SUBJECTS, MATERIALS, AND METHODS

Subjects

Medical history and ophthalmic assessment were obtained from 140 participants from 21 French families and two Algerian families, after informed consent according to French law. We focused our study on isolated bilateral high myopia. Families with unilateral high myopia, syndromes with high myopia, and myopia of prematurity were excluded.

For each patient, subjective refraction and keratometry were performed. Axial lengths were also measured for almost all of the subjects. Objective refraction was measured by automatic refractometry. The refraction defect in spherical equivalent was the criterion chosen to classify subjects into two groups, high myopes and unaffected persons. A subject was considered to be highly myopic if the refraction error in the lesser affected eye was −6 D or below. We considered low myopes (myopia between −6 and −1 D), emmetropes (refraction status between −1 and 1 D), and patients with hyperopia (refraction status greater than 1 D) as unaffected subjects. Details of refractive status are summarised in table 1.

DNA analysis/marker typing

Venous blood samples were collected in EDTA for DNA extraction according to standard methods.¹⁰ The genome screen used 400 highly polymorphic fluorescently labelled microsatellite markers, with an average spacing of 10 cM, from the ABI PRISM Linkage Mapping Set MD-10 (Perkin-Elmer, Warrington, UK). The map positions were generated from the CEPH genotype data used for the Généthon map. For fine mapping, we selected those regions with a two point lod score >1. This is clearly well below the threshold for statistical significance but provided a convenient cut off for identification of regions meriting higher density genotyping. Additional polymorphic markers (heterozygosity ≥75%) were selected from the Généthon and the CHLC genetic maps and were fluorescently labelled. All labels were either 6FAM, HEX, or NED 5` end labels.

All PCR reactions were carried out using 25 ng of genomic DNA as a template in a mixture of 1 × Perkin-Elmer PCR buffer, 2.5 mmol/l MgCl₂, 200 nmol/l of each dNTP, 5 pmol of each primer, and 0.1 μl of TaqGold polymerase (Perkin-Elmer, Warrington, UK) in a final volume of 15 μl. The thermocycling conditions were 95°C for 18 minutes, followed by 38 cycles at 94°C for one second, 55°C for 25 seconds, and 72°C for five seconds, followed by a 10 minute final extension step at 72°C. PCR products were pooled with regard to their size range and labelling, mixed with a formamide sample buffer, and electrophoresed through preheated 6% acrylamide/50% (W/V) urea gels on an ABI 373 DNA sequencer XL upgrade (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations. All amplimers were sized by the GeneScan Analysis 3.1 software (PE Applied Biosystems, Foster City, CA, USA) and scored by the Genotyper 2.1 software (PE Applied Biosystems, Foster City, CA, USA) with respect to the CEPH control genotype No 1347-02.

Statistical analysis

In parametric statistics, we assumed an autosomal dominant mode of inheritance with 58.4% penetrance and a myopia gene frequency of 0.013, as they were established in a previous study.⁷ Data were simulated using the program SLINK¹¹ in order to determine the power to detect linkage. Two point lod scores and maximum lod scores (Zmax) were calculated using the MLINK and ILINK routines of the FASTLINK program¹² and genetic heterogeneity was tested using the HOMOG program. Marker allele frequencies were estimated from the non-inbred founders in the data. The two Algerian pedigrees were included in the whole population to calculate a unique set of allele frequencies.

Multipoint lod score analysis of the genome screen was performed with the GENEHUNTER computer package¹³ under the parametric model previously defined (lod) and the non-parametric option. In addition, lod scores were calculated under the locus heterogeneity hypothesis (hlod). The proportion of the families linked (alpha) was allowed to vary until the hlod was maximised. Map order and genetic distances between markers were determined from the Généthon Human Linkage Map.¹⁴

RESULTS

Twenty-three families were included in the genome scan (fig 1). They were not selected a priori, in order to avoid a bias in favour of dominance. They only had to have at least one highly myopic subject in their pedigree. Fifty highly myopic patients with a sex ratio of 1 were studied. The mean refraction value was −13.05 (SD 4.92) D for high myopes versus −1.32 (SD 2.43) D for non-highly myopic subjects. The mean axial lengths were 27.87 (SD 2.92) mm and 23.52 (SD 1.26) mm for highly myopic and non-highly myopic subjects, respectively.

The simulation test found a power of 41.6% to obtain significant evidence for linkage (Zmax >3) and a power of 70.6% to obtain suggestive evidence of linkage (Zmax >2) when a recombination fraction (θ) of 0.01 between the marker tested and the disease locus was assumed.

Some markers implicated in familial high myopia on chromosome 12q (D12S351, D12S346, and D12S78) and chromosome 18p (D18S59 and D18S63) by Young et al^8,9 were tested during our initial genome scan and showed negative lod scores (data not shown). The multipoint analysis also excluded both regions and under the heterogeneity hypothesis no evidence for linkage to these regions was found (fig 2).

The initial 10 cM genome screen did not show any suggestive evidence of linkage even under the heterogeneity hypothesis. Indeed, no lod score above 2 was obtained. No significantdifferences were observed when allowing genetic heterogeneity in the initial screen. However, five chromosomal regions, on 4q, 5p, 7q, 13q, and 15q, were identified as having Zmax >1. In each region, additional highly polymorphic markers were genotyped and analysed by two point and multipoint linkage analysis. The new markers for chromosome 4q, 5p, 13q, and 15q gave similar or lower two point lod scores. The multipoint analysis confirmed the exclusion of these regions (fig 3). In contrast, the maximum two point lod score with chromosome 7q markers reached 1.87 at θ=0 (table 2). The multipoint analysis showed suggestive evidence of linkage with a maximum multipoint lod score of 2.81 (fig 3), whereas no locus heterogeneity could be detected. The implicated region extended from D7S798 to D7S2423, the latter marker being located in the immediate vicinity of the telomere. A recombination event between D7S798 and D7S2546 in family 17 (fig 4) allowed us to set this interval in an 11.7 cM region extending from D7S798 to the telomeric end of the chromosome.

DISCUSSION

In our previous study,⁷ we have shown that genetic factors were of decisive importance in the aetiology of familial high myopia. By segregation analysis, we showed that an autosomal dominant mode of inheritance with weak penetrance was largely favoured in the families that we studied. Nevertheless, some families (family 2) showed an autosomal recessive mode of transmission. The weak penetrance of a dominant allele may give the appearance of recessive transmission which could not be totally excluded in some families.

The pathophysiology of isolated high myopia remains unknown. Besides heredity, the trait depends on environmental factors and, moreover, the genetic component is also complex. Indeed, we found that the locus for familial high myopia on chromosome 18p11.31, recently reported by Young et al,⁸ was probably not implicated in our families. This indicates that the disease is genetically heterogeneous as there were no obvious phenotypic differences between our patients and the ones reported by these authors.

We now confirm the locus heterogeneity of the disease with the families we have studied by excluding the loci for familial high myopia on chromosomes 12q21-23 and 18p11.31. Moreover, the results obtained during our genome scan suggest the presence of a new susceptibility locus for familial high myopia on chromosome 7q36 within a 11.7 cM interval. Evidence for locus heterogeneity could not be detected, even if some families, when the data were examined in detail, did not seem to be linked. The added high resolution markers were informative in our population and gave results in keeping with the simulation test, which predicted a power of only 41.6% to obtain a lod score above 3. Furthermore, the complexity of the high myopia trait probably leads to the decrease in the power to detect significant linkage.

The non-parametric lod score curve of the genome scan (data not shown) showed the same variations as the lod score curve but remained less powerful. This strongly suggested that our parametric segregation model was correct and that our results were not due to a possible error in the model.

Myopia is commonly considered as a complex, multifactorial trait, where several genes could act together in a quantitative way. To date, in single families or limited familial series, single loci have been identified. Our data suggest that a new locus triggers or participates in familial high myopia. The computational search for genes and/or expressed sequence tags physically mapped between markers D7S798 and the telomere showed numerous unidentified transcripts, mRNAs for an open reading frame, and several genes. None of these appeared to be good candidate genes on the basis of their known function. In addition, there is no evidence of any closely related genes shared by the regions of interest on chromosomes 7q36, 12q21-23, and 18p11.31.

A majority of the families that we studied contained low myopic persons whom we considered as being unaffected, in order to preserve phenotypic homogeneity. The −6 diopter limit for high myopia that was chosen for the current clinical definition cannot be considered perfectly biologically accurate. Consequently, it would be interesting to analyse high myopia as a quantitative trait.

In summary, we provide suggestive evidence for the presence of a third autosomal locus for familial high myopia on chromosome 7q36. Studies are currently under way in our laboratory, in order to validate these results and to reduce the critical region for high myopia. The recruitment and analysis of new families and/or new members of the families already studied are needed before starting gene isolation experiments. The characterisation of the genes implicated in this common eye disorder will lead to a better understanding of the molecular mechanisms contributing to eye shape and growth.