in further linkage analyses (meiotic recombination). We therefore have devised a novel and innovative alternate approach using mitotic recombination in somatic cells. By enhancing the production of mitotic ally-generated recombinant progeny cells, this method will create a large cellular pseudo-family from a single BMP4 over-expressing "parent" FOP cell line. Somatic cell recombination will create a loss of the mutant chromosomal locus in a subset of the cellular "progeny", which will be identified by the loss of BMP4 over-expression (the marker for the FOP phenotype). The chromosomal site of recombination will be identified by the loss of heterozygosity (LOH) of chromosomal microsatellite markers within these "progeny" cells, and will facilitate a highly focused and targeted positional cloning approach to L identify the mutated gene locus in FOP patients.