Molecular analysis of cataract families in India: new mutations in the CRYBB2 and GJA3 genes and rare polymorphisms

Abstract

Purpose: The aim of the study was to resolve the genetic etiology in families having inherited cataracts.

Methods: Families afflicted with congenital/childhood cataracts were registered in Chennai and Orissa (India). Blood samples were collected from the probands and available family members. Selected functional candidate genes were amplified by polymerase chain reaction (PCR) and characterized by direct sequencing. Putative mutations were confirmed in healthy controls.

Results: We observed interesting new polymorphisms of ethnic specificity, some of frequent nature, such as a 3-bp deletion in intron 3 of CRYBB2 (encoding βB2-crystallin) and IVS1+9 c>t variation in HSF4 (encoding heat-shock factor 4). Some rare single nucleotide polymorphisms (SNPs) co-segregate with the respective phenotype such as IVS3+120c>a of CRYBB2, while M44V of CRYGD (encoding γD-crystallin), although found in association with blue dot opacity was seen in a few healthy controls too. We identified two new mutations co-segregating along with the respective cataract phenotype within the families that were not seen in healthy controls from India or Germany. These include two missense mutations; one in GJA3 (encoding gap junction protein α3, which is also referred to as connexin 46); the mutation affects codon 19 (T19M), and the corresponding phenotype is a posterior-polar cataract. The other missense mutation affects CRYBB2 (W59C; total cataract). Additionally, a cDNA variation (G54A) identified in a zonular cataract affects a highly conserved splice site of CRYBB2. This mutation, however, showed reduced penetrance in the family, which might be explained by different molecular consequences in the affected family members: nonsense-mediated decay of the mutated mRNA might have no clinical phenotype in heterozygotes, whereas the translation of the mutated mRNA is predicted to lead to a small hybrid protein (consisting of 16 amino acids of the βB2-crystallin and 18 new amino-acids), which might have a dominant-negative function in the lens.

Conclusions: This report identifies in families with childhood cataract some new alleles, which may be considered as causative for cataracts. Furthermore, we report some geographically restricted rare polymorphic sites, whose significance might be considered in some context as modifiers or alleles in sensitizing ocular lens toward cataractogenesis.

Figure 1

Cataract phenotype in family CBE21. Pedigree of the family CBE21 indicates consanguinity after one generation. The blue dot opacity appeared in the second generation and affects four members of the family. The deceased proband (IV-7) is indicated by an arrow. The numbers below the symbol indicate the laboratory number of the samples.

Figure 2

CRYGD mutation in the family CBE21. A: Sequence analysis of exon 2 of CRYGD indicates heterozygosity (arrow) for the mother of the proband (III.4). B: Comparison of the wild-type sequence (WT) with the proband’s mother’s sequence (CBE21) demonstrates that the C→T mutation at cDNA position 130 leads to an amino acid exchange from Met to Val at pos. 44 (M44V); moreover, it shows the creation of a new Alw21I restriction site (underlined) in two (III.4 and III.6) of the affected members of the family checked, while the same was absent in the proband’s grand mother (II.2). One of the proband’s uncle (III.5) could not be checked (in the cDNA sequence, the A of the ATG start codon is counted as #1; in the amino-acid sequence, the first Met is counted as #1). C: Restriction analysis with (+) or without (-) the enzyme Alw21I in the members of the core family leads to an additional fragment of 370 bp in the mutant DNA (red); it demonstrates the presence of the mutation in the affected mother (III.4) and the affected brother of the proband’s mother (III.6). The asterisks mark the additional band of 370 bp indicating the mutation; the band of 533 bp indicates the undigested DNA. WT represents an independent control from the laboratory; M=marker.

Figure 3

Cataract phenotype in family JEE13. A: A lamellar cataract was observed in the 6-year-old proband. B: The pedigree of a 3-generation family demonstrates that the cataract appeared in all three generations affecting both sexes; it indicates an autosomal dominant mode of inheritance. The proband is indicated by an arrow.

Figure 4

HSF4 mutation in family JEE13. A: Sequence analysis of HSF4 genomic DNA indicates heterozygosity at position +9 of the first intron (red arrow) of the proband’s HSF4 gene. B: Comparison of the wild-type sequence (AB029347) with the proband’s sequence demonstrates a C→T exchange at position 9 of intron 1. The mutation leads to a loss of a HaeIII restriction site (underlined). C: Restriction analysis with (+) or without (-) the enzyme HaeIII in members of the family leads to an additional fragment of 98 bp in the mutant DNA (red); it demonstrates the presence of the mutation in the affected mother (II.3) and in the proband (III.4). The asterisks mark the additional band of 98 bp indicating the mutation; the band of 262 bp indicates the undigested DNA. C40 and WT represent independent controls from our laboratories; M=marker.

Figure 5

CRYBB2 mutation in family DJC1. A: The pedigree of family DJC1 indicates that a zonular cataract appeared in the youngest daughter of healthy, but consanguineous parents. There is no other report of any type of cataract over three generations. B: Sequence analysis of CRYBB2 genomic DNA indicates heterozygosity for the proband at cDNA pos. 54 (red arrow). C: Comparison of the wild-type sequence (Z99916) with the proband’s sequence demonstrates that the G→A exchange at cDNA-position 54 leads to an altered splice site; it is predicted that the mutated mRNA contains at least part of intron 2 (the A of the ATG start codon is counted as #1; in the amino-acid sequence, the first Met is counted as #1). The mutation leads to a loss of an Eco130I restriction site. D: Restriction analysis using the enzyme Eco130I creates a larger fragment of 216 bp in the mutant DNA (red); it demonstrates the presence of the mutation in the proband (III.4), but surprisingly also in the healthy mother (II.6) and one healthy sister (III.3) of the proband; the asterisks mark the additional band of 216 bp indicating the mutation. Undigested PCR fragments of 393 bp are indicated by “-” or “+” are digested PCR products. The small fragment of 51 bp in the wild-type DNA is not visible; the two bands of 165 and 177 in the wild type are not separated under the conditions used here. C1 and C2 represent independent controls from the laboratory; M=marker.

Figure 6

Cataract phenotype in family JPM1. A: The eye picture shows a central opacity of both lenses of the female proband (III.2; 2.5 years). B: The central lens opacity appeared in 4 out of 7 siblings of seemingly healthy parents (generation I); one of the brothers (generation II) transmitted the cataract to both his offspring (generation III) indicating an autosomal dominant mode of inheritance. The mutation might have originated in the germ cells of the healthy grandparents (generation I).

Figure 7

CRYBB2 mutation in family JPM1. A: Sequence analysis of CRYBB2 genomic DNA indicates heterozygosity in the proband’s father (II.7) at cDNA pos. 177 (red arrow). B: Comparison of the wild-type sequence (Z99916) with the proband’s father sequence demonstrates that the G→C exchange at cDNA-position 177 leads to an exchange of Trp by Cys (W59C). The BseY1 restriction site in the wild-type sequence is underlined. C: Restriction analysis using the enzyme BseYI leads to an additional fragment of 137 bp in the mutant DNA (red); it demonstrates the presence of the mutation in the affected father (II.7) and the affected brother (III.1) of the proband (III.2). The asterisks mark the additional band of 137 bp indicating the mutation; “undig” is an undigested PCR fragment of 239 bp. WT represents an independent control from the laboratory; M=marker.

Figure 8

Cataract phenotype in family SEC18. A: The posterior polar cataract in the proband (9 years) is demonstrated in the left eye; the right eye is pseudoaphakic. B: Pedigree of family SEC18 indicates the occurrence of the cataract in the second generation of two unrelated families; it is fully transmitted to both sons in the third generation.

Figure 9

GJA3 mutation in family SEC18. A: Sequence analysis of GJA3 genomic DNA indicates heterozygosity (red arrow) in the proband. B: Comparison of the wild-type sequence with the proband’s sequence demonstrates a C→T exchange at position 56 leading to an amino acid alteration at codon 19 (T19M). C: Restriction analysis with (+) or without (-) the enzyme NcoI in members of the family leads to an additional fragment of 688 bp in the mutant DNA (red); it demonstrates the presence of the mutation in the affected father (II.2), in the proband (III.2) and in his brother (III.1). The asterisks mark the additional band of 688 bp indicating the mutation; the band of 732 bp indicates the undigested DNA. WT represents independent control from the laboratory; M=marker.