TSH and FT4 analyses

Measurements of TSH were performed at the laboratories of Montefiore Medical Center, Bronx, New York, using a solid- phase, two-site chemiluminescent immunometric assay (Immu- nolite 2000 Third Generation TSH; Siemens Corp., Tarrytown, NY; manufacturer’s reference limits, 0.4–4.0 mIU/liter). FT4 was measured using ELISA (Alpco Diagnostic, Salem, NH; man- ufacturer’s reference limits, 0.8–1.8 ng/dl). Because this method provides only an estimate of FT4 and not a direct measurement of FT4, all FT4 values represent estimated levels of FT4. Anti- thyroid antibodies were not determined because of insufficient availability of serum.

This project was part of the Longevity program at Albert Einstein College of Medicine, Bronx, New York.

Subjects and Methods

Population

We studied three distinct populations: 1) 232 Ashkenazi Jew- ish centenarians (166 women, median age, 97 yr; and 66 men, median age, 97 yr); 2) 366 Ashkenazi Jewish offspring of these centenarians (185 women, median age, 68 yr; and 181 men, median age, 69 yr); and 3) 111 of the offsprings’ Ashkenazi Jewish spouses (control group A; 51 women, median age, 66 yr; and 60 men, median age, 73 yr). An additional control group of 52 Ashkenazi Jews (control group B; 28 women, median age, 70 yr;and24men,medianage,75yr),whichwasnotfamily-related totheabovesubjectsbutwasfromthesamegeographicarea,was similarly assessed. Two control groups were used to exclude any possibility of environmental factors affecting the thyroid func- tiontestsincontrolgroupA(subjectslivedinthesamehousehold as their spouse, the offspring). However, all parameters tested were virtually identical in both control groups (data not shown). Thus, in our report, the controls represent control group A and control group B combined. All participants were recruited randomly.

Due to logistic and technical restrictions, serum FT4 was an- alyzed in 174 offspring and 172 controls. However, only 100 offspring and 134 controls had both serum TSH and FT4 deter- mined (inter- and intraassay coefficients of variation were 1.5 and 3% for TSH, and 3 and 6% for FT4).

Allblooddrawswereperformedinthemorningafteranover- night fast.

Analysis of single nucleotide polymorphisms (SNPs)

TheTSHgene,alsocalledTSH-B (TSH subunit- ), spans 4.5 kb with three exons and is located on the long arm of chro- mosome 1. We selected four SNPs (rs10494170, rs12046203, rs12740035, and rs7523360) of TSH-B gene and genotyped them in both Ashkenazi Jewish centenarians and controls. The allelicfrequenciesoftheseSNPsbetweentheAshkenaziJewishcen- tenarians and controls did not show significant differences. Due to the association between TSH receptor (TSHR) and TSH expres- sion, we further examined SNPs in this gene. SNPs (rs12050077 and rs10149689) were genotyped using the PSQ HS 96A Pyrose- quencer, according to the manufacturer’s recommendations (Py- rosequencing, Uppsala, Sweden; www.pyrosequencing.com). Briefly, a PCR product was generated from a primer pair that included one primer covalently coupled to biotin, the biotinyl- ated template was bound to streptavidin-coated Sepharose HP beads, and this mixture was then annealed to a sequencing primer. Stepwise elongation of a sequencing primer strand upon sequential addition of a specified sequence of deoxynucleotide triphosphates and the degradation of nucleotides by apyrase werecarriedoutsimultaneously.Asthesequencingreactionpro-

Stated age was verified by checking birth certificates or U.S. passports. Jewish religion was self-reported (mother and father Jewish), and subjects were considered Ashkenazi Jews if their four grandparents were originally Ashkenazi Jews. Information on medical history, demographic characteristics, and clinical data were obtained uniformly using a structured questionnaire. Allsubjectsunderwentaphysicalexaminationandwereaskedto provide a blood sample. Informed written consent was obtained in accordance with the policy of the Committee on Clinical In- vestigation of the Albert Einstein College of Medicine.

Exclusion criteria included acute or debilitating medical

gressed, the DNA strand was extended, and the sequence was determined from the measured signal output of light upon nu- cleotideincorporation.Theresultingpeaksinthepyrogramwere analyzed using Pyrosequencing software. Primer sequences are available from the authors upon request. Error rates based on blind replicates were estimated to be 0–5%.

was not different from controls (70 yr). However, the me- dian age of female controls, 67 yr [97.5% confidence in- terval(CI),52–80],wassignificantlylowerthanmalecon-   trols, 74 yr (97.5% CI, 59–80; P 0.002).

Serum TSH distribution

Statistical analysis

Serum TSH concentrations in the offspring were sig- nificantly higher compared with controls [median, 97.5% CI: 1.68 (0.65–4.79) and 1.55 (0.63–3.93) mIU/liter, re- spectively; P  0.02; Table 1). Moreover, serum TSH distribution was also shifted to higher TSH concentra- tions in offspring compared with controls, including the TSH concentrations at peak frequency (Fig. 1), suggest- ing a population shift phenomenon. TSH distribution in Ashkenazi Jewish centenarians, recently published (4) and shown only for comparative purposes, was further shifted to higher TSH concentrations (Fig. 1) compared with offspring, suggesting a genetic predisposition for higher TSH.

All analyses were conducted using Statistical Analysis Soft- ware(SASInstitute,CaryNC),version9.12.BecauseserumTSH and FT4 were not normally distributed, nonparametric tests in- cluding the Kruskal-Wallis one-way ANOVA by ranks and Mann-Whitney rank sum test were used for group comparisons.