Frequency and distribution of FCN2 and FCN3 functional variants among MBL2 genotypes.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.The six types of pattern recognition molecules (PRMs) that initiate complement via the lectin pathway (LP) comprise collectins and ficolins. The importance of having various PRMs to initiate the LP is currently unclear. Mannan-binding lectin (MBL) is a collectin member of the LP PRMs. MBL deficiency is common with mild clinical consequence. Thus, the lack of MBL may be compensated for by the other PRMs. We hypothesized that variants FCN2 + 6424 and FCN3 + 1637delC that cause gene-dose-dependent reduction in ficolin-2 and ficolin-3 levels, respectively, may be rare in MBL-deficient individuals due to the importance of compensation within the LP. The aim of this study was to investigate the distribution and frequency of these variants among MBL2 genotypes in healthy subjects. The allele frequency of FCN2 + 6424 and FCN3 + 1637delC was 0.099 and 0.015, respectively, in the cohort (n = 498). The frequency of FCN2 + 6424 tended to be lower among MBL-deficient subjects (n = 53) than among MBL-sufficient subjects (n = 445) (0.047 versus 0.106, P = 0.057). In addition, individuals who were homozygous for FCN2 + 6424 were sufficient MBL producers. The frequency of FCN3 + 1637delC did not differ between the groups. The frequency of FCN2 + 6424 was similar in FCN3 + 1637delC carriers (n = 15) versus wild type (n = 498). Furthermore, subjects that were heterozygote carriers of both FCN2 + 6424 and FCN3 + 1637delC were sufficient MBL producers. In conclusion, FCN2 + 6424 carriers with MBL deficiency tend to be rare among healthy individuals. MBL-deficient individuals with additional LP PRM defects may be at risk to morbidity.Icelandic Research Fund Unversity of Iceland Research Fund Landspitali University Hospital Research Fun

Normal neonatal TREC and KREC levels in early onset juvenile idiopathic arthritis

Objective: Dysregulated central tolerance predisposes to autoimmune diseases. Reduced thymic output as well as compromised central B cell tolerance checkpoints have been proposed in the pathogenesis of juvenile idiopathic arthritis (JIA). The aim of this study was to investigate neonatal levels of T-cell receptor excision circles (TRECs) and kappa-deleting element excision circles (KRECs), as markers of T- and B-cell output at birth, in patients with early onset JIA. Methods: TRECs and KRECs were quantitated by multiplex qPCR from dried blood spots (DBS), collected 2–5 days after birth, in 156 children with early onset JIA and in 312 matched controls. Results: When analysed from neonatal dried blood spots, the median TREC level was 78 (IQR 55–113) in JIA cases and 88 (IQR 57–117) copies/well in controls. The median KREC level was 51 (IQR 35–69) and 53 (IQR 35–74) copies/well, in JIA cases and controls, respectively. Stratification by sex and age at disease onset did not reveal any difference in the levels of TRECs and KRECs. Conclusion: T- and B-cell output at birth, as measured by TREC and KREC levels in neonatal dried blood spots, does not differ in children with early onset JIA compared to controls

A rapid real-time qRT-PCR assay for ovine beta-actin mRNA

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldBeta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells

Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldWe developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 x 10(7) copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV

Encapsidation Determinants Located Downstream of the Major Splice Donor in the Maedi-Visna Virus Leader Region

We investigated the role of the 5′-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computer-predicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (Δ3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (Δ5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The Δ5MSD mutant and a double mutant (DM) (Δ5MSD and Δ3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in Δ5MSD was dispensable in FOS cells, and the DM had the same REE as the Δ3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors

Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldWe developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 x 10(7) copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV

Familiality of benign and malignant paraproteinemias. A population-based cancer-registry study of multiple myeloma families

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldBACKGROUND AND OBJECTIVES: The occurrence of two or more cases of multiple myeloma (MM) in the same family has been reported from time to time. The current study is the first population- and cancer-registry-based survey to investigate familiality of premalignant or malignant B-cell proliferation. DESIGN AND METHODS: A family registry of 218 multiple myeloma cases was compared with the records of the Icelandic Cancer Registry in order to analyze the pedigrees for the occurrence of families with multiple cases of paraproteinemia and hematologic malignancies. RESULTS: The relative risk of developing monoclonal gammopathies of unknown significance (MGUS) was not increased among first-degree relatives of MM patients, but there was a significantly increased risk of developing MM for females separately (RR = 3.23, CI 1.17-7.01) and for males and females combined (RR = 2.33, CI 1.12-4.26). Analysis for all hematologic malignancies showed an increased risk for female relatives of MM patients (RR = 1.95, CI 1.10-3.20). Eight families were identified in which the propositus with MM had > 1 relatives with MGUS and > 1 with another hematologic malignancy, including 4 families with another relative with MM. In three families both myeloid and lymphoid malignancies occurred. INTERPRETATION AND CONCLUSIONS: Although inheritance does not appear to be a major risk factor for the development of paraproteinemias a significant risk of developing MM was found for female relatives. The occurrence of multiple cases of benign and malignant paraproteinemias in a few families does suggest a hereditary contribution. Further studies of such families might reveal clues on pathogenesis

Monoclonal gammopathy in Iceland: a population-based registry and follow-up

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldThe term monoclonal gammopathy (MG) signifies the benign or malignant clonal growth of B lymphocytes. In the present study, monoclonal gammopathy of unknown significance (MGUS) was defined as those patients with no identified haematological malignancy. A database was constructed of all 713 MG patients in Iceland between 1976 and 1997 and compared with the Icelandic Cancer Registry. The age-standardized incidence per 100 000 of MG was 10.3 for males and 8.6 for females, calculated for the whole period, rising steadily from 5.8 (men) and 4.9 (women) during the 5-year period 1976-80 to 14.7 (men) and 12.5 (women) during the last 5 year period. Age-standardized incidence rates were very low for subjects under 50 years of age, then increased with age from 11 and 17 per 100 000 at 50-54, to 169 and 119 per 100 000 at age 80-84, for men and women respectively. No association was detected between MG and non-haematological malignancies, neither retrospectively nor prospectively. Haematological malignancy was diagnosed in 209 (29.3%) cases before the recorded finding of MG or within the same calendar year, leaving 504 (70.7%) patients diagnosed with MGUS. Of these, 51 (10%) progressed to multiple myeloma or Waldenström's macroglobulinaemia after a mean interval of 3.8 years; mean follow-up was 7.4 years, median 6 years. The most common immunoglobulin (Ig) class was IgG (55%), followed by IgM (32%) and IgA (13%). MGUS was a highly significant risk factor for developing haematological malignancies and the risk was significantly greater for MG of the IgA class compared with either IgG or IgM