Disorders of striking genetic heterogeneity, such as BBS, represent a major diagnostic challenge as mutations at more than 12 loci have been shown to be necessary and sufficient for pathogenesis, rendering medical resequencing of >204 known exons laborious and expensive. Other viable alternatives, such as specific interrogation of known mutations by primer extension is practical, but can only capture less than 50% of the mutational load25–28
(our unpublished data). In the present study, we have demonstrated that whole genome homozygosity mapping followed by direct sequencing is an effective alternative means of identifying causative mutations in BBS. We have shown recently that homozygosity mapping can also be applied to individuals who reside within “outbred” populations but are “homozygous by descent“ from a remote ancestor at the locus of the disease-causing gene21
. We here successfully apply this approach to patients from both, inbred and outbred populations, confirming that there can be broad utility of homozygosity mapping in the absence of ancestry information21
. Specifically, in families A2875 and A2876 who derive from outbred populations (), we were able to detect the disease causing mutations (Supplementary Figure 1
Alternatively, the “homozygosity peaks” could be generated by hemizygosity, i.e. a large heterozygous deletion. In a recessive disease, a second mutant allele would be expected to be present in the gene affected by the disease-relevant heterozygous deletion giving rise to a compound heterozygous state. However, especially in individuals with known consanguinity of the parents, compound heterozygosity is unlikely, because consanguinity poses an a priory risk for the affected individual for homozygous mutations but not for compound heterozygous mutations. In molecular diagnostics the presence of a large heterozygous deletion would have the same practical consequence, i.e. to perform exon sequencing to detect the second recessive mutation.
Notably, none of the families studied had a sufficient number of affected individuals to allow mapping of a disease locus by classic linkage analysis, further emphasizing the fact that homozygosity mapping can solve an otherwise experimentally intractable problem, especially as the cost for total genome SNP analysis has considerably dropped recently. In the families who do not yield cZLR peaks, which were 13 families in this study, mutation analysis for compound heterozygous mutations will still have to be performed for all exons of all known BBS genes.
In conclusion, we increased the number of BBS-associated mutations by 5% (11/218) employing homozygosity mapping. Our study will likely catalyze the identification of additional BBS loci, since 12 families exhibited homozygous regions outside a known BBS locus or were negative upon mutation analysis (data not shown). Alternatively, these families may have compound heterozygous mutations in known BBS genes, because there is a possibility of false positive “homozygosity peaks” at a rate of less than 35% in individuals from “outbred” background (Hildebrandt et al. PloS Genet 5:e1000353, 2009). This rate was been determined by examining parents of children with recessive mutations for “homozygosity peaks”. However, as parents of children with mutations homozygous by descent have themselves a some what increased likelihood that their own parents are related, this rate of 35% may be somewhat of an overestimate.”
Ultimately, comprehensive analysis of the entire BBS-associated gene spectrum will be necessary, because of the presence of modifier alleles (even in consanguineous families) that can modify the penetrance and expressivity of BBS endophenotypes18,29,30
. Nonetheless, the rapid identification of the primary genetic lesion will have a clear and immediate benefit with regard to patient management and reproductive choices and is likely to prove useful in the study of other disorders of marked genetic heterogeneity such as retinitis pigmentosa, sensorineural deafness and others.