Marco Songini, Director

1
The Center for Immunology and
Transplantation University
of Florida, Gainesville, July 5th, 2005 type 1
diabetes and related ADs in Sardinia

• Marco Songini, Director
• Diabetes Unit - Azienda Ospedaliera G.Brotzu
• www.aob.it, marcosongini_at_aob.it
• Cagliari Sardinia, Italy

2
Cagliari
3

• Gene and diseases in the founder population of
  Sardinia

4
Sardinia and the genetic map of Europe According
to Cavalli-Sforza et al. 1994 Different colours
indicate genetic distances

• Genes
• Uniformly distributed
• Frequencies unique for Sardinia
• RH-? MNS-M? DIA2?, HLA B18?
• Cavalli-Sforza et al. (1994)

• Isolation
• Insularity and geographical position
• Physical geography
• Low population density

• Consanguinity
• Kinship
• 1880-1884 12.26
• 1945 8.21
• Today very rare
• Moroni et al (1972)

• Genetic drift
• Paleolitic 700-1800 inhabitants (14.000 b.C.
  about 750 generations ago)
• Small number of founders
• Same ancestral gene pool
• Skewing effect of selection
• Considerable drift

• Stable population
• About 300,000 inhabitants from 3rd 800 b.C.
  Phoenicians and
• century b.C. to 17th century
  Carthaginians
• No real colonization in terms of 238 b.C.
  Romans
• agent of demographic development 456 A.D.
  Vandals
• No large-scale admixture between 534 A.D.
  Eastern Roman populations from costal to
  internal empire (Byzantium)
• regions 1258 A.D. Pisans
• The invaders were not inclined 1323 A.D.
  Catalan-Aragoneses
• towards permanent inhabitation of 1479 A.D.
  Spanish
• the island 1720 A.D. Italians Piedmont
• Little genetic flow from invaders (Savoys)

• Language
• Romance (the most archaic
• neo-latin language spoken so far)
• Early Latinization
• Isolated location
• Resistance to innovation
• Any lexical innovation mostly regards some
  Southern and Northern areas
• Paulis (1995)

• Life expectancy
• The oldest man in the world lives currently in
  Sardinia
• at birth 1year
• 1930-32 54.9 60.5
• 1960-62 69.4 71.8
• 1970-72 72.9 73.9
• 1989-93 73.9 73.6
• (National Institute of Statistics)

• Matrimonial mobility
• (average distances of birth place of the spouses)
• 1850 13.3 Km
• 1911-1949 20 Km
• 1950-1965 39.9 Km
• Nowadays gt 40 lt 50 Km
• Gatti (1990)

• Stable population
• Struggle for survival i.e. not to become extinct
• Famine and malnutrition
• Endemic illness (Malaria and other major
  infections)

24,000 sq. Km popul. 1.654.470 Lat. 39-41
N Long.8-10 E
5
GENETIC DISTANCE AMONG POPULATIONS
DAN
SVE
LAP
NOR
FINLANDIA
DUT
1.40
IRI
SCO
FMG
GER
1.14
AUS
SWI
0.88
CZE
MAR
PUG
0.62
TOS
FRE
HUN
CAL
0.36
UMB
BAS
PIE
0.10
CAM
SPA
-0.16
SIC
EMI
POR
-0.42
LIG
VEN
LOM
-0.68
LAZ
SARDEGNA
GRE
-0.94
-1.20
0.12
0.38
0.64
0.90
1.16
1.42
-1.96
-1.70
-1.44
-1.18
-0.92
-0.66
-0.40
-0.14
6
Neighbor-joining tree of 16 European and
North-African populations using DRB1-DQA1-DQB1
haplotype frequencies
(Courtesy of F. Cucca)
ALG, Algerians BULG, Bulgarians CRE, Cretan
CZE, Czech FRA, French GB, Britons GRE,
Greeks ITA, Italians MOR, Moroccan NOR,
Norwegians ROM, Romanians SARD, Sardinians
SPA, Spanish TUN, Tunic TUR, Turks US-WHITE,
American Whites.
7
(Courtesy of F. Cucca)
8
(Courtesy of F. Cucca)
9


DRB1 DQA1 DQB1 haplotypes in 5 ethnic groups
25
20
20
Chinese
15
15
10
10
5
5
25
25
Blacks
20
20
15
15
10
10
5
5
25
25
Latin American
20
20
15
15
10
10
haplotype frequency ()
5
5
25
25
Caucasians
20
20
15
15
1501-0102-0602
0301-0501-0201
10
10
5
5
25
Sardinians
20
15
30 of Sardinians
10
5
0
0101-0101-0501
0102-0101-0501
0103-0101-0501
0301-0501-0201
0302-0401-0402
0401-0301-0301
0401-0301-0302
0402-0301-0301
0402-0301-0302
0403-0301-0302
0403-0301-0304
0403-0301-0305
0404-0301-0301
0404-0301-0302
0404-0301-0401
0405-0301-0201
0405-0301-0302
0405-0301-0401
0405-0501-0301
0406-0301-0302
0407-0301-0301
0407-0301-0302
0407-0301-0304
0408-0301-0301
0408-0301-0302
0409-0301-0401
0701-0201-0201
0701-0201-0303
0701-0301-0201
0801-0301-0302
0801-0401-0402
0802-0401-0402
0803-0103-0601
0803-0601-0301
0804-0401-0402
0804-0501-0301
0901-0301-0201
0901-0301-0303
1001-0101-0501
1101-0102-0602
1101-0501-0301
1102-0501-0301
1103-0501-0301
1104-0501-0301
1201-0101-0501
1201-0501-0301
1301-0103-0502
1301-0103-0603
1302-0102-0501
1302-0102-0604
1301-0102-0605
1303-0201-0201
1303-0501-0301
1305-0201-0201
1401-0101-0501
1401-0101-0503
1402-0501-0301
1403-0501-0301
1501-0102-0501
1501-0102-0602
1502-0101-0501
1502-0103-0601
1503-0101-0501
1503-0102-0602
1601-0102-0501
1601-0102-0502
1601-0102-0603
1602-0102-0502
1602-0501-0301
Lampis, Cucca et al. 2001
10
Genetic clusters for T1D in Sardinians
Genetic tree of the Sardinian and Italian
populations for HLA
Genetic tree for the 21 linguistic domains in
Sardinia for 12 polymorphisms
Vona et al. 1997
Cappello et al. 1996
11
Genetic diseases G6PD deficiency (favism).
Prevalence () among males
Silvetti et al. 1969
12
Genetic diseases Beta thalassemia in Sardinia

• Epidemiology of beta-thalassemia (95
  beta39founder effect)
• 13 beta-thalassemia carrier rate
• 170 couple is at risk for
• 1250 newborns would be affected but they are
  reduced to 14,000 thanks to prenatal diagnosis

Rosatelli et al. 1992 Cao et al. 1996
13
Genetic diseases Wilsons Disease
Incidence Sardinia 1-210,000 Other
countries 1-2100,000 Heterozygous in Sardinia
3.4 Heterozygous in other countries 1.1
Loudianos et al 1999
14
Genetic diseases Wilsons Disease mutations in
Sardinian population (founder effect)
Loudianos et al. 1999
15

• Other diabetes-related autoimmune diseases in
  Sardinia

16
Other autoimmune diseases APECED
(APS1) Autoimmune Polyendocrinopathy Candidiasis
Ectodermal Dystrophy
Autosomal recessive disorder Incidence Finns
125,000 Iranian Jews 19,000 Sardinians
114,400 Genetic Mutation of AIRE (autoimmune
regulator) gene R139X (18/20 indipendent
mutations found are R139X founder
effect) Heterozygous in Sardinia for R139X 1.7
Rosatelli et al. 1998
17
Other autoimmune diseases Celiac disease (CD) in
background population
Population Number Prev. (95 C.I. )
Ref. North Sardinia 1,607 1.06
(0.6-1.6) Meloni et al. 1999
Schoolchildren Italy - Marche
3,351 0.33 (0.1-0.5) Catassi
et al. 1994 Schoolchildren Finnish sibs
550 1.1 (0.4-2.3)
Saukkonen et al 2001 of T1D (mean age
12yrs) Finnish adults 1,070 0.8
(0.3-1.5) Kolho et al. 1998
biopsy proven
18
Other autoimmune diseases CD among T1D patients
Population Number Prev. (95 C.I. )
Ref. NorhtSardinian 650 4.1
(2.7-6.0) Frongia et al. (unpubl.) Schoolchi
ldren T1D Italian 4,514
2.7 (2.2-3.1) Pocecco et al.
1989 Schoolchildren 498 3.2
(1.8-5.2) Barera et al. 1991 Sardinian
adults 394 3.8 (2.1-6.2)
Songini et al. 1998 T1D patients
Italian adults 383 2.6
(1.3-4.7) Sategna-Guidetti et al. 1994 T1D
patients 820 7.0 (5.4-9.0)
De Vitis et al. 1996 biopsy proven
19
Other autoimmune diseases Myasthenia gravis (MG)
in background adult populations
Population Number Prev. Incid.
Ref. North Sardinia 270.000 11.1
0.8 Aiello et al.1997
Whole Sardinia 1.5mil 4.5
0.25 Giagheddu et al.
1995 Denmark 2.8mil 7.8
0.5 Christensen et al.
1993 Whole Norway 4.5mil 9
0.24 Storm-Mathisen et al.
1984 Italian adults BO 815.000 4.9
DAlessandro et
al. 1991 FE 381.118 9.7
x100.000
20
Other autoimmune diseases Thyroid autoantibodies
(TA) in background population
Population Number Prev. (95 C.I.
) Ref. Sardinian 8,484 3.7
(3.3-4.1) Mariotti et al. 2000
Schoolchildren Italian 419 2.4
(1.1-4.3) Aghini et al. 1999
Schoolchildren 142 4.3
(1.6-9.0) Fenzi et al. 1986 Sardinian
mothers 2,500 11.8
(10.5-13.1) Olivieri et al. 2000 at delivery
Mothers at 4,022 5.5
(4.8-6.2) Sakaihara et al. 2000 delivery
(Japan, USA) 552 19.6
(18.1-21.1) Stagnaro et al.1990
21
Other autoimmune diseases Thyroid autoantibodies
among T1D patients
Population Number Prev. (95 C.I. )
Ref. T1D Sardinian 650 17.6
(14.6-20.5) Ricciardi et al. Schoolchildren
(unpubl.) T1D Italy/Europe 212
16.5 (11.5-21.5) Lorini et al.
1996 Schoolchildren 495 22.0
(18.4-25.7) Holl et al. 1999 T1D Sardinian
399 28.3 (23.9-22.6)
Songini et al. 1998 adults patients T1D
European 258 19.8
(14.9-24.6) Mangendre et al. 2000 adults patients
111 27.9 (19.6-36.3)
Fernandez-Castaner et al, 1999
22
The new concept of T1D as a cluster of
autoimmune-related diseasesthe example of MS
and T1D in Sardinia
Sardinia Giants tomb
23
43
Prevalence of MS x 100.000 in Europe (MS pts 2.5
mil ww)
J Cl Nurology and Neurosurgery 104 (002) 182-91
24
Other autoimmune diseases Multiple Sclerosis (MS)
and T1D
Population Number MS (Prev)
(95 C.I. ) Reference Sardinians
270,000 0.16 (0.14-0.17)
Granieri et al. 2000 (NW,all ages) Italians (all
ages) 0.05
Granieri et al. 1997 Sardinian T1D 424
0.47 (0.05-1.69) Songini et al.
(unpubl) adults (15-50 yrs) Sardinian T1D
650 0.77 (0.25-1.79) Chessa
et al. (unpubl) schoolchildren Population
Number T1D (Prev) (95 C.I. )
Reference Sardinian 1,053 2.40
(1.54-3.49) Marrosu et al. 2000 MS pts
(9-65 yrs) Sardinian pop 6,255 0.37
(0.22-0.52) Muntoni et al. 1988 (20-59yr)
25
Other autoimmune diseases Genetic association of
MS with
In Northern Europeans DRB11501-DQB10602
(DR2) Protective for T1D then T1D and MS
occur together very rarely In
Sardinians DRB10301-DQB10201
(DR3) DRB10405-DQB10301 (DR4) Also
predisposing to T1D, DR2 is very rare then
T1D and MS may coexist
Marrosu et al. 2000
26
Multiple Sclerosis (MS) and T1D in Sardinia
Population Number MS (Prev)
(95 C.I. ) Reference Sardinians
270,000 0.16 (0.14-0.17)
Granieri et al. 2000 (NW,all ages) Italians (all
ages) 0.05
Granieri et al. 1997 Sardinian T1D 424
0.47 (0.05-1.69) Songini et al.
(unpubl) adults (15-50 yrs) Sardinian T1D
650 0.77 (0.25-1.79) Chessa
et al. (unpubl) schoolchildren Population
Number T1D (Prev) (95 C.I. )
Reference Sardinian 1,053 2.40
(1.54-3.49) Marrosu et al. 2000 MS pts
(9-65 yrs) Sardinian pop 6,255 0.37
(0.22-0.52) Muntoni et al. 1988 (20-59yr)
27
Genetic association of MS with T1D
In Northern Europeans DRB11501-DQB10602
(DR2) Protective for T1D then T1D and MS
occur together very rarely In
Sardinians DRB10301-DQB10201
(DR3) DRB10405-DQB10301 (DR4) Also
predisposing to T1D, DR2 is very rare then
T1D and MS may coexist
Marrosu et al. 2000
28
MS with T1D together at last
(Allegheny County, US)
Characteristics T1D T1D Siblings Siblings Parents Parents
Characteristics Males (n148) Females (n149) Males (n143) Females (n187) Males (n84) Females (n140)
Age (yrs) 41.4 42.6 42.4 42.3 69.0 67.2
Duration T1D (yrs) 35.2 35.4 ---- ---- ---- ----
Observed MS prev. 0 2.0 0 0.5 0 0
Expected MS prev. n/a 0.1 n/a 0.1 n/a n/a
Relative increased risk of MS n/a 20fold n/a 5 fold n/a n/a
plt0.01 unaffected with T1D n/a non applicable
Dorman JS, Songini M et al. Diabetes
Care,november 2003
29
The two Sardinian provinces under study Sassari
and Nuoro the south-western to north-eastern
gradient of MS prevalence in the province of
Sassari (Pugliatti et al., 2002b), correspond to
significant differences of genetic and linguistic
domains (prevalence is expressed as number of
cases per 100,000 population for each commune of
the province black communes gt150, grey communes
gt130 and lt150, blank communes lt 130 MS cases). B.
Polymorphisms of mitochondrial genes in Barbagia
(central, archaic Sardinia), Gallura (norther
Sardinia), Corse (insular France) and Tuscany
(mainland Italy) (Morelli et al., 2000) and
subdivision of the island in 23 linguistically
and genetically domains (Cappello et al., 1996)
Mediterranean sea

Tuscany 15
A
SASSARI
Corse (F) 20
Gallura 20
NUORO
Barbagia 50
2
CAGLIARI
1
4
3
7
9
22
6
5
8
11
10
13
12
15
14
16
17
18
19
21
20
B
MS prevalence gradient
Gallurese-speaking communes
23
30
(No Transcript)
31
A
Figure 4. A. Temporal trends of MS incidence
rates in the province of Nuoro, Sassari and
Ferrara, in the period 1965 to 1993.
32
1) Average annual incidence rates (per 100,000
per year, right Y axis) of MS in Macomer,
Sardinia, from 1912 through 1981 (5-years
periods). Left Y axis expresses the population
growth (per 1,000). Adapted from reference 12.
33


DRB1 DQA1 DQB1 haplotypes in 5 ethnic groups
25
20
20
Chinese
15
15
10
10
5
5
25
25
Blacks
20
20
15
15
10
10
5
5
25
25
Latin American
20
20
15
15
10
10
haplotype frequency ()
5
5
25
25
Protetctive T1DM
MS
Caucasians
20
20
15
15
10
10
5
5
MS
MS and T1DM
25
20
15
Sardinians
10
5
0
0301-0501-0201
1501-0102-0602
0101-0101-0501
0102-0101-0501
0103-0101-0501
0302-0401-0402
0401-0301-0301
0401-0301-0302
0402-0301-0301
0402-0301-0302
0403-0301-0302
0403-0301-0304
0403-0301-0305
0404-0301-0301
0404-0301-0302
0404-0301-0401
0405-0301-0201
0405-0301-0302
0405-0301-0401
0406-0301-0302
0407-0301-0301
0407-0301-0302
0407-0301-0304
0408-0301-0301
0408-0301-0302
0701-0201-0201
0701-0201-0303
0701-0301-0201
0801-0301-0302
0801-0401-0402
0802-0401-0402
0803-0103-0601
0803-0601-0301
0804-0401-0402
0804-0501-0301
0901-0301-0201
0901-0301-0303
1001-0101-0501
1101-0102-0602
1101-0501-0301
1102-0501-0301
1103-0501-0301
1104-0501-0301
1201-0101-0501
1201-0501-0301
1301-0103-0502
1301-0103-0603
1302-0102-0501
1302-0102-0604
1301-0102-0605
1303-0201-0201
1303-0501-0301
1305-0201-0201
1401-0101-0501
1401-0101-0503
1402-0501-0301
1403-0501-0301
1501-0102-0501
1502-0101-0501
1502-0103-0601
1503-0101-0501
1503-0102-0602
1601-0102-0501
1601-0102-0502
1601-0102-0603
1602-0102-0502
1602-0501-0301
0409-0301-0401
0405-0501-0301
Lampis,R et al. 2001
34
HLA allele T1DM effect MS effect
DRB11501 (DR2) DQA1 0102- DQB1 0602 Dominant protection Predisposition in Caucasians
DRB1 0301(DR3) DQA1 0501 - DQB1 0201- Predisposition Predisposition in Sardinians
other loci outside the MHC region may be shared
between MS and T1DM and contribute to the
susceptibility to both the diseases.
35
The strong effect of having multiple MS affected
relatives suggests that genetic variability in
risk for diabetes is linked to genetic loading
for MS, which is substantially increased in these
individuals. These findings suggest that loci or
genes predisposing to familial MS aggregation
also contain an enrichment of diabetes
predisposing genes.
36

• Taken together, these data suggest
• that common genes, either within the HLA region
  or outside it, influences susceptibility to both
  MS and T1DM
• that genetic structure of Sardinians might
  contain a particular combination of etiologic or
  a lack of protective genes for these diseases.
• Thus, Sardinians appears to be a very suitable
  population for studying what genes contribute to
  trigger and sustain autoimmunity in humans.

37
Conclusions
38
To sum up (1)

• T1D has started to rise around the middle of the
  20th century and keeps increasing ww in younger
  ages mainly in low risk countries (catch-up
  phenomenon)
• Whether its an earlier presentation or a true
  increase its not clear yet
• Sardinia is confirmed to be a hot spot for
  clinical T1D and islet-related autoimmunity, as
  for some other autoimmune-related diseases (CD,
  MS, not TD)
• An increasing T1D incidence in the Island has
  been found rising dramatically since the years
  60 as in other countries
• No defined geographical clustering for T1D was
  found across the island

39
To sum up (2)

• The combination of more than 1 islet-related
  autoantibody (rather than which) is the best
  predictor for the development of T1D in the
  Sardinian background population (SC)
• None of the classical ecological variables
  considered so far has shown any firm influence
  towards the etiopathogenesis of T1D in Sardinia
  however other variables need to be further
  investigated
• Migrants data seems to support a different role
  of genetic vs environmental determinants
• Theres still a long way in front of us to
  possibly prevent type 1 diabetes

40
What we are doing now with the help of so many (1)

• Trying to further improve the prediction of T1D
  in the general population by studying the
  immunological and genetic markers for T1D and
  other autoimmune diseases in Sardinian population
  and migrants and their relatives
• Investigating other putative environmental
  factors which can play a role towards the
  etiopathogenesis of T1D (e.g., chemicals, toxins,
  vaccinations, viral infections, etc.)

41
What we are doing now... (2)

• Broadening the original investigation for T1D on
  the prevalence of others autoimmune diseases and
  to study their associations
• Comparing data from Sardinia and other areas by
  new collaborative studies(RIDI,TRIGR,ENDIT,
  SARDINIAN DIABFIN)
• Sensitizing Sardinian Health Autorities
  population towards primary prevention of T1D and
  autoimmune diseases ..and by this way...
• improving the current care of diabetes in
  Sardinia!

42
Acknoledgements
Anthropology G. Vona (Sardinia-Italy) Other
autoimmune diseases GF Bottazzo and coworkers
(Rome-Italy) S. Mariotti and coworkers
(Cagliari-Italy) T. Meloni and coworkers
(Sardinia-Italy) M. Marrosu and coworkers
(Sardinia-Italy) Epidemiology The Sardinian
Epidemiology Study Group Eurodiab TIGER Study
Group IDA Study Group RIDI Study Group Z.
Laron, I. Askenazi (Israel) L. Bernardinelli
and C. Pascutto (Pavia-Italy) P. Contu, L.
Minerba (Cagliari-Italy) Genetic F. Cucca and
coworkers (Sardinia-Italy) E.A.M. Gale and
coworkers (UK) I. Loudianas (Sardinia-Italy)
43
Acknoledgements
Echological studies G.F. Bottazzo
(Rome-Italy) M. Fadda (Sardinia-Italy) B.
Elliot (New Zeland) P. Pozzilli
(Rome-Italy) A. Contu, M. Carlini
(Sardinia-Italy) Prediction Study G.F. Bottazzo
M. Locatelli (Rome- Italy) S. Carta, M.
Sorcini, A. Olivieri (Rome-Italy) GF Bottazzo,
A. Loviselli and the Sardinian SchooIchildren
Study Group (Sardinia-Italy) GF Bottazzo,R.
Cirillo and the Sardinian Newborn Study Group
(Sardinia-Italy) A. Dolei, G. Delitala
(Sardinia-Italy) M.T. Tenconi, G. Devoti
(Pavia-Italy) The League of Sardinian Migrant
Associations ASRIS (Association for Study and
Research of IDDM in Sardinia) M. Porceddu
(secretary) Tina Carboni President G.F.
Bottazzo VicePresident M.Songini C. Putzu, A.
Casu R.
Cavallo R.A.S. (Regione Autonoma della
Sardegna) European Union Italian Ministry of
Healh, ADCT National Institute of
Health GPsPediatricians from Sardinia.and many
more!
44
My Diabetes staff thanks you