臺灣博碩士論文加值系統

由於副甲狀腺素受體下游包括刺激型G蛋白的α次單元之GNAS基因發生異常，導致細胞受體經副甲狀腺素的刺激後，無法將訊號正常傳遞，而引起偽副甲狀腺功能低下症（pseudohypoparathyroidism, PHP）。GNAS是個高複雜性的印記基因（imprinted gene），可透過不同的選擇性啟動子（alternative promoters）及選擇性剪接（alternative splicing）產生不同的轉錄產物（transcripts），其中最具有特色的轉錄產物就是由GNAS基因產生的Gsα，在大部份的組織中GNAS基因會同時以兩個對偶基因來表現Gsα，但少部份的組織（例如：近端腎小管）則是只表現母系那條對偶基因的Gsα。

偽副甲狀腺功能低下症1a型是由於母系GNAS對偶基因的外顯子發生突變，使近端腎小管對副甲狀腺素有阻抗性，但大部份的組織中，其具有功能之Gsα蛋白單倍劑量不足（haploinsufficiency），而造成遺傳性骨發育不全（Albright hereditary osteodystrophy, AHO）的臨床表徵。偽假性副甲狀腺功能低下症是由於父系GNAS對偶基因的外顯子發生突變性，患者雖不會會副甲狀腺素有抗性，但其仍會有遺傳性骨發育不全的臨床症狀。而偽副甲狀腺功能低下症1b型患者是由於GNAS基因差異甲基化區域的甲基化情形發生異常而造成患者的腎近端小管對副甲狀腺素有阻抗性，其臨床表現少有遺傳性骨發育不全特徵。

目前臺大醫院陳沛隆醫師實驗室已經建立以聚合酶連鎖反應（polymerase chain reaction, PCR）及桑格定序（Sanger sequencing）來檢測分析GNAS基因13個外顯子之序列。但這個流程費時又費力，故我們將利用探針捕獲（probe capture）待測區域及次世代定序（next-generation sequencing）一次將GNAS基因13個外顯子進行分析，以減緩臨床檢測時效性的壓力，且能提高檢測的精準度。另外，在臺灣偽副甲狀腺功能低下症1b型的基因檢測方式仍尚未建立，故我們將建立藉由解析度高的次世代定序方法來檢測GNAS基因之差異甲基化區域的甲基化情況，以提供臨床上疑似偽副甲狀腺功能低下症1b型患者進行分子診斷確診，目前我們看到在偽副甲狀腺功能低下症1b型患者患者之差異甲基化區域（exon A/B）的甲基化情況有別於未生病的正常控制組，其甲基化情況與我們預期偽副甲狀腺功能低下症1b型皆無甲基化相符合。除此之外，亦建立以探針捕獲差異甲基化區域的方法檢測偽副甲狀腺功能低下症1b型。

Pseudohypoparathyroidism (PHP) is caused by pathogenic variants and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3. GNAS encodes for the alpha-subunit of the stimulatory G protein (Gsα) and several other alternative splicing products. GNAS is paternally imprinted in certain tissues, which means that Gsα expression of the paternal allele is absent in those tissues (including proximal renal tubules). Consequently, in the presence of an inactivating mutation in the maternal GNAS gene, there is little or no Gsα protein, leading to PTH-resistant hypocalcemia and hyperphosphatemia. The inactivating variant in the maternal GNAS gene causes PHP1a, while the paternal GNAS pathogenic variant ends up in pseudo-PHP (PPHP). And loss of methylation at a differentially methylated region (DMR) of the GNAS locus, exon A/B, has been observed in patients with PHP1b. Patients with PHP1a often present with additional hormonal resistance and show characteristic physical features that are collectively termed Albright’s hereditary osteodystrophy (AHO). These features are also present in PPHP, but patients affected by this disorder do not show hormone resistance. Patients with PHP1b have hypocalcemia and hyperphosphatemia due to renal PTH-resistance, but lack physical features of AHO.

In Taiwan, genetic testing of PHP1b has not yet been established, so we established a testing method using next-generation sequencing (NGS) for diagnosis of PHP1b. Furthermore, we also successfully applied the NGS-based methods for the genetic diagnosis of all types of PHPs.

口試委員會審定書 i
致謝 ii
中文摘要 iii
英文摘要 v
圖目錄 ix
表目錄 xi
第一章 研究背景與動機 1
1.1 胞外訊號傳遞 2
1.1.1 訊號分子 2
1.1.2 細胞表面受體 3
1.1.3 G蛋白家族 5
1.1.4 GNAS gene 6
1.2 表觀遺傳學 10
1.3 基因印記 11
1.4 偽副甲狀腺功能低下症 12
1.4.1 疾病分型及其臨床表現 13
1.4.2 疾病遺傳學 14
1.4.3 臨床症狀 16
1.4.4 疾病臨床檢驗方法 17
1.4.5 疾病治療方法 18
1.4.6 建立分子診斷之目的 19
第二章 研究方法 20
2.1 受試者條件 20
2.2 實驗前檢體前處理 20
2.3.1 DNA萃取 20
2.3.2 DNA品質確認 21
2.3 PHP type1a及PPHP之GNAS基因編碼區域序列定序 21
2.3.1 實驗流程 21
2.3.2 實驗方法與步驟 21
2.4 PHP type1b之GNAS基因甲基化區域定序分析 24
2.4.1 實驗流程 24
2.4.2 實驗方法與步驟 24
2.5 次世代定序方法檢測偽副甲狀腺功能低下症 28
2.5.1 實驗流程 28
2.5.2 實驗方法與步驟 28
第三章 結果 30
3.1 受試者 30
3.2 GNAS基因編碼區域序列定序結果 30
3.3 GNAS基因甲基化區域定序分析結果 39
第四章 討論 48
第五章 參考文獻 50
附錄一：臨床試驗計劃書 56
附錄二：受試者本人同意書 62
附錄三：受試者家屬同意書 66
附錄四：兒童版受試者同意書 70

1.Maeda, S.S., et al., Hypoparathyroidism and pseudohypoparathyroidism. Arq Bras Endocrinol Metabol, 2006. 50(4): p. 664-73.

2.Cooper, M.S. and N.J. Gittoes, Diagnosis and management of hypocalcaemia. BMJ, 2008. 336(7656): p. 1298-302.

3.Tafaj, O. and H. Juppner, Pseudohypoparathyroidism: one gene, several syndromes. Journal of Endocrinological Investigation, 2017. 40(4): p. 347-356.

4.Mantovani, G., Clinical review: Pseudohypoparathyroidism: diagnosis and treatment. J Clin Endocrinol Metab, 2011. 96(10): p. 3020-30.

5.Quarles, L.D., Endocrine functions of bone in mineral metabolism regulation. J Clin Invest, 2008. 118(12): p. 3820-8.

6.Bruce Alberts, D.B., Karen Hopkin, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, Essentials of Cell Biology. 3 ed. 2010, Charlotte, North Carolina: Baker & Taylor Books.

7.Lodish, H.F., Berk, A., Zipursky, L.S., Matsudaira, P.T., Baltimore, D. and Darnell, J, Molecular cell biology. 4 ed. 1999, New York: Scientific American Books.

8.Cooper, G.M., The cell: a molecular approach. 2 ed. 2000, Sunderland (MA): Sinauer Associates.

9.Weinstein, L.S., et al., Studies of the regulation and function of the Gs alpha gene Gnas using gene targeting technology. Pharmacol Ther, 2007. 115(2): p. 271-91.

10.Wettschureck, N. and S. Offermanns, Mammalian G proteins and their cell type specific functions. Physiol Rev, 2005. 85(4): p. 1159-204.

11.Izzi, B., et al., A new approach to imprinting mutation detection in GNAS by Sequenom EpiTYPER system. Clin Chim Acta, 2010. 411(23-24): p. 2033-9.

12.Lemos, M.C. and R.V. Thakker, GNAS mutations in Pseudohypoparathyroidism type 1a and related disorders. Hum Mutat, 2015. 36(1): p. 11-9.

13.Richard, N., et al., A new deletion ablating NESP55 causes loss of maternal imprint of A/B GNAS and autosomal dominant pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab, 2012. 97(5): p. E863-7.

14.Linglart, A., et al., A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS. Am J Hum Genet, 2005. 76(5): p. 804-14.

15.Bastepe, M. and H. Juppner, GNAS locus and pseudohypoparathyroidism. Horm Res, 2005. 63(2): p. 65-74.

16.Hayward, B.E., et al., Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci U S A, 1998. 95(26): p. 15475-80.

17.Bastepe, M., et al., Autosomal dominant pseudohypoparathyroidism type Ib is associated with a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS. J Clin Invest, 2003. 112(8): p. 1255-63.

18.Bastepe, M., et al., Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit. Mol Endocrinol, 2002. 16(8): p. 1912-9.
19.Klemke, M., et al., Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties. J Biol Chem, 2000. 275(43): p. 33633-40.

20.Oczkowicz, M., et al., Expression and imprinting analysis of the NESP55 gene in pigs. Gene Expr Patterns, 2012. 12(1-2): p. 18-23.
21.Ischia, R., et al., Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem, 1997. 272(17): p. 11657-62.

22.Weinhaeusel, A., et al., PCR-based analysis of differentially methylated regions of GNAS enables convenient diagnostic testing of pseudohypoparathyroidism type Ib. Clin Chem, 2008. 54(9): p. 1537-45.

23.Chillambhi, S., et al., Deletion of the noncoding GNAS antisense transcript causes pseudohypoparathyroidism type Ib and biparental defects of GNAS methylation in cis. J Clin Endocrinol Metab, 2010. 95(8): p. 3993-4002.

24.Bird, A., Perceptions of epigenetics. Nature, 2007. 447(7143): p. 396-8.

25.Saxonov, S., P. Berg, and D.L. Brutlag, A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A, 2006. 103(5): p. 1412-7.

26.Gardiner-Garden, M. and M. Frommer, CpG islands in vertebrate genomes. J Mol Biol, 1987. 196(2): p. 261-82.

27.Khalil, A.M. and C. Wahlestedt, Epigenetic mechanisms of gene regulation during mammalian spermatogenesis. Epigenetics, 2008. 3(1): p. 21-8.

28.Lee, D. and C. Shin, MicroRNA-target interactions: new insights from genome-wide approaches. Ann N Y Acad Sci, 2012. 1271: p. 118-28.

29.Bajrami, E. and M. Spiroski, Genomic Imprinting. Open Access Maced J Med Sci, 2016. 4(1): p. 181-4.

30.Elhamamsy, A.R., Role of DNA methylation in imprinting disorders: an updated review. J Assist Reprod Genet, 2017. 34(5): p. 549-562.

31.Barlow, D.P. and M.S. Bartolomei, Genomic imprinting in mammals. Cold Spring Harb Perspect Biol, 2014. 6(2).

32.Takatani, R., et al., Analysis of Multiple Families With Single Individuals Affected by Pseudohypoparathyroidism Type Ib (PHP1B) Reveals Only One Novel Maternally Inherited GNAS Deletion. J Bone Miner Res, 2016. 31(4): p. 796-805.

33.Nakamura, A., et al., Complex Genomic Rearrangement Within the GNAS Region Associated With Familial Pseudohypoparathyroidism Type 1b. J Clin Endocrinol Metab, 2016. 101(7): p. 2623-7.

34.Liu, J., J.G. Nealon, and L.S. Weinstein, Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB. Hum Mol Genet, 2005. 14(1): p. 95-102.

35.Zeniya, S., et al., A 22-year-old woman with hypocalcemia and clinical features of albright hereditary osteodystrophy diagnosed with sporadic pseudohypoparathyroidism type Ib using a methylation-specific multiplex ligation-dependent probe amplification assay. Intern Med, 2014. 53(9): p. 979-86.

36.Saiki, R.K., et al., Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988. 239(4839): p. 487-91.

37.Sanger, F. and A.R. Coulson, A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol, 1975. 94(3): p. 441-8.

38.Darst, R.P., et al., Bisulfite sequencing of DNA. Curr Protoc Mol Biol, 2010. Chapter 7: p. Unit 7 9 1-17.

39.Millar, D., Y. Christova, and P. Holliger, A polymerase engineered for bisulfite sequencing. Nucleic Acids Res, 2015. 43(22): p. e155.

40.Tetzner, R., D. Dietrich, and J. Distler, Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA. Nucleic Acids Res, 2007. 35(1): p. e4.

41.Li, Y. and T.O. Tollefsbol, DNA methylation detection: bisulfite genomic sequencing analysis. Methods Mol Biol, 2011. 791: p. 11-21.

42.Ehrich, M., et al., A new method for accurate assessment of DNA quality after bisulfite treatment. Nucleic Acids Res, 2007. 35(5): p. e29.

43.Rochtus, A., et al., Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects. Clin Epigenetics, 2016. 8: p. 10.

44.Coffee, B., Methylation-specific PCR. Curr Protoc Hum Genet, 2009. Chapter 10: p. Unit 10 6.