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



  Studying a subset of 500,000 or 1 million is limitative


  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

. . A A . . . . . . . . A . . . . A . . .
. . G A . . . . . . . . C . . . . A . . .


3) Impute Missing Genotypes

C G G A G C T C T T T T C T T T T A 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

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

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