Epstein-Barr Virus in the Cerebrospinal Fluid and Blood Compartments of Patients With Multiple Sclerosis and Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood. METHODS: CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls. RESULTS: One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls (p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations. DISCUSSION: EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome.

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70-80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, "Why this variation?" remains largely unanswered.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

Genomic and epidemiological report of the recombinant XJ lineage SARS-CoV-2 variant, detected in northern Finland, January 2022.

Recombinant sequences of the SARS-CoV-2 Omicron variant were detected in surveillance samples collected in north-western Finland in January 2022. We detected 191 samples with an identical genome arrangement in weeks 3 to 11, indicating sustained community transmission. The recombinant lineage has a 5'-end of BA.1, a recombination breakpoint between orf1a and orf1b (nucleotide position 13,296-15,240) and a 3'-end of BA.2 including the S gene. We describe the available genomic and epidemiological data about this currently circulating recombinant XJ lineage.

Introduction and Rapid Spread of SARS-CoV-2 Omicron Variant and Dynamics of BA.1 and BA.1.1 Sublineages, Finland, December 2021.

Multiple introductions of SARS-COV-2 Omicron variant BA.1 and BA.1.1. lineages to Finland were detected in early December 2021. Within 3 weeks, Omicron overtook Delta as the most common variant in the capital region. Sequence analysis demonstrated the emergence and spread through community transmission of a large cluster of BA.1.1 virus.

Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction.

Low protein levels and copy number variation (CNV) of the fourth component of human complement (C4A and C4B) have been associated with various diseases. High-throughput methods for analysing C4 CNV are available, but they commonly do not detect the most common C4A mutation, a silencing CT insertion (CTins) leading to low protein levels. We developed a SYBR® Green labelled real-time quantitative polymerase chain reaction (qPCR) with a novel concentration range approach to address C4 CNV and deficiencies due to CTins. This method was validated in three sample sets and applied to over 1600 patient samples. CTins caused C4A deficiency in more than 70% (76/105) of the carriers. Twenty per cent (76/381) of patients with a C4A deficiency would have been erroneously recorded as having none, if the CTins had not been assessed. C4A deficiency was more common in patients than a healthy reference population, (OR = 1.60, 95%CI = 1.02-2.52, p = 0.039). The number of functional C4 genes can be straightforwardly analyzed by real-time qPCR, also with SYBR® Green labelling. Determination of CTins increases the frequency of C4A deficiency and thus helps to elucidate the genotypic versus phenotypic disease associations.

HLA-DRB1 allele frequencies and C4 copy number variation in Finnish sarcoidosis patients and associations with disease prognosis.

Sarcoidosis is a multiorgan immune-mediated disease of unknown etiology with varying clinical pictures. We studied 3 genes in the major histocompatibility complex region (HLA-DRB1 and complement C4A and C4B) in patients with resolved disease after a 2-year follow-up (n = 90) and in patients whose disease was still active at that time point (n = 98) and compared them with controls (n = 150). Our primary aim was to detect genetic differences between the patient groups. We observed that the susceptibility allele for sarcoidosis was HLA-DRB1*15:01 (p = 0.011; odds ratio [OR] = 1.67) and the protective allele was HLA-DRB1*01:01 (p = 0.001; OR = 0.43). HLA-DRB1*03:01 was associated with resolving disease when compared with the persistent group (p = 0.011; OR = 2.22). The probability of having resolving disease was even greater if the patient had HLA-DRB1*03:01 and did not have extrapulmonary lesions (p = 0.001; OR = 3.39). By evaluating amino acid variants of the HLA-DRB1 gene, we determined that specific amino acids in pockets 4, 7, and 9 were associated with the prognosis of sarcoidosis. Our results support the importance of HLA-DRB1 as a predisposing gene for sarcoidosis. Particularly, HLA-DRB1*03:01 and polymorphisms of DRB1 pocket residues were associated with a favorable prognosis. Thus, accurate categorization of disease phenotype and HLA-DRB1 sequencing offer a basis for disease course estimation of sarcoidosis.

Beneficial effect of clarithromycin in patients with acute coronary syndrome and complement C4 deficiencies.

OBJECTIVES: We sought to examine the role of complement component C4 deficiencies on the effect of antibiotic treatment in patients with acute coronary syndrome (ACS). DESIGN: Patients with ACS (n=144) were randomly divided to receive a three-month treatment of clarithromycin or placebo and followed for major adverse coronary and cerebrovascular events (MACCEs) for 404.5 median days (range 138-924 days). The primary results indicated that clarithromycin prevented recurrent cardiovascular attacks. For the present study we performed serum C4 allotyping of C4A and C4B. The clarithromycin response was reanalyzed taking into account the deficiencies in the C4 allotypes. RESULTS: The prevalence of C4A deficiency, C4B deficiency or these combined were 29.2% (42/144), 39.6% (57/144) and 66.0% (95/144), respectively. In patients with C4 deficiencies clarithromycin treatment resulted in a reduced number of MACCEs and the best cumulative survival as compared with the placebo group (MACCE 18.8% versus 39.1%, respectively; Log rank test, p=0.015). CONCLUSIONS: Only patients with ACS and C4 deficiencies seem to benefit from antibiotic treatment. This may explain the controversial results of secondary prevention trials of coronary artery disease and possibly serve as a pharmacogenomic marker for clarithromycin treatment.

Helicobacter pylori infection induces a reversible expression of the CDX2 transcription factor protein in human gastric epithelium.

OBJECTIVE: The homeobox gene CDX2 is implicated in the appearance of intestinal metaplasia in Helicobacter pylori gastritis. The aim of this study was to investigate whether CDX2 expression in gastric mucosa occurs before the appearance of overt intestinal metaplasia in H. pylori gastritis, and whether or not this expression is reversible. MATERIAL AND METHODS: CDX2 was studied by immunohistochemistry in a cohort of 38 patients with H. pylori gastritis before and after eradication (mean follow-up 6.3 years) of H. pylori. A cohort of 49 individuals with healthy stomachs was analysed as a control. RESULTS: In the control group no immunostaining of CDX2 in the epithelial cells of the gastric body was found, while in 57% of the cases a mild, aberrant nuclear immunostaining of CDX2 in the non-metaplastic epithelial cells in antrum, designated as "positive staining of single cells" (PSSC), was found. In H. pylori gastritis, the PSSC was seen in antrum and corpus in 100% and 26% of the cases, respectively. The prevalence of antral PSSC was significantly increased (on average by 4-fold) in H. pylori gastritis as compared with controls. After eradication of H. pylori, the prevalence of PSSC decreased significantly in antrum but not in corpus. CONCLUSIONS: Expression of CDX2 at low intensity is common in the epithelium of normal antrum, and this expression is enhanced in H. pylori gastritis. Expression of CDX2 is reversible at least in antrum after eradication of H. pylori infection.

Can bladder adenocarcinomas be distinguished from schistosomiasis-associated bladder cancers by using array comparative genomic hybridization analysis?

Bladder cancer is the most common malignancy in many tropical and subtropical areas, correlating well with the endemicity of schistostomiasis. The majority of schistostomiasis-associated (SA) bladder cancers are squamous cell cancers, whereas the majority of non-SA cases in the Western world are transitional cell cancers, suggesting different carcinogenetic mechanisms. Approximately 6% of SA and 1% of non-SA cases are adenocarcinomas. To achieve fine-resolution information of DNA copy number changes in SA adenocarcinomas, 10 tumor samples were analyzed on an oligonucleotide-based CGH array. The frequency of aberrations ranged from 2 to 17, with an average of 10 alterations per sample. The most frequently gained regions were 20q and 8q (in 70 and 60% of the cases, respectively), whereas the most frequently lost regions were 5q and 8p (both in 40% of the cases). In addition, six regions of amplification were found in three samples, containing both well characterized and novel regions. Comparison of the DNA copy number profiles to previously reported profiles of SA transitional cell carcinoma and squamous cell carcinoma revealed similarities (e.g., gains at 5p and 8q), as well as differences (e.g., TCC- and SCC-associated losses at 18p and 20p, and adenocarcinoma-associated gains at 20q). The results suggest that although SA cancers share genetic features, there also exist histology-specific regions of gain and loss.

Molecular karyotyping in sarcoma diagnostics and research.

Conventional cytogenetic and molecular genetic studies have both clinical and biological significance in sarcomas. However, the resolution of these methods does not always suffice to screening of novel, specific genetic changes, such as small deletions, amplifications, and fusion genes. Tumor-specific chromosomal translocations revealed by cytogenetic and molecular methods play a decisive role in the differential diagnosis of sarcomas. The novel molecular karyotyping techniques have proven to be powerful in the screening of clinically and biologically relevant molecular changes in human neoplasias. A variety of platforms for molecular karyotyping is available, e.g., arrayed cDNA clones or oligonucleotides that can be used in microarray-based comparative genomic hybridization (CGH) and gene expression analysis. We review here the clinically most relevant cytogenetic and molecular changes in sarcomas and describe latest microarray techniques for screening of clinically relevant gene copy number and expression changes.

Characterizing genetically stable and unstable gastric cancers by microsatellites and array comparative genomic hybridization.

Gastric cancer (GCA) displays a variety of genomic aberrations, including DNA copy number alterations, microsatellite instability (MSI), and loss of heterozygosity (LOH). The main aim of the present work was to determine the copy number aberrations in tumors with and without MSI or LOH. Fifteen fresh-frozen GCA samples, 11 of the intestinal and 4 of the diffuse type, were grouped by microsatellite analysis into high-level MSI (MSI-H, n = 2), LOH (n = 5), and microsatellite stable, LOH not detected (MSS/LOH-N, n = 8) tumors. The DNA samples were subsequently analyzed by array comparative genomic hybridization with 16,000 cDNA clones. As expected, the LOH tumors showed more copy number changes; however, the frequency of small-size amplifications was similar across all tumor groups. In addition, the cDNA arrays detected two apparently single-gene amplicons, at 11q13 (CCND1) and 12p12.1 (K-RAS), the presence of which were confirmed using oligonucleotide arrays. A novel amplicon at 5q13.2 was found only in diffuse-type tumors, which were otherwise genetically stable. The results suggest that DNA copy number changes may also occur in gastric cancers that show genomic stability in microsatellite analysis.

Pathology and molecular biology of gastric cancer.

Several attempts to classify gastric cancer (GCA) have been made over the past decades. Most successful, and widely used, is the classification by Laurén, which distinguishes, by microscopical morphology alone, two main cancer pathogeneses, diffuse (DGCA) and intestinal (IGCA) subtypes, which appear clearly as dissimilar clinical and epidemiological entities. Here we review the main differences in epidemiology, histopathology, and molecular pathology of the two main subtypes of gastric carcinomas based on Laurén classification. In clinical practice, however, clinical staging, particularly in predicting the survival, still remains superior to all classifications of gastric cancer independent of cancer type. The existence of local precursor lesions or conditions of IGCA tumours, i.e. Helicobacter pylori gastritis, atrophic gastritis (AG), intestinal metaplasia (IM), adenoma, dysplasia, and intramucosal neoplasia, is firmly established. The links of DGCA with intestinal-type epithelium, AG or IM are poor, or do not exist. So far, H. pylori gastritis is the only universal precursor condition for DGCA. It implies that AG and achlorhydria are of minor significance and infrequent in the development of DGCA but are important steps in that of IGCA. Despite an increasing body of data, the overall view on molecular pathology of GCA remains fragmentary. No consistent differences in the molecular pathology of GCA subtypes to meet the Laurén classification have been established. With the exception of TP53, no gene mutation occurring regularly in both histological types of GCA has been reported. Chromosomal aberrations and loss of heterozygosity seem to be non-specific and do not follow any consistent route in the progression of GCA. Microsatellite instability is more commonly found in IGCA than in DGCA. The present epigenetic data suggest that most of the decrease (or loss) of gene expression may be explained by promoter hypermethylation which is more often found in IGCA. In DGCA specific genes such as CDH1 are more often hypermethylated. Compared with GCA, in premalignant condition lesions gene mutations and chromosomal aberrations are infrequent. Epigenetic dysregulation might also represent a major mechanism for altered gene expression in premalignant stages in gastric carcinogenesis.

DNA copy number aberrations in intestinal-type gastric cancer revealed by array-based comparative genomic hybridization.

Genomic instability can be divided into 2 categories: chromosomal instability (CIN) and microsatellite instability (MSI). CIN has been linked to aneuploidy and chromosomal aberrations, and high-level loss of heterozygosity (LOH-H) has been suggested to be an indicator of CIN. High-level MSI (MSI-H), which results from nonfunctional mismatch repair, has previously been suggested to be mutually exclusive with CIN. Four MSI-H and three LOH-H primary gastric tumors of intestinal histology were used for copy number analysis by array-based comparative genomic hybridization (aCGH) with 13,000 cDNA targets. The MSI-H group showed fewer gains (0-12, average 4.5) and losses (0-10, average 2.5) per tumor as compared to the LOH-H group (9-15 gains, average 11.6 and 1-6 losses, average 4). Two MSI-H tumors did not show any copy number changes and one showed only gains of whole chromosomes. The most common alterations were gains of 20q (5/7 samples), 1q, 8, and 10p (3/7 samples) and losses of 1p and 5p (3/7 samples). The minimal amplified regions in 1q and 20q were localized to 1q21.1 approximately q21.2, 1q21.3, 20q11.2, 20q13.12, and 20q13.3 approximately qter. No copy number change was found to be specific for MSI-H or LOH-H. The results suggest that the LOH-H phenotype revealed by microsatellite analysis predicts reliably copy number abnormalities on aCGH and that a subset of MSI-H and all LOH-H tumors share the CIN phenotype.

Genome-wide differences between microsatellite stable and unstable colorectal tumors.

Genomic copy number changes are frequently found in cancers and they have been demonstrated to contribute to carcinogenesis; and it is widely accepted that tumors with microsatellite instability (MSI) are genetically stable and mostly diploid. In the present study we compared the copy number alterations and the gene-expression profiles of microsatellite stable (MSS) and MSI colorectal tumors. A total number of 31 fresh-frozen primary tumors (16 MSS and 15 MSI) were used. Twenty-eight samples (15 MSS and 13 MSI) were analyzed with metaphase comparative genomic hybridization (CGH), nine of which plus one additional sample (4 MSS and 6 MSI) were further analyzed by cDNA-based array-CGH. Gene expression analysis was performed with six samples [3 MSS and 3 MSI, four of these used in metaphase CGH (mCGH) analysis] to identify differentially expressed genes possibly located in the lost or amplified regions found by CGH, stressing the biological significance of copy number changes. Metaphase and array-CGH analysis of two colon cancer cell lines (HTC116 and SW480, reported as MSI and MSS archetypes) gave comparable results. Alterations found by mCGH in MSS tumors were +20, +8q, -8p and -18q. Interestingly, 1p22, 4q26 and 15q21 were found deleted preferentially in MSS tumors, while 22q13 was found gained in MSI tumors. The regions of alterations identified by array-CGH were gains at 8q24, 16q24.3 and 20q13, and the loss of 5q21, appearing in the both types of tumors. Gene expression analysis revealed genes with specific associations with the copy number changes of the corresponding genomic regions. As a conclusion, colorectal cancer is a heterogeneous disease, demonstrated by the genomic profiles of individual samples. However, our data shows that copy number changes do not occur exclusively in the MSS phenotypes.

New Insights into the Cellular Pathways Affected in Primary Uterine Leiomyosarcoma.

Resistance to chemotherapeutic agents and radiotherapy has kept surgery the primary treatment of uterine leiomyosarcoma (ULMS). In search of leads for potential therapeutic targets, array CGH (aCGH) was used to obtain a genomewide pattern of ULMS-specific genetic imbalances and to define the affected biological processes. Fine-resolution genomewide aCGH analysis was performed using customised 16K cDNA microarrays on 18 primary ULMS cases. Furthermore, patterns of DNA copy number changes were assessed for associations with clinical parameters, i.e., tumour grade, tumour size and patient status at last follow-up. Our aCGH results demonstrated extensive DNA copy number changes in all chromosomes. Of the 10,590 gene loci included in the analysis, 4,387 were found to be affected by DNA copy number gains and 4,518 by DNA copy number losses in at least one case. Further analyses revealed that 231 of these were commonly gained, and 265 lost in at least 20% of the cases. The gains affected loci at 1p, 1q, 2p, 3p, 6p, 8q, 10q and 18q, whereas losses were observed at 2q, 4q, 6p, 6q, 7p, 7q, 13q, 14p, 16q, 19p, Xp and Xq. Enrichment analysis of biological processes revealed the gained genes to be involved in the G1/S transition of mitotic cell cycle, co-translational protein targeting to membrane, actin filament polymerisation and positive regulation of cytokine biosynthesis, whereas the genes affected by losses were associated with DNA replication, chromatin modification, telomere maintenance, meiosis, mitosis and angiogenesis. These biological processes featured prominently two well-established tumour suppressors (BRCA2, EREG) and one proto-oncogene (GFI1). No statistically significant associations were found between the aberration patterns and clinical variables. Analysis of gene pathways using aCGH uncovered the biological networks involved in malignant progression of ULMS.

Intrinsic structural variation of the complex microsatellite marker MYCL1 in Finnish and Somali populations and its relevance to gastrointestinal tumors

The structurally complex MYCL1 microsatellite marker is often used to determine microsatellite instability in colorectal cancers but the allelic variation of this marker has remained largely uncharacterized in both populations and in cancers. Our study describes the allelic distributions of MYCL1 in Finnish (n = 117) and Somali population samples (n = 61) of non-related individuals and compares this distribution with the instability pattern obtained from 61 gastrointestinal tumors.

Differences in genomic instability between intestinal- and diffuse-type gastric cancer.

BACKGROUND: Microsatellite instability (MSI) and loss of heterozygosity (LOH) are lesions in the genome found with different frequencies in gastric carcinomas (GCAs). Despite a great body of studies, no systematic approach to the detailed classification of MSI and LOH in the two major types of GCA has been published. METHODS: Thirty-seven advanced GCAs, 25 intestinal-type (IGCAs) and 12 diffuse-type (DGCAs), were assayed with 15 autosomal tetranucleotide markers on 14 chromosomal arms. The observed frequencies and types of microsatellite alterations allowed stratification into subgroups, i.e., high- and low-grade MSI (MSI-H, MSI-L) or microsatellite-stable (MSS), and high- or low-grade, or non-detectable LOH (LOH-H, LOH-L, LOH-N). RESULTS: Collectively, the markers detected MSI-H tumors with sensitivity equal to that of BAT-26 (a single marker highly specific for MSI-H). Likewise, the markers detected LOH at chromosomal arms 5q, 18q, and 21q with a sensitivity equal to markers used previously. Seven (19%) MSI-H and six (16%) LOH-H tumors were found, with a significant association (P = 0.027) with IGCA: 92% of MSI-H and LOH-H occurred in IGCA patients only. Conversely, in DGCA, a significantly higher prevalence of a stable (LOH-N/MSS) phenotype was found as compared with IGCA (75.1% vs 28.0%; P = 0.035). The MSI-L phenotype was found in 57.9% of non-MSI-H IGCA tumors and was associated significantly (P = 0.015) with LOH-H. CONCLUSION: A clear difference in genomic instability between IGCA and DGCA was found. In IGCA, the MSI and LOH pathways were more commonly involved, whereas in DGCA, a stable phenotype was predominant. As a novel finding, MSI-L as a true phenomenon and its association with LOH was observed in IGCA.

Typing of XY (male) genotype from malignant neoplastic tissue by the amelogenin-based sex test.

DNA profiling of a cancer tissue can be problematic because of genomic instability. Here we have analyzed gastrointestinal cancer specimens from 46 males, of which seven (15%) showed aberrations in determination of gender by the widely used amelogenin test. The X-type amelogenin allele in all cases remained intact. All male tumor samples showing frequent autosomal loss of heterozygosity had a decreased signal of the Y allele from the amelogenin marker. When tested with an alternate set of primers for the amelogenin locus, the Y-type allele showed loss of heterozygosity in the same seven cases. However, when amplified with 15 Y-specific STR primers, all the cancerous tissue Y chromosomes seemed to be intact. These results indicate when malignant neoplastic tissue specimens are used, that amelogenin-based gender determination should be carefully interpreted.