Genetics of inherited small vessel diseases

Dementia disorders are a major health challenge as the life expectancy of the population increases. Vascular cognitive impairment (VCI) is the second most common cause of dementia after Alzheimer's disease, based on clinical studies. Several different vascular changes can cause VCI, and genetic factors underlying the pathogenesis of VCI are largely unknown. The aim of this study was to examine the genetic background of VCI in Finnish patients and to investigate the genetic and clinical spectrum of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in Finland. The study subjects of the first and second studies included a total of 80 patients with VCI. Whole-exome sequencing (WES) was performed in both studies, and copy-number variant (CNV) analysis using SNP-array was performed on all samples in the second study. The studies revealed that in addition to NOTCH3 variants, variants in COL4A1, COL4A2, HTRA1 and TREX1 are also found in Finnish patients with VCI. Interestingly, the studies also identified several variants in genes linked to neurodegenerative disorders indicating that cerebrovascular pathology may be associated with neurodegeneration in the pathogenesis of VCI. The study showed WES to be useful in diagnosing patients with suspected inherited VCI. The third study showed a duplication in chromosome 13 including COL4A1 and COL4A2 genes in a Finnish family with an autosomal dominantly inherited small vessel disease. The study described the clinical features of the disease, and droplet digital PCR (ddPCR) showed that the duplication caused an upregulation of COL4A1 and COL4A2. The fourth study retrospectively examined the spectrum of NOTCH3 variants and clinical features in Finnish CADASIL patients (n=294). The study showed that in addition to the most common NOTCH3 variants (p.Arg133Cys and p.Tyr1069Cys) several other less common NOTCH3 variants were detected in Finnish CADASIL patients and were often associated with severe clinical features. In conclusion, this is the first study revealing the genetic and clinical spectrum of VCI and CADASIL in the Finnish population. In addition, this study revealed novel insights into the molecular basis of VCI. This thesis highlights the power of advanced sequencing technologies in identifying causal genetic variants, and provides tools for diagnostics and genetic counseling of patients with VCI.Muistisairautta aiheuttavat aivosairaudet ovat suuri terveydenhuollon haaste väestön elinajanodotteen noustessa. Kliinisisten tutkimusten perusteella vaskulaarinen kognitiivinen heikentymä eli verenkiertoperäinen muistisairaus on toiseksi yleisin dementian syy Alzheimerin taudin jälkeen. Verenkiertoperäinen muistisairaus voi aiheutua useista erilaisista aivoverenkiertohäiriöiden aiheuttamista aivomuutoksista, ja sen syntyyn vaikuttavat geneettiset tekijät ovat vielä suurelta osin epäselviä. Tämän tutkimuksen tavoitteena oli selvittää verenkiertoperäisten muistisairauksien geneettistä taustaa suomalaisilla potilailla sekä tutkia CADASIL-taudin (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) geneettistä ja kliinistä kirjoa Suomessa. Ensimmäisen ja toisen osatyön aineistot koostuivat yhteensä 80 verenkiertoperäistä muistisairautta sairastavasta potilaasta. Molemmissa osatöissä tehtiin eksomin sekvensointi ja toisessa osatyössä kaikista näytteistä tehtiin kopiolukumuutosanalyysi SNP-sirulla. Tutkimuksissa todettiin suomalaisilla verenkiertoperäistä muistisairautta sairastavilla potilailla COL4A1-, COL4A2-, HTRA1- ja TREX1-geenien muutoksia, NOTCH3-geenin geenivirheiden lisäksi. Tutkimuksissa myös tunnistettiin useita neurodegeneratiivisiin tauteihin liitettyjen geenien muutoksia viitaten siihen, että aivoverisuonimuutokset saattavat olla yhteydessä neurodegeneraatioon verenkiertoperäisen muistisairauden patogeneesissa. Tutkimus osoitti eksomin sekvensoinnin olevan hyödyllinen diagnostinen menetelmä epäiltäessä perinnöllistä verenkiertoperäistä muistisairautta. Kolmannessa osatyössä osoitettiin kromosomiin 13 paikantuvan COL4A1- ja COL4A2-geenit sisältävän duplikaation segregoivan suomalaisessa perheessä vallitsevasti periytyvän pienten suonten taudin kanssa. Lisäksi osatyössä kuvattiin tautiin liittyvä taudinkuva sekä osoitettiin droplet digital PCR –menetelmällä (ddPCR) duplikaation aiheuttavan COL4A1-ja COL4A2-geenien yliekspression. Neljännessä osatyössä tutkittiin retrospektiivisesti suomalaisten CADASIL-potilaiden (n=294) NOTCH3-geenivirheiden kirjoa ja taudinkuvaa. Tutkimus osoitti, että yleisimpien NOTCH3-varianttien (p.Arg133Cys and p.Tyr1069Cys) lisäksi suomalaisilla CADASIL-potilailla on todettu useita muita harvinaisempia NOTCH3-geenin variantteja, jotka liittyivät usein vakaviin kliinisiin oireisiin. Tämä tutkimus paljasti ensimmäistä kertaa verenkiertoperäisen muistisairauden ja CADASIL-taudin geneettistä ja kliinistä taustaa suomalaisilla potilailla, sekä toi uutta tietoa verenkiertoperäisen muistisairauden molekulaarisesta taustasta. Lisäksi tämä tutkimus korosti uusien sekvensointimenetelmien käytön tärkeyttä tautia aiheuttavien geenimuutosten tunnistamisessa. Tämä väitöskirja tarjoaa työkaluja verenkiertoperäisen muistisairauden diagnostiikkaan ja tautia sairastavien potilaiden perinnöllisyysneuvontaan

Clinical features and spectrum of NOTCH3 variants in Finnish patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Objectives Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease caused by pathogenic variants in the NOTCH3 gene. In Finland, the majority of CADASIL patients carry the pathogenic founder variant c.397C>T, (p.Arg133Cys), but the spectrum of other NOTCH3 variants has not been investigated previously. The aim of the study was to investigate the spectrum and prevalence of NOTCH3 variants Finnish CADASIL patients and to examine the clinical features associated with them. Materials and Methods The spectrum of NOTCH3 variants and the clinical features associated with them were retrospectively examined in 294 Finnish CADASIL patients tested during January 1996 to October 2021 in the Medical Genetics laboratory of Department of Genomics of Turku University Hospital, where practically all samples of patients with suspected CADASIL in Finland are investigated. Results The most common NOTCH3 variants in the study cohort were c.397C>T, (p.Arg133Cys) (68%) and c.3206A>G p.(Tyr1069Cys) (18%), but other less common NOTCH3 variants were detected in as many as 14% of the patients. Eight of the detected NOTCH3 variants were novel: c.520T>A,p.(Cys174Ser), c.836A>G,p.(Gln279Arg), c.1369T>G,p.(Cys457Gly), c.1338C>G,p.(Cys446Trp), c.1564T>G,p.(Cys522Gly), c.2848T>G,p.(Cys950Gly), c.6102dup,p.(Gly2035Argfs*60), and c.2410+6C>G. Other NOTCH3 variants than p.Arg133Cys and p.Tyr1069Cys were more often associated with more severe clinical features. Conclusion This study revealed the genetic and clinical spectrum of CADASIL in the Finnish population. Sequencing of the whole NOTCH3 gene performing a gene-panel or exome sequencing is recommended when suspecting CADASIL.Peer reviewe

Clinical features and spectrum of NOTCH3 variants in Finnish patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Objectives: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease caused by pathogenic variants in the NOTCH3 gene. In Finland, the majority of CADASIL patients carry the pathogenic founder variant c.397C>T, (p.Arg133Cys), but the spectrum of other NOTCH3 variants has not been investigated previously. The aim of the study was to investigate the spectrum and prevalence of NOTCH3 variants Finnish CADASIL patients and to examine the clinical features associated with them.Materials and methods: The spectrum of NOTCH3 variants and the clinical features associated with them were retrospectively examined in 294 Finnish CADASIL patients tested during January 1996 to October 2021 in the Medical Genetics laboratory of Department of Genomics of Turku University Hospital, where practically all samples of patients with suspected CADASIL in Finland are investigated.Results: The most common NOTCH3 variants in the study cohort were c.397C>T, (p.Arg133Cys) (68%) and c.3206A>G p.(Tyr1069Cys) (18%), but other less common NOTCH3 variants were detected in as many as 14% of the patients. Eight of the detected NOTCH3 variants were novel: c.520T>A,p.(Cys174Ser), c.836A>G,p.(Gln279Arg), c.1369T>G,p.(Cys457Gly), c.1338C>G,p.(Cys446Trp), c.1564T>G,p.(Cys522Gly), c.2848T>G,p.(Cys950Gly), c.6102dup,p.(Gly2035Argfs*60), and c.2410+6C>G. Other NOTCH3 variants than p.Arg133Cys and p.Tyr1069Cys were more often associated with more severe clinical features.Conclusion: This study revealed the genetic and clinical spectrum of CADASIL in the Finnish population. Sequencing of the whole NOTCH3 gene performing a gene-panel or exome sequencing is recommended when suspecting CADASIL.</p

Genetic analysis reveals novel variants for vascular cognitive impairment

Objectives The genetic background of vascular cognitive impairment (VCI) is poorly understood compared to other dementia disorders. The aim of the study was to investigate the genetic background of VCI in a well-characterized Finnish cohort. Materials & Methods Whole-exome sequencing (WES) was applied in 45 Finnish VCI patients. Copy-number variant (CNV) analysis using a SNP array was performed in 80 VCI patients. This study also examined the prevalence of variants at the miR-29 binding site of COL4A1 in 73 Finnish VCI patients. Results In 40% (18/45) of the cases, WES detected possibly causative variants in genes associated with cerebral small vessel disease (CSVD) or other neurological or stroke-related disorders. These variants included HTRA1:c.847G>A p.(Gly283Arg), TREX1:c.1079A>G, p.(Tyr360Cys), COLGALT1:c.1411C>T, p.(Arg471Trp), PRNP: c.713C>T, p.(Pro238Leu), and MTHFR:c.1061G>C, p.(Gly354Ala). Additionally, screening of variants in the 3 ' UTR of COL4A1 gene in a sub-cohort of 73 VCI patients identified a novel variant c.*36T>A. CNV analysis showed that pathogenic CNVs are uncommon in VCI. Conclusions These data support pathogenic roles of variants in HTRA1, TREX1 and in the 3 ' UTR of COL4A1 in CSVD and VCI, and suggest that vascular pathogenic mechanisms are linked to neurodegeneration, expanding the understanding of the genetic background of VCI.Peer reviewe

Genetic analysis reveals novel variants for vascular cognitive impairment

Objectives The genetic background of vascular cognitive impairment (VCI) is poorly understood compared to other dementia disorders. The aim of the study was to investigate the genetic background of VCI in a well-characterized Finnish cohort. Materials & Methods Whole-exome sequencing (WES) was applied in 45 Finnish VCI patients. Copy-number variant (CNV) analysis using a SNP array was performed in 80 VCI patients. This study also examined the prevalence of variants at the miR-29 binding site of COL4A1 in 73 Finnish VCI patients. Results In 40% (18/45) of the cases, WES detected possibly causative variants in genes associated with cerebral small vessel disease (CSVD) or other neurological or stroke-related disorders. These variants included HTRA1:c.847G>A p.(Gly283Arg), TREX1:c.1079A>G, p.(Tyr360Cys), COLGALT1:c.1411C>T, p.(Arg471Trp), PRNP: c.713C>T, p.(Pro238Leu), and MTHFR:c.1061G>C, p.(Gly354Ala). Additionally, screening of variants in the 3 ' UTR of COL4A1 gene in a sub-cohort of 73 VCI patients identified a novel variant c.*36T>A. CNV analysis showed that pathogenic CNVs are uncommon in VCI. Conclusions These data support pathogenic roles of variants in HTRA1, TREX1 and in the 3 ' UTR of COL4A1 in CSVD and VCI, and suggest that vascular pathogenic mechanisms are linked to neurodegeneration, expanding the understanding of the genetic background of VCI.</p

Oligogenic basis of sporadic ALS The example of SOD1 p.Ala90Val mutation

Objective To characterize the clinical and neuropathologic features of patients with amyotrophic lateral sclerosis (ALS) with the superoxide dismutase 1 (SOD1) p.Ala90Val mutation, as well as the mutation frequency and the role of oligogenic mechanisms in disease penetrance. Methods An index patient with autopsy-proven ALS was discovered to have the SOD1 p.Ala90Val mutation, which was screened in 2 Finnish ALS cohorts (n = 453). Additional contributing variants were analyzed from whole-genome or whole-exome sequencing data. Results Seven screened patients (1.5%) were found to carry the SOD1 heterozygous mutation. Allele-sharing analysis suggested a common founder haplotype. Common clinical features included limb-onset, long disease course, and sensory symptoms. No TDP43 pathology was observed. All cases were apparently sporadic, and pedigree analysis demonstrated that the mutation has reduced penetrance. Analysis of other contributing genes revealed a unique set of additional variants in each patient. These included previously described rare ANG and SPG11 mutations. One patient was compound heterozygous for SOD1 p.Ala90Val and p.Asp91Ala. Conclusions Our data suggest that the penetrance of SOD1 p.Ala90Val is modulated by other genes and indicates highly individual oligogenic basis of apparently sporadic ALS. Additional genetic variants likely contributing to disease penetrance were very heterogeneous, even among Finnish patients carrying the SOD1 founder mutation

Oligogenic basis of sporadic ALS The example of SOD1 p.Ala90Val mutation

Objective To characterize the clinical and neuropathologic features of patients with amyotrophic lateral sclerosis (ALS) with the superoxide dismutase 1 (SOD1) p.Ala90Val mutation, as well as the mutation frequency and the role of oligogenic mechanisms in disease penetrance. Methods An index patient with autopsy-proven ALS was discovered to have the SOD1 p.Ala90Val mutation, which was screened in 2 Finnish ALS cohorts (n = 453). Additional contributing variants were analyzed from whole-genome or whole-exome sequencing data. Results Seven screened patients (1.5%) were found to carry the SOD1 heterozygous mutation. Allele-sharing analysis suggested a common founder haplotype. Common clinical features included limb-onset, long disease course, and sensory symptoms. No TDP43 pathology was observed. All cases were apparently sporadic, and pedigree analysis demonstrated that the mutation has reduced penetrance. Analysis of other contributing genes revealed a unique set of additional variants in each patient. These included previously described rare ANG and SPG11 mutations. One patient was compound heterozygous for SOD1 p.Ala90Val and p.Asp91Ala. Conclusions Our data suggest that the penetrance of SOD1 p.Ala90Val is modulated by other genes and indicates highly individual oligogenic basis of apparently sporadic ALS. Additional genetic variants likely contributing to disease penetrance were very heterogeneous, even among Finnish patients carrying the SOD1 founder mutation.Peer reviewe

PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies

TREX1 p.A129fs and p.Y305C variants in a large multi-ethnic cohort of CADASIL-like unrelated patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and retinal vasculopathy with cerebral leukodystrophy and systemic manifestations (RVCL-S) are the most common forms of rare monogenic early-onset cerebral small vessel disease and share clinical, and, to different extents, neuroradiological and neuropathological features. However, whether CADASIL and RVCL-S overlapping phenotype may be explained by shared genetic risk or causative factors such as TREX1 coding variants remains poorly understood. To investigate this intriguing hypothesis, we used exome sequencing to screen TREX1 protein-coding variability in a large multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic CADASIL-like Caucasian patients from the USA, Portugal, Finland, Serbia and Turkey. We report 2 very rare and likely pathogenic TREX1 mutations: a loss of function mutation (p.Ala129fs) clustering in the catalytic domain, in an apparently sporadic 46-year-old patient from the USA and a missense mutation (p.Tyr305Cys) in the well conserved C-terminal region, in a 57-year-old patient with positive family history from Serbia. In concert with recent findings, our study expands the clinical spectrum of diseases associated with TREX1 mutations

TREX1 p.A129fs and p.Y305C variants in a large multi-ethnic cohort of CADASIL-like unrelated patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and retinal vasculopathy with cerebral leukodystrophy and systemic manifestations (RVCL-S) are the most common forms of rare monogenic early-onset cerebral small vessel disease and share clinical, and, to different extents, neuroradiological and neuropathological features. However, whether CADASIL and RVCL-S overlapping phenotype may be explained by shared genetic risk or causative factors such as TREX1 coding variants remains poorly understood. To investigate this intriguing hypothesis, we used exome sequencing to screen TREX1 protein-coding variability in a large multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic CADASIL-like Caucasian patients from the USA, Portugal, Finland, Serbia and Turkey. We report 2 very rare and likely pathogenic TREX1 mutations: a loss of function mutation (p.Ala129fs) clustering in the catalytic domain, in an apparently sporadic 46-year-old patient from the USA and a missense mutation (p.Tyr305Cys) in the well conserved C-terminal region, in a 57-year-old patient with positive family history from Serbia. In concert with recent findings, our study expands the clinical spectrum of diseases associated with TREX1 mutations