The Gene Dosage Changes of TPTE and BAGE2 and Breakpoint Analysis in Robertsonian Down syndrome

唐氏症是最常見導致智能障礙的疾病，只有百分之四左右的唐氏症屬於羅伯遜轉位型唐氏症。羅伯遜轉位發生在近端著絲粒染色體上，包含了第13，14，15，21和22號染色體。文獻上有幾種機制會產生羅伯遜轉位，最常見的是其中兩條染色體都斷在短臂，然後互相接合，接合後形成兩個中心體的新染色體。除了第21號染色體之外，所有的近端著絲粒染色體在短臂上面都沒有基因分佈。因此我們這個研究目標是當發生羅伯遜轉位時，希望可以找到在第21號染色體短臂上的可能斷點區域。我們從三個醫學中心，前瞻性且連續收集十個羅伯遜轉位型唐氏症患。十個個案中，有六個屬於原發性轉位，另外四個是因為父母親的遺傳。分析起來有四個der(21q;21q)，四個der(14q;21q)，一個der(13q;21q)與一個der(21q;22q)。在六個原發性羅伯遜轉位唐氏症中，五個是母源，只有一個是父源。另外有四個第21號染色體發生接合時, 都是屬於等臂染色體。10K微矩陣比較性基因體雜交技術應用在這十個案例上，發現所有第21號染色體的長臂都有複製，不過並沒有發現任何缺失或增加在其他染色體上。244K微矩陣比較性基因體雜交的解析度更高，在其中一個案例發現偵測21p的基因變化的結果，和即時定量聚合酵素連鎖反應的結果相符合。我們接著應用即時定量聚合酵素連鎖反應偵測每個案例在TPTE和BAGE2上面的基因劑量變化，這兩個基因都位在21p11，另外也偵測位於21q11上面的SAMSN1基因。第21號染色體短臂比長臂的基因拷貝數比例：第21號等臂染色體是1:3，其他三種羅伯遜轉位是2:3。因此，我們初步的研究結果顯示，當第21號染色體發生羅伯遜轉位時，其斷點極有可能介於中心粒與BAGE2之間。Down syndrome (DS) is the most common single known cause of intellectual disability. Only 4% of DS is Robertsonian translocation (ROB) from acrocentric chromosome rearrangement including chromosome 13, 14, 15, 21 and 22. There are some possible mechanisms of formation of the translocation. Union following breakages in both short arms results in a chromosome with two centromeres. Chromosome 21 is the only acrocentric chromosome that has known genes in short arm. The aim of this study was to define the possible breakage area in 21p when ROB occurs. We prospectively and consecutively collected 10 cases ROB DS from 3 medical centers. Of the 10 DS children, 6 were de novo (60%) and 4 were inheritance (40%). There were 4 der(21q;21q), 4 der(14q;21q), 1 der(13q;21q), and 1 der(21q;22q). The origin of the extra chromosome 21q was maternal in 5 of 6 de novo ROB and was paternal in one case. All of the four der(21q;21q) ROB DS were isochromosome. 10K array comparative genomic hybridization (CGH) was applied to 10 cases that showed the whole long arm amplification in chromosome 21. There was no microscopic nor subtelomeric deletion/duplication in other chromosomes. The result of gene dosage change by real-time quantitative polymerase chain reaction (PCR) was compatible with 244K array CGH in one case. We further used real-time PCR to detect the copy number of TPTE and BAGE2 that located in 21p11, SAMSN1 in 21q11. The ratio of copy number in 21p:21q was 1:3 in der(21q;21q), 2:3 in der(13q;21q), der(14q;21q), and der(21q;22q). Our preliminary results demonstrated breakpoint of chromosome 21 involving ROB might be between BAGE2 and centromere, located from 10.1 Mb to 12.3 Mb.Contents 致謝 摘要 Abstract 1. Introduction 1 1.1. General background of Down syndrome 1 1.2. Three genetic types of Down syndrome 1 1.3. Mechanism of Robertsonian translocation 2 1.4. Two genes in the short arm of chromosome 21 2 1.5. Advantage of array CGH 3 1.6. Aim of our study 3 2. Materials and Method 4 2.1. Case selection 4 2.2. Genomic DNA isolation 5 2.3. Molecular genetic analysis 5 2.4. Human Mapping 10K Array 5 2.5. 244k Array CGH experiment 6 2.6. Image Acquisition and Raw Data Processing. 7 2.7. PCR primers 8 2.8. Real-time Quantitative PCR and Calculation of Gene Dosage 8 3. Result 10 3.1. Parental origin and UPD study 10 3.2. Phenotype of Robertsonian Down syndrome 11 3.3. Result of 10K array CGH 11 3.4. Result of 244K array CGH and real-time PCR in one case 12 3.5. Real-time PCR for gene dosage change 12 4. Discussion 14 4.1. Parental origin of ROB DS and isochromosome 21 14 4.2. UPD study for Robertsonian translocation 14 4.3. Phenotype of ROB DS and free trisomy 21 was similar 15 4.4. Array CGH could detect microscopic changes 15 4.5. 10K array CGH showed whole 21q amplification 16 4.6. Probes of TPTE and BAGE2 on 244K array CGH 16 4.7. Real time PCR showed the breakpoint in 21p 17 4.8. Further studies could be planned in the future 18 5. Conclusion 19 6. Reference 20 7. Legends to figures 28 7.1. Figure 1 to Figure 10 28 7.2. Figure 11 28 7.3. Figure 12 28 7.4. Figure 13 29 7.5. Figure 14 29 8. Figures 30 8.1. Figure 1 30 8.2. Figure 2 31 8.3. Figure 3 32 8.4. Figure 4 33 8.5. Figure 5 34 8.6. Figure 6 35 8.7. Figure 7 36 8.8. Figure 8 37 8.9. Figure 9 38 8.10. Figure 10 39 8.11. Figure 11 40 8.12. Figure 12 41 8.13. Figure 13 42 8.14. Figure 14 43 9. Tables 44 9.1. Table 1. General characteristics of 10 cases Robertsonian Down syndrome 44 9.2. Table 2. The parental origin of 10 cases by informative small tandem repeat (STR) markers 45 9.3. Table 3. Phenotype of 10 cases Robertsonian Down syndrome 46 9.4. Table 4. Real-time PCR verification of gene dosage of TPTE, BAGE2, and SAMSN1 by array-CGH in case 2 47 9.5. Table 5. The result of gene dosage and DNA copy number ratio of 21p:21q by real-time PCR 48 10. Addendum 49 10.1. 台大醫院研究倫理委員會通過證明 49 10.2. 長庚醫院人體試驗倫理委員會通過證明 50 10.3. 長庚醫學研究計畫通過證明 51 10.4. Recent Publication 52 10.5. Research Project 5