Human HtrA1 in the archived eyes with age-related macular degeneration.
OBJECTIVE
HtrA1 belongs to the high temperature requirement factor A family of serine proteases, which are involved in protein quality control and cell fate. A single-nucleotide polymorphism (SNP), rs11200638, in the promoter of HtrA1 at chromosome 10q26 is reported as a likely causal variant for age-related macular degeneration (AMD). The SNP is located in the regulatory region and increases production of HtrA1 protein. This study investigates HtrA1 expression and SNP genotypes in archived ocular slides with AMD.


METHODS
Macular, nonretinal, and peripheral retinal cells were microdissected from archived slides from 57 eyes with AMD and 16 age-matched, non-AMD controls. HtrA1 rs11200638 SNP genotyping was performed using polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis. HtrA1 transcripts were measured using real-time reverse transcriptase-PCR. HtrA1 protein expression was evaluated using avidin-biotin complex immunohistochemistry.


RESULTS
HtrA1 (G/A) SNP was successfully genotyped in 52 AMD cases and 13 non-AMD subjects. The frequencies of the risk allele (A) were 55 of 104 (52.9%) and 8 of 26 (30.8%) in AMD and control groups, respectively. HtrA1 mRNA was detected in normal peripheral and macular retinas, higher in the periphery than maculae. HtrA1 mRNA was much higher in the macula and a lot lower in the periphery of the AMD eyes as compared to control eyes. HtrA1 protein was expressed in normal retinal vascular endothelia and retinal pigment epithelia. Intense immunoreaction against HtrA1 was found in AMD lesions, slightly more in wet than dry AMD lesions.


CONCLUSIONS
This study successfully analyzes HtrA1 SNP and transcript expression in microdissected cells from archived paraffin fixed slides. Up-regulation of HtrA1 is detected in the macular lesions of AMD eyes. The data further suggest that rs11200638 in HtrA1 promoter is associated with AMD development.
Chan
Chi-Chao
CC
Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
Shen
Defen
D
Zhou
Min
M
Ross
Robert J
RJ
Ding
Xiaoyan
X
Zhang
Kang
K
Green
W Richard
WR
Tuo
Jingsheng
J
D000368:Aged; D000369:Aged, 80 and over; D001344:Autopsy; D002386:Cataract / Q000150:complications; D004210:Dissection / Q000379:methods; D005123:Eye / Q000201:enzymology*; D005260:Female; D005838:Genotype; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D008268:Macular Degeneration / Q000201:enzymology / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D012016:Reference Values; D012697:Serine Endopeptidases / Q000235:genetics*
D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases