Mutational screening of six genes in Chinese patients with congenital cataract and microcornea

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: To identify mutations in 6 genes of 9 Chinese families with congenital cataract and microcornea.

Methods: Nine unrelated families with congenital cataract and microcornea were collected. Cycle sequencing was used to detect variants in the coding and adjacent regions of the crystallin alpha A (CRYAA), crystallin beta B1 (CRYBB1), crystallin beta A4 (CRYBA4), crystallin gamma C (CRYGC), crystallin gamma D (CRYGD), and gap junction protein alpha 8 (GJA8) genes.

Results: Upon complete analysis of the 6 genes, three mutations in 2 genes were detected in 3 families, respectively. These mutations were not present in 96 normal controls. Of the three mutations, two novel heterozygous mutations in GJA8, c.136G>A (p.Gly46Arg) and c.116C>G (p.Thr39Arg), were found in two families with congenital cataract and microcornea. The rest one, a heterozygous c.34C>T (p.Arg12Cys) mutation in CRYAA, was identified in three patients from a family with nuclear cataract, microcornea with axial elongation. No mutation in the 6 genes was detected in the remaining 6 families.

Conclusions: Mutations in GJA8 and CRYAA were identified in three families with cataract and microcornea. Elongation of axial length accompanied with myopia was a novel phenotype in the family with the c.34C>T mutation in CRYAA. Our results expand the spectrum of GJA8 mutations as well as their associated phenotypes.

Congenital cataract is a leading cause of childhood blindness accounting for about 10%~38% of blindness in children [1], with a prevalence around 0.006%~0.06% in live births [2,3]. It may occur alone or associated with other ocular or systemic abnormalities. Microcornea, one of the most frequent abnormalities associated with congenital cataract, results from the secondary damage of the lens maldevelopment or from mutations in some growth or transcription factors [4]. To date, around 200 genes and loci have been associated with cataracts [4,5]. Of these genes, mutations in at least 9 genes were reported to be responsible for congenital cataract associated with microcornea, including genes encoding crystallins (crystallin alpha-A [CRYAA], OMIM 123580; crystallin beta-A4 [CRYBA4], OMIM 123631; crystallin beta-B1 [CRYBB1], OMIM 600929; crystallin beta-B2 [CRYBB2], OMIM 123620; crystallin gamma-C [CRYGC], OMIM 123680; and crystallin gamma-D [CRYGD], OMIM 123690) [6-14], gap junction protein alpha 8 (GJA8, OMIM 600897) [6,15], v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF, OMIM 177075) [16,17], and solute carrier family 16 member 12 (SLC16A12, OMIM 611910) [18]. Analyses of individual gene in patients with cataract and microcornea have been frequently reported [8-16,18-21] but comprehensive analysis of all these genes in the same set of families is rare [6].

In this study, we performed mutational screening of 6 genes (CRYAA, CRYBB1, CRYBA4, CRYGC, CRYGD, and GJA8) in 9 Chinese families with congenital cataract and microcornea. Three mutations in GJA8 and CRYAA were identified in 3 families.

Upon complete analysis of the 6 genes, three heterozygous mutations in 2 genes were detected in 3 families (Figure 1), including c.34C>T (p.Arg12Cys) mutation in CRYAA, and c.116C>G (p.Thr39Arg) and c.136G>A (p.Gly46Arg) mutations in GJA8, where the last two mutations are novel. Both of the c.116C>G and c.136G>A mutations in GJA8 are predicted to be “probably damaging” by PolyPhen-2 and “intolerant” by SIFT. The p.Thr39Arg would change the Blosum62 score from 4 to −1 whereas the p.Gly46Arg would change the Blosum62 score from 6 to −2. The p.Thr39Arg and p.Gly46Arg variants involved residues that are conserved across different species (Figure 2).

The heterozygous c.34C>T mutation in CRYAA was identified in all three patients in a three-generation family (QT597), where all patients had congenital nuclear cataract and microcornea (Figure 3, Table 2). Myopic fundus change in both eyes were observed in the affected father (II:1) and affected grandfather (I:1; Figure 4). Ocular ultrasound recorded axial lengths of 24.47 mm for the right eye and 24.61 mm for the left eye of II:1 and that of 27.82 mm for the right eye and 26.35 mm for the left eye of I:1. The proband had −3.00D for both eyes at the age of 4 years old.

The c.136G>A mutation in GJA8 was identified in a two-generation family (QT204) with complete opacity of the lens and microcornea (Table 2). Horizontal cornea diameter was 9 mm for both eyes of the affected mother and 7 mm for both eyes of the affected daughter at the age of 5 years old.

The c.116C>G mutation in GJA8 was identified in a sporadic patient (QT895) of 7 years old with microcornea, complete opacity of lenses, and iris hypoplasia (Figure 5, Table 2). Horizontal corneal diameter was about 6 mm for both eyes.

In this study, we screened 6 genes for mutations in 9 Chinese families with congenital cataract and microcornea. Three mutations were identified in 3 of the 9 (30%) families, including a c.34C>T (p.Arg12Cys) in CRYAA, and a c.136G>A (p.Gly46Arg) and a c.116C>G (p.Thr39Arg) in GJA8, respectively.

CRYAA is located in 21q22.3 and encodes the α-A-crystallin in lens epithelial cells and fiber cells. α-A-crystallin is a member of small heat shock proteins with the chaperone activity which contributes to keeping lens transparent [6,10,27]. Up to now, there were eight mutations of CRYAA found in sixteen families most of which involved substitutions from or to arginine [5]. And the corresponding phenotypes of the mutations were related with congenital cataract with or without microcornea, microphthalmia, or iris coloboma.

We found a known c.34C>T (p.Arg12Cys) mutation in CRYAA of three patients from a family with congenital nuclear cataract and microcornea. Previously, this mutation has been identified in four families with nuclear or lamellar cataracts, and some patients accompanied with microcornea or microphthalmia [6,10,28,29]. Elongation of axial length or myopia has not been observed in previous studies.

GJA8 is located in chromosome 1q21.1 and encodes the gap junction proteins, connexin50. GJA8 is one of the most common genes causing congenital cataract with or without other ocular abnormalities. Previous studies showed that GJA8-knockout mice developed nuclear cataract and microphthalmia, from which it is considered that GJA8 plays a role not only in keeping lens transparent but in ocular growth [30,31]. Up to now, about twenty mutations in GJA8 have been associated with congenital cataracts in at least 21 families. Of these mutations, five were identified in five families with microcornea and two families accompanied with microphthamia [32,33].

In this study, we found two novel missense mutations c.136G>A and c.116C>G in GJA8 in two families with congenital cataract and microcornea. The c.136G>A mutation led to a substitution from glycine to arginine at the amino acid position 46, and the c.116C>G mutation led to a substitution from threonine to arginine at the amino acid position 39. Both the 46 and 39 positions are located in the first transmembrane domain. In a previous study, Minogue et al. [32] identified a c.137G>T (p.Gly46Val) mutation in GJA8 of a proband with early-onset total cataract accompanied with small eyes and pupils. Therefore, the three mutations may result in phenotype by the similar mechanism.

In summary, a known c.34C>T mutation in CRYAA and two novel mutation in GJA8 were identified in 3 of 9 families after comprehensive analysis of 6 genes known to cause cataract and microcornea. Our results expand the mutation spectrum of GJA8 and phenotypic variations associated with CRYAA mutations. Patients without mutation in the 6 genes are potential candidate for future study of additional causative genes for cataracts and microcornea.

The authors thank the family members for their participation. This study was supported by the National Science Fund for Distinguished Young Scholars (30725044 to Q.Z.) and National 973 Plan of China (2010CB529904 to Q.Z.).