The SardiNIA Project

Governance and current state of the Project
The governance of the Project began with its implementation under the auspices of the National Institute on Aging (NIA), leading to the name SardiNIA (i.e., Sardi + NIA). A contract to Giuseppe Pilia, M.D., Principal Investigator for the Italian National Research Council (CNR) Institute in Cagliari (now the Institute of Genetic and Biomedical Research (IRGB), with David Schlessinger, Ph.D., Chief of the Laboratory of Genetics at NIA, as Principal Investigator for NIA and the indispensable support of Antonio Cao, M.D., the Dean of human genetics studies in Italy. During the initial 5 years (mid-2001 to mid-2005), a clinic was set up in the town of Lanusei and a team of Investigators ("Progenia") assembled there to recruit participants and collect data. After the untimely death of Dr. Pilia, Dr. Manuela Uda, took over for the next 5-year period, and was succeeded in 2010 by Francesco Cucca, M.D, Professor of Medical Genetics at the University of Sassari and director of IRGB-CNR. Soon after the project commenced, Drs. Pilia and Schlessinger were joined by a senior statistical geneticist, Goncalo Abecasis, Ph.D., at the University of Michigan, to head up data analysis efforts and train several Sardinian geneticists, while other Sardinians grew to capable members of the team of Ph.D. analysts n Cagliari, Sardinia – they include Serena Sanna, Carlo Sidore, Maristella Steri and another analytic group, headed by Jun Ding, Ph.D., has been added at the Laboratory of Genetics of the NIA in Baltimore. Currently, the collaborative group comprising the leadership of the Project includes those individuals working with investigators at several U.S. Universities (especially, Stephen Montgomery, Ph.D., at Stanford University, and John Novembre, Ph.D., at the University of Chicago, as well as Dr. Abecasis at the University of Michigan) and heads of Intramural Program Laboratories at the NIA, including Edward Lakatta, M.D. (Cardiovascular Science), Behavioral Neuroscience (Susan Resnick, Ph.D.), Population Science (Lenore Launer), and the Translational Gerontology Branch (Luigi Ferrucci, M.D.).

Concerning distribution of effort in ongoing and planned work, data collection continues at the SardiNIA/ProgeNIA clinic in Lanusei, where the genotyping work at different level of resolution is also performed, and island-wide sites for the collection of disease-specific and blood bank control samples. Sequencing work is done in a joint effort at the Sardinian and U. Michigan collaborative sites.

NIA investigators, led by Dr. Schlessinger, are involved in choosing and analyzing traits and test protocols for frailty, cardiovascular, and psychological traits; in the maintenance of the master database and preparation of summaries of heritability and age- and gender-specific distributions for all traits; they also actively collaborate on immunosenescence studies performed in Sardinia; in the development of algorithms for targeted types of analyses, and in the preparation of all manuscripts.The SardiNIA investigators operating in Sardinia, led by Dr. Cucca, are the primary producers and analysts of all genetic data, including the design and coordination of the genotyping and sequencing work (in close collaboration with the University of Michigan team led by Dr. Abecasis) as well as of the phenotypic data, with the supervision of all clinical activities performed in the Center in Lanusei. They are also the primary investigators in traits and test protocols for hematological, inflammatory, immunological (both cellular and humoral), and anthropometric (including those related to internal organs) phenotypes, as well as those related to kidney function. They also participate actively in the design and analysis of cardiovascular traits, with special emphasis in Echocardiographic cardiovascular measures. They also take the lead in writing papers and obtaining grants for the traits of paramount relevance for this component of the project

The Core Project is supported by the ongoing contract from the National Institute on Aging with cost-sharing by the IRGB, aided by cost-sharing from the Italian National Research Council and awards and collaborative grants from other funding sources for specific topics. They include support for 1) extensive study of immune cells and molecules; 2) extensive DNA sequencing, with assessment of DNA methylation patterns; 3) RNA and methylation sequencing for white cells from the cohort; and 4) refined study of immune system cell types in cohort members.

Auxiliary studies that have continued to benefit from the cohort and population structure and repeated visits include analyses of human demographic history, involving the groups of Dr. John Novembre at the University of Chicago and Antonio Torroni at the Universita degli Studi di Pavia, and analyses of genetic and environmental effects on RNA levels with the team of Dr. Stephen Montgomery.

Case-control studies have concentrated on diseases strikingly common in Sardinia such as autoimmune multiple sclerosis and type 1 diabetes as well as thalassemia. They also include diseases of more global high prevalence such as common hypertension, chronic kidney disease and breast cancer.

For these case-control studies, the entire island remains the catchment to recruit enough cases from among well characterized clinical series collected by Sardinian physicians, compared with unaffected Sardinians represented by healthy blood donors collected in the main Transfusion Centers on the island.

The results of the case-control studies and those of related quantitative traits generated in the SardiNIA project are analyzed jointly to reveal coincident associations, indicative of molecules, cells and pathways primarily involved in the disease process. The results of these studies as well as those focusing on aging have the potential to translate into substantial improvement in clinical diagnosis and identification of new targets for treatment.

A complete list of the publications in whole or in part from the Project (as of January 1, 2016) is given under the PUBLICATIONS rubric at this WEB site, and is updated continuously there. The publications include epidemiological studies, mainly on cardiovascular and personality topics, and genetic studies, many of which are based mainly on this cohort and the rest from Consortia in which SardiNIA was a major contributor. The overall goals are

1) Measures of genetic variation, quantitative traits, and of longitudinal trends for traits and outcomes, focusing on the longitudinal dimension of the study, aiming at determining individual trajectories and features of the aging process, as well as the identification of longer-term health-related outcomes; and

2) Gene variation associated with phenotypic traits, prevalent diseases, and age-related trends, based on increasingly complete recovery of the range of trait variability and genetic variation in the population.

Specific Aim 1. Measures of genetic variation, quantitative traits, and of longitudinal data for traits and outcomes.

a) Measures of variation: genotyping and sequencing in an enlarged cohort

  •  Variation.

    Recruitment. The number of individuals in the overall datasets (combining the SardiNIA cohort and the various case-control studies) has increased to ~17,000.

    Genotyping and sequencing of DNA and RNA. The core group of ~6,000 individuals of the SardiNIA cohort has been supplemented with an additional ~700 volunteers from the same area to integrate those that could not participate in the various stages of the follow up with a targeted emphasis on individuals older than 90. Overall, ~6,700 individuals have had full-genome scans thorough 4 genotyping chips: immunochip, cardiometabochip, exome chip, and the Illumina OmniExpress Chip with 750,000 .common. SNPs that give comparable coverage across the genome. The case: control individuals have been studied with the OmniExpress chip to integrate their genomes into the cohort by imputation.

    In an extension of analyses, 3,400 individuals from the cohort, chosen to optimize the representation of cases and families, have had their DNA sequenced to 4-fold coverage across the whole genome. This has recovered a total of 17,500,000 single nucleotide variants, including the full range of 12,500,000 variants that occur with minor allele frequencies >0.4%. In addition, rare, personal, nonsense or nonsynonymous coding variants have also been recovered for further analyses. Using the family relationships, imputation has permitted the inference of all the variation useable for GWAS in all individuals in the cohort. DNA sequencing is thus completed for the cohort, and has permitted us to generate a Sardinian HapMap that increases the accuracy of imputation to > 99.9%.

    At the same time we have begun analysis of the levels of RNA transcripts in PBMC from the same core 3,400 individuals in the cohort. To date, poly(A)+ RNA from 605 individuals has been sequenced, and preliminary analyses working in collaboration with Dr. Steven Montgomery at Stanford University show the order of one cis-eQTL per locus across the genome.

  • Quantitative traits.

    We have maintained a primary concentration on risk factors for age-related diseases and traits/risk factors. For traits, we have specifically profited from the experience in long-standing epidemiological studies at the NIA (for example, incorporating frailty-related tests), and have made trait lists concordant with the lists of traits studied in the Baltimore Longitudinal Study of Aging and the AGES and InCHIANTI studies to facilitate cross-replication and meta-analyses among the studies sponsored by the NIA.

    The focus is on cardiovascular traits, personality traits, sensory capacity (retinal scans and hearing tests); levels of immune system cells, frailty-related traits (walking speed, grip strength, and bone density); and kidney ultrasound features.

  • Diseases

    Data collection has been completed for multiple sclerosis, Type1 Diabetes, and breast cancer cases and controls and efforts continue to assemble data to assess chronic kidney disease.

b. Epidemiological and longitudinal trends: outcome study

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.

Specific Aim 2. Analyses of gene variation associated with phenotypic traits and/or prevalent diseases, and of age-related trends and outcomes.

a) Loci and gene variation associated with traits

  • Heritability.This primary tool determines for us the relative contribution of epidemiological and genetic factors, including the .narrow. heritability due to genetic variation. Based on the results, we determine which traits can be usefully followed up with further GWAS or other analyses

  • Quantitative traits.

    Analyses. For all of the standard traits listed above (Specific Aim 1.b), including cardiovascular, personality, sensory capacity, frailty-related, and kidney ultrasound measures, full GWAS of all traits are being completed with the 12,500,000 SNP set and over 10,000 individuals.

    The analysis has proceeded furthest thus far for the sample trait of levels of immune system cells. GWAS analysis on 1,200 individuals with the 12,500,000 SNP set revealed 23 with a large effect on at least one cell type. For example, the SH2B3 variant, R262W, already found associated with several autoimmune diseases and negative regulation of hematopoiesis, was shown to particularly affect the numbers of CD4+ T cells, likely resulting in a loss of function. Extensions of this approach have now extended analyses to a much larger cohort, with up to 100 significant loci identified, and to additional types of cells, including B cell subtypes, and cytokines.

    Especially notable, a number of SNPs that affect the levels of particular cell types have also been associated with autoimmune diseases in Consortium studies; our analyses extend associations of variants with a disease to specify the cell types in which the alleles have their effects. This provides markers and potential cellular and molecular targets for further analyses and possible intervention.

  • Disease associations

    Case:control analyses are being completed for 2,000 cases of Type 1 diabetes, and 1,000 cases of breast cancer in the cohort. Multiple sclerosis, by contrast, has revealed several additional novel risk loci. GWAS with 3,000 patients and 3,000 controls studied with several million SNPs identified CBLB as associated with MS. We will also continue to analyze several age-related conditions that affect substantial numbers of the Core cohort (in conjunction with Consortium analyses; e.g., of blood pressure/hypertension and CKD).

b. Epidemiological and longitudinal trends: outcome study

With the completion of the fourth visit data analyses can start estimating age-related trajectories for most of the major traits and ask definitively 1) whether differential rates of change are predicted by polymorphisms in specific genes and 2) whether any such genes are the same that were found associated with same traits in the cross-sectional analysis. In a first example that used aggregate groups of individuals, a genetic score was generated from thirteen reported eGFR- and related loci. Both uni- and multivariable analyses were then assessed to query the relationship between clinical, ultrasound, and genetic variables with three outcomes: CKD, change in eGFR, and fast eGFR decline. Genetic risk score, in addition to classical factors, was determined to impose independent risk.