Since the amplicons designed for mutation analysis covered not only the corresponding exons but also included 50 to 100 bp of flanking intronic sequence (
Fig. 2A), several SNPs were genotyped as a byproduct of the mutation analysis. In our cohort of German patients and controls, we detected sequence variation at four SNP loci: rs41317515, rs9659061, rs12124948, and rs10687239 (
Fig. 3). However, the c.759_778del20 deletion always, without any exception, was accompanied by the haplotype C-A-T-ins4 at these SNPs (
Fig. 3A). To determine the frequency of the deletion-associated SNP alleles in our normal population, we genotyped 20 to 40 unrelated, healthy German individuals, either by allele-specific PCR (rs41317515) or by sequencing (rs9659061, rs12124948, and rs10687239). On average, the deletion-associated SNP alleles had a frequency of approximately 50% in the control cohort (
Fig. 3B). Since we did not have access to relatives of these controls, we could not construct haplotypes by segregation analysis. In our EL families, however, we detected various different haplotypes by segregation analysis. Thus, the deletion-associated haplotype C-A-T-ins4 is just one among several others that strongly suggest a founder for the c.759_778del20 deletion.
To estimate the time when the founder mutation took place, we genotyped microsatellite markers on chromosome 1 (
Table 1) in our EL patients as well as in a cohort of unrelated, healthy German individuals. For the nearest marker (TSL4CA, 50-kb distance to
ADAMTSL4) 100% of our EL patients carried the 209 allele (
Fig. 4, left), in which a CA-dinucleotide unit is repeated 14 times. In the control cohort, this allele had a frequency of only 37% (
Fig. 4, left), again strongly suggesting a founder of the c.759_778del20 deletion. Concerning the second closest marker (D1S498, 770-kb distance to
ADAMTSL4), the EL patient group showed some sequence variety (
Table 1,
Fig. 4, right), which is probably due to recombination events and suggests that the founder mutation is very old. However, there is still a predominant allele, which occurred in 75% of the EL cases but in only 20% of the control cohort (
Fig. 4, right). In conclusion, there is strong evidence of a founder mutation and the founder event appears to have occurred more than 100 generations ago.