Genetic Linkage Analysis by Mitotic Recombination

通过有丝分裂重组进行遗传连锁分析

• 批准号: 6441323
• 负责人: FREDERICK Samuel KAPLAN
• 金额: $ 7.93万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-28 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6441323
• 关键词: autosomal dominant trait; biomarker; biotechnology; bone morphogenetic proteins; cell line; connective tissue; disease /disorder etiology; flow cytometry; gene mutation; genetic mapping; genetic models; genetic recombination; human genetic material tag; loss of heterozygosity; lymphoblast; progressive myositis ossificans; technology /technique development; transfection

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.

描述(由申请者提供)：在开发方面的巨大进步 遗传性疾病的治疗和治疗将源于识别 导致这些疾病的基因突变。对于罕见的遗传性疾病， 家系很少，遗传连锁和位置克隆很少 困难、低效和/或不可能。 进行性骨化性纤维发育不良(FOP)，最致残的骨化性纤维发育不良 异位骨化是人类已知的一种非常罕见的常染色体显性遗传 生殖适合度低的遗传性疾病及其父母遗传 在家庭中生孩子是很少见的。FOP的特点是骨骼 拇指畸形和渐进性骨形成 在异地异地。Bmp4的mRNA和蛋白在淋巴细胞和 FOP和BMP4过度表达患者的皮损细胞可作为一种 该条件下可靠的分子标记。BMP4基因不会发生突变 FOP，并且BMP4基因已被排除与该疾病的连锁。 虽然FOP已通过遗传连锁与染色体4q27-31连锁 在四个小家庭中进行分析，这些数据在预测中的可靠性 连锁的遗传位点及突变基因的鉴定 轨迹尚不确定。多代家庭的匮乏也导致了 怀疑定位的保真度，因为遗传异质性可能 是存在的。 我们的FOP研究小组协调了一个完整的全球医生网络 谁负责全球FOP患者的护理。即使有了这个 资源，不太可能有更多的多代家庭 确定用于进一步的连锁分析(减数分裂重组)。我们 因此，我们设计了一种新颖而创新的替代方法，使用有丝分裂 体细胞中的重组。通过促进有丝分裂的产生 ALL生成的重组子代细胞，这种方法将产生大量的 来自单个过表达BMP4的亲本FOP细胞的细胞伪家族 排队。体细胞重组会造成突变染色体的丢失 在细胞“后代”的子集中的位置，它将由 BMP4过度表达缺失(FOP表型的标志)。这个 重组的染色体位置将通过丢失 这些染色体微卫星标记的杂合性(LOH) “子代”细胞，并将促进高度集中和有针对性的位置 L的克隆方法确定FOP患者突变的基因座。