Optimising blood glucose control with portioned meal box in type 2 diabetes mellitus patients: a randomised control trial.

Background: The impact of dietary factors on glycaemic control in type 2 diabetes mellitus (T2DM) is well established. However, the effectiveness of transforming portion control into a practical innovation for glycaemic control in T2DM has not yet been established for counselling in nutrition. The aim of this study was to compare the effect of general counselling in nutrition (GCN) and a portioned meal box (PMB) on fasting blood glucose, glycated haemoglobin (HbA1c) and body composition. Methods: A randomised, parallel intervention trial was conducted over 12 weeks, with GCN: carbohydrate portion control concept by using food exchange lists (n = 25) and PMB: portioned meal box was set by energy requirements (n = 25). Results: Both GCN and PMB demonstrated reductions in HbA1c levels at the 6th and 12th weeks compared to baseline. However, no significant difference in HbA1c was observed between GCN and PMB at either the 6th or 12th week. Using PMB at least four times a week significantly decreased HbA1c during the intervention period (p = 0.021 and p < 0.001 for weeks 6 and 12 when compared with baseline, respectively). Changes in body composition were observed: body weight decrease in PMB only, body fat decrease and constant muscle mass in both groups. Both methods tended to relieve hunger and increased satiety in both groups. The satisfaction evaluation showed that participants preferred to use PMB over GCN (p = 0.001). Additionally, participants consumed less energy, carbohydrate and fat in PMB (p = 0.001, p = 0.019, and p = 0.001, respectively) and less energy and fat in GCN (p = 0.006 and p = 0.001, respectively). Conclusion: A better diet, either through GCN or PMB, can play an important role in improving dietary intake compliance and controlling blood glucose.

Eight Weeks of Physical Training Decreases 2 Years of DNA Methylation Age of Sedentary Women.

Purpose: The acceleration of epigenetic age is a predictor of mortality and contributes to the increase in chronic diseases. Adherence to a healthy lifestyle is a strategy to reduce epigenetic age. The present study aimed to determine whether eight weeks of combined (aerobic and strength) training (CT) can influence the epigenetic age of women between 50 and 70 years old and the differences in sites and methylated regions. Methods: Eighteen women (AARLow: lower age acceleration residual, n = 10; AARHigh: higher age acceleration residual, n = 8) participated in a combined exercise training program (60 minutes, 3× a week) for eight weeks. DNA was extracted from whole blood using the salting out technique. DNA methylation was performed using the array technique (Illumina's Infinium Methylation BeadChip 850k). We used the DNA Methylation Age Calculator platform to calculate the biological epigenetic age. Two-way ANOVA followed by FISHER LSD posthoc was Applied, adopting p < .05. Results: After eight weeks of CT, there were no changes to the epigenetic age acceleration for the AARLow group (PRE: -2.3 ± 3.2 to POST: -2.3 ± 3.6). However, the AARHigh group significantly decreased the age acceleration (PRE: 3.6 ± 2.6 to POST: 2.2 ± 2.7) (group effect, p = .01; time effect, p = .31; group vs. time effect, p = .005). Conclusion: CT for eight weeks benefits the epigenetic clock of women with the most accelerated age.

Epigenetic Gene-Regulatory Loci in Alu Elements Associated with Autism Susceptibility in the Prefrontal Cortex of ASD.

Alu elements are transposable elements that can influence gene regulation through several mechanisms; nevertheless, it remains unclear whether dysregulation of Alu elements contributes to the neuropathology of autism spectrum disorder (ASD). In this study, we characterized transposable element expression profiles and their sequence characteristics in the prefrontal cortex tissues of ASD and unaffected individuals using RNA-sequencing data. Our results showed that most of the differentially expressed transposable elements belong to the Alu family, with 659 loci of Alu elements corresponding to 456 differentially expressed genes in the prefrontal cortex of ASD individuals. We predicted cis- and trans-regulation of Alu elements to host/distant genes by conducting correlation analyses. The expression level of Alu elements correlated significantly with 133 host genes (cis-regulation, adjusted p < 0.05) associated with ASD as well as the cell survival and cell death of neuronal cells. Transcription factor binding sites in the promoter regions of differentially expressed Alu elements are conserved and associated with autism candidate genes, including RORA. COBRA analyses of postmortem brain tissues showed significant hypomethylation in global methylation analyses of Alu elements in ASD subphenotypes as well as DNA methylation of Alu elements located near the RNF-135 gene (p < 0.05). In addition, we found that neuronal cell density, which was significantly increased (p = 0.042), correlated with the expression of genes associated with Alu elements in the prefrontal cortex of ASD. Finally, we determined a relationship between these findings and the ASD severity (i.e., ADI-R scores) of individuals with ASD. Our findings provide a better understanding of the impact of Alu elements on gene regulation and molecular neuropathology in the brain tissues of ASD individuals, which deserves further investigation.

Dopachrome tautomerase is a retinoblastoma-specific gene, and its proximal promoter is preferentially active in human retinoblastoma cells.

Purpose: Retinoblastoma (RB) is a malignant childhood intraocular tumor. Current treatment options for RB have undesirable side effects. A comprehensive understanding of gene expression in human RB is essential for the development of safe and effective new therapies. Methods: We reviewed published microarray and RNA sequencing studies in which gene expression profiles were compared between human RB and normal retina tissues. We investigated the expression of genes of interest using quantitative reverse transcription PCR. We examined the activities of cloned promoter DNA fragments with luciferase assay. Results: Dopachrome tautomerase (DCT) was among the most overexpressed genes in RB in published studies. We found that DCT was highly expressed in six of 13 samples microdissected from Thai RB tissues. Expression of DCT was absent or barely detected in retina tissues, various human ocular cells, and major organs. We also demonstrated that the -657 to +411 DCT promoter fragment efficiently directs RB cell-specific transcription of the luciferase reporter gene in cell lines. Conclusions: The present work highlights that DCT is one of the most RB-specific genes. The regulatory elements required for this cell-specific gene expression are likely located within its proximal promoter.

14-weeks combined exercise epigenetically modulated 118 genes of menopausal women with prediabetes.

Background: Pre-diabetes precedes Diabetes Mellitus (DM) disease and is a critical period for hyperglycemia treatment, especially for menopausal women, considering all metabolic alterations due to hormonal changes. Recently, the literature has demonstrated the role of physical exercise in epigenetic reprogramming to modulate the gene expression patterns of metabolic conditions, such as hyperglycemia, and prevent DM development. In the present study, we hypothesized that physical exercise training could modify the epigenetic patterns of women with poor glycemic control. Methods: 48 post-menopause women aged 60.3 ± 4.5 years were divided according to their fasting blood glucose levels into two groups: Prediabetes Group, PG (n=24), and Normal Glucose Group, NGG (n=24). All participants performed 14 weeks of physical exercise three times a week. The Infinium Methylation EPIC BeadChip measured the participants' Different Methylated Regions (DMRs). Results: Before the intervention, the PG group had 12 DMRs compared to NGG. After the intervention, five DMRs remained different. Interestingly, when comparing the PG group before and after training, 118 DMRs were found. The enrichment analysis revealed that the genes were related to different biological functions such as energy metabolism, cell differentiation, and tumor suppression. Conclusion: Physical exercise is a relevant alternative in treating hyperglycemia and preventing DM in post-menopause women with poor glycemic control.

LINE-1 and Alu methylation signatures in autism spectrum disorder and their associations with the expression of autism-related genes.

Long interspersed nucleotide element-1 (LINE-1) and Alu elements are retrotransposons whose abilities cause abnormal gene expression and genomic instability. Several studies have focused on DNA methylation profiling of gene regions, but the locus-specific methylation of LINE-1 and Alu elements has not been identified in autism spectrum disorder (ASD). Here we interrogated locus- and family-specific methylation profiles of LINE-1 and Alu elements in ASD whole blood using publicly-available Illumina Infinium 450 K methylation datasets from heterogeneous ASD and ASD variants (Chromodomain Helicase DNA-binding 8 (CHD8) and 16p11.2del). Total DNA methylation of repetitive elements were notably hypomethylated exclusively in ASD with CHD8 variants. Methylation alteration in a family-specific manner including L1P, L1H, HAL, AluJ, and AluS families were observed in the heterogeneous ASD and ASD with CHD8 variants. Moreover, LINE-1 and Alu methylation within target genes is inversely related to the expression level in each ASD variant. The DNA methylation signatures of the LINE-1 and Alu elements in ASD whole blood, as well as their associations with the expression of ASD-related genes, have been identified. If confirmed in future larger studies, these findings may contribute to the identification of epigenomic biomarkers of ASD.

Genomic targets and selective inhibition of DNA methyltransferase isoforms.

BACKGROUND: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. RESULTS: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δß > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (ß: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δß > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. CONCLUSIONS: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome.

Corrigendum: Novel Zinc-Related Differentially Methylated Regions in Leukocytes of Women With and Without Obesity.

[This corrects the article DOI: 10.3389/fnut.2022.785281.].

Novel Zinc-Related Differentially Methylated Regions in Leukocytes of Women With and Without Obesity.

Introduction: Nutriepigenetic markers are predictive responses associated with changes in "surrounding" environmental conditions of humans, which may influence metabolic diseases. Although rich in calories, Western diets could be linked with the deficiency of micronutrients, resulting in the downstream of epigenetic and metabolic effects and consequently in obesity. Zinc (Zn) is an essential nutrient associated with distinct biological roles in human health. Despite the importance of Zn in metabolic processes, little is known about the relationship between Zn and epigenetic. Thus, the present study aimed to identify the epigenetic variables associated with Zn daily ingestion (ZnDI) and serum Zinc (ZnS) levels in women with and without obesity. Materials and Methods: This is a case-control, non-randomized, single-center study conducted with 21 women allocated into two groups: control group (CG), composed of 11 women without obesity, and study group (SG), composed of 10 women with obesity. Anthropometric measurements, ZnDI, and ZnS levels were evaluated. Also, leukocyte DNA was extracted for DNA methylation analysis using 450 k Illumina BeadChips. The epigenetic clock was calculated by Horvath method. The chip analysis methylation pipeline (ChAMP) package selected the differentially methylated regions (DMRs). Results: The SG had lower ZnS levels than the CG. Moreover, in SG, the ZnS levels were negatively associated with the epigenetic age acceleration. The DMR analysis revealed 37 DMRs associated with ZnDI and ZnS levels. The DMR of PM20D1 gene was commonly associated with ZnDI and ZnS levels and was hypomethylated in the SG. Conclusion: Our findings provide new information on Zn's modulation of DNA methylation patterns and bring new perspectives for understanding the nutriepigenetic mechanisms in obesity.

Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3).

Natural antisense transcripts (NATs) constitute a significant group of regulatory, long noncoding RNAs. They are prominently expressed in testis but are also detectable in other organs. NATs are transcribed at low levels and co-expressed with related protein coding sense transcripts. Nowadays NATs are generally considered as regulatory, long noncoding RNAs without closer focus on the inevitable interference between sense and antisense expression. This work describes a cellular system where sense and antisense transcription of a specific locus (SLC34A1/PFN3) is induced using epigenetic modifiers and CRISPR-Cas9. The renal cell lines HEK293 and HKC-8 do not express SLC34A1/PFN3 under normal culture conditions. Five-day exposure to dexamethasone significantly stimulates sense transcript (SLC34A1) levels and antisense (PFN3) minimally; the effect is only seen in HEK293 cells. Enhanced expression is paralleled by reduced sense promoter methylation and an increase in activating histone marks. Expression is further modulated by cassettes that stimulate the expression of sense or antisense transcript but disrupt protein coding potential. Constitutive expression of a 5'-truncated SLC34A1 transcript increases sense expression independent of dexamethasone induction but also stimulates antisense expression. Concordant expression is confirmed with the antisense knock-in that also enhances sense expression. The antisense effect acts on transcription in cis since transient transfection with sense or antisense constructs fails to stimulate the expression of the opposite transcript. These results suggest that bi-directional transcription of the SLC34A1/PFN3 locus has a stimulatory influence on the expression of the opposite transcript involving epigenetic changes of the promoters. In perspective of extensive, previous research into bi-directionally transcribed SLC34A loci, the findings underpin a hypothesis where NATs display different biological roles in soma and germ cells. Accordingly, we propose that in somatic cells, NATs act like lncRNAs-with the benefit of close proximity to a potential target gene. In germ cells, however, recent evidence suggests different biological roles for NATs that require RNA complementarity and double-stranded RNA formation.

Elevated serum mitochondrial DNA in females and lack of altered platelet mitochondrial methylation in patients with Parkinson´s disease.

Purpose: Mitochondrial dysfunction has long been considered in the pathogenesis of Parkinson's disease (PD). This is evident from the presence of mitochondrial DNA deletions in substantia nigra neurons and respiratory chain abnormalities in the skeletal muscle of PD patients. However, the contributing factors that potentially cause oxidative stress in PD are still elusive. To a certain extent, the identification of acquired changes in circulating mitochondrial DNA (mtDNA) content in blood samples may mirror the mitochondrial (dys-) function. Therefore, herein, we investigated the mtDNA concentrations in serum and cerebrospinal fluid (CSF) of PD patients.Materials and methods: We performed quantitative analysis (qPCR) at two mitochondrial regions (D-Loop; ATPase6) and evaluated the platelet mtDNA methylation levels (MT-TL1 ,MT-CO1, MT-CO2 and MT-CO3) by bisulfite-PCR pyrosequencing.Results: Our quantitative analysis at two mitochondrial regions (D-Loop; ATPase6) revealed an increase in mtDNA serum concentrations in PD females compared to healthy females. Of particular interest, these altered concentrations were restricted to females serum only. Thus, in males as well as CSF of PD patients no increase was detected. Additionally, mtDNA methylation in platelets isolated from the plasma of PD patients showed no altered methylation levels in the mitochondrial MT-TL1 and MT-CO1 regions. Besides, a complete lack of platelet mtDNA methylation was observed at MT-CO2 and MT-CO3 mitochondrial sites.Conclusions: Taken together, we found an increased mtDNA serum concentration exclusively in PD females. As of yet, it is unclear whether this might reflect specific changes or characteristics of female PD pathobiology. However, in context to the ongoing debate about mtDNA methylation, we could show that the mitochondrial epigenome does harbor detectable CpG methylation sites in platelets-derived DNA.

DNA methylation patterns of LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes.

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 µg/day) and vitamin B12 (500 µg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD.

Psychosocial stress is associated with benign breast disease in young Chinese women: results from Project ELEFANT.

PURPOSE: Psychosocial stress, including bereavement and work-related stress, is associated with the risk of breast cancer. However, it is unknown whether it may also be linked with increased risk of benign breast disease (BBD). METHODS: Our study leveraged 61,907 women aged 17-55 years old from the Project ELEFANT study. BBD was diagnosed by clinician. Self-reported data on psychosocial stress over a 10-year period was retrospectively collected from questionnaires and categorised by cause (work, social and economic) and severity (none, low and high). Odd ratios (ORs) for the development of BBD were estimated using logistic regression. The model was adjusted for age, BMI, TSH levels, smoking, alcohol consumption, family history, age of menarche, oral contraceptive usage, education and occupation. RESULTS: Within our study, 8% (4,914) of participants were diagnosed with BBD. Work-related stress [OR 1.57, 95% confidence interval (CI) 1.46-1.69] and financial stress (OR 1.34, 95% CI 1.24-1.44) were significantly associated with BBD incidence, with a smaller but still significant association with social stress (OR 1.11, 95% CI 1.01-1.21). The associations remained significant after exclusion of participants with first- and second-degree family history of breast disease. The presence of multiple forms of stress did not synergistically increase risk. The neutrophil-lymphocyte ratio (NLR), a marker of systemic inflammation and prognostic marker for breast cancer, was not associated with BBD. CONCLUSIONS: Psychosocial stress, particularly work-related and financial stress, is associated with increased risk of benign breast disease among young Chinese women.

Dietary Intervention Modifies DNA Methylation Age Assessed by the Epigenetic Clock.

SCOPE: Alterations in DNA methylation patterns are correlated with aging, environmental exposures, and disease pathophysiology; the possibility of reverting or preventing these processes through dietary intervention is gaining momentum. In particular, methyl donors that provide S-adenosyl-methionine for one-carbon metabolism and polyphenols such as flavanols that inhibit the activity of DNA methyltransferases (DNMTs) can be key modifiers of epigenetic patterns. METHODS AND RESULTS: DNA methylation patterns are assessed in publicly available Illumina Infinium 450K methylation datasets from intervention studies with either folic acid + vitamin B12 (GSE74548) or monomeric and oligomeric flavanols (MOF) (GSE54690) in 44 and 13 participants, respectively. Global DNA methylation levels are increased in unmethylated regions such as CpG islands and shores following folic acid + vitamin B12 supplementation and decreased in highly methylated regions, including shelves and open-seas, following intervention with MOF. After supplementation with folic acid + vitamin B12, epigenetic age, estimated by the Horvath "epigenetic clock" model, is reduced in women with the MTHFR 677CC genotype. CONCLUSIONS: The effects of supplementation with folic acid + vitamin B12 and MOF on DNA methylation age are dependent upon gender and MTHFR genotype. Additionally, the findings demonstrate the potential for these dietary factors to modulate global DNA methylation profiles.

ChREBP Regulates Itself and Metabolic Genes Implicated in Lipid Accumulation in ß-Cell Line.

Carbohydrate response element binding protein (ChREBP) is an important transcription factor that regulates a variety of glucose-responsive genes in hepatocytes. To date, only two natural isoforms, Chrebpα and Chrebpß, have been identified. Although ChREBP is known to be expressed in pancreatic ß cells, most of the glucose-responsive genes have never been verified as ChREBP targets in this organ. We aimed to explore the impact of ChREBP expression on regulating genes linked to accumulation of lipid droplets, a typical feature of ß-cell glucotoxicity. We assessed gene expression in 832/13 cells overexpressing constitutively active ChREBP (caChREBP), truncated ChREBP with nearly identical amino acid sequence to Chrebpß, or dominant negative ChREBP (dnChREBP). Among multiple ChREBP-controlled genes, ChREBP was sufficient and necessary for regulation of Eno1, Pklr, Mdh1, Me1, Pdha1, Acly, Acaca, Fasn, Elovl6, Gpd1, Cpt1a, Rgs16, Mid1ip1,Txnip, and Chrebpß. Expression of Chrebpα and Srebp1c were not changed by caChREBP or dnChREBP. We identified functional ChREBP binding sequences that were located on the promoters of Chrebpß and Rgs16. We also showed that Rgs16 overexpression lead to increased considerable amounts of lipids in 832/13 cells. This phenotype was accompanied by reduction of Cpt1a expression and slight induction of Fasn and Pklr gene in these cells. In summary, we conclude that Chrebpß modulates its own expression, not that of Chrebpα; it also regulates the expression of several metabolic genes in ß-cells without affecting SREBP-1c dependent regulation. We also demonstrate that Rgs16 is one of the ChREBP-controlled genes that potentiate accumulation of lipid droplets in ß-cells.

Genome-Wide Analysis of ChREBP Binding Sites on Male Mouse Liver and White Adipose Chromatin.

Glucose is an essential nutrient that directly regulates the expression of numerous genes in liver and adipose tissue. The carbohydrate response element-binding protein (ChREBP) links glucose as a signaling molecule to multiple glucose-dependent transcriptional regulatory pathways, particularly genes involved in glycolytic and lipogenic processes. In this study, we used chromatin immunoprecipitation followed by next-generation sequencing to identify specific ChREBP binding targets in liver and white adipose tissue. We found a large number of ChREBP binding sites, which are attributable to 5825 genes in the liver, 2418 genes in white adipose tissue, and 5919 genes in both tissues. The majority of these target genes were involved in known metabolic processes. Pathways in insulin signaling, the adherens junction, and cancers were among the top 5 pathways in both tissues. Motif analysis revealed a consensus sequence CAYGYGnnnnnCRCRTG that was commonly shared by ChREBP binding sites. Putative ChREBP binding sequences were enriched on promoters of genes involved in insulin signaling pathway, insulin resistance, and tumorigenesis.

Impact of CCR2 and SDF1 polymorphisms on disease progression in HIV-infected subjects in Thailand.

BACKGROUND: The genotypic polymorphisms of CCR5, CCR2, and SDF1 were analyzed to determine their impact as potential confounders with regard to disease progression because of the role that host genetic factors appear to be involved in determining rates of disease progression. METHODS: Genomic DNA was extracted from Ethylenediaminetetraacetate whole blood using Qiagen DNA extraction kit. The amplification of CCR5, CCR2, and SDF1 genes was performed by PCR. RESULTS: Two hundred and twenty-one samples were genotyped for the CCR5, CCR2, and SDF1 mutation. Among these, all (100%) were identified as wild type for CCR5. All were then investigated considering the impact on CD4+ T-cell counts. Samples were divided into two groups based on the CD4+ T-cell numbers. It revealed that in the group of CD4+ T-cell counts ≥200 cells/µl, 15 were found for the homozygous for SDF1 gene (3'A/3'A) whereas one was found in the group of CD4+ T-cell counts <200 cells/µl. Homozygosity for the CCR2 polymorphisms (64I/64I) were five in the group of CD4+ T-cell counts ≥200 cells/µl and none were found in the group of CD4+ T-cell counts <200 cells/µl. These results demonstrated that there was a significant association between CD4+ T-cell numbers and CCR2 and SDF1 polymorphisms (P < 0.001). CONCLUSIONS: The mutation of CCR2 and SDF1 genes showed a significant difference in the distribution of CD4+ T-cell numbers (P < 0.001) whereas mutation of chemokine coreceptor CCR5 was not appeared to be associated with the impact of CD4+ T-cell counts.