Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus

Two polymorphisms of the human Ig(lambda) (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the C(lambda)2-C(lambda)3 region. The second polymorphism is the Mcg(-)Ke(+)Oz(-) isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic C(lambda)2 segment because of its high homology with the Mcg(-)Ke(-)Oz(-) C(lambda)2 isotype. It has been speculated that the Mcg(-)Ke(+)Oz(-) isotype might be encoded by a C(lambda) gene segment of the amplified C(lambda)2-C(lambda)3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-C(lambda)2 region and is likely to have originated from unequal crossing over of the J-C(lambda)2 and J-C(lambda)3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-C(lambda)2 per genome regions, leading to decreased Ig(kappa):Ig(lambda) ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg(-)Ke(+)Oz(-) isotype is encoded by a polymorphic C(lambda)2 segment that differs from the normal C(lambda)2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-C(lambda)2 amplification polymorphism and was not found in the J-C(lambda)2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.     

End stage renal disease patients have a skewed T cell receptor Vβ repertoire

Background

End stage renal disease (ESRD) is associated with defective T-cell mediated immunity. A diverse T-cell receptor (TCR) Vβ repertoire is central to effective T-cell mediated immune responses to foreign antigens. In this study, the effect of ESRD on TCR Vβ repertoire was assessed.

Results

A higher proportion of ESRD patients (68.9 %) had a skewed TCR Vβ repertoire compared to age and cytomegalovirus (CMV) – IgG serostatus matched healthy individuals (31.4 %, P?<?0.001). Age, CMV serostatus and ESRD were independently associated with an increase in shifting of the TCR Vβ repertoire. More differentiated CD8⁺ T cells were observed in young ESRD patients with a shifted TCR Vβ repertoire. CD31-expressing naive T cells and relative telomere length of T cells were not significantly related to TCR Vβ skewing.

Conclusions

ESRD significantly skewed the TCR Vβ repertoire particularly in the elderly population, which may contribute to the uremia-associated defect in T-cell mediated immunity.

     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA

Embryonic stem (ES) cell technology allows modification of the mouse germline from large deletions and insertions to single nucleotide substitutions by homologous recombination. Identification of these rare events demands an accurate and fast detection method. Current methods for detection rely on Southern blotting and/or conventional PCR. Both the techniques have major drawbacks, Southern blotting is time-consuming and PCR can generate false positives. As an alternative, we here demonstrate a novel approach of Multiplex Ligation-dependent Probe Amplification (MLPA) as a quick, quantitative and reliable method for the detection of homologous, non-homologous and incomplete recombination events in ES cell clones. We have adapted MLPA to detect homologous recombinants in ES cell clones targeted with two different constructs: one introduces a single nucleotide change in the PCNA gene and the other allows for a conditional inactivation of the wild-type PCNA allele. By using MLPA probes consisting of three oligonucleotides we were able to simultaneously detect and quantify both wild-type and mutant alleles.     

Transplacental Zika virus transmission in ex vivo perfused human placentas

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.     

Intelligence May Moderate the Cognitive Profile of Patients with ASD

BackgroundThe intelligence of individuals with Autism Spectrum Disorder (ASD) varies considerably. The pattern of cognitive deficits associated with ASD may differ depending on intelligence. We aimed to study the absolute and relative severity of cognitive deficits in participants with ASD in relation to IQ.MethodsA total of 274 children (M age = 12.1, 68.6% boys) participated: 30 ASD and 22 controls in the below average Intelligence Quotient (IQ) group (IQ<85), 57 ASD and 54 controls in the average IQ group (85<IQ<115) and 41 ASD and 70 controls in the above average IQ group (IQ>115). Matching for age, sex, Full Scale IQ (FSIQ), Verbal IQ (VIQ), Performance IQ (PIQ) and VIQ-PIQ difference was performed. Speed and accuracy of social cognition, executive functioning, visual pattern recognition and basic processing speed were examined per domain and as a composite score.ResultsThe composite score revealed a trend significant IQ by ASD interaction (significant when excluding the average IQ group). In absolute terms, participants with below average IQs performed poorest (regardless of diagnosis). However, in relative terms, above average intelligent participants with ASD showed the most substantial cognitive problems (particularly for social cognition, visual pattern recognition and verbal working memory) since this group differed significantly from the IQ-matched control group (p < .001), whereas this was not the case for below-average intelligence participants with ASD (p = .57).ConclusionsIn relative terms, cognitive deficits appear somewhat more severe in individuals with ASD and above average IQs compared to the below average IQ patients with ASD. Even though high IQ ASD individuals enjoy a certain protection from their higher IQ, they clearly demonstrate cognitive impairments that may be targeted in clinical assessment and treatment. Conversely, even though in absolute terms ASD patients with below average IQs were clearly more impaired than ASD patients with average to above average IQs, the differences in cognitive functioning between participants with and without ASD on the lower end of the IQ spectrum were less pronounced. Clinically this may imply that cognitive assessment and training of cognitive skills in below average intelligent children with ASD may be a less fruitful endeavour. These findings tentatively suggest that intelligence may act as a moderator in the cognitive presentation of ASD, with qualitatively different cognitive processes affected in patients at the high and low end of the IQ spectrum.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.