A prevalent mutation with founder effect in Spanish Recessive Dystrophic Epidermolysis Bullosa families

Dystrophic Epidermolysis Bullosa (DEB) is a rare disease, characterized by trauma induced-blistering and scarring [1]. This genodermatosis is a rare autosomal dominant (DDEB [MIM#131750, #131800]) or recessive (RDEB [MIM#226600]) disorder caused by mutations in COL7A1 gene [MIM*120120], encoding type VII collagen (protein component of anchoring fibrils) [2]. COL7A1 gene is an unusually complex gene with 118 exons. It has the second largest number of exons of all genes described to date [3]. So far, more than 500 mutations have been described in the COL7A1 gene http://​www.​hgmd.​cf.​ac.​uk[4]. Although COL7A1 genetic database indicates that most of the DEB mutations are family specific, with few recurrent mutations, in the Spanish cohort, a high recurrence of the c.6527insC pathogenic mutation has recently been reported by our group [5]. Accounting for 46.3% of alleles this is a level of recurrence for a single genetic defect hardly ever found for the COL7A1 gene [5, 6]. The c.6527insC mutation creates a premature codon termination (PTC), leading to nonsense mediated decay (NMD) that manifests as a complete absence of collagen protein (Fig. 1). This insertion mutation was detected mainly in families native of the southwest of the Iberian Peninsula (Fig. 2). The overrepresentation of a single mutation and the geographic clustering of the c.6527insC pedigrees indicate at least one founder effect or a mutational hotspot. We investigated the putative founder effect of the c.6527insC mutation through the construction of SNP haplotypes throughout the COL7A1 gene. A rare single haplotype in the Spanish population was present in all patients and relatives who carried the c.6527insC mutation, supporting the hypothesis that all chromosomes carrying the c.6527insC mutation arise from a single founder effect.

We identified 5 novel SNPs in the region corresponding to COL7A1 gene, NM-000094.3:c.11639C>T, 24558C>T, 25215C>T, 29056C>T, 31427C>A. The distribution of all SNPs in the control Spanish population did not deviate from HWE (?2-test; p > 0.05) (Additional file 1, Table S3). The NM-000094.3:c.11639C>T and 25215C>T were considered for the construction of haplotypes.

The existence of complete two-generation pedigrees in all our patients allowed the construction of different haplotypes formed by nine SNPs. Considering the two blocks of linkage LD located throughout the COL7A1 gene, we determined twelve haplotypes in DEB patients (from H1 haplotype to H7 haplotype, H9, H12, H14, H15 and H16 haplotypes) and thirteen haplotypes in the Spanish population (from H1 to H13). The overall distribution of the estimated haplotypes was significantly different between healthy controls and patients (see Table 1 and Table 2).

The total number of the alleles (n = 38) that carry the c.6527insC mutation cosegregated exclusively with H5 haplotype (CCGCTCAAA), namely all alleles that carry the c.6527insC mutation were CCGCTCAAA_6527insC. On the other hand, the frequency of H5 haplotype in the Spanish population was 5.81%. Consequently, the c.6527insC mutation in the Spanish cluster is a result of a single mutational event, and the affected pedigrees must descend from one genetic founder who exhibited the H5 haplotype. Out of 27 pedigrees bearing at least one CCGCTCAAA_6527insC allele, 26 stemmed from ancestors who had been living in the southern half of the Iberian Peninsula for at least 3 generations. The other lineage bearing the CCGCTCAAA_6527insC allele comes from ancestors who had been living in Northern Spain for over 3 generations. In addition, we analyzed a Portuguese pedigree carrier of the CCGCTCAAA_6527insC allele. These results reveal that the c.6527insC mutation is not the result of separate mutations occurring independently in different individuals, but is the result of a one-time mutation occurring in a common ancestor of all the families. Furthermore, we also found a pedigree where the chromosome carrying the mutation cosegregated with G2520V H5 haplotype. The other forty recessive pathogenic mutations cosegregate with other fifteen different haplotypes (Table 2).

High carrier rates are usually attributed to a founder effect in a population and are usually evidenced by conservation of haplotypes with directly associated markers [7]. We constructed fifteen different intragenic haplotypes throughout the COL7A1 gene to explore the origin of the alleles carrying the c.6527insC mutation and other mutations described in our DEB patients. All alleles carrying the c.6527insC mutation were CCGCTCAAA_6527insC, indicating a common origin. This hypothesis is supported by the diversity of haplotypes throughout the COL7A1 gene. Moreover, H5 haplotype is rare in the control population (5.81%).

The c.6527insC mutation is found at a high prevalence among patients from the southern half of the Iberian Peninsula. This mutation has previously been found in one patient in France [8] and in another in Germany [9]. However, a Spanish predecessor of those patients cannot be excluded, taking into account the large Spanish emigration to France that occurred after the Spanish civil war (1936-1939) and to Germany in the early 1960 s due to economic hard-ship.

The overall distribution of the estimated haplotypes was significantly different between patients and healthy controls. The absence of recurrence in the other mutations did not allow us to distinguish whether a single haplotype is associated with a mutation, or conversely, if one mutation is associated with general haplotypes suggesting multiple origins. The H1 and H2 haplotypes identified as cosegregating with other mutations are common in the background population. This finding is neither surprising nor unexpected. In terms of elementary probability, the probability of occurrence of a pathogenic mutation in a chromosome harboring a relatively common haplotype in the population is obviously high.

We found a single mutation, p.R525X, which cosegregated with two different haplotypes (H2 haplotype and H9 haplotype). Moreover this nonsense mutation has been described previously in other DEB patient cohorts [10]. This could indicate the presence of a mutational hotspot in codon 525 of COL7A1 gene. Codon 525 contains a CpG dinucleotide, which is the known site of DNA modification by cytosine methylation.

In summary, the conservation of a single haplotype surrounding the c.6527insC mutation suggested that this allele has a single origin. The finding of a founder effect in a highly recurrent mutation in a rare disease characterized by intrafamilial mutations is essential for the implementation of protocols for genetic diagnosis, for genetic counselling of affected pedigrees and is fundamental to search for new therapies.