METHODS

We ascertained a family with HBT from northern Italy. After obtaining informed consent, 20 family members and four spouses underwent a detailed dermatological examination and a blood sample was taken. Three affected individuals had a skin biopsy. Skin samples were stained with haematoxylin-eosin and examined by light microscopy.

Genomic DNA was extracted from blood samples following standard procedures.

The family was first tested for linkage to the genetic regions containing Endoglin and ALK1, the two known genes responsible for hereditary haemorrhagic telangiectasia, on chromosomes 9q (ORW1 – OMIM 187300) and 12q (ORW2 – OMIM 600376), respectively.

Before the reported linkage of CMC1 to chromosome 5q,¹² we analysed 380 microsatellite markers covering the whole genome, with an average distance between two adjacent markers of 10 cM (ABI PRISM linkage mapping set, version 2). Markers were run on a 3100 automated DNA sequencer (ABI PRISM) and analysed using Genescan and Genotyper software. Two-point LOD scores were generated with the FASTLINK version of the MLINK program,¹⁴ assuming equal male–female recombination rate, autosomal dominant inheritance, a gene frequency of 0.0001, equal allele frequencies for each marker, and reduced penetrance (0.50 to 0.95). Haplotypes were manually constructed and phase was assigned based on the smallest number of recombinants. Genetic distances between markers were taken from the genetic map of the Marshfield Centre for Medical Genetics (www.marshfieldclinic.org/research/genetics/), while physical distances were taken from the University of California Santa Cruz draft of the human genome, release “November 2002” (www.genome.ucsc.edu) (table 1).

To refine the upper and lower limit of the linked region, three novel polymorphic markers (arbitrarily named RST2, RST3, and RIT3) were selected from the human genome working draft using the tandem repeat finder software.¹⁵ These markers were amplified using the following pairs of primers: RST2-F: 5′-CCCTGCTGCTGTTTATTTTT-3′ and RST2-R: 5′-AGGAGCCATAGCCTCTCTTT-3′; RST3-F: 5′-ACTTCCTAGCAAACTCCCAACC-3′ and RST3-R: 5′-GGCAACTAATATGGCCTTTTCTT-3′; RIT3-F: 5′-CGCAACTGAGGAACCTTTTAAC-3′ and RIT3-R: 5′-TGAAGTTATAGCGCAGACCTGA-3′. For each pair, the forward primer was fluorescently labelled with either an FAM or a HEX dye. The position of these novel markers on the physical map of human chromosome 5 is also shown in table 1.

RESULTS

The pedigree of the HBT family is shown in fig 1. The inheritance is autosomal dominant, with no apparent lack of penetrance. Thirteen of 20 family members (four male and nine female) had a definite diagnosis of HBT. Table 2 summarises the clinical features of the affected individuals. The age of onset could not always be determined accurately, but some parents noted the onset of macular telangiectases in their sons during the first months of life. A large variability in size (1×1 to 6×4 cm) and number (1 to >10) of telangiectases was observed among the affected family members, but lesions invariably became paler with increasing age. Only one patient (IV:1) had mucous membrane involvement, with a lesion affecting the vermilion border of the upper lip. Histological examination showed normal epidermis and dilatation of the smallest blood vessels of the upper part of dermis (fig 2).

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Figure 1

Pedigree of the family and haplotypes of marker loci spanning the linked region on chromosome 5q14. Black symbols denote affected individuals, deceased members are marked with a diagonal bar. A thin horizontal line above symbols indicates members of the family who were examined clinically and had blood samples taken. The arrow indicates the proband. The black bar denotes the haplotype segregating with the disease in the family.

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Figure 2

Clinical and histological characteristics of cutaneous lesions in HBT family. Telangiectases distributed on the neck and thorax in subject IV:1 (A–E); large macular telangiectatic lesion on the armpit in subject III:1 (F). Haematoxylin-eosin staining of skin biopsy in patient III:1, showing dilated thin walled vessels (black arrows) lined by a single layer of endothelium within the upper dermis (G).

Linkage to ORW1 and ORW2 was excluded, with negative LOD scores across both regions, ruling out the hypothesis that HBT could represent a benign allelic variant of either endoglin or ALK1 genes. Analysis of the 380 markers from the genome-wide search produced negative or non-significant LOD scores for all tested loci, except for nine markers on chromosomes 5, 9, 16, 17, and 18. The regions surrounding these loci were saturated with more microsatellite markers and haplotypes were manually constructed. The segregation of different haplotypes in affected individuals and the negative LOD scores obtained allowed excluding all regions but an 11.5 cM interval on chromosome 5q, where a common haplotype was shared by all affected family members. All genotyped markers within this region generated positive LOD score values with a maximum pairwise LOD score of 5.27 for marker D5S641 (θ  =  0; penetrance  =  0.95) (table 1). Calculation of pairwise LOD scores assuming lower penetrance values (0.50 to 0.90) and under the assumption “affected individuals only” did not result in a significant change (data not shown). To determine the minimum linked interval, we then genotyped three novel polymorphic markers selected from the human genome working draft. The upper and lower boundaries of the region were determined by recombination events occurred in individuals III:2 and II:4, respectively, refining the locus to a 7 Mb interval flanked by markers RST2 and RIT3. Only one of eight unaffected individuals (IV:4, age 15 years) carried part of the disease haplotype, from D5S672 to RST3 (fig 1).

DISCUSSION

We report the assignment of a locus for HBT to chromosome 5q14 in a large kindred from northern Italy. This is the largest HBT family reported to date; the wide phenotypic variability observed among the 13 affected individuals runs through the spectrum of HBT lesions described so far.^2–3,5–10

While this work was in progress, the locus for capillary malformation (CMC1) was assigned to a 19 Mb region on 5q13–15.^12,13 The linked interval in the present family spans 7 Mb on chromosome 5q14 physical map and coincides to CMC1. Among cutaneous vascular anomalies, HBT and CM are usually classified in two distinct groups. In particular, while CM is considered a vascular malformation, HBT and other telangiectases (such as HHT) fall within the group of disorders caused by dilatation of pre-existing vessels.⁴

The typical cutaneous lesion in CM is congenital and appears as a single, cherry-red macular stain, larger in size than HBT lesions. CMs tend to darken slowly but progressively throughout life, becoming raised and thickened plaques (hence the name “port-wine stain”). These are usually unilateral with a fairly sharp midline cut-off. Associated eye and brain abnormalities occur in 8–15% of patients with facial port-wine stains (osseous malformations, glaucoma, Sturge-Weber and Klippel-Trenaunay syndromes).⁴ Although a typical port-wine stain was not observed in any of the 13 affected individuals in the present family, atypical lesions—difficult to frame within one or the other disorder—have been reported.^8,12 Moreover, both conditions share similar histopathological changes characterised by a dilatation of dermal thin walled blood vessels.¹ These observations suggest that HBT and CM represent variable clinical presentations of the same disorder and linkage of HBT to CMC1 corroborates this hypothesis. In this light, the 7 Mb region identified in the present family could significantly narrow the CMC1 locus, representing an important step towards the identification of the causative gene.

This region could be further narrowed to 6.4 Mb by considering individual IV:4, who is unaffected and carries part of the disease haplotype (fig 1). However, even if penetrance appears to be very high in this family, lack of penetrance in subject IV:4 cannot be excluded with certainty. For this reason, penetrance values used for LOD score calculations have been ranged up to 0.95.

The linked region contains 15 genes, some of which represent suitable candidates for cutaneous vascular anomalies. EDIL-3 (EGF-like repeats and discoidin I-like domains 3) encodes Del1, a protein expressed by endothelial cells during early embryogenesis.¹⁶ This protein has been shown to regulate the process of primary morphogenesis or vascular remodelling, both in vitro and in vivo.¹⁷ Versican is a modular proteoglycan involved in the control of cellular growth and differentiation. Although versican is generally known as a large chondroitin sulphate proteoglycan, the smallest splice variant (V3) consists only of the amino- and carboxy-terminal globular domains. If overexpressed, V3 has been shown to alter arterial smooth muscle cell adhesion, migration, and proliferation in vitro.¹⁸ Another interesting gene is RASA1 (RAS p21 protein activator-1), as mosaic mice composed of wild-type and RASA1 null cells show localised vascular defects at E15.¹⁹ The identification of the gene responsible for HBT and CM will help formulate genotype–phenotype correlations within the wide clinical spectrum of these conditions and give insight into the mechanisms leading to vascular malformations.