Thiopurine methyltransferase genotyping in Palestinian childhood acute lymphoblastic leukemia patients

Patients and sample collection

This is a descriptive study with Convenience sampling and definite time period. Fifty six children suffering from and being managed for acute lymphoblastic leukemia in European Gaza hospital, AL-Nasser hospital and Abd El Aziz Al Rantisi hospital were included in the period between July 2008 to august 2009. The study population included all patients attending these hospitals at time of the study. Other types of leukemia were excluded. Approximately 2.5 ml venous blood samples were collected, from the patients in EDTA tubes.

The procedures of the study were approved by the Helsinki research ethics committee of the Palestinian authority ministry of health according to the World Medical Association Declaration of Helsinki [11], and a written consent was obtained from the parents of each patient.

TPMT genotyping

DNA was extracted from 300 μl whole blood samples using a commercial kit (promega, USA), according to the manufacture recommendations, and based on nonorganic extraction procedure. The extracted DNA was resuspended in 100 μl of the provided alkaline hydration solution at 65°C.

A total of 56 DNA samples were analyzed. Total genomic DNA extracted from peripheral leucocytes was processed by PCR either immediately or stored at -20°C until being used.

An allele-specific PCR [12] was used to analyze the mutation c.238G>C in exon 5 (TPMT *2 allele), using sequence specific primers (wild type specific: 5′-GTA TGA TTT TAT GCA GGT TTG-3′, mutant specific: 5′- GTA TGA TTT TAT GCA GGT TTC-3′ and common: 5′-TAA ATA GGA ACC ATC GGA CAC-3′) in two separate 20 μl reactions containing 0.5 μM of each primer, 5 μl DNA 1X PCR master mix (promega, USA). The cycling conditions consisted 35 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s.

The mutations c.719A>G in exon 10 and c.460G>A in exon 7 (TPMT*3A, TPMT*3B and TPMT*3C alleles) were determined by polymerase-chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) using AccI and MwoI respectively (New England Biolabs, USA) [12]. The PCR amplifications were carried out in 20 μl final volume containing 0.5 μM of the primers (5′-AAG TGT TGG GAT TAC AGG TG-3′ and 5′-TCC TCA AAA ACA TGT CAG TGT G-3′ for c.719A>G; and 5′-GGG ACG CTG CTC ATC TTC T-3′ and 5′-GCC TTA CAC CCA GGT CTC TG-3′ for c.460G>A), 5 μl DNA and 1X of PCR master mix (promega, USA). The cycling conditions consisted of 35 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min for c.719A>G; and 35 cycles of 94°C for 1min, 59°C for 1 min, 72°C for 1 min for c.460G>A.

Data collection and analysis

Relevant medical information was collected from the patient’s records in the hospitals. Data included adverse effects such as leucopoenia (white blood cells count <3000/ cubic millimeter), thrombocytopenia (platelets < 100000/ cubic millimeter), abnormal liver function (elevation of ALT level of 2 or more times the upper limit of normal); and duration of 6-MP therapy at time of sampling and at the time of adverse effects appearance. Data were analyzed using the SPSS software (Version. 17) as needed. Chi square test and One-Way ANOVA were used and P-values of < 0.05 were considered statistically significant. Means were presented ± standard deviation.

Description of the study population

In this study, the sample included 56 pediatric patients suffering from ALL; 32 (57.1%) were males and 24 (42.9%) were females. Their ages at the time of diagnosis varied between 6 months to 12 years (mean 4.4 ± 2.6 years). The majority of patients were between 3.7 - 5.1 years with 95% Confidence interval. All patients were diagnosed as ALL patients by blood film, bone marrow aspiration and complete blood cell count (CBC).

In the present study the calculated incidence of ALL among children in Gaza strip during the specified period of samples collection was 2 patients per 100000 children. The incidence of ALL is higher in males than in females.

Management of patients

During the collection of the sample, most of the patients (75%) were given 6-MP for different periods of time and 25% of them finished having the 6-MP treatment.

Among the 42 patients who were receiving 6-MP at the time of the sample collection, 6 patients suffered from leucopoenia (white blood cells count <3000/cubic millimeter); 5 patients from leucopoenia and thrombocytopenia; 3 patients from leucopoenia , thrombocytopenia and liver toxicity and 3 patients had only liver toxicity. When such complications took place, the only intervention was 6-MP dose reduction. During the course of management, one patient had neuroblastoma and one patient had mild brain atrophy, which likely didn’t result from the 6-MP treatment. Bone marrow transplantation was performed for 2 patients besides the 6-MP treatment.

TPMT genotypes

Adverse effects

Collectively, 14 patients developed side effects associated with 6-MP therapy, characterized by rapidly developing severe myelosuppression expressed as leucopoenia alone or in combination with thrombocytopenia. Thirteen of those patients had wild type TPMT alleles and only one patient had a heterozygous TPMT*3A genotype.

The duration of 6-MP treatment doesn’t seem to significantly correlate with the occurrence of adverse effects by the time of sample collection (p-value = 0.87).

For the 14 patients with side effects, myelosuppression occurred at different time periods, ranging from 1–20 months after 6-MP administration (mean= 4.6 ± 5.2 months). On the other hand 6-MP treatment continued for 1–24 months (mean = 8.6 ±6.2) before samples were collected from the other patients and yet no evidence of myelosuppression was detected.