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1.  Allelic heterogeneity contributes to variability in ocular dysgenesis, myopathy and brain malformations caused by Col4a1 and Col4a2 mutations 
Human Molecular Genetics  2013;23(7):1709-1722.
Collagen type IV alpha 1 and 2 (COL4A1 and COL4A2) are present in nearly all basement membranes. COL4A1 and COL4A2 mutations are pleiotropic, affecting multiple organ systems to differing degrees, and both genetic-context and environmental factors influence this variable expressivity. Here, we report important phenotypic and molecular differences in an allelic series of Col4a1 and Col4a2 mutant mice that are on a uniform genetic background. We evaluated three organs commonly affected by COL4A1 and COL4A2 mutations and discovered allelic heterogeneity in the penetrance and severity of ocular dysgenesis, myopathy and brain malformations. Similarly, we show allelic heterogeneity in COL4A1 and COL4A2 biosynthesis. While most mutations that we examined caused increased intracellular and decreased extracellular COL4A1 and COL4A2, we identified three mutations with distinct biosynthetic signatures. Reduced temperature or presence of 4-phenylbutyrate ameliorated biosynthetic defects in primary cell lines derived from mutant mice. Together, our data demonstrate the effects and clinical implications of allelic heterogeneity in Col4a1- and Col4a2-related diseases. Understanding allelic differences will be valuable for increasing prognostic accuracy and for the development of therapeutic interventions that consider the nature of the molecular cause in patients with COL4A1 and COL4A2 mutations.
doi:10.1093/hmg/ddt560
PMCID: PMC3943517  PMID: 24203695
2.  COL4A1 mutations in patients with sporadic late-onset intracerebral hemorrhage 
Annals of Neurology  2012;71(4):470-477.
Objective
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, non-familial, intracerebral hemorrhages (ICHs).
Methods
We sequenced COL4A1 in 96 patients with sporadic ICH. The presence of putative mutations was tested in 145 ICH–free controls. The effects of rare coding variants on COL4A1 biosynthesis were compared to previously validated mutations that cause porencephaly, small vessel disease and HANAC syndrome.
Results
We identified two rare non–synonymous variants in ICH patients that were not detected in controls, two rare non–synonymous variants in controls that were not detected in patients and two common non–synonymous variants that were detected in patients and controls. No variant found in controls affected COL4A1 biosynthesis. Both variants (COL4A1P352L and COL4A1R538G) found only in patients changed conserved amino acids and impaired COL4A1 secretion much like mutations that cause familial cerebrovascular disease.
Interpretation
This is the first assessment of the broader role for COL4A1 mutations in the etiology of ICH beyond a contribution to rare and severe familial cases and the first functional evaluation of the biosynthetic consequences of an allelic series of COL4A1 mutations that cause cerebrovascular disease. We identified two putative mutations in 96 patients with sporadic ICH and show that these and other previously validated mutations inhibit secretion of COL4A1. Our data support the hypothesis that increased intracellular accumulation of COL4A1, decreased extracellular COL4A1, or both, contribute to sporadic cerebrovascular disease and ICH.
doi:10.1002/ana.22682
PMCID: PMC3335762  PMID: 22522439
3.  COL4A1 and COL4A2 mutations and disease: insights into pathogenic mechanisms and potential therapeutic targets 
Human Molecular Genetics  2012;21(R1):R97-R110.
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.
doi:10.1093/hmg/dds346
PMCID: PMC3459649  PMID: 22914737
4.  Endoplasmic Reticulum Stress as a Primary Pathogenic Mechanism Leading to Age-Related Macular Degeneration 
Age-related macular degeneration (AMD) is a multi-factorial disease and a leading cause of blindness. Proteomic and genetic data suggest that activation or de-repression of the alternate complement cascade of innate immunity is involved in end-stage disease. Several lines of evidence suggest that production of reactive oxygen species and chronic oxidative stress lead to protein and lipid modifications that initiate the complement cascade. Understanding the triggers of these pathogenic pathways and the site of the primary insult will be important for development of targeted therapeutics. Endoplasmic reticulum (ER) stress from misfolded mutant proteins and other sources are an important potential tributary mechanism. We propose that misfolded-protein-induced ER stress in the retinal-pigmented epithelium and/or choroid could lead to chronic oxidative stress, complement deregulation and AMD. Small molecules targeted to ER stress and oxidative stress could allow for a shift from disease treatment to disease prevention.
doi:10.1007/978-1-4419-1399-9_46
PMCID: PMC3068206  PMID: 20238041
5.  Mutant Myocilin Nonsecretion In Vivo Is Not Sufficient To Cause Glaucoma▿  
Molecular and Cellular Biology  2006;26(22):8427-8436.
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.
doi:10.1128/MCB.01127-06
PMCID: PMC1636791  PMID: 16954374
6.  Genetically Increasing Myoc Expression Supports a Necessary Pathologic Role of Abnormal Proteins in Glaucoma 
Molecular and Cellular Biology  2004;24(20):9019-9025.
Despite the importance of MYOC for glaucoma, the protein's 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.
doi:10.1128/MCB.24.20.9019-9025.2004
PMCID: PMC517885  PMID: 15456875
7.  Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: Relevance to ocular dysgenesis and hearing impairment 
BMC Medical Genetics  2004;5:17.
Background
Thirty-nine patients have been described with deletions involving chromosome 6p25. However, relatively few of these deletions have had molecular characterization. Common phenotypes of 6p25 deletion syndrome patients include hydrocephalus, hearing loss, and ocular, craniofacial, skeletal, cardiac, and renal malformations. Molecular characterization of deletions can identify genes that are responsible for these phenotypes.
Methods
We report the clinical phenotype of seven patients with terminal deletions of chromosome 6p25 and compare them to previously reported patients. Molecular characterization of the deletions was performed using polymorphic marker analysis to determine the extents of the deletions in these seven 6p25 deletion syndrome patients.
Results
Our results, and previous data, show that ocular dysgenesis and hearing impairment are the two most highly penetrant phenotypes of the 6p25 deletion syndrome. While deletion of the forkhead box C1 gene (FOXC1) probably underlies the ocular dysgenesis, no gene in this region is known to be involved in hearing impairment.
Conclusions
Ocular dysgenesis and hearing impairment are the two most common phenotypes of 6p25 deletion syndrome. We conclude that a locus for dominant hearing loss is present at 6p25 and that this locus is restricted to a region distal to D6S1617. Molecular characterization of more 6p25 deletion patients will aid in refinement of this locus and the identification of a gene involved in dominant hearing loss.
doi:10.1186/1471-2350-5-17
PMCID: PMC455682  PMID: 15219231
8.  COL4A2 mutation associated with familial porencephaly and small-vessel disease 
Familial porencephaly, leukoencephalopathy and small-vessel disease belong to the spectrum of disorders ascribed to dominant mutations in the gene encoding for type IV collagen alpha-1 (COL4A1). Mice harbouring mutations in either Col4a1 or Col4a2 suffer from porencephaly, hydrocephalus, cerebral and ocular bleeding and developmental defects. We observed porencephaly and white matter lesions in members from two families that lack COL4A1 mutations. We hypothesized that COL4A2 mutations confer genetic predisposition to porencephaly, therefore we sequenced COL4A2 in the family members and characterized clinical, neuroradiological and biochemical phenotypes. Genomic sequencing of COL4A2 identified the heterozygous missense G1389R in exon 44 in one family and the c.3206delC change in exon 34 leading to frame shift and premature stop, in the second family. Fragmentation and duplication of epidermal basement membranes were observed by electron microscopy in a c.3206delC patient skin biopsy, consistent with abnormal collagen IV network. Collagen chain accumulation and endoplasmic reticulum (ER) stress have been proposed as cellular mechanism in COL4A1 mutations. In COL4A2 3206delC fibroblasts we detected increased rates of apoptosis and no signs of ER stress. Mutation phenotypes varied, including porencephaly, white matter lesions, cerebellar and optic nerve hypoplasia and unruptured carotid aneurysm. In the second family however, we found evidence for additional factors contributing to the phenotype. We conclude that dominant COL4A2 mutations are a novel major risk factor for familial cerebrovascular disease, including porencephaly and small-vessel disease with reduced penetrance and variable phenotype, which might also be modified by other contributing factors.
doi:10.1038/ejhg.2012.20
PMCID: PMC3400734  PMID: 22333902
COL4A2; collagen-IV; porencephaly; small-vessel disease
9.  COL4A1 Mutations Cause Ocular Dysgenesis, Neuronal Localization Defects, and Myopathy in Mice and Walker-Warburg Syndrome in Humans 
PLoS Genetics  2011;7(5):e1002062.
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.
Author Summary
Muscle-eye-brain disease (MEB) and Walker-Warburg Syndrome (WWS) are devastating childhood diseases that belong to a subgroup of congenital muscular dystrophies (CMDs) characterized by ocular dysgenesis, neuronal migration defects, and congenital myopathy. Genetic studies have revealed a number of genes involved in the etiology of CMDs, and subsequent studies show that alterations in dystroglycan glycosylation underlie MEB/WWS. However, over half of MEB/WWS patients do not have mutations in known genes encoding glycosyltransferases, suggesting that other genes are involved. Here, we describe a novel and genetically complex mouse model for MEB/WWS and identify putative heterozygous mutations in COL4A1 in two MEB/WWS patients. We identify a novel gene implicated in the etiology of MEB/WWS, provide evidence of mechanistic heterogeneity for this subgroup of congenital muscular dystrophies, and develop an assay to test the functional significance of putative COL4A1 mutations. Our findings represent the first evidence for a dominant mutation leading to MEB/WWS–like diseases and expand the spectrum of clinical disorders resulting from Col4a1/COL4A1 mutations.
doi:10.1371/journal.pgen.1002062
PMCID: PMC3098190  PMID: 21625620
10.  A Male with Unilateral Microphthalmia Reveals a Role for TMX3 in Eye Development 
PLoS ONE  2010;5(5):e10565.
Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.
doi:10.1371/journal.pone.0010565
PMCID: PMC2868029  PMID: 20485507

Results 1-10 (10)