Fine mapping of the hereditary haemorrhagic telangiectasia (HHT)3 locus on chromosome 5 excludes VE-Cadherin-2, Sprouty4 and other interval genes

Abstract

Background: There is significant interest in new loci for the inherited condition hereditary haemorrhagic telangiectasia (HHT) because the known disease genes encode proteins involved in vascular transforming growth factor (TGF)-beta signalling pathways, and the disease phenotype appears to be unmasked or provoked by angiogenesis in man and animal models. In a previous study, we mapped a new locus for HHT (HHT3) to a 5.7 Mb region of chromosome 5. Some of the polymorphic markers used had been uninformative in key recombinant individuals, leaving two potentially excludable regions, one of which contained loci for attractive candidate genes encoding VE Cadherin-2, Sprouty4 and FGF1, proteins involved in angiogenesis.

Methods: Extended analyses in the interval-defining pedigree were performed using informative genomic sequence variants identified during candidate gene sequencing. These variants were amplified by polymerase chain reaction; sequenced on an ABI 3730xl, and analysed using FinchTV V1.4.0 software.

Results: Informative genomic sequence variants were used to construct haplotypes permitting more precise citing of recombination breakpoints. These reduced the uninformative centromeric region from 141.2-144 Mb to between 141.9-142.6 Mb, and the uninformative telomeric region from 145.2-146.9 Mb to between 146.1-146.4 Mb.

Conclusions: The HHT3 interval on chromosome 5 was reduced to 4.5 Mb excluding 30% of the coding genes in the original HHT3 interval. Strong candidates VE-cadherin-2 and Sprouty4 cannot be HHT3.

Figures

Figure 1

Previous HHT3 interval. A) HHT3 linked Family S pedigree (black symbols represent HHT-affected individuals; white symbols represent unaffected individuals; squares represent males and circles represent females. Individuals are denoted by their generation number, such that the oldest male is I.1, oldest female I.2). B) Published HHT3 interval on chromosome 5 between microsatellite markers D5S2011 and D5S2490 [35], identifying the region shared by all affected individuals (open interval between D5S402 and D5S436); regions where the designated individuals had inherited their affected parent's non disease-gene bearing allele (black bars), and regions in which the transition from the non disease-gene bearing allele (black bars) to disease-gene bearing allele (open interval) had occurred, but the exact sites of the meiotic recombination event was not definable using the markers studied in [35] (grey bars; uninformative markers). It was possible that the interval could be reduced to a minimum of D5S402-D5S436, according to where the recombination breakpoints had occurred in individuals II.1, II.3 and II.9. cen: centromere; tel: telomere; Mb: mega bases.

Figure 2

Informative genomic sequence variants used in fine mapping. Locations of genomic variants where individual I.2 was heterozygous, potentially allowing her disease associated and non-disease associated alleles to be tracked. Sequence variants are illustrated in relation to candidate gene exons (grey boxes) and original microsatellite markers. By chance, all five genes are on the reverse strand of chromosome 5, designated by reverse arrows. rs; as on NCBI dbSNP.

Figure 3

Genomic variant haplotypes in interval-defining members of the pedigree. A) Allele inheritance and haplotypes for the inheritance of the key microsatellite markers, and new genomic sequence variants, across PCDH12, SPRY4, FGF1 and NR3C1 genes in eight members of Family S. For each sequence variant, offspring inherit one allele from their mother and one from their father. Occasional alleles where it was not possible to determine parental origin (and which were therefore not used for haplotype construction) are denoted in non-bold italics. Individual II.5 demonstrates the disease haplotype across the interval, a haplotype which is now shared by II.1, but not II.3 and her descendants until rs17209237 in NR3C1. Therefore II.3 excludes the centromeric region between rs153148 and the novel SNP in FGF1. B) Haplotypes for the inheritance of the new genomic sequence variants across the PPP2R2B gene in nine members of Family S. Affected individuals II.1, II.3, II.5, III.3 and III.4 demonstrate the disease haplotype which is not shared by II.9 at rs 11958187, but is shared at rs12110022, rs 6879694, and the remainder of the HHT3 interval. Therefore II.9 excludes rs11958187 and beyond from the telomeric extreme of the interval.]: uninformative regions; bp: base pairs.

Figure 4

The refined HHT3 interval. The original HHT3 interval was between microsatellite markers D5S2011 and D5S2490, and uninformative between D5S2011-D5S402 and D5S2490-D5S436 (black dotted bars) in key individuals II.1, II.3 and II.9 [35]. Informative genomic variations used in fine mapping reduced the extent of the uninformative regions (grey bars, see Figure 1B). The refined HHT3 interval is sited between a novel SNP within FGF1 intron 1, and rs11958187 within PPP2R2B intron 4. Black bars indicate the definite recombination events that defined the interval in the denoted individuals. Fine mapping, by reducing the interval, excluded 12 interval genes as shown in the lower Ensembl genome browser image of candidate genes (genes no longer in the interval are denoted by orange dotted boxes; Mb: mega bases).