Genomic and epigenetic investigations of Silver-Russell syndrome and growth restriction

A combination of genes, their epigenetic regulation, and the environment control the phenotypes of an individual, such as how short or tall one grows. Epigenetics refers to chemical modifications of DNA and histones that regulate gene activity and genome stability, and can take the form of, for example, addition of a methyl group to DNA. A rare but illustrative example of growth restriction is Silver-Russel syndrome (SRS), which also features a relatively large, triangular head and asymmetry between body halves. Molecular studies have demonstrated that SRS is an interesting model for how growth is controlled by both genetic architecture and epigenetics, and recurring findings include DNA hypomethylation at an imprinted region on chromosome 11 (H19 ICR) in 20-65% of cases, maternal uniparental disomy of chromosome 7 (matUPD7) in 5-15%, and rare maternal duplications of chromosomes 7p and 11p. Imprinting is a rare but remarkable epigenetic phenomenon that describes parent-of-origin dependent gene activity, such that some genes are only expressed if they were inherited from, for example, the father. Differential DNA methylation (for example, maternal but not paternal methylation) is thought to regulate imprinting. All of the above mentioned molecular findings in SRS can cause dysregulation of imprinted genes. Interestingly, a large proportion of SRS patients remains molecularly unexplained. In this thesis we applied genome-wide genotyping and targeted epigenetic studies of imprinted genes to investigate the genetic nature of SRS and to disentangle the epi(genotype) and phenotype correlations in SRS and growth restriction. We devised a new approach to confirm UPD by the use of genotyping arrays and demonstrated a much increased resolution compared to the commonly used microsatellite markers. We further demonstrated the power of using genome-wide genotyping arrays in rare disorders such as SRS where UPD, copy number variants, or shared homozygosity might occur. We identified pathogenic submicroscopic events on chromosomes 15, 22, and X in molecularly unexplained SRS patients. A simple method for quantification of locus-specific DNA methylation is described and its accuracy and quantitative nature are demonstrated. In addition, reference distributions of DNA methylation at imprinted genes in controls are defined. This method was used to evaluate H19 ICR DNA methylation in SRS and isolated growth restriction, and 62% of SRS patients were hypomethylated. We further found a dose-response relationship between the degree of H19 ICR hypomethylation and phenotype severity in SRS and reported for the first time the association of specific anomalies of the spine, elbows, hands and feet, and genital defects in SRS with severe hypomethylation. In conclusion, we showed the utility of genotyping arrays to identify both UPD and submicroscopic genomic aberrations, and demonstrated that this genome-wide approach also enables the identification of important but unexpected events. Importantly, screens using genotyping arrays have the potential to detect the majority of genomic events in SRS. Through targeted epigenetic analysis we could conclude that H19 ICR methylation is clinically important as demonstrated by a strong correlation between the degree of hypomethylation and SRS phenotype severity and specifically associated clinical findings