Spanish families with cerebral cavernous angioma do not bear 742C→T Hispanic American mutation of the KRIT1 gene

Abstract

Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas, 1 and 65% of non-Hispanic American families have been linked to CCM1 with no founder effects. 2 The common mutation, 742C3 T transition, recently described in 16 of 21 families, 3 supports the previously described strong founder effect in Hispanic Americans. 4 We have recently reported on Spanish families with cerebral cavernous angioma linked to CCM1 locus, which do not match the Hispanic American CCM1 haplotype, 5 although 2 of 9 families partially shared the Hispanic American haplotype of CCM1. We have studied the possibility of an ancestor chromosome in a cohort of Spanish cavernous angioma families by a single screening test that detects the 742C3 T transition in exon VI of KRIT1 gene.The exon VI was analyzed in the probands of 39 nuclear Spanish families. A fragment of KRIT1 gene containing exon VI was PCR amplified with primers forward (5Ј TTGTTAG-ATTGTGATGTA) and reverse (5Ј AACATAATAAAAACT-TTC). Aliquots of the amplified DNA were initially screened by analysis of single-strand conformational polymorphism (SSCP). Polymerase chain reaction (PCR) products were electrophoresed in 10% acrylamide in the absence and in the presence of 10% glycerol as recently described. 1 We analyzed the 742C3 T transition by the formation of the sequence 5Ј TTAA that is the target for the restriction endonuclease MseI. The product of the PCR includes exon VI and an intronic MseI restriction site that served as internal control of the endonuclease digestion. The undigested PCR product consists of a 238-bp fragment, which, following the digestion with MseI, is divided in two fragments of 210 and 28 bp (Fig). The presence of the 742C3 T transition should split the PCR product in three fragments of 141, 69, and 28 bp.DNA sequencing was carried out with 5Ј 32 P-labeled primers and terminal dideoxynucleotides ( fmol kit of Promega, Lyon, France). The forward primer was 5Ј CGAATATA CAGAATGGATG, and the above-described oligonucleotide was the reverse primer.The results showed that only 1 of 39 probands gave a significant SSCP, but both the sequencing of the exon VI and the lack of the MseI restriction site demonstrated that the 742C3 T transition was not present in the affected chromosome. Given the great diversity of mutations recently described in KRIT1 gene, 1,3 the finding in the Spanish population of a single chromosome with the 742C3 T transition could support the existence of an ancestor chromosome. We failed to find this mutation in 39 unrelated patients, however, further supporting that the strong founder effect described in Mexican American families 4 with cerebral cavernous angioma are specific for this population.

Mutational analysis of 206 families with cavernous malformations

“…Including the 12 mutations reported in this series, all but one exist in familial cases. 1,2,4,6,15,17,20,21,23,24,27 In every instance except one, sequencing data have shown nonsense, point, splice-site, or frame-shift mutations leading to premature stop codons, distributed throughout the KRIT1 gene (Fig. 4).…”

“…14 Positional cloning experiments have ultimately identified one of the genes located in this region, KRIT1, as the CCM1 gene. 1,2,4,6,15,17,21,23,24,27 The function of the KRIT1 protein is unknown. The KRIT1 gene was incidentally cloned using a yeast two-hybrid screen with Krev1 (Rap1A) as bait.…”

KRIT1 , a gene mutated in cerebral cavernous malformation, encodes a microtubule-associated protein

“…CCM1 is located at 7q21 (17)(18)(19)(20)(21), CCM2 at 7p13-15 (18), and CCM3 at 3q25-27 (18). Apparent loss of function mutations in KRIT1 (Krev1 interaction trapped gene 1) have recently been shown to be the cause of disease mapping to CCM1 (22)(23)(24)(25)(26)(27); the genes underlying CCM2 and CCM3 have not yet been identified.…”

Study of cerebral cavernous malformation in Spain and Portugal

“…An exception is the 742T fi C transition of the Krit1 gene, a highly prevalent mutation in the HispanicMexican population [12] which was not detected in patients of Spanish and Portuguese descent [11].…”