Functional characterisation of <i>MeCP2</i> mutations found in male patients with X linked mental retardation

igh myopia often appears as a familial disease.It is usually defined as a refraction error equal to or below -6 diopters (D) in each eye. 1 Highly myopic patients represent 27-33% of the myopic population. 2 The prevalence of the disease in the general population varies according to the country, from 2.1% in the USA, 2 to 3.2% in France, 3 and up to 9.6% in Spain.3 High myopia is also termed "pathological" myopia because of its potential complications.The highly myopic eye is usually characterised by an abnormal lengthening and a posterior staphyloma.It is often accompanied by glaucoma, cataracts, macular degeneration, and retinal detachment, leading to blindness when the damage to the retina is extremely severe.Both genetic and environmental factors, such as close work, are known to play a role in the aetiology of high myopia.The inheritance of the disease is equivocal.Several genealogical studies have shown autosomal dominant or autosomal recessive modes of inheritance.4 5 Rare cases of sex linked transmission have been observed.6 In a previous study, 7 we showed that, assuming a single gene model, autosomal dominant transmission with weak penetrance was largely present in the families that we studied.Young et al have recently reported linkage of familial high myopia to chromosome regions 18p 8 and 12q.9 We previously found no evidence for linkage to the former chromosomal region in the families of our study.Several putative candidate loci were excluded as well in these families, such as the locus for Stickler syndrome types 1 and 2, versican and aggregan genes, Marfan 1 syndrome, and a Marfan-like disorder localised to 3p24.2-p25.In order to find new loci implicated in high myopia, we conducted a genome screen in 23 families following an autosomal dominant mode of inheritance with weak penetrance.Here, we provide further evidence for genetic heterogeneity by excluding the chromosome 12q and 18p regions, previously linked to familial high myopia, 8 9 and report suggestive evidence for the presence of a third autosomal locus on chromosome 7q. SUBJECTS, MATERIALS, AND METHODS SubjectsMedical 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 typingVenous 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 MgCl 2 , 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.

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