A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies

Large population‐based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 participants in the Health and Retirement Study (HRS) are described. The extensive quality control involved in a large‐scale immunophenotyping epidemiological study is also outlined. The existing literature on the effect of cryopreservation on various immune cell subsets including T, B, NK cells, monocytes, and dendritic cells is provided. © 2018 by John Wiley & Sons, Inc.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143631/1/cpcy35.pd

Prevalence of clinically actionable disease variants in exceptionally long-lived families

BACKGROUND: Phenotypic expression of pathogenic variants in individuals with no family history of inherited disorders remains unclear. METHODS: We evaluated the prevalence of pathogenic variants in 25 genes associated with Mendelian-inherited disorders in 3015 participants from 485 families in the Long Life Family Study (LLFS). Boot-strapping and Fisher\u27s exact test were used to determine whether allele frequencies in LLFS were significantly different from the allele frequencies reported in publicly available genomic databases. RESULTS: The proportions of pathogenic autosomal dominant mutation carriers in BRCA1 and SDHC in LLFS study participants were similar to those reported in publicly available genomic databases (0.03% vs. 0.0008%, p = 1 for BRCA1, and 0.08% vs. 0.003%, p = 0.05 for SDHC). The frequency of carriers of pathogenic autosomal recessive variants in CPT2, ACADM, SUMF1, WRN, ATM, and ACADVL were also similar in LLFS as compared to those reported in genomic databases. The lack of clinical disease among LLFS participants with well-established pathogenic variants in BRCA1 and SDHC suggests that penetrance of pathogenic variants may be different in long lived families. CONCLUSION: Further research is needed to better understand the penetrance of pathogenic variants before expanding large scale genomic testing to asymptomatic individuals

Low risk for developing diabetes among the offspring of individuals with exceptional longevity and their spouses

Little is known about the risk of type 2 diabetes (T2D) among the offspring of individuals with exceptional longevity. We determined the incidence of and potential risk and protective factors for T2D among the offspring of probands and offspring\u27s spouses (mean age=60 years, range 32-88 years) in the Long Life Family Study (LLFS), a multicenter cohort study of 583 two-generation families with a clustering of healthy aging and exceptional longevity. Incident T2D was defined as fasting serum glucose ≥126 mg/dl, or HbA1c of ≥6.5%, or self-reported with doctor diagnosis of T2D, or the use of anti-diabetic medication during a mean follow-up 7.9 ± 1.1 years. Among offspring (n=1105) and spouses (n=328) aged 45-64 years without T2D at baseline visit, the annual incident rate of T2D was 3.6 and 3.0 per 1000 person-years, respectively, while among offspring (n=444) and spouses (n=153) aged 65+ years without T2D at baseline, the annual incident rate of T2D was 7.2 and 7.4 per 1000 person-years, respectively. By comparison, the annual incident rate of T2D per 1000 person-years in the U.S. general population was 9.9 among those aged 45-64, and 8.8 among those aged 65+ years (2018 National Health Interview Survey). Baseline BMI, waist circumference, and fasting serum triglycerides were positively associated with incident T2D, whereas fasting serum HDL-C, adiponectin, and sex hormone binding globulin were protective against incident T2D among the offspring (all P\u3c0.05). Similar associations were observed among their spouses (all P\u3c0.05, except sex hormone binding globulin). In addition, we observed that among spouses, but not offspring, fasting serum interleukin 6 and insulin-like growth factor 1 were positively associated with incident T2D (P\u3c0.05 for both). Our study suggests that both offspring of long-living individuals and their spouses, especially middle-aged, share a similar low risk for developing T2D as compared with the general population. Our findings also raise the possibility that distinct biological risk and protective factors may contribute to T2D risk among offspring of long-lived individuals when compared with their spouses. Future studies are needed to identify the mechanisms underlying low T2D risk among the offspring of individuals with exceptional longevity, and also among their spouses

Genetic association analysis of the cardiovascular biomarker: N-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP)

BACKGROUND: NT-proBNP is a biomarker of cardiovascular disease (CVD). Little is known about the heritability and genetic variants associated with NT-proBNP. Therefore, we estimated the heritability of and examined genetic associations of SNPs in the BNP gene region with circulating NT-proBNP and prevalent CVD in 4,331 participants from the Long Life Family Study (LLFS). METHODS AND RESULTS: Genotypes of 10 SNPs from the NPPB and NPPA regions that encode BNP and A-type natriuretic peptide, respectively, were tested for association with NT-proBNP and prevalent cardiovascular disease and risk factors. We performed analyses using the Sequential Oligogenic Linkage Analysis (SOLAR) program to account for family relatedness, and adjusted all models for age, sex, and field center. The mean age of the LLFS was 69.8 years (range 24-110) with 55.4% females. NT-proBNP was significantly heritable (h2 = 0.21; P = 4x10-14), and the minor alleles of rs632793 (p\u3c0.001) and rs41300100 (p = 0.05) were independently associated with higher serum NT-proBNP levels. Additionally, the minor allele of rs632793 was significantly and consistently associated with lower prevalent CVD, including blood pressures, independent of NT-proBNP level (all P\u3c0.05). Results for prevalent CVD, but not NT-proBNP levels, showed significant interaction by familial generation. CONCLUSION: In this family-based study of subjects with exceptional longevity, we identified several allelic variants in the BNP gene region associated with NT-pro-BNP levels and prevalent CVD

Serum Adiponectin is Associated with Adverse Outcomes of Asthma in Men but Not in Women

Background: Murine studies suggest a beneficial effect of systemic adiponectin on asthma. Our objective was to determine the association between serum adiponectin concentrations and asthma control/severity outcomes in men and women separately. Methods: Cross-sectional and longitudinal analyses of data from years 10, 15, and 20 examinations of the prospective coronary artery risk development in young adults study in the United States were performed. Asthma was defined by self-reported provider diagnosis at or prior to year 15 examination. Outcomes included presence of active disease, number of respiratory symptoms, and number of asthma medications; as well as longitudinal decline in absolute FEV1. Year 15 serum adiponectin concentration was the predictor variable. Results: In a multivariable analysis of 411 eligible subjects, after adjusting for body mass index and covariates, higher serum adiponectin concentrations were associated with more frequent active disease (including more frequent use of any asthma medication), and greater number of respiratory symptoms and asthma medications among men but not among women with asthma (p for interactions between sex and adiponectin for all analyses < 0.05). Conclusions: Higher serum adiponectin concentrations may be independently associated with adverse clinical outcomes of asthma in men but not in women. If biological effect is confirmed in future studies, modification of systemic adiponectin concentrations may open up newer ways to treat asthma in men

Prevalence of clinically actionable disease variants in exceptionally long-lived families

Background Phenotypic expression of pathogenic variants in individuals with no family history of inherited disorders remains unclear. Methods We evaluated the prevalence of pathogenic variants in 25 genes associated with Mendelian-inherited disorders in 3015 participants from 485 families in the Long Life Family Study (LLFS). Boot-strapping and Fisher’s exact test were used to determine whether allele frequencies in LLFS were significantly different from the allele frequencies reported in publicly available genomic databases. Results The proportions of pathogenic autosomal dominant mutation carriers in BRCA1 and SDHC in LLFS study participants were similar to those reported in publicly available genomic databases (0.03% vs. 0.0008%, p = 1 for BRCA1, and 0.08% vs. 0.003%, p = 0.05 for SDHC). The frequency of carriers of pathogenic autosomal recessive variants in CPT2, ACADM, SUMF1, WRN, ATM, and ACADVL were also similar in LLFS as compared to those reported in genomic databases. The lack of clinical disease among LLFS participants with well-established pathogenic variants in BRCA1 and SDHC suggests that penetrance of pathogenic variants may be different in long lived families. Conclusion Further research is needed to better understand the penetrance of pathogenic variants before expanding large scale genomic testing to asymptomatic individuals

African Ancestry Gradient Is Associated with Lower Systemic F 2

Context. Low levels of systemic F2-isoprostanes (F2-IsoP) increase the risk of diabetes and weight gain and were found in African Americans. Low F2-IsoPs could reflect an unfavorable metabolic characteristic, namely, slow mitochondrial metabolism in individuals with African ancestry. Objective. To examine differences in plasma F2-IsoPs in three groups with a priori different proportion of African ancestry: non-Hispanic Whites (NHWs), US-born African Americans (AAs), and West African immigrants (WAI). Design. Cross-sectional study. Setting. Georgia residents recruited from church communities. Participants. 218 males and females 25–74 years of age, who are self-identified as NHW (n=83), AA (n=56), or WAI (n=79). Main Outcome Measure(s). Plasma F2-IsoPs quantified by gas chromatography-mass spectrometry. Results. After adjustment for age, gender, obesity, and other comorbidities, WAI had lower levels of plasma F2-IsoP than AA (beta-coefficient = −9.8, p<0.001) and AA had lower levels than NHW (beta-coefficient = −30.3, p<0.001). Similarly, among healthy nonobese participants, F2-IsoP levels were lowest among WAI, followed by AA, and the highest levels were among NHW. Conclusion. Plasma F2-IsoPs are inversely associated with African ancestry gradient. Additional studies are required to test whether optimization of systemic F2-IsoP levels can serve as means to improve race-specific lifestyle and pharmacological intervention targeted to obesity prevention and treatment

Sex Differences in Associations of Adiposity Measures and Insulin Resistance in US Hispanic/Latino Youth: The Hispanic Community Children's Health Study/Study of Latino Youth (SOL Youth)

Context: US Hispanic/Latino youth are disproportionally affected by the obesity and diabetes. Objective: We examined associations of adiposity measures with insulin resistance (IR) and hyperglycemia and the influences of sex and pubertal development on these associations. Design, Setting, and Participants: We performed a cross-sectional analysis of 1223 8- to 16-year-old Hispanic/Latino youth from a community-based study in the United States (SOL Youth). Main Outcome Measures: We measured IR (≥75th percentile of sex-specific Homeostatic Model Assessment of Insulin Resistance) and hyperglycemia (fasting glucose ≥100 mg/dL or hemoglobin a1c ≥5.7%). Results: In boys, body mass index (BMI) showed the strongest association with IR [prevalence ratio (PR), 2.10; 95% confidence interval (CI), 1.87 to 2.36 per standard deviation], which was not statistically different compared with body fat percentage (%BF) (PR, 2.03; 95% CI, 1.81 to 2.29) and waist circumference (WC) (PR, 1.89; 95% CI, 1.67 to 2.13) but was significantly stronger compared with fat mass index (FMI) (PR, 1.79; 95% CI, 1.63 to 1.96), waist-to-hip ratio (WHR) (PR, 1.32; 95% CI, 1.21 to 1.44), and waist-to-height ratio (WHtR) (PR, 1.76; 95% CI, 1.54 to 2.01) (P for difference, <0.05). In girls, %BF (PR, 2.73; 95% CI, 2.34 to 3.20) showed a significantly stronger association with IR compared with BMI (PR, 1.48; 95% CI, 1.29 to 1.70), FMI (PR, 1.71; 95% CI, 1.49 to 1.95), WC (PR, 1.96; 95% CI, 1.70 to 2.27), WHR (PR, 1.95; 95% CI, 1.70 to 2.23), and WHtR (PR, 1.79; 95% CI, 1.53 to 2.09) (P for difference, <0.003). Associations between adiposity measures and IR were generally stronger among children in puberty versus those who had completed puberty, with significant interactions for WC and WHtR in boys and for BMI in girls (P for interaction, <0.01). Adiposity measures were modestly associated with hyperglycemia (PR, 1.14 to 1.25), with no interactions with sex or pubertal status. Conclusions: Sex and puberty may influence associations between adiposity measures and IR in US Hispanic/Latino youth. Multiple adiposity measures are needed to better assess IR risk between boys and girls according to pubertal status

Leukocyte telomere length is unrelated to cognitive performance among non-demented and demented persons: An examination of long life family study participants

OBJECTIVE: Leukocyte telomere length (LTL) is a widely hypothesized biomarker of biological aging. Persons with shorter LTL may have a greater likelihood of developing dementia. We investigate whether LTL is associated with cognitive function, differently for individuals without cognitive impairment versus individuals with dementia or incipient dementia. METHOD: Enrolled subjects belong to the Long Life Family Study (LLFS), a multi-generational cohort study, where enrollment was predicated upon exceptional family longevity. Included subjects had valid cognitive and telomere data at baseline. Exclusion criteria were age ≤ 60 years, outlying LTL, and missing sociodemographic/clinical information. Analyses were performed using linear regression with generalized estimating equations, adjusting for sex, age, education, country, generation, and lymphocyte percentage. RESULTS: Older age and male gender were associated with shorter LTL, and LTL was significantly longer in family members than spouse controls (p \u3c 0.005). LTL was not associated with working or episodic memory, semantic processing, and information processing speed for 1613 cognitively unimpaired individuals as well as 597 individuals with dementia or incipient dementia (p \u3c 0.005), who scored significantly lower on all cognitive domains (p \u3c 0.005). CONCLUSIONS: Within this unique LLFS cohort, a group of families assembled on the basis of exceptional survival, LTL is unrelated to cognitive ability for individuals with and without cognitive impairment. LTL does not change in the context of degenerative disease for these individuals who are biologically younger than the general population