Il sequenziamento dei genomi sardi al CRS4 Francesco Cucca (University of Sassari and INN-CNR)

Il sequenziamento dei genomi sardi al CRS4 Francesco Cucca INN-CNR 1

•  Humans and other living organisms all contain a digital project constituted by a linear sequence of different combinations of 4 small chemical compounds, named nucleotides, which together constitute their DNA. • Particular combinations of nucleotides specify the key qualitative and quantitative instructions for the synthesis of essential structural and operative components of the cell formed by different combinations of 20 molecules named amino acids • In turn amino acids are linked to each other to form more complex molecules named proteins.

U U U Phe U C U Ser U A U Tyr U G U Cys U U C Phe U C C Ser U A C Tyr U G C Cys U U A Leu U C A Ser U A A STOP U G A STOP U U G Leu U C G Ser U A G STOP U G G Trp C U U Leu C C U Pro C A U His C G U Arg C U C Leu C C C Pro C A C His C G C Arg C U A Leu C C A Pro C A A Gln C G A Arg C U G Leu C C G Pro C A G Gln C G G Arg A U U Ile A C U Thr A A U Asn A G U Ser A U C Ile A C C Thr A A C Asn A G C Ser A U A Ile A C A Thr A A A Lys A G A Arg A U G Met A C G Thr A A G Lys A G G Arg G U U Val G C U Ala G A U Asp G G U Gly G U C Val G C C Ala G A C Asp G G C Gly G U A Val G C A Ala G A A Glu G G A Gly G U G Val G C G Ala G A G Glu G G G Gly

•  While the basic composition of both DNA and protein building blocks and the translational system of one chemical language into the other is conserved, there is wide variation in the order of these block units in different organisms and individuals. • This is because the DNA and deriving protein products are not a static entity. Instead, DNA is subjected to a variety of different types of heritable change known as mutation. •  Mutations often arise as copying errors during DNA replication. Although the fidelity of DNA replication is strikingly high, misincorporation occurs at a given frequency, known as mutation rate.

•  Modern humans originated ~100,000 years ago from pre- modern humans and represent a relatively homogenous species which has experienced a dramatic expansion during its recent evolutionary history. •  Two unrelated human individuals on our planet are identical for about 99.9% and thus differ for about 0.1% of their DNA content. •  This means that there is approximately one change every 1000 nucleotides (our genome has an overall content of about two copies of 3.3 billion nucleotides) when comparing the DNA from two unrelated individuals.

This genetic variation has important medical consequences: In simple mendelian traits, the relationship between the causal genetic variant (genotype) and the disease state is deterministic. In a complex trait such as MS, the disease state results from interactions between multiple genotypes and the environment. The influence of any individual causal allele tends to be modest and the relationship between the causal variant and the disease state is probabilistic.

Qualitative trait Quantitative trait 9

theoretical framework R. A. Fisher, 1890-1962 10

POSSIBILI SIGNIFICATI DI UN’ASSOCIAZIONE ASSOCIAZIONE PRIMARIA CON LA VARIANTE CAUSALE ASSOCIAZIONE SECONDARIA DOVUTA A CONTIGUITA’ ASSOCIAZIONE SPURIA DOVUTA A SUBSTRUTTURA DI POPOLAZIONE

Why a sequencing project? 12

The imperfect genome-wide search   All Gene Chip Arrays contain SNPs chosen based on linkage disequilibrium (LD) observed in HapMap populations, a catalogue of ~ 3 million SNPs genotyped in Europeans, Asians, and Africans

The imperfect genome-wide search   All Gene Chip Arrays contain SNPs chosen based on linkage disequilibrium (LD) observed in HapMap populations, a catalogue of ~ 3 million SNPs genotyped in Europeans, Asians, and Africans   Studying a subset of 500,000 or 1 million is limitative

The imperfect genome-wide search   All Gene Chip Arrays contain SNPs chosen based on linkage disequilibrium (LD) observed in HapMap populations, a catalogue of ~ 3 million SNPs genotyped in Europeans, Asians, and Africans   Studying a subset of 500,000 or 1 million is limitative Power to detect disease associations Causative Tested at a locus inversely correlates with variant variant the r2 between typed(tested) and untyped (causative) SNPs

Why a sequencing project in Sardinia? CROATIA UKRAINE SARDINIA HUNGARY POLAND CATALONIA BASQUE COUNTRY GEORGIA ANDALUSIA CORSICA SICILY NORTH-CENTRAL ITALY ALBANIA CALABRIA GREECE LEBANON TURKEY

Why a sequencing project in Sardinia? SAAMI UDMURT MARI CZECH AND SLOVAKIAN DUTCH UKRAINIAN FRENCH POLISH HUNGARIAN CROATIAN GEORGIAN CENTRAL-NORTHERN ITALIAN MACEDONIAN ALBANIAN SPANISH BASQUES CALABRIAN TURKISH SYRIAN ANDALUSIAN GREEK LEBANESE MOROCCO 17

What samples to sequence in Sardinia? •  ProgeNIA study •  Case-Control studies •  Future work

ProgeNIA 6.148 volontari Arzana Elini Ilbono Lanusei 19

ProgeNIA/SardiNIA project  6,148 individuals - aged 14-102 y. 95% are known to have all grandparents born in Sardinia  711 pedigrees up to 5 generations deep Largest family: 625 phenotyped individuals  >34,000 relatives pairs Pilia et al. PLoS Genet. 2006

> 150 quantitative traits   Anthropometric Measurements Height, Weight, Hip, Waist, BMI   Blood Chemistry Components LDL, HDL, TG, Insulin, RBC, MCH, MCV, Bilirubin, hsCRP, MCP-1, IL-6, etc.   Cardiovascular Traits HR, SBP, DBP, PP, PWV, IMT, QT, etc   Personality Facets Neuroticism, Extraversion, Openess, Agreeableness, Coscientiousness, etc.   New traits will be added soon (immunological traits).  Cytokines

Case-control samples •  The special case of autoimmune diseases 22

10 42 36 21 26 20 23 10 6 22 8 7 13 13 6 19 13 7 6 10 15 12 12 7 8 8 9 9 10 6 7 19 11 9.8 12 8 7 14 12 5 8 15 6 42 6 9 12 7 15 *Adapted from EURODIAB 18 8 5

119 165 74 93 120 153 186 187 60 56 112 55 135 126 76 35 55 86 83 62 50 112 83 81 50 21 47 55 42 65 39 55 39 6 10 140 31 61 Pugliatti et al (EBC), Eur J Neurol 2006 10 68 7 17 29

70 70 70 60 60 60 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10 0 0 0 Pazienti Controlli Pazienti Controlli Pazienti Controlli

How many samples to sequence? • Is it necessary to sequence all people analysed? 28

•  Observed genotypes • Inferred DNA stretches sharing along chromosome • Inferred missing genotypes according to chromosome sharing Chen and Abecasis AJHG 2008 Burdick et al. Nat. Genet. 2006

1) Identify Match Among Reference Individuals in study sample . . A A . . . . . . . . A . . . . A . . . . . G A . . . . . . . . C . . . . A . . . Observed HapMap Chromosomes C G A G A T C T C C T T C T T C T G T G C C G A A A T C T C C C G A C C T C A T G G C C A A G C T C T T T T C T T C T G T G C C G A A G C T C T T T T C T T C T G T G C C G A G A C T C T C C G A C C T T A T G C T G G A A T C T C C C G A C C T C A T G G C G A G A T C T C C C G A C C T T G T G C C G A G A C T C T T T T C T T T T A T A C C G A G A C T C T C C G A C C T C G T G C C G G A G C T C T T T T C T T C T G T G C

2) Phase Chromosome Individuals in study sample . . A A . . . . . . . . A . . . . A . . . . . G A . . . . . . . . C . . . . A . . . Observed HapMap Chromosomes C G A G A T C T C C T T C T T C T G T G C C G A A A T C T C C C G A C C T C A T G G C C A A G C T C T T T T C T T C T G T G C C G A A G C T C T T T T C T T C T G T G C C G A G A C T C T C C G A C C T T A T G C T G G A A T C T C C C G A C C T C A T G G C G A G A T C T C C C G A C C T T G T G C C G A G A C T C T T T T C T T T T A T A C C G A G A C T C T C C G A C C T C G T G C C G G A G C T C T T T T C T T C T G T G C

3) Impute Missing Genotypes Individuals in study sample C G A A A T C T C C C G A C C T C A T G G C G G A G C T C T T T T C T T T T A T G C Observed HapMap Chromosomes C G A G A T C T C C T T C T T C T G T G C C G A A A T C T C C C G A C C T C A T G G C C A A G C T C T T T T C T T C T G T G C C G A A G C T C T T T T C T T C T G T G C C G A G A C T C T C C G A C C T T A T G C T G G A A T C T C C C G A C C T C A T G G C G A G A T C T C C C G A C C T T G T G C C G A G A C T C T T T T C T T T T A T A C C G A G A C T C T C C G A C C T C G T G C C G G A G C T C T T T T C T T C T G T G C

Recent updates   We used whole-genome sequences of 52 Europeans available from the 1,000 Genomes Project to infer ~6.6 million markers in individuals typed with the higher density chip…..   …. then with imputation method we inferred the 6.6 million markers to all individuals and performed a GWAS   This :  Provides a fine mapping for previously discovered loci  May show new loci that were poorly tagged by the previous set of SNPs

GWAS finding Mostly all of the loci detected by GWAS only explain a small fraction of the heritability Trait Heritability So far explained HbF ~60% ~17% Height ~80% ~4% BMI ~40% ~1% Smaller is the effect size, larger is the sample size required to maintain adequate power

Shankar Balasubramanian David Klenerman 38

ProgeNIA Team Lanusei-Cagliari Manuela Uda Monica Lai Serena Sanna Anna Cau Eleonora Porcu Barbara Deiana Ilenia Zara Monica Balloi Carlo Sidore Maria Grazia Piras Maristella Steri Gianluca Usala Marco Masala Antonella Mulas Gianmauro Cuccuru Andrea Maschio Angelo Scuteri Fabio Busonero Marco Orrù Sandra Lai Maria Grazia Pilia Mariano Dei Danilo Fois Liana Ferreli Laura Crisponi Francesco Loi Silvia Naitza Caterina Flore Simona Foddi Giuseppe Pilia, Ideatore e Fondatore del Progetto ProgeNIA

Acknowledgements: Paolo Zanella Chris Jones Roman Tirler Antonio Cao Giuseppe Pilia David Schlessinger Goncalo Abecasis John Todd

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Il sequenziamento dei genomi sardi al CRS4 Francesco Cucca (University of Sassari and INN-CNR)

by CRS4 Research Center in Sardinia on Mar 25, 2010

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Il sequenziamento dei genomi sardi al CRS4 Francesco Cucca (University of Sassari and INN-CNR) — Presentation Transcript