以先天中樞性換氣不足症 為疾病模式建立新生兒基因篩檢技術

Abstract

研究目的 近幾年來，隨著基因診斷技術的快速發展，有愈來愈多的疾病被證實與基因突變有關。由於要從傳統的新生兒篩檢血片中萃取高品質的DNA以供後續的分析使用並不容易，所以台灣目前的新生兒篩檢，仍局限於先天代謝性與內分泌性疾病，對於單一基因疾病之篩檢則付之闕如。所以，如何從新生兒篩檢血片中萃取高品質的DNA並建立新生兒基因篩檢技術，成了刻不容緩的重要課題。 先天中樞性換氣不足症（Congenital central hypoventilation syndrome；CCHS）是一種罕見的遺傳疾病，其特徵為睡眠時出現不正常之自主神經系統所控制之呼吸行為，使患者陷入缺氧危機而不自知。由於其發病最常發生於新生兒時期，且大部分沒有家族史，故某些嬰兒猝死個案可能與此症有關。過去此疾病的診斷方法，主要依據為︰1999年由美國胸腔科醫學會(American Thoracic Society)針對此症提出之臨床診斷方法。近年來，在神經發育時期之轉錄因子PHOX2B基因之突變，被認為是先天中樞性換氣不足症的致病基因。其遺傳模式為自體顯性遺傳，其突變方式可分為兩大類︰第一類為polyalanine repeat expansion mutation （PARM）佔92%，在正常的allele上會有一段20個alanines 之重覆片段，而突變的allele上其alanines之重覆次數將增加為24至33次（genotypes : 20/24-20/33）；第二類 為non-PARM（NPARM）約佔8%，包含missense、nonsense、frameshift突變。由於此病症若能及早診斷及早予以呼吸輔助治療，待患者長大後，病症將漸漸緩解。而若能將基因診斷提早至新生兒時期，將可拯救寶貴的生命，並大幅改善預後。故我們希望能以先天中樞性換氣不足症為疾病模式，從新生兒篩檢血片中萃取穩定的DNA，建立新生兒基因篩檢技術。 研究方法 第一部份 首先，我們進行了60個來自台灣各醫院依臨床症狀被懷疑為先天中樞性換氣不全症之患者或出生後發生缺氧甚至因而死亡之新生兒﹔及其中被確定為具PHOX2B基因突變患者之27名親屬﹔其血液檢體之PHOX2B基因檢測。利用聚合酶連鎖反應（Polymerase Chain Reaction : PCR）、毛細管電泳(capillary electrophoresis : CE)以及直接基因定序，研究是否可找到PHOX2B基因的突變點位及台灣是否有特定好發之突變區域。 第二部份 我們以1520名正常人血液檢體進行PHOX2B基因之PARM檢測。採用PCR、CE及直接基因定序之分生技術來完成，用以測試PARM篩檢技術的穩定性。 第三部份 我們完成了3480 個新生兒篩檢血片之PARM檢測，這其中包含QIAcard 、Whatman 903、及IDBS這三種市售標榜可萃取DNA之新生兒基因篩檢血片。應用磁珠分離萃取微量DNA的技術，結合PCR、CE及直接基因定序之分生方法，研究新生兒基因篩檢的可行性。 結果 在60個以臨床病症被懷疑為先天中樞性換氣不全症之患者或出生後發生缺氧甚至因而死亡之新生兒中，10名有PARM突變，2名有NPARM 之frameshift突變。其PHOX2B基因突變陽性率為20% (12/60)。而在1520名正常人及3480名新生兒共5000人中，沒有發現任何PARM突變，但有一部份人存在polyalanine contraction；其中(GCN)20佔95.01%，(GCN)15佔4.25%，(GCN)14佔0.04%，(GCN)13佔0.55%，(GCN)7佔0.14%，(GCN)6佔0.01%。 結論 我們成功的建立了台灣在先天中樞性換氣不足症的新生兒基因篩檢技術，將基因診斷的時機點提前至新生兒時期，若能即早診斷即早治療，將可大幅改善此症之預後。我們找到的所有PHOX2B基因突變點位皆位於polyalanine repeat附近的區域，這顯示此區域為台灣族群PHOX2B基因突變的熱區。我們的先天中樞性換氣不足症之新生兒基因篩檢方法，主要針對polyalanine repeat附近的區域進行篩檢，這可以檢測出所有具有PARM的患者，及位於此區域之NPARM患者。此外，QIAcard 、Whatman 903、IDBS﹔這三種市售新生兒篩檢血片皆可以提供高品質、高穩定度的DNA樣本來源，供後續的萃取與分析使用，且可被長期保存再使用。這項新生兒基因篩檢技術將來可以被廣泛的應用於其它的單一基因疾病，例如耳聾基因等發生率更高的疾病。

Introduction : In recent years, with the rapid development of genetic diagnostic technologies, more and more diseases have been linked to genetic mutation. Because isolation of high quality DNA from dried blood spots on traditional filter paper blotters for subsequent DNA analysis is not easy, Taiwan's current newborn screening is still limited to congenital metabolic and endocrine diseases. Newborn genetic screening is still not available now. So, how to create a newborn genetic screening technology, has become an important issue without delay. Congenital central hypoventilation syndrome (CCHS) is a rare neurological disorder characterized by abnormal autonomic central nervous system control of breathing during sleep. In the past, CCHS was diagnosed on the basis of clinical criteria proposed by the American Thoracic Society in 1999. CCHS is characteristically diagnosed in the newborn period almost without family history. Some cases of sudden infant death may be related to this disease.The paired-like homeobox gene PHOX2B, which is active during neuronal development, is the disease-defining gene for CCHS. Approximately 92% of individuals with the CCHS phenotype will be heterozygous for a polyalanine repeat expansion mutation (PARM); the normal allele will have 20 alanines and the affected allele will have 24–33 alanines (genotypes 20/24–20/33). The remaining 8% of individuals with CCHS will have a non-PARM (NPARM) in the PHOX2B gene; these will be missense, nonsense, or frameshift. CCHS is inherited in an autosomal dominant manner with a stable mutation.

Early detection of CCHS is important because of the significant morbidity, especially neurologic consequences, and the risk of death in the undiagnosed subject. We hope to use congenital central hypoventilation syndrome as a disease model to establish newborn genetic screening technology. Materials and Methods Part 1 First, genomic DNA was collected from peripheral whole blood of 60 clinical suspected CCHS patients; newborn babies with hypoxia even death and their 27 family members from the National Taiwan University Hospital and the referring hospitals. We used PCR, CE, and direct sequencing to find out mutation points of PHOX2B gene. Part 2 1520 health individuals’ peripheral whole blood was obtained and analyzed for PARM, using PCR, CE, and direct sequencing techniques, to test the stability of our PARM screening techniques and to investigate polyalanine polymorphism in normal population. Part 3 Finally, We completed 3480 PARM tests of DNA from dried blood spots on filter paper blotters including QIAcard, Whatman 903, and IDBS; these three commercially available cards. We adopted magnetic beads separation of trace DNA techniques, combined with PCR, CE, and direct sequencing to study the feasibility of newborn genetic screening.

Results In 60 clinical suspected CCHS patients; newborn babies with hypoxia even death, 10 individuals have polyalanine expansions, and 2 individuals have frameshift mutations. The PHOX2B mutation rate is 20% (12/60). The polyalanine expansion mutation is not found in 1520 health individuals and 3480 newborn babies in our population study. There are some types of polyalanine contraction found in these 5000 individuals. With a frequency of 95.01% in the sample population, (GCN)20 is the most common allele in the 20-residue polyalanine domain while (GCN)15 is second in the order of allele prevalence with a frequency of 4.25%. Additionally, the (GCN)14, (GCN)13, (GCN)7 and (GCN)6 alleles ( 0.04%,0.55%,0.14%,and 0.01%, respectively ) are also identified in the sample.

Conclusion We successfully establish the newborn genetic screening technology in CCHS to advance genetic diagnosis to the time point of the neonatal period. With early treatment of this disease, the prognosis will be greatly improved. We found all the PHOX2B gene mutation points are located in the area around the polyalanine repeats, which shows this area as the Taiwanese population PHOX2B gene mutation hot spot. Our newborn PHOX2B screening methods focus on the polyalanine repeat region, which can detect all patients with PARM and the NPARM patients located in this region. In addition, QIAcard, Whatman 903, IDBS; these three commercially available filter paper blotters can provide both high quality and high stability of the DNA sample source for the extraction and subsequent analysis, and can be stored for a long time for re-use. This newborn genetic screening technology will be widely used in other single gene disorders in the future, such as a higher incidence of deafness genes.