A Molecular Vision Short Report
Mol. Vis. 2 : 10, 1996 <http://www.emory.edu/molvis/v2/danciger>

A Homozygous PDE6B Mutation in a Family with Autosomal Recessive Retinitis Pigmentosa

Michael Danciger,^1,2,* Vickie Heilbron,¹ Yong-Qing Gao,¹ Dan-Yun Zhao,¹ Samuel G. Jacobson,³ and Debora B. Farber.¹

¹Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA
²Loyola Marymount University, Los Angeles, CA
³Scheie Eye Institute, U. of Pennsylvania, Philadelphia, PA 19104

*To whom correspondence should be addressed (E-mail: danciger@ucla.edu)

Based on average estimates of the prevalence of non-syndromic retinitis pigmentosa (RP) at 1/4,000, there are approximately 1.5 million people in the world with this inherited, progressive, degenerative disease of the retinal photoreceptor cells which often leads to blindness (1,2,3). About 50% of these cases are inherited in an autosomal recessive manner (AR).

With the approach of screening the exons of candidate genes in large numbers of unrelated ARRP probands, mutations associated with disease have been found in several candidate genes expressed in rod photoreceptors at very low frequency: RHO, encoding rhodopsin, 1/126 patients screened (4); PDE6B, encoding the beta-subunit of rod cGMP-phosphodiesterase, 4/88 patients (5); PDE6A, encoding the alpha-subunit of rod cGMP-phosphodiesterase, 2/173 patients (6); and CNCG, encoding the alpha-subunit of the rod cGMP-gated channel, 3/173 patients (7). With the approach of linkage analysis in large families, two ARRP loci have so far been discovered: 1q31-q32.1 (8,9) and 6p, distal to RDS-peripherin (10). Also, linkage analysis of an informative consanguineous family led to the discovery of a second homozygous RHO mutation (11).

ARRP tends to appear most often in small nuclear families that by themselves are not informative enough to yield significant linkage data, and screening all the exons of candidate genes like PDE6A and PDE6B (each with 22 exons) in large numbers of unrelated probands is costly and time consuming. Therefore, we have taken the approach of studying candidate genes in small nuclear ARRP families with a double screening protocol. Linkage analyses of markers close to the loci of the candidate genes are performed first, and any families where a gene locus clearly does not segregate with disease are ruled out from further study of that gene. DNAs of the probands from the remaining families (where the gene locus cannot be ruled out from segregating with disease) are then screened for mutations in the exons of the candidate gene by SSCPE (single strand conformation polymorphism electrophoresis) and DGGE (denaturing gradient gel electrophoresis). Any exonic variants found are sequenced directly and analyzed within the corresponding family to see if they appear to segregate with disease. With this approach we studied 24 families with inherited retinal degenerations (14 with typical RP) for mutations in the genes PDE6B, MYL5, PDE6C, CNCG, RHO, ROM1 and RDS-peripherin. We have reported two typical ARRP families where the affecteds uniquely inherited compound heterozygous mutations in the PDE6B gene (12). With a similar approach, homozygous mutations also have been found in PDE6B in the affecteds of two other ARRP families (13,14).

We have now completed analysis of PDE6B in the original 24 families and extended our study to a total of 30 nuclear families of patients with autosomal recessive inheritance of retinal degeneration; 19 with typical RP and 11 with various forms of cone-involved disease. In each of the families neither parent had disease, and in 26 of the families there were two or more affected siblings; in the 4 families with only one affected sibling either the parents were related or a relative not in the direct line of descent was affected with similar disease. In the 8 families where all of the affecteds were males, no X-linked carrier signs were observed in the mother, and/or the parents were related. We report here our findings involving the PDE6B gene.

Blood was drawn in 10 ml tubes from members of the 30 families after informed consent was given, and DNAs were extracted from the leukocytes. The DNAs were analyzed for the alleles of a dinucleotide repeat marker ("PDEB") adjacent to the PDE6B gene at chromosome 4p16.3 (15,16). Genomic DNAs were amplified by PCR in the presence of [alpha-³²P]dCTP, electrophoresed in 7% acrylamide denaturing gels along with a size marker, and exposed to X-ray film as described previously (12). All members of any particular family were run side by side in the same gel. Of the 30 families tested for the marker, 21 could be ruled out from segregating with disease. The DNAs of the probands of the 9 remaining families were screened for sequence variants in the PDE6B gene by SSCPE and DGGE (12). Exons containing variants were sequenced directly by the standard dideoxy method using [alpha-³²P]dCTP as label and the fmol sequencing kit (Promega, Madison,WI).

Along with the two families we previously reported, in which the affecteds have typical RP and carry compound heterozygous mutations in the PDE6B gene, we now report a third typical RP family with a novel homozygous mutation in PDE6B. The mutant allele segregates with disease in this small family (Figure 1a), and contains a nonsense point mutation which converts the Cysteine 270 codon in exon 4 to a stop codon (TGC to TGA; Cys270X) (Figure 1b). This predicts a protein truncated by more than 500 amino acids including the complete loss of the catalytic site.

The patient had a history of night vision disturbances for many years and more recent difficulty with peripheral vision. Neither her parents, who were first cousins, nor her two siblings had any visual symptoms. At age 25 the proband was given a complete eye examination, Goldman kinetic visual fields, dark-adapted two color (500 and 650 nm stimuli) static threshold perimetry, and full field electroretinograms (ERGs). Perimetry and ERG methods have been published (17). She showed the ophthalmologic hallmarks of RP including attenuated retinal vessels, waxy-appearing optic nerve heads, and pigmentary changes in the midperipheral retina. Her kinetic visual fields with the V-4e target showed midperipheral loss, most prominently in the nasal and superior fields; with the I-4e target, there was only a central island of vision (Figure 2a). Dark-adapted perimetry indicated that only cone function remained and that it was abnormally reduced (Figure 2b). ERGs were mainly not detectable; the only response that remained was a very reduced and delayed cone ERG to white light flashes on a white background (Figure 2c).

The mouse homolog of PDE6B has been shown to be the site of the mutation responsible for the inherited retinal degeneration of the rd mouse (18,19) and verified by transgenic rescue (20). Two mutations were found in the rd gene in every strain of mouse carrying it: a homozygous nonsense mutation in exon 7 (21) and a homozygous 8.5 kb murine leukemia provirus intronic insertion (22). Additionally, retinal degeneration in the Irish setter dog was first shown to be caused by a defect in the canine PDE6B gene (23), and later identified as a homozygous null mutation (24). Considering the evidence in animal models it is extremely likely that the homozygous nonsense mutation in exon 4 of the proband presented in this paper is responsible for her disease. Another point of support is the fact that the affecteds in the now 9 small families in which either compound heterozygous or homozygous mutations in PDE6B have been found (5,12,13,14 and this paper) all have similar although not unique disease signs and symptoms, including long-standing night blindness, slow progression of peripheral vision loss, and preservation of visual acuity until late in the disease course. Additionally, typical features of RP are seen with ophthalmoscopy, and early loss of rod function with abnormal but detectable cone function (becoming undetectable later) is found with electroretinography.

We have now discovered mutations in the PDE6B gene in 3 of 19 ARRP families (~16%). As already mentioned, in other studies of this gene, 4/88 unrelated ARRP patients (~4.5%) were shown to carry compound heterozygous mutations that cosegregated with disease in their respective families (5) and the affecteds of 1/19 small families (~5%) were found to have a homozygous mutation (13). Our finding of 3/19 typical RP families with mutations in PDE6B reflects a higher prevalence than 4/88 or 1/19. However, the numbers are too small to have a statistically significant difference at the 0.05 level, and the populations studied may be different. Furthermore, any prevalence data for RP associated with this or other genes has to be qualified by the possiblity of undetected mutations in regions not tested by exon screening such as regulatory sites of the 3'UTR, or intronic regions. Further study of many more families will be necessary to determine a more accurate prevalence of exonic PDE6B mutations in ARRP, and new approaches will be necessary to evaluate the non-exonic regions of this and other candidate genes.

Acknowledgements

This research was supported by grants from the Foundation Fighting Blindness (DBF, MD and SGJ), from the George Gund Foundation (DBF and MD), the NIH (DBF, EY08285 and EY00331; SGJ, EY05627), and a Research to Prevent Blindness Unrestricted Grant (DBF). DBF is the recipient of a Research to Prevent Blindness Senior Scientific >Investigators Award. We thank Mr. G. Regunath for help with visual function data analysis.

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