Haplotype study of microsatellites flanking the t(15;17) breakpoint in acute promyelocytic leukemia patients from North Portugal

Leukemia volume 16, pages1353–1357(2002)Cite this article

Abstract

A higher frequency of acute promyelocytic leukemia (APL) has been noted in countries of Southern Europe and among ‘Latino’ patients of the United States with acute myeloid leukemia (AML). In order to discover whether there is any genetic predisposition to the disease, we analyzed microsatellites flanking PML and RARα genes in 29 t(15;17) APL patients from North Portugal and compared them with a control group of 123 healthy individuals. Fluorescent PCR products were analyzed using an automated capillary electrophoresis system and allele and haplotype frequencies of the two populations were determined. No significant differences were found, suggesting the same genetic origin of patients and healthy individuals. As suggested by the four microsatellites screened, MSI (microsatellite instability) does not explain the increased incidence of t(15;17) APL in this Portuguese population. These results intend to be a new approach to the study of APL, reflecting the particularity of the disease.

Introduction

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized in more than 95% of cases by the fusion of PML (15q22) and RARα (17q12) genes.1,2 The different types of PML-RARα mRNA fusion transcripts formed by the balanced reciprocal translocation t(15;17) and the other chromosomal variants have been intensively characterized,3,4 but the causes favoring interchromosomal recombination, if any, are largely unknown.

Geographical distribution of APL seems to be somewhat peculiar since the discovery of the increased frequency of the disease among ‘Latino’ patients with AML in the US. In Northern Europe and the United States, the incidence of APL among AML patients in several large studies has been reported to be between 5% and 13%.5,6,7,8 Although no clear definition of ‘Latino’ was presented by Douer et al9 in that US population, this frequency can be as high as 37.5%. Considering that the study was done in Southern California, it is our understanding that the authors are probably refering to Central American patients who will be mostly of Indian rather than South European genetic background. In Southern Europe, however, APL incidence also seems to be increased. In our hospital, APL cases were 18% of total AML patients, which is a much higher frequency than those reported for German and French groups: 5% and 10%, respectively.5,10 There is no knowledge, so far, of environmental or genetic factors explaining this uneven distribution. Nevertheless, one can hypothesize that certain populations may be more prone to chromosomal breakage in the region involved in the t(15;17) translocation. Recently, a study on intergene distances of chromosomes in the cellular cycle has shown a close association of PML with RARα at the transition between S and G2 phases of the cell cycle, which persists during G2 and prophase. This evidence suggests that chromosomal recombinations may not occur entirely at random.11

In order to investigate the possibility of a genetic predisposition towards APL in the Portuguese population, we studied highly polymorphic microsatellite loci flanking PML and RARα genes in families of t(15;17) APL patients, as well as in a control population. This study also allowed the detection of intergenerational genomic instability.

Materials and methods

Samples

Peripheral blood samples were collected from 123 unrelated healthy individuals and 69 mother– and/or father–child pairs from North Portugal. Additionally, samples from 29 APL t(15;17) patients and their families were also collected. Blood was obtained in FTA cards and DNA was extracted by the chelating resin method.12

Microsatellites

All the microsatellites selected are CA repeats. The selection was based on heterozygosity and proximity to PML and RARα genes. Because of the ambiguity in the reverse primer of the D15S216 marker, described in the literature, we have used a different sequence (Table 1).

Table 1 Microsatellites used in this study
Full size table

DNA amplification

The four microsatellites were amplified in two duplex PCR reactions (D15S216–D17S1818 and D15S114–D17S1861). One primer per locus was 5′-end labelled with a fluorescent tag that emits light of a specific wavelength. We used 6-FAM (6-carboxyfluorescein) to tag D15S216 and D17S1861, and HEX (4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) to tag D15S114 and D17S1818. The two amplification reactions were carried out in a total volume of 25 μl containing 6 μl template DNA, 0.25 μM of each primer, 10 mM Tris HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP and 1 U Taq polymerase (PE Applied Biosystems, Foster City, CA, USA). Reactions were performed on DNA thermal cycler 2400 (PE Applied Biosystems) under the following conditions: initial denaturation step at 94°C for 2 min; 35 cycles consisting of 94°C for 1 min, 53°C (on duplex D15S216–D17S1818) or 55°C (on duplex D15S114–D17S1861) for 1 min and 72°C for 1 min; followed by a final extension of 7 min at 72°C.

Product analysis

DNA fragment length analysis was performed on the ABI-Prism 310 Genetic Analyzer laser-induced fluorescence capillary electrophoresis system (PE Applied Biosystems), using GENESCAN-500 TAMRA (N,N,N′,N′-tetramethyl-6-carboxyrhodamine) as standard. The analysis of the DNA fragments was carried out by the GeneScan Analysis 3.1 software (PE Applied Biosystems).

Recombination rate

The recombination rate represents the relative frequency of crossing-over between the two markers of the same chromosome. It was conservatively estimated by simply dividing the number of gametes that could not be found in parent's haplotypes by the total number of gametes. Given the short distances under consideration, 1 cM was considered equivalent to 1% recombination.

Statistical analysis

Allele and haplotype frequencies were estimated by gene/haplotype-counting method. The Markov test in Arlequin 1.1 software13 and the GENEPOP14 were used to test the differentiation between two populations in haplotypic and allelic data, respectively.

Results

Genotyping

The comparison of D15S216 allele frequencies between APL patients and the control population did not show significant differences (P = 0.91706 ± 0.00802) (Table 2A). In the D15S114 marker, the differentiation rate was 0.8781599 ± 0.00701 (Table 2B). Also the analysis of allelic frequencies in chromosome 17 did not detect significant differences, with P = 0.6855 ± 0.01750 in D17S1818 (Table 2C) and P = 0.26036 ± 0.01768 in D17S1861 (Table 2D).

Table 2 Allele frequencies of (A) D15S216, (B) D15S114, (C) D17S1818 and (D) D17S1861 markers in APL patients and control population from North Portugal (n = No. chromosomes; bp = base pairs)
Full size table

Haplotypes are the combination of two alleles from different loci (D15S216 and D15S114 for chromosome 15; and D17S1818 and D17S1861 for chromosome 17) of the same chromosome. In this study, the chosen loci were upstream and downstream of the chromosome 15 and 17 breakpoints. In some mother– and/or father–child pairs and in one APL family it was not possible to establish parental haplotypes due to double heterozygosity and/or small number of offspring. These non-informative cases were not considered for further analysis.

Comparative haplotypic frequencies for 15 and 17 chromosome markers are displayed in Figures 1a and b, respectively. The analysis of chromosome 15 haplotypic data showed a tri-modal distribution in both populations. Modal values were obtained in short and medium weight alleles. In chromosome 17, a multimodal distribution was observed with modal values spread along the 90 haplotypes found. More relevant differences between APL patients and the control population were observed in 123/103, 127/95 and 127/105 haplotypes. These haplotypes are represented in APL patients in a frequency that is three-fold, six-fold and six-fold higher than in the control population, respectively.

Figure 1
figure1

Haplotype frequencies in APL patients (black columns) and control population (grey columns) from North Portugal. (a) D15S216/D15S114 haplotypes (patients, n = 56; control population, n = 200). (b) D17S1818/D17S1861 haplotypes (patients, n = 56; control population, n = 216). Although differentiation does not reach a significance level, note that haplotypes 123/103, 127/95 and 127/105 (signalled by arrows) are much commoner in the APL than in the control population (three-fold, six-fold and six-fold, respectively). Allele designation represents the fragment size (bp).

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However, the Markov test results did not show significant differences between APL patients and the control population in either D15S216/D15S114 (P = 0.20615 ± 0.0595; 20 000 Markov steps) and D17S1818/D17S1861 (P = 0.26825 ± 0.0423; 20 000 Markov steps) haplotypes. Even using a conservative estimation method (as described in the Materials and methods section), the recombination rates found in our family material were higher than previously reported (Table 1) for both chromosome 15 (2.97% vs 2.3%) and chromosome 17 (5.98% vs 2.8%) markers.

Microsatellite instability

We also noted that one patient showed an allele (103 bp long) at D17S1861 locus, absent from both parents. The same situation was found in one of his sisters at the same locus (Figure 2). Additionally, the patient is homozygous in respect to D17S1818 (123–123) but this allele is not present in his father. Microsatellite instability (MSI) was absent from chromosome 15 markers. The biological kinship of the patient and of his younger sister were tested with standard markers (GenePrint Fluorescent STR Systems, Promega; AmpFeSTR Profiler Plus, PE Applied Biosystems) and corresponding paternity probabilities were above 99.9999%.

Figure 2
figure2

Chromosome 17 markers in a family with microsatellite instability. (a) Pedigree representing affected individual (II:3) and his sister (II:5) with alleles absent from their father. (b) Electropherogram of the amplification products of D17S1861 (left) and D17S1818 (right) loci (RFU = relative fluorescence units). Shaded peaks represent the lengths of alleles amplified. The others result from replication slippage because this procedure is plagued by slipped strand mispairing which generates stutter bands peaks.

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Discussion

Studies dealing with microsatellites have been used in population genetics, as they may provide an important source of information about a wide range of evolutionary situations, due to their high polymorphic rate. In the present study we analyzed microsatellites flanking PML and RARα genes in order to define non-allelic combinations (haplotypes) and to compare APL patients from North Portugal with a control group from the same region. This study was prompted by the recognition of the geographical variation of APL incidence, which may arise from genetic as well as from other epidemiological factors. If one was able to find a different genetic background in APL patients, one would be able to postulate that some haplotypes might be associated with the likelihood of the appearance of the t(15;17) translocation in that cohort.

The analysis of D15S216 and D15S114 markers in chromosome 15 and D17S1818 and D17S1861 in chromosome 17, have not shown significant differences in allelic or haplotypic frequencies between the patient and control populations. However, given the high degree of polymorphism of the markers, to attain significant departures would require unattainable sample sizes. In fact, some haplotypes, particularly for chromosome 17, show quite different frequencies in control and patient samples. These markers were chosen due to their heterozygosity and proximity to the PML and RARα breakpoints. The results suggest that affected and healthy populations are not from a different genetic background. However, we cannot rule out, at this point, that the population of North Portugal might have a distinct genetic profile regarding the regions flanking the t(15;17) translocation. Further studies comparing this population with populations from other genetic background need to be done.

We found MSI in an APL patient and in his younger sister. In the chromosome 17 markers, the patient has one allele per locus absent from both parents. His sister has only MSI in one allele of the D17S1861 marker. If it is the case that MSI in the reported family is APL related, then the presence of MSI in his younger sister might in someway predispose to the disease. MSI and/or generation of new alleles is known already to be disease related,15 but there is no previous evidence in APL of this type of genetic instability. Indeed, a retrospective study of AML patients has suggested that DNA mismatch repair deficiency is not a common factor in leukemogenesis.16 The occurrence of MSI simultaneously in the two loci analyzed may suggest that it can be associated with regional chromosome instability. It is also curious to note that chromosome 17 breaks, where this MSI occurs, are present in other translocations associated with APL, like t(11;17)(q23;q21),17 t(5;17)(q32;q12)18 and t(11;17)(q13;q21).19

In summary, our results do not show different haplotypic frequencies of the chromosomes 15 and 17 flanking polymorphisms between APL patients and a control population. However, not showing widespread chromosome instability, we cannot rule out entirely the hypothesis that microsatellite instability may play a role in the pathogenesis of APL, as one case of chromosome 17 MSI was found in a patient. Because APL is a unique and well-defined disease entity, it would be interesting to deepen this study to determine if chromosome 17 breakage has a higher probability of occurring in certain populations. The comparison of the genetic profile of populations with increased and low frequencies of APL might help the search for predisposition to chromosome 15 and 17 breakage.

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Acknowledgements

This research was supported by Ministry of Health, Project 226/1999, Portugal, and by the program POCTI (Programa Operacional Ciência, Tecnologia e Inovação).

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  1. IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
    • S Martins
    • , L Azevedo
    • , JE Guimaraes
    •  & A Amorim
  2. Serviço de Hematologia Clínica, Hospital de São João, Porto, Portugal
    • F Trigo
    • , MJ Silva
    •  & JE Guimaraes
  3. Faculdade de Medicina, Universidade do Porto, Porto, Portugal
    • JE Guimaraes
  4. Faculdade de Ciências, Universidade do Porto, Porto, Portugal
    • A Amorim
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Martins, S., Trigo, F., Azevedo, L. et al. Haplotype study of microsatellites flanking the t(15;17) breakpoint in acute promyelocytic leukemia patients from North Portugal. Leukemia 16, 1353–1357 (2002). https://doi.org/10.1038/sj.leu.2402525

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Keywords

  • acute promyelocytic leukemia
  • haplotype
  • linkage
  • microsatellite instability

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