原發性肉鹼缺乏症之新生兒篩檢個案的SLC22A5 (OCTN2)基因R254X突變分析

Abstract

原發性肉鹼缺乏症為OCTN2基因突變所引起。目前篩檢原發性肉鹼缺乏症的方式為分析血片游離肉鹼濃度；但是由於受到母體之影響，患嬰體內之肉鹼濃度在出生第二至三天篩檢進行時可能還沒有降的很低，而沒有被篩檢出來。文獻指出，南方中國人有OCTN2基因之常見突變c.981C>T (p.R254X)，因此我們想利用基因分析來提高新生兒篩檢原發性肉鹼缺乏症之檢出率。 本研究之方法為檢測血片游離肉鹼濃度偏低檢體之OCTN2基因p.R254X突變，以協助篩檢的進行。本研究利首先分析348件隨機DNA檢體，探索p.R254X在台灣族群之盛行率；本研究接著分析48件游離肉鹼濃度偏低(≦11µM)之新生兒篩檢血片，進行OCTN2基因p.R254X之突變偵測。 本研究首先發現，血片利用Methanol法、煮沸法、或QIAamp法抽取genomic DNA時，以QIAamp法萃取之實驗結果最好。p.R254X基因分析顯示348件控制組檢體中有2名為p.R254X之異型合子；48件實驗組血片中也有2名為異型合子。因此，控制組及實驗組之帶因頻率分別為1/174及1/24；與文獻報告的帶因頻率相比，控制組無差異，但實驗組帶因率較高(p=0.01996)。 游離肉鹼濃度偏低的新生兒，如果為p.R254X帶因時，其為原發性肉鹼缺乏症之機率將提高。這些新生兒應作進一步追蹤以了解是否罹病。因此，此項方法將可在不提高篩檢偽陽性率的條件下，增加新生兒篩檢偵測原發性肉鹼缺乏症之敏感度。

Mutations in the SLC22A5 gene, which encodes the plasma membrane carnitine transporter OCTN2, cause primary carnitine deficiency (PCD). Currently, PCD is screened in newborns using free carnitine level as a marker. However, owing to the high free carnitine level in the maternal circulation, the affected babies may not have a free carnitine level low enough to be picked up by screening. Previously, the OCTN2 gene c.981 C>T (p.R254X) mutation was found to be common in the Southern Chinese population. Therefore, in this study we want to see if molecular diagnosis could enhance newborn screening of PCD. In this study, we analyzed blood spot DNA OCTN2 gene p.R254X mutation. We first analyzed 348 random anonymous control DNA samples to see the prevance of this mutation in the population. We then analyzed 48 blood spot samples in which free carnitine level was lower than 11µM (the standard cut off for free carnitine was lower than 6.44 μΜ). We found that among the three methods for blood spot DNA extreaction (the methanol method, boiling method, and QIAamp method); the QIAmp method gave the best result. We then found two p.R254X heterozygotes in the 348 control DNA (1in 174); another two p.R254X heterozygotes in the 48 samples with low free carnitine level (1 in 24). In comparison to previous data, newborns with low free carnitine level did have a higher prevalence of OCTN2 gene p.R254X mutation (p=0.01996). Babies who had both low free carnitine and OCTN2 mutation should have a higher chance to be a patient of primary carnitine deficiency, and their disease status should be determined. This method, therefore, increases the sensitivity of detecton of PCD without increase the false positive rate of the screening.