ANALYSIS OF THE haplotype associated mutation in fanca Pau Castillo FA is a rare autosomal recessive genetic syndrome There are at least 13 complementation

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.

A59%

B2%

C9%

D13%

D23%

E2%

F2%

G8%

I6%

J3%

L1%M1%

N1%

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)

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?

ANALYSIS OF THE HAPLOTYPE ASSOCIATED WITH 3788-3790delTCT MUTATION IN FANCA

Aims of the study

FANCA gene

1 3 4 8 13 23 26 28 30 32 41 43

3788-3790delTCT

IVS

6+74

G/A

IVS

7-12

G/A

79

6 G

/A

1501

G/A

IVS

18+

82T

/C

IVS

23+

8T/C

2426

G/A

IVS

33-4

2 G

/A

IVS

39-1

6 T

/C

IVS

42+

29T

/C

IVS

25-7

5 G

/A

Heterozygous frequency between 0.237 and 0.499

Chromosome 16q24.3

80 Kb and 43 exons

FANCA gene is highly polymorphic

11 SNPs were chosen for the analysis

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

Each sample obtained from the patients was analyzed using the Denaturing High Performance Liquid Chromatography (DHPLC)

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


Non-porous poly styrene-divinilbenzene (PS-DVB)

Triethylammonimum acetate (TEAA)

Positive charged binds to negatively charged phosphate groups on the DNA


Non-porous poly styrene-divinilbenzene (PS-DVB)

Triethylammonimum acetate (TEAA)

Acetonitril (ACN)

Abs

orpt

ion

260

nm

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

Patient?

Control heterozygous

T/C

Control homozygous1

C/C

Control homozygous2

T/T

• SNP exon 18 (IVS18+82C/T):– Steps 1 and 2: PCR amplification and de/re.

Example

• 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


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 26 IVS25-75 G/A G G

Exon 26 2426 G/A G G


Exon 38 3788-3790delTCT Del Del

Exon 40 IVS39-16 C/T C C


All eight patients analysed are homozygous for all the SNPs and share the same haplotype

Haplotype 1Haplotype 1


• 8 Brazilian patients homozygous for 3788-3790delTCT mutation (2 black, 3 caucasian, 3 unknown)

• It seems that all the patients have the same ancestor.


Canarian IslandsLa 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)


No DNA

No DNA

FAMILY 1 FAMILY 2 FAMILY 3

FAMILY 4 FAMILY 5


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 Del

Exon 40 IVS39-16 C/T C

Exon 42 IVS42+29T/C T Haplotype 1Haplotype 1

The haplotype assoiated with 3788-3790delTCT is the same in all 5 families, and is the same as the one seen

in Brazilian patients


14 homo or heterozygous patients for 3788-3790delTCT from...


Exon 6 IVS6+74 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 26 IVS25-75 G A

Exon 26 2426 G/A G A G


Exon 38 3788-3790delTCT Del Del Del

Exon 40 IVS39-16 C/T C T C


Haplotype 2 Haplotype 2 1 patient 1 patient (Africa)(Africa)

Haplotype 3 Haplotype 3 1 patient 1 patient

(Germany)(Germany)

Haplotype 1 Haplotype 1 12 patients12 patients

All together...Haplotype 1

Haplotype 2

Haplotype 3

27 patients

Haplotype frequencies general population (HapMap)

Haplotype 1

Haplotype 2Haplotype 3 not found in HapMap data

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

CEU

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

YRI

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

CHB+JPT

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%).


• 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.


• 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.


• 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.


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

FANCAD16S3121 D16S3407 D16S303

380Kb 320Kb

D16S3026

Heterozygous frequency between 0.431 and 0.77


18 patients

Patients analyzed


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 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


d16s3121 68 72 72 72

d16s3026 198 202 200 202

Haplotype 3Haplotype 3Haplotype 1Haplotype 1

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

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ANALYSIS OF THE haplotype associated mutation in fanca Pau Castillo FA is a rare autosomal recessive genetic syndrome There are at least 13 complementation