Douglas B. Gould

The forkhead/winged helix gene Mf1 is disrupted in the pleiotropic mouse mutation congenital hydrocephalus.

Mf1 encodes a forkhead/winged helix transcription factor expressed in many embryonic tissues, including prechondrogenic mesenchyme, periocular mesenchyme, meninges, endothelial cells, and kidney. Homozygous null Mf1lacZ mice die at birth with hydrocephalus, eye defects, and multiple skeletal abnormalities identical to those of the classical mutant, congenital hydrocephalus. We show that congenital hydrocephalus involves a point mutation in Mf1, generating a truncated protein lacking the DNA-binding domain. Mesenchyme cells from Mf1lacZ embryos differentiate poorly into cartilage in micromass culture and do not respond to added BMP2 and TGFbeta1. The differentiation of arachnoid cells in the mutant meninges is also abnormal. The human Mf1 homolog FREAC3 is a candidate gene for ocular dysgenesis and glaucoma mapping to chromosome 6p25-pter, and deletions of this region are associated with multiple developmental disorders, including hydrocephaly and eye defects.

Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly

Background: Porencephaly (cystic cavities of the brain) is caused by perinatal vascular accidents from various causes. Several familial cases have been described and autosomal dominant inheritance linked to chromosome 13q has been suggested. COL4A1 is an essential component in basal membrane stability. Mouse mutants bearing an in-frame deletion of exon 40 of Col4a1 either die from haemorrhage in the perinatal period or have porencephaly in survivors. A report of inherited mutations in COL4A1 in two families has shown that familial porencephaly may have the same cause in humans. Objective: To describe three novel COL4A1 mutations. Results: The three mutations occurred in three unrelated Dutch families. There were two missense mutations of glycine residues predicted to result in abnormal collagen IV assembly, and one mutation predicted to abolish the traditional COL4A1 start codon. The last mutation was also present in an asymptomatic obligate carrier with white matter abnormalities on brain magnetic resonance imaging. Conclusions: This observation confirms COL4A1 as a major locus for genetic predisposition to perinatal cerebral haemorrhage and porencephaly and suggests variable expression of COL4A1 mutations.

COL4A1 and COL4A2 Mutations and Disease: Insights into Pathogenic Mechanisms and Potential Therapeutic Targets

Heterotrimers composed of collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) constitute one of the most abundant components of nearly all basement membranes. Accordingly, mutations in COL4A1 or COL4A2 are pleiotropic and contribute to a broad spectrum of disorders, including myopathy, glaucoma and hemorrhagic stroke. Here, we summarize the contributions of COL4A1 and COL4A2 mutations in human disease, integrate knowledge gained from model organisms and evaluate the implications for pathogenic mechanisms and therapeutic approaches.

Genetically Increasing Myoc Expression Supports a Necessary Pathologic Role of Abnormal Proteins in Glaucoma

ABSTRACT Despite the importance of MYOC for glaucoma, the proteins normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.

COL4A2 Mutations Impair COL4A1 and COL4A2 Secretion and Cause Hemorrhagic Stroke

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

COL4A1 Mutations Cause Ocular Dysgenesis, Neuronal Localization Defects, and Myopathy in Mice and Walker-Warburg Syndrome in Humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.

COL4A1 Mutations in Patients with Sporadic Late-Onset Intracerebral Hemorrhage

Mutations in the type IV collagen alpha 1 gene (COL4A1) cause dominantly inherited cerebrovascular disease. We seek to determine the extent to which COL4A1 mutations contribute to sporadic, nonfamilial, intracerebral hemorrhages (ICHs).

Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma.

ABSTRACT Glaucoma is a leading cause of blindness, affecting over 70 million people worldwide. Vision loss is the result of death of the retinal ganglion cells. The best-known risk factor for glaucoma is an elevated intraocular pressure (IOP); however, factors leading to IOP elevation are poorly understood. Mutations in the MYOC gene are an important cause of open-angle glaucoma. Over 70 MYOC mutations have been identified, and they lead to approximately 5% of all primary open-angle glaucoma cases. Nevertheless, the pathogenic mechanisms by which these mutations elevate IOP are presently unclear. Data suggest that a dominant interfering effect of misfolded mutant MYOC molecules may be pathogenic. To test this hypothesis, we have generated mice carrying a mutant allele of Myoc that is analogous to a human mutation that leads to aggressive glaucoma in patients. We show that mutant MYOC is not secreted into the aqueous humor. Instead of being secreted, mutant MYOC accumulates within the iridocorneal angle of the eye, consistent with the behavior of abnormally folded protein. Surprisingly, the accumulated mutant protein does not activate the unfolded protein response and lead to elevated intraocular pressure or glaucoma in aged mice of different strains. These data suggest that production, apparent misfolding, and nonsecretion of mutant MYOC are not, by themselves, sufficient to cause glaucoma in vivo.

Abnormal Expression of Collagen IV in Lens Activates Unfolded Protein Response Resulting in Cataract

Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. Using two different mouse models, we show that the accumulation of collagen chains in the secretory pathway activates the stress signaling pathway termed unfolded protein response (UPR). Transgenic mice expressing ectopic Col4a3 and Col4a4 genes in the lens exhibited activation of IRE1, ATF6, and PERK associated with expansion of the endoplasmic reticulum and attenuation of general protein translation. The expression of the transgenes had adverse effects on lens fiber cell differentiation and eventually induced cell death in a group of transgenic fiber cells. In Col4a1+/Δex40 mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results provide in vivo evidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum.

Clinical and brain MRI follow-up study of a family with COL4A1 mutation

Objective: To better delineate the clinical spectrum and the natural history of COL4A1 mutations, a newly defined genetic cause of small vessel disease including the brain and retina. Methods: Clinical and brain MRI follow-up study of a family with COL4A1 mutation. Results: During a 7-year period, two affected members died from intracranial hemorrhage. Four other members had a COL4A1 mutation (age ranges 25 to 74 years). None reported stroke or retinal hemorrhage or hematuria and none had dementia according to Diagnostic and Statistical Manual of Mental Disorders-IV criteria. Follow-up brain MRI showed grade 3 diffuse leukoencephalopathy in three out of four patients. All had dilated perivascular spaces and three out of four had silent microbleeds mainly in the deep white matter. MRI signal abnormalities did not change in severity, number, or location between baseline and follow-up imaging. Conclusions: COL4A1 mutation carriers have great diversity in the clinical expression of the disease within the same family. Some affected family members may remain asymptomatic during several years of follow-up and have no evidence of progression of vascular changes on brain MRI. GLOSSARY: FSE = T2 fast spin echo; GE = gradient echo; PVS = perivascular spaces; TE = echo time; TI = inversion time; TR = repetition time.