This study will systematically assess epigenome methylation changes in participants of the Israel multi-ethnic centenarian study cohort, which includes citizens of Israel aged 95 years and older, compared to their offspring and younger controls.

The number of men and women reaching advanced age is rapidly increasing in developed countries and life expectancy has already doubled since the beginning of the last century. In parallel, the proportion of those who have survived to advanced old age without major health complications has become increasingly common. This phenomenon has led to concerted research efforts to understand the correlates and mechanisms of extended human longevity and healthy aging. For decades the public health perspective has emphasized environmental and life style factors as having beneficial effects on health and disease prevention in the general population. Important biological insights have been reached by examining the genetic basis of longevity and healthy aging, especially among individuals who demonstrate a favorable healthy aging phenotype. Epigenetics, defined as heritable changes in gene function or phenotypes, in the absence of DNA sequence changes, has revolutionized the concept of how genetics affect disease risk. Epigenetics have been shown to be associated with cancer risk and autoimmunity disorders. Moreover, epigenetic changes at specific sites appear to increase with age, hence they may serve as indices for chronological age. This background has led us to our central hypotheses that: i) epigenetic changes associated with healthy aging could serve as markers for a healthy life span, and ii) the epigenetic changes may represent one of the central mechanisms by which aging predisposes to many age-related diseases, and therefore affects healthy aging. We propose to test these hypotheses within the Israel multi-ethnic centenarian study cohort (which includes all citizens of Israel>95 years old; an estimated 1 in 5000 of the population). We will systematically assess genomic methylation changes in three major sub-groups which signifies three major points in human lifespan: Age 60±5 years (leading age of onset of age associated diseases and also considered to represent the beginning of old age, age 80±5 years (average life expectancy in the western world), and age 100±5 years (exceptional longevity). Our centenarians includes 3 subgroups: SURVIVORS: those who survive and become centenarians in spite of early onset of cognitive impairment, functional disability or major age associated diseases, such as diabetes, vascular disease or cancer, at the beginning of old age, i.e., age of 60±5 years (thus long life span but short healthy lifespan). DELAYERS: those who develop age related diseases or cognitive/physical disability much later than the control population i.e., at the age of 80±5 years instead of 60 years (therefore have a longer healthy life span). DODGERS: those who fail to develop age-related illnesses or cognitive/physical disability throughout their life span. We will compare these groups to healthy controls with no family history of longevity in two reference groups, namely 60±5 years and 80±5 years, comparing them to the survivors and delayers groups, respectively. We predict that subjects will exhibit differences in methylation at sites distinct from each other, in contrast to healthy subjects.

Accordingly our proposed goals are:

1. To characterize the Israel multi-ethnic centenarian study cohort (I-MECS).
2. To screen, catalog and annotate epigenome methylation variants in five different groups (a. Survivors, b. Delayers, c. Dodgers, d. 60±5YO healthy control, and e. 80±5YO healthy controls).
3. To validate differential methylated loci by ranking and prioritizing using Sequenom's MassARRAY.
4. Exploratory goal (depend on time and funding availability) - To establish expression patterns using a qRT-PCR (Polymerase Chain Reaction) Roche light-cycler of the candidate epigenetic loci identified in Aims 1, 2 and 3 as well as specific genes (such as deoxyribonucleic acid methyltransferase and tumor suppressor genes) in blood cells of the initially screened population.

To test our hypotheses, we propose to employ a novel high-throughput genome-wide methylation assay, namely HELPtag. Additionally, we will utilize a combination of epigenome-wide association studies (EWAS) to identify the most distinctive epigenetic loci that show greatest differential methylation. We will then perform multi-locus validation for methylation status using MassARRAY.

Inclusion Criteria:

• Aged 95 years and older.
• Provide verbal consent for participation in the study.
• Cognitively able to provide consent and answer a structured questionnaire.

Exclusion Criteria:

• Not able to provide consent for participation in the study.
• Impaired consciousness.
• Unable to communicate.

Subjects aged 95 years and older will form the study cohort. Control group will comprise children of the older subjects and age-matched controls.

Individual Participant Data (IPD) will not be shared