Molecular Vision 2006; 12:1496-1498 <http://www.molvis.org/molvis/v12/a170/> Received 22 May 2006 | Accepted 5 October 2006 | Published 4 December 2006

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A screen for mutations in the transducin gene GNB1 in patients with autosomal dominant retinitis pigmentosa

Geetha H. Mylvaganam, Terri L. McGee, Eliot L. Berson, Thaddeus P. Dryja
 
 

Ocular Molecular Genetics Institute and the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA

Correspondence to: Thaddeus P. Dryja, Ocular Molecular Genetics Institute, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA, 02114; Phone: (617) 573-3319; FAX: (617) 573-3168; email: thaddeus_dryja@meei.harvard.edu

Abstract

Purpose: To search for mutations in the GNB1 gene (coding for the transducin β1-subunit protein) in patients with autosomal dominant retinitis pigmentosa.

Methods: We screened 185 unrelated patients with autosomal dominant retinitis pigmentosa (ADRP) using direct genomic sequencing of the three non-coding exons and 9 coding exons, along with immediately flanking intron DNA.

Results: We found 2 polymorphisms, one in intron 1 with a minor allele frequency of 24%, and one in intron 6 with a minor allele frequency of 12% among the 185 patients. Two rare variants (minor allele frequency <1%) were found in the 3' untranslated region of exon 12. No changes were found in the open reading frame (exons 3-11) or in the noncoding exons 1 and 2.

Conclusions: No likely pathogenic GNB1 mutations have been found in any of 185 unrelated patients with ADRP. This result would be expected if hemizygosity for GNB1 does not result in ADRP or is a rare cause of ADRP.

Introduction

Transducin is a heterotrimeric G-protein that responds to activated opsin and in consequence activates cGMP-phosphodiesterase; it thus mediates an intermediary step in phototransduction [1-3]. Mammalian rod and cone photoreceptors have distinct transducins, referred to as rod transducin and cone transducin. A missense mutation was reported in the gene encoding the α-subunit of rod transducin in the Nougaret form of congenital stationary night blindness [4]. Another group has recently reported that the autosomal dominant retinal degeneration in the mouse strain Rd4 is associated with an inversion in the gnb1 gene, which normally encodes the β-subunit of rod transducin [5]. The inversion would interrupt the 5' untranslated region and likely create a null gnb1 allele. It is possible that the retinal degeneration in the Rd4 mice is related to the hemizygous loss of gnb1 with a corresponding 50% reduction in rod transducin. If so, it is possible that a similar hemizygous loss of GNB1 in humans might lead to a dominant photoreceptor degeneration. We explored this possibility by screening a large set of patients with autosomal dominant retinitis pigmentosa (ADRP) for mutations in GNB1. We did not evaluate patients with recessive RP because the mouse gnb1 mutant is dominant and because the mutation is lethal in homozygous mice, [5] making it unlikely that a retina-specific phenotype would occur in humans with null GNB1 mutations affecting both alleles.

Methods

The human GNB1 locus extends from base 1812355 (5' end) to 1706589 (3' end) in the genomic sequence of chromosome 1p (NCBI). The gene has 12 exons. There is an open reading frame in exons 3 to 11 with a coding sequence corresponding to the human cDNA sequence of the β-subunit of rod transducin (accession number BC004186) [6]. This human cDNA sequence encodes a polypeptide with a sequence identical to the β-subunit of rod transducin in both cattle (accession number BC105260) and mice (accession number NM_008142) [5-7]. We developed PCR-based methods to amplify individually each of the 12 exons of the human GNB1 gene. The primers used for PCR are in Table 1. Each set of primers amplified an entire exon as well as 10 to 72 bp of flanking intron DNA or untranslated region. We amplified the individual exonic fragments from genomic leukocyte DNA from 185 unrelated patients with ADRP. These patients came from families with at least two affected generations with RP; many families had three or more affected generations. Almost all patients live in the United States and Canada; their ethnicity reflects the mixed ethnic make-up of RP patients in those countries. Many of the patients had been screened for mutations in other dominant RP genes; those with identified mutations were excluded from this study.

Each amplification reaction contained 20 ng of leukocyte DNA, 10 mM tris pH 8.6, 1.5 mM MgCl₂, 50 mM KCl, 1.5 M Betaine, 0.2 mM of each dNTP, 1 μM of each primer, and 0.5 units Taq polymerase in a volume of 20 μl. Denaturation was carried out at 95°C for 30 s. The annealing step of the amplification cycle was as follows: for exon 1, 62 °C for 45 s; exon 2, 54 °C for 30 s; exons 3-8, 58 °C for 30 s; exons 9 and 10, 56 °C for 30 s; and exon 12, 54 °C for 45 s. Polymerization was carried out at 72 °C for 45 s. Unused primer molecules and dNTPs were removed from 10 μl of the PCR reaction volume by adding 1.5 μl of ExoSAP-IT (USB, Cleveland, Ohio) and 3.5 μl water. Sequencing was performed using 1 μl of BigDye Terminator v. 3.1 cycle Ready Reaction Mix, 1 μl of BigDye Terminator v. 3.1 cycle Sequencing Buffer (Applied Biosystems, Foster City, CA), 3.2 picomoles of sequencing primer, and 6 μl of Exo-SAP-IT purified PCR product in a total volume of 10 μl. The sense direction primer was used to sequence exons 1-2, 4-6, and 8-12, and the antisense primer was used for exons 3 and 7. All detected sequencing changes were confirmed by sequencing the respective samples in the opposite direction. Sequencing was performed using an ABI 3100 Sequencer (Applied Biosystems, Foster City, CA).

Results

In intron 6 we found two sequence changes in perfect disequilibrium (i.e., all who carried one change carried the other also). We presumed the changes were always in cis and designate these changes as forming one allele: (IVS6+19C>T; IVS6+14G>A). The minor allele frequency was 12% based on our analysis of the 185 patients (146 wild type, 33 heterozygous, and 6 homozygous). Another polymorphism was found in intron one: IVS1+50G>A; its minor allele frequency was also 24% (106 wild type, 69 heterozygous, and 10 homozygous). Two rare variants were found in the 3' untranslated region of exon 12; 3'UTR+83C>T (found in one heterozygous patient) and 3'UTR+285A>G (found in two heterozygous patients). None of these three changes is expected to create or destroy splice donor or acceptor sites based on splice-site prediction software (NN SPLICE, version 0.9) [8]. The distribution of patients who were wild-type, heterozygotes, and homozygotes was not statistically significantly different from what was expected for Hardy-Weinberg equilibrium among the set of 185 patients.

Discussion

Our analysis of 185 patients with ADRP did not reveal any likely pathogenic mutations in the GNB1 gene encoding the β-subunit of rod transducin. It is possible that we missed one or more patients with a large deletion of the gene, since our PCR-based methods for screening for mutations would fail to detect deletions in heterozygotes. However, at human loci where null alleles cause disease, most of the null alleles are nonsense mutations, splice-site mutations, and small deletions or insertions (frameshift mutations) that would be detected with the exon-by-exon analysis that was used in this study. For example, only about 15-22% of dominant, null mutations at the retinoblastoma (RB1) locus are large deletions [9-11]. Assuming that null mutations arise at the GNB1 locus with a spectrum similar to that at other human loci, it is likely that most null alleles would be detected by our study. The fact that we did not discover mutations in 185 patients suggests that GNB1 is not a cause of ADRP or it is a very rare cause of ADRP. It is possible that null GNB1 alleles cause a form of retinal degeneration in humans that is clinically distinct from ADRP. In conclusion, the human counterpart for the Rd4 mouse with dominant retinal degeneration associated with an interruption of the murine gnb1 gene remains unknown.

Acknowledgements

This work was supported by grants from the Foundation Fighting Blindness (TPD, ELB) and the National Eye Institute (EY00169, EY08683, P30 EY014104).

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