Limits...
A novel human CRYGD mutation in a juvenile autosomal dominant cataract.

Roshan M, Vijaya PH, Lavanya GR, Shama PK, Santhiya ST, Graw J, Gopinath PM, Satyamoorthy K - Mol. Vis. (2010)

Bottom Line: AluI restriction site analysis confirmed the transversion mutation.Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation.The same was found to be absent in the ethnically matched controls (n=100) studied.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka state, India.

ABSTRACT

Purpose: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India.

Methods: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site.

Results: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein.

Conclusions: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.

Show MeSH

Related in: MedlinePlus

Hydrophobicity profile of wild-type and R77S mutant γD-crystallin protein. Dotted circle represent the shift in the hydrophobicity around the mutant site. The prediction was done by ProtScale program at Expasy server.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875257&req=5

f6: Hydrophobicity profile of wild-type and R77S mutant γD-crystallin protein. Dotted circle represent the shift in the hydrophobicity around the mutant site. The prediction was done by ProtScale program at Expasy server.

Mentions: Both normal and mutant proteins were analyzed for their structure. The R77S (in the processed protein) is situated in second Greek key motif in the linker region as the last amino acid before start of the next beta sheet. The isoelectric point (pI) was found to be almost same for both wild type (7.0) and mutant (6.58) proteins. Molecular weight of the mutant (20,669 Da) protein was similar to that of wild-type protein (20,738 Da). There was an increase in hydrophobicity at the mutant site and its neighborhood (Figure 6).


A novel human CRYGD mutation in a juvenile autosomal dominant cataract.

Roshan M, Vijaya PH, Lavanya GR, Shama PK, Santhiya ST, Graw J, Gopinath PM, Satyamoorthy K - Mol. Vis. (2010)

Hydrophobicity profile of wild-type and R77S mutant γD-crystallin protein. Dotted circle represent the shift in the hydrophobicity around the mutant site. The prediction was done by ProtScale program at Expasy server.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875257&req=5

f6: Hydrophobicity profile of wild-type and R77S mutant γD-crystallin protein. Dotted circle represent the shift in the hydrophobicity around the mutant site. The prediction was done by ProtScale program at Expasy server.
Mentions: Both normal and mutant proteins were analyzed for their structure. The R77S (in the processed protein) is situated in second Greek key motif in the linker region as the last amino acid before start of the next beta sheet. The isoelectric point (pI) was found to be almost same for both wild type (7.0) and mutant (6.58) proteins. Molecular weight of the mutant (20,669 Da) protein was similar to that of wild-type protein (20,738 Da). There was an increase in hydrophobicity at the mutant site and its neighborhood (Figure 6).

Bottom Line: AluI restriction site analysis confirmed the transversion mutation.Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation.The same was found to be absent in the ethnically matched controls (n=100) studied.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka state, India.

ABSTRACT

Purpose: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India.

Methods: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site.

Results: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein.

Conclusions: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.

Show MeSH
Related in: MedlinePlus