為單基因血脂異常疾病建立以次世代定序為基礎之基因檢測平台

Abstract

背景及目的：家族性高膽固醇血症（Familial hypercholesterolemia, FH）是一種體染色體顯性遺傳疾病，其發病特徵為患者血漿中總膽固醇與低密度脂蛋白膽固醇(low-density lipoprotein cholesterol)濃度顯著上升，從而導致動脈粥狀硬化及增加患者罹患冠狀動脈性心臟病(coronary heart disease, CHD)的風險。然而，由於缺乏可靠且符合成本效益的基因檢測，在台灣正式確診的家族性高膽固醇血症患者不到1%。高三酸甘油脂血症(Hypertriglyceridemia)是一種常見的脂質代謝疾病,嚴重的高三酸甘油脂血症會導致復發性急性胰腺炎也會增加心血管疾病的風險。我們的目標是為家族性高膽固醇血症及其他單基因血脂異常疾病建立以次世代定序(Next generation sequencing, NGS)為基礎之基因檢測平台。 方法：在家族性高膽固醇血症基因診斷方面，我們設計針對LDLR全基因以及針對APOB和PCSK9基因編碼序列(coding sequence)的探針，並使用Illumina MiSeq次世代定序平台進行雙端定序（2×300 bps）。13個已知疾病相關變異點的DNA樣品（包含3個大片段重複和2個大片段缺失）用於確效整個實驗流程。隨後我們招募了28位無相關的新收指標患者，其收案標準為血清總膽固醇濃度≥ 8.28 mmol/l、低密度膽固醇濃度(LDL-C)≥ 6.37 mmol/l。此外，我們也設計針對20個已知與單基因血脂異常疾病(monogenic dyslipidemia)有關的致病基因的NGS套組，並招募了17位嚴重高三酸甘油脂血症的患者，其收案標準為血中三酸甘油脂濃度測量至少兩次高於11.3 mmol/l。指標患者家屬接受家族基因篩檢(genetic cascade screening)。 結果：我們正確識別了13個用於確效的DNA樣品中所有疾病相關變異點，包含大片段重複及大片段缺失。在28位新收指標患者中，21位患者發現帶有疾病相關變異位點(75%檢出率)；其中5位患者被發現攜帶未被報導過的LDLR c.1186 + 2T> G剪接位點變異。將此五位指標患者及其親屬基因型與表現型比較發現：攜帶LDLR c.1186 + 2T> G組（n = 26）其低密度脂蛋白膽固醇濃度為7.82±2.13 mmol / l與未攜帶組別（n = 25）低密度脂蛋白膽固醇濃度3.18±1.36 mmol / l相比顯著提高( p value <0.0001)。單套型分析 (haplotype analysis)顯示患者LDLR c.1186 + 2T> G起源於共同祖先，揭示了台灣獨特的LDLR突變譜。在嚴重高三酸甘油脂血症基因診斷方面，17位新收指標患者中，13位患者發現帶有疾病相關變異位點(76.5%檢出率)；其中八位指標患者帶有LPL p.L279V (47.1%)，七位患者帶有APOA5p.G185C (41.2%)。 結論：此研究為首次針對家族性高膽固醇血症使用以覆蓋LDLR全基因區域的捕獲探針為基礎的NGS基因檢測，因此能有效地用於偵測結構變異。該家族性高膽固醇血症基因檢測套組可全面且可靠地檢測FH患者LDLR，APOB和PCSK9的致病變異點位。此外，單基因血脂異常疾病的NGS套組亦能有效的偵測多種遺傳性血脂異常疾病，同時使我們更加了解台灣族群高三酸甘油脂血症的遺傳背景。

Background and aims: Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized with high levels of total cholesterol and low-density lipoprotein (LDL) cholesterol in the plasma, causing FH patients to suffer from atherosclerosis and increased risk of coronary heart disease (CHD). However, less than 1% FH patients in Taiwan were formally diagnosed, partly due to the lack of reliable cost-effective genetic testing. Hypertriglyceridemia is a common disorder. Severe hypertriglyceridemia can cause recurrent pancreatitis and also increase risk of cardiovascular disease. We aimed to establish a next-generation sequencing (NGS) platform as the clinical genetic testing method for FH and other monogenic dyslipidemias. Methods: We designed probes to capture the whole LDLR gene and all coding sequences of APOB and PCSK9, and then sequenced with Illumina MiSeq platform (2 × 300 bps). The entire pipeline was tested on 13 DNA samples with known causative variants (including 3 large duplications and 2 large deletions). Then we enrolled a new cohort of 28 unrelated FH patients with serum cholesterol levels ≥8.28 mmol/l and LDL-C ≥6.47 mmol/l. In addition, we also designed another NGS panel for detecting 20 disease-causing genes known to be associated with monogenic dyslipidemia, and recruited 17 patients with severe hypertriglyceridemia. Severe HTG was defined as at least two lipid measurements with TG ≥11.3 mmol/l. Results: From the 13 validation samples, we correctly identify all the variants, including big duplications and deletions. From the new cohort, we identified the causative variants in 21 of the 28 unrelated probands (75% detection rate); five of them carrying a novel splice site variant c.1186+2T>G in LDLR. Among the family members, the concentration of LDL cholesterol was 7.82±2.13 mmol/l in LDLR c.1186+2T>G carrier group (n = 26), and was significantly higher than 3.18±1.36 mmol/l in the non-carrier group (n = 25) (p value <0.0001). Haplotype analysis shows that LDLR c.1186+2T>G originates from a common ancestor, revealing the specific LDLR mutation spectrum in Taiwan. In the diagnosis of severe hypertriglyceridemia, 13 of the 17 patients were detected to have causative variants (76.5% detection rate). Eight of them had LPL p.L279V (47.1%) and seven patients had APOA5 p.G185C (41.2%). Relatives were included in the genetic cascade screening. Conclusion: This is the first capture-based NGS testing for FH to cover the whole LDLR genomic region, and therefore making reliable structural variation detection. This panel can comprehensively detect disease-causing variants in LDLR, APOB, and PCSK9 for FH patients. In addition, the NGS panel we designed for monogenic dyslipidemia can also effectively detect various dyslipidemia-causing genes, and at the same time, we can better understand the genetic background of hypertriglycerideemia in Taiwan population.