Type IV procollagen missense mutations associated with defects of the eye, vascular stability, the brain, kidney function and embryonic or postnatal viability in the mouse, Mus musculus: an extension of the Col4a1 allelic series and the identification of the first two Col4a2 mutant alleles

Abstract

The basement membrane is important for proper tissue development, stability, and physiology. Major components of the basement membrane include laminins and type IV collagens. The type IV procollagens Col4a1 and Col4a2 form the heterotrimer [alpha1(IV)]2[alpha2(IV)], which is ubiquitously expressed in basement membranes during early developmental stages. We present the genetic, molecular, and phenotypic characterization of nine Col4a1 and three Col4a2 missense mutations recovered in random mutagenesis experiments in the mouse. Heterozygous carriers express defects in the eye, the brain, kidney function, vascular stability, and viability. Homozygotes do not survive beyond the second trimester. Ten mutations result in amino acid substitutions at nine conserved Gly sites within the collagenous domain, one mutation is in the carboxy-terminal noncollagenous domain, and one mutation is in the signal peptide sequence and is predicted to disrupt the signal peptide cleavage site. Patients with COL4A2 mutations have still not been identified. We suggest that the spontaneous intraorbital hemorrhages observed in the mouse are a clinically relevant phenotype with a relatively high predictive value to identify carriers of COL4A1 or COL4A2 mutations.

Figure 1.—

Slit lamp microscopy documenting variable eye phenotypes associated with Col4a1 and Col4a2 mutations. Eyes from 11-month-old mice are shown. (A) C3H wild type. (B) Col4a2^ENU4003 +/− expressing microphthalmia and total lens opacity. (C) Col4a2^ENU4003 +/− expressing buphthalmos. (D) Col4a2^ENU4020 +/− with total lens opacity, eye size normal. (E) Col4a1^ENU911 +/− with microphthalmia. (F) Contralateral eye to E expressing corneal opacity, hyperplasia, and neovascularization (arrowhead). (G) Col4a1^ENU4004 +/− with total lens opacity, eye size normal. (H) Col4a1^ENU4004 +/− with total lens opacity and intraorbital hemorrhage (arrowhead). All eyes are photographed at 20× magnification. Bar in B, 1 mm.

Figure 2.—

Histology and lens morphology documenting the variable eye phenotypes associated with Col4a1 and Col4a2 mutations. (A–C) Eyes of E14 embryos. (A) Wild type with well-developed cornea (cor), lens (le), and retina (ret); (B) Col4a2^ENU415 −/+ eye of normal size, thickened cornea, and reduced distance separating the cornea and the anterior surface of the lens (arrowhead); (C) Col4a2^ENU415 −/− eye expressing microphthalmia and the lumen of the lens has failed to fill with primary lens fibers (arrowhead). (D–F) Eyes of P21 Col4a2^ENU4020 −/+ mutant mice. (D) Area from the anterior surface of the lens showing disorganization of the lens epithelial layer (ep) and vacuoles in the underlying secondary fiber cell region (arrowhead). (E) Corneal-lens adhesion (arrowhead). (F) Preretinal blood vessel (arrowhead). (G–I) Eyes of P21 Col4a2^ENU4003 −/+ mutant mice. (G) Preretinal blood vessel (arrowhead) in an eye diagnosed to have intraorbital hemorrhage and red floaters. (H) Anterior section of the same eye as in G demonstrating a hernia in the corneal basement (arrowhead) and the anterior chamber is filled with red blood cells. (I) Higher magnification of the same eye as in G demonstrating the disrupted preretinal blood vessel (arrowhead) and a plume of red blood cells in the posterior chamber. (J–L) Lenses of P35 Col4a1 or Col4a2 −/+ mutant mice. (J) Total lens opacity in a Col4a2^ENU415 −/+ heterozygote. Eye size is normal and the lens is intact. (K) Lens of a Col4a1^F247 −/+ carrier is totally opaque and disrupted. Eye size is normal. (L) Lenses from both eyes of a Col4a1^D456 −/+ carrier. To the left is an intact and totally opaque lens from a normal-size eye. To the right is the lens from the contralateral eye of the same carrier expressing total opacity and microphthalmia. Bars in A–C, 100 μm. Bar in D, 20 μm. Bars in E–I, 50 μm.

Figure 3.—

Phenotypes in E14 or E15 embryos associated with the Col4a2^ENU415 mutation. (A) +/+ (left), −/+ (middle), and −/− (right) E15 embryos. The heterozygous mutant expresses microphthalmia and a small hemorrhage on the right hind paw. The homozygous mutant expresses microphthalmia and extensive hemorrhaging over the entire body. (B) E14 −/+ embryo expressing total cataract. Eye size is normal. (C) E14 −/+ embryo expressing microphthalmia and extensive hemorrhages over the entire body. (D) E15 −/+ embryo expressing microphthalmia and hemorrhaging in the eye and body. (E) E15 −/+ embryo with hemorrhaging in a normal-sized eye. (F) E15 −/+ embryo with a normal eye and hemorrhaging in the skin.

Figure 4.—

Brain histology in wild-type and heterozygous mutant Col4a1 or Col4a2 mouse embryos. (A) Coronal section of the dorsal telencephalon of an E15 +/+ embryo showing a well-developed arachnoid (ar), an intact and continuous lamina I (arrow) below the subarachnoid space, normal organization of the underlying laminae II–VI, and the lateral ventricular lumen (vl). (B) Dorsal telencephalon in an E17 Col4a2^ENU415 heterozygote. There are multiple porencephalic lesions (*), a floating blood vessel (arrowhead) within a porencephalic lesion, disruptions of lamina I, and focal adhesions of lamina I to the arachnoid (arrow) with attachments of neuroblasts. (C) Thalamus of an E17 Col4a2^ENU4003 heterozygote with extensive diapedesis of erythrocytes through the vascular wall (arrowhead). (D) Coronal section of the dorso-lateral telencephalon in an E17 Col4a2^ENU4003 heterozygote. There is a large pseudocyst (*) situated between lamina I and the underlying cerebral cortex, clustering of migrating neurons at the margins of the pseudocyst (arrowhead), and disruptions in lamina I with a focal adhesion to the arachnoid (arrow). (E) Coronal section of the thalamic region in an E16 Col4a1^D456 +/− embryo. There is a large hemorrhagic necrosis (*) at the lateral surface of the epithalamus. Nonpathological blood-filled meningeal vessels are shown (arrow). tha, thalamus; tel, telencephalon; vl III, third ventricular lumen. (F) Coronal section of the thalamic region in an E15 Col4a1^F247 +/− embryo. There is disruption in the ventricular layer extending laterally through the neural parenchyma (arrowhead). A–E, 200× magnification. F, 400× magnification. Bars in B and F, 50 μm.

Figure 5.—

Hematology and clinical chemistry analysis of plasma and urine of wild-type and heterozygous mutant Col4a1 or Col4a2 mice. Each bar represents the mean with ±SEM from an approximately equal number of male and female mice. WT represents the pooled wild-type littermates from all Col4a1 and Col4a2 mutant lines (129 animals). Col4a1 consists of the pooled heterozygous carriers of the procollagen Col4a1 mutant lines (115 animals). Col4a2 represents the pooled heterozygous carriers from the procollagen Col4a2 mutant lines (57 animals). (A) Red blood cell count, showing a slight but consistent reduction in heterozygous mutants as compared to wild type. (B) Hematocrit, showing a reduction in the mutants as compared to wild type. (C) Hemoglobin, showing a reduction in the heterozygous mutants as compared to wild type. (D) Total protein in urine, showing an increase in the mutant lines as compared to wild type. (E) Albumin in urine, with no differences among the genotypes. (F) Plasma urea, showing a slight but significant increase in the mutant lines as compared to wild type.

Figure 6.—

Predicted location of signal peptide cleavage sites in the mouse Col4a2 wild-type and Col4a2^ENU415 (Val31Phe) amino-terminal sequences. The sequence of the first 50 amino acids was submitted to SignalP 3.0 for evaluation. Red bars indicate the cleavage site scores for each amino acid position. (A) The wild-type sequence was predicted to have a signal peptide cleavage site between amino acid positions 33 and 34, with a cleavage site probability of 0.786. (B) Col4a2^ENU415 sequence. The Val31Phe amino acid substitution disrupts the predicted cleavage site between positions 33 and 34. Low cleavage-site scores were obtained between positions 28 and 29, 30 and 31, and 32 and 33, with the maximum cleavage site probability of 0.252 between positions 28 and 29.