李宋豬先天眼球缺陷及基因型鑑定研究

Abstract

先天性眼球缺陷包含了：無眼症、小眼症及貓眼症，是造成人類幼年失明的主因之一。在美國，每十萬人約有三人會受到此類眼球缺陷疾病的影響。包括了小鼠、斑馬魚及爪蟾等的許多模式動物可被用來研究先天性眼球缺陷的發生機制，然而這些因其體型、解剖、生理以及遺傳距離上的差異，並無法忠實的模擬發生於人類上的疾病。而豬（Sus scrofa）是除了非人類靈長類之外，與人類有許多相同之處的大型哺乳類動物，因此豬便可能是個良好的模式動物來進行眼球發育的研究。自1983年起在臺灣開發了可供作為生醫研究用途的小型豬種：李宋豬，然而在2008年於李宋豬保種族群中發現了自發性的眼球缺陷，包括了：無眼症、小眼症與無虹膜症。這些帶有眼球缺陷的李宋豬提供了一個新穎的模式動物來進行眼球發育的研究。因此本試驗的目的為建立穩定帶有眼球缺陷的李宋豬族群，以及篩選調控其缺陷機制的候選基因。 為了建立眼球缺陷之李宋豬族群，本試驗以雙眼無眼症之公母豬進行配種，結果顯示其雜交後代有超過五成帶有無眼症缺陷（第一胎：54.5％，第二胎：55.6％）。以正常族群與眼球缺陷族群進行系譜分析發現，這些眼球缺陷為遺傳性狀，且可能受到數個基因的調控。為了要瞭解不同缺陷個體的眼球構造差異，以核磁共振掃瞄正常、單眼無眼症以及雙眼無眼症之李宋豬頭部影像，結果顯示眼球缺陷個體完全喪失了發育出完整眼球的能力，顯示此缺失應發生於眼球發育的初期。此外單眼無眼症個體雖仍發育出一側眼球，但其眼球大小以及晶狀體的結構也受到了缺損。調控眼球發育的基因在不同物種間具有相當高的保留性，在人類及其他物種中，已有許多突變位點已被證實與眼球缺陷有關，本試驗檢測了14個與先天性眼球缺陷有關之基因，包括了BMP4、BMP7、HES1、LHX2、MAB21L2、MITF、OTX2、PAX2、PAX6、RAX、SHH、SOX2以及VSX2，然而於李宋豬中卻未發現與眼球缺陷相關的突變位點。為了獲得尚未被發現之位點，本試驗挑選包括正常、雙眼無眼症、雙眼無虹膜症、單眼無眼症單眼無虹膜症、小眼無虹膜症等五個性狀個體進行次世代高通量定序，並透過比較不同個體之全基因序列，獲得不同個體間的SNP位點。序列資料分析分為兩個部分，第一，將定序結果組裝成完整的全基因組序列；第二，將四個眼球缺陷個體與正常個體之序列進行比對，結果顯示總共得出185、247、207以及195個會造成胺基酸序列改變的SNP。並挑選來自雙眼無眼症及單眼無眼症單眼無虹膜症之個體的SNP進行驗證，總共挑選出88個SNP位點，共55個基因。目前已完成20個基因，但並無與缺陷性狀相關的位點產生。 總上所述，透過本試驗之配種策略，本研究室已建立帶有眼球缺陷之李宋豬族群，包括了無眼症、小眼症以及無虹膜症。但影響其缺陷產生的基因仍需要進一步的釐清。

Congenital eye malformations, including microphthalmia, anophthalmia and coloboma (MAC), act as certain major causes in human blindness. These diseases affected 3 per 100,000 newborns in the United States. Many animal models such as rodents, xenopus and zebra fish were used for investigating the pathogenesis of MAC. However, these animal models are not faithfully to mimic the relevant human diseases due to its body size, physiology, anatomy or genetic differences between human. Apart from non-human primates, Pig (Sus scrofa) is one of the large mammals that share many similarities with human. Therefore, pigs may serve as an ideal alternative model for eye development study. In Taiwan, a miniature breed called Lee-Sung pig (LS pig) was developed as laboratory model since 1983. LS pigs with spontaneous eye defects, including anophthalmia and aniridia from one family were identified from the conserved population in 2008. These defected LS pig provides a novel mammal model for eye development research. The aim of this study was to establish a population of Lee-Sung pigs with microphthalmia and anophthalmia, and to screen candidate genes that control eye development. In order to establish a population of LS pigs with eye defects, a sow with bilateral anophthalmia (An/An) and bred with a wild type boar and 4 female and 1 male LS pigs were delivered. One female LS pig showed aniridia (Ani/Ani). Full-sib mating of the pigs was carried out and a male LS pig with bilateral anophthalmia (An/An) was obtained. We then crossed two LS pigs with anophthalmia. The result showed that more than 50% of their offspring expressed eye defects (parity 1: 54.5%, parity 2: 55.6% in anophthalmia). With pedigree analysis and compare with control herd, the defects in LS pig were an inherited trait and controlled by a few genes. To understand the eye structure between different phenotype of LS pigs, the Magnetic Resonance Imaging (MRI) was conducted from three LS pigs, including wild type, bilateral and unilateral anophthalmia. Results showed that defected LS pig lost the ability to form a normal eye structure and this defect might occur in early stage of eye development. Moreover, the size of the eye ball and structure of lens might be disrupted in a unilateral anophthalmia LS pig. Genes that control the eye development are highly conserved among species, as many mutations in genes which are critical for eye development were identified in human or some other species. The SNPs related to MAC from twelve candidate genes, including BMP4, BMP7, HES1, LHX2, MAB21L2, MITF, OTX2, PAX2, PAX6, RAX, SHH, SOX2 and VSX2 in LS pigs were examined by PCR and Sanger sequencing. However, the published SNP sites were not relevant to MAC phenotypes in LS pig. In order to obtain the novel SNP sites that relate to LS pig MAC defects, five LS pigs with different phenotypes were chosen (wild type, AN/AN, Ani/AN, Ani/Ani and M/M+Ani respectively) to perform the Next-Generation Sequencing (NGS). Through NGS, whole genome of LS pig was obtained from this large-scale sequencing, and the SNP sites between different phenotypes could be discovered. The sequencing data analyses were separated into two parts, one was to assemble the reads into complete LS pig genome, and the other was to map the reads from five LS pigs to reference genome. Non-synonymous SNPs were called from four defected LS pigs (185, 247, 207 and 195 SNPs; 124, 155, 150 and 128 genes respectively). Totally, 144 candidate genes were selected, and assigned into different gene clusters which include JAK-STAT pathway (7 genes respectively), immune system process (27 genes respectively) and 26 genes contribute to MAC diseases. Further validation of these SNPs will examine by PCR and Sanger sequencing to confirm the accuracy of these sites. Taking together, through our breeding strategy, we have established a population with eye defects which include anophthalmia, microphthalmia and aniridia LS pigs stably. But the genes from LS pig involved in MAC need to be explored in the next step.