Whole-genome methylation scan in ICF syndrome: hypomethylation of non-satellite DNA repeats D4Z4 and NBL2

The ICF (immunodeficiency, centromeric instability and facial abnormalities ) syndrome is a rare recessive disease characterized by immunodeficiency, e xtraordinary instability of certain heterochromatin regions and mutations i n the gene encoding DNA methyltransferase 3B, In this syndrome, chromosomes 1 and 16 are demethylated in their centromere-adjacent (juxtacentromeric) heterochromatin, the same regions that are highly unstable in mitogen-treat ed ICF lymphocytes and B cell lines, We investigated the methylation abnorm alities in CpG islands of B cell lines from four ICF patients and their una ffected parents, Genomic DNA digested with a CpG methylation-sensitive rest riction enzyme was subjected to two-dimensional gel electrophoresis. Most o f the restriction fragments were identical in the digests from the patients and controls, indicating that the methylation abnormality in ICF is restri cted to a small portion of the genome. However, ICF DNA digests prominently displayed multicopy fragments absent in controls. We cloned and sequenced several of the affected DNA fragments and found that the non-satellite repe ats D4Z4 and NBL2 were strongly hypomethylated in all four patients, as com pared with their unaffected parents. The high degree of methylation of D4Z4 that we observed in normal cells may be related to the postulated role of this DNA repeat in position effect variegation in facioscapulohumeral muscu lar dystrophy and might also pertain to abnormal gene expression in ICF, In addition, our finding of consistent hypomethylation and overexpression of NBL2 repeats in ICF samples suggests derangement of methylation-regulated e xpression of this sequence in the ICF syndrome.