CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis

Background

22q11.2 deletion syndrome (22q11.2DS) is a common microdeletion syndrome, which occurs in approximately 1:4000 births. Familial autosomal dominant recurrence of the syndrome is detected in about 8-28% of the cases. Aim of this study is to evaluate the intergenerational and intrafamilial phenotypic variability in a cohort of familial cases carrying a 22q11.2 deletion.

Methods

Thirty-two 22q11.2DS subjects among 26 families were enrolled.

Results

Second generation subjects showed a significantly higher number of features than their transmitting parents (212 vs 129, P?=?0.0015). Congenital heart defect, calcium-phosphorus metabolism abnormalities, developmental and speech delay were more represented in the second generation (P?<?0.05). Ocular disorders were more frequent in the parent group. No significant difference was observed for the other clinical variables. Intrafamilial phenotypic heterogeneity was identified in the pedigrees. In 23/32 families, a higher number of features were found in individuals from the second generation and a more severe phenotype was observed in almost all of them, indicating the worsening of the phenotype over generations. Both genetic and epigenetic mechanisms may be involved in the phenotypic variability.

Conclusions

Second generation subjects showed a more complex phenotype in comparison to those from the first generation. Both ascertainment bias related to patient selection or to the low rate of reproductive fitness of adults with a more severe phenotype, and several not well defined molecular mechanism, could explain intergenerational and intrafamilial phenotypic variability in this syndrome.     

CCL3L1 and CCL4L1: variable gene copy number in adolescents with and without human immunodeficiency virus type 1 (HIV-1) infection

As members of the chemokine family, macrophage inflammatory protein 1 alpha (MIP-1alpha) and MIP-1beta are unique in that they both consist of non-allelic isoforms encoded by different genes, namely chemokine (C-C motif) ligand 3 (CCL3), CCL4, CCL3-like 1 (CCL3L1) and CCL4L1. The products of these genes and of CCL5 (encoding RANTES, i.e., regulated on activation, normal T expressed and secreted) can block or interfere with human immunodeficiency virus type 1 (HIV-1) infection through competitive binding to chemokine (C-C motif) receptor 5 (CCR5). Our analyses of 411 adolescents confirmed that CCL3 and CCL4 genes occurred invariably as single copies (two per diploid genome), whereas the copy numbers of CCL3L1 and CCL4L1 varied extensively (0-11 and 1-6 copies, respectively). Neither CCL3L1 nor CCL4L1 gene copy number variation showed appreciable impact on susceptibility to or control of HIV-1 infection. Within individuals, linear correlation between CCL3L1 and CCL4L1 copy numbers was moderate regardless of ethnicity (Pearson correlation coefficients=0.63-0.65, P<0.0001), suggesting that the two loci are not always within the same segmental duplication unit. Persistently low serum MIP-1alpha and MIP-1beta (in the pg/ml range) compared with high CCL5 concentration (ng/ml range) implied that multi-copy genes CCL3L1 and CCL4L1 conferred little advantage in the intensity of expression among uninfected or infected adolescents.     

Gene copy number regulates the production of the human chemokine CCL3-L1

Genetic variation in the chemokine system is likely to affect responses to infection, and influence the course of autoimmune and inflammatory disease. We and others have shown that the human beta-chemokine CCL3-L1, unlike its related non-allelic isoform CCL3, has high affinity for the chemokine receptors D6, CCR3 and CCR5. Moreover, CCL3-L1, but not CCL3, is susceptible to cleavage by CD26, creating a truncated -2 form with enhanced affinity for CCR1 and CCR5. Strong interaction with CCR5 means that CCL3-L1, and particularly its -2 variant, are by far the most potent natural HIV entry inhibitors described to date. Here, using real-time PCR we have shown that CCL3-L1 and a novel CCL4 isoform (termed CCL4-L1) can vary from 1-6 copies per diploid genome (pdg) in Caucasians and are occasionally completely absent. The other isoforms (CCL3 and CCL4) remain at two copies per dpg. Importantly, in a model system of pro-inflammatory chemokine production (LPS-activated monocytes)higher gene copy number correlates with an increased ratio of CCL3-L1 versus CCL3 mRNA, and enhanced chemokine production. Supernatants from samples with high copy number are able to more potently chemoattract CCR5-expressing cells, an effect blocked with anti-CCL3/CCL3-L1 antibodies. As a result of these studies, we hypothesize that genetic variation in CCL3-L1 gene copy number may affect the susceptibility to, or the progression or severity of, diseases in which this chemokine plays a role.     

Association study of copy number variants in CCL3L1, FCGR3A and FCGR3B genes with risk of ankylosing spondylitis in a West Algerian population

Numerous single nucleotide polymorphisms (SNPs) were explored in the Algerian population to evaluate associated ankylosing spondylitis (AS) genetic risk factors, but no study has identified the impact of copy number variations (CNVs). The aim of the study was to determine whether CNVs of CCL3L1, FCGR3A and FCGR3B genes were also associated with the susceptibility of AS disease in Algerian population. The data set of the current study is composed of 81 patients with AS and 119 healthy controls. All samples were genotyped by digital droplet PCR (ddPCR). Chi‐square test and OR calculation were used to evaluate association between CNVs and AS and the risk associated with copy numbers (CN). In results, FCGR3A CN less than two copies (<2) was significantly increased in spondylitis patients (p = .0001, OR = 7.74 [2.32–25.74]). Additionally, FCGR3A CN < 2 copies association was present only in HLA‐B27 (‐) patients. We have concluded that FCGR3A deletions have an independent effect on AS regarding HLA‐B27 status. This is the first study that investigated the CCL3L1 CNVs in relation to AS risk disease. It reveals that CCL3L1 and FCGR3B CNVs may not be involved in susceptibility to AS risk in the Algerian population.     

Host CCL3L1 gene copy number in relation to HIV-1-specific CD4+ and CD8+ T-cell responses and viral load in South African women

HIV-specific T-cell responses play an important role in control of infection. Because CCL3 has immune modulatory and antiviral activities, we hypothesized that host CCL3 genotype (CCL3L1 gene duplications) would influence the development of effective HIV-specific immune responses. Copy numbers of CCL3L1 were determined for 71 HIV-infected women, and HIV-specific CD4 and CD8 T-cell responses to overlapping peptide pools spanning the HIV-1 subtype C genome were simultaneously measured by an interferon-gamma and interleukin-2 whole-blood flow cytometric assay. Host CCL3L1 copy number correlated negatively with viral load (r=-0.239, P=0.045), as did magnitudes of Gag CD4 (r=-0.362, P=0.002) and CD8 (r=-0.261, P=0.028) T-cell responses. Patients with a Gag CD4 response (P=0.002) or dominant Gag CD8 (P=0.006) response had significantly lower viral loads than those whose dominant response targeted another region of the genome, whereas a dominant Nef-specific CD8 T-cell response was associated with higher HIV viral load. CCL3L1 copy number greater than or equal to the population median of 5 was significantly associated with increased magnitude of CD4 Gag responses (P=0.017), and women who had CD4 and CD8 Gag-specific responses had significantly lower viral loads (P=0.004) and higher CCL3L1 copy number (P=0.015) than those women with only CD8 Gag-specific responses.     

Population-specific GSTM1 copy number variation

As one of the major glutathione conjugation enzymes, GSTM1 detoxifiesa number of drugs and xenobiotics. Its expression and activityhave been shown to correlate both with cancer risks and drugresistance. Through a genome-wide association study, we identifieda significant association between HapMap SNP rs366631 and GSTM1expression. In this study, utilizing lymphoblastoid cell linesderived from International HapMap Consortium CEU and YRI populations,we designed and performed site-specific genotyping assays forboth rs366631 and a highly homologous GSTM1 upstream site. Copynumber variation (CNV) assays were performed for three differentregions of the GSTM1 gene. We demonstrated that HapMap SNP rs366631is a non-polymorphic site. The false genotyping call arisesfrom sequence homology, a common GSTM1 region deletion and anon-specific genotyping platform used to identify the SNP. However,the HapMap call for rs366631 genotype is an indicator of GSTM1upstream region deletion. Furthermore, this upstream deletioncan be used as a marker of GSTM1 gene deletion. Using a novelGSTM1 CNV assay, we showed a population-specific CNV in thisregion upstream of the gene. More than 75% of the Caucasian(CEU) samples exhibit GSTM1 deletion and none contain two copiesof GSTM1. In contrast, up to 25% of African (YRI) samples werefound to have two copies of GSTM1. In conclusion, HapMap rs366631is a pseudo-SNP that can be used as a GSTM1 deletion marker.Both the pseudo-SNP allele frequency and GSTM1 upstream regionCNV show population-specific patterns between CEU and YRI samples.     

CCL3L1 基因拷贝数目变异与慢性乙肝的易感性相关

目的: 研究我国汉族人群 CCL3L1 基因拷贝数的分布,及其与非静脉吸毒者慢性乙肝的相关关系。 方法: 选1 477例慢性乙肝病人及1 023例健康人。用qPCR方法检测 CCL3L1 基因的拷贝数;用qPCR及ELISA方法分别检测 CCL3L1 基因拷贝数与其mRNA、蛋白质的定量关系,并用Transwell法分析了不同拷贝数目与其趋化效力的关系。用Pearson's卡方检验和Logistic 回归方法检测 CCL3L1 基因拷贝数目变异与乙肝易感性的统计学相关性。 结果: CCL3L1 基因拷贝数目在我国汉族正常人为0-9 (中位数=3),与其mRNA、蛋白质产物呈正相关,也与其趋化性呈正相关关系。低拷贝数目者比健康对照更易患乙肝(P<0.01)。结论: 在中国汉族人群中, CCL3L1 基因拷贝数目变异与乙型病毒性肝炎易感性存在显著相关,低拷贝数目者更容易患乙肝。     

Functional and clinical consequences of Fc receptor polymorphic and copy number variants

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leucocytes, thereby linking the humoral and cellular components of immunity. Indeed, engagement of Fc receptors by immunoglobulins initiates a range of immunoregulatory processes that might also play a role in disease pathogenesis. In the circulation, five main types of immunoglobulins (Ig) exist – namely IgG, IgA, IgE, IgM and IgD and receptors with the ability to recognize and bind to IgG (Fcγ receptor family), IgE (FcεRI and CD23), IgA (CD89; Fcα/µR) and IgM (Fcα/µR) have been identified and characterized. However, it is astonishing that nearly all the known human Fc receptors display extensive genetic variation with clear implications for their function, thus representing a substantial genetic risk factor for the pathogenesis of a range of chronic inflammatory disorders.     

Identification of new variants within the two functional genes CCL3 and CCL3L encoding the CCL3 (MIP‐1α) chemokine: implications for HIV‐1 infection

The CC chemokine CCL3 is encoded by two functional genes, namely CCL3 and CCL3L, and has been identified as a key chemokine in HIV‐1 susceptibility and disease progression. The complete CCL3 and CCL3L genes and core promoters of 43 African mother–infant pairs (86 samples) and 28 Caucasian adults in South Africa were sequenced and extensively analysed for genetic variations. Africans were found to be more polymorphic in both genes with 25 single nucleotide polymorphisms (SNPs) in the CCL3 gene and 14 gene copy number single nucleotide polymorphisms (gcnSNPs) in the CCL3L gene, compared to nine CCL3 SNPs and eight CCL3L gcnSNPs in Caucasians. A total of 14 polymorphisms across the two genes were newly identified in this study, most (12/14) of which were exclusive to the African population. In addition, two indels were identified and characterized in the CCL3 and CCL3L genes of a small number of individuals. Of the numerous unique intragenic haplotypes found in the two genes, none were shared by the two population groups. A newly identified five‐SNP CCL3 haplotype (Hap‐C1) found in a high frequency in Caucasians, however, seems to be evolutionarily related to the most prevalent newly identified African seven‐SNP CCL3 haplotype (Hap‐A1). Hap‐A1 also includes an SNP in the core promoter region and previous CCL3 haplotypes that have been reported to be associated with HIV‐1 infection appear to be smaller haplotypes within Hap‐A1. We thus propose Hap‐A1 as a likely candidate for influencing levels of CCL3 production and in turn outcomes of HIV‐1 infection.     

Distribution of the D15Z1 copy number polymorphism

Using fluorescent in situ hybridization (FISH) with the probe p15 (D15Z1), we investigated the distribution of the polymorphic 15p signal which has been reported to occur on acrocentric chromosomes in addition to chromosome 15. The short arm of chromosome 15 has a characteristic signal pattern when hybridized with the FISH probe D15Z1. However, the D15Z1 signal can occasionally be seen on the short arm of other acrocentric chromosomes. We studied the distribution of the D15Z1 probe in 1657 patients consisting both of individuals with a normal karyotype and those with a variety of chromosome abnormalities involving the acrocentric chromosomes. Our results show that one in six individuals, regardless of their patient ascertainment category or karyotypic status, had one or more additional D15Z1 signals, and that there were no significant differences in the distribution of extra signals among the patient groups.     

人趋化因子CCL3L1融合蛋白表达和活性分析

目的人趋化因子CCL3L1进行融合蛋白原核表达和真核表达，纯化后活性分析。方法克隆人类CCL3L1eDNA，构建两种CCL3L1表达载体，获得两个CCL3L1融合蛋白，一个在BL21大肠杆菌表达的GST-CCL3L1融合蛋白，另一个在跎果蝇细胞表达的His-CCL3L1融合蛋白。同时克隆了pcDNA3.1-flag-CCR5表达载体，培养了稳定表达flag-CCR5的细胞株，进行人趋化因子CCL3L1活性分析。结果成功构建人趋化因子CCL3L1融合蛋白原核表达载体pGEX-4T和真核表达载体pMT／BiP／V5-His，免疫沉淀法检测和Westernblot法分析His-CCL3L1蛋白在浓度1nmol／L到50nmol／L存在剂量依赖性，浓度50nmol／L到100nmol／L没有剂量依赖性。纯化的His-CCL3L1蛋白能特异性结合CCR5受体。结论成功表达了融合蛋白GST-CCL3L1和His-CCL3LI，果蝇细胞表达的His-CCL3L1蛋白具有与天然CCL3L1相同的生物学活性，为进一步制备CCL3L1单克隆和多克隆抗体及研究CCL3L1影响HIV-1感染的机制提供基础资料。     

High-resolution copy number arrays in cancer and the problem of normal genome copy number variation

High-resolution techniques for analysis of genome copy number (CN) enable the analysis of complex cancer somatic genetics. However, the analysis of these data is difficult, and failure to consider a number of issues in depth may result in false leads or unnecessary rejection of true positives. First, segmental duplications may falsely generate CN breakpoints in aneuploid samples. Second, even when tumor data were each normalized to matching lymphocyte DNA, we still observed copy number polymorphisms masquerading as somatic alterations due to allelic imbalance. We investigated a number of different solutions and determined that evaluating matching normal DNA, or at least using locally derived normal baseline data, were preferable to relying on current online databases because of poor cross-platform compatibility and the likelihood of excluding genuine small somatic alterations.     

Genome-wide copy number analysis using copy number inferring tool (CNIT) and DNA pooling

Copy number variation (CNV) has become an important genomic structure element in the human population, and some CNVs are related to specific traits and diseases. Moreover, analysis of human genomes has been potentiated by the use of high-resolution microarrays that assess single nucleotide polymorphisms (SNPs). Although many programs have been designed to analyze data from Affymetrix SNP microarrays, they all have high false-positive rates (FPRs) in copy number (CN) analyses. Copy number analysis tool (CNAT) 4.0 is a recently developed program that offers improved CN estimation, but small amplifications and deletions are lost when using the smoothing procedure. Here, we propose a copy number inferring tool (CNIT) algorithm for the 100K SNP microarray to investigate CNVs at 29.6-kb resolution. CNIT estimated SNP allelic and total CN with reliable P values based on intensity data. In addition, the hidden Markov model (HMM) method was applied to predict regions having altered CN by considering contiguous SNPs. Based on a CN analysis of 23 unrelated Taiwanese and 30 HapMap Centre d'Etude du Polymorphisme Humain (CEPH) trios, CNIT showed higher accuracy and power than other programs. The FPRs and false-negative rates (FNRs) of CNIT were 0.1% and 0.16%, respectively. CNIT also showed better sensitivity for detecting small amplifications and deletions. Furthermore, DNA pooling of 10 and 30 normal unrelated individuals were applied to the 100K SNP microarray, respectively, and 12 common CN-variable regions were identified, suggesting that DNA pooling can be applied to discover common CNVs.     

Human SULT1A1 gene: copy number differences and functional implications

SULT1A1, which catalyzes the sulfate conjugation of a wide variety of natural and synthetic compounds, is genetically polymorphic. Biochemical and pharmacogenetic studies have demonstrated that individual variation in the level of enzyme activity is inherited. Common single-nucleotide polymorphisms (SNPs) located in the open reading frame and in the 5'-flanking region (5'-FR) may account for a portion of this individual variation. In this study, we demonstrate the presence of SULT1A1 gene deletions and duplications, representing an additional source of variability in the metabolic activity of this enzyme. A quantitative multiplex PCR assay was used to measure the extent of copy number differences and the frequency of these events in different populations. An analysis of DNA from 362 Caucasian-American and 99 African-American showed the presence of 1 to approximately 5 copies of SULT1A1 in individual samples: 5% of Caucasian subjects contained a single copy of the gene and 26% had three or more copies, while 63% of African-American subjects had three or more copies. Analysis of the genomic region surrounding the SULT1A1 gene in three separate cases with a deletion demonstrated that the entire SULT1A1 gene was affected. Reporter assays, constructed for each of the various 5'-FR SNP haplotypes, suggest that these may also play a role in SULT1A1 activity. However, the variability in the level of enzyme activity among 23 human platelet and 267 human liver samples was best explained by gene copy number differences when all sources of genetic variability were considered (P < 0.0001). Overall, these observations have obvious implications for the effectiveness of SULT1A1 as a drug and hormone metabolizing enzyme and its potential role as a risk factor for disease.     

Germline copy number variations in BRCA1-associated ovarian cancer patients

We investigated characteristics of germline copy number variations (CNV) in BRCA1-associated ovarian cancer patients by comparing them to CNVs present in sporadic ovarian cancer patients. Germline CNVs in 51 BRCA1-associated, 33 sporadic ovarian cancer patients, and 47 healthy women were analyzed by both signal intensity and genotyping data using the Affymetrix Genome-Wide Human SNP Array 6.0. The total number of CNVs per genome was greater in the sporadic group (median 26, range 12-34) than in the BRCA1 group (median 21, range 11-35; post hoc P < 0.05) or normal group (median 20, range 7-32; post hoc P < 0.05). While the number of amplifications per genome was higher in the sporadic group (median 13, range 7-26) than in the BRCA1 group (median 8, range 3-23; post hoc P < 0.001), the number of deletions per genome was higher in the BRCA1 group (median 12, range 6-24) than in the sporadic group (median 9, range 3-17; post hoc P < 0.01). In addition, 31 previously unknown CNV regions were present specifically in the BRCA1 group. When we performed pathway analysis on the 241 overlapping genes mapped to these novel CNV regions, the 'purine metabolism' and '14-3-3-mediated signaling' pathways were over-represented (Fisher's exact test, P < 0.01). Our study shows that there are qualitative differences in genomic CNV profiles between BRCA1-associated and sporadic ovarian cancer patients. Further studies are necessary to clarify the significance of the genomic CNV profile unique to BRCA1-associated ovarian cancer patients.     

Profiling genomic copy number changes in retinoblastoma beyond loss of RB1

Loss of both RB1 alleles is rate limiting for development of retinoblastoma (RB), but genomic copy number gain or loss may impact oncogene(s) and tumor suppressor genes, facilitating tumor progression. We used quantitative multiplex polymerase chain reaction to profile "hot spot" genomic copy number changes for gain at 1q32.1, 6p22, and MYCN, and loss at 16q22 in 87 primary RB and 7 cell lines. Loss at 16q22 (48%) negatively associated with MYCN gain (18%) (Fisher's exact P = 0.031), gain at 1q32.1 (62%) positively associated with 6p "hot spot" gain (43%) (P = 0.033), and there was a trend for positive association between 1q and MYCN gain (P = 0.095). Cell lines had a higher frequency of MYCN amplification than primary tumors (29% versus 3%; P = 0.043). Novel high-level amplification of 1q32.1 in one primary tumor, confirmed by fluorescence in situ hybridization, strongly supports the presence of oncogene(s) in this region, possibly the mitotic kinesin, KIF14. Gene-specific quantitative multiplex polymerase chain reaction of candidate oncogenes at 1q32.1 (KIF14), 6p22 (E2F3 and DEK), and tumor suppressor genes at 16q22 (CDH11) and 17q21 (NGFR) showed the most common gene gains in RB to be KIF14 in cell lines (80%) and E2F3 in primary tumors (70%). The patterns of gain/loss were qualitatively different in 25 RB compared with 12 primary hepatocellular carcinoma and 12 breast cancer cell lines. Gene specific analysis of one bone marrow metastasis of RB, prechemotherapy and postchemotherapy, showed the typical genomic changes of RB pretreatment, which normalized after chemotherapy.     

Maintenance and copy number control of ARS1 plasmids in Saccharomyces cerevisiae

Summary Following mating of a and isogenic haploids we observe that the frequency of plasmid bearing cells, during selective growth, increases three fold. By examining the mitotic stability, the frequency of plasmid bearing cells during the cell cycle and the copy number of ARS1 plasmids in isogenic haploid and diploid cells, we show that the apparent stability of circular ARS1 plasmids in a/ cells is largely due to a diminished copy number in these cells. This observation is fully comprehensible with the model for plasmid segregation as presented by Murray and Szostak (1983). In order to account for the differences in copy numbers, a and a/a isogenic strains were compared. Likewise a number of mating type nonspecific sterile mutants were compared with the parental Ste⁺ strain. It seems that a diminished copy number is established when the MATa1/MAT2 regulatory system (Klar et al. 1981) is switched on, since the effect is observed in Sir^– strains only.     

Determination of SMN1 and SMN2 copy number using TaqMan technology

Infantile spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of the SMN1 gene in more than 90% of patients. Identification of carriers for the SMN1 deletion is important for diagnostic purposes and for genetic counseling. The current practical implications of SMN2 copy number determination are limited but may be important, for example, for future drug trials. Here we present a new rapid and reliable approach to determine the copy numbers of the SMN1 and SMN2 genes: For differentiation of the two genes, we developed a quantitative test on the basis of TaqMan technology using minor groove binder (MGB) probes. To evaluate the approach in respect to detection of SMN1 deletion carriers, we tested 40 putative carriers as well as 100 controls. We confirmed the carrier status in all individuals; furthermore, the distribution of SMN1 and SMN2 copies in the control cohort corresponded to that published previously. In total, a clear-cut differentiation between the different copy number ranges could be observed for both genes. This distinct differentiation is based on the exact specificity of MGB probes and the parallel analysis of an external reference locus that circumvents the problem of unavoidable deviations in DNA concentrations. The simplicity and reproducibility of the TaqMan assay presented here should facilitate its establishment in molecular diagnostic laboratories. Nevertheless, the applicability of quantitative analyses of SMN copy numbers requires knowledge about its options and limitations, based on the complex nature of the SMN region and the clinical variability of SMA. Therefore, determination of SMN1 and SMN2 copy numbers should only be offered after careful consideration in each case.