We report three new mutations in COL4A1 in three unrelated Dutch families with dominant porencephaly. Two of the mutations affect highly conserved glycines of Gly‐X‐Y repeats within the collagenous domain of the protein which is known to interact with COL4A2 to form a collagen IV triple helix.
A mouse model for perinatal cerebral haemorrhage and porencephaly shows an in‐frame deletion of exon 40 of Col4a1
. Mutations in conserved Gly‐X‐Y repeats of COL4A1
were subsequently found in two other families with autosomal dominant porencephaly.16
Immunohistochemistry and electronmicroscopy of Col4a1
mutants indicate impaired secretion of both COL4A1 and COL4A2. Similarly, a mutation of the C elegans
orthologue of Col4a1 (let‐2)
results in impaired secretion and intracellular accumulation of both let‐2
and the Col4a2
Mice homozygous for Col4a1Δex4016
or a null allele20
are not viable, and heterozygous null mice have no apparent phenotype.20
However, 50% of heterozygous Col4a1+/Δex40
mice die following parturition and 18% of the survivors show obvious porencephalic lesions. This suggests that synthesis of a mutant protein in Col4a1+/Δex40
mice has a negative effect on survival. Experimentally, a negative effect of Col4a1+/Δex40
mutation was demonstrated on collagen IV triple helix assembly and secretion.16
Mutations in highly conserved Gly‐X‐Y domains have been shown in several collagen proteins, leading to a dominant negative effect.25,26
Based on these observations, synthesis of an abnormal protein can be predicted in our families B and C.
The consequence of the mutation in family A is more difficult to predict. One possibility is that this is an effective null allele and a transcript is only produced from the normal allele. However, evidence from model organisms shows no obvious phenotype in heterozygotes for null alleles and suggests that dominant interfering proteins are necessary for pathogenesis. Thus a null mutation is not expected to be pathogenic and it is unlikely that the mutation in family A is a null allele. Following the conserved initiation codon, the next in‐frame start codon contains a pyrimidine at the ‐3 position, which is highly conserved with respect to translation initiation sites and suggests that this start site might be used.27
Initiation of translation at this second site would result in synthesis of a protein with a 64 amino acid N‐terminal truncation but with an intact NC1 domain and collagenous domain. We predict that the NC1 domain of the mutant trimer is able to initiate assembly of heterotrimers but that the heterotrimers would be structurally or functionally abnormal because of the N‐terminal truncation. Further insights into the effect of this mutation will result from the introduction of this start codon mutation in mice, or functional studies on the COL4A1/2 protein in patients from family A. Future in vitro expression studies of the mutant protein by our group are also aimed at confirming this assumption.
The gliotic lesions in our families indicate that the time of onset of porencephaly is related to hypoxic‐ischaemic events occurring in a late stage of pregnancy (after the 20th week).13
This is compatible with the specific function of COL4A1 in the formation of α1.α1.α2.(IV) protomer, which is expressed in early embryonic development (from the 32 to 64 cell stage of the mouse embryo) but is not essential for basement membrane deposition. Instead, its essential function involves the maintenance of the structural integrity of the basement membranes at later stages—that is, later fetal life.20
Its total ablation leads to embryonic lethality only at E10.5–11.5 because of impaired basement membrane stability.20
In this respect, localisation of the white matter lesions in areas draining from the vena terminalis in our families A and C and in the watershed area between the anterior and medial cerebral arteries in patient B‐II‐413
(table 2) are compatible with abnormalities of the vascular basement membrane as a result of COL4A1
Our findings also confirm that in asymptomatic carriers white matter lesions on MRI can be considered an expression of (and perhaps a risk factor for) COL4A1 mutation.
A history of strokes in middle age in patients A‐II‐3 and B‐II‐2 could not be correlated with COL4A1 abnormalities as these patients refused DNA tests. However, recurrent strokes in COL4A1
related porencephaly have also been observed by Gould et al
and additional patients need to be tested to prove this relation. Further evidence is needed to determine whether other neurological complaints in our families, such as the carotid artery aneurysm in patient A‐II‐2,13
recurrent migraine in family A, and mental retardation in family B, are also related to COL4A1
Porencephaly in our families occurs in areas where traumatic perinatal arterial bleeding (watershed areas)14,15
or venous thrombotic events also occur,13
and white matter lesions are seen in asymptomatic carriers of COL4A1
mutations. This suggests that trauma28,29
could be factors influencing the occurrence of cerebral bleeding in COL4A1
mutants. In this case genetic testing for COL4A1
mutations in families at risk could aid counselling or suggest the need for additional perinatal care to avoid a traumatic delivery.