台灣兒童先天性高胰島素血症之研究

Abstract

先天性高胰島素血症是一群具多種臨床表現和遺傳機轉的疾病，其特徵是低血糖時仍有失調性持續分泌胰島素的現象。目前至少有 12 個基因已被證明與單基因性先天性高胰島素血症相關，其中ABCC8 和 KCNJ11 基因上的致病性變異是最重要的病因。在胰臟 β 細胞中，ATP 敏感鉀離子 (KATP) 通道在血中葡萄糖濃度變化導致細胞膜電位改變和胰島素釋放的一連串步驟中發揮關鍵作用。 KATP 通道由四個形成孔道的鉀離子通道亞基 (Kir6.2) 和四個磺醯尿素(sulfonylureas) 受體 1 (SUR1) 亞基組成，分別由 KCNJ11 和 ABCC8 基因編碼。目前已知先天性高胰島素血症在組織學上可分為瀰漫型、局部型或非典型。 瀰漫型先天性高胰島素血症者通常與 ABCC8 和 KCNJ11 基因的隱性功能喪失變異有關，他們通常對diazoxide沒有反應，需要廣泛的胰臟切除術來改善低血糖。染色體 11p15.5 區域是一個印記中心，包含母系表達的腫瘤抑制基因 H19 和 CDKN1C 以及父系表達的生長因子基因 IGF2，局部型先天性高胰島素血症通常是由父系遺傳的單一等位基因 KATP 通道致病變異合併體細胞性母源染色體11p15 區域缺失引起的，臨床上難以區分的瀰漫型和局部型形式可以透過使用氟18 L-3,4-二羥基苯丙胺酸正子斷層掃描 (18F-FDOPA PET) 掃描來確定，手術切除局部型病變即可治癒患者。過去報告ABCC8 和 KCNJ11 基因的顯性突變，以及其他先天性高胰島素血症相關基因的致病變異，臨床表現常為輕度對diazoxide治療有反應的先天性高胰島素血症。由於台灣缺乏對先天性高胰島素血症的研究，我們旨在描述台灣先天性高胰島素血症兒童的臨床表現並闡明其遺傳病因，並分析其基因型與表型相關性。 我們對 13 名被診斷出患有最嚴重對diazoxide治療無反應的兒童進行了研究，所有患者在藥物治療失敗後均接受了胰臟切除術，我們的研究首次展示了台灣先天性高胰島素血症兒童的臨床特徵和長期結果，我們的結果表明，出生時為巨嬰和低血糖神經性症狀是診斷先天性高胰島素血症的重要臨床線索，低血糖發生的年齡反映了先天性高胰島素血症的嚴重程度，另外，低血糖期間 I/G 比值 >0.3–0.5 作為先天性高胰島素血症的診斷標準，並不像先前報導的那麼可靠，因此，謹慎的做法是對低血糖發作期間所有可檢測到血中胰島素濃度的患者進行檢查，以防止先天性高胰島素血症的延遲診斷。 我們結合了桑格式定序與全外顯子定序 (WES) 方法，發現 KATP 通道中的致病性變異是 13 名台灣兒童對diazoxide無反應先天性高胰島素血症的最常見致病原因，我們證明 13名病患中最常出現的ABCC8 基因p.T1042Qfs*75 變異是台灣的奠基者突變，我們並針對病患中發現的三個罕見SUR1 蛋白變異做功能性研究，發現 p.L366F會造成KATP 通道門控缺陷而p.R797Q 和 p.R1393C會造成KATP 通道運輸缺陷，這樣的缺失是造成病患對diazoxide無反應的原因，我們的研究也證實GCK 基因的 p.I211F 變異會導致對diazoxide治療無反應的先天性高胰島素血症，攜帶p.I211F 變異的患者需要胰臟切除手術，才能維持正常血糖。 已知多個症候群也與先天性高胰島素血症有關。Beckwith-Wiedemann 症候群 (BWS) 是一種過度生長和容易出現胚胎性腫瘤的症候群，是最常見與先天性高胰島素血症相關的症候群， BWS 與表觀遺傳或基因組異常有關，這些異常會改變染色體 11p15.5 上印記基因的表達。我們提出了一個罕見的病例，這個病患由於胎盤間質發育不良而被懷疑為 BWS，儘管病患外觀並沒有明顯 BWS 的特徵，如出生時巨嬰、巨舌症、單側肥大或臍膨出現象，其周邊血DNA中BWS 關鍵區域的甲基化模式也並未發現異常，在她兩個月大時出現巨大的胰臟母細胞瘤並伴有高胰島素性低血糖後，我們進行的分子研究顯示鑲嵌性父系全基因組單倍體導致嬰兒胰臟母細胞瘤，因此病患符合BWS 類群的診斷。我們的結果強調了雄性細胞鑲嵌體與非典型 BWS 和高胰島素性低血糖之間關聯的重要性。 自2015 年以後，本院開始可以進行18F-FDOPA PET 掃描辨別臨床上diazoxide無反應的先天性高胰島素血症患者的局部病變，我們報告了兩名患者均存在父系遺傳的單一等位基因 ABCC8 致病性變異，且胰臟組織存在鑲嵌性染色體 11p15.5 區域父系單親二倍體，兩位病患的18F-FDOPA PET 掃描結果均低估了局部病變的範圍，待收集更多病例，未來將進一步研究分析18F-FDOPA PET診斷局部型先天性高胰島素血症的價值。我們也將繼續應用次世代定序的方法於基因診斷，特別是全基因組定序來識別那些具有未知遺傳病因的患者，由此基礎，可以開發個人化的治療方法和發展新的治療藥物，使先天性高胰島素血症患者能有更好的預後。

Congenital hyperinsulinism (CHI) is a group of clinically and genetically heterogeneous disorders characterized by dysregulated insulin secretion during hypoglycemia. At least twelve genes have been proved to be associated with monogenic CHI and pathogenic variants in ABCC8 and KCNJ11 genes account for the most important underlying etiology. In pancreatic β cells, the ATP-sensitive potassium (KATP) channel plays a critical role in coupling changes in the plasma glucose concentration to electrical excitability and insulin release. The KATP channel consists of four subunits of the pore-forming potassium channel subunit (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits, which are encoded by KCNJ11 and ABCC8 genes, respectively. CHI can be histologically characterized into diffuse, focal or atypical forms. Patients with diffuse CHI is generally associated with recessive loss-of-function variants in the ABCC8 and KCNJ11 genes. They are usually unresponsive to diazoxide, and extensive pancreatectomy is required to ameliorate hypoglycemia. Chromosome 11p15.5 region is an imprinting center that harbors maternally expressed tumor suppressors, H19 and CDKN1C, and a paternally expressed growth factor gene, IGF2. Focal CHI is usually caused by paternally inherited monoallelic KATP channel mutation with somatic loss of the maternal 11p15 region. The clinically indistinguishable diffuse and focal forms can be determined by scanning using fluorine 18 L-3, 4- dihydroxyphenyalanine positron emission tomography (18F-FDOPA PET) and removal of the focal lesion provides cure for the patient. Dominant mutations in ABCC8 and KCNJ11 genes, as well as disease-causing variants in other CHI-related genes, have been frequently associated with mild and diazoxide-responsive CHI. Because the study of CHI is lacking in Taiwan, we aim to describe the clinical manifestations and to elucidate genetic etiologies of Taiwanese children with CHI and analyze their genotype-phenotype correlations. Thirteen children diagnosed with the most severe diazoxide-unresponsive CHI were studied. All received pancreatectomy after failure of initial medical treatment. Our study demonstrates clinical characteristics and long-term outcome of Taiwanese children with CHI for the first time. Our results showed that macrosomia and irritability of the nervous system were important clinical clues to the diagnosis of CHI. The result indicated the age at onset of hypoglycemia reflects the severity of CHI. Our data showed that the I/G ratio >0.3–0.5 during hypoglycemia, used as a diagnostic criterion of CHI, was not so reliable as previously reported. Therefore, it is prudent to work up on patients with detectable serum insulin levels during the episode of hypoglycemia in order to prevent delayed diagnosis of CHI. By combining the Sanger sequencing with whole exome sequencing (WES), we found that pathogenic variants in the KATP channel were the most common underlying cause of diazoxide-unresponsive CHI in thirteen Taiwanese children. We demonstrated that the high frequency of the p.T1042Qfs*75 variant in the ABCC8 gene is likely due to a founder effect in Taiwan. Our study demonstrated that patients carrying three rare SUR1 mutants with diazoxide-unresponsive phenotype are due to KATP channel gating defect (p.L366F) or trafficking defect (p.R797Q and p.R1393C). Our study also confirmed that the p.I211F variant of the GCK gene results in diazoxide-unresponsive CHI. The result suggests additional management, including near-total pancreatectomy, may be required to maintain euglycemia in patients with the p.I211F variant. A variety of syndromes are also known to be associated with CHI. Beckwith-Wiedemann syndrome (BWS), an overgrowth and embryonic tumors predisposition syndrome, is the most frequent syndromic condition associated with CHI. BWS is linked to epigenetic or genomic abnormalities that alter the expression of imprinted genes on chromosome 11p15.5. We present a rare case in whom BWS was first suspected because of placental mesenchymal dysplasia although no stigmata characteristic of BWS such as macrosomia, macroglossia, hemi-hypertrophy, or omphalocele was noted. The peripheral blood methylation pattern over BWS critical region was normal. Molecular study done after she developed massive pancreatoblastoma with hyperinsulinemic hypoglycemia revealed mosaic paternal haploidy associated with pancreatoblastoma which was compatible with the diagnosis of BWS spectrum. Our result highlights the importance of the association of androgenetic cell mosaicism with atypical BWS and hyperinsulinemic hypoglycemia. 18F-FDOPA PET scan has been implemented to identify focal lesion in diazoxide-unresponsive CHI patients in our hospital since 2015. Two patients had molecular study confirmed paternally inherited monoallelic ABCC8 pathogenic variant with mosaic somatic paternal uniparental isodisomy over chromosome 11p15.5 region. Their surgical pathology revealed more extensive focal islet change than 18F-FDOPA PET image showed. Further study for analysis of the diagnostic value of 18F-FDOPA PET for focal CHI will be done. We will continue to apply next generation sequencing-based approach for genetic diagnosis, especially whole genome sequencing to identify those with unknown genetic etiology. Accordingly, personalized approach to treatment and novel therapy can be developed in a hope to improve better outcome in children with CHI.