Bisulphite sequencing of the transient neonatal diabetes mellitus DMR facilitates a novel diagnostic test but reveals no methylation anomalies in patients of unknown aetiology

Transient neonatal diabetes mellitus (TNDM) is associated with overexpression of an imprinted locus on chromosome 6q24; this locus contains a differentially methylated region (DMR) consisting of an imprinted CpG island that normally allows expression only from the paternal allele of genes under its control. Three types of abnormality involving 6q24 are known to cause TNDM: paternal uniparental disomy of chromosome 6 (pUPD6), an isolated methylation defect of the imprinted CpG island at chromosome 6q24 and a duplication of 6q24 of paternal origin. A fourth group of patients has no identifiable anomaly of 6q24. Bisulphite sequencing of the DMR has facilitated the development of a diagnostic test for TNDM based on ratiometric methylation-specific polymerase chain reaction. We have applied this method to 45 cases of TNDM, including 12 with pUPD6, 11 with an isolated methylation mutation at 6q24, 16 with a duplication of 6q24 and six of unknown aetiology, together with 29 normal controls. All were correctly assigned. The method is therefore capable of detecting all known genetic causes of TNDM at 6q24, although pUPD6 and methylation mutation cases are not distinguished from one another. In addition, we have carried out bisulphite sequencing of the DMR to compare its methylation status between six TNDM patients with a known methylation mutation, six patients with no identifiable 6q24 mutation and six normal controls. Whereas methylation mutation patients showed a near-total absence of DNA methylation at the TNDM locus, the patients with no identified molecular anomaly showed no marked methylation variation from controls.