For the Project (called Progenia for the Sardinian public), this poster shows one of the participants , who lived to over 100 through a vigorous old age

Welcome to SardiNIA Project

Finding longevity genes is only one of many goals for gerontologists. An equally important mission is unraveling the genetic processes involved in age-related traits and diseases to promote healthspan as well as lifespan. NIA and Italian investigators have been focusing their attention on the Mediterranean island of Sardinia. Why? We quote directly from the rationale and background for the initiation of the study in 2001, which also applies to follow-up studies in course:

Rationale: To identify genetic bases for prominent age-associated changes, including, for example, cardiovascular risk factors and determinants of personality traits, in a founder population. The results of the study will extend the studies of aging-associated conditions of outbred populations. Ongoing reports from the Baltimore Longitudinal Study on Aging (BLSA, http://www.blsa.nih.gov/) and other population studies have identified epidemiological and some genetic risk factors for aging-associated diseases; but the majority of studies have been observational, determining the range of values for a phenotype as a function of aging. These analyses have been increasingly supplemented in recent years by genomic analyses and genetic studies based on powerful genome-wide association studies in numerous general populations. Nevertheless, considerable attention has focused on the promise of 'founder populations' for the simplification of analysis of complex traits and aging-related diseases. Such rare populations arise from a delimited group living in a defined region for many centuries with minimal admixture from outside populations. The Sardinian population is one of the few that is both large and accessible, and one of the most extreme in its relative lack of heterogeneity. It can thus be studied for a wide variety of both frequent and relatively rare traits. The targeted region for the present study, Ogliastra, has a particularly isolated population of 60,000, in an area enclosed by two mountain ranges and the sea; and for the study of diseases, the entire catchment of 1,500,000 Sardinians across the island can furnish enough cases and controls to investigate genetic factors for a wide range of conditions and maladies.

The SardiNIA study has decisively established that analyses on the island can indeed help to analyze both complex traits and diseases, often providing information that was difficult or impossible to obtain in other population studies.

Concerning the choice and progress of the Project, its positive features include:

• Relatively constant environment (in rural towns for the core study)
• High level of altruism and interest in sharing genetic patrimony
• Support of the local Bishop, Mayors, and Health Authorities
• Local clinic location that facilitates rapid and repeated visits; for example, 4,000 participants were recalled in 1 year for fractionation of white cells into 95 subtypes within 90 min of blood draw, and for a subsequent second blood draw to extend studies to more cell types, cytokines, and autoantibodies.
• Structures of more than 1,000 families embedded in a large cohort of individuals, permitting accurate estimateo Structures of more than 1,000 families embedded in a large cohort of individuals, permitting accurate estimates of genetic heritability. s of genetic heritability.
• o Lower cost sequencing and genotyping coverage: 2,000 individuals sequenced at 4-fold coverage yielded the imputed DNA sequence for the entire cohort.
• Reliable detection of rare DNA sequence variants.
• Assessment of diagnostic/prognostic strength of genetic factors in a longitudinal study with medical outcomes.
• Straightforward formulation and testing of imputation algorithms.
• Excellent coverage of genetic variation in Europe.
• Easier discovery of interactions/pleiotropy of traits.

In a first survey, the project team recruited over 6,100 subjects from a catchment area including four towns in east-central Sardinia and assessed a first list of about 200 traits. The baseline survey has been advanced by follow-up visits that collected longitudinal data on the same traits collected at baseline but with added assessments of frailty-related traits, namely measures of bone density and geometry, muscle strength, and gait speed, and additional cardiovascular measures (see below). In the course of subsequent visits, along with the expansion of the cohort and the addition of more traits (see below), increases in testing efficiency and additional cost-sharing funds from Sardinian sources permitted the completion of Fourth and Fifth Visits for the entire cohort. Also, DNA sequencing has recovered essentially all of the genetic variation in the cohort, and further arrangements have also been made for an Outcome Study to be implemented. The infrastructure for the clinic and phenotypic testing has been stable, with stringent quality control, which is reflected in the high quality of the database. The initial sample cohort included over 62% of the eligible population living in the region (age 14-102 years), and at least 96% of the initial cohort have all grandparents born in the same province. The initial group included 4,933 phenotyped sib pairs, 4,266 phenotyped parent-child pairs, 4,069 phenotyped cousin pairs and 6,459 phenotyped avuncular pairs. Additional recruitment has increased the cohort substantially, and results have consistently shown that, for essentially every trait, most of the associated genes and variants would be involved in determining variance in both young and old and in men and women. Thus, genetic analyses can draw on data from all ages and both genders.

The added value of studying a founder population has also been demonstrated by the extension of DNA analysis to the full range of variation by sequencing. The population has proven to contain the great bulk of variation found in other populations, but during its isolation over many thousands of years, many variants rare elsewhere have risen to relatively high levels on the island by drift or selection and others have newly arisen as Sardinian-specific. These have provided extensive new information about a wide range of traits and pathways (e.g., Nature Genetics, November 2015 articles and Editorial). The study has also fostered the development of new analytic tools for genetic analysis, including a widely adopted program for measuring mitochondrial DNA copy number in cells (Ding J et al., 2017); a sensitive program to detect pleiotropic interactions of genetic variants with traits (Meirelles et al., in submission); and a program that reintroduces linkage mapping as a tool to add to genome-wide association analysis and increase the information about genetic factors affecting traits and diseases (Zajac G et al., 2023).

Sardinia also offers a special entree to the genetics of specific diseases that are especially prevalent in the founder population. This includes the anomalously high incidence of autoimmune diseases including Multiple Sclerosis and Type 1 diabetes, which interrupt the high-to-low gradient of incidence from Northern to Southern Europe with the highest levels seen anywhere in the world. Again, this has fostered novel findings about causation and pathophysiology.

The founder population itself also contains within its DNA a record of human demography through history, which has permitted the inference of the timing of human population movements based on analyses of mitochondrial and Y chromosomes.

Regarding the course and mechanism of aging, the longitudinal study, now in its 22nd year, has focused on residents of the cluster of towns to collect longitudinal information on more than 800 age-related quantitative traits ("endophenotypes" or "quantitative risk-related genetic or environmental factors") that can be scored on a continuous scale, as well as over 200 dichotomous traits (including major diseases and risk factors such as smoking). The use of quantitative traits permits the study of the entire range of allelic variation in a population, with particular interest in a range of cardiovascular risk factors, anthropometric measurements, blood test values, facets of personality, and bone-density and frailty-related variables. The longitudinal study of a broad range of phenotypes in a founder population is distinctive in this study, and stable environmental/epidemiological factors combined with the simplification of genetic analyses also aid in joint investigations of relative risk. Furthermore, because we are collecting risk factor data, we can also analyze, in an Outcome Study, the prognostic power and/or pathophysiological relevance of earlier predictors for the onset of serious risk factors [e.g., increases in pulse wave velocity as a function of earlier (predictor) lipid and inflammatory markers].

To identify health-related events that can be correlated with genetic and environmental risk factors and their interactions, a system has been set up to ascertain major health-related outcomes. The sources of information start with the self-reports of individuals at visits, but depend more on three more reliable sources: 1) medical records from local primary care physicians (every Sardinian has a local physician); 2) an extensive database maintained by the Sardinia Region for all health and social services delivered by the health care system, including hospitalization, treatment, and all pharmaceuticals taken by each individual; and 3) death certificates on file locally. The sixth visits now in progress are part of this Outcome Study.

In the most recent 5-year iteration of the study, now in its second year, an additional arm has been added to investigate genetic factors in Alzheimer/dementia. Once again, unique features of the island population support the justification of the study. They notably include the occurrence of the lowest reported levels of risk allele ApoE4 and ApoE2 in the population, so that over 90% of Sardinians are ApoE3-ApoE3; thus, fundamental etiology, including genetic factors, can be investigated in a constant ApoE background. Similar to previous studies of Multiple Sclerosis and Type 1 Diabetes, the study is based on coordinated recruitment at each of the 6 major sites across the island where AD/dementia patients are seen. Furthermore, the longitudinal study cohort provides 300 cases with blood and cell samples dating back over 20 years, permitting extensive studies of biomarkers and progression of pathophysiology. Finally, the ongoing extensive analysis of genetic factors affecting the immune system and immunosenescence in Sardinians promises to provide insights into the increasingly central involvement of immune factors in AD/dementia. 

The study of AD/dementia is one of three initiatives that provide the study with increasing power. It aims to recruit up to 8,000 cases and controls in the 5th contract period of the Project (extending up to mid-2025). Neuropsychologists and neurologists involved in the study of neurodegenerative diseases and auxiliary laboratory personnel have been recruited at the sites seeing patients, and using a protocol developed with Dr. Lenore Launer and Dr. Ann Sill at NIA in conjunction with Sardinian collaborators, data are being entered into a tailored electronic medical record. 

A second initiative is a continuation of substantial "auxiliary" efforts by the Sardinian scientists, especially those directed toward analyses of the immune system and autoimmune disease on the island. All of these have been supported by studies and investigators independent of the NIA-sponsored efforts but synergistic with them. They include, as specific examples, analyses of genetic factors affecting risk of Multiple Sclerosis; genetic factors affecting the levels of 118 subtypes of immune system cells and a battery of inflammatory molecules and autoantibodies; and the genetic factors affecting immunosenescence of those cells and molecules. In addition to their intrinsic interest, it is these studies that promise to reinforce and extend the now-ongoing study of Alzheimer/dementia. Finally, in another auxiliary extension of the studies, the study of genetic and epidemiological factors at the clinic in Lanusei is being extended to enroll at least 100,000 Sardinians over the next several years at the two largest hospitals on the island, one in Cagliari and the other in Sassari. This study is funded independently from NIA or CNR, but once again, it will interact synergistically – for example, providing more power for studies of genome-wide association of genetic variants with traits and diseases.

 Finally, in another auxiliary extension of the studies, the study of genetic and epidemiological factors at the clinic in Lanusei is being extended to enroll at least 100,000 Sardinians over the next several years at the two largest hospitals on the island, one in Cagliari and the other in Sassari. This study is funded independently from NIA or CNR, but once again, it will interact synergistically – for example, providing more power for studies of genome-wide association of genetic variants with traits and diseases.