Figure 4: Family 3 and patient P4.

(a) Pedigree of family 3 showing autosomal dominant inheritance of constitutional MLH1 epimutation. The two alleles of individuals who were tested for MLH1 methylation are depicted, with wild-type allele(s) in gray and methylated allele in purple. The two probands are indicated by an arrow. CRC, colorectal cancer. (b) Methylation analysis of MLH1 C-region for family 3 patient III-1: pyrogram showing low methylation level. (c) Haplotype analysis using microsatellites in family 3 and for patient P4 and her daughter. The different alleles of each microsatellite were numbered according to their increasing numbers of repeats. The haplotype associated with the epimutation (Me, methylation) is highlighted in yellow for family 3 and in orange for patient P4. Other haplotypes shared by two first-degree relatives are indicated by different shades of gray. (d) and (e) Methylation analysis of C- and D-regions for both alleles and after allele-specific amplification of the c.116+106G and the c.116+106A alleles (as shown by gel electrophoresis) (NTC, no template control). Positive results are the mean of at least three independent experiments (separate bisulfite conversions and polymerase chain reaction (PCR) amplifications) and standard deviations are indicated. The high variability between replicates and the nonquantitative results observed for the c.116+106A allele may be explained by the requirement for a degenerated reverse primer (primer overlapping 2CpG). (d) Family 3 (numbers refer to the ones indicated on family 3 pedigree). (e) Patient P4. (f) Patient P4: pyrograms showing methylation level of the C-region for both alleles and after allele-specific amplification of the c.-93G allele or the c.-93A allele. The c.-93A and the c.116+106A variants are located on the same allele (same reads in next-generation sequencing results). (g) and (h) Methylation analysis of intron 1 for both alleles and after allele-specific pyrosequencing of the c.116+106G and the c.116+106A alleles. Positive results are the mean of at least three independent experiments (separate bisulfite conversions and PCR amplifications) and standard deviations are indicated. (g) Family 3 (numbers refer to the ones indicated on family 3 pedigree). (h) Patient P4.