Major Histocompatibility Complex Genes : Method of Analysis, Association with Acute Coronary Syndromes and Effect on Immune Reactions

Atherosclerosis is a chronic, inflammatory, lifestyle-disease that is also influenced by genetic factors. Coronary atherosclerosis manifests as silent, chronic and acute forms. The acute forms, acute coronary syndromes (ACS) are an important cause of death due to coronary atherosclerosis. The major histocompatibility complex (MHC) region is a collection of genes on chromosome 6 that are associated with immune response. The genes in this region are usually inherited as a strongly linked genetic combination, a haplotype. Certain alleles and haplotypes of the MHC region have been linked with different forms of coronary atherosclerosis. The Thesis was set to study the role of selected MHC genes/alleles in ACS. First aim of the Thesis was to develop a novel quantitative polymerase chain reaction (qPCR) method for complement component C4 analyses. A qPCR method with a novel concentration range approach and SYBR® Green dye was developed (I). It was validated by applying it to over 1600 patient samples with available C4 protein data and by analysing 129 samples that were also assessed with other methods for C4 gene analysis. The results indicated that the developed method can be used to reliably assess C4 copy number variation. The second aim was to confirm the role of selected MHC genes and haplotypes in different clinical forms of ACS. Based on previous studies, four MHC markers that had been reported to be associated with coronary atherosclerosis were selected (HLA-DRB1*01, HLA-B*35, deficiency of complement component C4A or C4B). A haplotype with DRB1*01, with neither C4 deficiencies nor B*35 was more frequently detected in patients suffering from ACS than in healthy controls (11 vs. 5%; III). None of the markers alone was associated with a significantly increased risk of ACS (II-IV). However, the power was too low to exclude a weak association of C4 deficiency and ACS (II, IV). If only men were studied, both DRB1*01 alone and the DRB1*01-haplotype were significantly associated with ACS. The third aim was to evaluate the possible inflammatory mechanisms through which the association of MHC genes/haplotypes and ACS could be mediated. The DRB1*01-haplotype was associated with higher hsCRP levels (III). C4 deficiency segregated patients that benefitted from macrolide treatment in secondary prevention of recurrent ACS (II). C4 deficiency was also associated with elevated heat shock protein 60 IgA autoantibody levels, which, in turn, was shown to be associated with ACS and recurrent cardiovascular end points (IV). C4 deficiency was also increased in patients suffering from recurrent infections (I). The genetic polymorphism in MHC may be linked with ACS by affecting the inflammatory responses. These data are observational and thus do not indicate causality. However, these data might help to elucidate the complex interplay of inflammatory reactions in ACS and in directing patient care.Sepelvaltimotauti on kehittyneiden maiden yleisimpiä kuolinsyitä. Sydänkohtaus (sydäninfarkti tai epävakaa rintakipu) on sepelvaltimotaudin pelätyimpiä ilmentymiä. Vaikka sydänkohtauksen tarkkaa syntymekanismia ei tiedetä, on sille tunnistettu useita riskitekijöitä. Näistä tunnetuimpia ovat korkea verenpaine, tupakointi, sokeritauti, miessukupuoli, korkea ikä ja sukulaisilla olleet sepelvaltimotautikohtaukset. Riskitekijöiden lisäksi geenien tiedetään aiheuttavan osan sydänkohtauksista. Väitöskirjassa tarkasteltiin MHC-geenejä, niiden analysointia ja niiden roolia sydänkohtauksissa. MHC-geenit ovat joukko geenejä kromosomissa 6, jotka liittyvät mm. tulehdusvasteeseen. Ensimmäinen väitöskirjan tavoite oli kehittää uusi menetelmä MHC-geenien analysointiin. Tulokset osoittivat, että kehittämämme menetelmä on yhtä luotettava MHC-geenien analysoinnissa kuin vanhat menetelmät (I). Toinen väitöskirjan tavoite oli varmentaa MHC-geenien rooli sydänkohtauksissa. Valitsimme tiettyjä MHC-geeneiä (HLA-DRB1*01, HLA-B*35 ja komplementtitekijä C4n puutokset) tarkempaan tarkasteluun. Aiemmissa tutkimuksissa nämä on liitetty suurentuneeseen riskiin saada sydänkohtaus. Tulokset osoittivat, että HLA-DRB1*01-geeniyhdistelmä löytyi merkittävästi useammin sydänkohtauksen sairastaneilta potilailta kuin terveiltä verrokeilta. Potilaista yhdellätoista prosentilla, mutta verrokeista vain viidellä prosentilla oli kyseinen geeniyhdistelmä (III). Tehdyissä tutkimuksissa komplementti C4n puutoksen rooli sydänkohtauksissa jäi epäselväksi (II-IV). Väitöskirjan kolmas tavoite oli selvittää, millä mekanismeilla MHC-geenit voisivat aiheuttaa sydänkohtauksia. Tutkimme yleistä tulehdusarvoa (III), antibioottihoidon vaikuttavuutta (II) sekä HSP60-vasta-aineita elimistön omia rakenteita vastaan (IV). Antibioottitutkimuksessa sydänkohtauksen sairastaneille potilaille annettiin kolmen kuukauden ajan antibioottia, jonka toivottiin estävän uusiutuvat sydänkohtaukset. Tulokset osoittivat, että DRB1*01-geeniyhdistelmän kantajilla oli korkeampi tulehdusarvo kuin niillä, joilla sitä ei ollut (III). Antibioottitutkimuksessa havaittiin, että potilaat, joilla oli komplementtitekijä C4n puutos, hyötyivät antibioottihoidosta. Heillä riski saada uusi sydänkohtaus oli yli puolet pienempi kuin niillä, jotka saivat lumelääkettä. Niillä potilailla, joilla ei ollut geenipuutosta, ei antibiootista ollut hyötyä (II). Tämän lisäksi potilailla, joilla oli komplementtitekijä C4n puutos, oli korkeampi HSP60-vasta-ainetaso. Kohonnut HSP60-vasta-ainetaso liittyi lisääntyneeseen riskiin saada sydänkohtaus (IV). MHC-geenialue näyttää liittyvän toistuvasti sydänkohtauksiin, mahdollisesti tulehduksellisten reaktioiden kautta. Tämä assosiaatio vahvistaa tulehdusreaktion tärkeyttä sydänkohtausten taustatekijänä. Löydökset vaativat jatkotutkimuksia, ennen kuin niitä voidaan käyttää potilaiden hoidossa. Jos löydökset osoittautuvat toistettavaksi, ne voivat olla apuna potilaan räätälöidyssä hoidossa sekä sydänkohtausten mekanismien selvittelyssä

Soluble HLA-DR serum levels are associated with smoking but not with acute coronary syndrome

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Left ventricular non-compaction as a potential source for cryptogenic ischemic stroke in the young : A case-control study

Background Up to 50% of ischemic strokes in the young after thorough diagnostic work-up remain cryptogenic or associated with low-risk sources of cardioembolism such as patent foramen ovale (PFO). We studied with cardiac magnetic resonance (CMR) imaging, whether left ventricular (LV) non-compaction-a possible source for embolic stroke due to sluggish blood flow in deep intertrabecular recesses-is associated with cryptogenic strokes in the young. Methods Searching for Explanations for Cryptogenic Stroke in the Young: Revealing the Etiology, Triggers, and Outcome (SECRETO; NCT01934725) is an international prospective multicenter case-control study of young adults (aged 18-49 years) presenting with an imaging-positive first-ever ischemic stroke of undetermined etiology. In this pilot substudy, 30 cases and 30 age- and sex-matched stroke-free controls were examined with CMR. Transcranial Doppler (TCD) bubble test was performed to evaluate the presence and magnitude of right-to-left shunt (RLS). Results There were no significant differences in LV volumes, masses or systolic function between cases and controls; none of the participants had non-compaction cardiomyopathy. Semi-automated assessment of LV non-compaction was highly reproducible. Non-compacted LV mass (median 14.0 [interquartile range 12.6-16.0] g/m(2)vs. 12.7 [10.4-16.6] g/m(2), p = 0.045), the ratio of non-compacted to compacted LV mass (mean 25.6 +/- 4.2% vs. 22.8 +/- 6.0%, p = 0.015) and the percentage of non-compacted LV volume (mean 17.6 +/- 2.9% vs. 15.7 +/- 3.8%, p = 0.004) were higher in cases compared to controls. In a multivariate conditional logistic regression model including non-compacted LV volume, RLS and body mass index, the percentage of non-compacted LV volume (odds ratio [OR] 1.55, 95% confidence interval [CI] 1.10-2.18, p = 0.011) and the presence of RLS (OR 11.94, 95% CI 1.14-124.94, p = 0.038) were independently associated with cryptogenic ischemic stroke. Conclusions LV non-compaction is associated with a heightened risk of cryptogenic ischemic stroke in young adults, independent of concomitant RLS and in the absence of cardiomyopathy.Peer reviewe

Copy Number Analysis of Complement C4A, C4B and C4A Silencing Mutation by Real-Time Quantitative Polymerase Chain Reaction

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Right atrium and cryptogenic ischaemic stroke in the young : A case-control study

Publisher Copyright: © 2021 BMJ Publishing Group. All rights reserved.Background Recent studies suggest left atrial (LA) dysfunction in cryptogenic stroke. We studied the dynamics of right atrium (RA) and right atrial appendage (RAA) in young adults with cryptogenic stroke. We hypothesised that bi-atrial dysfunction and blood stagnation might contribute to thrombosis formation in patients with patent foramen ovale (PFO), as deep venous thrombosis is detected only in the minority of patients. Methods Thirty patients (aged 18-49) with a first-ever cryptogenic stroke and 30 age-matched and sex-matched stroke-free controls underwent cardiac magnetic resonance (CMR) imaging. An approach to estimate the RAA volume was developed, using crista terminalis and pectinate muscles as anatomical landmarks. Atrial expansion indices were calculated as (maximal volume - minimal volume) ×100%/minimal volume. Total pulmonary to systemic blood flow ratio (Qp/Qs) was based on phase contrast CMR. Right-to-left shunt (RLS) was evaluated with transoesophageal echocardiography in 29 patients and transcranial Doppler in 30 controls, moderate-to-severe RLS considered as clinically significant. Results We found that RA and RAA volumes were similar between patients and controls. Also, RA expansion index was similar, but RAA (95.6%±21.6% vs 108.7%±25.8%, p=0.026) and LA (126.2%±28% vs 144.9%±36.3%, p=0.023) expansion indices were lower in patients compared with controls. Seven (24%) of 29 patients had an RLS compared with 1 (3%) of 30 controls (p=0.012). Among 59 study subjects, RLS was associated with lower RA (81.9%±15.9% vs 98.5%±29.5%, p=0.030), RAA (84.7%±18% vs 105.6%±24.1%, p=0.022), LA (109.8%±18.6% vs 140.1%±33.7%, p=0.017) and LAA (median 102.9% (IQR 65.6%-121.7%) vs 229.1% (151.8%-337.5%], p=0.002) expansion indices and lower Qp/Qs ratio (0.91±0.06 vs 0.98±0.07, p=0.027). Conclusions This study suggests bi-atrial dysfunction in young adults with cryptogenic stroke, associated with moderate-to-severe RLS. Dysfunction of the atria and atrial appendages may be an additional mechanism for PFO-related stroke. Trial registration number NCT01934725.Peer reviewe

Diversity of Extended HLA-DRB1 Haplotypes in the Finnish Population

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Complement C4 Deficiency - A Plausible Risk Factor for Non-Tuberculous Mycobacteria (NTM) Infection in Apparently Immunocompetent Patients

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Hydroxychloroquine reduces interleukin-6 levels after myocardial infarction : The randomized, double-blind, placebo-controlled OXI pilot trial

Objectives: To determine the anti-inflammatory effect and safety of hydroxychloroquine after acute myocardial infarction. Method: In this multicenter, double-blind, placebo-controlled OXI trial, 125 myocardial infarction patients were randomized at a median of 43 h after hospitalization to receive hydroxychloroquine 300 mg (n = 64) or placebo (n = 61) once daily for 6 months and, followed for an average of 32 months. Laboratory values were measured at baseline, 1, 6, and 12 months. Results: The levels of interleukin-6 (IL-6) were comparable at baseline between study groups (p = 0.18). At six months, the IL-6 levels were lower in the hydroxychloroquine group (p = 0.042, between groups), and in the on-treatment analysis, the difference at this time point was even more pronounced (p = 0.019, respectively). The high-sensitivity C-reactive protein levels did not differ significantly between study groups at any time points. Eleven patients in the hydroxychloroquine group and four in the placebo group had adverse events leading to in-terruption or withdrawal of study medication, none of which was serious (p = 0.10, between groups). Conclusions: In patients with myocardial infarction, hydroxychloroquine reduced IL-6 levels significantly more than did placebo without causing any clinically significant adverse events. A larger randomized clinical trial is warranted to prove the potential ability of hydroxychloroquine to reduce cardiovascular endpoints after myocar-dial infarction. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).Peer reviewe

MiR-185-5p regulates the development of myocardial fibrosis

Background: Cardiac fibrosis stiffens the ventricular wall, predisposes to cardiac arrhythmias and contributes to the development of heart failure. In the present study, our aim was to identify novel miRNAs that regulate the development of cardiac fibrosis and could serve as potential therapeutic targets for myocardial fibrosis. Methods and results: Analysis for cardiac samples from sudden cardiac death victims with extensive myocardial fibrosis as the primary cause of death identified dysregulation of miR-185-5p. Analysis of resident cardiac cells from mice subjected to experimental cardiac fibrosis model showed induction of miR-185-5p expression specifically in cardiac fibroblasts. In vitro, augmenting miR-185-5p induced collagen production and profibrotic activation in cardiac fibroblasts, whereas inhibition of miR-185-5p attenuated collagen production. In vivo, targeting miR-185-5p in mice abolished pressure overload induced cardiac interstitial fibrosis. Mechanistically, miR-185-5p targets apelin receptor and inhibits the anti-fibrotic effects of apelin. Finally, analysis of left ventricular tissue from patients with severe cardiomyopathy showed an increase in miR-185-5p expression together with pro-fibrotic TGF-beta 1 and collagen I. Conclusions: Our data show that miR-185-5p targets apelin receptor and promotes myocardial fibrosis.Peer reviewe

Clinical features of patients with homozygous complement C4A or C4B deficiency

Introduction Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. Material and methods Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. Results Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%Cl = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%Cl = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%Cl = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%Cl = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%Cl = 1.22-4.88, p = 0.010). Conclusion This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.Peer reviewe