ANALYSIS OF THE haplotype associated mutation in fanca Pau Castillo

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ANALYSIS OF THE haplotype associated mutation in
fancaPau 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.

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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)

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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?
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FANCA gene
Chromosome 16q24.3 80 Kb and 43 exons FANCA gene
is highly polymorphic 11 SNPs were chosen for the
analysis
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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

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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
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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)
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Analysis of SNPs DHPLC
Triethylammonimum acetate (TEAA)
Acetonitril (ACN)
Absorption 260 nm
Non-porous poly styrene-divinilbenzene (PS-DVB)
Elution Time
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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
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Example

• SNP exon 18 (IVS1882C/T)
• Steps 1 and 2 PCR amplification and de/re.

Control heterozygous T/C
Control homozygous1 C/C
Control homozygous2 T/T
Patient?
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• SNP exon 18 (IVS1882C/T)
• Steps 3 and 4 Mix patient sample with controls
  at 11 ratio of PCR product. De/Re.

Patient Control homozygous1 (C/C)
Patient Control homozygous2 (T/T)
Conclusion Patient is IVS1882T/T
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1. Brazilian patients. One or more ancestors?
All eight patients analysed are homozygous for
all the SNPs and share the same haplotype
Exon 6 IVS674 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 IVS1882T/C T T
Exon 23 IVS238T/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 IVS4229T/C T T
Haplotype 1
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1. Brazilian patients. One or more ancestors?

• 8 Brazilian patients homozygous for
  3788-3790delTCT mutation (2 black, 3 caucasian, 3
  unknown)
• It seems that all the patients have the same
  ancestor.

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2. La Palma patients. Is there a common ancestor
for La Palma and Brazilian populations?
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)
Canarian Islands
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2. La Palma patients. Is there a common ancestor
for La Palma and Brazilian populations?
FAMILY 1
FAMILY 2
FAMILY 3
FAMILY 4
FAMILY 5
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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 IVS674 G/A G
Exon 8 IVS7-12 G/A A
Exon 9 796 A/G A
Exon 16 1501G/A G
Exon 18 IVS1882T/C T
Exon 23 IVS238T/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 Del
Exon 40 IVS39-16 C/T C
Exon 42 IVS4229T/C T
Haplotype 1
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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...
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3. Patients from all over the world. How many
different haplotypes are associated with
3788-3790delTCT?
Exon 6 IVS674 G/A G A G
Exon 8 IVS7-12 G/A A G A
Exon 9 796 A/G A A
Exon 16 1501G/A G A G
Exon 18 IVS1882T/C T T
Exon 23 IVS238T/C T T
Exon 26 IVS25-75 G A
Exon 26 2426 G/A G A G
Exon 33 IVS33-42 G/A G G
Exon 38 3788-3790delTCT Del Del Del
Exon 40 IVS39-16 C/T C T C
Exon 42 IVS4229T/C T T
Haplotype 2 1 patient (Africa)
Haplotype 3 1 patient (Germany)
Haplotype 1 12 patients
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All together...
27 patients
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Haplotype frequencies general population (HapMap)
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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).

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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.

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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.

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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.

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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).

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Microsatellite analysis
Heterozygous frequency between 0.431 and 0.77
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Microsatellite analysis
Patients analyzed
18 patients
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Microsatellite analysis
Haplotype 3
Haplotype 1
d16s303 116 116 116 116
d16s3407 198 198 198 198
Exon 6 IVS674 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 IVS1882T/C T T T T
Exon 23 IVS238T/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 IVS4229T/C T T T T
d16s3121 68 72 72 72
d16s3026 198 202 200 202
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Haplotype analysis
5 different haplotypes found
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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).

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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 worlds 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