非侵入性產前胎兒顯性遺傳疾病篩檢平台之建立

Abstract

傳統的產前檢查是透過侵入性的方式取得胎兒羊水、絨毛，進行染色體或基因檢測。這樣的檢查準確性高也是產前檢查的標準方式。然而侵入性的採樣可能有流產的風險，因此許多研究致力於尋找更安全的產檢途徑。母血唐氏症篩檢的開發，達成了檢測安全的目標，但準確率約為80% ~ 85%。1997年盧煜明教授在懷孕婦女的血液中發現含有胎兒的游離DNA (cell-free fetal DNA, cffDNA)，並搭配次世代定序 (Next Generation Sequence, NGS) ，在2008年建立了非侵入性產前染色體篩檢 (Non-invasive prenatal screening, NIPS)。NIPS對染色體非整倍體的檢查具有高度準確性，可用於妊娠早期篩檢，且無風險。而染色體數目異常、染色體結構異常及單基因缺陷皆會造成胎兒先天性異常，現行NIPS局限於染色體數目及微片段缺失的篩檢。因此，本實驗研究目的是建立以聚合酶連鎖反應之增幅序列 (Amplicon-PCR based) 與搭配NGS平台，應用於非侵入性產前胎兒顯性遺傳疾病的篩檢。試驗對象為父親具有體染色體顯性遺傳疾病之胎兒，或是胎兒因超音波異常，疑似患有自發性突變之顯性遺傳疾病。採集父母血液之後，利用Amplicon-PCR based NGS定序，並使用生物資訊分析數據判讀檢驗結果，再透過常規之產前單基因檢驗 (使用sanger sequence平台) 進行胎兒突變點位確認。本研究顯示母血血漿DNA (Plasma DNA) 試驗結果與胎兒檢體之檢驗結果具有一致性。依據本研究試驗者的檢測因素，提供孕婦客製化的非侵入性產前胎兒家族顯性遺傳疾病篩檢，以及開發非侵入性產前胎兒骨骼發育異常篩檢之檢驗平台，為有檢驗顯性遺傳疾病需求之孕婦，帶來早期篩檢、安全又準確的檢驗方法；並給予個案家庭更充裕的時間因應胎兒的狀況。

To obtain adequate amniotic fluid or villus samples usually needs an invasive procedure for the prenatal diagnosis. The risk for miscarriage or preterm premature rupture of membranes (PPROM) after the procedure is a challenge for a safe genetic test. The detection rate of non-invasive maternal serum screening for Down syndrome is around 80 ~ 85%. In 1997, Lo et al. identified cell free fetal DNA (cffDNA) from the maternal plasma. In combination with next generation sequencing (NGS), they established a platform, non-invasive prenatal screening (NIPS) for early non-invasive genetic test subsequently. NIPS can detect the fetal aneuploidies, chromosome imbalance, copy number variations and even single gene disorders, which cause a significant phenotype. Currently, the chromosome aneuploidies and microdeletions could be detected with high accuracy. Based on the principle of NIPS, the study tried to combine the amplicon-PCR based NGS to detect a sequence variant from autosomal dominant diseases. In addition, the platform was also employed to detect a mutation in fetus with abnormalities in ultrasonography such as bony defects. As soon as the variant was found, the confirmation sequencing using the samples from amniotic fluid, villus and cord blood from the same fetus or mother were performed. In all our families with an affected father, the results from both the NIPS and the conventional sequencing are consistent. Although the parents were healthy, NIPS can also detect the mutations from fetus with achondroplasia, crouzon syndrome, or osteogenesis imperfecta. Moreover, the NIPS findings can further be validated by conventional sequencing. Thus, we highly recommend that NIPS combining amplicon-PCR with NGS is a useful and safe platform for prenatal diagnosis to detect a fetal mutation from the affected parents or a de novo mutation in the candidate genes, which are selected in accordance with the fetal phenotypes.