Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Abstract

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.

Figure 1

Genealogical trees of the 20 families with CCMs. Family numbers are indicated above each pedigree. Black symbols show affected individuals; question marks indicate unknown status; empty symbols depict healthy individuals with normal MRI results. Probands are designated by an arrow. Identified mutations have been indicated above the corresponding pedigrees.

Figure 2

Parental noncontribution within family C052 at the CCM3 locus. The genealogical tree for family C052, with CCM3 haplotypes, is shown. A null allele was detected at the D3S3673, D3S1264, and D3S3622 markers (boxed), which strongly suggested the existence of a deletion; this deletion was confirmed by additional genotyping (fig. 3).

Figure 3

A, Genetic map of the CCM3 locus. The two markers that bracketed the previously published CCM3 interval—D3S1763 and D3S1262—are indicated in bold. Some of the microsatellite markers used to screen families for putative null alleles are shown. B, CCM3 deletion in pedigree C052. Microsatellites used to identify and refine the deletion in the proband from family C052 are shown. Dots denote markers for which apparent non-Mendelian inheritance was observed in the C052 proband. Vertical bars denote heterozygosities at given markers. A de novo deletion that encompasses D3S1763 was detected in the C052 proband. The novel critical interval, now bracketed by D3S1763 and D3S1614, is blackened. C, Genes mapped within the novel CCM3 interval. The five genes identified within this interval are schematized as arrows.

Figure 4

PDCD10 point mutations. A, PDCD10 genomic organization. The 10 PDCD10 exons are numbered and are indicated by vertical hatches. The ATG initiator codon is included in the 4th exon, and the TGA stop codon is in the 10th (last) exon. The locations of the different PDCD10 point mutations are indicated by arrows. B, Mutated alleles in families harboring mutations. Family numbers are given above the mutation. Sequence chromatograms of genomic DNA are shown for all probands, except for the C043 proband, whose chromatogram is of cDNA. Families C030 and C077 harbored the same mutation; therefore, we show only the chromatogram obtained in the proband of pedigree C030. F = forward primer; R = reverse primer.

Figure 5

PDCD10 northern blot analysis. Human adult MTN (Human 12-lane MTN Blot [Clontech]) was hybridized with a PDCD10 (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; and lane 12, peripheral blood leukocytes.