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. 2007 Jan;80(1):69-75.
doi: 10.1086/510439. Epub 2006 Nov 14.

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Christina L Liquori  1 , Michel J Berg, Ferdinando Squitieri, Tracey P Leedom, Louis Ptacek, Eric W Johnson, Douglas A Marchuk
Affiliations

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Christina L Liquori et al. Am J Hum Genet. 2007 Jan.

Abstract

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.

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Figures

Figure 1.
Figure 1.
Representative chromatograms from MLPA analysis of CCM1 and CCM2. A, Results for control individual with peaks corresponding to CCM1 and CCM2 exonic probes. There are two probes to CCM2 exon 2 (5′ and 3′) and one probe to a region 690 bp upstream of CCM2 exon 1 (“upstream”). All unlabeled peaks represent the control peaks resulting from the amplification of probes located on different chromosomes. B, Chromatogram from CCM-affected proband 254, showing the relative reduction in peak area of probes hybridizing to exons 5 and 6 of CCM2. Arrows mark the deleted CCM2 exons. C, Chromatogram from CCM-affected proband 186, showing the relative reduction in peak area of probes hybridizing to exons 2–10 of CCM2. This represents the common CCM2 deletion spanning CCM2 exons 2–10. Arrows mark the deleted CCM2 exons.
Figure 2.
Figure 2.
Segregation of deletion with disease in family 186. Blackened symbols show affected individuals; blackened dots indicate carrier status (by microsatellite marker disease haplotype); unblackened symbols depict healthy individuals with normal MRI results. The top band is a PCR product through use of primers spanning the CCM2 exons 2–10 deletion, and only those individuals who harbor the deletion will show this band. The bottom band is a control PCR product (CCM1 exon 2), to ensure appropriate DNA quality and quantity in the multiplex PCR. A 100-bp ladder was used for sizing, with the brighter band indicating 600 bp.
Figure 3.
Figure 3.
PCR across the deletion of CCM2 exons 2–10. PCR by use of primers in intron 1 and distal to exon 10 resulted in the same size band in all eight probands who had a deletion of CCM2 exons 2–10 as detected by MLPA. The top band (839 bp) is a PCR product through use of primers spanning the deletion, and only those individuals who harbor the deletion will show this band. The bottom band is a control PCR product (CCM1 exon 2), to ensure appropriate DNA quality and quantity in the multiplex PCR. A 100-bp ladder was used for sizing, with the brighter band indicating 600 bp. ctrl = Control.
Figure 4.
Figure 4.
Large deletions within CCM2. A, Schematic diagram of the CCM2 gene and the 14 CCM2 deletions that were identified. The CCM2 gene is depicted with each exon shown as a white box with the corresponding exon number and each intron shown as a line. Thick black lines below the gene depict each of the CCM2 deletions, with the solid portion showing the minimal extent of the deletion and the broken portion showing the maximal extent of the deletion. SINE and LINE elements located near the breakpoints of the common exons 2–10 deletion are depicted as gray boxes above the CCM2 gene, with orientation of each element indicated by the arrowhead. The black triangles indicate the location of the breakpoints in the common deletion spanning CCM2 exons 2–10. Not drawn to scale. B, Schematic diagram of the recombinant Alu element in the common CCM2 deletion and surrounding region. The recombinant Alu element is shown as a box, with the AluSx and AluSg halves indicated. Nearby SNPs are also shown. A more detailed version of the recombinant Alu element is shown below. White indicates regions where the sequence is specific to the AluSx, black indicates regions where the sequence is specific to the AluSg, and gray indicates regions where the sequence is identical between the AluSx and the AluSg. Base-pair positions are shown above the box. C, Sequence homology between the parental AluSx (chr7:45024885–45025183) and AluSg (chr7:45101654–45101952). White boxes indicate identical base pairs, gray boxes indicate different base pairs, and black boxes indicate the location of two SNPs. The carat represents a SNP in AluSx (chr7:45025035G→A), and the asterisk (*) represents SNP rs6955183 in AluSg.
Figure 5.
Figure 5.
Schematic representation of affected haplotypes for families with the CCM2 exons 2–10 deletion. Each haplotype is represented by an unblackened bar, with the affected alleles for each marker shown within the bar. The location of the deletion is shown as “del” with a blackened triangle above the bar. Marker names are listed above their associated alleles, and family numbers are listed to the left of their associated haplotypes. For marker D7S2528, allele 2 is 306 bp. For marker D7S2427, allele 10 is 222 bp. For marker D7S2436, allele 4 is 262 bp. For marker D7S621, allele 5 is 274 bp and allele 7 is 266 bp. For marker CL7N4, allele 1 is 240 bp and allele 3 is 236 bp. For marker CL7N5, allele 2 is 191 bp and allele 6 is 183 bp. For marker D7S626, allele 6 is 371 bp, allele 7 is 367 bp, allele 8 is 363 bp, and allele 11 is 351 bp. The distance (in kb) of each marker from the deletion is shown at the top. Not drawn to scale.
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References

Web Resources

    1. dbSNP, http://www.ncbi.nlm.nih.gov/SNP/
    1. International HapMap Project, http://www.hapmap.org/
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for CCM1, CCM2, and CCM3)
    1. University of California–Santa Cruz Human Genome Bioinformatics, http://genome.ucsc.edu/ (for the March 2006 assembly)

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