Krit1 missense mutations lead to splicing errors in cerebral cavernous malformation

Abstract

At least 40% of families affected with cerebral cavernous malformation have a mutation in Krit1. We previously identified two point mutations in Krit1 leading to changes in amino acids (D137G and Q210E) in two different families. Further RNA analysis reveals that both point mutations actually activate cryptic splice-donor sites, causing aberrant splicing and leading to a frameshift and protein truncation. To date, no simple missense mutations have been detected in Krit1.

Figure 1

A, Pedigree of IFCAS-41. The blackened symbols denote affected individuals, and the unblackened square denotes an individual not known to be affected. Asterisks denote mutations, and the small unblackened circle denotes an absence of mutation. B, Genomic DNA sequences of unaffected (1) and affected (2) individuals. The affected individual carries an A→G substitution at nucleotide position 410 of the coding sequence. C, cDNA migration pattern of the normal (a) and mutated (b) alleles, for each member of the IFCAS family. D, cDNA sequences of the normal and mutated alleles. The mutated allele causes cryptic splicing, as is illustrated in the diagram.

Figure 2

A, Pedigree of IFCAS-28. Definitions of symbols are the same as in figure 1. B, Genomic DNA sequences of a normal (3) and affected (1) individuals. The affected individual carries a C→G substitution at nucleotide position 601 of the coding sequence. C, cDNA migration pattern of the normal (a) and mutated (b) alleles, for each member of the IFCAS family. D, cDNA sequences of the normal and mutated alleles. The mutated allele causes cryptic splicing, as is illustrated in the diagram.

Figure 3

Splice-donor site consensus sequence. The A→G substitution in IFCAS-41 changed the first nucleotide of the intron, whereas the C→G substitution in IFCAS-28 changed the third nucleotide of the intron.