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. 2011 Jul;13(7):662-6.
doi: 10.1097/GIM.0b013e318211ff8b.

A founder mutation in the Ashkenazi Jewish population affecting messenger RNA splicing of the CCM2 gene causes cerebral cavernous malformations

Carol J Gallione  1 , Ann Solatycki, Issam A Awad, James L Weber, Douglas A Marchuk
Affiliations

A founder mutation in the Ashkenazi Jewish population affecting messenger RNA splicing of the CCM2 gene causes cerebral cavernous malformations

Carol J Gallione et al. Genet Med. 2011 Jul.

Abstract

Purpose: Cerebral cavernous malformations can occur sporadically or are caused by mutations in one of three identified genes. Cerebral cavernous malformations often remain clinically silent until a mutation carrier suffers a stroke or seizure. Presymptomatic genetic testing has been valuable to follow and manage cerebral cavernous malformation mutation carriers. During routine diagnostic testing, we identified a two base pair change in seven unrelated people of Ashkenazi Jewish heritage. Because of the location of the variant beyond the invariant splice donor sequence, the change was reported as a variant of unknown significance. In this study, we determined whether this change was a disease-causing mutation and whether it represents a founder mutation in the Ashkenazi Jewish population.

Methods: Transcripts arising from the normal and mutant alleles were examined by reverse transcription-polymerase chain reaction from affected and unaffected Ashkenazi Jewish cerebral cavernous malformation family members. A synthetic splicing system using a chimeric exon was used to visualize the effects of the change on splice donor site utilization.

Results: The two base pair change in CCM2, c.30 + 5_6delinsTT, segregated with affected status in the study families. Reverse transcription-polymerase chain reaction revealed loss of the transcript allele that was in phase with the mutation. The two base pair change, when tested in an in vitro synthetic splicing system, altered splice donor site utilization. Resequencing of the genomic region proximal and distal to the CCM2 gene mutation revealed a common single-nucleotide polymorphism haplotype in affected individuals.

Conclusions: The two base pair change in CCM2, c.30 + 5_6delinsTT, disrupted proper splice donor utilization leading to a degraded transcript. Single nucleotide polymorphism haplotype analysis demonstrated that this mutation was due to a founder in the Ashkenazi Jewish population. These data have the potential to simplify genetic testing for cerebral cavernous malformation in the Ashkenazi Jewish population.

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Figures

Figure 1
Figure 1
A. DNA sequence traces from unaffected and affected individuals. The nucleotides at position c.30+5_6 are boxed. B. Pedigrees of two Ashkenazi Jewish families in this study. Genders are obscured in order to protect patient confidentiality. Clear diamonds indicate unaffected, dark diamonds indicate clinically affected, speckled diamonds indicate uncertain disease status. Probands are indicated with arrows. The genotypes at c.30+5_6 and the exon 2 SNP (rs2107732) are noted. C. The CCM2 AJ affected SNP haplotype is shown with the rare alleles shown in bold.
Figure 2
Figure 2
A. Schematic diagram of CCM2 exons 1 through 4 including the alternate exon 1. The boxes represent the exons, the primary transcription start codon in exon 1, secondary transcription start site in alternate exon 1, and the reporter SNP in exon 2 (rs2107732) are indicated, and the arrows below the exons indicate the primers used for RT-PCR. B. The top tracing for each column is the sequence of genomic DNA from an unaffected individual (left) and an affected individual (right) both also heterozygous at SNP rs2107732 in exon 2. The second set of tracings show RT-PCR products from the same unaffected and affected individuals using primers in exons 1 and 4 (black arrows). Transcripts from both alleles are present in the unaffected family member, but only one transcript is seen in the affected individual, with the transcript derived from the mutant allele being absent. The last tracing shows the RT-PCR product from the affected individual using primers in exons alternate exon 1 and 3 (red arrows). Transcripts from both alleles are present in the affected individual when transcription initiates downstream of the mutation.
Figure 3
Figure 3
A. RT-PCR products from the synthetic splicing system containing a chimeric exon. B: Schematic representations of the chimeric exon cloned into the pSPL3 vector (top) and the three different resulting RT-PCR products. The wild type sequence is above the line and the mutant sequence is below. 1. Full length transcript containing the vector-supplied exon 1 (V1), the vector-supplied terminal exon (Vt) of the splicing vector pSPL3, and the full sequence of the chimeric exon. 2. The product of the vector-supplied first and terminal exons spliced to a smaller fragment of the chimeric exon. The arrow indicates the position of the cryptic splice site used to generate the truncated transcript. 3. Transcript containing only V1 and Vt. M= 100 bp marker, WT = vector with chimeric exon containing wild type splice donor sequence, Mut = vector with chimeric exon containing mutant splice donor sequence. Mut1 and Mut2 are from duplicate transfections of the mutant version of the construct.
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