The advent of genome scanning tools allows comprehensive analysis of chromosomal rearrangements, dependent on the resolution of the experimental approach. The aCGH experiment using genomic BACs provided extensive coverage of the genome at a resolution of ~1 Mb, so any smaller chromosomal aberrations would not be detected. SNP arrays not only provide genotypes for thousands of SNPs, but can also be used to detect copy number changes based on hybridization signal intensities. Genotype data is useful for detecting loss of heterozygosity in chromosomal regions with deletions or uniparental disomy. At an average inter-marker distance of ~60 kb and ~12 kb for the 50K and 250K GeneChips respectively, and additional genotyping through the Illumina SNP panel, our analysis provide sufficient resolution to detect submicroscopic structural variants that are defined in the range of ~10 kb to 3 Mb (Feuk et al., 2006
). Several samples were interrogated using more than one method (), thus allowing data comparison and verification.
Our study using aCGH and genotyping arrays did not reveal any genomic alterations within MZ twins discordant for nonsyndromic CLP. Genomic alterations could be confined to specific tissues depending on the timing of the mutational event, resulting in mosaicism. Since acquisition of DNA samples from lip and palatal tissues is difficult, our analysis was limited to analysis of DNA samples collected from peripheral blood lymphocytes, which may not accurately represent the target tissues affected in CLP. Additionally, our experimental approaches were incapable of detecting balanced variants such as those resulting from inversions and translocations, and chromosomal aberrations that involve regions with only homozygous alleles. Routine karyotyping which can generally detect such chromosomal abnormalities could not be performed due to unavailability of living cells from a blood sample.
We are unaware of definitive reports of postzygotic genomic rearrangements underlying MZ twin discordance. A recent report of copy number variants arising between MZ twins during somatic development is consistent with our rationale for using discordant MZ twins in disease gene identification (Bruder et al., 2008
). Changes in the somatic genome are well recognized as a source of diversity within the immunoglobulin and T-receptor genes (Kastern and Kryspin-Sorensen, 1988
). In disease states, rearrangements can induce a phenotype by directly interrupting a gene sequence, altering gene dosage, or gene expression through position effects (Lupski and Stankiewicz, 2005
). Mitotic recombination is especially relevant in tumor development, since it can lead to the expression of recessive tumor suppressor genes and/or amplification of protooncogenes (Gupta et al., 1997
). Analysis of MZ twins concordant for cancer can reveal DNA rearrangements that are common to both twins as potential candidates for susceptibility loci (el-Rifai et al., 1999
). In addition to oncogenesis, somatic mutations mediated through LOH can potentially contribute to other biological processes such as aging (Grist et al., 1992
Phenotypic discordance is a common observation in complex genetic diseases, and a postzygotic somatic change is only one of several proposed mechanisms for discordance. Since CLP is a congenital defect, non-genetic intrauterine environmental factors such as unequal cell allocation at twinning and disproportionate placental blood supply may contribute to discordance (Gringras and Chen, 2001
). If such developmental influences alone were responsible for MZ twin discordance, then one would expect an excess of CLP in MZ twins compared to singletons. This is not the case as studies in Denmark have demonstrated no significant difference in prevalence of CLP in twins versus singletons (Christensen and Fogh-Andersen, 1993a
; Christensen and Fogh-Andersen, 1993b
). The absence of genomic differences between MZ twins underscores their use as matched case-controls particularly in studies exploring the environmental component in complex traits. With the availability of array-based techniques to simultaneously scan the whole genome, it is now possible to map genomic alterations at a high resolution and additional experimental tools such as quantitative PCR and multiplex ligation-dependent probe amplification (MLPA) can be applied to independently confirm any observed genomic alterations. This straightforward approach can be applied to other cases of discordant MZ twins where identification of genomic alterations can reveal potential candidate genes, or provide evidence of a gene’s involvement in disease etiology.