Family-based analysis identified CD2 as a susceptibility gene for primary open angle glaucoma in Chinese Han population

Abstract

Primary open angle glaucoma (POAG) is characterized by optic disc cupping and irreversible loss of retinal ganglion cells. Few genes have been detected that influence POAG susceptibility and little is known about its genetic architecture. In this study, we employed exome sequencing on three members from a high frequency POAG family to identify the risk factors of POAG in Chinese population. Text-mining method was applied to identify genes associated with glaucoma in literature, and protein-protein interaction networks were constructed. Furthermore, reverse transcription PCR and Western blot were performed to confirm the differential gene expression. Six genes, baculoviral inhibitors of apoptosis protein repeat containing 6 (BIRC6), CD2, luteinizing hormone/choriogonadotropin receptor (LHCGR), polycystic kidney and hepatic disease gene 1 (PKHD1), phenylalanine hydroxylase (PAH) and fucosyltransferase 7 (FUT7), which might be associated with POAG, were identified. Both the mRNA expression levels and protein expression levels of HSP27 were increased in astrocytes from POAG patients compared with those from normal control, suggesting that mutation in CD2 might pose a risk for POAG in Chinese population. In conclusion, novel rare variants detected by exome sequencing may hold the key to unravelling the remaining contribution of genetics to complex diseases such as POAG.

Keywords: Chinese population; exome sequencing; primary open angle glaucoma.

Figure 1

The glaucoma genetic map for Li family. ‘/’ indicates that persons had passed away; ‘○’ represents female; ‘□’ represents male. The sequence marked in red represents the susceptibility genes inherited from his (her) ancestor. The persons marked in black represent glaucoma sufferer, and persons without black marker do not suffer from glaucoma.

Figure 2

The result of quality control. The central red line is the median value; the yellow box represents the inter-quartile range (25–75%); the upper and lower whiskers represent the 10% and 90% points; the blue line represents the mean quality; the y-axis on the graph shows the quality scores. The higher the score, the better the base call. The background of the graph divides the y-axis into very good quality calls (green), calls of reasonable quality (orange) and calls of poor quality (red).

Figure 3

The protein–protein interaction (PPI) network in which the proteins of the mutated gene and its neighbour proteins are involved. The yellow nodes are the proteins of mutated genes and the pink nodes are their interactive proteins.

Figure 4

Sequence alignment of CD2 gene. The A at position 596 was replaced by G and the G at position 666 was replaced by C.

Figure 5

SDS-PAGE of proteins from non-transfected samples and transfected samples. The lane 1 represents the non-transfected samples (control), the lane 2 represents the normal cells transfected CD2 gene (pCD2-1) and the lane 3 represents the primary open angle glaucoma (POAG) cells transfected CD2 gene (pCD2-2).

Figure 6

The mRNA expression of HSP27 in primary open angle glaucoma (POAG) cells and normal cells. (A) RT-PCR was used to measure expression levels of HSP27 at 24, 48 and 72 hrs after pCD2 transfection using total RNA from POAG cells and normal cells. (B) The relative expression level of HSP27. Expression values were normalized to β-actin.

Figure 7

The protein expression of HSP27 in primary open angle glaucoma (POAG) cells and normal cells. (A) Western blot was used to measure expression levels of HSP27 at 24, 48 and 72 hrs after pCD2 transfection using proteins from primary open angle glaucoma (POAG) cells and normal cells. (B) The relative expression level.