RB患者及家庭成员Rb基因突变和患病风险的基因诊断及遗传咨询

目的建立Ｒｂ基因突变的基因诊断方法，正确估计视网膜母细胞瘤（ＲＢ）患者的预后及其家庭成员的患病风险。方法综合利用Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交、ＳＳＣＰ分析、直接ＤＮＡ序列测定等多种分子生物学技术作染色体单体型分析及Ｒｂ基因点突变的直接检测。结果７９个有Ｒｂ基因突变的ＲＢ家系中２５例先证者仅查出体细胞起源的Ｒｂ基因突变，其余５４例存在生殖细胞起源的Ｒｂ基因突变，其中３６例突变是新产生的，１５例突变由亲代遗传而来，此外，尚有３例Ｒｂ基因突变嵌合体。结论直接检测Ｒｂ基因点突变可不依赖于患者家庭成员ＲＢ发病情况的遗传背景资料诊断患者是否属于遗传型，正确估计患者的预后；并能在肿瘤发生前甚至产前检出Ｒｂ基因突变携带者，正确估计患病风险

GENE DIAGNOSIS AND GENETIC COUNSELLING OF Rb GENE MUTATIONS IN RETINOBLASTOMA PATIENTS AND THEIR FAMILY MEMBERS

Objective To develop a diagnostic test for direct identification of disease causing mutation in the patients with retinoblastoma and correct prediction of carrier status in unaffected adults and newborns in the RB kindred.Methods Southern blot hybridized by Rb cDNA and other intragenic probes were used to detect big detetions or rearrangements at Rb gene locus. SSCP analysis and direct sequencing of primer directed enzymatic amplification to identify point mutations as small as a single nucleotide change. RFLPs and VNTRs within the Rb gene were used as genetic markers for haplotype analysis.Results The probands from 79 RB kindreds were identified to have Rb gene mutation, including 25 somatic mutations and 54 germline mutations (36 new germline mutations, 15 inherited mutations and 3 mosaicisms). The WBC DNAs from their family members were also analyzed for determining origin and carrier of mutation.Conclusion The direct identification of causing cancer mutations by combining SSCP analysis and direct DNA sequencing showed many advantages than other indirect methods such as haplotype analysis. It can distinguish hereditary RB from nonhereditary RB and identify the unaffected carriers without family history and informes affected family member. This method is helpful in gene diagnosis and genetic counselling.