Involvement of germline DDX1–MYCN duplication in inherited nephroblastoma

This report concerns a 3-year-old girl with prenatal bilateral nephroblastomatosis and a family history of nephroblastoma. This girl had a chromosome 8 pericentric inversion inherited from her father. This inversion was observed in healthy individuals of the family and was absent in other individuals suffering from embryonic kidney tumor. We then supposed that another genetic anomaly predisposed her to tumorogenesis. Additional cryptic imbalances are reported in cases of apparently balanced chromosomal rearrangements with an abnormal phenotype. Array-CGH analysis showed a 569 kb duplication at 2p24.3 including the DDX1 and MYCN genes. This duplication was inherited from the patient's father who also had a nephroblastoma. A link between germline MYCN duplication and the occurrence of other embryonic cancers such as neuroblastoma has already been described. We supposed that germline DDX1–MYCN duplication could also be involved in the apparition of nephroblastomas.     

Investigation of gene–gene interactions between CD40 and CD40L in Polish multiple sclerosis patients

CD40–CD40L interaction is necessary for the activation of both humoral and cellular immune response and has been suggested to play a role in the pathogenesis of multiple sclerosis (MS). Therefore, we analyzed the combined influence of the CD40 and CD40L variants on MS susceptibility and progression on well-defined Polish population. Our investigation revealed that CT individuals in rs1883832 locus of CD40 possessed almost 1.5-fold higher risk for MS than CC individuals (OR = 1.44; 95%CI = 1.03–2.1; p = 0.032), while this risk for TT individuals was almost 2.5-fold higher (OR = 2.36; 95%CI = 1.19–4.78; p = 0.014).     

Association analysis of GRIN1 and GRIN2B polymorphisms and Parkinson's disease in a hospital-based case–control study

Hyperactivation of N-methyl-d-aspartate receptors (NMDARs) leads to neuronal excitotoxicity and is suggested to play a role in many brain disorders, including Alzheimer's disease and schizophrenia. However, the association between polymorphisms in the genes that code for NMDAR subunits, N-methyl-d-aspartate 1 and 2B (GRIN1 and GRIN2B) and Parkinson's disease (PD) remains unclear. In a hospital-based case–control study of PD, DNA samples were collected from 101 PD patients and 205 healthy controls. Genotyping assays were used to screen for polymorphisms in the GRIN1 (rs2301364 T > C, rs28489906 T > C, and rs4880213 T > C) and GRIN2B (C366G, C2664T, and rs1805476 T > G) genes, and logistic regression analysis was then used to assess the association between these single nucleotide polymorphisms (SNPs) and PD susceptibility. None of the 6 SNPs were significantly associated with PD risk on their own. However, in conjunction with putative low-risk genotypes for the GRIN1 gene, the GRIN2BC366G variant was significantly associated with reduced PD risk compared with the homozygous genotype 366CC (OR = 0.38, 95%CI = 0.17–0.93, P = 0.033). A synergistic effect on risk reduction was observed in subjects who carried multiple polymorphisms of GRIN1 and the GRIN2BC366G polymorphism (OR = 0.78, 95%CI = 0.59–1.02, P_trend = 0.073). Our results suggest that polymorphisms in the GRIN1 and GRIN2B genes may serve as potential biomarkers for a reduced risk of PD among the Chinese population in Taiwan.     

Blood–brain barrier cell line PBMEC/C1-2 possesses functionally active P-glycoprotein

The blood–brain barrier (BBB) maintains the homeostasis between the central nervous system and the blood circulation. One of the main efflux transporter proteins at the BBB is P-glycoprotein (P-gP) also known as ABCB1 or MDR1. Due to the important role of P-gP for the transport barrier function of the BBB, the presence and functionality of P-gP was investigated in porcine cell line PBMEC/C1-2. Presence of P-gP was confirmed on the protein level by western blotting and immunofluorescence microscopy as well as on the mRNA level by qPCR. Functional assessment was accomplished by an established 96-well uptake assay using Rhodamine 123 and Doxorubicin as P-gP substrates and Verapamil as moderate P-gP inhibitor. In this regard, fluorescence microscopy confirmed a significant higher uptake of Rhodamine 123 into PBMEC/C1-2 cells when preincubated with Verapamil. Finally, knock-down of P-gP by antisense oligonucleotides revealed an increase of Rhodamine 123 uptake indicating decreased P-gP functionality. In summary, the presence and functionality of P-gP in the immortalised cell line PBMEC/C1-2 was proven with several techniques and assays. Thus, this cell line could be used for P-gP studies in the context of BBB relevant issues.     

An association study between the genetic polymorphisms within GnRHI, LHβ and FSHβ genes and central precocious puberty in Chinese girls

Objectives: Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are three hypothalamic–pituitary–gonadal axis expressing hormones. They play critical roles in the onset of puberty. Here we report the relationship between the three hormones and Central Precocious Puberty (CPP) in Chinese Han girls. Methods: We analyzed the single nucleotide polymorphisms (SNPs) of 5′-flanking regions of these genes by DNA sequencing in 27 CPP samples. Then the SNPs sites were genotyped by ligase detection reaction in a total of 283 Chinese Han CPP cases and 284 matched controls. Distributions of the polymorphisms and haplotypes were calculated for statistical evaluation. Results: Nine SNPs (One in GnRHI gene: −2003 C/T; Five in LHβ gene: −1456 C/G, −1424 C/G, −238 G/A, −164 G/A and −34 T/A; Three in FSHβ gene: −1825 T/C, −261 G/T and −132 T/A.) were found. A quantitative genetic association study was made. −1825 T/C in FSHβ gene was related with CPP with a weak effect (P = 0.025). A haplotype in the 5′-flanking region of LHβ gene was significantly associated with CPP in Chinese Han girls (P = 8.25 × 10⁻⁰⁹). However, analysis software showed that none of SNP was found in the regulating control element of these genes. Conclusions: Our finding implies that the polymorphisms in the 5′-flanking regions of FSHβ gene and LHβ gene probably were related to the puberty onset time of these girls. Further studies on the polymorphisms are needed for the exact mechanism.     

Role for the fibrinogen-binding proteins Coagulase and Efb in the Staphylococcus aureus–Candida interaction

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus–Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus–Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.     

Plasmodium falciparum origin recognition complex subunit 1 (PfOrc1) functionally complements Δsir3 mutant of Saccharomyces cerevisiae

Telomere position effect efficiently controls silencing of subtelomeric var genes, which are involved in antigenic variation in human malaria parasite Plasmodium falciparum. Although, PfOrc1 has been found to be associated with PfSir2 in the silencing complex, its function in telomere silencing remained uncertain especially due to an apparent lack of BAH domain at its amino-terminal region. Here we report that PfOrc1 possesses a Sir3/Orc1 like silencing activity. Using yeast as a surrogate organism we have shown that PfOrc1 could complement yeast Sir3 activity during telomere silencing in a Sir2 dependent manner. By constructing a series of chimera between PfOrc1 and ScSir3 we have observed that the amino-terminal domain of PfOrc1 harbors silencing activity similar to that present in the amino-terminal domain of ScSir3. We further generated several amino-terminal deletion mutants to dissect out such silencing activity and found that the first seventy amino acids at the amino-terminal domain are dispensable for its activity. Thus our results strongly supports that PfOrc1 may have a role in telomere silencing in this parasite. This finding will help to decipher the mechanism of telomere position effect in P. falciparum.     

Alleles and genotypes of polymorphisms of IL-18, TNF-α and IFN-γ are associated with a higher risk and severity of hepatocellular carcinoma (HCC) in Brazil

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver. We evaluated the association of alleles and genotypes of polymorphisms of IL-18 (-607C/A and -137G/C), IFN-γ (+874T/A) and TNF-α (-238G/A and -308G/A) with the risk and severity of HCC. One-hundred-and-twelve patients with HCC and 202 healthy controls were studied. Single nucleotide polymorphisms (SNPs) were amplified by PCR with specific primers and the products were submitted to polyacrylamide gel electrophoresis and stained with silver. We evaluated tumor presentation, tumor size and presence of metastasis. Significant higher risk of HCC was associated with: alleles IL-18 -607^∗A (P = 0.0235; OR = 1.48; 95%CI = 1.06–2.08); TNF-α -238^∗A (P = 0.0025; OR = 2.12; 95%CI = 1.32–3.40) and TNF-α -308^∗A (P = 0.0351; OR = 1.82; 95%CI = 1.07–3.08); and genotypes IL-18–607AA (P = 0.0048; OR = 3.03; 95%CI = 1.40–6.55); TNF-α -238GA (P = 0.0011; OR = 2.44; 95%CI = 1.45–4.12); and TNF-α -308GA (P = 0.0031; OR = 2.51; 95%CI = 1.39–4.51). Significant association was found between multinodular HCC and IL-18 -607^∗C allele (P = 0.029; OR = 2.40, 95%CI: 1.09–5.28), and IL-18 -607CC genotype (P = 0.028; OR = 3.5, 95%CI: 1.24–9.86). Diffuse HCC was significantly associated with IFN-γ +874TA genotype (P = 0.044; OR = 3.6, 95%CI: 1.03–12.47). The IL-18 -137^∗C allele showed a significant association with the presence of metastasis. Thus, IL-18 -607^∗A and TNF-α (-238^∗A and -308^∗A) alleles may confer susceptibility to HCC, while IL-18 -607^∗C and -137^∗C alleles more severe disease.     

GSK3β polymorphisms, MAPT H1 haplotype and Parkinson's disease in a Greek cohort

To determine whether polymorphisms in the microtubule-associated protein tau (MAPT) and/or glycogen synthase kinase-3β (GSK3β) genes underpin susceptibility to Parkinson's disease (PD), we conducted a case-control association study in a Greek cohort of 196 PD cases and 163 healthy controls. In our study, the MAPT H1 haplotype was found to be significantly associated with PD, no association was detected between the intronic rs6438552 (−157 T/C) GSK3β polymorphism and PD, whereas the C/C genotype of the promoter rs334558 (−50 T/C) GSK3β polymorphism was found to exert a protective role. The C/C genotype of the rs334558 GSK3β polymorphism was also found to have an additional protective role in our MAPT H1/H1 PD subgroup. Haplotype analysis revealed that, the T-T haplotype of both GSK3β polymorphisms was over-represented in PD patients compared to controls, and this association was independent of MAPT H1 haplotype.     

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate–cysteine ligase

Glutamate–cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate–cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51 kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35 dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate–cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.     

The interleukin-6 gene −572C/G promoter polymorphism modifies Alzheimer's risk in APOE ɛ4 carriers

Inflammatory response is involved in the etiopathology of Alzheimer's disease (AD). Interleukin-6 (IL-6), a pleiotropic inflammatory cytokine, was reported to be associated with both increased Aβ aggregation and the appearance of hyperphosphorylated tau in AD brain. To explore the association of genetic variants in the promoter of IL-6 gene with sporadic AD (SAD), a case–control study was conducted in a North Chinese Han population. A systematic screening of IL-6 promoter was performed using direct sequencing and two polymorphisms were obtained including −572C/G (rs1800796) and −384A/T (rs7802308). Definitive genotyping of these markers and apolipoprotein E (APOE) polymorphism were surveyed in 341 SAD patients and 421 controls. The results revealed no significant differences in the distributions of alleles or genotypes between SAD and control groups. However, there was an interaction between −572C/G and APOE genotypes (P = 0.016) using logistic analysis. In the subjects with APOE ?4, there were significant differences in the allele (P = 0.004) and genotype (P = 0.004) distributions of −572C/G polymorphism between SAD and control groups. The −572CC genotype increased risk for AD by 3.301-fold (Wald = 11.093, adjust OR = 3.301, 95% CI = 1.635–6.665, P = 0.001) compared to CG + GG genotype. The present results suggest the −572 polymorphism could modify the risk for SAD in APOE ?4 carriers.     

Host–pathogen interactions in epidermolysis bullosa patients colonized with Staphylococcus aureus

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus. We therefore determined the S. aureus colonization rates in EB patients from the Netherlands by collecting swabs from their anterior nares, throats and wounds. Within a period of ∼2 years, more than 90% of the sampled chronic wounds of EB patients were found to be colonized by S. aureus. Molecular typing revealed that EB patients were not colonized by a single S. aureus type. Rather the S. aureus population structure in the sampled EB patients mirrored the local S. aureus population structure within the Netherlands. Furthermore, multiple types of S. aureus were found in close proximity to each other within individual chronic wounds, indicating that these S. aureus types are not mutually exclusive. Over time, strong fluctuations in the S. aureus types sampled from individual EB patients were observed. This high exposure to different S. aureus types is apparently reflected by high plasma levels of antistaphylococcal IgG's, especially in patients carrying multiple S. aureus types. It remains to be determined to what extent this strong immune response protects EB patients against serious staphylococcal infections. Lastly, further research is needed to define the impact of staphylococcal colonization of chronic wounds on the development, exacerbation and healing of such wounds in patients with EB.