Recurrent single-CpG methylation differences and incomplete segregation in familial type 1 diabetes

Type 1 diabetes (T1D) clusters within families, yet shared genetic susceptibility does not fully explain discordance among relatives. We investigated whether single CpG methylation differences recur independently across multiplex T1D families and whether methylation at these loci segregates with T1D status or relates to HLA class II risk haplotype dosage.

Seventeen participants from three multiplex Arab families, including nine with T1D, underwent targeted bisulfite sequencing. Data from two sequencing runs were combined. Each pedigree was analysed independently. Unadjusted within-family analyses defined the cross-family recurrent set, requiring q<0.01, |Δβ|≥25% and concordant direction in all three families; age- and sex-adjusted analyses were then used to prioritise recurrent candidates. Candidate loci underwent pyrosequencing; public T1D transcriptomic datasets provided context for annotated genes. HLA genotyping determined risk haplotype dosage, and donor-level models examined associations with pyrosequencing methylation while accounting for family structure.

Unadjusted within-family analyses identified 22 recurrent CpGs with concordant methylation direction across the three pedigrees. Six of these retained the prespecified criteria after adjustment for age and sex and were prioritised for subsequent analyses. These mapped to MYRIP, DLGAP2, ULK4, MTCL1, LINC00701 and CCNG1. Pyrosequencing of MYRIP, ULK4 and MTCL1 revealed distinct methylation distributions, with affected and unaffected relatives occupying overlapping states or ranges, consistent with incomplete within-family segregation. HLA risk haplotype dosage showed a gradient in T1D occurrence across the families. MYRIP methylation was nominally lower in participants with T1D in the unadjusted donor-level analysis, but did not survive multiple testing correction. In exploratory adjusted donor-level models, higher HLA risk dosage was associated with lower MYRIP methylation.

These findings identify recurrent single-CpG methylation differences across multiplex T1D pedigrees but show incomplete individual-level segregation with disease. The six prioritised loci should therefore be considered exploratory methylation candidates whose temporal relationship with T1D and reproducibility require evaluation in larger independent and longitudinal family cohorts.