A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract,on chromosome 1q.

CZP1, a locus for autosomal dominant "zonular pulverulent" cataract, previously had been linked with the Duffy blood-group-antigen locus on chromosome 1q. Here we report genetic refinement of the CZP1 locus and show that the underlying mutation is present in GJA8, the gene for connexin50. To map the CZP1 locus we performed linkage analysis using microsatellite markers on two distantly related branches of the original Ev. pedigree, which now spans eight generations. Significantly positive two-point LOD score (Z) values were obtained for markers D1S2669 (maximum Z [Zmax] = 4.52; maximum recombination frequency [thetamax] = 0) and D1S514 (Zmax = 4.48; thetamax = 0). Multipoint analysis gave Zmax = 5.22 (thetamax = 0) at marker D1S2669. Haplotyping indicated that CZP1 probably lies in the genetic interval D1S2746-(20.6 cM)-D1S2771. Sequence analysis of the entire protein-coding region of the GJA8 gene from the pedigree detected a C-->T transition in codon 88, which introduced a novel MnlI restriction-enzyme site that also cosegregated with the cataract. This missense mutation is predicted to result in the nonconservative substitution of serine for a phylogenetically conserved proline (P88S). These studies provide the first direct evidence that GJA8 plays a vital role in the maintenance of human lens transparency and identify the genetic defect believed to underlie the first inherited disease to be linked to a human autosome.     

A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q

Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans.     

Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3)

We describe a four-generation family with fully penetrant, autosomal dominant, congenital cataracts (ADCC), presenting with morphologically homogeneous "zonular pulverulent" cataracts (CZP) and typical early-onset phenotype. Linkage analysis was performed with a panel of polymorphic markers mapped to all genomic regions of ADCC susceptibility. Contiguous significant two-point lod scores were generated at autosomal region 13q11-q13 and further linkage and haplotype studies confined the disease locus to 13q11, supporting a previous linkage of CZP (specifically CZP3) to 13q11. Mutations in a gap-junction protein, connexin 46 (alphaa3 subunit or GJA3), have recently been reported as being linked to the 13q11 region. Mutational analysis of connexin 46 in our family revealed a C-->T at position 560 (P187L) of the cDNA sequence creating a novel MnlI restriction site that segregated with affected members of the pedigree. This family represents a second report of CZP3 linkage to 13q and is associated with a novel mutation in the connexin 46 (GJA3) gene.     

A New Locus for Dominant “Zonular Pulverulent” Cataract, on Chromosome 13

Inherited cataract is a clinically and genetically heterogeneous disease that most often presents as a congenital autosomal dominant trait. Here we report the linkage of a new locus for dominant “zonular pulverulent” cataract (CZP) to chromosome 13. To map the CZPlocus we performed molecular-genetic linkage analysis using microsatellite markers in a five-generation English pedigree. After exclusion of eight known loci and several candidate genes for autosomal dominant cataract, we obtained significantly positive LOD scores (Z) for markers D13S175 (maximum Z [Z_max] å 4.06; maximum recombination frequency [u_max] å 0) and D13S1236 (Z_max å 5.75, u_max å 0). Multipoint analysis gave Z_maxå 6.62 (u_max å 0) at marker D13S175. Haplotype data indicated that CZP probably lies in the centromeric region of chromosome 13, provocatively close to the gene for lens connexin46.     

Connexin46 mutations in autosomal dominant congenital cataract

Loci for autosomal dominant "zonular pulverulent" cataract have been mapped to chromosomes 1q (CZP1) and 13q (CZP3). Here we report genetic refinement of the CZP3 locus and identify underlying mutations in the gene for gap-junction protein alpha-3 (GJA3), or connexin46 (Cx46). Linkage analysis gave a significantly positive two-point LOD score (Z) at marker D13S175 (maximum Z [Zmax]=>7.0; maximum recombination frequency [thetamax] =0). Haplotyping indicated that CZP3 probably lies in the genetic interval D13S1236-D13S175-D13S1316-cen-13pter, close to GJA3. Sequencing of a genomic clone isolated from the CZP3 candidate region identified an open reading frame coding for a protein of 435 amino acids (47,435 D) that shared approximately 88% homology with rat Cx46. Mutation analysis of GJA3 in two families with CZP3 detected distinct sequence changes that were not present in a panel of 105 normal, unrelated individuals. In family B, an A-->G transition resulted in an asparagine-to-serine substitution at codon 63 (N63S) and introduced a novel MwoI restriction site. In family E, insertion of a C at nucleotide 1137 (1137insC) introduced a novel BstXI site, causing a frameshift at codon 380. Restriction analysis confirmed that the novel MwoI and BstXI sites cosegregated with the disease in families B and E, respectively. This study identifies GJA3 as the sixth member of the connexin gene family to be implicated in human disease, and it highlights the physiological importance of gap-junction communication in the development of a transparent eye lens.     

Detection of c.139G&gt;A (D47N) mutation in <Emphasis Type="BoldItalic">GJA8</Emphasis> gene in an extended family with inheritance of autosomal dominant zonular cataract without pulverulent opacities by exome sequencing

The aim of this study was to identify the gene causing bilateral autosomal dominant zonular congenital cataract (ADZCC) without pulverulent opacities in an extended Muslim family by exome sequencing and subsequent analysis. An extended family of 37 members (14 affected and 23 unaffected) who belong to different nuclear families was screened for causative gene. Proband and her unaffected son were screened for causative variant by exome sequencing followed by Sanger sequencing of the proband’s entire nuclear family. The rest of the members were further screened for variants detected, by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS PCR). Review of exome sequencing data of the proband and her unaffected son for 40 known genes causing congenital nonsyndromic cataracts revealed two variants, namely c.139G>A (p.Asp47Asn; D47N) in the GJA8 gene and c.2036C>T in the FYCO1 gene to be potentially pathogenic. Further, validation of these two variants in the entire family showed cosegregation of c.139G>A variant in GJA8 with ADZCC without pulverulent opacities. Variation of c.2036C>T in FYCO1 was not associated with disease in the family. The mutation c.139G>A in the GJA8 gene detected in the present study was also previously reported in Caucasian and Chinese families but with different phenotypes, i.e. nuclear and nuclear pulverulent cataracts. Thus, the mutation c.139G>A in GJA8 appears to exhibit marked interfamilial phenotypic variability.     

A 5-base insertion in the γC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract

A seven-generation family with 30 members affected by highly variable autosomal dominant zonular pulverulent cataracts has been previously described. We have localized the cataracts to a 19-cM interval on chromosome 2q33-q35 including the gamma-crystallin gene cluster. Maximum lod scores are 4.56 (theta=0.02) with D2S157, 3.66 (theta=0.12) with D2S72, and 3.57 (theta=0.052) with CRYG. Sequencing and allele-specific oligonucleotide analysis of the pseudo gammaE-crystallin promoter region from individuals in the pedigree suggest that activation of the gammaE-crystallin pseudo gene is unlikely to cause the cataracts in the family. In addition, base changes in the TATA box but not the Sp1-binding site have been found in unaffected controls and can be excluded as a sole cause of cataracts. In order to investigate the underlying genetic mechanism of cataracts in this family further, exons of the highly expressed gammaC- and gammaD-crystallin genes have been sequenced. The gammaD-crystallin gene shows no abnormalities, but a 5-bp duplication within exon 2 of the gammaC-crystallin gene has been found in one allele of each affected family member and is absent from both unaffected family members and unaffected controls. This mutation disrupts the reading frame of the gammaC-crystallin coding sequence and is predicted to result in the synthesis of an unstable gammaC-crystallin with 38 amino acids of the first "Greek key" motif followed by 52 random amino acids. This finding suggests that the appropriate association of mutant betagamma-crystallins into oligomers is not necessary to cause cataracts and may give us new insights into the genetic mechanism of cataract formation.     

A novel form of "central pouchlike" cataract, with sutural opacities, maps to chromosome 15q21-22

Congenital cataract is a clinically and genetically highly heterogeneous eye disorder, with autosomal dominant inheritance being most common. We investigated a large seven-generation family with 74 individuals affected by autosomal dominant congenital cataract (ADCC). The phenotype in this family can be described as "central pouchlike" cataract with sutural opacities, and it differs from the other mapped cataracts. We performed linkage analysis with microsatellite markers in this family and excluded the known candidate genes. A genomewide search revealed linkage to markers on chromosome 15, with a maximum two-point LOD score of 5.98 at straight theta=0 with marker D15S117. Multipoint analysis also gave a maximum LOD score of 5.98 at D15S117. Multipoint and haplotype analysis narrowed the cataract locus to a 10-cM region between markers D15S209 and D15S1036, closely linked to marker D15S117 in q21-q22 region of chromosome 15. This is the first report of a gene for a clinically new type of ADCC at 15q21-22 locus.     

A CRYGC gene mutation associated with autosomal dominant pulverulent cataract

Purpose

To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation.

Methods

One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing.

Results

DNA sequencing in the affected members revealed the c.143G>A mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143G>A mutation. No other polymorphisms or mutations were found to be present.

Conclusion

In the present study, we described a family with pulverulent congenital cataract that segregated the c.143G>A mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a disease-causing mutation and not an SNP.     

Assignment of congenital cataract Volkmann type (CCV) to chromosome 1p36

Congenital cataract, type Volkmann (McKusick no 115665, gene symbol CCV) is an autosomal dominant eye disease. The disease is characterized by a progressive, central and zonular cataract, with opacities both in the embryonic, fetal and juvenile nucleus and around the anterior and posterior Y-suture. We examined blood samples from 91 members of a Danish pedigree comprising 426 members, by using highly informative short tandem repeat polymorphisms and found the closest linkage of the disease gene (CCV) to a (CA) _n dinucleotide repeat polymorphism at locus D1S243 (Zmax = 14.04 at _M = 0.025 _F = 0.000), at a penetrance of 0.90. Using two additional chromosome 1 markers, we were able to map the CCV gene in the sequence 1pter-(CCV, D1S243)-D1S468-D1S214. The (enolase 1) gene has been mapped to this area; however, a mutation described in this gene did not give eye disease.