HANAC Col4a1 Mutation in Mice Leads to Skeletal Muscle Alterations due to a Primary Vascular Defect.

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 Col4a1G498V 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.

Col4a1 mutation generates vascular abnormalities correlated with neuronal damage in a mouse model of HANAC syndrome.

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.

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease.

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.

Porencephaly in a fetus and HANAC in her father: variable expression of COL4A1 mutation.

COL4A1-associated disorders encompass a wide range of hereditary vasculopathy, including porencephaly and HANAC (adult-onset hemorrhagic stroke with cerebral aneurysm and retinal arterial tortuosity, renal cysts, and thenar muscle cramp). It remains elusive whether or not porencephaly and HANAC are molecularly distinctive disorders due to different classes of mutations. We report on a girl with porencephaly and an episode of microangiopathic hemolysis in infancy and her father with HANAC, both of whom had a heterozygous missense mutation of COL4A1 (c.3715G>A, p.G1239R). The current observation implies phenotypic diversities of COL4A1 mutations.

ADRA2A and IRX1 are putative risk genes for Raynaud's phenomenon.

Raynaud's phenomenon (RP) is a common vasospastic disorder that causes severe pain and ulcers, but despite its high reported heritability, no causal genes have been robustly identified. We conducted a genome-wide association study including 5,147 RP cases and 439,294 controls, based on diagnoses from electronic health records, and identified three unreported genomic regions associated with the risk of RP (p < 5 × 10-8). We prioritized ADRA2A (rs7090046, odds ratio (OR) per allele: 1.26; 95%-CI: 1.20-1.31; p < 9.6 × 10-27) and IRX1 (rs12653958, OR: 1.17; 95%-CI: 1.12-1.22, p < 4.8 × 10-13) as candidate causal genes through integration of gene expression in disease relevant tissues. We further identified a likely causal detrimental effect of low fasting glucose levels on RP risk (rG = -0.21; p-value = 2.3 × 10-3), and systematically highlighted drug repurposing opportunities, like the antidepressant mirtazapine. Our results provide the first robust evidence for a strong genetic contribution to RP and highlight a so far underrated role of α2A-adrenoreceptor signalling, encoded at ADRA2A, as a possible mechanism for hypersensitivity to catecholamine-induced vasospasms.

Primary biliary cirrhosis in a genetically homogeneous population: disease associations and familial occurrence rates.

BACKGROUND: Primary biliary cirrhosis (PBC) is a disease with genetic and environmental pathogenetic background. Chemicals, infectious agents, hormone therapy, reproductive history and surgical interventions have been implicated in the induction of PBC. Familial PBC has been documented in first degree relatives (FDR). Most cohort studies are genetically heterogeneous. Our study aimed to determine eventual lifestyle or disease associations and familial occurrence rates in a genetically homogeneous and geographically defined population of PBC patients. METHODS: 111 consenting PBC patients, were compared with 115 FDR and 149 controls matched for age, sex, Cretan origin and residence. All participants completed a questionnaire regarding demographics, lifestyle, medical, surgical and reproductive history. Significant variables on the univariate analysis were analyzed by multivariate analysis using a forward step-wise logistic regression model. RESULTS: Dyslipidaemia was found in 69.4% of patients, 60% of FDR and 40.9% of controls (p < 0.0001 and p = 0.003 respectively), autoimmune diseases in 36.9% of patients, 30.4% of FDR and 13.4% of controls (p < 0.0001 and p = 0.011 respectively). Hashimoto's disease (p = 0.003), Raynaud syndrome (p = 0.023) and Sjögren syndrome (p = 0.044) were significantly associated with PBC. On multivariate analysis statistically significant associations were found with primary educational level (AOR 2.304, 95% CI 1.024-5.181), cholecystectomy (AOR 2.927, 95% CI 1.347-6.362) and the presence of at least another autoimmune disease (AOR 3.318, 95% CI 1.177-6.22). Cancer history was more frequent in patients than in controls (p = 0.033). Familial PBC was found to be 9.9%. CONCLUSIONS: Dyslipidaemia and autoimmune diseases were significantly increased not only in patients as expected but also in their FDR. An increased prevalence of malignancies was found in patients. Primary educational level, cholecystectomy and the presence of at least another autoimmune disease were found as putative risk factors for PBC. No association was found with smoking, urinary tract infection or reproductive history. The reported high familial occurrence of PBC could imply screening with AMA of FDR with at least another autoimmune disease.

Heritability of vasculopathy, autoimmune disease, and fibrosis in systemic sclerosis: a population-based study.

OBJECTIVE: To investigate the familiality of systemic sclerosis (SSc) in relation to Raynaud's phenomenon (RP) (a marker of vasculopathy), other autoimmune inflammatory disease, and fibrotic interstitial lung disease (ILD). METHODS: A genealogic resource, the Utah Population Database (UPDB), was used to test heritability of RP, other autoimmune disease, and ILD. Diseases were defined by International Classification of Diseases, Ninth Revision codes and identified from statewide discharge data, the University of Utah Health Science Center Enterprise Data Warehouse, and death certificates and were linked to the UPDB for analysis. Familial standardized incidence ratio (FSIR), relative risks (RRs) to first-, second-, third-, and fourth-degree relatives for SSc, RP, other autoimmune disease, and ILD (with 95% confidence intervals [95% CIs]), and population attributable risk (PAR) were calculated. RESULTS: A software kinship analysis tool was used to analyze 1,037 unique SSc patients. Fifty SSc families had significant FSIRs, ranging from 2.07 to 17.60. The adjusted PAR was approximately 8%. The RRs were significant for other autoimmune disease in the first-degree relatives (2.49 [95% CI 1.99-3.41], P = 2.42 x 10(-15)) and second-degree relatives (1.48 [95% CI 1.34-2.39], P = 0.002), for RP in first-degree relatives (6.38 [95% CI 3.44-11.83], P = 4.04 x 10(-9)) and second-degree relatives (2.39 [95% CI 1.21-4.74], P = 0.012), and for ILD in first-degree relatives (1.53 [95% CI 1.04-2.26], P = 0.03), third-degree relatives (1.47 [95% CI 1.18-1.82], P = 0.0004), and fourth-degree relatives (1.2 [95% CI 1.06-1.35], P = 0.004). CONCLUSION: These data suggest that SSc pedigrees include more RP, autoimmune inflammatory disease, and ILD than would be expected by chance. In SSc pedigrees, genetic predisposition to vasculopathy is the most frequent risk among first-degree relatives.

Novel COL4A1 mutations associated with HANAC syndrome: a role for the triple helical CB3[IV] domain.

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.

Manganese superoxide dismutase Ala-9Val mitochondrial targeting sequence polymorphism in systemic lupus erythematosus in Poland.

Systemic lupus erythematosus (SLE) is a chronic and progressive autoimmune disease in which reactive oxygen species contribute to pathogenesis. We analysed the distribution of manganese superoxide dismutase (MnSOD2) 47C>T (Ala-9Val) functional polymorphic variants within the mitochondrial targeting sequence in SLE patients (n = 102) and controls (n = 199). We did not find significant differences in the distribution of MnSOD2 47C>T polymorphic variants in SLE patients and controls. However, we found that MnSOD2 Val/Val genotype (recessive model) showed a significant association with Raynaud's phenomenon, odds ratio (OR) = 12.000 [95% confidence interval (CI) = 2.315-62.193], p = 0.0015. We also found that the MnSOD2 Val/Val genotype contributes to immunologic manifestations, OR = 2.957 (95% CI = 1.207-7.243), p = 0.0222, and anti-dsDNA antibody presence OR = 3.365 (95% CI = 1.364-8.304), p = 0.0107, in patients. Our observations indicate that MnSOD2 Val/Val variant can be linked to some clinical manifestations in patients with SLE.

Association of Raynaud's phenomenon with a polymorphism in the NOS1 gene.

BACKGROUND: Raynaud's phenomenon (RP) describes the phenomenon of recurrent vasospasm of digital arteries, associated with skin colour changes: pallor, cyanosis and erythema. Twin studies have indicated a genetic predisposition for RP; however, the precise aetiology of RP remains unknown. It is thought that genetic variation in temperature-responsive or vasospastic genes might underlie RP so performed a candidate gene study in a large, population based sample. We assessed the association between RP and single nucleotide polymorphisms (SNPs) in the TRPA1, TRPM8, CALCA, CALCB and NOS1 genes. METHODS: Analysis included a total of 4276 individuals from the TwinsUK database. RP status had been determined using validated, self-administered questionnaires and was diagnosed in 640 individuals (17.6%). 66 tag SNPs across the candidate genes were tested for association with RP status using a linear regression model, accounting for covariates. Adjustment was made for multiple testing. RegulomeDB and GTEx databases were used to assess possible functional effects of the polymorphisms. RESULTS: Nominally significant associations between RP and four SNPs in NOS1 and one in CALCB were identified. After permutation testing, rs527590 SNP in NOS1 passed the significance threshold. RegulomeDB scores indicated an unlikely functional effect of this variant, while the survey of the GTEx database found the SNP and several variants in linkage disequilibrium to be cis-eQTLs in skin. CONCLUSION: Results indicate that RP is associated with variation in gene NOS1. This finding may be related to the observation that the significant SNP in NOS1 is known to exhibit functional influence on the gene expression.

Angiotensin-converting enzyme gene polymorphism in Kuwaiti patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the frequency of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism genotypes in patients with systemic lupus erythematosus (SLE), and to study the correlation between I/D polymorphism of the ACE gene and the clinical manifestations of SLE, especially vascular involvement, lupus nephritis and disease severity. METHODS: The frequency of ACE gene I/D polymorphism genotypes was determined in 92 patients with SLE from Kuwait, and compared to that in 100 ethnically matched healthy controls using the polymerase chain reaction. RESULTS: The distribution of ACE I/D polymorphism and allele frequencies in SLE patients was not significantly different from controls. Further analyses of SLE patients showed that there was a significant association between DD genotype and Raynaud's phenomenon (p=0.008, odd ratio=5.4, 95% confidence interval: 1.6-18.6). However, there was no significant association between the ACE genotype and lupus nephritis or disease severity. CONCLUSION: No difference was found between the distribution of the ACE genotype in SLE patients and the general pop-ulation in Kuwait. However, the presence of the DD genotype may confer susceptibility to the development of vascular morbidity.

[Heredity and genetic aspects of Raynaud's disease]. / Aspects héréditaires et génétiques de la maladie de Raynaud.

The pathophysiology of primary Raynaud's phenomenon (Raynaud's disease) remains uncertain but the transmission of this primary microcirculatory dysregulation seems strongly influenced by genetic factors. For a long time, physicians have found that the hereditary factor plays an important role in the genesis of Raynaud's disease. Familial analysis and twin studies have confirmed the role of an hereditary factor. It seems heterogeneous but pedigree analysis indicates the possibility of an autosomal dominant transmission influenced by sex, in some families, allowing an approach called "reverse genetic" based on linkage analysis. Such an approach has focused on few loci but sequencing of candidate genes for genetic mutations remains negative. Given the supposed heterogeneity of the genetic transmission of Raynaud's disease, diversification of strategies in molecular genetics is suitable with reference to techniques applied to multifactorial heredity.

HTR1B gene variants associate with the susceptibility of Raynauds' phenomenon in workers exposed hand-arm vibration.

OBJECTIVE: To explore whether polymorphic variants of the HTR1B gene are associated with the susceptibility of Raynauds' Phenomenon (RP) coursed by vibration. METHODS: 148 subjects exposed to vibration for more than 2 years were classified into either induced white finger (VWF) group (n = 72), or non-VWF group (n = 76). Vibration exposure levels were measured and assessed following ISO 5349-1:2001 protocol. All workers were genotyped by sequencing for the single nucleotide polymorphisms (SNPs) in the 5'-flanking and coding region of HTR1B. Genetic characteristics and linkage disequilibrium (LD) were analyzed with Haploview. Serum serotonin levels of each subject were detected using ELISA. The association between the susceptibility of vascular damage and genotype was analyzed via logistic regression. RESULTS: 7 known SNPs were obtained and their allele frequencies were inserted into the Hardy-Weinberg equilibrium. rs6297 variant genotype had an increased risk of VWF compared with wild genotype (OR = 2.14, 95% CI = 1.04- 4.58, P < 0.05). rs6298 mutant type (AG+GG) was found to have a significant interaction on vibration exposure LN(CEI), accounting for VWF occurrence. LN(5-HT) level is significantly different between the VWF group (x¯±s= 1.99±1.09 ng/mL) and the non-VWF group (x¯±s= 2.72±1.47 ng/mL). CONCLUSIONS: Serotonin levels may affect the progression of secondary RP. Polymorphic variants of the HTR1B gene are associated with the susceptibility of secondary RP in vibration-exposed occupational populations of Chinese Han people.

Familial clustering of Leiomyomatosis peritonealis disseminata: an unknown genetic syndrome?

BACKGROUND: Leiomyomatosis peritonealis disseminata (LPD) is defined as the occurrence of multiple tumorous intraabdominal lesions, which are myomatous nodules. LPD is a rare disease with only about 100 cases reported. The usual course of LPD is benign with the majority of the patients being premenopausal females. Only two cases involving men have been reported, no syndrome or familial occurrence of LPD has been described. CASE PRESENTATION: We describe a Caucasian-American family in which six members (three men) are diagnosed with Leiomyomatosis peritonealis disseminata (LPD) and three deceased family members most likely had LPD (based on the autopsy reports). Furthermore we describe the association of LPD with Raynaud's syndrome and Prurigo nodularis. CONCLUSION: Familial clustering of Leiomyomatosis peritonealis disseminata (LPD) has not been reported so far. The etiology of LPD is unknown and no mode of inheritance is known. We discuss possible modes of inheritance in the presented case, taking into account the possibility of a genetic syndrome. Given the similarity to other genetic syndromes with leiomyomatosis and skin alterations, we describe possible similar genetic pathomechanisms.

Case of H syndrome with massive skin involvement, retroperitoneal fibrosis and Raynaud's phenomenon with a novel mutation in the SLC29A3 gene.

We describe a case of H syndrome with massive skin involvement, retroperitoneal fibrosis and Raynaud's phenomenon. A 48-year-old man with parents of a consanguineous marriage, first appeared with decreased urine output, skin sclerosis on his inner thighs and short stature (142 cm, 47 kg). The patient had suffered from hearing loss since the age of 1 year, and his secondary sexual characteristics had not developed. Computed tomography showed periaortic fibrosis, bilateral ureteral stenosis, hydronephrosis and sclerosis of the germinal cords. A biopsy from the retroperitoneal mass revealed remarkable fibrosis with chronic inflammatory cells. Biopsies from the skin lesion showed thick collagen bundles through the dermis and lymphohistiocytic infiltration with numerous plasma cells. Serum inflammatory markers, such as C-reactive protein, vascular endothelial factor, transforming growth factor-ß and soluble interleukin-2 receptor, were elevated. Prednisolone was effective in treating skin lesions and in lowering serum inflammatory markers. After a long period of follow up, genomic DNA of the patient was obtained, and we identified a homozygous mutation in exon 5, c.625G>A, which caused transition of glycine to arginine, p.Gly208Arg, in the patient, but not in DNA samples from another 50 healthy individuals. This is the first case of H syndrome with Raynaud's phenomenon and retroperitoneal fibrosis, and the first Japanese case of H syndrome reported in the English published work with a novel mutation in the SLC29A3 gene.

A common genetic factor underlies hypertension and other cardiovascular disorders.

BACKGROUND: Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm. METHODS: We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis. RESULTS: Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions. CONCLUSIONS: We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm.