Homozygosity mapping of achromatopsia to chromosome 2 using DNA pooling
Homozygosity mapping of achromatopsia to chromosome 2 using DNA poolingNancy C. Arbour1, Joel Zlotogora3, Robert G. Knowlton5, Saul Merin4, Ada Rosenmann3, Adam B. Kanis1, Tatiana Rokhlina1, Edwin M. Stone2 and Val C. Sheffield1,*
1Department of Pediatrics and 2Department of Ophthalmology, University of Iowa, Iowa City, IA 52242, USA, 3Department of Human Genetics and 4Department of Ophthalmology, Hadassah Medical Center, Jerusalem 91120, Israel and 5Thomas Jefferson University, Philadephia, PA 19107-6731, USA
Received November 25, 1996;Revised and Accepted February 26, 1997
Achromatopsia is an autosomal recessive disease of the retina, characterized clinically by an inability to distinguish colors, impaired visual acuity, nystagmus and photophobia. A genome-wide search for linkage was performed using an inbred Jewish kindred from Iran. To facilitate the genome-wide search, we utilized a DNA pooling strategy which takes advantage of the likelihood that the disease in this inbred kindred is inherited by all affected individuals from a common founder. Equal molar amounts of DNA from all affected individuals were pooled and used as the PCR template for short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected members of the kindred was used as a control. A reduction in the number of alleles in the affected versus control pool was observed at several loci. Upon genotyping of individual family members, significant linkage was established between the disease phenotype and markers localized on chromosome 2. The highest LOD score observed was 5.4 ([theta] = 0). When four additional small unrelated families were genotyped, the combined peak LOD score was 8.2. Analysis of recombinant chromosomes revealed that the disease gene lies within a 30 cM interval which spans the centromere. Additional fine-mapping studies identified a region of homozygosity in all affected individuals, narrowing the region to 14 cM. A candidate gene for achromatopsia was excluded from this disease interval by radiation hybrid mapping. Linkage of achromatopsia to chromosome 2 is an essential first step in the identification of the disease-causing gene.
Achromatopsia (total color blindness) is a hereditary disease of the retina which afflicts ~1 in 100 000 people (1 ,2 ). It is relatively common among Jewish Moroccan, Iranian and Iraqi inbred kindreds (3 ). The clinical features of achromatopsia include an inability to discriminate colors, nystagmus, photophobia, and impaired visual acuity in ordinary light. In patients with the disease, the retina has a normal appearance. The underlying cause of the disease is primarily at the photoreceptor level, such that the retinal cones responsible for normal trichromatic color vision are either absent or defective.
The molecular defects that cause achromatopsia have not been identified. Genetic heterogeneity is suggested by the fact that partial achromatopsia exists, in which only two of the three cone pigment types are absent or defective. One form of incomplete achromatopsia, blue cone monochromatism, is an X-linked recessive disorder caused by mutations in photoreceptor pigment genes (4 ,5 ). Complete congenital achromatopsia, or rod monochromacy, is inherited as an autosomal recessive disease. This disorder has not been genetically mapped, although a locus on chromosome 14 has been suggested based on a patient with maternal isodisomy through a Robertsonian 14;14 translocation (6 ).
In the present study, an inbred Iranian Jewish kindred with total color blindness was evaluated. A genome-wide linkage search was performed which was facilitated by pooling DNA from affected and unaffected individuals. This strategy relies on the assumption that there is a high probability that individuals from an inbred population affected with an autosomal recessive disease inherited both copies of the disease gene from a single common ancestor (7 -9 ). DNA pools were screened with polymorphic markers across the genome to identify loci which were homozygous in affected individuals. Genetic markers which displayed a shift toward homozygosity in the affected DNA pool compared to the unaffected parental and sibling pools were used to genotype each individual member of the kindred. Using this approach, an achromatopsia locus was identified on 2p11-q12.
Linkage analysis of the Iranian Jewish kindred (Fig. 1 ) initially involved an examination of markers on chromosome 14, since Pentao et al. (6 ) have identified a maternal isodisomy of chromosome 14 in a patient afflicted with rod monochromacy. Two point maximum likelihood calculations between 18 chromosome 14 markers and the disease phenotype revealed an absence of statistically significant linkage (Table 1 ).
After failing to identify linkage to chromosome 14, a genome-wide linkage study was initiated by using a pooling technique (7 -9 ). DNA samples from the Iranian Jewish kindred were segregated into groups based on phenotype (affected, unaffected parents and unaffected siblings) and PCR amplified with short tandem repeat polymorphic markers (STRPs) (10 ). The amplification products were subsequently separated by electrophoresis on polyacrylamide gels and allele distributions between affected and unaffected DNA pools compared for each marker. Relative intensity of a given allelic band correlates with its frequency within the pooled DNA population. STRPs not linked to the disease locus show similiar band intensities in affected and unaffected DNA pools. With linked STRPs, a reduction in allele distribution is observed in the affected DNA pool, presumably due to homozygosity at the disease locus.
The pooled DNA samples were screened with 300 STRPs to search for linkage with the disease. Four autosomal loci were identified which showed a reduction in allele frequencies in the affected pool when compared with the unaffected parental and sibling pools. These loci included: D2S441 and D2S436 (chromosome 2), D12S395 (chromosome 12) and D19S254 (chromosome 19). Individual members of the Iranian Jewish kindred were genotyped with these markers. Two point maximum likelihood calculations were performed and the following results obtained: D2S441 (LOD 1.9, [theta] = 0.1), D2S436 (LOD 2.2, [theta] = 0.1), D12S395 (LOD 0.0, [theta] = 0.5) and D19S254 (LOD 0.4, [theta] = 0.2). Linkage of achromatopsia to chromosome 2 was confirmed by genotyping additional STRPs near D2S441 and D2S436. The individual genotypes for the loci on chromosome 2 are summarized in Figure 1 . Two point maximum likelihood calculations were performed, resulting in a maximum lod score of 5.4 at [theta] = 0 with markers D2S373 and D2S293 (Table 2 ).
Based upon observed recombination events in affected individuals (III-3 and III-8) in the kindred, the disease interval is defined by markers GATA6E12 and GATA123B03, a distance of ~30 cM. However, a 14 cM region of homozygosity in all affected individuals in the large pedigree is observed between markers D2S388 and D2S373, defining the disease interval based upon ancestral recombination events. Markers within the 14 cM interval were used to genotype four additional small pedigrees shown inFigure 2 . When the two point maximum likelihood calculations were combined with the data obtained from the largest kindred, a maximum LOD score of 8.2 was obtained with marker D2S373 (Families C-E, [theta] = 0, Family B, [theta] = 0.17). Genotypes for all of the affected individuals are shown in Table 3 and the genetic map showing the relationship of markers used in this study is shown in Figure 3 .
Genotypes of all individuals in Families A-E affected with achromatopsia
The numbers above the genotypes refer to the corresponding individuals in the pedigrees. The solid line indicates the region of homozygosity defined by markers D2S388 and D2S373 in the largest kindred.
A promising candidate gene for achromatopsia, visinin (VSNL1), has been genetically mapped to 2p (11 ). Visinin is expressed in the retina and is cone cell-specific (12 ). Radiation hybrid mapping studies were performed to determine the relative order of VSNL1 and flanking markers for the achromatopsia interval on chromosome 2. Two independent analyses were performed, both of which indicated that VSNL1 maps ~400 cR on the proximal side of the achromatopsia locus (Fig. 4 A). Linked markers within the disease gene interval were also radiation hybrid-mapped to determine their relative order (Fig. 4 B).
Mapping of autosomal recessive achromatopsia to chromosome 2 was accomplished by using a DNA pooling strategy. This technique has been successfully applied to linkage mapping of several recessive disorders (8 ,9 ,13 ,14 ). DNA pooling has proven especially effective in linkage studies in genetically inbred populations such as the Iranian Jewish kindred used in the present study. There is no evidence for linkage to chromosome 14, previously associated with rod monochromacy based upon a patient with uniparental isodisomy (6 ).
Analysis of observed recombination events between linked markers and the disease locus in affected individuals indicates that the disease-causing gene lies in a 30 cM interval between markers GATA6E12 and GATA123B03 on the short and long arm of chromosome 2, respectively. A region of homozygosity shared by all affected individuals with markers within the linked interval narrows the achromatopsia interval to ~14 cM. Based upon current genetic and radiation hybrid maps available in the public domain, this region of observed homozygosity is localized at 2p11-q12. Evidence for linkage was also found with the smaller unrelated families. The affected individuals from the smaller pedigrees are clinically indistiguishable from those from the larger Iranian Jewish pedigree. There was no evidence of extensive haplotype sharing between any of the families. This suggests the possibility of allelic heterogeneity, or alternatively, that the families are only distantly related, in which case shared haplotypes would only be observed at markers very near the disease gene.
A possible eye disease-related gene, VSNL1, has been genetically mapped to 2p (11 ). VSNL1 is a human homologue of the rat visinin-like peptide and codes for a cone-specific calcium-binding protein (12 ). Its involvement in phototransduction has been implicated (15 ). Radiation hybrid mapping studies excluded VSNL1 as the cause of achromatopsia by mapping the gene outside of the disease interval.
A Jewish family from Iran consisting of 22 individuals was evaluated. Four additional small families were examined, two of which are Moroccan Jews (Families B and C, Fig. 2 ) and two of which are Palestinian Arabs (Families D and E). All of the affected individuals displayed classic clinical symptoms associated with achromatopsia, including complete absence of color vision, photophobia, low visual acuity and nystagmus. All of the patients were examined by one of the authors who confirmed the diagnosis. Most of the Jewish patients had previously participated in a clinical and electroretinographical study of achromatopsia (16 ).
Blood samples were collected from all individuals and DNA prepared using a standard non-organic procedure (17 ). DNA concentrations were determined by spectrophotometry readings at 260 nm and the samples diluted to 20 ng/[mu]l. The samples were then used as templates in control PCR DNA amplification reactions to confirm equal amplification of each individual sample prior to pooling. Equivalent amounts (1 [mu]g) of DNA from each individual in the large Iranian pedigree were combined into three separate pools: affected patients, unaffected parents and unaffected siblings. Each pool contained either seven or eight DNA samples.
Tri- and tetra-nucleotide STRPs developed by the CHLC (10 ) and commercially available as STRP screening set 6 (Research Genetics) were used for PCR amplification. PCR amplification reactions were prepared with 40 ng of pooled DNA by combining 1.25 [mu]l of PCR buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl2, 0.01% w/v gelatin), 200 [mu]M of dCTP, dGTP, dATP and dTTP, 2.5 pmol of each primer, and 0.25 U Taq polymerase (Boehringer Mannheim) in a final volume of 8.4 [mu]l. Samples were subjected to 35 cycles of amplification (94oC, 30 s; 55oC, 30 s; 72oC, 30 s), the products separated by electrophoresis on 6% denaturing polyacrylamide gels (7.7 M urea) and visualized by silver staining (18 ). Individual samples were also subjected to PCR amplification in an identical manner using 20 ng genomic DNA from each patient as template.
Markers linked to the disease interval and a candidate gene for achromatopsia were mapped on chromosome 2 using radiation hybrid mapping (19 ,20 ) with the GeneBridge 4 radiation hybrid panel (Research Genetics, Huntsville, AL). DNA from each radiation hybrid clone (25 ng) was dispensed into 96-well plates and PCR amplification reactions were prepared as previously described for microsatellite markers. Amplification of a 125 bp segment of the VSNL1 gene (accession no. U14747) was performed using oligonucleotide primers that were selected with the PRIMER program (version 0.5). The sequences of the PCR primers were: VSNL1F 5'-AAG TGA TGG AGG ACC TGG TG-3' and VSNL1R 5'-ATA GAG CTG CTG AAA TTC CTC G-3'. PCR products were separated by electrophoresis on 1% agarose gels and visualized with ethidium bromide. The data were submitted to the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) for assignment of these STSs relative to the radiation hybrid map of chromosome 2.
Individual genotypic data were analyzed for each marker and linkage assessed using the LOD score method (21 ). The criterion for significant linkage was considered to be a LOD score of 3.0 or greater. Analysis was performed with the LINKAGE and FASTLINK programs (22 ,23 ). Marker allele frequencies were assumed to be equal. The reference genetic map used for this study was generated by CHLC and can be accessed electronically via anonymous FTP at http://www.chlc.org. Information on primer sequences, PCR product sizes and genetic distances between markers was obtained from the CHLC (http://www.chlc.org/), Genome Data Base (GDB, http://gdbwww.gdb.org; 24 ), Genethon (http://www.genethon.fr/genethon_en.html; 25 ,26 ), and the Utah Marker Development Group (http://www.genetics.utah.edu/totalmap/index.html; 27 ).
We would like to thank the family members for their willingness to participate in this study and Neena Haider, Anne Kwitek-Black, Theresa Brennan and Darryl Nishimura for their helpful discussions. This work was supported by NIH grant #HG00457 (V.C.S.), the Roy J. Carver Charitable Trust (V.C.S. and E.M.S.) and a grant from the Chief Scientist, Israel Ministry of Health (J.Z).
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*To whom correspondence should be addressed. Tel: +1 319 335 7311; Fax: +1 319 335 7588; Email: val-sheffield@uiowa.edu
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