Polymerase Chain Reaction-based DNA Research Articles

Development and successful clinical application of preimplantation genetic haplotyping for Herlitz junctional epidermolysis bullosa

Herlitz junctional epidermolysis bullosa (HJEB) is a severe, life-threatening, autosomal recessive blistering skin disease for which no cure is currently available. Prenatal diagnosis for couples at risk is feasible through fetal skin biopsy or analysis of DNA extracted from chorionic villi, but these methods can be applied only after pregnancy has been established. An alternative approach, which involves the analysis of single cells from embryos prior to establishment of pregnancy, is preimplantation genetic diagnosis (PGD). Until now, its clinical uptake has been hindered by lengthy delays in establishing mutation-specific protocols, and by the small amount of template DNA that can be obtained from a single cell. A new method that addresses these problems, preimplantation genetic haplotyping (PGH), relies on whole genome amplification followed by haplotyping of multiple polymorphic markers using standard DNA-based polymerase chain reaction (PCR) assays. To design and validate a generic PGH assay for HJEB and to transfer this into clinical practice. We established a multiplex PCR-based PGH assay involving 16 markers within and flanking the LAMB3 gene (the most frequently mutated gene in HJEB). The assay was then validated in 10 families with at least one previously affected offspring. After licensing by the Human Fertilisation and Embryology Authority (HFEA), the new test was used for PGD in a couple at risk of HJEB. The chromosome 1 LAMB3 markers within the assay were shown to be of sufficient heterogeneity to have widespread application for preimplantation testing of HJEB. In one couple that were heterozygous carriers of nonsense mutations in LAMB3, we used the new assay to identify unaffected embryos in a series of PGD cycles. Pregnancy was established in the third PGD cycle and a healthy, unaffected child was born. DNA analysis of cord blood confirmed the predicted single-cell mutation status of wild-type LAMB3 alleles. PGH represents a major step forward in widening the scope and availability of preimplantation testing for serious mapped single-gene disorders. We have established a generic test that is suitable for the majority of couples at risk of HJEB.

Unique histological characteristics of Scedosporium that could aid in its identification

Scedosporium prolificans has been increasingly recognized as an etiological agent of disseminated mycelial infections in profoundly immunocompromised patients. Reported herein is a case of disseminated S. prolificans infection in a patient undergoing anti-neoplastic chemotherapy for acute myeloid leukemia. Antemortem blood culture yielded S. prolificans, which was confirmed on conventional morphological examination and polymerase chain reaction-based DNA sequencing targeting internally transcribed spacer regions. Histopathology of autopsy specimens indicated fungal infection in the heart, lungs, liver, kidneys, spleen, pancreas and gastrointestinal tract, with the development of hemorrhagic and ischemic necrosis. The infecting fungus had developing septate hyphae and was identified as belonging to the genus Scedosporium, on in situ hybridization of tissue. The combination of haphazardly branching hyphae and lemon-shaped conidia appeared to be the most useful distinguishing features to allow differentiation of this fungus from other filamentous fungi in tissue. Three other unique histopathological characteristics of the fungus were noted: (i) parallel hyphae bridged at right angles to produce letter-H patterns; (ii) intravascular conidiation; and (iii) purple conidia in tissue, though these are usually described as brown in most text books. Precise histopathology, in addition to other techniques such as in situ hybridization, can aid in the identification of etiological fungi.

Inflammatory cytokine gene polymorphisms increase the risk of atrophic gastritis and intestinal metaplasia

To investigate the effects of interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF) gene polymorphisms, Helicobacter pylori (H. pylori) infection, on the risk of developing severe chronic atrophic gastritis (SCAG) and intestinal metaplasia (IM). A total of 372 cases were selected from a cohort study in Linqu County, a high risk area for gastric cancer (GC) in northern China. To obtain a sufficient group size, patients with normal or superficial gastritis were included. Based on an average follow-up period of 56 mo, the 372 cases were divided into no progression group (no histological progression from normal or superficial gastritis, n = 137), group I (progressed from normal or superficial gastritis to SCAG, n = 134) and group II (progressed from normal or superficial gastritis to IM, n = 101). IL-8, MIF gene polymorphisms were detected by polymerase chain reaction-based denaturing high-performance liquid chromatography analysis and DNA sequencing. An increased risk of SCAG was found in subjects with IL-8-251 AA genotype [odds ratio (OR) = 2.62, 95% CI: 1.23-5.72] or IL-8-251 A allele carriers (AA + AT) (OR = 1.81, 95% CI: 1.06-3.09). An elevated risk of IM was found in subjects with IL-8-251 AT genotype (OR = 2.27, 95% CI: 1.25-4.14) or IL-8-251 A allele carriers (OR = 2.07, 95% CI: 1.16-3.69). An increased risk of SCAG was found in subjects with MIF-173 GC genotype (OR = 2.36, 95% CI: 1.38-4.02) or MIF-173 C allele carriers (GC + CC) (OR = 2.07, 95% CI: 1.21-3.55). An elevated risk of IM was found in subjects with MIF-173 CC genotype (OR = 2.27, 95% CI: 1.16-4.46) or MIF-173 C allele carriers (OR = 3.84, 95% CI: 1.58-9.34). The risk of SCAG and IM was more evident in subjects carrying IL-8-251 A allele (OR = 6.70, 95% CI: 1.29-9.78) or MIF-173 C allele (OR = 6.54, 95% CI: 2.97-14.20) and positive for H. pylori infection. IL-8-251 and MIF-173 gene polymorphisms are significantly associated with the risk of SCAG and IM in a population with a high risk of GC in Linqu County, Shandong Province, China.

A new genetic lesion in B‐CLL: a NOTCH1 PEST domain mutation

We recently demonstrated that Notch1 and Notch2 constitutive activation plays a critical role in survival and apoptosis resistance of B-chronic lymphocytic leukemia (B-CLL) cells (Rosati et al, 2009). B-CLL cells also constitutively over-expressed Jagged1 and Jagged2 ligands, suggesting autocrine/paracrine loops for constitutive Notch signalling activation (Rosati et al, 2009). Notch activation mechanisms appear different in B-CLL and T-cell acute lymphoblastic leukemia (T-ALL) (Osborne, 2009). In B-CLL the altered generation of Notch1- and Notch2-intracellular (IC) domains, which is a crucial event for Notch signalling activation (Kopan, 2002), depends on ligand interactions (Rosati et al, 2009). In T-ALL, the aberrant Notch1-IC generation occurs in a ligand-independent fashion and is due to NOTCH1 activating mutations (Weng et al, 2004; O’Neil et al, 2006) which involve the heterodimerization (HD) domain, facilitating Notch1-IC proteolytic cleavage and/or the negative regulatory PEST domain, increasing Notch1-IC half-life (Monsour et al, 2006). To determine whether NOTCH1 activating mutations are present in B-CLL we studied 43 B-CLL patients (34 males/9 females, median age 59 years, range 44–79; median follow-up 84 months, range 12–204). ZAP70 expression and IGHV mutational status (Rassenti et al, 2008) were also determined (25 ZAP-70 positive; 10 ZAP-70 negative, 8 undetermined; 26 unmutated IGHV, 17 mutated IGHV) (Table I). Mutations of NOTCH1 exons 26, 27 and 34 were investigated by DNA-based polymerase chain reaction as described elsewhere (Weng et al, 2004) and frequencies were determined using the National Center for Biotechnology Information (NCBI) database. Cycling conditions were: 95°C for 5 min (one cycle); 95°C for 45 s, 58°C for 45 s, 72°C for 1 min for 36 cycles; 72°C for 6 min (one cycle). After direct sequencing from both strands, purified amplicons were compared with corresponding germline sequences, and germline polymorphisms in the NCBI SNP database were excluded. NOTCH1 sequencing did not detect mutations at the HD cleavage site. A PEST domain mutation i.e. 7544_7545 DEL (CT) was found in 2/43 patients (4.6%) (Fig 1). The mutation was confirmed by independent amplification of the amplicon. Chromatogram of NOTCH1 mutation in B-CLL as identified by direct sequencing. The arrow indicates the identified deletion (ΔCT). As suggested for T-ALL, deletions of the COOH-terminal sequences in the PEST domain, which regulates protein turnover by targeting proteins to the ubiquitin-proteosome complex for subsequent degradation, could enhance Notch-IC stability and signalling. Unlike the various mutations in T-ALL, the same NOTCH1 PEST domain mutation was found in these B-CLL patients. All had unmutated IGHV, were ZAP-70 positive and had a very poor prognosis. Both developed progressive disease from diagnosis, became resistant to more than three lines of chemotherapy and one died soon after the development of diffuse large B-cell lymphoma. In attempting to reconcile this first evidence of a NOTCH1 PEST mutation in a minority of B-CLL patients (4.6%), all with constitutively activated Notch1 and Notch2 (Rosati et al, 2009), one can only speculate that NOTCH1 mutations participate in Notch activation in B-CLL, at least in the subgroup carrying the mutation. They could be cooperating events and other genetic lesions may be involved. In fact, Notch2 plays a role in the development and maintenance of selected B cell subsets including marginal zone and B1 B cells (Witt et al, 2003) and was mutated in both HD and PEST domains in 5% of Marginal Zone Lymphoma cases (Troen et al, 2008). Additional studies are needed to clarify whether and how NOTCH mutations contribute to altered Notch signalling and oncogenesis in B-CLL. We would like to thank Dr Geraldine Anne Boyd for her help. This work was supported by ‘Associazione Umbra Leucemie e Linfomi’, Perugia Italy, by ‘Associazione Italiana Leucemie, Linfomi e Mielomi’, L’Aquila Section, L’Aquila, Italy and by ‘Fondazione Cassa di Risparmio di Perugia-2009’, Perugia, Italy.

Does the frequency of Prevotella intermedia increase during pregnancy?

The former Bacteroides intermedius, currently including Prevotella intermedia and Prevotella nigrescens, has been associated with hormone-induced pregnancy gingivitis. The aim of the present longitudinal study was to determine whether only P. intermedia or P. nigrescens, or both species, are involved in the demonstrated microbial shift during pregnancy. Subgingival plaque and saliva samples, collected from 30 healthy pregnant women and 24 healthy non-pregnant women as their controls, were examined for the presence of pigmented gram-negative anaerobes. Altogether 2628 isolates were preliminarily identified as P. intermedia sensu lato, based on phenotypic testing. Their further identification was performed by using a 16S ribosomal DNA-based polymerase chain reaction (PCR). A mean of 8.3 P. intermedia sensu lato isolates from each subject/sampling was examined. During the second trimester, the mean number of P. intermedia sensu lato in plaque increased along with increasing signs of pregnancy gingivitis, and then both decreased. After delivery, gingival inflammation still decreased while the number of P. intermedia sensu lato transiently increased both in plaque and saliva. In the present study, the vast majority of isolates (95.3%) proved to be P. nigrescens and 2.5% were P. intermedia. The remaining 2.2% of the isolates could not be identified with PCR as P. intermedia or P. nigrescens. The corresponding percentages in the control population were 94.2%, 5.5%, and 0.3%. In the oral cavity of relatively young women without periodontitis, P. nigrescens, unlike P. intermedia, is a frequent finding. Conceivably, pregnant women harbor increasing numbers of P. nigrescens associated with pregnancy gingivitis.

DNA-based Polymerase Chain Reaction for Detecting FUS-CREB3L2 in Low-grade Fibromyxoid Sarcoma Using Formalin-fixed, Paraffin-embedded Tissue Specimens

Detection of the tumor-specific fusion gene, FUS-CREB3L2, is useful in the diagnosis of low-grade fibromyxoid sarcoma (LGFMS), especially by reverse transcription (RT)-polymerase chain reaction (PCR) using formalin-fixed, paraffin-embedded (FFPE) tumor tissues. Because FUS and CREB3L2 gene segments with their break points within exons are fused without interposed introns in many LGFMSs, DNA-based PCR may also be applicable to detect the FUS-CREB3L2 fusion gene using FFPE specimens. In this study, DNA and RNA were extracted from FFPE specimens of 16 and 19 LGFMSs, respectively, and PCR and RT-PCR were performed using primers that specifically amplify most of the junctional regions of the FUS-CREB3L2 fusion gene. RT-PCR analysis revealed FUS-CREB3L2 fusion transcripts in 16 of the 19 (84%) LGFMSs. In 14 informative examples, including 11 with detectable fusion transcripts by RT-PCR, DNA-based PCR amplified the FUS-CREB3L2 fusion gene in 9 (64%) examples. Nucleotide sequence analysis confirmed that PCR products were identical to the respective RT-PCR products in 8 cases. Although the sensitivity was not as high as RT-PCR, DNA-based PCR can be used as an alternative molecular approach for detecting the FUS-CREB3L2 fusion gene.

Lung transplantation complicated by graft‐versus‐host disease and confounded by incidental transfusion‐associated macrochimerism

Passenger lymphocyte-mediated graft-versus-host disease (GVHD) in solid organ transplantation (SOT-GVHD) is considered a rare complication, particularly among recipients of lung allografts. The risk of transfusion-associated GVHD (TA-GVHD) in solid organ transplants is also considered rare. The suspicion of either may be heralded by signs and symptoms of GVHD in the company of a population of passenger lymphocytes in excess of 1 percent (microchimerism). This case report illustrates the challenge of a patient who presented with macrochimerism both from the lung transplant allograft and from transfusions. Chimerism assessments of the pre- and posttransplant donor lung, and the recipient's aplastic marrow, were made using DNA-based polymerase chain reaction testing. Macrochimerism was observed in both the posttransplant aplastic host marrow and the engrafted donor lung, with the former predominantly consisting of lung donor lymphocytes and the latter a mixture of lung and presumably transfusion source donor lymphocytes. The pretransplant donor lung exhibited no GVHD-like pathology. This case demonstrates SOT-GVHD, with the unusual feature of concomitant macrochimerism from transfusions. SOT-GVHD likely predisposed this patient to the observed transfusion-associated macrochimerism. However, the dissociation between transfusion-attributable macrochimerism and attributable pathology is intriguing. Furthermore, the risk spectrum of transfusion-associated macrochimerism and TA-GVHD in solid organ transplant recipients with and without the complication of SOT-GVHD is unknown and warrants further study.

Detection ofChlamydia trachomatisOcular Infection in Trachoma-Endemic Communities by rRNA Amplification

Trachoma remains the leading infectious cause of blindness worldwide. The World Health Organization (WHO) recommends mass antibiotic distributions in its strategy to eliminate blinding trachoma. To determine the most effective antibiotic treatment strategy, it is essential to have a diagnostic test that can correctly measure the true status of ocular Chlamydia trachomatis infection in individuals, particularly after treatment. A newer ribosomal ribonucleic acid (rRNA)-based amplification test was compared with the current DNA-based polymerase chain reaction (PCR) for the detection of C. trachomatis. An rRNA-based assay and PCR were performed on swab specimens taken from the right upper tarsal conjunctiva of 240 children aged 1 to 5 years living among 16 endemic villages in the Gurage Zone, Ethiopia. The rRNA-based test detected ocular C. trachomatis infection in 142 (59%) subjects compared with 67 (28%) detected by PCR (McNemar's test, P < 0.0001). The rRNA-based test gave positive results for all subjects who were positive by PCR and detected infection in 75 (31%) additional subjects. The rRNA-based test appears to have significantly greater sensitivity than PCR for the detection of ocular C. trachomatis infection in children in trachoma-endemic villages. The increased sensitivity of the rRNA-based test may be due to its ability to detect low levels of C. trachomatis infection in individuals, which can occur especially after antibiotic treatment. Data from past studies in which PCR was used to assess the prevalence of infectious trachoma after community-wide antibiotic treatments could have underestimated the true prevalence of infection.

CTLA-4 +49 A/G genotype and HLA-DRB1 polymorphisms in Turkish patients with Henoch–Schönlein purpura

The pathogenesis of Henoch-Schönlein purpura (HSP) remains unknown; however, it is generally considered to be an immune complex-mediated disease. Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is expressed on activated T cells, and, thus, it is critically involved in the immune response. We aimed to investigate the possible influence of CTLA-4 polymorphisms for susceptibility to HSP and determine if there were associations with human leukocyte antigen (HLA)-DRB1 genotypes. Using polymerase chain reaction-based DNA genotyping, we investigated the polymorphisms located in the genes encoding CTLA-4 in 100 patients with HSP and 156 ethnically matched healthy controls. When CTLA-4 +49 A/G polymorphism of HSP patients and control group was compared, no associations with joint, gastrointestinal or renal manifestations, or susceptibility to HSP, were observed. However, patients with nephrotic proteinuria had higher HLA-DRB1*13 positivity [odds ratio (OR) = 3.76, 95% confidence interval (95%CI) = 1.25-11.23, P = 0.025]. When the patients were stratified according to CTLA-4 polymorphism, a significant association between nephrotic proteinuria patients and carriage of the AG genotype was also found (OR = 15.42, 95%CI = 1.59-148.82, P = 0.008). These results suggested that CTLA-4 +49 A/G polymorphism does not contribute to susceptibility to HSP; however, the presence of CTLA-4 AG genotype and HLA-DRB1*13 could be a risk factor for developing nephrotic-range proteinuria in these patients.

Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)

Intron F G79a polymorphism of the protein Z gene in cancer patients with and without thrombosis

Venous thromboembolism (VTE) is well-recognized complication of cancer; however its pathogenesis is not entirely established. Protein Z (PZ) is a member of the coagulation cascade. We aimed to investigate whether intron F G79A polymorphism of PZ gene is associated with risk of VTE in cancer patients. The study consisted of 55 cancer patients who developed thrombosis (group 1) and 115 cancer patients without VTE (group 2). Intron F G79A of PZ gene, factor V Leiden (FVL) and prothrombin (PT) G20210A polymorphisms were determined by the method of polymerase chain reaction-based DNA analysis. The prevalence of FVL was significantly greater in group 1 compared with group 2 (27.3% vs. 3.5%, P < 0.0001). No differences were seen in genotype and allele frequencies of PZ gene between two groups (P > 0.05). When the analysis was also made after excluding FVL positive patients, the same insignificant result was found. Intron F G79A polymorphism of PZ gene does not contribute to meaningful diagnostic investigation of thrombophilia in cancer patients.

Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens

Determining the effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broilers. 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling, DNA sequencing and real-time quantitative PCR techniques were used. The richness of both ileal and caecal microbiota increased with chicken age. The microbiota from those birds of the same age demonstrated relatively similar PCR-DGGE profiles and tended to form closely related clusters in the relatedness analyses. Dietary treatment with bacitracin (50 mg kg(-1)) and access to range did not change the richness but altered the composition of the microbiota. The impact of bacitracin was particularly obvious in 3-day-old chicks. Lactobacilli were abundant in the caecal microbiota of 3-day-old chicks regardless of the dietary treatment with bacitracin. The access to range enriched Bifidobacterium in both the ileum and caeca. Bacitracin, bird age and access to range all influenced bacterial microbiota in the ileum and caeca of broilers, with bird age having the greatest apparent effect. Providing useful information for the development of antibiotic replacement therapy for poultry production.

SERUM LEVELS OF MYCN AMPLIFICATION IN NEUROBLASTOMA WITH TUMOR-RELEASED DNA BY REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION

INTRODUCTION: MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma and is routinely assayed for therapy stratification. OBJECTIVE: We aimed to develop a diagnostic tool to predict MYCN status by using serum DNA, which, in patients with cancer, predominantly originates from tumor-released DNA. METHODS: Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as a MYCN/NAGK ratio in 87 patients with neuroblastoma whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified neuroblastoma, and 70 had nonamplified neuroblastoma. RESULTS: The serum MYCN/NAGK ratio in the MNA group (median: 199.32; range: 17.1–901.6 [99% confidence interval: 107.0–528.7]) was significantly (P &amp;lt; .001) higher than that in the non-MNA group (median: 0.87; range: 0.25–4.6 [99% confidence interval: 0.82–1.26], Mann-Whitney U test). The sensitivity and specificity of the serum MYCN/NAGK ratio as a diagnostic test were both 100% when the serum MYCN/NAGK ratio cutoff was set at 10.0. Among 6 patients in the MNA group whose clinical courses were followed, the serum ratios decreased to within the normal range in the patients in remission (n = 3), but they rose to high levels in the patients who had a relapse (n = 2) or failed to achieve remission (n = 1). The serum MYCN/NAGK ratio in the MNA group is likely to be the more sensitive tumor marker than conventional urinary vanillylmandelic and homovanillic acid markers and neuron-specific enolase markers to predict patients' clinical course. CONCLUSIONS: Measurement of the serum MYCN/NAGK ratio seems to be a promising method for accurately assessing MYCN status in neuroblastoma.

Diagnostic value of p16INK4a in squamous intraepithelial lesion in gynecologic cytology

To study the diagnostic value of p16(INK4a) in squamous intraepithelial lesion in gynecologic cytology and its relationship with types of human papillomavirus (HPV). 88 liquid-based gynecologic cytology cases with histologic correlation, including 20 cases of cervicitis, 18 cases of low-grade squamous intraepithelial lesion (LSIL), 34 cases of high-grade squamous intraepithelial lesion (HSIL) and 16 cases of squamous cell carcinoma (SCC), were enrolled into the study. Immunocytochemistry for p16(INK4a) protein and polymerase chain reaction-based HPV DNA testing (for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, as well as HPV types 6, 11, 42, 43 and 44) were performed. The rate of expression of p16(INK4a) protein was 0, 27.8%, 100% and 100% in the cervicitis group, LSIL group, HSIL group and SCC group, respectively. The expression was significantly higher in the latter 3 groups than that in the cervicitis group (P < 0.01). Besides, the expression was significantly higher in cases associated with high-risk HPV genotypes (96.4%) than in cases associated with low-risk HPV genotypes (7.7%). p16(INK4a) is a valuable biomarker for detection of HPV-related dysplastic squamous cells, with high sensitivity and specificity.

Characterization of three new imatinib-responsive fusion genes in chronic myeloproliferative disorders generated by disruption of the platelet-derived growth factor receptor gene

We sought to identify new fusion genes with involvement of the platelet-derived growth factor receptor beta gene (PDGFRB) in three patients presenting with various subtypes of chronic myeloproliferative disorders associated with chromosomal aberrations involving chromosome bands 5q31-33. We performed 5 rapid amplification of cDNA ends (5 -RACE)-polymerase chain reaction (PCR) with RNA/cDNA derived from a patient (case #1) with a t(5;12)(q31-33;q24) and a second patient (case #2) with a complex rearrangement involving chromosomes 1, 5 and 11. A newly developed DNA-based long-distance inverse PCR (LDI-PCR) was performed on a third patient (case #3) with a t(4;5;5)(q23;q31;q33). In cases #1 and #2, we identified mRNA fusions between GIT2 exon 12 and GPIAP1 exon 7, respectively, and PDGFRB exon 11. In case #3, LDI-PCR revealed a fusion between PRKG2 exon 5 and a truncated PDGFRB exon 12. The region encoding the catalytic domain of PDGFRbeta is retained in all three cases, with the partner contributing a coiled-coil domain (GPIAP1, PRKG2) or an ankyrin protein interaction motif (GIT2) that may potentially lead to dimerization and constitutive activation of the fusion proteins. Treatment with imatinib (400 mg/day) has led to sustained complete hematologic remission in all three patients. These data provide further evidence that numerous partner genes fuse to PDGFRB in BCR-ABL negative chronic myeloproliferative disorders. Although these fusion genes occur rarely, their identification is essential in order to detect patients in whom targeted treatment with tyrosine kinase inhibitors is likely to be successful.

Microsatellite Instability Is Uncommon in Lymphoepithelioma-like Carcinoma of the Lung

Primary lymphoepithelioma-like carcinoma of the lung (LELC) shares some morphologic and clinical characteristics with malignancies associated with microsatellite instability (MSI). The aims of our study were to determine the MSI status in LELC and compare these findings with stage I non-small cell lung carcinoma (NSCLC) with marked lymphocytic host response (MLHR). We assessed MSI by a DNA-based polymerase chain reaction assay using mononucleotide (BAT25 and BAT26) and dinucleotide (D2S123, D5S346, and D17S250) repeats. MSI was detected in 2 (29%) of 7 LELC cases with only 1 marker (D17S250), and in 3 (19%) of 16 NSCLC cases with MLHR with only 2 markers (1D2S123 and 2 D17S250). Loss of heterozygosity (LOH) was detected at 1 or 2 of 3 dinucleotide repeats in 11 NSCLC cases (69%) with MLHR and 3 LELC cases (43%) (P = .36). The overall frequencies of LOH in NSCLC with MLHR were 29% and 19% in LELC (P = .55). MSI is very uncommon in LELC, indicating that MSI is not an important event in carcinogenesis for this tumor subtype. The presence of LOH suggests a probable role of tumor suppressor genes in LELC carcinogenesis.

Prevalence of Factor V 1691 G–A (Leiden) and prothrombin G20210A polymorphisms and the risk of venous thrombosis among cancer patients

Cancer patients have an increased risk for thromboembolism (TE). Factor V 1691 G-A(Leiden) (FVL) and prothrombin (PT) G20210A mutation are common inherited risk factor for TE. The aim of the study is to evaluate the prevalence of FVL and PT G20210A polymorphism in cancer patients with and without TE as compared to patients with TE without malignancy and healthy control. We have studied 43 cancer patients who developed TE during cancer treatment (group 1); 81 cancer patients without TE (group 2); 100 patients with TE without malignancy (group 3); 100 healthy control (group 4). FVL and PT G20210A polymorphisms were determined by the method of polymerase chain reaction-based DNA analysis. The prevalence of FVL was significantly greater in cancer patients with TE (13 of 43, 30.2%) compared with other groups: 3.7% in group 2; 18% in group 3; 8% in group 4 (P < 0.0001). There was no significant difference in the prevalence of PT G20210A among the groups (P > 0.05). The study suggested that cancer patients with TE should be evaluated for FVL but PT G20210A was not contributing factor to the development of TE during cancer therapy.

Establishment of an HPA‐1‐ to ‐16‐typed platelet donor registry in China

In order to determine gene frequencies of human platelet antigen (HPA) and establish a panel of accredited HPA-1a, -2a, -4a, -5a and -6a-negative donors as well as an HPA-typed platelet donor registry, a total of 1000 Chinese donors of Han nationality (500 from north China and 500 from south China) were typed for HPA-1 through -16 using a DNA-based polymerase chain reaction with sequence-specific primers genotyping method. The gene frequencies of HPA-1b, -2b, -3b, -4b, -5b, -6bw, -10bw and -15b were 0.0060, 0.0485, 0.4055, 0.0045, 0.0140, 0.0135, 0.0005 and 0.4680, respectively. The HPA-7bw, -8bw, -9bw, -11bw, -12bw, -13bw, -14bw and -16bw alleles were not found. The HPA-2b and -5b homozygous donors were detected at low frequencies. The HPA mismatch probabilities potentially leading to alloimmunization in random platelet transfusion vary with a region from 0.1% to 37% depending on the distribution patterns of common and less common alleles in each system. This study provides a useful HPA-typed plateletpheresis donor registry in China and could improve platelet antibody detection and HPA-matched platelet transfusion in alloimmune thrombocytopenic patients.

Diversity and expression of nitrogenase genes (nifH) from ectomycorrhizas of Corsican pine (Pinus nigra)

The diversity of bacterial nitrogenase genes (nifH) and their mRNA transcription in ectomycorrhizas of Corsican pine (Pinus nigra) were examined. DNA and RNA were extracted from surface-sterilized and non-sterilized Corsican pine roots colonized by the ectomycorrhizal (ECM) fungi, Suillus variegatus and Tomentellopsis submollis. DNA-derived nifH polymerase chain reaction (PCR) products were obtained from all samples, but only a few reverse transcription PCRs for nifH mRNA were successful, suggesting that nitrogenase genes were not always transcribed. Several different nifH sequences were detected and the bacteria actively transcribing nifH were different from those whose genes were detected through DNA-based PCR. Putative nitrogenase amino acid sequences revealed that more than half of the nifH products were derived from methylotrophic bacteria, such as Methylocella spp. The next most frequent sequence types were similar to those from Burkholderia.

Human Leukocyte Antigen Concordance and the Transmission Risk via Breast‐Feeding of Human T Cell Lymphotropic Virus Type I

We examined the association between mother-to-child human T cell lymphotropic virus type I (HTLV-I) transmission and human leukocyte antigen (HLA) class I types. In 1989, children born to HTLV-I-infected mothers in Jamaica were enrolled and prospectively evaluated for HTLV-I infection. HLA class I types in mothers and children were determined by DNA-based polymerase chain reaction methods. Associations between HLA class I types and transmission of HTLV-I were analyzed using proportional-hazards regression models adjusted for the duration of breast-feeding. Transmission risk in children still breast-feeding at 12 months was determined using actuarial methods. Of 162 children, 28 (17%) became infected. After Bonferroni's adjustment for multiple comparisons, the transmission risk was not influenced by any specific HLA class type or the A2 supertype. However, compared with children who shared 3 HLA class I types with their mothers (the minimum number possible), the transmission risk increased 1.8-fold with 4 shared types and 3.0-fold with 5 or 6 shared types (Ptrend = .039; 1.75-fold increase for each additional concordant HLA type). This association was independent of maternal HTLV-I proviral level, antibody titer, and household income. We found a significant dose-response relationship between HTLV-I transmission via breast-feeding and mother-child HLA class I type concordance. Immunological interactions between a child's cells and maternal cells may influence the risk of HTLV-I infection by breast-feeding, perhaps because antigens on maternal cells are seen by the child as being "self."