基因醫學在小兒內分泌疾病之應用

The application of genetic medicine in pediatric endocrine diseases

童怡靖(Yi-Ching Tung)

指導教授 ： 蔡文友

共同指導教授 ： 江伯倫(Bor-Luen Chiang)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2019年)

https://doi.org/10.6342/NTU201902417

摘要

遺傳疾病指的是由於基因的錯誤，導致疾病的產生。在內分泌疾病領域，我們各以單基因疾病的身材矮小及多基因疾病的第1型糖尿病，作為遺傳醫學在兒童內分泌疾病研究的探討模式。

在單基因身材矮小的部分，選擇了SHOX基因缺乏症，因為該基因於1997年發現後，病患給予生長激素治療有較好的身高預後。我們將診斷為特發性矮小(idiopathic short stature)及不成比例的身材矮小的病童，根據修改自Rappold的表現型評分系統大於7分的病患，進一步進行SHOX基因檢驗。病患抽取的DNA，進行多重連線探針擴增技術MLPA偵測基因缺失等拷貝數變異，未發現基因拷貝數病變的檢體，我們進行第二階段定序檢查SHOX基因及其調控區域。由於SHOX基因與肢體中段骨骼以及生長板軟骨內骨化(endochondral ossification)調控有關，所以病患前臂及手部腕關節可能是檢視的重要關鍵。我們的研究加入表現型評分系統，SHOX基因缺乏症檢出率為26%，病患都符合手臂跨度與身高的比值小於96.5%的標準，且均有前臂明顯短小的特徵，這將是臨床醫師懷疑該疾病的重點之一。腕骨影像上，包括遠端橈骨透亮化(radiolucency)，骨骺三角化(triangularization)，以及腕骨錐狀化(pyramidalization)，尺骨錯位(Madelung deformity)皆為強烈懷疑該疾病的特徵。基因檢驗方面，我們的SHOX基因缺乏症病患中86%是由於不等大小的基因缺失，其中16%僅發生在調節增強區域，也僅有一位是點突變造成。由於該基因位於偽體染色體區域(PAR)，當缺失片段較大時，可能會發生連續基因缺失症候群(contiguous gene deletion syndrome)，甚至跨越PAR及一般性染色體基因區域，可能影響的基因可能有不同的遺傳模式。SHOX基因缺乏症是我們研究身材矮小的第一個候選基因，希望了解台灣SHOX基因缺乏症病患的基因表現型及遺傳模式，以作為後續身材矮小病患基因檢查的依據。

在多基因疾病中，則選擇自體免疫破壞的第1型糖尿病，其致病原因包括先天的遺傳基因，加上後天環境，包括飲食習慣，感染，化學物質等的誘發，使得免疫細胞攻擊β細胞，終究導致剩餘胰島素功能嚴重不足，甚至發生高血糖危症之酮酸中毒症。我們希望先由剩餘胰島素功能及胰島相關自體抗體的研究，界定第1型糖尿病患的正確診斷，進一步研究基因部分的影響。根據之前國外的研究，HLA基因占了遺傳影響的大部分，我們先進行第1型糖尿病HLA基因在台灣第1型糖尿病的風險性的研究。將104為第1型糖尿病病患，抽取白血球的分析，在HLA-A、-B、-C、-DRB1、-DQB1及 -DPB1定序分型法(sequence-based typing)分析其HLA基因型，並與504位對照組加以比較分析。23個對偶基因中在第1型糖尿病的病患組和對照組在下列四種檢定上(Armitage trend test，the allelic test，the genotypic test以及and the dominant model)有顯著上的差異，並以連鎖不平衡檢定發現三個易罹病的基因型DR3 (DRB1*03:01-DQB1*02:01)，DR4 (DRB1*04:05-DQB1*04:01)及DR9 (DRB1*09:01-DQB1*03:03)，與一個保護性基因型(DRB1*08:03-DQB1*06:01)，該保護型單套型與自體免疫第1型糖尿病的關聯，為第一次發現。另外DQB1*03:02本身就是一個常見的易感基因，而DQB1*03:01則具有相當的保護性。這些資訊對於台灣本土第1型糖尿病研究相當重要。另外我們也發現DRB1*12:02對第1型糖尿病具有保護性，有趣的是我們之前在葛瑞夫氏症的研究DRB1*12:02也是一個保護的對偶基因。是否某些對偶基因可同時減少自體免疫過程的發生，更值得進一步研究探討。根據我們研究的結果，可以做為第1型糖尿病遺傳致病機轉研究的基石。在多基因疾病中，加上其他風險基因或是環境、飲食及感染、等分析，可以做為疾病預測模式的參考。

運用基因醫學的進步，不論是在單基因或多基因的兒童內分泌疾病，精準醫學能在診斷及治療有決定性的幫助。期待我們在身材矮小及糖尿病疾患建立的基因診斷模式，也能成為未來相關疾病研究的基石。

關鍵字

SHOX基因 ； 多重連線探針擴增技術MLPA ； 第1型糖尿病 ； β細胞 ； HLA基因 ； 單套型 ； 連鎖不平衡

並列摘要

A genetic disorder is a genetic problem caused by one or more abnormalities in the genome. A monogenic disorder is the result of a single mutated gene and multiple gene disorders are associated with the combination of the effects of multiple genes, epigenetic and environmental factors. We chose monogenic short stature and type 1 diabetes as interests of the study. We hope to demonstrate the application of genetic medicine in pediatric endocrine diseases.

A deficiency in the SHOX gene (short stature homeobox-containing gene) is a newly defined cause of short stature found in 1997. Growth hormone therapy is proved to use in such patients for the better height outcome. Twenty-three patients with idiopathic short stature or disproportionately short stature were enrolled in this study. The phenotype scoring system, modified from Rappold et al. was used for the selection of patients and patients with more than 7 points were screened for SHOX deficiency. A multiplex ligation-dependent probe amplification (MLPA) assay was used to identify the DNA copy number of SHOX gene and its regulatory regions. Sequencing of the SHOX gene was performed if no deletions were detected by MLPA. We found that SHOX gene deletion or mutation was detected in 26% of the disproportionately short children. All patients with SHOX deficiency had an arm-span-to-height ratio less than 96.5%, and a short forearm phenotype; these were the most striking features in patients with SHOX deficiency and it provides the clinicians to consider the SHOX gene mutation. The striking radiological findings of wrist joint include “lucency” of the distal radius, “triangularization” of the distal radial epiphysis and “Madelung deformity” attributed to premature epiphyseal fusion of the distal radius related to a disturbance in the programmed cell death of hypertrophic chondrocytes. Eighty-six percent of patients with SHOX deficiency in our study had a variable deletion size. One deletion (16%) was detected only in the regulatory enhancer region. Another patient had a missense mutation of the SHOX gene. Contiguous gene deletion syndrome involving the SHOX gene is not rare. SHOX gene is the first candidate gene in our study for monogenic short stature.

Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by T-cell-mediated pancreatic β-cell destruction. The pathogenesis of T1D features a complex interaction of genetic, environmental, and immunological factors. In the genetic aspect, the HLA genes that encode molecules that bind and present peptide antigens to T-cells accounted for approximately half the genetic risk of T1D. We performed direct comprehensive genotyping of 6 classical HLA loci (HLA-A, -B,
-C, -DPB1, -DQB1, and -DRB1) to 4-digit resolution in 104 unrelated T1D patients and 504 controls. Genomic DNA from these subjects were extracted and performed HLA sequence-based typing. Of these, 23 alleles exhibited frequency differences between T1D cases and controls in the Armitage trend test, the allelic test, the genotypic test and the dominant model. We analyzed the linkage disequilibrium (LD) between these 23 alleles and found three susceptibility haplotypes of DR3 (DRB1*03:01-DQB1*02:01), DR4 (DRB1*04:05-DQB1*04:01), and DR9 (DRB1*09:01-DQB1*03:03) and one protective haplotype (DRB1*08:03-DQB1*06:01). DRB1*08:03-DQB1*06:01 is the first report to associated with autoimmune type 1 diabetes. DQB1*03:02 was itself a susceptibility allele. The DRB1*12:02 in our study seems to protect against T1D. Intriguingly, in our previous study in patients with Graves’ disease, DRB1*12:02 was also identified as a protective allele. Maybe it implies that some alleles will protect from or exert the autoimmune process. Our study has yielded useful information on the combined effects of different HLA loci and alleles on T1D in Taiwan. In such multifactorial disorders, we might collect the data of environment exposures in addition to the genotypes of associated genes to establish the disease prediction model.

Based on the genetic diagnosis, the health practitioners can make precision medicine in such pediatric endocrine disorders, including monogenic and multifactorial diseases. Our work in monogenic short stature and type 1 diabetes may be helpful clinically and could be a reference of genetic studies in the future.

並列關鍵字

SHOX gene ； multiplex ligation-dependent probe amplification (MLPA) ； type 1 diabetes (T1D) ； β cell ； HLA gene ； haplotype ； linkage disequilibrium (LD)

參考文獻

Aly TA, Ide A, Jahromi MM, Barker JM, Fernando MS, Babu SR, Yu L, Miao D, Erlich HA, Fain PR, Barriga KJ, Norris JM, Rewers MJ, and Eisenbarth GS. Extreme genetic risk for type 1A diabetes. Proc Natl Acad Sci U S A. 2006; 103(38): 14074-9.

American Diabetes Association. Summary of revisions for the 2010 Clinical Practice Recommendations. Diabetes Care. 2010; 33 Suppl 1: S3.

American Diabetes Assciation. Type 2 diabetes in children and adolescents.. Pediatrics. 2000; 105(3 Pt 1): 671-80

Andrade AC, Jee YH, and Nilsson O. New Genetic Diagnoses of Short Stature Provide Insights into Local Regulation of Childhood Growth. Horm Res Paediatr. 2017; 88(1): 22-37.

Arnqvist HJ, Littorin B, Nystrom L, Schersten B, Ostman J, Blohme G, Lithner F, and Wibell L. Difficulties in classifying diabetes at presentation in the young adult. Diabet Med. 1993; 10(7): 606-13.