DNA samples

The possible association between 110A > C polymorphism in HSP70-1 gene and successful/ unsuccessful aging was studied on DNA samples collected from a cohort of 200 DZ twin pairs aged 70–91 years, from LSADT study. These samples form a part of the Danish Twin Registry (Skytthe et al. 2002), which is the first nationwide twin reg- istry in the world. The LSADT is an ongoing lon- gitudinal study of Danish twins (Christensen et al. 1999) who are more than 70 years old (about 4000 individuals). With an interval of two years these individuals have been offered an examination at their home, which includes both a traditional health-related interview and a number of objective and cognitive tests, and a collection of blood sample. Till date five rounds have been performed in the years 1995, 1997, 1999, 2001, and 2003. The DZ twins from LSADT-1999 wave are the basis of this investigation. These twins are between the ages 70 and 91 years, and have been categorised according to the absence or presence of various age-related diseases viz. lung disease, osteoporosis, arthritis, osteoarthrosis, cardiovascular diseases (ischaemic heart disease (AMI/angina), heart fail- ure and stroke), hypertension, hyperthyroidism, cancer and diabetes, and for various age-related parameters including sex- and age-adjusted scores of physical and cognitive tests (Frederiksen et al. 2002b). Various parameters which have been used as measures of aging include scorings of the phys- ical function such as ‘hand grip strength’, ‘activity of daily living (ADL) strength’ and ‘ADL endur- ance’ (Nybo et al. 2001); scorings of the mental state of the individual viz. ‘cognititive composite score’ and ‘mini mental state examination’ (MMSE) (Frederiksen et al. 2002b). Furthermore, ‘self-rated health’ (scored both in absolute terms, measured in the scale of 1–5, with the possible re- sponses, ‘very good’, ‘good’, ‘fair’, ‘poor’, ‘very poor’; and in relation to others of the same age,

In a recent cross-sectional study on samples from Calabria, a south Italian region, Altomare et al. (2003) have reported that the presence of an allele carrying adenine (allele A), in the ) 110A > C polymorphism present in the 5¢ flanking region, 3 base pairs upstream of the heat shock element (HSE) of the promoter region of HSP70-1 gene, was unfavourable for longevity in women. In another association study between polymorphism (2437T > C) in the coding region of HSP70-Hom with human aging, done on the Irish population (Ross et al. 2003), the frequency of T allele has been shown to increase in the old population (80–90 years) as compared to the young controls (19–45 years). However, as will be discussed later, there are confounding factors which occur while performing such cross-sectional studies, where allele frequencies in a group of young controls are compared against the allele frequencies in the oldest-old. Taking this into ac- count, we report the results of our study per- formed on DNA samples collected from Danish dizygotic (DZ) twin cohort of 400 individuals aged 70–91 years (mean ¼ 75.6), from the Longitudinal

cytosine (C). Hence on performing the melting peak analysis, the high or low melting peak corre- sponded to the presence of cytosine or adenine respectively (Figure 1). LightCycler reaction was performed on 3 ll DNA in the reaction mix of 10 ll containing the following reagents: 10· Titanium Taq PCR buffer (1 ll), ADV Taq 50· (0.25 ll), 10 mM dNTPs (0.13 ll), Primer HSP70-1-forw (5 pmol/ll; 1.0 ll), Primer HSP70-1-rev (5 pmol/ll; 1.0 ll), Anchor probe (5 pmol/ll; 0.2 ll), Sensor probe (5 pmol/ll; 0.2 ll), DMSO (0.5 ll) and dou- ble distilled water (2.72 ll). The sequence of the primers and probes used is given in Table 1. The program used for the LightCycler run can be available from the authors on request.

A substantial genetic component has been found for all the above traits, although the extent of the genetic component differs (Frederiksen and Chris- tensen, 2003). For example, a quarter of the vari- ation in self-rated health score has been explained by genetic factors for the groups 60–69 years and 70 years and older (Christensen et al. 1999). Also, ‘hand grip strength’ has been shown to be a suitable phenotype for identifying genetic variants of importance to mid- and late-life physical func- tioning and has a heritability of 52% (Frederiksen et al. 2002a). Once heritability is defined to all these age-related traits the next step lies in associating these traits with the specific genes. Based on these scorings we have analysed the association of parameters correlated with human longevity with ) 110A > C polymorphism in HSP70-1 gene.

Statistical analysis

We grouped the participants according to their HSP70-1 genotype and compared these groups with respect to various aging phenotypes. Con- tinuous phenotypes (physical ability scores, grip strength, depression and cognitive scores) were compared across the genotypes using the one way ANOVA test. Discrete variables (diseases – see Table 3) were compared across the genotypes using Pearson’s v2 test. Bonferroni correction on re- peated testing was applied.

Genotyping

Genotyping was done using the real-time PCR on the LightCycler system (Roche Applied Sciences), which helps to monitor the amplification of PCR product simultaneously, using fluorescent labelled sequence specific oligonucleotide probes. Probes were designed such that the oligonucleotides were complementary to the allele carrying the nucleotide

Fluorescence -d(F2/F1)/dT   eat GT GG Gia Ee Cie LIM LInEaE eae et520 640 660 580 600 620 640 660 680 700 720 740 760 780 600 820 940 860

NT en fkSke ale pene ee een Re eR eee dee oko athe Rae ae620 40 860 68.0 000 620 640 080 680 700 720 740 760 780 900 820 940 800