NF1 mutation analysis in a child homozygous for MLH1 mutation

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant disease characterized by an inherited predisposition to early onset colorectal cancer and an increased risk of certain other cancers. Cancer susceptibility is due to a heterozygous germ line mutation in one of five mismatch repair (MMR) genes, with the majority of mutations found in MLHl and MSH2. This study focused on a child with a homozygous germ line mutation in MLHl [C676T—>Arg226Stop] inherited from consanguineous parents (Ricciardone ef a i, 1999). This child presented with neurofibromatosis type 1 and hematological malignancy, instead of the colorectal cancer phenotype usually seen in HNPCC individuals. This severe tumorigenic syndrome most likely resulted from a downstream mutation in the NFl gene that could not be repaired due to complete absence of DNA MMR activity. To confiiTn this hypothesis and possibly clarify the genetic mechanism involved in this phenotype, exons in the functional domain o(NFl that contained mononucleotide or dinucleotide repeat sequences were analyzed by single strand conformation polymorphism (SSCP) and DNA sequence analysis for the presence of mutations. SSCP analysis of the child’s DNA identified altered mobility of NF'l exon 22. Subsequent DNA sequence analysis revealed a heterozygous C3721T transition mutation. Taql restriction digestion o f the exon fragment confirmed loss of the restriction site, thus, verifying the sequencing results. The C3721T mutation results in substitution of a stop codon for an arginine codon at position 1241. The resulting NFl gene product is truncated at the beginning of the functional GTPase activation domain. A putative somatic mutation in the wild-type allele caused complete loss of neurofibromin function. The resulting impaired regulation of Ras*GTP signaling probably contributed to tumor development -- neurofibromatosis and hematological malignancy. This NFl mutation was not present in the mother, father or sibling, indicating that this was a de novo mutation that occurred in early embryogenesis, most probably as a downstream consequence of constitutional MMR deficiency.