Circadian regulation gene polymorphisms are associated with sleep disruption and duration, and circadian phase and rhythm in adults with HIV

<div><p>Genes involved in circadian regulation, such as circadian locomotor output cycles kaput [CLOCK], cryptochrome [CRY1] and period [PER], have been associated with sleep outcomes in prior animal and human research. However, it is unclear whether polymorphisms in these genes are associated with the sleep disturbances commonly experienced by adults living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Thus, the purpose of this study was to describe polymorphisms in selected circadian genes that are associated with sleep duration or disruption as well as the sleep–wake rhythm strength and phase timing among adults living with HIV/AIDS. A convenience sample of 289 adults with HIV/AIDS was recruited from HIV clinics and community sites in the San Francisco Bay Area. A wrist actigraph was worn for 72 h on weekdays to estimate sleep duration or total sleep time (TST), sleep disruption or percentage of wake after sleep onset (WASO) and several circadian rhythm parameters: mesor, amplitude, the ratio of mesor to amplitude (circadian quotient), and 24-h autocorrelation. Circadian phase measures included clock time for peak activity (acrophase) from actigraphy movement data, and bed time and final wake time from actigraphy and self-report. Genotyping was conducted for polymorphisms in five candidate genes involved in circadian regulation: CLOCK, CRY1, PER1, PER2 and PER3. Demographic and clinical variables were evaluated as potential covariates. Interactions between genotype and HIV variables (i.e. viral load, years since HIV diagnosis) were also evaluated. Controlling for potentially confounding variables (e.g. race, gender, CD4+ T-cell count, waist circumference, medication use, smoking and depressive symptoms), CLOCK was associated with WASO, 24-h autocorrelation and objectively-measured bed time; CRY1 was associated with circadian quotient; PER1 was associated with mesor and self-reported habitual wake time; PER2 was associated with TST, mesor, circadian quotient, 24-h autocorrelation and bed and wake times; PER3 was associated with amplitude, 24-h autocorrelation, acrophase and bed and wake times. Most of the observed associations involved a significant interaction between genotype and HIV. In this chronic illness population, polymorphisms in several circadian genes were associated with measures of sleep disruption and timing. These findings extend the evidence for an association between genetic variability in circadian regulation and sleep outcomes to include the sleep–wake patterns experienced by adults living with HIV/AIDS. These results provide direction for future intervention research related to circadian sleep–wake behavior patterns.</p></div

Differences between the lymphedema and no lymphedema groups.

<p>A – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous for the common allele (AA) or heterozygous or homozygous for the rare allele (AG+GG) for rs315721 in lymphocyte cytosolic protein 2 (LCP2). B – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous or heterozygous for the common allele (TT+TG) or homozygous for the rare allele (GG) for rs849530 in neuropilin-2 (NRP2). C – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous or heterozygous for the common allele (AA+AT) or homozygous for the rare allele (TT) for rs158689 in protein tyrosine kinase (SYK). D – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygous for the common allele (CC) or heterozygous or homozygous for the rare allele (CT+TT) for rs3176861 in vascular cell adhesion molecule 1 (VCAM1).</p

VEGFC Gene Structure and Linkage Disequilibrium.

<p>An ideogram of vascular endothelial growth factor C (VEGFC) is presented above the white bar that represents the physical distance along human chromosome 4 (chr4: 177,841,685–177,950,889; genome assembly 36.3, NM_005429.2). Exons are represented as thick bars. Gray lines connecting the exons represent introns. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for VEGFC was rendered with FancyGene 1.4. The correlation statistics (r² and D') are provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' values (range: 0–1, inclusive) were rendered in color, with darker red diamonds representing D' values approaching 1.0. When the r² values (range of 0–100, inclusive) are not equal to 0 or 100, they are provided in a given diamond. The 2-SNP haplotype associated with LE consists of one rare and one common allele of two SNPs (rs3775202 “G” rare allele, rs3775195 “C” common allele) located in intron 4 of the gene. Of note, the strong linkage disequilibrium estimates observed in public databases (i.e., HapMap) resulted in the selection of 8 SNPs that tagged the entire coding region of the VEGFC gene.</p

Multiple logistic regression analyses for FOXC2. LCP2, NRP2, SYK, VCAM1, and VEGF-C genotypes and halotypes to predict the development of lymphedema.

<p>For each model, the first three principal components identified from the analysis of ancestry informative markers as well as self-report race/ethnicity (i.e., White, Black, Asian/Pacific Islander, Hispanic/Mixed ethnic background/Other) were retained in all models to adjust for potential confounding due to race or ethnicity (data not shown). Predictors evaluated in each model included genotype (FOXC2 haplotype A03 composed of the rs34221221 “C” allele and the rs1035550 “C” allele; LCP2 rs315721: AA versus AG+GG; NRP2 rs849530: TT+TG versus GG; NPR2 haplotype F01 composed of the rs849530 “G” allele, the rs950219 “G” allele, and the rs3771052 “G” allele; SYK rs158689: AA+AT versus TT; VCAM1 rs3176861: CC versus CT + TT; VEGFC haplotype B03 composed of the rs3775202 “G” allele and the rs3775195 “C” allele), BMI (kilograms/meter squared), stage of disease, SLNB, number of lymph nodes removed, receipt of chemotherapy prior to or following surgery, and receipt of radiation therapy following surgery.</p><p>Abbreviations: BMI  =  body mass index; CI  = confidence interval; FOXC2  =  Forkhead box protein C2; LCP2  =  Lymphocyte cytosolic protein 2; NRP  =  neuropilin-2; SLNB  =  sentinel lymph node biopsy; SYK  =  spleen tyrosine kinase; VCAM1  =  vascular cell adhesion molecule 1; VEGFC  =  vascular endothelial growth factor-C.</p

NRP2 Gene Structure and Linkage Disequilibrium.

<p>An ideogram of neuropilin 2 (NRP2) is presented above the white bar that represents the physical distance along human chromosome 2 (chr2: 206,255,469–206,371,102; genome assembly 36.3, NM_201266.1). Exons are represented as thick bars. Gray lines connecting the exons represent introns. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for NRP2 was rendered with FancyGene 1.4. The correlation statistics (r² and D') are provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' values (range: 0–1, inclusive) were rendered in color, with darker red diamonds representing D' values approaching 1.0. When the r² values (range of 0–100, inclusive) are not equal to 0 or 100, they are provided in a given diamond. The 3-SNP haplotype associated with LE consists of one rare and two common alleles of three SNPs (rs849530 “G” rare allele, rs950219 “G” common allele, rs3771052 “G” common allele) located in intron 1 of the gene.</p

FOXC2 Gene Structure and Linkage Disequilibrium.

<p>An ideogram of forkhead box C2 (FOXC2) is presented above the white bar that represents the physical distance along human chromosome 16 (chr16: 85,158,358–85,160,040; genome assembly 36.3, NM_005251.2). Exons are represented as thick bars. Reference sequence identifiers (rsID) for each single nucleotide polymorphism (SNP) are plotted both in terms of their physical distance (i.e., the white bar at the top of the figure) and equidistantly to render the pairwise linkage disequilibrium (LD) estimates that were calculated and visualized with Haploview 4.2. The gene structure for FOXC2 was rendered with FancyGene 1.4. The correlation statistic (r² and D') is provided in the heatmap. LD-based haplotype block definition was based on the D' confidence interval method. The haploblock is indicated in a bolded triangle and its component SNPs are rendered in bold font. Pairwise D' value (range: 0–1, inclusive) was rendered in color, with darker red diamond representing D' value approaching 1.0. When the r² value (range of 0–100, inclusive) is not equal to 0 or 100, it is provided in a given diamond. The 2-SNP haplotype associated with LE is composed of one rare and one common allele of two SNPs located in the immediate early promoter (rs34221221; rare “C” allele) and immediately downstream of the FOXC2 coding region (rs1035550; common “C” allele).</p

Differences in demographic and clinical characteristics between patients with (n = 155) and without (n = 387) lymphedema.

<p>Abbreviations: kg  =  kilograms, m² – meter squared, NS  =  not significant, SD  =  standard deviation.</p

Differences in Baseline Demographic and Clinical Characteristics Between the Two Latent Classes for General Sleep Disturbance Scale Total Score.

<p>Differences in Baseline Demographic and Clinical Characteristics Between the Two Latent Classes for General Sleep Disturbance Scale Total Score.</p

Multiple Logistic Regression Analyses for Interleukin 6 (IL6) rs35610689 and Nuclear Factor Kappa Beta 2 Subunit (NFKB2) rs7897947 to Predict Higher Sleep Disturbance Class.

<p>For each model, the first three principle components identified from the analysis of ancestry informative markers as well as self-report race/ethnicity (White, Asian/Pacific Islander, Black, Hispanic/Mixed background/Other) were retained in all models to adjust for potential confounding due to race or ethnicity (data not shown). Predictors evaluated in the model included genotype (IL6 rs35610689: AA versus AG+GG; NFKB2 rs7897947: TT versus TG + GG), age (5 year increments), and functional status (KPS score, 10 point increments). Patient versus family caregiver (FC) status could not be included in the regression analyses because no FCs were included in the higher sleep disturbance class.</p

GMM parameter estimates for general sleep disturbance scale latent class^a solution with 7 assessments, with dyad as a clustering variable.

*<p>p<.05, **p<.01, ^***p<.001.</p>a<p>Trajectory group sizes are for classification of individuals based on their most likely latent class probabilities.</p>b<p>Growth mixture model estimates were obtained with robust maximum likelihood, with dyad as a clustering variable to account for dependency between patients and caregivers within the same dyad. Quadratic slope variances were fixed at zero to improve estimation.</p><p>Abbreviations: GMM  =  Growth mixture model; S.E.  =  standard error.</p