ANALYSIS OF THE haplotype associated
mutation in fanca
Pau Castillo
• FA is a rare autosomal recessive genetic syndrome
• There are at least 13 complementation groups each one connected with
a distinct disease gene. The FANCA is the most common one (59% of all
FA cases). More than 200 different mutations have been described for this
gene.
B
2%
C
9%
D1
3%
D2
3%
E
2%
F
2%
G
8%
I
6%
J
3%
L
1%
M
1%
N
1%
A
59%
Brief introduction to the Fanconi Anemia (FA) disease
•The prevalence is 1 per 5 million and the estimated carrier frequency 1 in 200/300
in the general population
• Some ethnic groups have higher prevalence of FA due to founder effects and
isolation, for example the Spanish Gypsies or the citizens of La Palma island.
• In the present study we will analyse the deletion 3788-3790delTCT. This deletion
is one of the most frequent mutations found in the FANCA gene (between 5% to
10% of the FANCA mutations)
•It is found in many populations all over the world, but is particularly frequent
among the Brazilian population (50% of FANCA mutations)
• It is well known that 3788-3790delTCT is associated to, at least, 2 different
haplotypes (Levran et. al., 1997)
ANALYSIS OF THE HAPLOTYPE ASSOCIATED
WITH 3788-3790delTCT MUTATION IN FANCA
Aims of the study
Haplotype analysis associated with 3788-3790delTCT mutation in:
1. Brazilian patients. One or more ancestors?
2. La Palma patients. Is there a common ancestor for La
Palma and Brazilian populations?
3. Patients from all over the world. How many different
haplotypes are associated with 3788-3790delTCT?
FANCA gene
Chromosome 16q24.3
80 Kb and 43 exons
FANCA gene is highly polymorphic
3788-3790delTCT
30
32
41
Heterozygous frequency between 0.237 and 0.499
43
IVS42+29T/C
28
IVS33-42 G/A
26
IVS25-75 G/A
2426 G/A
23
IVS23+8T/C
13
IVS18+82T/C
8
1501G/A
4
IVS7-12 G/A
796 G/A
3
IVS6+74 G/A
1
IVS39-16 T/C
11 SNPs were chosen for the analysis
Each sample obtained from the patients was
analyzed using the Denaturing High Performance
Liquid Chromatography (DHPLC)
For each SNP:
1. PCR amplification
2. Denaturation and renaturation. Check homo or heterozygosis on
DHPLC and quantification of PCR product
3. If homozygous: Mix patient with homozygous 1 / homozygous 2
4. Denaturation and renaturation. DHPLC analysis
Analysis of SNPs: DHPLC
DHLPC: Denaturing High-Performance
Liquid Chromatography
Developed in 1995 by Oefner and Underhill
Detection of single-base substitutions, small
insertions and deletions in 150-1500bp
DNA fragments
Analysis of SNPs: DHPLC
Triethylammonimum
acetate (TEAA)
Positive charged
binds to negatively
charged
phosphate groups
on the DNA
Non-porous poly
styrene-divinilbenzene
(PS-DVB)
Analysis of SNPs: DHPLC
Triethylammonimum
acetate (TEAA)
Non-porous poly
styrene-divinilbenzene
(PS-DVB)
Absorption 260 nm
Acetonitril (ACN)
Elution Time
Elution at different times
• Depends on:
– Fragment length.
– Heteroduplexes elute sooner than
corresponding homoduplexes at apropiated
temperature (partially denatured
heteroduplexes).
99.8% of sequence variants
can be detected
Example
• SNP exon 18 (IVS18+82C/T):
– Steps 1 and 2: PCR amplification and de/re.
Patient
?
Control
Control
Control
heterozygous homozygous1 homozygous2
T/C
C/C
T/T
• SNP exon 18 (IVS18+82C/T):
– Steps 3 and 4: Mix patient sample with
controls at 1:1 ratio of PCR product. De/Re.
Patient + Control
homozygous1 (C/C)
Patient + Control
homozygous2 (T/T)
Conclusion: Patient is IVS18+82T/T
1. Brazilian patients. One or more ancestors?
All eight patients analysed are homozygous for all the
SNPs and share the same haplotype
Exon 6
IVS6+74 G/A
G
G
Exon 8
IVS7-12 G/A
A
A
Exon 9
796 A/G
A
A
Exon 16
1501G/A
G
G
Exon 18
IVS18+82T/C
T
T
Exon 23
IVS23+8T/C
T
T
Exon 26
IVS25-75 G/A
G
G
Exon 26
2426 G/A
G
G
Exon 33
IVS33-42 G/A
G
G
Exon 38
3788-3790delTCT
Del
Del
Exon 40
IVS39-16 C/T
C
C
Exon 42
IVS42+29T/C
T
T
Haplotype 1
1. Brazilian patients. One or more ancestors?
• 8 Brazilian patients homozygous for 37883790delTCT mutation (2 black, 3
caucasian, 3 unknown)
• It seems that all the patients have the
same ancestor.
2. La Palma patients. Is there a common ancestor for La
Palma and Brazilian populations?
Canarian Islands
La Palma Island
High incidence of FA (1 in
each 10000)
All patients belong to FA-A
complementation group
67% of the mutations found:
3788-3790delTCT (6
patients)
2. La Palma patients. Is there a common ancestor for La
Palma and Brazilian populations?
FAMILY 1
FAMILY 2
FAMILY 3
No DNA
FAMILY 4
No DNA
FAMILY 5
2. La Palma patients. Is there a common ancestor for La
Palma and Brazilian populations?
The haplotype assoiated with 3788-3790delTCT is the
same in all 5 families, and is the same as the one seen
in Brazilian patients
Exon 6
IVS6+74 G/A
G
Exon 8
IVS7-12 G/A
A
Exon 9
796 A/G
A
Exon 16
1501G/A
G
Exon 18
IVS18+82T/C
T
Exon 23
IVS23+8T/C
T
Exon 26
IVS25-75 G/A
G
Exon 26
2426 G/A
G
Exon 33
IVS33-42 G/A
G
Exon 38
3788-3790delTCT
Exon 40
IVS39-16 C/T
C
Exon 42
IVS42+29T/C
T
Del
Haplotype 1
3. Patients from all over the world. How many different
haplotypes are associated with 3788-3790delTCT?
14 homo or heterozygous patients for 3788-3790delTCT from...
3. Patients from all over the world. How many different
haplotypes are associated with 3788-3790delTCT?
Exon 6
IVS6+74 G/A
G
A
G
Exon 8
IVS7-12 G/A
A
G
A
Exon 9
796 A/G
A
Exon 16
1501G/A
G
Exon 18
IVS18+82T/C
T
T
Exon 23
IVS23+8T/C
T
T
Exon 26
IVS25-75
G
A
Exon 26
2426 G/A
G
Exon 33
IVS33-42 G/A
G
Exon 38
3788-3790delTCT
Exon 40
Exon 42
A
A
A
G
G
G
Del
Del
Del
IVS39-16 C/T
C
T
C
IVS42+29T/C
T
Haplotype 1
12 patients
T
Haplotype 2
1 patient
(Africa)
Haplotype 3
1 patient
(Germany)
All together...
Haplotype 1
Haplotype 2
Haplotype 3
27 patients
Haplotype frequencies general
population (HapMap)
0,7
0,6
0,35
0,5
0,3
0,4
0,25
0,3
0,2
0,2
0,15
0,1
0,1
0,05
0
CHB+JPT
0
CEU
0,35
0,3
0,25
0,2
0,15
0,1
0,05
Haplotype 1
Haplotype 2
0
YRI
Haplotype 3 not found in HapMap data
Conclusions: SNPs analysis
• The haplotype associated with mutation
3788-3790delTCT in FANCA gene, in
Brazilian and La Palma patients is the
same (Haplotype1). It is likely then, that
the mutation was introduced to both
populations from a common ancestry.
However, we cannot disregard the
possibility of two different ancestries, as
the frequency of this haplotype in the
studied populations is quite high (31.7%).
Conclusions: SNPs analysis
• Haplotype1 was identified as associated
with mutation 3788-3790delTCT in most of
the patients analyzed from European and
American populations, and also in a
patient from Pakistan (Asia). In a patient
from Germany, however, a variation of this
haplotype was observed (Haplotype3),
which differs from haplotype1 in one of the
SNPs analyzed in exon 26 of FANCA
gene.
Conclusions: SNPs analysis
• In the only patient from Africa analyzed, a
different haplotype that was found associated
with mutation 3788-3790delTCT (Haplotype 2).
In this case, we can affirm that the mutation was
originated from a different ancestry. This
observation supports the idea of a hot-spot in
this position. Furthermore is well known that the
slipped-strand mispairing and Alu-mediated
recombination are the two major mechanisms
for FANCA mutagenesis.
Conclusions: SNPs analysis
• The results of this study indicate that the
mutation 3788-3790delTCT in FANCA
gene was not introduced to Brazil and
other American countries from African
populations but, most probably, from the
European countries.
3. Patients from all over the world. How many different haplotypes are
associated with 3788-3790delTCT?
More than 2 ancestries?
• Haplotype1 is quite common in caucasian
population (31.7%).
• Check highly polymorphic microsatellites
flanking FANCA (more variable than
SNPs).
Microsatellite analysis
D16S3026
FANCA
D16S3121
380Kb
D16S3407
D16S303
320Kb
Heterozygous frequency between 0.431
and 0.77
Microsatellite analysis
Patients analyzed
18 patients
Microsatellite analysis
Haplotype 1
Haplotype 3
d16s303
116
116
116
116
d16s3407
198
198
198
198
Exon 6
IVS6+74 G/A
G
G
G
G
Exon 8
IVS7-12 G/A
A
A
A
A
Exon 9
796 A/G
A
A
A
A
Exon 16
1501G/A
G
G
G
G
Exon 18
IVS18+82T/C
T
T
T
T
Exon 23
IVS23+8T/C
T
T
T
T
Exon 26
IVS25-75
G
G
G
A
Exon 26
2426 G/A
G
G
G
G
Exon 33
IVS33-42 G/A
G
G
G
G
Exon 38
3788-3790delTCT
Del
Del
Del
Del
Exon 40
IVS39-16 C/T
C
C
C
C
Exon 42
IVS42+29T/C
T
T
T
T
d16s3121
68
72
72
72
d16s3026
198
202
200
202
Haplotype analysis
5 different haplotypes found
Conclusions
• It seems that the deletion 3788-3790delTCT found in the FANCA gene
has, at least, two different origins: the African one and the European one.
(It would be nice to increase our data with more African patients)
• The SNP found in the German patient is not described in the HapMap
data neither in the SNP NCBI database. Actually, it was found per chance
during the sequence analysis of another SNP so we can hypothesise that
is a rare punctual mutation.
• Most probably the European mutation was spread to America during the
migrations in the 15th -16th century. The distance in cM of the
microsatellites analysed is arround 2 cM, so we can hypothesize that the
differences between the microsatellites length is cause of the
recombination frequency. This hypothesis is supported by the fact that we
cannot find differences between the closest microsatellites analyzed
(d16s303 and d16s3407).
Bibliography
• O. Levran et al. 2005. Spectrum of Sequence Variations in the FANCA Gene:
An International Fanconi Anemia Registry (IFAR) Study. Human mutation
• E. Callén et al. 2005. A common founder mutation in FANCA underlies the
world’s highest pervalence of Fanconi anemia in Gypsy families from Spain.
Blood
• O. Levran et al. 1997. Sequence variation in the Fanconi anemia gene FAA.
PNAS
• The International HapMap Consortium. 2005. A haplotype map of the Human
genome. Nature
• A.J. Tipping et al. 2001. Molecular and genealogical evidence for a founder
effect in Fanconi anemia families of the Afrikaner population of South Africa.
PNAS