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Identification of an ancient haemophilia A splice site mutation
Thrombosis Research 130 (2012) 445–450
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Identification of an ancient haemophilia A splice site mutation Sylvia Reitter-Pfoertner a,⁎, 1, Arndt von Haeseler b, Birgit Horvath c, Raute Sunder-Plassmann c, Vera Tiedje a, Ingrid Pabinger a, Christine Mannhalter c a b c
Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Austria Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, University of Veterinary Medicine Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Austria
a r t i c l e
i n f o
Article history: Received 29 December 2011 Received in revised form 9 February 2012 Accepted 10 February 2012 Available online 6 March 2012 Keywords: Splie site mutation Haemophilia Founder
a b s t r a c t Introduction: To date, numerous mutations resulting in haemophilia A are known and recorded at HAMSTeRS. We identified a new splice site mutation in intron 6 of the F8 gene (T to G transition at position −14; c.788-14T>G) in seven not knowingly related patients, who all suffer from mild haemophilia A. RNA analysis of blood cells indicated that this mutation leads to the preferred generation of a transcript lacking the complete exon 7 (without frameshift). Methods: To determine whether the mutation represented a founder mutation we analyzed intragenic (intronic) and extragenic short tandem repeat (STR) regions and constructed haplotypes in the 7 patients and 128 apparently healthy male control individuals. Results: In the 128 healthy control individuals, 109 different haplotypes were found. Surprisingly, also the 7 patients carried 3 different haplotypes. However, by genealogy reconstruction using BATWING we could identify an ancestral haplotype on which the mutation apparently occurred. This haplotype - DXS9897:12DXS1073:21-HA472:64-DXS1108:26 - was frequent and was found in three patients, but was also present in four control individuals who did not carry the splice site mutation. Conclusion: Our data indicate that the splice site mutation occurred in an individual carrying a relatively common haplotype. While the mutation was passed on through generations, the haplotypes identified in the seven patients derived from this founder haplotype but were changed by later mutations in the STR regions. © 2012 Elsevier Ltd. All rights reserved.
Introduction Haemophilia A, a recessive X-linked bleeding disorder characterized by factor VIII (FVIII) deficiency, is generally caused by mutations in the F8 gene. The gene is located at the guanine-cytosine-rich terminal reverse band on the long arm of the X-chromosome at Xq28. The whole gene spans a length of 186 kb, consists of 26 exons, has a coding region of 9 kb and encodes a mature protein of 2,332 amino acids [1]. Overall, a large number (>1,200) of different mutations in the F8 gene have been found to cause haemophilia. These are intron inversions, stop and missense mutations, deletions, insertions and splice site mutations. Causative mutations are recorded at HAMSTeRS [2], which serves as a reference data base for haemophilia A diagnostic laboratories all over the world. The most common mutation causing approximately 40 – 50% of severe haemophilia A is the intron 22 inversion, an inversion created by homologous recombination of intron 22 and related
⁎ Corresponding author at: Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna. Tel.: +43 1 40400 2757; fax: +43 1 40400 4030. E-mail address: [email protected] (S. Reitter-Pfoertner). 1 Dr. Reitter-Pfoertner is a recipient of the Bayer Haemophilia Clinical Training Award. 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.02.008
sequences outside the F8 gene [3]. The second most common mutation is the inversion in intron 1 with a prevalence of about 5% [4]. Other mutations, such as missense mutations, nonsense mutations, deletions and insertions are reported in all regions of the F8 gene. A number of individual mutations have been located in introns of the F8 gene. Recently, 80 new, intragenic and extragenic STR loci on Xq28 were published [5]. Furthermore, several genetic variants have been identified in the F8 gene – some single nucleotide polymorphisms and some microsatellites. These polymorphic STR loci located in intragenic and extragenic regions, both upstream and downstream of the F8 gene, have been reported and validated for indirect carrier-tracking in haemophilia A. They are a useful tool for monitoring inheritance of genetic alterations and are still used for FVIII linkage analysis which, until recently, was the most commonly used technique for carrier detection. Now, this approach has been superseded by direct mutation analysis [6]. However, the highly polymorphic STR markers are still helpful to discriminate whether a mutation represents a mutation hotspot or a founder mutation, and they can also be used to trace patients' ancestry. A study among 240 Austrian patients on the spectrum of haemophilia A mutations identified over 50 thus far not reported mutations [7]. One of these was a novel splice site mutation in intron 6 of the F8 gene (c.788-14T>G), which appeared in seven not knowingly related patients, all suffering from mild haemophilia A.
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In the present study, we investigated whether this new mutation represents a thus far unrecognized mutation hotspot or appeared as a result of common ancestor genes (founder mutation). Patients and Methods Patients and Controls Our study included seven patients with mild haemophilia A (FVIII:C 0.05-0.25 IU/ml), who were referred for mutation analysis and in whom a novel splice site mutation in intron 6 was identified as the causative mutation. All patients had a mild phenotype with no relevant bleeding events; in particular, one patient was diagnosed at the age of >50 when surgery became necessary. In the patients' family history, no close relatives with bleeding tendency have been reported. According to patient interviews, the patients were not knowingly related. We have also tested 128 male control individuals without known bleeding problems. All patients and control individuals gave their written informed consent to DNA analysis. The study was approved by the ethics committee of the Medical University of Vienna. Methods DNA was extracted from venous blood anticoagulated with EDTA using the MagNA Pure DNA-isolation system (Roche Diagnostics, Mannheim, Germany). Total RNA was isolated from leucocytes with the RNeasy® Mini Kit (Qiagen, Hilden, Germany) on a QIAcube instrument (Qiagen, Hilden, Germany). In both cases, the manufacturer's instructions were followed. RNA was reversely transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (ABI, Foster City, CA, USA). For cDNA transcription, 2 μg of RNA were used. The reaction was performed with the following temperature protocol: first, RNA was incubated at 22 °C, then a temperature of 37 °C was maintained for 45 minutes, followed by 5 minutes at 85 °C. Nested RT-PCR was performed essentially as described in El-Maarri et al. [8]. in an Eppendorf Cycler (Eppendorf, Hamburg, Germany) using 5 μl cDNA for the first PCR and 2 μl of the resulting PCR product for the second PCR. For the first PCR, the PCR program was set to initial denaturation at 95 °C for 10 min., followed by 30 cycles of amplification with 30 seconds of denaturation at 95 °C, 30 seconds annealing at 52 °C, 1 minute elongation at 72 °C and 7 minutes final extension at 72 °C. For the second PCR, the same PCR protocol was used. The annealing temperature for the second PCR, however, was 58 °C and there were 35 cycles of amplification. The amplicons were evaluated on a 5% Criterion gel (Bio-Rad Laboratories GmbH, Munich, Germany). Sequencing was performed as described below. All primers used for PCR amplifications were synthesized and PAGEpurified by VBC Biotech (Vienna, Austria). The primer sequences and the PCR protocols used in our laboratory can be provided upon request.
Regarding the primers used for sequencing of exon 7 (genomic DNA) as well as for sequencing of the cDNA, see Table 1. All coding regions as well as the exon/intron boundaries and part of the promoter region were analyzed. PCR products generated from genomic DNA and cDNA were sequenced following treatment with ExoSAP-it (USB, Cleveland, OH, USA). The sequencing reaction was performed with the Big Dye Terminator cycle sequencing kit (ABI, Foster City, CA, USA) using 5 pmol of each primer by applying an ABI 3130xl Genetic Analyzer. The best practice of DNA sequencing [9] was followed. Analysis of Short Tandem Repeats (STRs) Patients and control individuals were tested for the following previously published extragenic STRs (in 3′ ➔ 5′ order): DXS 9897, DXS 1073, HA 472 and DXS 1108. Regarding the specific localisation of these STRs, see Table 1. All STRs were repeatedly tested in order to confirm the repeat number. For each PCR, 1.7 pmol forward and reverse primer (Eurofins MWG Operon, Ebersberg, Germany) were used, respectively. The PCR program was set to initial denaturation at 95 °C for 10 min., followed by 28 cycles of amplification with 45 seconds of denaturation at 95 °C, 45 seconds annealing at 57 °C, 45 seconds elongation at 72 °C and 7 minutes final extension at 72 °C. The amplicons were tested on a Spreadex EL400 Wide Mini gel (Elchrom Scientific AG, Cham, Switzerland). A 1.5 μl aliquot of the PCR product was added to 10 μl of Hi-Di Formamide (ABI, Foster City, California, USA). Then, 0.3 μl size standard GeneScan 400HD (ABI, Foster City, California, USA) was added and the mixture was heated at 95 °C for 2 minutes to denature the amplicon. Fragments were analysed at 60 °C on an ABI Prism 3100 GeneAnalyzer using a 50-cm capillary array and POP 6 polymer (all from ABI, Foster City, California, USA). The Genotyper software version 3.7 (ABI, Foster City, California, USA) was used for data analysis. Statistical Analysis The analysis is based on four STRs. We followed the approach outlined by Zivelin et al. [10,11]. However, the chi-square (χ2)-test was adapted to multiple alleles. To check the reliability of the χ2-approximation, we drew 10,000 times random samples with replacement of seven (corresponding to the number of patients) from the allele frequency distribution obtained in the 128 control individuals to approximate the chi-square distribution. The p-value of the observed χ2-value was then estimated from the simulated distribution. Finally, the data was subject to a genealogy reconstruction using BATWING [12]. From the resulting 5,000 trees a consensus tree was computed. Using a maximum parsimony approach [13], the number of mutations leading to the different repeat numbers of the four STRs found in the patients and control individuals could be evaluated. A symmetric step-wise mutation model was used, where each STR gains or loses one repeat unit.
Table 1 Intragenic (intronic) and extragenic markers used for linkage analysis with the respective primers. Locus name
Concensus pattern
Forward sequence (5′ ➔ 3′)1
Reverse sequence (5′ ➔ 3′)1
Physical position
Int1 Int6 Int 9.1 Int 22 Int 25.3 DXS 9897 DXS 1073 HA 472 DXS 1108
AC TG AG GT TG CTAT TG CTT CA
CTG CCC TTG GAC ATA AGC AT TTC TCC TGC TTC AGC CTC TC AGA TTC GAG CGA TTC TCC TG AAG ACC CTT AGC TGT TTC ATA AGC TCC AAG ATC AAG GGG TAG GC TTC TGC TGT GCA ATA CAT CTG A AAG AAT GCC CTC TCC GAG TT GCT CCT TTG ATT GGA TAA TTT CA GGG AGA TAG GAA TGA TGG AGT G
CCA TAT GAT CCA GCA ACT CG AGC ATA TCC ACC CTC ACC AC CAG TCA TTG CTG TGG GTT TG TTC ATA CAG TGG GAT CAT TCA TT GCC TGG ACT ACA GAG GGA GA CAG CAG ACA TTA TTG AGG GAG A ATT GGT GGC CTT TGA AAC AC TGC CTC AAC ATC AGA ATA GAC C TAT TTT CTG GGC CAT CTT GG
within the F8 gene within the F8 gene within the F8 gene within the F8 gene within the F8 gene approx. 1596 kb 3′ of the F8 gene approx. 235 kb 3′ of the F8 gene approx. 163 kb 3′ of the F8 gene approx. 610 kb 5′ of the F8 gene
1 Machado FB, Medina-Acosta E. High resolution combined linkage physical map of short tandem repeat loci on human chromosome Xq28 for indirect haemophilia A carrier detection. Haemophilia (2009), 15, 297-308.
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Results Mutation Analysis By sequencing of the F8 gene we identified a missense mutation (T➔G) at position −14 in intron 6 of the F8 gene in proximity to the acceptor splice site (c.788-14T>G) in seven patients. This mutation is not listed at HAMSTeRS and to the best of our knowledge, has not been published elsewhere. We did not find any other mutation or any polymorphism in the coding region of the F8 gene. In 5 patients sequence analysis of mRNA/cDNA from leucocytes could be performed. The data indicated that the T ➔ G mutation at position −14 in intron 6 (c.788-14T>G) leads to preferred use of another splice site and results in the loss of the complete exon 7 (without frameshift). Analysis of the leucocyte-derived cDNA in these 5 patients showed no expression of full-length mRNA (ie. mRNA containing exon 7).
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therefore not be included in the further analysis. As pointed out by Cochran [14] the approximation of the χ 2 distribution may not be adequate, because some expected STR counts are less than one. Thus, we carried out a bootstrap procedure to obtain a simulated χ 2 distribution for each STR. Regarding the detailed results of this analysis, see Table 2. From the simulated χ 2-square distribution (as outlined in the Methods section) the following p-values have been obtained: for DXS9897 0.114, for DXS1073 b 1∙10 − 5, for HA472 0.029, for DXS1 108 b 1∙10 − 5, respectively. Table 3 compares the results for the χ 2-value, the p-value and the simulated p-value for the four different STRs. Three of the four STR allele frequencies found in the patients clearly differ from the expected distributions derived from the findings in the control individuals. This is reflected by simulated p-values below 0.05, which is regarded as statistically significant. Merely, the locus DXS9897 (the most remote STR on the 3′-side of the F8 gene) does not show a significant deviation from the expected distribution.
Analysis of STRs Haplotypes in Patients and Controls Regarding the analyzed intragenic (intronic) STRs (for details see Table 1), all seven patients carried the same genotype. Following the advice of our statistician, these markers were not included in the genealogic analyses. Regarding details of the four analysed extragenic STRs (concensus pattern, sequence and physical position) see Table 1. Fig. 1 displays the allele frequencies of these STRs among patients and controls. For further analysis, we calculated the expected numbers for the different alleles of the four STRs and compared these to the observed numbers in our patient cohort. Interestingly, two theoretically possible alleles of the locus DXS1108 were not observed and could
Based on the four extragenic STRs, haplotypes for patients as well as for control individuals were generated. In the patients, three different haplotypes characterized by the following repeat numbers could be identified; namely, 12-22-63-26, 12-22-64-26 and 12-21-64-26 (see Table 2). At the locus DXS1073, two different alleles were found in the patients – the 21-repeat allele in three patients and the 22-repeat allele in four patients. Remarkably, the haplotypes with 22 repeats at locus DXS1073 were not identical but belonged to two different clades. Also, for the locus HA472, two different alleles were found in the 7 patients: 63 repeats in three patients and 64 repeats in four patients.
Fig. 1. a. Allele frequencies of DXS9897. b. Allele frequencies of DXS1073. c. Allele frequencies of HA472. d. Allele frequencies of DXS1108. x-axis: number of repeats; y-axis: number of patients and control individuals, respectively.
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Table 2 Observed number of alleles in the patients and expected number of alleles inferred from allele frequencies from the healthy individuals. DXS9897
DXS1073
HA472
DXS1108
Allele / product length
Observed Expected Allele /product length
Observed Expected
Allele /product length
Observed Expected
Allele /product length
Observed Expected
10-repeat / 224 bp 11-repeat 228 bp 12-repeat 232 bp 13-repeat 236 bp 14-repeat 240 bp
0 0 7 0 0
0 0 3 4 0 0 0 0 0
57-repeat /241 bp 58-repeat /244 bp 59-repeat / 247 bp 60-repeat /250 bp 61-repeat /253 bp 62-repeat 256 bp 63-repeat /259 bp 64-repeat /262 bp 65-repeat /265 bp 66-repeat /268 bp 67-repeat /271 bp 68-repeat / 274 bp 69-repeat / 277 bp 70-repeat / 280 bp 71-repeat / 283 bp 72-repeat / 286 bp 73-repeat / 289 bp 74-repeat / 292 bp
0 0 0 0 0 0 3 4 0 0 0 0 0
18-repeat /112 bp 19-repeat /114 bp 20-repeat /116 bp 21-repeat /118 bp 22-repeat /120 bp 23-repeat / 122 bp 24-repeat /124 bp 25-repeat /126 bp 26-repeat /128 bp 27-repeat /130 bp
0 0 0 0 0 0 0 7 0 0
0.164 1.695 3.50 1.422 0.219
19-repeat /130 bp 20-repeat /132 bp 21-repeat / 134 bp 22-repeat /136 bp 23-repeat /138 bp 24-repeat /140 bp 25-repeat /142 bp 26-repeat /144 bp 27-repeat /146 bp
0.2734375 2.078125 3.4453125 0.0546875 0.21875 0.0546875 0.0546875 0.7109375 0.109375
In the 128 male control individuals, 109 different haplotypes were detected. Haplotype 12-21-64-26 is the most frequent in the whole sample and was observed in three patients and four control individuals. Surprisingly, the haplotypes 12-22-63-26 and 12-22-64-26, which were found in four of seven patients, were not detected in the control cohort. Reconstruction of the Ancestral Haplotype To illustrate the genealogical relationships and ancestry of the patients, we inferred a genealogy of the samples and established a genealogical tree. The patient samples comprised three haplotypes, which form two distinct clusters in the genealogy. A consensus tree indicated that the patients form one common haplotype group. By computation we determined the minimum number of mutations necessary to explain the haplotype diversity in the sample. Fig. 2 shows the resulting haplotype genealogy for the patient samples – the circle size is proportional to the number of haplotypes found. The haemophilia A causing mutation occurred on the most frequent haplotype D1 (12-21-64-26). The mutation 21 ➔ 22 at the DXS1073 locus led to haplotype D2 (12-22-64-26), which was found in one patient, and subsequently the mutation 64 ➔ 63 on locus HA472 generated the third patient haplotype D3 (12-22-63-26), which was found in three patients. Haplotype D1 is the one linked to the remaining control haplotypes. From the genealogy we conclude that the haemophilia A causing mutation originated on the genetic background of haplotype D1, and mutations of STRs led to the different haplotypes D2 and D3 present in haemophilia patients with the same mutation. Discussion The splice site mutation present in seven unrelated haemophilia A patients is a not yet described T ➔ G missense mutation at position −14 Table 3 Results for the χ2-value, the p-value and the simulated p-value for the four different STRs. STR
χ2-value
p-value
simulated p-value (100,000 runs)
DXS9897 DXS1073 HA472 DXS1108
7.0 288.2 37.41 61.92
0.136 0.000 0.002 6 10− 11
0.114 b 1 10− 5 0.029 b 1 10− 5
0.0546875 0.1640625 0.546875 0.875 0.546875 0.65625 0.3828125 0.765625 0.8203125 0.765625 0.328125 0.6015625 0.109375
0.21875 1.640625 0 0 0.109375 0.4375 1.5859375 2.2421875 0.7109375 0.0546875
in intron 6 (c.788-14T>G). To our knowledge, sequencing of the F8 gene usually does not cover this part of intron 6 and thus this mutation may have escaped detection in patients with mild haemophilia and minor clinical symptoms. It is known that mutations causing haemophilia A can occur in introns of the F8 gene. It may be necessary to analyze gene regions beyond the coding region and the exon/intron boundaries at least in selected patient groups. Analysis of cDNA to evaluate the effect of this splice site mutation confirmed that this mutation leads to the preferred generation of mRNA lacking exon 7. The predominant presence of RNA lacking exon 7 seems to be responsible for the reduced FVIII activity and the mild haemophilia A observed in the seven patients. Previously, the deletion of exon 7 from genomic DNA has been reported to cause a severe haemophilia A phenotype [15]. We speculate that the mild phenotype observed in our patients with a splice site mutation may be due to the presence of a small amount of regularly spliced FVIII mRNA in liver cells which was below the detection limit in mRNA obtained from leucocytes. Efforts to detect minute amounts of correctly spliced mRNA in blood cells were unsuccessful, and we did not have access to liver samples, as none of the patients needed a liver biopsy for medical reasons. To distinguish whether the mutation represented a mutation hot spot or a founder mutation we analyzed intragenic (intronic) and extragenic short tandem repeat polymorphisms (STRs). Statistical analysis of the allele frequency distribution for the analyzed STRs in the patients and 128 individuals without any bleeding diathesis showed that the STR-allele distribution of the patients shows less variation compared to the control sample. The haplotype distribution found in patients differed from the one expected on the basis of the haplotypes identified in the controls. DXS9897 and DXS1108 were monomorphic among the patients. In this regard, it should be pointed out that the genetic length between DXS9897 and DXS1108 is 3.22 cM [16], an interval, which covers 81 genes and 13 pseudogenes [5]. Genetic disequilibrium can be eroded by recombination events, which break down the ancestral haplotypes. This leads to a linkage equilibrium over time [17]. DXS9897 lies approximately 1596 kb 3′ of the F8 gene. Recombination events likely occur in DNA stretches between STRs; more frequently if these stretches are longer. Probably, the STR locus DXS9897 is more frequently affected by recombination events due to the large distance to the FVIII gene. Overall, four healthy control individuals belong to the same haplotype cluster as our patient cohort, which means that they have the same ancestral haplotype. We could analyze one healthy male with haplotype D1. He had a normal FVIII:C level (155%) and never
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Fig. 2. Genealogy of the patient cohort (relevant part of the consensus tree in concise illus-tration). Each node represents a different haplotype. Circle areas are proportional to haplotype frequency. “C” stands for control individuals and “D” for patients. Filled circles represent haplo types not found in the samples. The dotted lines and circles indicate the link to the remainingnetwork of control individuals (not shown in this figure). The haplotype groups C1 and D1 are patients and control individuals sharing the same haplotype shown in parenthe sis. The labels at the branches connecting the circles specify the mutations at the respective loci leading to the different haplotypes. The repeat number on the left side of the equivalence sign repre- sents the repeat number of the left haplotype or the haplotype above. This information suf- fices to reconstruct the other haplotypes.
experienced bleeding events. Sequencing proved that this person did not carry the intron 6 splice site mutation found in the patients. This indicates that the missense mutation occurred once in a healthy individual with haplotype D1 and stayed in the population. Further support for a single mutational event is the absence of the D2 and D3 haplotype in the control population and the low frequency of the D1 haplotype in the control population. However, we note that we identified a very large number of haplotypes – 109 in 125 individuals. This might indicate that the STRs are unstable and/or that that there is a high rate of recombination in the population. It also indicates that the control population used was probably not large enough to enable an accurate estimation of the frequency of individual haplotypes. Due to the unknown mutation rates of the STRs our data does not provide accurate information on the age of the mutation. However, we can conclude that the splice site mutation occurred in one founder. From the number of STR mutations on the patient-D1 haplotype we can assume that the origin of the mutation dates back at least 500 years (25 generations), if we assume a mutation rate as high as 0.075 per marker per generation [18]. Nowadays, life expectancy of patients with mild haemophilia equals the life expectancy of the normal population [19]. Also in earlier days,
when no effective treatment was available, patients with mild haemophilia usually did not die at a very young age but reached adulthood [20]. This explains why the missense mutation found in our patient cohort, although based on a founder effect, was passed on over several generations and many years. Conflict of Interest Statement None of the authors has a conflict of interest to declare. Acknowledgements The authors would like to thank Renate Freitag for her contribution concerning the mutation analysis of the haemophilia A patients. Further, we would like to acknowledge the financial support rendered through the Anniversary Fund of the Oesterreichische Nationalbank (OeNB, project number AP12208ONB). Arndt von Haeseler also thanks the Vienna Science and Technology Fund (WWTF) for financial support. In addition, Sylvia Reitter-Pfoertner thanks Bayer Healthcare for receipt of the Bayer Haemophilia Clinical Training Award, which enabled her to
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work on this project at the Department of Laboratory Medicine at the Medical University of Vienna. References [1] Bicocchi MP, Pasino M, Lanza T, Bottini F, Boeri E, Mori PG, et al. Analysis of 18 novel mutations in the factor VIII gene. Br J Haematol 2003;122(5):810–7. [2] The Haemophilia A Mutation, Structure, Test and Resource Site. 12–3–2010.Ref Type: Internet Communication. [3] Lakich D, Kazazian Jr HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993;5(3):236–41. [4] Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002;99(1): 168–74. [5] Machado FB, Medina-Acosta E. High-resolution combined linkage physical map of short tandem repeat loci on human chromosome band Xq28 for indirect haemophilia A carrier detection. Haemophilia 2009;15(1):297–308. [6] Keeney S, Mitchell M, Goodeve A. The molecular analysis of haemophilia A: a guideline from the UK haemophilia centre doctors' organization haemophilia genetics laboratory network. Haemophilia 2005;11(4):387–97. [7] Reitter S, Sturn R, Horvath B, Freitag R, Male C, Muntean W, et al. Spectrum of causative mutations in patients with haemophilia A in Austria. Thromb Haemost 2010;104(1): 78–85. [8] El-Maarri O, Herbiniaux U, Graw J, Schroder J, Terzic A, Watzka M, et al. Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene. J Thromb Haemost 2005;3(2):332–9. [9] CMGS website. Sequencing Best Practice Guidelines. http://www.cmgs.org/BPGs/ best_practice_guidelines.htm20-9-2010 Ref Type: Internet Communication.
[10] Zivelin A, Griffin JH, Xu X, Pabinger I, Samama M, Conard J, et al. A single genetic origin for a common Caucasian risk factor for venous thrombosis. Blood 1997;89(2):397–402. [11] Zivelin A, Rosenberg N, Faier S, Kornbrot N, Peretz H, Mannhalter C, et al. A single genetic origin for the common prothrombotic G20210A polymorphism in the prothrombin gene. Blood 1998;92(4):1119–24. [12] Wilson IJ, Weale ME, Balding DJ. Inferences from DNA data: population histories, evolutionary processes and forensic match probabilities. J R Statist Soc A 2003;166(Part 2):155–201. [13] Fitch WM. Rate of change of concomitantly variable codons. J Mol Evol 1971;1(1): 84–96. [14] Cochran W. Some methods for strengthening the common Chi2 test. Biometrics 1954;10:417–51. [15] Citron M, Godmilow L, Ganguly T, Ganguly A. High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation. Hum Mutat 2002;20(4):267–74. [16] Matise TC, Chen F, Chen W, De LV, De La Vega FM, Hansen M, He C, et al. A second-generation combined linkage physical map of the human genome. Genome Res 2007;17(12):1783–6. [17] Sabatti C, Risch N. Homozygosity and linkage disequilibrium. Genetics 2002;160(4): 1707–19. [18] Ballantyne K. GMFRSOLOWA. Mutability of Y-Chromosomal Microsatellites: Rates, Characteristics, Molecular Bases, and Forensic Implications. Am J Hum Genet 2010;87:341–53. [19] Reitter S, Waldhoer T, Vutuc C, Lechner K, Pabinger I. Survival in a cohort of patients with haemophilia at the haemophilia care center in Vienna, Austria, from 1983 to 2006. Haemophilia 2009;15(4):888–93. [20] Larsson SA. Life expectancy of Swedish haemophiliacs, 1831–1980. Br J Haematol 1985;59(4):593–602.
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Regular Article
Identification of an ancient haemophilia A splice site mutation Sylvia Reitter-Pfoertner a,⁎, 1, Arndt von Haeseler b, Birgit Horvath c, Raute Sunder-Plassmann c, Vera Tiedje a, Ingrid Pabinger a, Christine Mannhalter c a b c
Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Austria Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, University of Veterinary Medicine Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Austria
a r t i c l e
i n f o
Article history: Received 29 December 2011 Received in revised form 9 February 2012 Accepted 10 February 2012 Available online 6 March 2012 Keywords: Splie site mutation Haemophilia Founder
a b s t r a c t Introduction: To date, numerous mutations resulting in haemophilia A are known and recorded at HAMSTeRS. We identified a new splice site mutation in intron 6 of the F8 gene (T to G transition at position −14; c.788-14T>G) in seven not knowingly related patients, who all suffer from mild haemophilia A. RNA analysis of blood cells indicated that this mutation leads to the preferred generation of a transcript lacking the complete exon 7 (without frameshift). Methods: To determine whether the mutation represented a founder mutation we analyzed intragenic (intronic) and extragenic short tandem repeat (STR) regions and constructed haplotypes in the 7 patients and 128 apparently healthy male control individuals. Results: In the 128 healthy control individuals, 109 different haplotypes were found. Surprisingly, also the 7 patients carried 3 different haplotypes. However, by genealogy reconstruction using BATWING we could identify an ancestral haplotype on which the mutation apparently occurred. This haplotype - DXS9897:12DXS1073:21-HA472:64-DXS1108:26 - was frequent and was found in three patients, but was also present in four control individuals who did not carry the splice site mutation. Conclusion: Our data indicate that the splice site mutation occurred in an individual carrying a relatively common haplotype. While the mutation was passed on through generations, the haplotypes identified in the seven patients derived from this founder haplotype but were changed by later mutations in the STR regions. © 2012 Elsevier Ltd. All rights reserved.
Introduction Haemophilia A, a recessive X-linked bleeding disorder characterized by factor VIII (FVIII) deficiency, is generally caused by mutations in the F8 gene. The gene is located at the guanine-cytosine-rich terminal reverse band on the long arm of the X-chromosome at Xq28. The whole gene spans a length of 186 kb, consists of 26 exons, has a coding region of 9 kb and encodes a mature protein of 2,332 amino acids [1]. Overall, a large number (>1,200) of different mutations in the F8 gene have been found to cause haemophilia. These are intron inversions, stop and missense mutations, deletions, insertions and splice site mutations. Causative mutations are recorded at HAMSTeRS [2], which serves as a reference data base for haemophilia A diagnostic laboratories all over the world. The most common mutation causing approximately 40 – 50% of severe haemophilia A is the intron 22 inversion, an inversion created by homologous recombination of intron 22 and related
⁎ Corresponding author at: Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna. Tel.: +43 1 40400 2757; fax: +43 1 40400 4030. E-mail address: [email protected] (S. Reitter-Pfoertner). 1 Dr. Reitter-Pfoertner is a recipient of the Bayer Haemophilia Clinical Training Award. 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.02.008
sequences outside the F8 gene [3]. The second most common mutation is the inversion in intron 1 with a prevalence of about 5% [4]. Other mutations, such as missense mutations, nonsense mutations, deletions and insertions are reported in all regions of the F8 gene. A number of individual mutations have been located in introns of the F8 gene. Recently, 80 new, intragenic and extragenic STR loci on Xq28 were published [5]. Furthermore, several genetic variants have been identified in the F8 gene – some single nucleotide polymorphisms and some microsatellites. These polymorphic STR loci located in intragenic and extragenic regions, both upstream and downstream of the F8 gene, have been reported and validated for indirect carrier-tracking in haemophilia A. They are a useful tool for monitoring inheritance of genetic alterations and are still used for FVIII linkage analysis which, until recently, was the most commonly used technique for carrier detection. Now, this approach has been superseded by direct mutation analysis [6]. However, the highly polymorphic STR markers are still helpful to discriminate whether a mutation represents a mutation hotspot or a founder mutation, and they can also be used to trace patients' ancestry. A study among 240 Austrian patients on the spectrum of haemophilia A mutations identified over 50 thus far not reported mutations [7]. One of these was a novel splice site mutation in intron 6 of the F8 gene (c.788-14T>G), which appeared in seven not knowingly related patients, all suffering from mild haemophilia A.
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In the present study, we investigated whether this new mutation represents a thus far unrecognized mutation hotspot or appeared as a result of common ancestor genes (founder mutation). Patients and Methods Patients and Controls Our study included seven patients with mild haemophilia A (FVIII:C 0.05-0.25 IU/ml), who were referred for mutation analysis and in whom a novel splice site mutation in intron 6 was identified as the causative mutation. All patients had a mild phenotype with no relevant bleeding events; in particular, one patient was diagnosed at the age of >50 when surgery became necessary. In the patients' family history, no close relatives with bleeding tendency have been reported. According to patient interviews, the patients were not knowingly related. We have also tested 128 male control individuals without known bleeding problems. All patients and control individuals gave their written informed consent to DNA analysis. The study was approved by the ethics committee of the Medical University of Vienna. Methods DNA was extracted from venous blood anticoagulated with EDTA using the MagNA Pure DNA-isolation system (Roche Diagnostics, Mannheim, Germany). Total RNA was isolated from leucocytes with the RNeasy® Mini Kit (Qiagen, Hilden, Germany) on a QIAcube instrument (Qiagen, Hilden, Germany). In both cases, the manufacturer's instructions were followed. RNA was reversely transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (ABI, Foster City, CA, USA). For cDNA transcription, 2 μg of RNA were used. The reaction was performed with the following temperature protocol: first, RNA was incubated at 22 °C, then a temperature of 37 °C was maintained for 45 minutes, followed by 5 minutes at 85 °C. Nested RT-PCR was performed essentially as described in El-Maarri et al. [8]. in an Eppendorf Cycler (Eppendorf, Hamburg, Germany) using 5 μl cDNA for the first PCR and 2 μl of the resulting PCR product for the second PCR. For the first PCR, the PCR program was set to initial denaturation at 95 °C for 10 min., followed by 30 cycles of amplification with 30 seconds of denaturation at 95 °C, 30 seconds annealing at 52 °C, 1 minute elongation at 72 °C and 7 minutes final extension at 72 °C. For the second PCR, the same PCR protocol was used. The annealing temperature for the second PCR, however, was 58 °C and there were 35 cycles of amplification. The amplicons were evaluated on a 5% Criterion gel (Bio-Rad Laboratories GmbH, Munich, Germany). Sequencing was performed as described below. All primers used for PCR amplifications were synthesized and PAGEpurified by VBC Biotech (Vienna, Austria). The primer sequences and the PCR protocols used in our laboratory can be provided upon request.
Regarding the primers used for sequencing of exon 7 (genomic DNA) as well as for sequencing of the cDNA, see Table 1. All coding regions as well as the exon/intron boundaries and part of the promoter region were analyzed. PCR products generated from genomic DNA and cDNA were sequenced following treatment with ExoSAP-it (USB, Cleveland, OH, USA). The sequencing reaction was performed with the Big Dye Terminator cycle sequencing kit (ABI, Foster City, CA, USA) using 5 pmol of each primer by applying an ABI 3130xl Genetic Analyzer. The best practice of DNA sequencing [9] was followed. Analysis of Short Tandem Repeats (STRs) Patients and control individuals were tested for the following previously published extragenic STRs (in 3′ ➔ 5′ order): DXS 9897, DXS 1073, HA 472 and DXS 1108. Regarding the specific localisation of these STRs, see Table 1. All STRs were repeatedly tested in order to confirm the repeat number. For each PCR, 1.7 pmol forward and reverse primer (Eurofins MWG Operon, Ebersberg, Germany) were used, respectively. The PCR program was set to initial denaturation at 95 °C for 10 min., followed by 28 cycles of amplification with 45 seconds of denaturation at 95 °C, 45 seconds annealing at 57 °C, 45 seconds elongation at 72 °C and 7 minutes final extension at 72 °C. The amplicons were tested on a Spreadex EL400 Wide Mini gel (Elchrom Scientific AG, Cham, Switzerland). A 1.5 μl aliquot of the PCR product was added to 10 μl of Hi-Di Formamide (ABI, Foster City, California, USA). Then, 0.3 μl size standard GeneScan 400HD (ABI, Foster City, California, USA) was added and the mixture was heated at 95 °C for 2 minutes to denature the amplicon. Fragments were analysed at 60 °C on an ABI Prism 3100 GeneAnalyzer using a 50-cm capillary array and POP 6 polymer (all from ABI, Foster City, California, USA). The Genotyper software version 3.7 (ABI, Foster City, California, USA) was used for data analysis. Statistical Analysis The analysis is based on four STRs. We followed the approach outlined by Zivelin et al. [10,11]. However, the chi-square (χ2)-test was adapted to multiple alleles. To check the reliability of the χ2-approximation, we drew 10,000 times random samples with replacement of seven (corresponding to the number of patients) from the allele frequency distribution obtained in the 128 control individuals to approximate the chi-square distribution. The p-value of the observed χ2-value was then estimated from the simulated distribution. Finally, the data was subject to a genealogy reconstruction using BATWING [12]. From the resulting 5,000 trees a consensus tree was computed. Using a maximum parsimony approach [13], the number of mutations leading to the different repeat numbers of the four STRs found in the patients and control individuals could be evaluated. A symmetric step-wise mutation model was used, where each STR gains or loses one repeat unit.
Table 1 Intragenic (intronic) and extragenic markers used for linkage analysis with the respective primers. Locus name
Concensus pattern
Forward sequence (5′ ➔ 3′)1
Reverse sequence (5′ ➔ 3′)1
Physical position
Int1 Int6 Int 9.1 Int 22 Int 25.3 DXS 9897 DXS 1073 HA 472 DXS 1108
AC TG AG GT TG CTAT TG CTT CA
CTG CCC TTG GAC ATA AGC AT TTC TCC TGC TTC AGC CTC TC AGA TTC GAG CGA TTC TCC TG AAG ACC CTT AGC TGT TTC ATA AGC TCC AAG ATC AAG GGG TAG GC TTC TGC TGT GCA ATA CAT CTG A AAG AAT GCC CTC TCC GAG TT GCT CCT TTG ATT GGA TAA TTT CA GGG AGA TAG GAA TGA TGG AGT G
CCA TAT GAT CCA GCA ACT CG AGC ATA TCC ACC CTC ACC AC CAG TCA TTG CTG TGG GTT TG TTC ATA CAG TGG GAT CAT TCA TT GCC TGG ACT ACA GAG GGA GA CAG CAG ACA TTA TTG AGG GAG A ATT GGT GGC CTT TGA AAC AC TGC CTC AAC ATC AGA ATA GAC C TAT TTT CTG GGC CAT CTT GG
within the F8 gene within the F8 gene within the F8 gene within the F8 gene within the F8 gene approx. 1596 kb 3′ of the F8 gene approx. 235 kb 3′ of the F8 gene approx. 163 kb 3′ of the F8 gene approx. 610 kb 5′ of the F8 gene
1 Machado FB, Medina-Acosta E. High resolution combined linkage physical map of short tandem repeat loci on human chromosome Xq28 for indirect haemophilia A carrier detection. Haemophilia (2009), 15, 297-308.
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Results Mutation Analysis By sequencing of the F8 gene we identified a missense mutation (T➔G) at position −14 in intron 6 of the F8 gene in proximity to the acceptor splice site (c.788-14T>G) in seven patients. This mutation is not listed at HAMSTeRS and to the best of our knowledge, has not been published elsewhere. We did not find any other mutation or any polymorphism in the coding region of the F8 gene. In 5 patients sequence analysis of mRNA/cDNA from leucocytes could be performed. The data indicated that the T ➔ G mutation at position −14 in intron 6 (c.788-14T>G) leads to preferred use of another splice site and results in the loss of the complete exon 7 (without frameshift). Analysis of the leucocyte-derived cDNA in these 5 patients showed no expression of full-length mRNA (ie. mRNA containing exon 7).
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therefore not be included in the further analysis. As pointed out by Cochran [14] the approximation of the χ 2 distribution may not be adequate, because some expected STR counts are less than one. Thus, we carried out a bootstrap procedure to obtain a simulated χ 2 distribution for each STR. Regarding the detailed results of this analysis, see Table 2. From the simulated χ 2-square distribution (as outlined in the Methods section) the following p-values have been obtained: for DXS9897 0.114, for DXS1073 b 1∙10 − 5, for HA472 0.029, for DXS1 108 b 1∙10 − 5, respectively. Table 3 compares the results for the χ 2-value, the p-value and the simulated p-value for the four different STRs. Three of the four STR allele frequencies found in the patients clearly differ from the expected distributions derived from the findings in the control individuals. This is reflected by simulated p-values below 0.05, which is regarded as statistically significant. Merely, the locus DXS9897 (the most remote STR on the 3′-side of the F8 gene) does not show a significant deviation from the expected distribution.
Analysis of STRs Haplotypes in Patients and Controls Regarding the analyzed intragenic (intronic) STRs (for details see Table 1), all seven patients carried the same genotype. Following the advice of our statistician, these markers were not included in the genealogic analyses. Regarding details of the four analysed extragenic STRs (concensus pattern, sequence and physical position) see Table 1. Fig. 1 displays the allele frequencies of these STRs among patients and controls. For further analysis, we calculated the expected numbers for the different alleles of the four STRs and compared these to the observed numbers in our patient cohort. Interestingly, two theoretically possible alleles of the locus DXS1108 were not observed and could
Based on the four extragenic STRs, haplotypes for patients as well as for control individuals were generated. In the patients, three different haplotypes characterized by the following repeat numbers could be identified; namely, 12-22-63-26, 12-22-64-26 and 12-21-64-26 (see Table 2). At the locus DXS1073, two different alleles were found in the patients – the 21-repeat allele in three patients and the 22-repeat allele in four patients. Remarkably, the haplotypes with 22 repeats at locus DXS1073 were not identical but belonged to two different clades. Also, for the locus HA472, two different alleles were found in the 7 patients: 63 repeats in three patients and 64 repeats in four patients.
Fig. 1. a. Allele frequencies of DXS9897. b. Allele frequencies of DXS1073. c. Allele frequencies of HA472. d. Allele frequencies of DXS1108. x-axis: number of repeats; y-axis: number of patients and control individuals, respectively.
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Table 2 Observed number of alleles in the patients and expected number of alleles inferred from allele frequencies from the healthy individuals. DXS9897
DXS1073
HA472
DXS1108
Allele / product length
Observed Expected Allele /product length
Observed Expected
Allele /product length
Observed Expected
Allele /product length
Observed Expected
10-repeat / 224 bp 11-repeat 228 bp 12-repeat 232 bp 13-repeat 236 bp 14-repeat 240 bp
0 0 7 0 0
0 0 3 4 0 0 0 0 0
57-repeat /241 bp 58-repeat /244 bp 59-repeat / 247 bp 60-repeat /250 bp 61-repeat /253 bp 62-repeat 256 bp 63-repeat /259 bp 64-repeat /262 bp 65-repeat /265 bp 66-repeat /268 bp 67-repeat /271 bp 68-repeat / 274 bp 69-repeat / 277 bp 70-repeat / 280 bp 71-repeat / 283 bp 72-repeat / 286 bp 73-repeat / 289 bp 74-repeat / 292 bp
0 0 0 0 0 0 3 4 0 0 0 0 0
18-repeat /112 bp 19-repeat /114 bp 20-repeat /116 bp 21-repeat /118 bp 22-repeat /120 bp 23-repeat / 122 bp 24-repeat /124 bp 25-repeat /126 bp 26-repeat /128 bp 27-repeat /130 bp
0 0 0 0 0 0 0 7 0 0
0.164 1.695 3.50 1.422 0.219
19-repeat /130 bp 20-repeat /132 bp 21-repeat / 134 bp 22-repeat /136 bp 23-repeat /138 bp 24-repeat /140 bp 25-repeat /142 bp 26-repeat /144 bp 27-repeat /146 bp
0.2734375 2.078125 3.4453125 0.0546875 0.21875 0.0546875 0.0546875 0.7109375 0.109375
In the 128 male control individuals, 109 different haplotypes were detected. Haplotype 12-21-64-26 is the most frequent in the whole sample and was observed in three patients and four control individuals. Surprisingly, the haplotypes 12-22-63-26 and 12-22-64-26, which were found in four of seven patients, were not detected in the control cohort. Reconstruction of the Ancestral Haplotype To illustrate the genealogical relationships and ancestry of the patients, we inferred a genealogy of the samples and established a genealogical tree. The patient samples comprised three haplotypes, which form two distinct clusters in the genealogy. A consensus tree indicated that the patients form one common haplotype group. By computation we determined the minimum number of mutations necessary to explain the haplotype diversity in the sample. Fig. 2 shows the resulting haplotype genealogy for the patient samples – the circle size is proportional to the number of haplotypes found. The haemophilia A causing mutation occurred on the most frequent haplotype D1 (12-21-64-26). The mutation 21 ➔ 22 at the DXS1073 locus led to haplotype D2 (12-22-64-26), which was found in one patient, and subsequently the mutation 64 ➔ 63 on locus HA472 generated the third patient haplotype D3 (12-22-63-26), which was found in three patients. Haplotype D1 is the one linked to the remaining control haplotypes. From the genealogy we conclude that the haemophilia A causing mutation originated on the genetic background of haplotype D1, and mutations of STRs led to the different haplotypes D2 and D3 present in haemophilia patients with the same mutation. Discussion The splice site mutation present in seven unrelated haemophilia A patients is a not yet described T ➔ G missense mutation at position −14 Table 3 Results for the χ2-value, the p-value and the simulated p-value for the four different STRs. STR
χ2-value
p-value
simulated p-value (100,000 runs)
DXS9897 DXS1073 HA472 DXS1108
7.0 288.2 37.41 61.92
0.136 0.000 0.002 6 10− 11
0.114 b 1 10− 5 0.029 b 1 10− 5
0.0546875 0.1640625 0.546875 0.875 0.546875 0.65625 0.3828125 0.765625 0.8203125 0.765625 0.328125 0.6015625 0.109375
0.21875 1.640625 0 0 0.109375 0.4375 1.5859375 2.2421875 0.7109375 0.0546875
in intron 6 (c.788-14T>G). To our knowledge, sequencing of the F8 gene usually does not cover this part of intron 6 and thus this mutation may have escaped detection in patients with mild haemophilia and minor clinical symptoms. It is known that mutations causing haemophilia A can occur in introns of the F8 gene. It may be necessary to analyze gene regions beyond the coding region and the exon/intron boundaries at least in selected patient groups. Analysis of cDNA to evaluate the effect of this splice site mutation confirmed that this mutation leads to the preferred generation of mRNA lacking exon 7. The predominant presence of RNA lacking exon 7 seems to be responsible for the reduced FVIII activity and the mild haemophilia A observed in the seven patients. Previously, the deletion of exon 7 from genomic DNA has been reported to cause a severe haemophilia A phenotype [15]. We speculate that the mild phenotype observed in our patients with a splice site mutation may be due to the presence of a small amount of regularly spliced FVIII mRNA in liver cells which was below the detection limit in mRNA obtained from leucocytes. Efforts to detect minute amounts of correctly spliced mRNA in blood cells were unsuccessful, and we did not have access to liver samples, as none of the patients needed a liver biopsy for medical reasons. To distinguish whether the mutation represented a mutation hot spot or a founder mutation we analyzed intragenic (intronic) and extragenic short tandem repeat polymorphisms (STRs). Statistical analysis of the allele frequency distribution for the analyzed STRs in the patients and 128 individuals without any bleeding diathesis showed that the STR-allele distribution of the patients shows less variation compared to the control sample. The haplotype distribution found in patients differed from the one expected on the basis of the haplotypes identified in the controls. DXS9897 and DXS1108 were monomorphic among the patients. In this regard, it should be pointed out that the genetic length between DXS9897 and DXS1108 is 3.22 cM [16], an interval, which covers 81 genes and 13 pseudogenes [5]. Genetic disequilibrium can be eroded by recombination events, which break down the ancestral haplotypes. This leads to a linkage equilibrium over time [17]. DXS9897 lies approximately 1596 kb 3′ of the F8 gene. Recombination events likely occur in DNA stretches between STRs; more frequently if these stretches are longer. Probably, the STR locus DXS9897 is more frequently affected by recombination events due to the large distance to the FVIII gene. Overall, four healthy control individuals belong to the same haplotype cluster as our patient cohort, which means that they have the same ancestral haplotype. We could analyze one healthy male with haplotype D1. He had a normal FVIII:C level (155%) and never
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Fig. 2. Genealogy of the patient cohort (relevant part of the consensus tree in concise illus-tration). Each node represents a different haplotype. Circle areas are proportional to haplotype frequency. “C” stands for control individuals and “D” for patients. Filled circles represent haplo types not found in the samples. The dotted lines and circles indicate the link to the remainingnetwork of control individuals (not shown in this figure). The haplotype groups C1 and D1 are patients and control individuals sharing the same haplotype shown in parenthe sis. The labels at the branches connecting the circles specify the mutations at the respective loci leading to the different haplotypes. The repeat number on the left side of the equivalence sign repre- sents the repeat number of the left haplotype or the haplotype above. This information suf- fices to reconstruct the other haplotypes.
experienced bleeding events. Sequencing proved that this person did not carry the intron 6 splice site mutation found in the patients. This indicates that the missense mutation occurred once in a healthy individual with haplotype D1 and stayed in the population. Further support for a single mutational event is the absence of the D2 and D3 haplotype in the control population and the low frequency of the D1 haplotype in the control population. However, we note that we identified a very large number of haplotypes – 109 in 125 individuals. This might indicate that the STRs are unstable and/or that that there is a high rate of recombination in the population. It also indicates that the control population used was probably not large enough to enable an accurate estimation of the frequency of individual haplotypes. Due to the unknown mutation rates of the STRs our data does not provide accurate information on the age of the mutation. However, we can conclude that the splice site mutation occurred in one founder. From the number of STR mutations on the patient-D1 haplotype we can assume that the origin of the mutation dates back at least 500 years (25 generations), if we assume a mutation rate as high as 0.075 per marker per generation [18]. Nowadays, life expectancy of patients with mild haemophilia equals the life expectancy of the normal population [19]. Also in earlier days,
when no effective treatment was available, patients with mild haemophilia usually did not die at a very young age but reached adulthood [20]. This explains why the missense mutation found in our patient cohort, although based on a founder effect, was passed on over several generations and many years. Conflict of Interest Statement None of the authors has a conflict of interest to declare. Acknowledgements The authors would like to thank Renate Freitag for her contribution concerning the mutation analysis of the haemophilia A patients. Further, we would like to acknowledge the financial support rendered through the Anniversary Fund of the Oesterreichische Nationalbank (OeNB, project number AP12208ONB). Arndt von Haeseler also thanks the Vienna Science and Technology Fund (WWTF) for financial support. In addition, Sylvia Reitter-Pfoertner thanks Bayer Healthcare for receipt of the Bayer Haemophilia Clinical Training Award, which enabled her to
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