Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita

Abstract

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

Figure 1

Pedigree of the Dutch Family Affected by DC The proband (III:12) is marked by an arrow.

Figure 2

Sequence Analysis of TERC Sequence analysis of TERC revealed a heterozygous 4 bp deletion, n.54_57del (RefSeq accession number NR_001566.1), in the proband (III:12). The wild-type allele was observed more than the mutated allele in DNA isolated from peripheral blood cells from the father (II:7) and an uncle (II:1) of the proband. Analysis of DNA from the father's lung tissue and from the uncle's cultured fibroblasts revealed that the mutation was heterozygous. These data are in concordance with somatic events in the hematopoietic compartments of II:7 and II:1; these events resulted in loss of the germline mutation in a considerable fraction of the peripheral blood cells.

Figure 3

Mitotic Recombination on Chromosome 3q Results in Isodisomy of TERC B-allele frequencies (y axis) of chromosome 3 of proband III:12 (his DNA was derived from the liver), his father, II:7, and his uncle, II:1 (their DNA was derived from peripheral blood). This analysis revealed UPD of the long arm of chromosome 3 in subject II:7. This is likely the result of a mitotic recombination event in the centromeric region (indicated by the triangle) of chromosome 3q. In subject II:1, mosaic UPD was observed, and two adjacent regions of chromosome 3q had different degrees of mosaicism. This suggests the presence of two subpopulations of cells (marked as 1 and 2) resulting from independent mitotic-recombination events with different breakpoints.

Figure 4

Additional DC-Affected Individuals Show Revertant Somatic Mosaicism SNP-array analysis revealed stretches of UPD on chromosome 3q in peripheral blood DNA of subjects 7 and 15, as observed in the B-allele-frequency plot. The starting points of these homologous stretches are indicated by a triangle. No indication of UPD was observed in the SNP-array data of chromosome 3q in subjects 4 and 14. However, quantitative analysis of the SNP data revealed an allelic imbalance in these samples (Table S2), indicating the presence of a significant population of cells homozygous for a region on 3qter. The sequence data were scored for an imbalance in peak heights between the wild-type and mutant peaks, as summarized in Table 2. All four samples showed an overrepresentation of higher wild-type peaks.