Methods  X chromosome genotyping, fine-point mapping, and haplotype analysis of the DNA from 22 Minnesota family individuals (8 affected males and 5 carrier females) and 6 members of the original family with BED were performed. Haplotype comparisons and mutation screening of the red-green cone pigment gene array were performed on DNA from both kindreds.

Results  Significant maximum logarithm of odds scores of 3.38 and 3.11 at = 0.0 were obtained with polymorphic microsatellite markers DXS8106 and DXYS154, respectively, in the Minnesota family. Haplotype analysis defined an interval of 34.4 cM at chromosome Xq27.3-Xq28. Affected males had a red-green pigment hybrid gene consistent with protanopia. We genotyped Xq27-28 polymorphic markers of the family with BED, and narrowed the critical interval to 6.8 cM. The haplotypes of the affected individuals were different from those of the Minnesota pedigree. Bornholm eye disease–affected individuals showed the presence of a green-red hybrid gene consistent with deuteranopia.

Conclusions  Because of the close geographic origin of the 2 families, we expected affected individuals to have the same haplotype in the vicinity of the same mutation. Mapping studies, however, suggested independent mutations of the same gene. The red-green and green-red hybrid genes are common X-linked color vision defects, and thus are unrelated to the high myopia and other eye abnormalities in these 2 families.

Clinical Relevance  X-linked high myopia with possible cone dysfunction has been mapped to chromosome Xq28 with intervals of 34.4 and 6.8 centimorgan for 2 families of Danish origin.

X-linked myopia transmission associated with other ocular findings, either as a possible secondary effect or as part of a syndrome, is more common. The association of X-linked transmission with congenital stationary night blindness and retinitis pigmentosa has been well described.^4-5 Forsius and Eriksson⁶ described the Åland eye disease, with X-linked myopia, albinism of the fundus, markedly impaired vision, abnormal dark adaptation, dyschromatopsia classified as protanomaly, foveal hypoplasia, and nystagmus. Francois et al⁷ described an X-linked atypical achromatopsia combined with myopia, impaired vision, foveal aplasia, nystagmus, and photophobia. Mäntyjärvi et al⁸ described a family with high myopia and cone dysfunction that they mapped to Xp11.4-q13.1 and designated as progressive cone-rod dystrophy locus 3 (COD3), which may be the same X-linked progressive cone-rod dystrophy with myopia recently mapped by Jalkanen et al.⁹

The Bornholm eye disease (BED) was mapped to Xq28 by Schwartz et al in 1990¹⁰ by linkage analysis using 3 restriction fragment length polymorphic markers. No haplotyped interval was provided and no gene mutations have been reported for this disorder. This locus has been designated the MYP1 locus by the Human Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature) (Online Mendelian Inheritance in Man entry 310460). The original 5-generation family studied from the island of Bornholm, Denmark, was initially described by Haim et al in 1988.¹¹ Affected males had an early-onset (1.5 to 5 years of age) X-linked form of myopia ranging from –6.75 to –11.25 D accompanied by several other ophthalmologic features. Six of 8 examined affected males had astigmatism of 1 D or more. Their ophthalmoscopic examinations showed temporal conus of the optic disc, as well as peripapillary atrophy of the retinal pigment epithelium, giving the appearance of moderate optic nerve hypoplasia. The choroidal vessels were described as visible in the posterior pole, and no macular or lattice degenerations were noted. No patients sustained a retinal detachment. Electroretinogram (ERG) testing (performed in 4 of the 8 affected individuals) demonstrated subnormal flicker function and normal results of scotopic testing. The refractive error, visual acuity, and results of ERG testing and color vision testing all remained stable in most affected males after age 16 years. All affected males had a color vision defect of deuteranopia.

We ascertained a large Minnesota family originating from the nearby Danish islands of Møn and Zealand with X-linked high myopia and phenotypic features similar to those described for BED. However, all affected individuals in the Minnesota family had a protanopic color vision defect. Thus, all affected individuals in the Minnesota and BED families had a color vision deficiency—albeit of different types—of protanopia and deuteranopia, respectively. This raised the question of whether color vision deficiency was part of the syndrome. We hypothesized that mutations in either the red or green pigment genes might have disrupted structure and function to cause cone dysfunction and the high myopia associated with it. A few such mutations had previously been described, such as the C203R substitution in the green pigment gene.^12-14 We investigated the gross structure and sequence of the red and green color vision genes in both the original BED family and the present Minnesota family. We examined the possibility that the phenotype of the 2 families mapped to the same locus, and whether mutations in the visual pigment genes might be responsible for the ophthalmologic phenotype. Fine-point mapping and candidate region mutation screening were performed in an effort to further refine the mapped interval, and to uncover the genetic mechanisms associated with this disorder.

METHODS

The family studied in this investigation was a 6-generation family originating from Denmark (Figure 1). Individual 1 was from the island of Møn, and his wife, individual 2, was from Vortenberg on the island of Zealand. There was no known consanguinity. Eight affected males and 5 female carriers were studied. In total, the DNA of 22 people underwent genotyping with polymorphic microsatellite markers of the X chromosome (Figure 1).

View larger version (53K): [in this window] [in a new window] Figure 1. Pedigree and haplotypes of a Minnesota 6-generation family with X-linked high myopia and protanopia. Circles and squares denote females and males, respectively; solid symbols denote affected individuals; and symbols with slashes denote deceased individuals. Obligate female carriers are denoted with a circle containing a dot. Unknown phenotype status is denoted with a circle or square containing a question mark. Each individual studied (plus sign) has alleles shown for X chromosome markers in descending marker order from the telomere of the p arm to the telomere of the q arm. Haplotypes were constructed on the basis of the minimum number of recombinations between markers. The chromosome assumed to carry the disease allele is blackened. Only essential matings are shown; nonparticipating family members are not shown. Haplotype analysis indicates crossovers in individuals 24 and 34, which defines the chromosomal region of interest. This region spans 34.4 centimorgans from GATA31E08 to q-ter. Inferred and undetermined genotypes are denoted with parentheses and question marks, respectively.

All Minnesota family participants underwent extensive ophthalmologic evaluation, including Snellen visual acuity testing, slitlamp examination, intraocular pressure testing, cycloplegic refraction, and detailed funduscopy. In addition, fundus photography, ultrasound axial length, and keratometry (corneal curvature) measurements were performed on most participants.

Full-field, single-flash, and flicker ERG responses were recorded (UTAS-E 2000 system; LKC Technologies Inc, Gaithersburg, Md) according to the recommendations of the International Society for Clinical Electrophysiology of Vision.¹⁵ Burian-Allen bipolar electrodes were used. Mixed rod and cone responses were obtained by means of stimulation with flashes of maximum intensity (0 dB) in dark-adapted conditions. Dark-adapted scotopic rod responses were recorded by means of full-field white flashes of relatively low intensity (24-dB attenuation). Single white-flash, dark-adapted oscillatory potentials measuring scotopic rod responses were recorded with high-intensity 0-dB stimulation. Photopic cone responses were elicited in light adaptation to white background (480 lumen/m²), and with maximum flash stimulation (0 dB). Flicker ERG responses (30 Hz) were recorded in light adaptation (480 lumen/m²), using an averaging technique (n = 10) and stimulation with maximum-intensity flashes.

Color vision was tested under standardized conditions, with pseudoisochromatic plates (Ishihara test for color blindness, 38-plate edition, 1988), the desaturated panel D-15 test, and the Farnsworth Munsell 100-hue test. The anomaloscope Nagel type II test was performed on 2 affected male subjects, 24 and 25.

PATIENTS (BED FAMILY)

Individuals with genomic DNA samples from the original BED family are shown in Figure 2. Individuals BED-16 and BED-25 had deuteranopia only, and not the full clinical spectrum of eye findings in the BED, such as myopia, photopic ERG abnormalities, and optic nerve hypoplasia. Individuals BED-26 and BED-28 were affected males, individual BED-22 was a carrier female, and individual BED-27 was an unaffected male offspring of BED-22. Individual BED-22 was also the monozygotic twin of the mother of individual BED-26 (BED-20).

View larger version (22K): [in this window] [in a new window] Figure 2. Limited version of a branch of the original Bornholm eye disease pedigree with haplotypes of chromosome Xq27-q28 markers. Symbols are explained in the legend to Figure 1. Individual 3 has only deuteranopia, which is unrelated to Bornholm eye disease (see the last paragraph of the "Molecular Structure of Color Vision Genes" subsection of the "Results" section).

This research was performed according to the Declaration of Helsinki and was approved by the local institutional review boards.

X CHROMOSOME MARKER TYPING AND FINE MAPPING

The DNA was isolated from peripheral-blood lymphocytes by standard techniques. The genome screen used polymorphic microsatellite markers from the Weber 4a, 8a, and 9a sets (Research Genetics Inc, Huntsville, Ala).¹⁶ For fine mapping, additional markers were selected from the Généthon (http://www.genethon.fr) and Marshfield (http://www.marshfieldclinic.org/research/genetics/) genetic map databases of chromosome Xq27 and Xq28. The 5' marker of each primer set was modified with a special M13 sequence that allows for fluorescent detection.¹⁷

Polymerase chain reactions (PCRs) were prepared in 96-well plates with 2.0mM magnesium chloride, 50mM potassium chloride, 10mM Tris hydrochloride (pH 9.0), 0.1% Triton X-100, 200µM of each dNTP, 1.0 pmol of each marker primer, 0.08 pmol of M13 primer, 0.4 U of Taq DNA polymerase (AmpliTaq or AmpliTaq Gold; Perkin Elmer, Norwalk, Conn), and 20 ng of DNA. Annealing temperatures were adjusted according to the specific characteristics of each marker. After amplification of 30 cycles, aliquots of amplification products were mixed with 6X Ficoll buffer and separated by electrophoresis through a preheated 23-cm, 6% polyacrylamide, 7M urea denaturing gel. A dual-dye infrared DNA analyzer was used (Li-Cor DNA 4200; Li-Cor Inc, Lincoln, Neb). Allele sizes were determined from computer images by means of restriction fragment length polymorphism (RFLP) scan software (Scanalytics Inc, Fairfax, Va). Alleles were visualized as an autoradiogram-like image on a computer, and size was determined with RFLP scan software (Scanalytics Inc). Allele sizes were then directly imported to a database (Filemaker Pro Inc, Santa Clara, Calif), which was then used for logarithm of odds (LOD) score determination.

LINKAGE ANALYSIS

Linkage analysis was performed with the C version of the LINKAGE package, FASTLINK^18-19 and the utility programs Makeped, LINKAGE control program, and LINKAGE report program from LINKAGE 5.1.^19-22 Marker allele frequency estimates were based on the frequencies of alleles in married unrelated individuals in the families. Two-point LOD scores were also calculated with all alleles set at equal frequencies to control for allele frequency effects. Standard marker databases were used for intermarker recombination frequencies, marker order, and marker distances.

Linkage analysis was performed with a myopia gene frequency of 0.01 at 100% penetrance of the gene. For initial data analysis, high myopia was assumed to have an X-linked inheritance mode, and the MLINK program^{18, 22} was used to calculate 2-point LOD scores between myopia and each marker. All affected individuals and informative spouses were included in the linkage analysis. The LODSCORE program was used to calculate maximum LOD scores at the lowest recombination frequency.

MUTATION SCREENING OF THE LOCUS CONTROL REGION

Primers with a 19–base pair M13 tail were designed by using a published DNA sequence of the locus control region (LCR) of the cone pigment genes on Xq28.^23-24 The PCR reactions were performed as previously described,^23-24 and the sequencing products were mixed with 6X Ficoll buffer and electrophoresed through a preheated 39-cm, 7% polyacrylamide, 7M urea denaturing gel in a DNA analyzer (Li-Cor Inc) as described in the preceding section. The base pair sequence was visualized on a computer with the use of Gene Imager software (Li-Cor Inc). The sequence was compared with that of unaffected controls and with the published sequence.

MOLECULAR ANALYSIS OF THE RED AND GREEN VISUAL PIGMENT GENES

The gross structure of the red and green visual pigment genes was determined by means of quantitative PCR amplification followed by single-stranded conformational polymorphism analysis (SSCP) as described in detail elsewhere.^25-26 This procedure allowed determination of the ratio of red to green promoters, which gives the total number of genes, as well as those of exons 2 to 5 (exons 1 and 6 are identical between the red and green pigment genes). In addition to ratios, SSCP detects nucleotide variants.²⁷

The primers used to cover the promoter, coding regions, and exon-intron junctions are described in detail elsewhere.^25-26 The following primers were used: 32 (antisense primer in exon 1) for the promoter region, 130 (an antisense primer in intron 1) for exon 1, 174 (a sense primer in intron 1) for exon 2, 2A (a sense primer in exon 3) and 2Z (an antisense primer in exon 3) for exon 3, 30 (a sense primer in exon 4) and 2B (an antisense primer in exon 4) for exon 4, 3C (a sense primer in exon 5) and 79G (an antisense primer in exon 5) for exon 5, and 34 (an antisense primer in 3'-UTR) for exon 6. First, 12-kilobase (kb) segments extending from the promoter to exon 5 of the red and green pigment genes were amplified separately. The forward primer was either red promoter–specific (169RF) or green promoter–specific (171GF). The reverse primer in exon 5 (79G) was green-specific (5'), as the arrays were found not to contain red exon 5 (presence of red-green hybrid gene in the first position).

The PCR reaction contained, in a total volume of 25 or 50 µL, 0.25 or 0.5 µg of total genomic DNA, 0.2µM of each primer, 400µM of dNTPs, 1 x LA PCR buffer II (Applied Biosystems, Foster City, Calif) (magnesium chloride), and 1 or 2 U of Taq DNA polymerase (Takara LA; Takara Biotechnology, Shiga, Japan). The amplification conditions were as follows: 1 minute of denaturation at 94°C was followed by 14 cycles of 98°C for 10 seconds and 68°C for 12 minutes; 16 cycles of 98°C for 10 seconds and 68°C for 12 minutes plus 15 s/cycle; and 72°C for 10 minutes. Second, a segment encompassing exon 5, intron 5, and exon 6 was amplified with forward primers 3C and 34 (5'-GCAGTGAAAGCCTCTGTGACT-3') primers.²² Third, the PCR products were gel purified with a gel extraction kit (QIAquick; Qiagen, Valencia, Calif) and the desired portions were directly sequenced by means of a kit (BigDye Terminator Cycle Sequencing kit; Perkin Elmer, Foster City, Calif) (total, 40 cycles) and a DNA sequencer (ABI Prism; Applied Biosystems).

SOUTHERN BLOT ANALYSIS

Southern analysis was performed with a 1.3-kb complementary DNA (cDNA) clone of the red cone pigment gene using standard techniques.^{13, 23-24} This hs7 probe was cut from its pUC19 vector, gel purified, and labeled with -³²P-dCTP. Patient sample genomic DNA was digested with EcoRI and BamHI (Gibco BRL, Gaithersburg, Md). Each reaction contained 7 µg of genomic DNA, 25 U of each enzyme in a total volume of 50 µL, and was incubated for 2 hours at 37°C. The digested products were separated by electrophoresis on a 0.7% SeaKem agarose gel (FMC; BioProducts, Rockland, Md). After separation, the cut DNA was capillary blot transferred onto a membrane overnight and blot fixed by UV crosslinking. Prehybridization and hybridization steps were performed with the labeled cDNA. The hybridized bands were visualized after overnight exposure of the membrane to radiographic film at –80°C.

CYTOGENETIC METHODS

A peripheral blood sample for chromosome analysis of Minnesota participant 25 was processed according to standard techniques, and prometaphase preparations were analyzed by means of 850 high-resolution G-banding.²⁸

RESULTS

Three generations of affected males were clinically studied in the Minnesota kindred. The youngest affected male studied was 5 months of age (subject 37); the oldest was 37 years of age (subject 25). Myopia was diagnosed at 6 years of age or younger (13 months to 6 years) in the affected males. Corrected visual acuity was excellent to good in all affected males (range of 20/20 to 20/40). On review of patients' ophthalmologic records, the visual acuity and degree of myopia was stable after puberty (age 16-20 years). We followed up all participants for 5 years, and the clinical features have been stable for all affected males.

No patient had photophobia, nystagmus, night blindness, or paradoxical pupillary response. Affected adult individuals had high myopia (mean spherical refractive component of –13.18 D; range, –10.25 to –18.25 D). The average adult keratometry reading for astigmatic cylinder of 43.78 D (range, +41.50 to +44.62 D) was not significantly higher (P = .18, 2-tailed t test) than the published mean ± SD adult normal value of 43.1 ± 1.62 D.²⁹ The average adult axial length of 28.39 mm (range, 26.84-31.73 mm) was significantly greater (P = .009) than the published mean ± SD adult normal value of 24.2 ± 0.85 mm.²⁹ Intraocular pressures were normal in all tested participants.

All affected males had fundus findings of optic nerve temporal conus and posterior pole retinal pigment epithelial thinning with prominent choroidal pattern. Optic nerve head caliber was normal. The peripheral fundus had less retinal pigment epithelial thinning with more pigmentation relative to the posterior pole. No peripheral retinal degenerative changes were noted. A tapetal reflex was not observed. No macular changes (bull's eye, granular, etc) were noted for any members. The vitreous was normal in all examined participants.

The ERG testing of all affected males in the Minnesota family showed normal rod-dominated scotopic waveforms (representative rod response b wave, 174/172 µV and 100/113.5 milliseconds; normal, >120 µV and <108.5 milliseconds). The mixed rod-cone b-wave amplitude responses were normal in some and depressed in others, with normal implicit times (representative maximal rod and cone response b wave, 179/277 µV and 44.5/44 milliseconds; normal, >320 µV and <53.5 milliseconds). The cone responses showed subnormal b-wave amplitudes and normal implicit times (representative cone response b wave, 40/29 µV and 32.5/35.5 milliseconds; normal, >65 µV and <32.5 milliseconds). All affected males had reduced amplitudes of the 30-Hz flicker ERG recordings with increased implicit times (representative flicker response b wave, 37/20 µV and 32.5/35 milliseconds; normal, >42 µV and <30 milliseconds). All carrier females and unaffected males or females had normal ERG recordings. Comparative ERG findings between an affected subject, an obligate carrier, and an unaffected male control are shown in Figure 3. While these findings are suggestive of a cone dysfunction, ERG amplitude may also be influenced by increasing axial length.^30-33 Westall et al³³ reported significant differences in ERG amplitude between subjects with high myopia and those with small refractive errors. Implicit times, the ratio of b to a wave, and semisaturation constants showed no significant differences. These authors and others found these results to apply to rod and cone responses. However, cone responses were more affected, presumably because of change in geometry, which could distort the axis of photoreceptor segments or reduce the number of cones per unit area.^30-33 In the latter case, it would not matter if all photoreceptors were included in the ERG measurements. In addition, subnormal flicker ERG findings noted in the BED kindred may be suggestive of cone dysfunction but, in view of high myopia, are not diagnostic.

View larger version (91K): [in this window] [in a new window] Figure 3. Comparative electroretinogram responses between an affected Minnesota family participant (person 24) (some blink artifact), obligate carrier (person 33), and an unrelated normal male control. A, Mixed rod and cone responses obtained with flashes of maximum intensity (0 dB) show a subnormal b-wave amplitude in the affected participant, but not in the carrier or control. B, Low-intensity (24-dB) dark-adapted scotopic rod responses showed no definite abnormality among the participants. C, Oscillatory potentials, single white-flash, dark-adapted (scotopic rod) responses recorded with the use of high-intensity 0-dB stimulation showed no definite abnormality among the participants. D, Maximum-flash (0-dB), photopic cone responses show marked reduction of both a- and b-wave amplitudes in the affected participant, compared with normal amplitudes for the carrier and control recordings. E, The affected individual has, in contrast to the carrier and control, grossly delayed and reduced amplitudes of his 30-Hz-flicker photopic cone electroretinogram responses.

All affected males of the Minnesota family failed the Ishihara color-plate screening test and had color vision testing results consistent with protanopia, unlike the BED phenotype, which had deuteranopia as a clinical feature. The Rayleigh match range determined by type II Nagel anomaloscope was 73, indicating dichromatic color vision consistent with a severe protan defect. Automated static perimetry using programs 30-2 and 30-1 (Humphrey Field Analyzer; Humphrey Instruments, Inc, San Leandro, Calif) of the central visual fields and formal Goldmann visual field testing in individuals 24 and 25 were normal. Pertinent clinical eye findings are listed in Table 1.

View this table: [in this window] [in a new window] Table 1. Ophthalmologic Characteristics of Affected Individuals

All carrier females, noncarrier females, and unaffected males had normal refractive errors, ophthalmologic evaluations, ERG results, and color testing results.

MAPPING STUDIES

The Minnesota pedigree was studied with 23 markers spanning the entire X chromosome (Table 2 and Figure 4). One or multiple recombination events occurred between each of the markers outside but not within the Xq27-q28 region and the putative disease gene. Haplotypes displaying the segregation marker alleles in the chromosome Xq27-28 region in the family are shown in Figure 1.

View this table: [in this window] [in a new window] Table 2. Two-Point Linkage Analysis Between High Myopia With Protanopia and Microsatellite Markers on Chromosome X

View larger version (56K): [in this window] [in a new window] Figure 4. Schematic representation of the X chromosome and the Xq27/Xq28 region. Marker order and genetic distances were determined by reference to the National Center for Biotechnology Information genetic map, the Weber 9 Set, and the Généthon genetic map. The position of the markers relative to the chromosomal bands are approximate. Opsin 1 indicates red-green cone pigment gene array; cM, centimorgan.

The status of individual 37 was designated as unknown primarily because of his age. He was 5 months old at the time of his first examination and had a cycloplegic refractive error of +1.00 +1.00 at 90° OU. A repeat ophthalmologic examination at 9 months of age showed a reduction of 1.00 D in hyperopic sphere in both eyes, indicating a non–age-appropriate myopic shift. Results of fundus examination were normal. Haplotype analysis shows that he inherited the disorder (Figure 1). More clinical information is needed and will be available when this subject is old enough to undergo further testing.

Close linkage without recombination was found between the putative gene and markers DXS8106 at Xq27.3, DXS8103 at Xq28, and DXYS154 at Xq28, with maximum LOD scores of 3.38, 3.01, and 3.11 at = 0.0, respectively. Analysis of the chromosome Xq27-28 haplotypes (Figure 1) suggests that the disease locus in the Minnesota family is located in the chromosomal region between the marker loci GATA31E08 and the q-ter, due to informative recombination events between markers GATA31E08 and DXS8106 for individuals 24 and 34. This region defines a 34.4-cM interval (Figure 4).

The BED family phenotype had a provisional assignment to the distal part of the X chromosome at Xq28 with the use of a limited number of markers originally. No critical region was defined within Xq28. We performed genotyping of 6 members of the BED family with additional markers. We observed that the disorders of the Minnesota and BED families of Danish origin map to the same locus (Figure 2 and Table 3), suggesting that these families carry the same mutation. Since the 2 families originated from villages that are only 100 miles apart, we explored the degree of identity in genotypes and haplotypes at various Xq28 markers. The markers studied were DXS8106, DXS8028, DXS998, DXS8069, DXS8103, DXS8061, DXS8087, DXS1073, and DXYS154. Our haplotype analysis of the 6 participating members of the BED family with the use of chromosome Xq27.3-28 polymorphic markers shows that the region of the X chromosome associated with high myopia and presumable cone dysfunction has different ancestral origins in the Minnesota and BED families (Table 3). Some markers could not be typed, probably because of older DNA (DXS8028, DXS998, DXS8069, and DXS8103). We found evidence of a recombination event that occurred in subject BED-20 (probable fraternal twin), inferred from the haplotype of her affected son BED-26, between markers DXS8103 and DXS8061. This defines a critical interval of 6.8 cM for the site of the basic defect. The results are shown in Figure 2 and Table 3.

View this table: [in this window] [in a new window] Table 3. Haplotype Analysis of Selected Members From the Bornholm Eye Disease (BED) Family*

MOLECULAR STRUCTURE OF COLOR VISION GENES

Figure 5A shows a diagram of a typical X-linked visual pigment gene array of a male with normal color vision. Quantitative SSCP analysis showed that the ratio of green to red pigment gene promoters for affected Minnesota subjects 29 and 35 was 3, indicating that each of their arrays contained 4 genes (Figure 5A). Sequence analysis of exons 2 to 5 showed that the arrays of both subjects were composed of 1 red-green hybrid gene with a fusion point in intron 4 (R4G5) followed by 3 normal green pigment genes. Exon 3 of both the hybrid and normal green pigment genes had alanine at the polymorphic amino acid residue 180.²⁴ Therefore, it is inferred that the pigment encoded by this hybrid gene is identical in spectral properties to that encoded by the green pigment gene, and is consistent with protanopia. The SSCP showed no abnormal sequence variants in these 2 individuals. This gene array is typically found among subjects with severe protan color vision defects and without high myopia.^12-13,34-35

View larger version (25K): [in this window] [in a new window] Figure 5. A, Genomic structure of the normal human red and green pigment array. The red and green pigment genes span 15.2 kilobase (kb) and 13.3 kb, respectively, with a 24.0-kb separation between these 2 genes. Additional copies of the green pigment gene arranged in tandem at 24.0-kb intervals are found in 60% of the general white population. B, Relationship of the position of the 3'-red-green-5' (R-G) hybrid gene in the visual-pigment gene array found in the Minnesota (MN) family. Hybrids can occur because of the high degree of homology of these 2 genes (98%) (see the first paragraph of the "Molecular Structure of Color Vision Genes" subsection of the "Results" section). Individuals have a variable number of green pigment genes. This family has 3 additional normal green pigment genes. C, Genomic structure of the human red and green pigment array from DNA samples of individuals 26 and 28 with Bornholm eye disease (BED). Analyses of DNA samples showed a normal red pigment gene in the first position, followed by a green-red hybrid and 2 intact green pigment genes. The fusion point in the hybrid gene was in intron 4 (G4R5). This gene array is typically found among subjects with deutan color vision defects. D, Their unaffected male cousin, BED-27, showed a normal red-green pigment array.

We also sequenced the 0.6-kb LCR of the visual pigment gene array located 3.6 kb upstream from the first cone pigment gene (usually the red pigment gene).²⁴ This conserved 5' LCR interacts with either the red or green pigment gene promoters and directs gene expression. We found no mutations in affected individuals, obligate carriers, and unaffected individuals in both kindreds.

Similar analyses of BED family DNA samples from 2 affected individuals (BED-26 and BED-28) showed a normal red pigment gene in the first position, followed by a green-red hybrid and 2 intact green pigment genes (Figure 5C). A fusion point in the hybrid gene was in intron 4 (G4R5). This gene array is typically found among subjects with deutan color vision defects.^34-35 Their unaffected male cousin, BED-27, showed a normal red-green pigment array (Figure 5D). It is noteworthy that 2 gene arrays associated with deutan color vision segregate in this family. One is genetically linked to the locus associated with the ophthalmic findings, and the other—subject 3 in Figure 2—did not have the ophthalmic syndrome and, on the basis of DNA marker analysis, had married into the BED family.¹⁰

To rule out the existence of rearrangements within the color vision gene array, we performed Southern blot analysis using EcoRI and BamHI restriction enzymes.¹³ Southern blot analysis using the cDNA probe for the red pigment gene did not show deletions or rearrangements. Five individuals from the Minnesota pedigree were included in this analysis: 2 affected (subjects 29 and 35), 2 unaffected males (subjects 11 and 32 [an unrelated marry-in]), and 1 obligate carrier (subject 19). All of the individuals tested, affected and unaffected, showed the same bands, consistent with the published results for the wild-type sequence in previous studies using this probe.¹³ These data rule out the presence of insertions, deletions, or inversions at this locus (data not shown).

CYTOGENETIC STUDIES

High-resolution cytogenetic analysis was performed on DNA from affected Minnesota male 24. No abnormalities were detected (karyotype not shown).

COMMENT

Red-green color vision defects are common and were among the first recognized X-linked traits.^35-38 The molecular genetics of the visual pigments mediating normal and defective color vision have been meticulously studied in recent years.^{12, 24-26,34-35} The red-green pigment gene complex maps to a subterminal site on the long arm of the X chromosome. The red-green gene arrays are composed of a single red pigment gene (6 exons) and 1 or more green pigment genes (6 exons) located downstream (3') of the red gene. The close homology of the red and green opsin genes, including introns, makes them prone to unequal crossing over and accounts for the numerical polymorphism of the green genes, usually ranging between 1 and 3. However, only the single proximal red pigment gene and only 1 proximal green pigment gene are expressed in the retina.³⁵ Deuteranomaly is the most common defect in Caucasian populations (4% to 5% of males). The other defects (protanopia, protanomaly, and deuteranopia) have frequencies of approximately 1% each among males. Myopia and ERG changes are not associated with the common red-green color deficits.

The finding of an almost identical ophthalmologic phenotype mapping to the Xq27-28 region in 2 different families of Danish origin from islands in proximity suggests the same mutation. Genotyping with DNA markers in the critical X chromosome region, however, showed differences between affected members of the 2 families, arguing against an identical mutation. Intuitively, the most likely model would be that of a single unequal crossover between the red and green pigment gene in ancestral chromosomes that could have produced both red-green (Minnesota) and green-red (BED) hybrid genes associated with deutan and protan color deficiency, respectively, in affected members of the 2 families. Such an unequal crossover event would have had to occur in a woman homozygous for the underlying ophthalmologic defect but with normal color vision genes. Furthermore, haplotypic differences between affected members in the 2 families were inconsistent with this hypothesis.

A recent report describes a large 6-generation Asian Indian pedigree with high-grade myopia (mean of –13.33 D) transmitted in an X-linked fashion that maps to the pseudoautosomal region of chromosome Xq28.³⁹ The authors obtained a maximum LOD score of 3.99 at = 0 with marker DXYS154. Little information was provided regarding associated clinical features, however, making comparisons with the Minnesota and BED pedigrees difficult. The affected individuals do not have a color vision deficiency, however (Uppala Radhakrishna, PhD, oral communication, November 18, 2002), which provides greater evidence that color vision deficiency is not part of the phenotype. This report lends support to an X-linked locus for myopia at chromosome Xq28.

The disease segregating in these pedigrees exhibits some clinical similarities to other reported types of X-linked cone dystrophy; however, the dissimilarities point to a novel disorder. These types include COD1, which maps to Xp21.1-p11.2^40-42; COD2, which maps to Xq27⁴³; blue cone monochromacy, which maps to Xq27²³; and a cone dystrophy reported by Reichel et al⁴⁴, which maps to Xq28 and does not have an assigned gene name. The disease locus for our pedigrees mapped to chromosome Xq, rather than chromosome Xp, so it is unlikely that our pedigrees represent a COD1 disorder. Furthermore, with COD1 the affected males have reduced visual acuity, progressive macular atrophy, and progressive color degeneration with ultimate achromatopsia. Our pedigrees also do not match the phenotypic and genetic characteristics described for COD2 (and blue cone monochromacy) because of the progressive nature of those disorders, including reduced rod ERG in later stages and carrier female abnormalities.^{23, 43} The phenotype of blue cone monochromacy includes nystagmus, incomplete achromatopsia, and mutations in the LCR.^{23, 43} The Xq28 mapped cone dystrophy described by Reichel et al⁴⁴ also does not fit with the Minnesota pedigree. Their affected participants exhibited reduced visual acuity, progressive macular atrophy, deteriorating color vision progressing to central acquired achromatopsia, and a deletion in the red-cone pigment gene detected by Southern blot analysis using the same hs7 cDNA probe. Table 4 shows comparative clinical features with the Minnesota pedigree, and those of others with X-linked cone dysfunction.^{10, 23, 40-47}

View this table: [in this window] [in a new window] Table 4. Main Clinical Characteristic Comparisons of X-Linked Myopia With Published X-Linked Cone Dystrophies

A chromosome Xq28-linked cone dystrophy in 3 English families was recently described by Johnson et al.¹⁴ The affected members had protanopia with an associated progressive cone dystrophy and were found to have point mutations in the X-linked red pigment gene that encodes the opsin gene and results in photoreceptor death, rather than the benign red-green hybrid gene found in the common protan defects.

A search for genes and/or expressed sequence tags physically mapped between marker GATA31E08 and q-ter shows 48 regulatory or structural genes, 81 unidentified transcripts, 16 messenger RNAs, and 2 open reading frames (Integrated X Chromosome Database [http://ixdb.molgen.mpg.de/maps.html]; National Center for Biotechnology Information database [http://www.ncbi.nlm.nih.gov/genemap/map.cgi]). Other close loci include those for blue cone monochromacy (mentioned herein), myotubular myopathy, adrenoleukodystrophy, dyskeratosis punctata, X-linked dominant chondrodysplasia punctata, glucose-6-phosphate dehydrogenase, cardiac valvular dysplasia 1, Emery-Dreifuss muscular dystrophy, factor VIII–associated gene, hemophilia A, fragile X disorders type E and F sites, and biglycan. Biglycan, also termed proteoglycan I, is a small, leucine-rich proteoglycan expressed during chondrogenesis in cartilage and sclera.⁴⁸ It functions in connective tissue metabolism by binding to collagen fibrils and transforming growth factor and may promote neuronal survival. Biglycan appeared to be a relevant candidate gene and underwent mutational screening analysis. No mutations were found (data not shown). Previous mutational analysis of this 8-exon gene was performed by Das et al,⁴⁹ who excluded it as a candidate for X-linked dominant chondrodysplasia punctata, dyskeratosis congenita, and incontinentia pigmenti.

In conclusion, we report the molecular genetic findings of 2 families originating from Denmark with high-grade myopia and red-green color vision defects. All affected males from the Minnesota family had an early onset and seemingly nondegenerative high-grade myopia, myopic fundus changes, severe protanomaly, and reduced cone function on ERG testing. The cone abnormalities suggested by the ERG findings, however, might be secondary to or are accentuated by the severe myopia. Two-point LOD score analysis confirms linkage of the phenotype to the telomeric end of the X chromosome. The haplotypes at the Xq27-28 region in the Minnesota and BED families are different, suggesting that independent mutational events led to the phenotype. In addition, the red and green pigment gene arrays of the affected individuals in the 2 families are different, one containing a red-green hybrid gene (Minnesota family) consistent with the demonstrated protanopia, and the other containing a green-red hybrid gene (BED family) consistent with the demonstrated deuteranopia. These findings strongly suggest that the color vision defects are unrelated to the underlying disease and do not play a role in causing cone dysfunction. Both phenotypes appear to be novel forms of X-linked myopia and nonprogressive cone dysfunction. Definitive identification of the DNA defect causing myopia and cone dysfunction in these families should resolve the exact nature of the basic genetic defect(s).

From the Departments of Ophthalmology (Drs Young, Ronan, and King and Mss Alvear and Holleschau) and Genetics (Drs Young, Dewan, Atwood, Oetting, and King, and Ms Brott), University of Minnesota Medical School, Minneapolis; Division of Ophthalmology, The Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia (Dr Young and Mr Scavello and Ms Paluru); Departments of Medicine (Medical Genetics) and Genome Sciences, University of Washington, Seattle (Drs Deeb, Hayashi, and Motulsky); Department of Ophthalmology, Health Partners Inc, Arden Hills, Minn (Dr Benegas); Section of Clinical Genetics, Department of Pediatrics, University Hospital, Rigshospitalet, Copenhagen, Denmark (Dr Schwartz); and National Eye Clinic for the Visually Impaired, Hellerup, Denmark (Dr Rosenberg). The authors have no relevant financial interest in this article.

REFERENCES

1. Brückner A, Franceschetti A. Myopie im Kindesalter. Arch Augenheilkd. 1932;105:1-12. 2. Wold KC. Hereditary myopia. Arch Ophthalmol. 1949;42:225-230. WEB OF SCIENCE | PUBMED 3. Bartsocas CS, Kastrantas AD. X-linked form of myopia. Hum Hered. 1981;31:199-200. FULL TEXT | WEB OF SCIENCE | PUBMED 4. Kaplan J, Pelet A, Martin C, Delrieu O, Ayme S, Bonneau D. Phenotype-genotype correlations in X-linked retinitis pigmentosa. J Med Genet. 1992;29:615-623. FREE FULL TEXT 5. Lyness A, Ernst W, Quinlan M, Clover G, Arden G, Carter R. A clinical, psychophysical, and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Br J Ophthalmol. 1985;69:326-339. FREE FULL TEXT 6. Forsius H, Eriksson AW. Ein neues Augensyndrom mit X-chromosomaler Transmission. Klin Monatsbl Augenheilkd. 1964;144:447-457. PUBMED 7. Francois J, Verriest G, Matton-van Leuven MT, De Rouck A, Manavian D. Atypical achromatopsia of sex-linked recessive inheritance. Am J Ophthalmol. 1966;61:1101-1108. WEB OF SCIENCE | PUBMED 8. Mäntyjärvi M, Katajakunnas M, Vanttinen S. High myopia with cone dysfunction. Acta Ophthalmol (Copenh). 1991;69:155-161. 9. Jalkanen R, Demirci FY, Bech-Hansen T, et al. A new genetic locus for X-linked progressive cone-rod dystrophy. J Med Genet. 2003;40:418-423. FREE FULL TEXT 10. Schwartz M, Haim M, Skarsholm D. X-linked myopia: Bornholm eye disease: linkage to DNA markers on the distal part of Xq. Clin Genet. 1990;38:281-286. WEB OF SCIENCE | PUBMED 11. Haim M, Fledelius HC, Skarsholm D. X-linked myopia in a Danish family. Acta Ophthalmol (Copenh). 1988;66:450-456. 12. Winderickx J, Sanocki E, Lindsey D, Teller DY, Motulsky AG, Deeb SS. Defective colour vision associated with missense mutation in the human green visual pigment gene. Nat Genet. 1992;1:251-266. FULL TEXT | WEB OF SCIENCE | PUBMED 13. Nathans J, Maumenee IH, Zrenner E, et al. Genetic heterogeneity among blue-cone monchromats. Am J Hum Genet. 1993;53:987-1000. WEB OF SCIENCE | PUBMED 14. Johnson S, Halford S, Mollon JD, Moore AT, Hunt DM. Molecular basis of cone dystrophy associated with protanopia. Invest Ophthalmol Vis Sci. 2001;42(suppl):3442. 15. Marmor MF, Zrenner E, International Society for Clinical Electrophysiology of Vision. Standard for clinical electroretinography (1999 update). Doc Ophthalmol. 1998-1999;97:143-156. FULL TEXT | PUBMED 16. Dubovvsky J, Sheffield V, Duyk G, Weber J. Sets of short tandem repeats polymorphisms for efficient linkage screening of the human genome. Hum Mol Genet. 1995;4:449-452. FREE FULL TEXT 17. Oetting WS, Lee H, Flanders D, Weisner G, Sellers T, King RA. Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics. 1995;30:450-458. FULL TEXT | WEB OF SCIENCE | PUBMED 18. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computation. Am J Hum Genet. 1993;53:252-263. WEB OF SCIENCE | PUBMED 19. Schaffer AA, Gupta SK, Shriram K, Cottingham RW Jr. Avoiding recomputation in genetic linkage analysis. Hum Hered. 1993;44:225-237. FULL TEXT | WEB OF SCIENCE 20. Lathrop GM, Lalouel J-M, Julier C, Ott J. Strategies for multilocus analysis in humans. Proc Natl Acad Sci U S A. 1984;81:3443-3446. FREE FULL TEXT 21. Lathrop GM, Lalouel J-M. Easy calculations of LOD scores and genetic risks on small computers. Am J Hum Genet. 1984;36:460-465. WEB OF SCIENCE | PUBMED 22. Lathrop GM, Lalouel J-M, White RL. Construction of human genetic linkage maps: likelihood calculations for multilocus analysis. Genet Epidemiol. 1986;3:39-52. FULL TEXT | WEB OF SCIENCE | PUBMED 23. Nathans J, Davenport CM, Maumenee IH, et al. Molecular genetics of blue cone monochromacy. Science. 1989;245:831-838. FREE FULL TEXT 24. Wang Y, Macke JP, Merbs SL, et al. A locus control region adjacent to the human red and green visual pigment genes. Neuron. 1992;9:429-440. FULL TEXT | WEB OF SCIENCE | PUBMED 25. Yamaguchi T, Motulsky AG, Deeb SS. Visual pigment gene structure and expression in human retinae. Hum Mol Genet. 1997;6:981-990. FREE FULL TEXT 26. Deeb SS, Hayashi T, Winderickx J, Yamaguchi T. Molecular analysis of human red/green visual pigment gene locus: relationship to color vision. Methods Enzymol. 2000;316:651-670. WEB OF SCIENCE | PUBMED 27. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymophisms. Proc Natl Acad Sci U S A. 1989;86:2766-2770. FREE FULL TEXT 28. Rooney DE, ed, Czepulkowski BH, ed. Human Cytogenetics: A Practical Approach. Vol II. Oxford, England: IRL Press; 1992. 29. Sorsby A, Leary GA, Richards MJ. Correlation ametropia and component ametropia. Vision Res. 1962;2:309-313. FULL TEXT | WEB OF SCIENCE 30. Perlman I, Meyer E, Haim T, Zonis S. Retinal function in high refractive error assessed electroretinographically. Br J Ophthalmol. 1984;68:79-84. FREE FULL TEXT 31. Yamamoto S, Nitta K, Kamiyama M. Cone electroretinogram to chromatic stimuli in myopic eyes. Vision Res. 1997;37:2157-2159. FULL TEXT | WEB OF SCIENCE | PUBMED 32. Kawabata H, Adachi-Usami E. Multifocal electroretinogram in myopia. Invest Ophthalmol Vis Sci. 1997;38:2844-2851. FREE FULL TEXT 33. Westall CA, Dhaliwal HS, Panton CM, et al. Values of electroretinogram responses according to axial length. Doc Ophthalmol. 2001;102:115-130. FULL TEXT | PUBMED 34. Deeb SS, Lindsey DT, Hibiya Y, et al. Genotype-phenotype relationships in human red/green color-vision defects: molecular and psychophysical studies. Am J Hum Genet. 1992;51:687-700. WEB OF SCIENCE | PUBMED 35. Hayashi T, Motulsky AG, Deeb SS. Position of a "green-red" hybrid gene in the visual pigment array determines colour-vision phenotype. Nat Genet. 1999;22:90-93. FULL TEXT | WEB OF SCIENCE | PUBMED 36. Gouras P. The history of colour vision. In Gouras P, ed. Vision and Visual Dysfunctions, Vol 6: The Perception of Colour Vision. Boca Raton, Fla: CRC Press; 1992:1. 37. Dalton J. Extra-ordinary facts relating to the vision of colours with observations. Mem Lit Philos Soc Lond. 1798;5:28. 38. Bell J, Haldane JBS. The linkage between the genes for colour-blindness and haemophilia in man. Proc R Soc (Lond). 1937;B123:119. FREE FULL TEXT 39. Radhakrishna U, Raval R, Morris MA, et al. A locus for a severe form of X-linked myopia maps to the pseudoautosomal region of Xq28 [abstract]. Am J Hum Genet. 2000;67(4, suppl):1734. 40. Jacobson DM, Thompson HS, Bartley JA. X-linked progressive cone dystrophy. Ophthalmology. 1989;96:885-895. WEB OF SCIENCE | PUBMED 41. Hong H-K, Ferrell RE, Gorin MB. Clinical diversity and chromosomal localization of X-linked cone dystrophy (COD1). Am J Hum Genet. 1994;55:1173-1181. WEB OF SCIENCE | PUBMED 42. Meire FM, Bergen AAB, de Rouck A, Leys M, Delleman JW. X-linked progressive cone dystrophy: localization of the gene locus to Xp21.1-p11.1 by linkage analysis. Br J Ophthalmol. 1994;78:103-108. FREE FULL TEXT 43. Bergen AAB, Pinckers AJLG. Localization of a novel X-linked progressive cone dystrophy gene to Xq27: evidence for genetic heterogeneity. Am J Hum Genet. 1997;60:1468-1473. FULL TEXT | WEB OF SCIENCE | PUBMED 44. Reichel E, Bruce AM, Sandberg MA, Berson EL. An electrographic and molecular genetic study of X-linked cone degeneration. Am J Ophthalmol. 1989;108:540-547. WEB OF SCIENCE | PUBMED 45. Verdoorn C, Pinckers AJLG. X-linked cone dystrophy. Doc Ophthalmol. 1988;70:195-198. FULL TEXT | WEB OF SCIENCE | PUBMED 46. Keunen JE, van Everdingen JA, Went LN, Oosterhuis JA, Norren van D. Color matching and foveal densitometry in patients and carriers on an X-linked progressive cone dystrophy. Arch Ophthalmol. 1990;108:1713-1719. FREE FULL TEXT 47. Heckenlively JR, Weleber RG. X-linked recessive cone dystrophy with tapetal-like sheen: a newly recognized entity with Mizuo-Nakamura phenomemon. Arch Ophthalmol. 1986;104:1322-1328. FREE FULL TEXT 48. Fisher LW, Termine JD, Young MF. Deduced-protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several non-connective tissue proteins in a variety of species. J Biol Chem. 1989;264:4571-4576. FREE FULL TEXT 49. Das S, Metzenberg A, Pai GS, Gitschier J. Mutational analysis of the biglycan gene excludes it as a candidate for X-linked dominant chondrodysplasia punctata, dyskeratosis congenita, and incontinentia pigmenti. Am J Hum Genet. 1994;54:922-925. WEB OF SCIENCE | PUBMED

SECTION EDITOR: THADDEUS P. DRYJA, MD

CiteULike    Connotea    Delicious    Digg    Facebook    Reddit    Technorati    Twitter     What's this?

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES

Refinement of the X-linked Nonsyndromic High-Grade Myopia Locus MYP1 on Xq28 and Exclusion of 13 Known Positional Candidate Genes by Direct Sequencing
Ratnamala et al.
IOVS 2011;52:6814-6819.
ABSTRACT | FULL TEXT  

Nonsyndromic High Myopia in a Chinese Family Mapped to MYP1: Linkage Confirmation and Phenotypic Characterization
Guo et al.
Arch Ophthalmol 2010;128:1473-1479.
ABSTRACT | FULL TEXT  

An International Collaborative Family-Based Whole-Genome Linkage Scan for High-Grade Myopia
Li et al.
IOVS 2009;50:3116-3127.
ABSTRACT | FULL TEXT  

Genomewide Linkage Scans for Ocular Refraction and Meta-analysis of Four Populations in the Myopia Family Study
Wojciechowski et al.
IOVS 2009;50:2024-2032.
ABSTRACT | FULL TEXT  

Evaluation of the X-Linked High-Grade Myopia Locus (MYP1) with Cone Dysfunction and Color Vision Deficiencies
Metlapally et al.
IOVS 2009;50:1552-1558.
ABSTRACT | FULL TEXT  

Complex Trait Genetics of Refractive Error
Young et al.
Arch Ophthalmol 2007;125:38-48.
ABSTRACT | FULL TEXT  

Confirmation of Linkage to Ocular Refraction on Chromosome 22q and Identification of a Novel Linkage Region on 1q
Klein et al.
Arch Ophthalmol 2007;125:80-85.
ABSTRACT | FULL TEXT  

Novel locus for X linked recessive high myopia maps to Xq23-q25 but outside MYP1.
Zhang et al.
J. Med. Genet. 2006;43:e20-e20.
ABSTRACT | FULL TEXT  

Identification of a Novel Locus on 2q for Autosomal Dominant High-Grade Myopia
Paluru et al.
IOVS 2005;46:2300-2307.
ABSTRACT | FULL TEXT