Genetic Heterogeneity and Protein Function in DBA

Diamond Blackfan贫血（DBA）是一种先天性贫血，发生在出生时或出生后不久，是由于红细胞及其前体的生产失败，骨髓和血小板谱系正常或接近正常。患者可能完全依赖皮质类固醇或对治疗产生耐药性，然后需要定期输血，或者如果有组织相容的兄弟姐妹供体，则需要进行骨髓移植。DBA患者患白血病和其他恶性肿瘤的风险增加。DBA在大约10- 15%的病例中是遗传的，主要是常染色体显性遗传。最近的遗传学研究令人惊讶地发现，在大约25%的家族性和散发性病例（DBA1）中，染色体19q13.2上的核糖体蛋白基因RPS19发生了突变。对多重DBA家族的连锁分析显示，在大约40%的家族中存在8p染色体上的另一个基因（DBA2），而其他谱系没有显示与8p或19q染色体有连锁的证据，这表明进一步的遗传异质性。本研究的长期目标是鉴定和分离DBA2基因。因此，具体目标是：(1)通过确定更多的家族来进一步确定染色体8p遗传图谱，通过连锁和单倍型分析来搜索侧翼区域的重组，并通过细胞遗传学技术筛选缺失和易位；(2)利用基因的cDNA阵列和关键区域的表达序列标签（est），通过检测候选基因在红细胞中的表达模式来确定候选基因，并将正常和患者基因组DNA的杂交与这些阵列进行比较，寻找杂合性的缺失；(3)通过SSCP、PCR杂合性筛选和序列分析，检测目标1和/或目标2在8号染色体连锁家族突变中的进展所鉴定的候选基因。对引起DBA的其他基因的了解可能为红细胞生成的分子调控和干细胞向红系谱系的过程提供新的见解，并且染色体8p上基因编码的蛋白质可能通过一种新的途径与RPS19相互作用。此外，这些患者患恶性肿瘤的风险增加表明该蛋白可能具有肿瘤抑制作用。因此，分离引起DBA的基因可能不仅对设计针对这些患者的新治疗方法很重要，而且对更好地了解红细胞生成和恶性肿瘤发展的调控也很重要。

Diamond Blackfan anemia (DBA) is a congenital anemia that develops at birth or soon after, and is due to failure of production of erythrocytes and their precursors, with normal or near normal myeloid and platelet lineages. Patients can emit completely on corticosteroids or may become resistant to treatment, and then require regular blood transfusions, or bone marrow transplant if a histocompatible sibling donor is available. DBA patients are at increased risk of developing leukemia and other malignancies. DBA is inherited in about 10-15 percent of cases, mostly as an autosomal dominant. Recent genetic studies have led to the the surprising identification of mutations in a ribosomal protein gene, RPS19, on chromosome 19q13.2, in about 25 percent of both familial and sporadic cases (DBA1). Linkage analysis in multiplex DBA families shows strong evidence for another gene on chromosome 8p (DBA2) in about 40 percent of families, and other pedigrees do not show evidence for linkage to either chromosome 8p or 19q, indicating further genetic heterogeneity. The long term objective of this proposal is to identify and isolate the DBA2 gene. Therefore the specific aims are to (1), further define the chromosome 8p genetic map by ascertaining more families to search by linkage and haplotype analysis for recombinations in the flanking regions, and screen by cytogenetic techniques for deletions and translocations; (2) use cDNA arrays of the genes and expressed sequence tags (ESTs) in the critical region to define candidate genes by examining their pattern of expressed RNAs in erythroid cells, and by comparing the hybridization of normal and patient genomic DNA to these arrays to look for loss of heterozygosity; and (3), test candidate genes identified either as a result of progress in aim 1 and/or aim 2 for mutations in chromosome 8 linked families by SSCP, PCR heterozygosity screening and sequence analysis. Knowledge of additional genes that cause DBA may offer new insights into the molecular regulation of erythropoiesis and the process of stem cell commitment to the erythroid lineage, and it is possible that the protein encoded by the gene on chromosome 8p interacts with RPS19 in a novel pathway. Furthermore, the increased risk of malignancy in these patients suggests that the protein may act as a tumor suppressor. Thus isolating the genes that cause DBA may be important not only for devising new treatment for these patients but also for a better understanding of the regulation of erythropoiesis and the development of malignancy.