Diapositiva 1

1
WP n.3 GENOTYPING Objectives genotyping of a
set of families (peculiar design of pedigrees)
for candidate loci (regions) in order to
identify the associations between POLYMORPHIC
MARKERS and FRAILTY-FUNCTIONAL ACTIVITY
INDEX(ES)
2
selection of candidate genes 1. LONGEVITY
studies in MODEL ORGANISMS (H-RAS1, SIRT-3)
2. Markers for RATE and QUALITY of AGEING
(IL-6, APOE) 3. Markers for INDIVIDUAL
CAPABILITY to cope with the STRESS FACTORS
(HSPs, TH) .selection of candidate
regions
3
CHROMOSOME 11

• H-Ras1

11p15.5

• SIRT3

(INSULIN, H19, IGF-2)

• TH

PARENTAL IMPRINTING SEX-SPECIFIC
IMPRINTING PARENTAL DISTORTION
4
HRAS1 proto-oncogene responsible for cell
growth and differentiation It belongs to an
evolutionary-conserved family of GTPases that
controls the response to physical stress from
yeast to humans
VNTR 0,9 3 Kb
INTRON 1
5
3
Multiallelic 28bp repeat (30-110 repeat)
6bp
P2 P3 P1
Hexanucleotide polymorphic site
About 4Kb
5
3 flanking region VNTR located 1kb downstream of
the gene coding sequences Minisatellite locus
composed of 30 to 110 units of a 28 bp consensus
sequence 4 MOST COMMON ALLELES a1-a2-a3-a4
(80-90 of alleles in whites)

unstable region
!!!!!!???????? a large number of RARE
ALLELES Bringing differences in
number of repeats internal structure of repeats
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ASSOCIATION between RARE ALLELES and many forms
of CANCER (Kontiris et al. NEJM 1993) Mechanism
alteration of interaction with transcription
factors belonging to the rel/NF-kB family
HYPERVARIABILITY
ENHANCING ACTIVITY
CHANGED INTERACTION PATTERN
7
In a previous study (Bonafè et al. , Gene 2001) a
decreased in the a3 common allele was observed
with ageing
Analysis performed by Yashin et al. (still
unpublished !!!!!!!!)
8
HRAS1 3 VNTR allele frequency distribution in
centenarians and young people from Italian
population
Difference in the overall allele frequency
distributions was found when young individuals
and centenarians were compared (MC?29.959,df4,P
0.041). Inspection of adjusted residuals (AR),
which quantify the magnitude of the differences
between observed and expected allele frequency,
revealed a decreased frequency of a3 allele in
centenarians (AR3.1).
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HRAS1 3 VNTR allele frequency distribution in
centenarians and young people from Southern Italy
This finding suggests a role for the allele a3 as
a FRAILTY ALLELE
10
Allele frequency distribution for COMMON ALLELES
at HRAS1 VNTR 3 in the ECHA samples
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Allele frequency distribution for RARE ALLELES
at HRAS1 VNTR 3 in ECHA samples
12
0.97
2.00
1.45
2.50 Kb
a4
a3
a1
a2
a2r
a3r
a4r
a1r
13

• INTRON 1 Hexanucleotide polymorphic site
• GGGCCT consensus sequence
• P1 allele ? 4 repeats
• P2 allele ? 2 repeats
• P3 allele ? 3 repeats
• This polymorphic site was found in linkage
  disequilibrium with the VNTR region at the 3 end
  

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ALLELE distribution of HRAS1 hexanucleotide
repeat in young individuals and centenarians from
Italian population
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GENOTYPE frequency distribution of HRAS1
hexanucleotide repeat in young individuals and
centenarians from Italian population
No significant differences were found between
young individuals and centenarians for ALLELE and
GENOTYPE distributions of HRAS1 INTRON 1
polymorphism
16
ALLELE frequency distribution of HRAS1
hexanucleotide repeat in young individuals and
centenarians in the ECHA SAMPLES
17
GENOTYPE distribution of HRAS1 hexanucleotide
repeat in young individuals and centenarians in
the ECHA SAMPLES
18
Haplotype analysis in young individuals and
centenarians from Italian population
19
Table of standardized disequilibrium values
(DD/Dmax)
Table of Chi-square P values for D
20
0.97
1.45
2.00
2.50 Kb
a4
a2
a3
a1
a2r
a3r
a4r
a1r
P2 P3 P1
21
IL-6
Multifunctional pro-inflammatory cytokine
involved in the pathogenesis of chronic diseases
associated whit ageing, which is characterized
by an unbalance of pro-inflammatory status
In many Population-based studies, the magnitude
of IL-6 serum level has been identified as a
RELIABLE MARKER FOR FUNCTIONAL DISABILITY In
fact, elevated IL-6 serum level are associated
with diseases, disability and mortality in the
ELDERLY
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CHROMOSOME 7
IL-6 gene
5
37bp repeat
3
VNTR 0.608-0.822 Kb
-174 C/G
CA repeat
23
-174 C/G locus base transition located in the
promoter region which affects the rate of IL-6
gene transcription Associated with different
IL-6 plasma levels IL-6 in vitro production 3
VNTR locus Repeated region associated with
autoimmune diseases muscle mass !!!!!!!
(sarcopenia) (Bonafe et al ms in preparation)
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3 VNTR locus Repeated region associated with
autoimmune diseases muscle mass !!!!!!!
(sarcopenia) (Bonafe et al ms in preparation)
4 common alleles (B1, B2, B3, B4). and 8 rare
alleles Strong linkage disequilibrium with
174C/G Allele disequilibrium 174C ----IL-6
VNTR B4 (haplotype)
25
In a previous study (Bonafè et al. Eur.J.Immunol.
2001) was observed that the proportion of
homozygotes for the G allele decreases in
centenarians males, but not in centenarians
females, and that only among males the
homozygotes for the G allele show higher IL-6
serum level
Percentage of the C allele and C/C- individuals
in men
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-174 C/G locus Percentage of the C allele and
C/C- individuals in women
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ECHA study -174 C/G locus
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ECHA study
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Centenarians n. 38 (16M22F)
ECHA study 174 C/G locus
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Young people n. 70 (32M38F)
ECHA study 174 C/G locus
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CA repeat (Italian population)
Young people
Elderly
Centenarians
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POLYMORPHISMS IN TGF-b1 GENE

• Recently, several reports have provided evidence
  that POLYMORPHISMS in TGF-b1
• gene lead to increased TGF-b1 production
• and have been linked to fibrosis and
  hypertension. Moreover, polymorphisms leading to
  decreased TGF-b1 production
• have been linked to osteoporosis
• it could be expected that polymorphisms in
  TGF-b1 gene may play a role in
• SUCCESSFUL AGEING and LONGEVITY.

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350 people from Northern and Central Italy
(ranging from 20-102 years of age)
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PRINCIPAL FUNCTIONAL PROPERTIES OF THE TGF-b1
PROMOTER REGION POLYMORPHISMS
-509 C/T is located in a negative regulatory
region (-731 to -453) -509 T allele is
associated with higher transcriptional activity
(Luedecking et al., 2000 Schulte et al.,
2001) -800 G/A is located in a region having
enhancer-like function -800 A is expected to
reduce the affinity to CREB (Luedecking et al.,
2000 Grainger et al., 1999).
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Exon 1, codon 10 (Leu10Pro) affects protein
transport across the endoplasmic reticulum
(Hoffmann et al., 2001). Exon 1, codon
25 (Arg25Pro) regulates gene transcription
(Hoffmann et al., 2001). The presence of the
Arg25 allele is associated with increased TGF-b1
production, fibrosis (El Gamel et al, 1998) and
hypertension (Li et al., 1998). Exon 5, codon
263 It is conceivable that the codon 263
polymorphism is involved in the activation
process (cleavage) of TGF-beta1. (Cambien et
al., 1996).
36
(No Transcript)
37
No significant differences in allele and
genotypedistributions of each polymorphisms
were found between sexes within the centenarians
and the younger controls. Males and females were
therefore combined to increase the statistical
power in the analyses. In all the samples, the
observed genotype of each polymorphism fitted
with those expected at Hardy-Weinberg Equilibrium
(Pgt0,05)