Genetic Diagnosis and Counseling of Retinoblastoma

視網膜母細胞瘤（Retinoblastoma，RB）為癌症的抑制基因RB1基因發生突變而導致在視網膜產生惡性腫瘤。為了有利於患者的分類及提供最適當的遺傳諮詢我們建立多步驟檢測流程以檢測RB1基因之突變。多步驟檢測流程包含以DNA sequencing 針對RB1基因的檢測27個exon及其兩側intron區域的突變；Multiplex Ligation-dependent Probe Amplification（MLPA）相對定量的技術平台檢測RB1基因的基因劑量變異；及以MS-MLPA（methylation specific-MLPA）進行RB1基因啟動子甲基化偵測。我們總共收集了63位患者，只提供血液檢體的有41位患者，檢測出RB1基因生殖細胞突變在17位雙側的患者中有16位（94.1%）; 在24位單側的患者中有5位（20.8%）；同時提供腫瘤組織及血液檢體的有22位患者，檢測出RB1基因生殖細胞突變在5位雙側的患者中有5位（100%），在17位單側的患者中有3位為生殖細胞突變7位為體細胞突變（58.8%）。在22個腫瘤組織檢體中，其中有13個（59.1%）檢測出有異型合子性狀消失（Loss of Heterozygosity，LoH）;有一個（4.5%）腫瘤組織檢體檢測出有RB1基因啟動子的甲基化。我們檢測出RB1基因的兩個對偶基因突變，且其中有部分檢體突變符合雙擊假說；另有些檢體檢測出RB1一個對偶基因的突變；仍有檢體沒有檢測出突變。然而，經由MS-MLPA的結果，我們推測這沒有檢測到RB1基因突變的單側腫瘤檢體，還有其他的遺傳機制或基因導致RB腫瘤的產生。藉由基因分析結果給予視網膜母細胞瘤的患者最適當的遺傳諮詢。Retinoblastoma (RB) is a neoplasm of retinal origin caused by mutations in RB1 gene, the retinoblastoma tumor suppressor gene. To facilitate patient classification and to provide proper genetics counseling, we adopted a multistep molecular screening protocol for detecting RB1 mutations. This protocol included DNA sequencing to identify mutations within coding exons and immediate flanking intronic regions, Multiplex Ligation-dependent Probe Amplification (MLPA) to detect RB1 gene deletions or duplications, MS-MLPA (methylation specific-MLPA) to detect methylation the promoter of RB1 gene. We have collected samples from a total of 63 RB patients. In the 41 patients who have provided peripheral blood, we identified germline RB1 mutations in 16 out of 17 bilateral RB patients (94.1%), and 5 out of 24 unilateral patients (20.8%). In the 22 patients who provided both peripheral blood and tumor tissues, we identified germline RB1 mutations in 5 out of 5 bilateral RB patients (100%), 3 out of 17 unilateral patients, and somatic RB1 mutations in 7 out of 17 unilateral patients (58.8%). In the 22 tumor tissues provided, a total of 13 patients (59.1%) showed loss of heterozygosity (LoH), while only one patient was detected with promotor methylation of RB1 gene (4.5%). We have identified biallelic RB1 gene inactivation, and some conformed with "Two-Hits Hypothesis". In some of the samples, only one of the two RB1 mutations was detected. However, there remained some samples with no mutations detected. Nevertheless, from the MS-MLPA result, the absence of detectable RB1 mutations in the unilateral tumors is presumed to be caused by other genetic mechanisms or genes in the development of RB. Through this genetic diagnosis, we hope to provide the most suitable genetic counseling for Retinoblastoma patients