Hereditary Angiopathy Research Articles

Hereditary Angiopathy with Nephropathy, Aneurysms, and Muscle Cramp (HANAC) Syndrome: There Is More to Explore!

Hereditary Angiopathy with Nephropathy, Aneurysms, and Muscle Cramp (HANAC) Syndrome: There Is More to Explore!

COL4A1 Mutation: Hereditary Angiopathy with Nephropathy, Aneurysms, and Muscle Cramps (HANAC) Syndrome, an ADPKD Mimicker

COL4A1 Mutation: Hereditary Angiopathy with Nephropathy, Aneurysms, and Muscle Cramps (HANAC) Syndrome, an ADPKD Mimicker

#4440 PREVALENCE OF GENES INVOLVED IN HEREDITARY KIDNEY DISEASE AS A TOOL FOR THE DEVELOPMENT OF A GENE PANEL

Abstract Background and Aims Genetic study is the fundamental tool for the diagnosis of Hereditary Kidney Diseases (HKD). The proliferation of its use has led to an increase in diagnostic complexity due to phenotypic overlap and genotypic heterogeneity. The aim of this study is to evaluate the prevalence of genes in our series of adult patients with suspected monogenic kidney disease in order to develop the basis for the elaboration of a gene panel that will make its usefulness in our routine clinical practice profitable. Method A retrospective descriptive observational study was performed with the collection of all genetic studies that were positive in the period between October 2014 and December 2020 in our center. With a description of the causative genes involved, as well as the clinical data of the proband patients who turned out to be carriers of these variants. Descriptive results of continuous variables are expressed as mean and standard deviation (SD) or median and interquartile range (IQR) according to their distribution. For categorical data, frequency and percentage are reported. Results The results of a total of 77 genetic studies on probands were collected, of whom 51% were women with a mean age at diagnosis of 46 years (SD ± 13.4) and 64.9% had a family history of kidney disease. Renal function measured by mean estimated glomerular filtration rate (eGFR) using the CKD-EPI (CKD Epidemiology Collaboration) was 90.1 ml/min/1,73 m2 (SD ± 29.64) with a mean proteinuria quantified in 24-hour urine of 627.9 mg (SD ±43.7) and 7.7% were on renal replacement therapy. (Table 1) 40.25% of probands were carriers of a mutation for Alport syndrome, in this group the most frequent gene was COL4A3 in 50% of cases. The second most frequent entity was autosomal dominant polycystic kidney disease (ADPKD) with 37% of the studies and among these the great majority were carriers of a variant in the PKD1 gene with 96.5%. The remaining diagnostic entities in order of frequency were Fabry disease, autosomal dominant tubulointerstitial nephropathy (ADTKD), congenital anomalies of the kidney and urinary tract (CAKUT), nephrotic syndrome, hereditary angiopathy with nephropathy, aneurysm and cramps syndrome (HANAC) and tuberous sclerosis (Figure 1). Conclusion Alport syndrome was the most frequently identified entity in patients with suspected monogenic renal disease in whom the genetic study was performed. Based on the results of our series, a proposal has been made for the genes to be included in a panel for hereditary renal diseases in our center. The development of these panels increases the efficiency and increases the cost-effectiveness of genetic studies in the diagnosis of these patients.

A clinical case of CADASIL syndrome in a patient after a new coronavirus infection COVID-19

CADASIL-cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy is the most common form of hereditary cerebral angiopathy and is characterized by recurrent subcortical ischemic strokes, migraine with aura, and cognitive impairment. The article presents clinical case of the female patient with newly diagnosed CADASIL syndrome, who was admitted to the clinic for a course of rehabilitation treatment three months after the onset of an acute cerebrovascular accident caused by a combination of congenital and infectious angiopathy. The combination of CADASIL syndrome and coronavirus infection COVID-19 caused by the SARS-CoV-2 manifested by an acute cerebrovascular accident and the occurrence of focal neurological signs. There was a positive trend in the form of a partial regression of neurological signs against the background of repeated courses of rehabilitation treatment, including physical exercises, mechanotherapy, physiotherapy and acupuncture.

116 Hereditary Angiopathy with Nephropathy, Aneurysms and Muscle Cramps (HANAC) Syndrome in a Patient with Polycystic Kidney Disease

116 Hereditary Angiopathy with Nephropathy, Aneurysms and Muscle Cramps (HANAC) Syndrome in a Patient with Polycystic Kidney Disease

Structural and functional analysis of retinal vasculature in HANAC syndrome with a novel intronic COL4A1 mutation.

Mutations of the COL4A1 gene, a major structural protein of vessels, may cause hereditary angiopathy with nephropathy, aneurysms and muscle cramps (HANAC) syndrome. The vascular structure and function of patients with HANAC is poorly known. Here, we report a family with HANAC syndrome associated to a previously unreported mutation in COL4A1. The structure and function of retinal vessels were detailed by adaptive optics ophthalmoscopy (AOO) and optical coherence tomography (OCT) angiography. Clinical data from six affected individuals (43 to 72years old) from a single family comprising two generations were collected. Imaging charts including conventional fundus imaging, OCT-angiography and AOO in static and dynamic (flicker) mode were reviewed. DNA sequencing was done in the proband. DNA sequencing of the proband revealed a heterozygous deletion of COL4A1 (NM_001845) at position 1120 in the intron 20 resulting in the loss of splicing donor site for exon 20 (c.1120+2_1120+8del heterozygote). Four patients had arterial hypertension, and three had kidney dysfunction, one of which under dialysis. By fundus examination, five had typical retinal arteriolar tortuosity with arteriolar loops. Wall-to-lumen ratio of arteries was within normal limits, that is, lower than expected for hypertensive patients. Several foci of arteriolar irregularities were noted in the two oldest patients. In three affected subjects, evaluation of the neurovascular coupling showed a higher flicker-induced vasodilation than a control population (6% to 11%; n<5%). Structural and dynamic analysis of retinal vessels in a HANAC family bearing a previously unreported intronic COL4 mutation was done. In addition to arteriolar tortuosity, we found reduced wall-to-lumen ratio, arteriolar irregularity and increased vasodilatory response to flicker light. These abnormalities were more marked in the oldest subjects. This abnormal flicker response affected also non-tortuous arteries, suggesting that microvascular dysfunction extends beyond tortuosity. Such explorations may help to better vascular dysfunction related to HANAC and hence better understand the mechanisms of end-organ damage.

AN INHERITABLE STROKE DISORDER PRESENTING WITH UNIPOLAR PSYCHOTIC DEPRESSION: CASE REPORT OF A RARE ENTITY

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant rare hereditary angiopathy that causes stroke in young patients. Most frequent clinical presentations include ischemic strokes, recurrent transient ischaemic attacks and migraine with aura. Psychiatric manifestations are conspicuously rare and especially if present as the only presenting manifestation. Here is a case of a female patient in her fties who was diagnosed with major depressive disorder with psychotic features and was on medication for the same for one and a half years with no improvement in symptoms. She was subsequently admitted due to an increased tendency to self-harm and on day 6 of admission had an episode of seizures, which led us to look for organic causes. After a battery of tests and ruling out various differentials, she underwent an MRI which revealed T2 hyperintense signal in bilateral frontal, temporal, parieto-occipetal, and periventricular white matter. Based on MRI ndings, family history of her father passing away due to a stroke at age 68 and poor response to antidepressants, she was suspected to have CADASIL. She underwent genetic testing which conrmed the same. The clinical course and distinct facts of the case are divulged below.

Structural and functional exploration of the retinal vasculature in HANAC syndrome associated with a novel intronic COL4A1 mutation

AbstractPurpose: Mutations of the COL4A1 gene may cause hereditary angiopathy with nephropathy, aneurysms and muscle cramps (HANAC) syndrome. The vascular structure and function of patients with HANAC is poorly known. Here, we report a family with HANAC syndrome associated to a previously unreported mutation in COL4A1. The structure and function of retinal vessels were detailed by adaptive optics ophthalmoscopy (AOO) and optical coherence tomography (OCT) angiography.Methods: Clinical data from six affected individuals from a single family over two generations were collected. Imaging charts including conventional fundus imaging, OCT‐angiography and AOO in static and dynamic (flicker) mode were reviewed. DNA sequencing was done in the proband.Results: DNA sequencing of the proband revealed a heterozygous deletion of COL4A1 (NM_001845) at position 1120 in the intron 20 resulting in the loss of splicing donor site for exon 20 (c.1120 + 2_1120 + 8del heterozygote). Four had arterial hypertension, and three had kidney dysfunction, one of which under dialysis. Examination of retinal vessels showed that all patients had retinal arteriolar tortuosity which peaked in precapillary arterioles but spared capillaries and veins. Wall‐to‐lumen ratio of arteries was within normal limits, that is, lower than expected for hypertensive patients. Several foci of arteriolar irregularities were noted I the oldest patients. In three affected subjects, evaluation of the neurovascular coupling showed a high flicker‐induced vasodilation (from 6% to 10.8%; n &lt; 5%).Conclusions: Structural and dynamic analysis of retinal vessels in a HANAC family bearing a novel intronic COL4 mutation showed reduced parietal thickness, arteriolar irregularity and increased dilatory response to flicker light. This affected as well non tortuous arteries, suggesting that HANAC causes diffuse microvascular dysfunction. Our findings may help to better understand end‐organ damage related to HANAC.

Atypical Macular Presentation in a FRAT Case and HANAC Syndrome, A Case Report

Purpose: Report an atypical Familial Retinal Arterial Tortuosity (FRAT) case associated with Hereditary Angiopathy with Nephropathy, Aneurysm and Cramps (HANAC). Methods: The authors report the case of a female patient with FRAT and HANAC, and an asymmetric ocular presentation which is unusual in Familial Retinal Arterial Tortuosity patients. Conclusion: These findings help in better understanding this rare disease. HANAC Syndrome is not a common issue; thus, it is essential to understand it to make the right diagnosis. Careful ophthalmological examination plays a key role in this process since, in the herein reported case, it could have helped to diagnose a disease capable of affecting a patient’s health, although it was an atypical ocular impairment.

Insights in the pathophysiology of haemorrhagic strokes in a sub-Sahara African country, an epidemiological and MRI study.

Intra-cerebral Haemorrhage (ICH) seems more prevalent in sub-Saharan Africa (SSA) than in High-Income Countries (HIC) with poorer clinical outcome. Higher impact of hypertension and/or amyloid angiopathy could account for this disproportion. Here, we sought to (i) retrospectively compare ICH clinical and imaging patterns in Belgium and Guinea and in a subsequent cohort (ii) prospectively compare brain MRI characteristics to seek evidence for a different proportion of amyloid angiopathy patterns. Ninety one consecutive patients admitted for spontaneous ICH at Brussels Erasme-ULB Hospital and at Conakry Ignace Deen-UGANC were retrospectively compared in terms of ICH volume estimated with the ABC/2 method, clinical characteristics and modified ranking (mRS) score at 30days. mRS was dichotomised as good outcomes (≤3) and poor outcomes (>3). A prospective cohort of 30 consecutive patients with ICH admitted at CHU Conakry Ignace Deen-UGANC was prospectively included to undergo brain MRI. Results of the Guinean MRI were compared to 30 patients randomly selected from Brussels' initial cohort. Paired Student's t-test and Mann-Whitney u-test were used for group comparisons. Age of ICH onset was higher in Belgium (68±17years vs. 56±14years, P<0.01) while ICH volume and 30-day mortality rate were higher in Guinea (20ml vs. 11ml, P<0.01 and mortality 33% vs. 10 %, P<0.01). ICH burden in survivors in Conakry and Brussels showed respectively good outcomes in 56.7% and 60.4% (P=0.09) and poor outcomes in 10.3% vs. 29.6% (P<0.001). MRI analysis of the prospective cohort failed to disclose significant differences regarding brain MRI characteristics. Intra-cerebral Haemorrhage affected patients 15years younger in Guinea with larger haematoma volumes and higher mortality than in Belgium. MRI findings did not show more prevalent amyloid angiopathy pathology suggesting that better primary prevention of hypertension could positively impact ICH epidemiology in Guinea.

Pathophysiological Mechanisms and Potential Therapeutic Targets in Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy (CADASIL).

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a hereditary small-vessels angiopathy caused by mutations in the NOTCH 3 gene, located on chromosome 19, usually affecting middle-ages adults, whose clinical manifestations include migraine with aura, recurrent strokes, mood disorders, and cognitive impairment leading to dementia and disability. In this review, we provide an overview of the current knowledge on the pathogenic mechanisms underlying the disease, focus on the corresponding therapeutic targets, and discuss the most promising treatment strategies currently under investigations. The hypothesis that CADASIL is an appropriate model to explore the pathogenesis of sporadic cerebral small vessel disease is also reviewed.

Abstract WP233: Distinct Vascular Profile in Middle-Aged Migraineurs: Higher Pulse Wave Velocity, Putaminal État Criblé and Incomplete Posterior Circle of Willis

Background: Migraineurs may develop stroke and cardiovascular diseases (CVD). We evaluated vascular findings in middle-aged migraineurs without CVD risks. Methods: Migraine was diagnosed according to ICHD-3 by direct interview and neurological examination. Migraine was classified into migraine with aura (MA) and migraine without aura (MO). Frequency of migraine attacks were denoted as &lt; 1 /mo or ≥ 1 /mo. Severity of migraine was scaled by Headache Impact Test-6. Usual CVD risk factors of smoking, obesity, hypertension, diabetes mellitus and dyslipidemia were examined. Brachial-ankle pulse wave velocity (baPWV) and ankle brachial index (ABI) were measured using a plethysmography. Brain magnetic resonance imaging (MRI) and angiography (MRA) were performed. Circle of Willis (CW) on MRA was morphologically classified into 2 types: complete posterior CW defined as the presence of both posterior communicating arteries and both P1 segments of posterior cerebral arteries, and other patterns of incomplete posterior CW (Ikeda et al. Headache 2007). Results: A total of 317 migraineurs (235 women and 82 men) were diagnosed. Thirty patients with CVD risk factors were excluded, and the study was completed in 287 migraineurs (215 women and 72 men), MA (n=56) and MO (n=231). Mean age (SD) was 44.0 (9.3) years. Consecutive 300 age- and sex-matched controls without migraine and CVD risks were prepared during the study. Compared to controls, baPWV was increased in migraineurs (P&lt;0.05) and MA patients (P&lt;0.01). ABI did not differ between migraineurs and controls. Other clinical findings of migraineurs were not linked to baPWV and ABI. MRI disclosed higher frequency of putaminal état criblé in migraineurs (P&lt;0.05) and MA patients (P&lt;0.01) compared to controls. Three MA patients were diagnosed with hereditary cerebral angiopathy of CADASIL (n=1), Moyamoya disease (n=1) and cavernous angioma (n=1). Morphological CW studies lower frequency of complete posterior CW in migraineurs (P&lt;0.05) and MA patients (P&lt;0.01) compared to controls. Conclusion: The present study suggested vascular changes of baPWV, putaminal état criblé and CW shape in migraineurs, especially in MA sufferers. The distinct vascular profile could contribute to higher risk of stroke and CVD in migraineurs.

BNS Epilepsy, Global Developmental Delay, Optic Coloboma, and Vesicoureteral Reflux as Leading Symptoms of COL4A1 Mutation: A Case Report

Background: Heterozygous mutations of COL4A1 gene lead to aut.-dom. handed diseases with variable clinical spectrum. Due to connective tissue disruption different organic systems are involved. A complex of symptoms including developmental disorders, cerebral, ocular and renal malformations, seizures as well as perinatal intracranial hemorrhage of unknown origin in term newborns could indicate COL4A1-associated diseases. In addition, autosomal dominant familiar porencephalia and hereditary angiopathy with nephropathy, aneurysm und muscle cramps (HANAC syndrome) belong to the spectrum of these mutations. Here, we report signs and clinical course of a novel COL4A1 deletion in an infant.

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.

A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

BackgroundHereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts.MethodsWe used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses.ResultsWe identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested.ConclusionsMissense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans.

Tortuosité artériolaire dans le cadre du syndrome HANAC (angiopathie héréditaire, néphropathie, anévrisme et crampe musculaire)

Tortuosité artériolaire dans le cadre du syndrome HANAC (angiopathie héréditaire, néphropathie, anévrisme et crampe musculaire)

Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BM functions due to aberrant COL4A1 protein, we have carried out a series of tests using DrosophilaDTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, DiptericinB, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.

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.

Two families with novel missense mutations in COL4A1: When diagnosis can be missed

Mutations in COL4A1, encoding one of the six collagen type IV proteins, cover a wide spectrum of autosomal dominant overlapping phenotypes including porencephaly, small-vessel disease and hemorrhagic stroke, leukoencephalopathy, hereditary angiopathy with nephropathy, aneurysms and muscle cramp (HANAC) syndrome, and Walker–Warburg syndrome. Over 50 mutations are known, mainly being missense changes. Intra- and inter-familial variability has been reported.We studied two Italian families in which the proband had a clinical diagnosis of COL4A1-related disorder. We found two novel mutations (c.1249G>C; p.Gly417Arg and c.2662G>C; p.Gly888Arg). Both involved highly conserved amino acids and were predicted as being deleterious by bioinformatics tools. The c.1249G>C (p.Gly417Arg) segregated in four subjects with variable neurological phenotypes, namely leukoencephalopathy with muscle symptoms, brain small-vessel disease, and mild infantile encephalopathy. A fourth case was a carrier of the mutation without any neurological symptoms and an MRI with a specific white matter anomaly. The c.2662G>C (p.Gly888Arg) mutation was de novo in the proband. After a temporary motor impairment at age 14, the subject complained of mild imbalance at age 30, during the third trimester of her twin pregnancy, when an anomaly of the left brain hemisphere was documented in one fetus. Both her male dizygotic twins presented a severe motor delay, early convulsions, and leukoencephalopathy, and were carriers of the mutation.In summary, we confirm that high intra-familial variability of COL4A1 mutations with very mild phenotypes, the apparent incomplete penetrance, and de novo changes may become a “dilemma” for clinicians and genetic counselors.

Spectroscopic and SAXS Studies of Human Cystatin C Mutants - Early Stages of Amyloid Formation Process

Human cystatin C (HCC) is a cysteine protease inhibitor. This protein in pathological conditions, forms dimers via a “domain swapping” mechanism. HCC is also associated with two types of amyloid deposition diseases - hereditary amyloid angiopathy (related to the Leu68Gln mutation) and wild-type cystatin C co-precipitation.The aim of our studies was the characterisation of the self-assembling properties of native and mutated (at positions 57 or 68) forms of human cystatin C in solution. The structure, overall conformation and secondary structure changes in solution were studied by Fourier transformed infrared spectroscopy (FTIR), circular dichroism spectroscopy (CD), dynamic light scattering (DLS) and time resolved small angle scattering of synchrotron radiation (TR-SAXS).SAXS data for native and mutated HCC were subjected to analysis by using SVD and MCR-ALS methods as well as the low resolution structure determination. Besides the monomeric forms of human cystatin C, also dimers and higher oligomers were formed even after short (50-ms) exposure on synchrotron radiation. In addition we observed for first time, formation of domain swapped dimers of human cystatin C induced by irradiation. The spectroscopic studies confirmed conformational changes.Authors acknowledge also the partial support by HARMONIA3 grant (Project No. 2012/06/M/ST4/00036) from National Science Centre (Poland).