Mutations within the MGC4607 gene cause cerebral cavernous malformations

Abstract

Cerebral cavernous malformations (CCM) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and focal neurological deficits due to cerebral hemorrhages. CCM loci have already been assigned to chromosomes 7q (CCM1), 7p (CCM2), and 3q (CCM3) and have been identified in 40%, 20%, and 40%, respectively, of families with CCM. Loss-of-function mutations have been identified in CCM1/KRIT1, the sole CCM gene identified to date. We report here the identification of MGC4607 as the CCM2 gene. We first reduced the size of the CCM2 interval from 22 cM to 7.5 cM by genetic linkage analysis. We then hypothesized that large deletions might be involved in the disorder, as already reported in other hamartomatous conditions, such as tuberous sclerosis or neurofibromatosis. We performed a high-density microsatellite genotyping of this 7.5-cM interval to search for putative null alleles in 30 unrelated families, and we identified, in 2 unrelated families, null alleles that were the result of deletions within a 350-kb interval flanked by markers D7S478 and D7S621. Additional microsatellite and single-nucleotide polymorphism genotyping showed that these two distinct deletions overlapped and that both of the two deleted the first exon of MGC4607, a known gene of unknown function. In both families, one of the two MGC4607 transcripts was not detected. We then identified eight additional point mutations within MGC4607 in eight of the remaining families. One of them led to the alteration of the initiation codon and five of them to a premature termination codon, including one nonsense, one frameshift, and three splice-site mutations. All these mutations cosegregated with the disease in the families and were not observed in 192 control chromosomes. MGC4607 is so far unrelated to any known gene family. Its implication in CCMs strongly suggests that it is a new player in vascular morphogenesis.

Figure 1

Genealogical trees of the 30 analyzed families with CCM. The family numbers are indicated above each pedigree. The five families used for genetic linkage analysis were families C004, C020, C039, C049, and C116.

Figure 2

D7S2427 parental noncontribution within family C116. The family C116 genealogical tree and CCM2 haplotypes are shown; markers D7S516 and D7S1818 flanking the published 22-cM CCM2 interval are indicated, as well as D7S691, the new telomeric boundary. A null allele was detected at the D7S2427 marker (boxed) within the haplotype cosegregating with CCM (gray), strongly suggesting the existence of a deletion that was confirmed by a distinct D7S2427-specific primer set and by additional genotyping (fig. 3).

Figure 3

Large deletions within the CCM2 interval. A, D7S2427 deletion in family C116. The 12 microsatellites located between D7S691 and D7S1818 are shown. Marker D7S2427, showing a null allele in family C116, is underlined. Markers D7S478 and D7S621 are boxed. Affected individual genotypes in family C116 were heterozygous for those two markers. Overlapping BACs containing the region of interest are represented with schematic bars. B, CCM2 deletion refinement. MS004847 and MS004844 are indicated, as are the 42 SNP (“rs”) markers retrieved from annotations of genomic sequence contigs. Dots denote null alleles detected with SNP markers; circled dots denote null alleles detected with markers D7S2427 and MS004847; vertical bars denote heterozygosities at given markers. Analysis of all 30 families with markers MS004844 and MS004847 (underlined) identified an additional null allele in family C127. Family C116 and C127 deletions were overlapping in a 12–29-kb region; the region of overlap contains the first exon of MGC4607.

Figure 4

MGC4607 point mutations. A, MGC4607 genomic organization. The 10 MGC4607 exons are numbered and are indicated by vertical hatches. The ATG initiator codon is included in the first exon, and the TGA stop codon in the 10th (last) exon. The locations of the eight different MGC4607 point mutations are indicated by arrows. B, Genomic wild-type and mutated alleles in families harboring point mutations. Family numbers are given above the mutation. F = forward primers; R = reverse primers.

Figure 5

MGC4607 expression analysis through use of RT-PCR. The expression of MGC4607 transcripts was analyzed by RT-PCR through use of the cDNA MTC1 panel (Clontech). Upper panel, MGC4607. Lower panel, β-actin. Lane 1,100-bp ladder. Lane 2, Brain. Lane 3, Heart. Lane 4, Skeletal muscle. Lane 5, Kidney. Lane 6, Liver. Lane 7, Pancreas. Lane 8, Lung. Lane 9, Placenta. In all tested cDNAs, two populations of MGC4607 transcripts existed, although at a different level, containing exon 2 (fragment from exons 1–6: 755 bp) or not containing exon 2 (fragment without exon 2: 581 bp).

Figure 6

MGC4607 northern blot analysis. Human adult MTN (human, 12 lanes [Clontech]) was hybridized with an MGC4607 (upper panel) and β-actin (lower panel) human cDNA probe. Lane 1, Brain. Lane 2, Heart. Lane 3, Skeletal muscle. Lane 4, Colon. Lane 5, Thymus. Lane 6, Spleen. Lane 7, Kidney. Lane 8, Liver. Lane 9, Small intestine. Lane 10, Placenta. Lane 11, Lung. Lane 12, Peripheral blood leukocytes. Expression of ∼1.8-kb MGC4607 transcripts was detected in all tissues studied.