Spanish families with cerebral cavernous angioma do not bear 742C→T Hispanic American mutation of the KRIT1 gene
✍ Scribed by Miguel Lucas; Francisca Solano; María D. Zayas; Jose M. García-Moreno; Miguel A. Gamero; Alzenira F. Costa; Guillermo Izquierdo
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 542 KB
- Volume
- 47
- Category
- Article
- ISSN
- 0364-5134
No coin nor oath required. For personal study only.
✦ Synopsis
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.