Developing Molecular Signatures for Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays a great clinical heterogeneity, with many patients having an indolent disease that will not require intervention for many years, while others present an aggressive and symptomatic leukemia requiring immediate treatment. Although there is no cure for CLL, the disease is treatable and current standard chemotherapy regimens have been shown to prolong survival. Recent advances in our understanding of the biology of CLL have led to the identification of numerous cellular and molecular markers with potential diagnostic, prognostic and therapeutic significance. We have used the recently developed digital multiplexed gene-expression technique (DMGE) to analyze a cohort of 30 CLL patients for the presence of specific genes with known diagnostic and prognostic potential. Starting from a set of 290 genes we were able to develop a molecular signature, based on the analysis of 13 genes, which allows distinguishing CLL from normal peripheral blood and from normal B cells, and a second signature based on 24 genes, which distinguishes mutated from unmutated cases (LymphCLL Mut). A third classifier (LymphCLL Diag), based on a 44-gene signature, distinguished CLL cases from a series of other B-cell chronic lymphoproliferative disorders (n = 51). While the methodology presented here has the potential to provide a "ready to use" classification tool in routine diagnostics and clinical trials, application to larger sample numbers are still needed and should provide further insights about its robustness and utility in clinical practice.     

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes

Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.     

嗜肺军团菌毒力岛分子分型及其致病性研究进展

嗜肺军团菌是引起社区获得性和医院内感染性肺炎的重要病原体,中央空调冷凝塔水系统是引发军团菌病的重要传染源,在国内外时有暴发流行,病死率较高。嗜肺军团菌的致病性与其毒力岛基因组密切相关。简要概述了嗜肺军团菌毒力岛、分子分型及其致病性。     

Molecular Characterization of Legionella Populations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops

The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.     

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.     

军团菌分子分型及其流行病学调查中应用

在军团病爆发流行时,为了解环境水源和病人标本中分离的军团菌株的遗传关系,需要对分离的军团菌进行分型和分类,以确定军团病发生、传播和流行之间的关系。对目前用于军团菌分型的4种常用分子分型技术:随机扩增多态性DNA(Random Amplified Polymorphic DNA,RAPD)、脉冲场凝胶电泳(Pulsed-FieldGel Electrophoresis,PFGE)、扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)、核酸测序分型(sequence-based typing,SBT)及其分型的分辨能力、重复性、符合率和实用性作了比较分析与探讨。     

Molecular Diagnostic and Prognostic Subtyping of Gliomas in Tunisian Population

It has become increasingly evident that morphologically similar gliomas may have distinct clinical phenotypes arising from diverse genetic signatures. To date, glial tumours from the Tunisian population have not been investigated. To address this, we correlated the clinico-pathology with molecular data of 110 gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR amplification were distributed in different glioma histological groups. However, 1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression were more common within high-grade gliomas. Furthermore, survival statistical correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact, significant lower overall survival (OS) was detected within GB that overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a markedly survival advantage. Interestingly, the association of IDHR132H mutation and EGFR normal status, as well as the association of differentiation markers, defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR amplification. Additionally, GB presenting p53-negative staining associated with CDKN2A loss or p21 positivity represented a subtype with short survival. Thus, distinct molecular subtypes with individualised prognosis were identified. Interestingly, we found a unique histone mutation in a poor survival young adult GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a remarkable methylation profile, it emphasised the oncogenic power of G34R mutation connecting gliomagenesis and chromatin regulation.     

大肠癌转移相关分子标签的筛选

目的:筛选与转移相关的大肠癌分子标签.方法:本文通过对大肠癌表达谱数据进行分析,按照表达谱中大肠癌转移情况,组织学分化程度以及患者生存时间进行显著性分析,将与大肠癌转移相关的具有显著性意义的基因群进行聚类,通过主成分分析以及自组织映射的方法计算出差异表达基因中,起着主体分类作用的基因群.结果:筛选出与肿瘤组织分化程度以及患者生存时间相关的差异表达基因,进行功能富集,并筛选出了一批与大肠癌转移密切相关的重要基因,这些基因对大肠癌早期诊断,及时治疗,预后评估有着重要意义.结论:细胞代谢,趋化因子信号通路和细胞因子受体等分子事件与大肠癌分化程度密切相关,是否发生转移与大肠癌的预后生存期密切相关.     

Phylogenetic Framework and Molecular Signatures for the Main Clades of the Phylum Actinobacteria

Summary: The phylum Actinobacteria harbors many important human pathogens and also provides one of the richest sources of natural products, including numerous antibiotics and other compounds of biotechnological interest. Thus, a reliable phylogeny of this large phylum and the means to accurately identify its different constituent groups are of much interest. Detailed phylogenetic and comparative analyses of >150 actinobacterial genomes reported here form the basis for achieving these objectives. In phylogenetic trees based upon 35 conserved proteins, most of the main groups of Actinobacteria as well as a number of their superageneric clades are resolved. We also describe large numbers of molecular markers consisting of conserved signature indels in protein sequences and whole proteins that are specific for either all Actinobacteria or their different clades (viz., orders, families, genera, and subgenera) at various taxonomic levels. These signatures independently support the existence of different phylogenetic clades, and based upon them, it is now possible to delimit the phylum Actinobacteria (excluding Coriobacteriia) and most of its major groups in clear molecular terms. The species distribution patterns of these markers also provide important information regarding the interrelationships among different main orders of Actinobacteria. The identified molecular markers, in addition to enabling the development of a stable and reliable phylogenetic framework for this phylum, also provide novel and powerful means for the identification of different groups of Actinobacteria in diverse environments. Genetic and biochemical studies on these Actinobacteria-specific markers should lead to the discovery of novel biochemical and/or other properties that are unique to different groups of Actinobacteria.     

Subtyping for TNFa microsatellite sequence variation

The microsatellite locus TNFa is frequently used as an additional genetic marker in studies of the major histocompatibility complex (MHC). Novel sequence variations at the TNFa locus have been described, and which may have implications for genetic analyses. In this study, we set up a nested polymerase chain reaction-sequence-specific primer (PCR-SSP) approach to type for these TNFa sequence variations. First, sequencing analysis of workshop B lymphoblastoid cell lines (n=13) showed the presence of three sequence variations upstream of the dinucleotide repeat at TNFa. Using nested PCR-SSP, we were able to detect these variations in a larger B lymphoblastoid cell line panel (n=34). Furthermore, we were able to show that TNFa alleles a7 and a10 are present in two distinct conformations leading to "splitting" of TNFa alleles exhibiting identical fragment lengths. To establish the frequency of the TNFa alleles and their variants, we performed microsatellite typing of a large panel of random individuals from the Dutch population (n=272). Subsequent nested PCR-SSP typing showed the presence of three previously described sequence variations in the Dutch population. Furthermore, the presence of a fourth subtype was established. The described variations of allele TNFa7 and TNFa10 are present in the random population with significant frequencies. Haplotyping analysis between HLA-DR, TNFa, and HLA-B showed that allele TNFa7.2 is present in an extended DR7-TNFa7.2-B13 haplotype. In this way, we were able to show that the additional sequence variations behave like distinct TNFa alleles.