A high-density SNP map for the FRAX region of the X chromosome

Abstract

otide repeat (FRAXA) located in the 5Ј untranslated region of the FMR1 gene gives rise to the fragile X syndrome, an inherited disorder associated with mental retardation (Fu et al. 1991;Verkerk et al. 1991). Distal to FMR1 (approximately 0.77 Mb) is FMR2, which contains a GCC triplet repeat (FRAXE) in the 5Ј untranslated region. Full mutations (over 200 repeats) in FRAXE are rare (Sutherland and Baker 1992) but appear to be associated with nonsyndromic mental impairment (Knight et al. 1994).Instability at FRAXA is thought to be due at least in part to loss of an AGG interspersion from the CGG repeat, resulting in slippage during replication (Eichler et al. 1994). However, haplotype studies using microsatellites suggest that other factors may also be important determinants of risk for FRAXA expansion (Murray et al. 1997). Eichler et al. (1996) proposed three independent mechanisms for repeat expansion: (1) loss of 3Ј stabilizing AGG interspersion followed by rapid expansion to a pre-or full mutation, (2) slow expansion from common to pre-and full mutation without loss of an AGG interspersion, and (3) generalized instability that affects other polymorphic microsatellite markers. Instability at FRAXE usually appears to increase with increasing repeat number. In contrast to FRAXA, the repeat does not contain any interspersions, suggesting a simple relationship between size of repeat and risk of expansion. However, work by Ennis et al. (2001) suggested that, although triplet repeat size was a significant predisposing factor for FRAXE expansion, there were other genetic determinants involved. For example, they described a significant association between unusual FRAXA and unusual FRAXE alleles. FRAXA repeat sizes of more than 50 were positively associated with FRAXE repeats of less than 11. Although these associations occurred on similar haplotype backgrounds, founder effects could not be confirmed without looking at comparative data from different ethnic backgrounds.With the near completion of the human genome sequencing project, there has been much interest in the use of single-nucleotide polymorphisms (SNPs) for studying genetic factors associated with complex disease traits. SNPs G. Brightwell (*) · R. Wycherley · G. Potts · A. Waghorn Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire SP2 8BJ, UK Tel. ϩ44-1722-425047; Fax ϩ44-1722-338095 e-mail: galebrightwell@hotmail.com Abstract Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation within the human genome, occurring approximately once every kilobase. However, for association studies, SNPs are not as informative as microsatellite markers and a large number of SNPs and substantial population sizes are required for linkage and mapping studies. A SNP map was generated for the FRAX region of the X chromosome, approximately 0.8 Mb proximal and 1.8 Mb distal to the FRAXA repeat, at a density of at least 1 SNP every 100 kb. SNPs were identified in a population of 28 women with a FRAXA expansion (includi...

Closely linkedcis‐acting modifier of expansion of the CGG repeat in high riskFMR1haplotypes

“…We used a panel of 36 females with one expansion and one normal chromosome and amplified 400–bp regions of DNA distributed at approximately 20–kb intervals over a 650–kb genomic region that included the FMR1 gene (GenBank accession number L29074.1) and 400 kb of upstream and 250 kb of downstream sequence from the FRAXA CGG repeat. The 400–bp PCR fragments formed heteroduplexes for analysis on the Transgenomic WAVE Machine (Transgenomic, Inc., Omaha, NE) and samples with shifted peaks were sequenced to confirm and characterize the variant [ Brightwell et al, 2002 ]. SNPs with minor allele frequencies >5% (i.e., found in two or more individuals) were chosen for genotyping in our male panel.…”

“…If no SNP was detected in a particular fragment, an adjacent 400–bp fragment was analyzed. Genotyping was by allele–specific PCR analyzed on ethidium bromide–stained agarose gels [ Brightwell et al, 2002 ] (Primer sequences available in Supplementary Tables S1a and S1b ; available online at http://www.interscience.wiley.com/jpages/1059-7794/suppmat ). Hardy–Weinberg equilibrium could not be tested in our male hemizygous samples but duplicate samples included for quality control purposes were concordant.…”

Single Nucleotide Polymorphism and FMR1 CGG Repeat Instability in Two Basque Valleys

“…Although instability seems to depend on three cis ‐acting factors, the size of the CGG repeat, the AGG interspersion pattern, and the STR haplotype, these factors alone do not completely describe the molecular basis for the linkage disequilibrium between normal and fragile X chromosomes. Some studies suggest that more stable markers, such as single nucleotide polymorphisms (SNPs) could provide an important contribution to study the origin of the CGG repeat instability (Gunter et al, 1998; Crawford et al, 2000a; Mathews et al, 2001; Brightwell et al, 2002a; Brightwell et al, 2002b; Curlis et al, 2005; Zhou et al, 2006; Ennis et al, 2007). Results obtained by Gunter et al (1998) with a SNP (ATL1) suggests that allele state at DXS548 and FRAXAC1 may not be relevant for repeat stability because chromosomes with the same haplotype for these two markers show clearly different transition rates to full mutation status.…”

“…Results obtained by Gunter et al (1998) with a SNP (ATL1) suggests that allele state at DXS548 and FRAXAC1 may not be relevant for repeat stability because chromosomes with the same haplotype for these two markers show clearly different transition rates to full mutation status. According to Brightwell et al (2002b), it is important to study populations of different genetic identity. These authors suggest that SNPs could provide a useful resource for investigating the genetic mechanisms behind instability and expansion of FRAXA triplet repeat.…”

“…1). These SNPs are WEX28 (rs17312728:G>T) located at (–184165) base pairs proximal to FRAXA (Brightwell et al, 2002b), WEX70 (rs45631657:C>T) located at (–53907) base pairs proximal to FRAXA (Ennis et al, 2007), WEX1 (rs10521868:A>C) located at (–2064) base pairs proximal to FRAXA (Brightwell et al, 2002b), ATL1 (rs4949:A>G) located in the first intron of the FMR1 gene (Gunter et al, 1998), FMRb (rs25707:A>G) located in the exon 5 of the FMR1 gene (Kunst & Warren 1994), WEX17 (rs12010481:C>T) located at (107335) base pairs proximal to FRAXA (Brightwell et al, 2002b), and WEX10 (ss71651741:C>T) located at (254752) base pairs proximal to FRAXA (Brightwell et al, 2002b).…”