HTRA1 Promoter Polymorphisms Are Associated With Age-related Macular Degeneration

Objective: To investigate the association of 9 single nucleotidepolymorphisms (SNPs) around the HTRA1 promoter region atChromosome 10q26 with different phenotypes of Age-related MacularDegeneration (AMD). Immunohistochemistry was used to determingHTRA 1 expression in the human eyes.Methods: 1. All participants were recruited from a Caucasian cohortin Utah. DNA was extracted from their blood. 2. 9 SNPs at Chromosome10q26 centered on rs11200638 were genotyped in AMD cases and ageand ethnicity matched normal controls. The chi-squared test for trend forthe additive model over alleles was performed to assess evidence forassociation. 3. The total RNA was extracted from normal human bloodand converted into first strand cDNA by SuperScript^TMâ…¢reversetranscriptase. The coding sequence of HTRA1 was cloned by PCR andwas inserted into pET32a vector after enzyme digestion. The recombinantplasmid was transformed into BL21 after sequencing confirmation, andthen induced by IPTG to get high-level expression of recombinantHTRA1 protein. The induced bacteria were lysed by lysis buffer. Therecombinant protein was purified from the bacteria lysate by Ni-NTAprotein purification columns. 4. Immunohistochemistry was used todeterming HTRA1 expression in the human eye by using the rabbitanti-human HTRA1 polyclonal antibody.Results: 1. HTRA1 promoter region SNP rs11200638 contributesequally to both forms of advanced AMD (GA and wet AMD). It also confers risk to soft confluent drusen, although to a lesser extent.rs10490924 was found to have a significant association with differentphenotypes of AMD, but inferior to rs11200638. 2. The HTRA1 codingsequence was cloned by two steps RT-PCR. After enzyme digestion andligation, the coding sequence was inserted into pET32a vector. The BL21,transformed with recombinant plasmid, showed high-level expression ofHTRA1 protein after ITPG induction. The high purity recombinantprotein was purified due to specific binding between His.Tag and Ni-NTA.The Western Blot showed that dialysised protein still has its nativeantigen. 3. The drusen and choroidal neovascularization wereimmunolabeled by HTRA 1 antibody in AMD patients.Conclusions: 1. rs11200638 in the promoter region of HTRA1remains to be the most significantly associated variant for GA and wetAMD in the Utah population and is a major risk factor for GA and softconfluent drusen, in addition to wet AMD. It may play an inportant rolein pathogenesis of advanced AMD and also confers risk to soft confluentdrusen. 2. The high purity and large volume protein is the most importantthing for protein structural and functional study. Those optimized Tags inpET32a vector help to produce soluble fusion protein and His.Tag makeit easy to purify the fusion protein from bacteria lysate. The HTRA1coding sequence was cloned by RT-PCR and inserted into pET32a vector.The recombinant plasmid shows high-level expression of fused HTRA1protein after transformed into BL21 and IPTG induction. The high purityHTRA1 protein with native antigen was purified and ready for furtherstudy. 3. HTRA1 may play an important role in neovascularization and drusen formation. The two processes, may not be completely independent,rather, they may converge.