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Mutations in the α1 (COL4A1) or α2 (COL4A2) chains of collagen type IV, a major component of the vascular basement membrane, cause intracerebral haemorrhages with variable expressivity and reduced penetrance by mechanisms that remain poorly understood. Here we sought to investigate the cellular mechanisms of COL4A1-related intracerebral haemorrhage and identify a marker for haemorrhage risk stratification. A combination of histological, immunohistochemical, and electron microscopy analyses were used to analyse the brain parenchyma, cerebrovasculature, and retinal vessels of mice expressing the disease-causing COL4A1 p.G498V mutation. Mutant mice developed cerebral microhaemorrhages and macroscopic haemorrhages (macrohaemorrhages), the latter with reduced penetrance, mimicking the human disease. Microhaemorrhages that occurred in early postnatal life were associated with a transient, generalized increase in blood-brain barrier permeability at the level of capillaries. Macrohaemorrhages, which occurred later in life, originated from deep brain arteries with focal loss of smooth muscle cells. Similar smooth muscle cell loss was detected in retinal arteries, and a time-course analysis of arterial lesions showed that smooth muscle cells are recruited normally in arterial wall during development, but undergo progressive apoptosis-mediated degeneration. By assessing in parallel the extent of these retinal arterial lesions and the presence/absence of macrohaemorrhages, we found that the arterial lesion load in the retina is strongly correlated with the burden of macrohaemorrhages. We conclude that microhaemorrhages and macrohaemorrhages are driven by two distinct mechanisms. Moreover, smooth muscle cell degeneration is a critical factor underlying the partial penetrance of COL4A1-related macrohaemorrhages, and retinal imaging is a promising tool for identifying high-risk patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The HANAC syndrome is caused by mutations in the gene coding for collagen4a1, a major component of blood vessel basement membranes. Ocular symptoms include an increase in blood vessel tortuosity and occasional hemorrhages. To examine how vascular defects can affect neuronal function, we analyzed the retinal phenotype of a HANAC mouse model. Heterozygous mutant mice displayed both a thinning of the basement membrane in retinal blood vessels and in Bruch's membrane resulting in vascular leakage. Homozygous mice had additional vascular changes, including greater vessel coverage and tortuosity. This greater tortuosity was associated to higher expression levels of vascular endothelial growth factor (VEGF). These major changes to the blood vessels were correlated with photoreceptor dysfunction and degeneration. The neuronal damage was associated with reactive gliosis in astrocytes and Müller glial cells, and by the migration of microglial cells into the outer retina. This study illustrates how vascular changes can trigger neuronal degeneration in a new model of HANAC syndrome that can be used to further study dysfunctions of neurovascular coupling., Summary Statement: This study provides a phenotypic analysis of a novel mouse model of HANAC syndrome focusing on the retinal aspect. It recapitulates most of the aspects of the human disease and is therefore a great tool to study and to address this condition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Collagen IV is a major component of basement membranes (BMs). The α1(IV) chain, encoded by the COL4A1 gene, is expressed ubiquitously and associates with the α2(IV) chain to form the α1α1α2(IV) heterotrimer. Several COL4A1 mutations affecting a conformational domain containing integrin-binding sites are responsible for the systemic syndrome of hereditary angiopathy, nephropathy, aneurysms, and cramps (HANAC). To analyze the pathophysiology of HANAC, Col4a1 mutant mice bearing the p.Gly498Val mutation were generated. Analysis of the skeletal muscles of Col4a1 G498V mutant animals showed morphologic characteristics of a muscular dystrophy phenotype with myofiber atrophy, centronucleation, focal inflammatory infiltrates, and fibrosis. Abnormal ultrastructural aspects of muscle BMs was associated with reduced extracellular secretion of the mutant α1α1α2(IV) trimer. In addition to muscular dystrophic features, endothelial cell defects of the muscle capillaries were observed, with intracytoplasmic accumulation of the mutant α1α1α2(IV) molecules, endoplasmic reticulum cisternae dilation, and up-regulation of endoplasmic reticulum stress markers. Induction of the unfolded protein response in Col4a1 mutant muscle tissue resulted in an excess of apoptosis in endothelial cells. HANAC mutant animals also presented with a muscular functional impairment and increased serum creatine kinase levels reflecting altered muscle fiber sarcolemma. This extensive description of the muscular phenotype of the Col4a1 HANAC murine model suggests a potential contribution of primary endothelial cell defects, together with muscle BM alterations, to the development of COL4A1-related myopathy., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman's capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44,α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman's capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders., (Copyright © 2016 by the American Society of Nephrology.)

The COL4A1 gene encodes the α1-chain of type IV collagen, which is ubiquitously expressed in basement membranes. Mutations in COL4A1 have been reported in autosomal-dominant porencephaly and in patients with symptomatic small vessel brain disease, inconstantly associated with eye defects. We have previously reported three COL4A1 mutations associated with a systemic phenotype that we called HANAC (Hereditary Angiopathy, Nephropathy, Aneurysms, and Cramps). We carried out a clinical and genetic study of three families presenting with characteristic features of HANAC syndrome. Common systemic signs included arterial retinal tortuosity and muscle cramps, with a variable combination of small vessel brain disease, Raynaud phenomena, and kidney defects. Three novel COL4A1 missense substitutions are described, which affect highly conserved glycine residues within the collagenous domain of the protein. All six known mutations associated with the HANAC phenotype are localized within the CB3[IV] fragment of COL4A1, which encompasses major integrin-binding sites. Our results confirm that HANAC syndrome is a distinct clinical entity within the COL4A1-related disorders, which is characterized by systemic involvement and usually asymptomatic brain disease. The restricted distribution of COL4A1 mutations within the CB3[IV] region is a characteristic of the reports of patients with HANAC, which suggests that abnormal cell-type IV collagen interactions may underlie the systemic defects observed in this syndrome., (Copyright © 2010 Wiley-Liss, Inc.)

Background: COL4A1 mutations cause familial porencephaly, infantile hemiplegia, cerebral small vessel disease (CSVD), and hemorrhagic stroke. We recently described hereditary angiopathy with nephropathy, aneurysm, and muscle cramps (HANAC) syndrome in 3 families with closely localized COL4A1 mutations. The aim of this study was to describe the cerebrovascular phenotype of HANAC., Methods: Detailed clinical data were collected in 14 affected subjects from the 3 families. MRI and magnetic resonance angiography (MRA) were performed in 9 of them. Skin biopsies were analyzed by electron microscopy in affected subjects in the 3 families., Results: Only 2 of 14 subjects had clinical cerebrovascular symptoms: a minor ischemic stroke at age 47 years and a small posttraumatic hemorrhage under anticoagulants at age 48 years. MRI-MRA showed cerebrovascular lesions in 8 of 9 studied subjects (mean age 39.4 years, 21-57 years), asymptomatic in 6 of them. Unique or multiple intracranial aneurysms, all on the carotid siphon, were observed in 5 patients. Seven patients had a CSVD characterized by white matter changes (7/7) affecting subcortical, periventricular, or pontine regions, dilated perivascular spaces (5/7), and lacunar infarcts (4/7). Infantile hemiplegia, major stroke, and porencephaly were not observed. Skin biopsies showed alterations of basement membranes at the dermoepidermal junction associated with expansion of extracellular matrix between smooth vascular cells in the arteriolar wall., Conclusion: The cerebrovascular phenotype in hereditary angiopathy with nephropathy, aneurysm, and muscle cramps syndrome associates a cerebral small vessel disease and a large vessel disease with aneurysms of the carotid siphon. It is consistent with a lower susceptibility to hemorrhagic stroke than in familial porencephaly, suggesting an important clinical heterogeneity in the phenotypic expression of disorders related to COL4A1 mutations.

Since 2005, several neurological diseases, including porencephaly, leukoencephalopathy and intracerebral hemorrhage without hypertension, and retinal arteriolar tortuosity have been linked to mutations in the COL4A1 gene, which encodes the alpha1 chain of type IV collagen, the main constituent of basement membranes. In three families, we observed a new syndrome that we called HANAC, for hereditary angiopathy with nephropathy, aneurysms and muscle cramps, which is associated with morphological alterations of cutaneous and renal basement membranes. This novel "basalopathy" is caused by glycine mutations in COL4A1 exons 24 and 25. We discuss phenotype-genotype correlations and the implications of the HANAC syndrome for the diagnosis of autosomal dominant hematuria, cystic kidney disease, intracranial aneurysms, and muscle cramps.

Background: COL4A3, COL4A4, and COL4A5 are the only collagen genes that have been implicated in inherited nephropathies in humans. However, the causative genes for a number of hereditary multicystic kidney diseases, myopathies with cramps, and heritable intracranial aneurysms remain unknown., Methods: We characterized the renal and extrarenal phenotypes of subjects from three families who had an autosomal dominant hereditary angiopathy with nephropathy, aneurysms, and muscle cramps (HANAC), which we propose is a syndrome. Linkage studies involving microsatellite markers flanking the COL4A1-COL4A2 locus were performed, followed by sequence analysis of COL4A1 complementary DNA extracted from skin-fibroblast specimens from the subjects., Results: We identified three closely located glycine mutations in exons 24 and 25 of the gene COL4A1, which encodes procollagen type IV alpha1. The clinical renal manifestations of the HANAC syndrome in these families include hematuria and bilateral, large cysts. Histologic analysis revealed complex basement-membrane defects in kidney and skin. The systemic angiopathy of the HANAC syndrome appears to affect both small vessels and large arteries., Conclusions: COL4A1 may be a candidate gene in unexplained familial syndromes with autosomal dominant hematuria, cystic kidney disease, intracranial aneurysms, and muscle cramps., (Copyright 2007 Massachusetts Medical Society.)

Background: Autosomal-dominant forms of hematuria have been mostly related to mutations in the COL4A3/COL4A4 genes. Patients with thin basement membrane (BM) disease do not have extrarenal manifestations, while those with Alport syndrome often present with hearing loss, anterior lenticonus, and dot-and-fleck retinopathy., Methods: We performed a phenotypic study and a candidate gene approach in a four-generation family presenting with autosomal-dominant hematuria associated with extrarenal manifestations. Renal biopsy was analyzed for determination of BM thickness and expression of chains of type IV collagen. Linkage to 18 candidate genes/loci was investigated using polymorphic microsatellite markers., Results: In all affected patients, hematuria without proteinuria was associated with muscular contractures and retinal arterial tortuosities responsible for retinal hemorrhages. Cardiac arrhythmia, Raynaud phenomena, and brain MRI abnormalities were also observed. Despite the presence of red cells in tubule sections, no glomerular abnormalities were found by electron microscopy. Expression of type IV collagen chains and glomerular BM thickness was normal. We searched for a molecular defect affecting either BM or angiogenesis. Linkage analyses of genes encoding BM components (COL4A3/COL4A4, COL6A1, COL6A2, COL6A3, FBLN1), and angiogenic factors or their receptors (VHL, ANPT1, ANPT2, TIE, TEK, NOTCH2, NOTCH3, NOTCH4, DLL4, JAG1, JAG2) and of the facio-sapulo-humeral dystrophy and 3q21 loci failed to show segregation of the disease with those gene loci., Conclusion: We have identified a new inherited hematuria syndrome associated with retinal vessel tortuosities and contractures. We recommend performing a fundus examination in patients with familial hematuria and episodes of visual impairment, as well as a urinary analysis in patients with retinal arterial tortuosity or congenital muscular contractures.

Background: Anti-glomerular basement membrane (GBM) nephritis is a rare disease induced by antibodies directed against alpha3(IV)NC1, the Goodpasture antigen. We report a patient with Crohn's disease who developed anti-GBM nephritis and the skin blistering disorder bullous pemphigoid, owing to distinct autoantibodies., Methods: Frozen sections of skin and kidney biopsies were incubated with antisera specific for human IgG, IgA, IgM, fibrin, and C3. Reactivity of the patient's serum with GBM antigens was studied by Western blot using bovine solubilized type IV collagen and by enzyme-linked immunosorbent assays using alpha1(IV), alpha3(IV), and alpha5(IV)NC1 recombinant proteins. Reactivity studies against skin antigens were done by Western blot using human keratinocyte and dermal extracts and three recombinant forms of the bullous pemphigoid antigen180 (BP180, also called BPAG2 or type XVII collagen). The patient's serum was affinity fractionated on a (IV)NC1 column, and the bound and unbound fractions were analyzed for their reactivity against GBM and skin antigens., Results: The patient had deposits of IgG along the GBM and the epidermal basement membrane zone. Circulating IgG antibodies against alpha3(IV)NC1 were detected. The patient's autoantibodies immunoblotted the intracellular domain but not the extracellular domain of BP180. Reactivity of the patient's IgG with BP180 was found only in the unbound fraction of the serum., Conclusion: The simultaneous development of a rare renal and skin autoimmune disorder, resulting from non-cross-reactive autoantibodies, suggests that a common triggering event could be responsible for the autoimmune injury., (Copyright 2002 by the National Kidney Foundation, Inc.)