Figure 2: Family 1.

(a) Pedigree of family 1 showing autosomal dominant inheritance of constitutional MLH1 epimutation. The two alleles of individuals who were tested for MLH1 methylation are depicted, with wild-type (WT) allele(s) in gray and methylated allele in purple. The proband is indicated by an arrow; roman numbers are for generations and Arabic numbers are for individuals; squares are males; circles are females; black shading indicates individuals affected by cancer as listed below, with the age of onset also indicated (CRC, colorectal cancer); crossed squares or circles are for deceased individuals. (b) Sequence of exon 1 with insertion of an AluYc sequence. The sequence is in capital letters, with exon 1 in gray and the inserted sequence in bold. Alignment to AluYc sequence using RepeatMasker (http://www.repeatmasker.org/) is depicted in lowercase letters below the sequence (“i” indicates a transition and “−” a deletion). The c.1 nucleotide is the A of the translational initiation codon (GenBank NM_000249.3). The target site duplication (TSD) is indicated in red arrows. The new splicing donor site, as identified by RNA analysis, is indicated by a star. (c) Sequencing of complementary DNA generated from RNA extracted from patient IV-1 lymphoblastoid cell line. Electropherogram shows splicing from a donor site located within the Alu sequence (the last five nucleotides of the forward primer used for polymerase chain reaction (PCR) amplification are complementary to the first five nucleotides of the inserted sequence; the reverse primer is located in exon 4) with expression of a major transcript, and of a minor transcript lacking the first five nucleotides of exon 2. (d) Methylation analysis of C- and D-regions for both alleles and after allele-specific amplification of the WT and the mutant alleles. Positive results are the mean of three independent experiments (three separate bisulfite conversions and PCR amplifications) and standard deviations are indicated. Amplification of the mutant allele is restricted to patients exhibiting the insertion (II-2, III-2, and IV-1), as shown by gel electrophoresis (NTC, no template control). Numbers refer to the ones indicated on the pedigree of family 1.