胚胎著床前基因診斷與遺傳性疾病帶因者篩檢及新生兒基因檢測之臨床應用

Abstract

研究目的 隨著基因診斷技術的快速發展，近幾年來有愈來愈多的遺傳性疾病被證實與單一基因變異有關。因此，基因檢測就變得非常重要，可以提供個人基因或是染色體的資訊。目前常見的基因檢測有產前篩檢、帶因者篩檢、胚胎著床前檢測、法醫檢測與新生兒檢測等。然而因為要建立一套標準的基因檢測流程並不容易，所以台灣目前的基因篩檢，仍然侷限於傳統的產前染色體檢查與新生兒先天代謝性與內分泌性疾病基因篩檢等，對於部分普及的基因檢測則仍欠缺。所以如何建立最新的基因篩檢流程並於應用於臨床，成了刻不容緩的重要課題。 研究方法 第一部分：胚胎著床前基因診斷於染色體轉位之應用 本研究收集自2011年至2014年， 46對在第一孕期或第二孕期習慣性與自發性重複流產的夫妻，且夫妻其中一方帶有染色體結構平衡性異常。個案在台灣各地人工生殖中心經過醫師與遺傳諮詢師討論後，接受試管嬰兒療程並執行胚胎著床前基因診斷。這期間總共執行76個週期、365個胚胎切片，利用微陣列比較式基因體雜交分析技術進行胚胎著床前基因診斷。 第二部分：脊髓性肌肉萎縮症帶因者篩檢 本研究為大規模前瞻性的世代人群研究，自2005年至2014年，共10年的時間從台灣25縣市收集139,404位懷孕婦女檢體。利用雙平台基因檢測技術，包括DNA 片段突變分析儀與多重連接探針擴增技術，進行SMN1/SMN2基因定量分析。此帶因者篩檢流程共分成三階段：（1）篩檢懷孕婦女是否為脊髓性肌肉萎縮症帶因者；(2) 若懷孕婦女為脊髓性肌肉萎縮症帶因者，則請配偶進行脊髓性肌肉萎縮症帶因者篩檢；(3) 若夫妻雙方皆為脊髓性肌肉萎縮症帶因者，則建議進行產前診斷。 第三部分：胚胎著床前基因診斷於脊髓性肌肉萎縮症之應用 針對一對雙方皆為脊髓性肌肉萎縮症帶因者的夫妻，利用囊胚期胚胎滋養層細胞切片、全基因體放大技術、微測序基因診斷技術與3個微衛星標記進行連鎖分析，針對1個週期、18個胚胎進行脊髓性肌肉萎縮症基因的胚胎著床前基因診斷，並選擇基因型正常的胚胎植入。 第四部分：遺傳性聽損新生兒基因檢測 本研究針對2011年至2014年於台灣各醫院診所出生的26,764位新生兒，並經由設計專一性引子對進行聚合酵素鏈鎖反應，利用螢光共振能量傳遞與即時核酸定量分析儀進行基因分析。根據台灣群族遺傳性聽損之基因檢體庫，進行常見的四個感覺神經性聽損基因突變點位之基因分析，四個突變點位分別為GJB2基因c.109G>A、GJB2基因c.235delC、SLC26A4基因c.919-2A>G與12S rRNA基因m.1555A>G。 結果 第一部分：胚胎著床前基因診斷於染色體轉位之應用 總共76個週期、365個胚胎進行基因診斷中，其胚胎成功放大率約為89.04% (325/365)。其中315個胚胎進行微陣列比較式基因體雜交分析，染色體劑量為整倍體的比例為26.67% (84/315)，染色體劑量異常的比例為73.66% (231/315)。共有49個週期可提供胚胎植入，平均每個週期可提供植入胚胎為1.11 顆。後續追蹤32對夫妻52個週期，胚胎植入後有15位婦女成功受孕，治療週期懷孕率為28.85% (15/52)。 第二部分：脊髓性肌肉萎縮症帶因者篩檢 我們統計在139,404位懷孕婦女中，共有2,859位為脊髓性肌肉萎縮症帶因者，約每49個人就有一位是脊髓性肌肉萎縮症帶因者，帶因率為2.05%。其中共有2,504位帶因者的配偶回診進行基因篩檢，有58位男性配偶同樣為脊髓性肌肉萎縮症帶因者，帶因率約為 2.32%。這58個具有生下脊髓性肌肉萎縮症患者的高風險家庭中，有49位懷孕婦女進行產前診斷，且有13個胎兒診斷為脊髓性肌肉萎縮症患者。因此在我們研究的台灣族群中，脊髓性肌肉萎縮症的盛行率約為1/10,723。 第三部分：胚胎著床前基因診斷於脊髓性肌肉萎縮症之應用 在單一週期中，胚胎成功放大率約為77.78% (14/18)，其中有10顆胚胎診斷為正常 (71.43%, 10/14)，2顆胚胎為異常 (14.29%, 2/14)，2顆胚胎因為實驗過程中等位基因遺失而造成無法判讀 (14.29%, 2/14)。後續選擇2個正常胚胎植入後成功受孕，並產下一位正常帶因者女孩。 第四部分：遺傳性聽損新生兒基因檢測 在26,764位新生兒中，共有5,253個新生兒 (19.63%) 具有至少一個聽損基因點位突變。總共399個新生兒 (1.49%) 帶有同型合子、複合性同型合子或同質體的基因突變，將有可能導致聽損。在這之中，298個新生兒 (1.11%) 為GJB2基因c.109 G>A同型合子，2個新生兒 (0.01%) 為GJB2基因c.235 delG同型合子、5個新生兒 (0.02%) 為SLC26A4基因c.919-2A>G同型合子、28個新生兒 (0.11%) 為GJB2基因c.109 G>A與c.235delC複合型同型合子，及66個新生兒 (0.25%) 為粒線體12S rRNA基因m.1555A>G同質體或異質體。 結論 在此我們建立四種嶄新的基因篩檢平台，包括胚胎著床前基因診斷於染色體轉位、脊髓性肌肉萎縮症帶因者篩檢、胚胎著床前基因診斷於單基因遺傳疾病及遺傳性聽損新生兒基因檢測。隨著新的基因診斷之不斷更新，檢驗技術成本逐漸降低，基因檢測將可以成為分子診斷的有力工具，期待這些檢測應用於臨床可以幫助更多病人及其家屬。

Introduction With the rapid development of genetic diagnostic technologies in recent years, there are more and more inherited diseases have been linked to single gene variation. Hence, the genetic testing is very important that can provide information about an individual’s genes or chromosomes. Recently, the common genetic tests include prenatal screening, carrier screening, preimplantation testing, forensic testing, newborn screening, and so on. However, because establishment of a standard protocol for genetic testing is not easy, the current genetic testing in Taiwan, still limited to traditional platforms, such as prenatal diagnosis by karyotyping and newborn screening for congenital metabolic and endocrine diseases. Some widespread genetic tests are still not available now. So, how to create the latest genetic screening protocol for clinical use has become an important issue without delay. Materials and Methods PART 1 Preimplantation genetic diagnosis for chromosomal translocation The study included 46 couples with recurrent first-trimester or second-trimester spontaneous miscarriage, who visited the fertility centers in Taiwan from 2011 to 2014, and one of the partners, carries a balanced structural chromosomal anomaly. After discussing with the doctor and genetics counselor, couples underwent in vitro fertilization (IVF), and a total of 365 embryos in 76 IVF cycles were biopsied for preimplantation genetic diagnosis (PGD) by array comparative genome hybridization (array CGH) screening. PART 2 Carrier screening for spinal muscular atrophy A prospective large population-based cohort study of 139,404 pregnancies was investigated in 25 counties of Taiwan during a 10 year period, 2005 through 2014. Two different validated platforms were used for parallel SMN1/SMN2 gene quantification: denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA). A three-stage screening program was used: (1) pregnancies were tested for SMA heterozygosity, (2) if a pregnancy was heterozygous for SMA (carrier status), the paternal partner was then tested, and (3) if both partners were SMA carriers, a prenatal diagnosis was offered. PART 3 Preimplantation genetic diagnosis for spinal muscular atrophy In this part, we report data to identify the SMN1 gene deletion on eighteen clinical embryos obtained from one participating couple, where both partners are heterozygous SMA carriers with 1-SMN1/3-SMN2 genotype. We validated and applied protocol clinically for PGD through the use of blastocyst biopsy, whole genome amplification, mini-sequencing genotype coupling with genetic linkage of SMN gene involving three informative microsatellite markers, and thawed embryo transfer. PART 4 Newborn genetic screening for hereditary hearing impairment This is a prospective population-based cohort study of 26,764 newborns in Taiwan conducted during the period 2011 to 2014. Based on Taiwanese genetic database of hereditary hearing impairment, the newborn genetic screening targeted four deafness-associated mutations commonly found in the Taiwanese population, including c.109G>A of the GJB2 gene, c.235delC of the GJB2 gene, c.919-2A>G of the SLC26A4 gene, and mitochondrial m.1555A>G of the 12S rRNA gene. The newborn genetic screening were performed using polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridization probes in a real-time PCR detection system. Results PART 1 Preimplantation genetic diagnosis for chromosomal translocation A total of 365 embryos from 76 cycles were analyzed, and the overall diagnostic efficiency was 89.04% (325/365). Then 315 embryos were analyzed by aCGH, as well as the euploidy rate and aneuploidy rate were 26.67% (84/315) and 73.33% (231/315), respectively. 49 cycles of thawed embryo transfer (ET) were carried out, and the average transfer embryo was 1.11 pieces. According our record for 52 IVF cycles of 32 couples, there were 15 women got pregnant and pregnancy rate per cycle was 28.85% (15/52). PART 2 Carrier screening for spinal muscular atrophy We found 2,859 individuals with one copy of the SMN1 genotype, recognized to be SMA carriers, among the 139,404 pregnancies screened. The carrier rate in our population was approximately 1 in 49 (2.05%). Of these individuals, 58 couples were at high risk for having offspring with SMA after testing of partners or spouses 2,504 who were also determined to be SMA carriers. Prenatal diagnoses were determined for 49 pregnancies (84.48%), of which 13 (26.53%) fetuses were diagnosed with SMA; the prevalence of SMA in our population was 1 in 10,723. PART 3 Preimplantation genetic diagnosis for spinal muscular atrophy Approximately 77.78% (14/18) of blastocysts were successfully amplified in a single PGD cycle. Among these embryos, ten (72%, 10/14) were diagnosed as unaffected, two (14%, 2/14) as affected, and two embryos (14%, 2/14) had no conclusive diagnosis due to allele drop-out (ADO). Two unaffected embryos were thawed and transferred in the next cycle resulting in a singleton pregnancy, and the birth of a healthy girl who carries the 1-SMN1/3-SMN2 genotype. PART 4 Newborn genetic screening for hereditary hearing impairment Of the 26,764 newborns, a total of 5,253 (19.63%) babies were found to have at least 1 mutated allele on the newborn genetic screening for hearing impairment. A total of 399 newborns (1.49%) carrier either homozygous, compound heterozygous or homoplasmic mutations in targeted gene, who may potentially have hearing loss. 293 (1.11%) of whom were homozygous for GJB2 c.109 G>A, 2 (0.01%) were homozygous for GJB2 c.235delG, 5 (0.02%) were homozygous for SLC26A4 c.919-2A>G, 28 (0.11%) compound heterozygous for GJB2 c.109 G>A and c.235delC, and 66 (0.25%) homoplasmic or heteroplasmic for m.1555A>G in 12SrRNA gene. Conclusion Here we established four novel genetic testing platforms, including PGD for chromosomal translocation, carrier screening for SMA, PGD for single gene disorder and newborn genetic screening for hereditary hearing impairment. With constantly improved technology and drastically reduced cost, genetic testing can be a powerful tool for achieving molecular diagnosis. We hope the application to clinical can help more and more patients and families.