INVESTIGATION OF IVS 14 + 1 G > A POLYMORPHISM OF DPYD GENE IN A GROUP OF BOSNIAN PATIENTS TREATED WITH 5-FLUOROURACIL AND CAPECITABINE

Adverse drug reactions still pose an important clinical problem. Dihydropyrimidine dehydrogenase (DPD) is an enzyme that regulates -FU quantities available for anabolic processes and hence aff ects its pharmacokinetics, toxicity and effi cacy. X ere are several studies describing a hereditary (pharmacogenetic) disorder in which individuals with absent or signifi cantly reduced DPD activity may even develop a life-threatening toxicity following exposure to -FU. X e most common mutation is known as the DPYD*A or as the splice-site mutation (IVS + G A) leading to creation of a dysfunctional protein. An objective behind the study was to ascertain existence of the IVS+ G A mutation among the population of Bosnia and Herzegovina. Our research has undeniably attested to existence of one heterozygote for the DPYD gene mutation, i.e. one heterozygote for IVS +  G > A, DPYD*A mutation.


Introduction
Adverse drug reactions still pose an important clinical problem.erein, it was estimated that in the last decade alone they caused over , deaths annually in the United States.As such, these reactions were the fourth leading cause of deaths in the U.S., immediately after hearth diseases, malignancies and strokes ().
ere is a direct link between therapeutical eff ects and toxicity of -FU and the drug's anabolic process related to nucleotides.In turn, this may inhibit activities of thymidylate synthetase or incorporate -FU into RNA and/or DNA.More than  of the -FU dosage administered to human subjects was degraded through catabolic pathways, meaning that only app. of the dosage was left to the anabolic process.In the past ten years it became clear that the dihydropyrimidine dehydrogenase (DPD) is an enzyme that regulates -FU quantities available for anabolic processes and hence affects its pharmacokinetics, toxicity and efficacy.The -FU anabolism leads to creation of  fluoro'deoxyuridine-'-monophosphate (FdUMP), which is a cytotoxic product of a multilevel activation pathway of the -FU.FdUMP acts as an inhibitor of the thymidylate synthetase (TS), thus causing an intracellular accumulation of the deoxyuridine monophosphate (dUMP) and a reduction in a level of deoxythymidine monophosphate (dTMP).Finally, this induces an arrest of a DNA synthesis.e initial and rate-limiting enzyme in the catabolism of the -FU is dihydropyrimidine dehydrogenase (DPD) that catalyses a -FU reduction into ,-dihydrofl uorouracil (DHFU).en on, DHFU is degraded down to fluoro-β-ureido propionic acid (FUPA) and flouro-β-alanine (FBAL) ().
ere are several studies describing a hereditary (pharmacogenetic) disorder in which individuals with absent or significantly reduced DPD activity may even develop a life-threatening toxicity following exposure to -FU.Here we need to consider the non-functional catabolic pathway due to a DPD inability to metabolise.Many studies, of which the most important are the ones by Van Kuilenburg and Johnson, have indicated that a high rate of the -FU toxicity is attributable to the reduced DPD activity ().Van Kuilenburg has found that  of oncological patients with -FU toxicity have also shown signs of reduced DPD activity, while Johnson () saw this frequency to be , thereof app. having profound and app. partial DPD defi ciency.These patients are likely to develop an unanticipated toxic reaction subsequent to being treated with the -FU.When given in standard doses and coupled with altered pharmacokinetics, -FU may lead to severe toxic reactions like mucositis, granulocytopenia, neuropathy, diarrhea and even death in the worst-case scenario.A cause to this toxicity seems to be in a decreased drug clearance hence resulting in an extended -FU exposure.DPD-defi cient patients exhibit a normal phenotype until such time they have been administered with the -FU.It has been estimated that nearly - of the general population have the DPD activity below  (<, nmol/min/mg for frozen samples) of the lower limit for the normal population ().It has been reported that  of patients with reduced DPD activity suffers from the grade  neutropenia, as opposed to  of patients with normal DPD activity (P = ,) ().Moreover, the toxicity occurs much earlier with regards to patients with low DPD activity (, ± , versus , ± , days, P < ,).e patients suff ering from the severe -FU toxicity have exhibited more than  mutations in the DPYD gene to include one G to A mutation in the '-splice recognition site of intron  that resulted in a -bp deletion that correspond to exon . is mutation is known as the DPYD*A as per defi ned nomenclature or as the splice-site mutation (IVS + G A) leading to creation of a dysfunctional protein ().e mutation was not only the fi rst one to be analysed, but also the most frequently observed of all mutations in the population studies (app.) ().

Materials and Methods
The study was approved by the local ethics committee, and written informed cons e n t w a s o b t a i n e d f r o m e a c h p a t i e n t .
is experimental research was designed as a prospective, open and translation study.The study was performed among patients of the Oncology Clinic of the University of Sarajevo Clinics Centre treated in the period from -.An objective behind the study was to ascertain existence of the IVS + G o A mutation among the population of Bosnia and Herzegovina.A test group observed in the subject-matter research consisted of  subjects -patients at the Oncology of the University of Sarajevo Clinics Centre that have undertaken chemotherapy and been treated with -FU or Capecetabine in the period from  to .erein, we have done a genomic analysis of DPYD gene for these patients.These patients were also divided into two subgroups regarding presence of toxicity grade  or  or without toxicity after administration of FU.All genotype analysis were conducted on samples of the patients' peripheral blood.e blood samples were taken by means of a venipuncture of a cubital vein that appears full and stored in vacutainers (BD biosciences) of ml with Na-EDTA anticoagulant. is was preceded by a written and informed consent by patients to participate in this study.e blood samples were refrigerated for a short period of time on °C (not beyond  hours) prior to their extraction.is was necessary as to preserve the quality and quantity of the isolated DNA.The DNA samples were stored on a temperature of -°C until we moved onto t h e n e x t p r o c e s s s t e p i n o u r a n a l y s i s .Determining a target DPYD gene structure was done by the Institute for Genetic Engineering and Biotechnology in Sarajevo (INGEB) by means of the following methods and procedures: A genotyping methods applied in the study relied on the PCR technology.We have amplified a genotyping-relevant sequence by applying an adequate primer pair subsequent to which we subjected it to an appropriate gene expression profiling (an automatic or a manual fragment analysis).Genomic DNA was extracted from leucocytes with the standard techniques and actual genotyping was done by means of the PCR-RFLP.The DPYD exon  and its flanking ' donor intronic region was amplified with the PCR.
Primers used in the amplifi cation of the exon  and its marginal regions (including newly formed Ndel restriction sites) are: F: ' ATC ˝AGG ACA TTG TGA CAT ATG TTT C ' R: ' CTT GTT TTA GAT GTT AAA TCA CAC ATA ' In our study, we have applied a method of highly specific typisation of the DNA sequence previously replicated during the PCR process.The PCR method followed the above outlined primer sequences as to ensure genotyping of DPYDA* polymorphisms in the DPYD gene.Relevant PCR products were tested by an agarose gel electrophoresis and then treated with the restriction enzyme Ndel (endonuclease) in an appropriate buffer solution on °C.During the RFLP processing, we have incubated ml of the PCR products overnight on °C with U endonuclease Ndel.Restriction fragments were separated by the  agarose gel electrophoresis stained with ethidium bromide.The genotyping, i.e. assessment of the size of restriction fragments, was done on a visual basis by identifying the wild-type allele (IVS+G) by means of splicing  bp with the PCR product to  bp and  bp fragments.Conversely, a mutant allele produced three fragments subsequent to Ndel digestion:  bp, , bp and  bp.Relevant results were recorded by taking photographs of gels under an UV light.

Results
e human DPD gene (DPYD) is present as a single copy gene on the chromosome p and consists of  exons.Table .provides an overview of all patients who participated in our study, their respective characteristics and responses to treatments vs. the subject-matter mutation.

Toxicity and lethal outcome
A toxic events analysis was done in accordance with the Common Toxicity Criteria (CTC).
We have factored in only those toxic events that could be interpreted by means of either laboratory tests or clinical examinations.There were also other cases like a neurological toxicity, but we have not taken it into consideration as we were unable to draw a clear line between this toxicity and the -FU.
We have described three types of toxicities exhibited by our patients: diarrhea, neutropenia with a resulting leukopenia and mucositis (to include stomatitis as well) (Chart .) Majority of cases, i.e.  subjects, had neutropenia, which is nearly  of all symptoms, while remaining subjects had diarrhea and mucositis.Not one of these events had a statistical signifi cance over the other two.
While the grade  mostly involves diarrhea, the grade  neutropenia, so it is evident that the only  lethal outcomes occurred with patients with the grade  neutropenia.This is to say that this occurrence bears a statistical significance of p=, according to the Fisher's exact test.
During our research, we had two lethal outcome cases that belonged to the group of subjects with toxicity subsequent to the -FU treatment.Therein, the significance level p was ,.Both of these cases occurred after the grade  neutropenia, so this event bears a statistical significance of p= , according to the Fisher's exact test.

IVS +  G > A, DPYD*A MUTATION
Our research has undeniably attested to existence of one heterozygote for the DPYD gene mutation, i.e. one heterozygote for IVS +  G > A, DPYD*A mutation.
We have determined existence of the heterozygote for the subject-matter mutation with the patient No.  of our test group.This patient was  years of age when she was diagnosed with a breast cancer with a liver metastasis as per relevant x-ray and CT scan imaging.After convening a consultation team, it was opted against conducting any surgery due to the stage of the illness.Instead, it was decided to start with a chemotherapy protocol with Capecitabine.
Seven days after beginning the Capecitabine treatment, i.e. during the fi rst cycle of treatment, the patient was admitted as an emergency case by our Emergency Department after being administered with a Capecitabine dosage of , mg / m twice a day.Attending physician's exam and relevant laboratory tests have indicated to leukopenia and neutropenia -grade  (L , /l), as well as the grade  mucositis (oral cavity was covered with white coating and crusts) and the grade ¾ diarrhea (over  liquid stools within  hours).
T h e p a t i e n t w a s p r o s t r a t e d , h y p o t e n s i v e and exhibited signs of hepatic insufficiency.She was then treated with GSF (Neupogen), antibiotics (Amoxicillin, clavulanic acid and Ciprofloxacin), intravenous solutions and other symptomatic therapy.After  days, despite all measures of supportive therapy in accordance with the NCNC Guide, the patient fell in a comatose state.In a matter of hours, exitus letalis occurred.
Find below is an UV photograph ( Figure .) taken after staining the  agarose gel with ethidium bromide.e image displays wild-type DPYD in lane  and heterozygote mutation in lane . e latter is IVS +  G > A, DPYD*A that appeared after restriction, while the wild-type allele was identified by splicing  bp with a PCR product on  bp and  bp fragments.Conversely, the mutant allele produced three fragments after Ndel digestion:  bp, , bp and  bp.

Discussion
Although it was synthesised over  years ago, the -FU remains one of the most prescribed cytostatic agents in treatment of various malignant diseases.Adverse drug reactions still remain one of the most signifi cant clinical problems.This, coupled with the meta-analysis results involving , patients treated with the -FU, indicate that grades  and  toxicities occur with - of patients and have a lethality rate of , ().Although we are well aware now that many human illnesses are caused by gene mutations, there is a lesser awareness of a fact that known gene variances can affect a patient's response to certain drugs (,).
The importance of the DPD deficiency and severe -FU-related toxicity is of even greater significance considering the wide range of the -FU administration.e meta-analysis covering a group of over , patients suggests that more than  of patients treated with the -FU experienced a major toxic event prompted by this medication.A frequency of low DPD enzyme activity in the general population was initialy evaluated to be somewhere between -.
However, further studies have proven a significant variability between different ethnic groups (Table ).
e human DPD gene (DPYD) is present as a single copy gene on the chromosome p and consists of  exons.The Table .provides an insight into results of certain studies (ours included) that dealt with determining relevant prevalence rate with patients of different ethnic groups.As we can see, none of the neighboring countries, has had anything published regarding this type of research on patients administered with the -fluorouracil, as far as we know.Hence, this makes our study that more important as it practically substantiates the only example of the DPYD A mutation in the region.In Bosnia and Herzegovina, more precisely in the Federation of Bosnia and Herzegovina, there were , patients treated with the -FU during the Y, while in the same period there were  patients administered with Capecetabine (source: FB&H Solidarity Insurance Fund).To certain extent, these figures apply to our study as well, as we do not have specific information on the -FU-related toxicity.
is is to say there is no exact method of monitoring these patients and recording of toxic events, so we can only speculate on the issue at the country level.Given these numbers, i.e. considering that in the Y alone there were altogether , patients treated with the -FU and Capecetabine and considering that our study indicated to  mutation frequency, this means that there are over , patients every year that is prone to a severe toxic response and possibly lethal outcome subsequent to the -FU administration.e root cause here is the DPD gene mutation.Another fact to consider is that exon -skipping accounts for nearly  of the DPD defi ciency.erefore, the aforesaid number of patients would have been much greater even based solely on changes in one gene in the -FU metabolism.
Another interesting information here is that all heterozygous patients with proven mutations in all mentioned studies, have had the grade  neutropenia.If we take the fi ndings of our study and compare it against the grade  neutropenia alone (wherein relevant toxicity rate was above  for patients with the DPYD A* mutation and ended in a lethal outcome) and considering that it indicates that  of our test group with the grade  neutropenia symptoms had the subject-matter mutation, then our results are very similar with such studies.Of course, we take due note of the earlier mentioned limitations imposed on this research.In conclusion, during our study, the lethal outcome occurred with two patients suffering from the grade  neutropenia.It is pertinent to note here that the said mutation (IVS+G>A), in any case, does not represent a sole cause of death in patients developing the life-threatening toxicity after the -FU administration.So far, the analysed gene displayed over  mutations and other metabolic pathways that may have caused the said lethality.
The deficient DPD activity is a pharmacogenetic syndrome with a possible fatal outcome following the -FU therapy.Although molecular defects of DPYD leading to the defi cient DPD activity can be root causes of the -FU syndrome, actual DPD regulatory mechanisms have not been clearly outlined.This calls for highly specific techniques for screening of the entire DPYD gene and for measuring DPD activity.Resultantly, this would enable us to draw clear conclusions of the relationship between the genotype and phenotype of the pharmacogenetic syndrome in question.
Certainly, this is only a speculative statement on our part, as more elaborate and definite conclusions can be reached only after conducting a greater scale of research.Still, it provides us with indicative information.
Even major countries have not yet solved a matter of routine screening of patients, but there is an increasing tendency of such tests.is is rooted in a pharmaco-economic aspect as treatment costs for patients experiencing severe toxicity have already proven to be much greater than screening costs.Needless to say, new screening methods are reducing inherent costs even further down.

Conclusion
Our study has indicated to the presence of the DPD mutation on the exon  (IVS+G>A).is is the fi rst time such a case was reported with respect to the Bosnian population.(DPYD*A frequency is nearly ).Furthermore, we attest to IVS+G>A DPYD mutation being responsible for a signifi cant proportion of the life-threatening toxicity in the -FU administration.is is based on a sample of four patients suff ering from the CTC-defi ned grade  myelosupression after being treated with the -FU.

Introduction
Obesity epidemic is one of the most important health problems of modern age.Over the past two decades, obesity prevalence in European countries has tripled.About  of adults have excessive weight while / of European population is obese.Even, - of European children and adolescents are overweight (, ).Statistical data by USA Centre for Disease Control and Prevention (CDC) also testify that the overweight population has tripled over the past two decades.Also,  of children and adolescents between  and  years of age are overweight (, , and ).
Approximately - overweight children grow up into obese adults, who lead to earlier and more frequent development of chronic non-infectious diseases: hypertension, early atherosclerosis, Diabetes mellitus Type , orthopaedic, endocrine and psycho-social deviations (, ).
Although hereditary and hormonal factors may be significant in overweight development in children, excessive food consumption and low physical activity are undoubtedly the major causes (, ).
Sedentary time spent with TV set and computer coinciding with the consumption of calories-rich food and sweet beverages in the long term leads to an imbalance between energy intake and expenditure.e result of such imbalance is excessive body weight.Body mass index BMI > p  is considered obese (, , ).
An alarming trend of obesity epidemic expansion, and increase in prevalence in young population in particular, represent a problem of major economical and social consequences for every community (, ).
European charter on counteracting obesity adopted in  proposes global measures for obesity prevention in all European countries.Precise epidemiological data on the number of obese children and youth and information of their eating habits and activities are prerogative for designing effi cient action plan for the prevention of obesity development in each country (, ).

Aims of the study
e aims of the study were: -estimate the prevalence of excessive body weight in children and youth in Sarajevo Canton, -delineate major causes of this condition and to propose the strategy for its effi cient prevention.

Materials and Methods
The study was conducted during  and .It included representative sample of Sarajevo Canton elementary and secondary schools, which were randomly selected.The number of subjects per school class (elementary - and secondary -) was balanced.
The students were requested to fill in a questionnaire.The questionnaire was originally designed for this study and includes questions that pertain to eating habits (frequency, quantity and types of consumed food), consumption of liquids, and physical activity (frequency and intensity).e questionnaire addressed subjects' social status regarding the total number of family members that are sustained from the same source per employed individual.The questionnaires for elementary and secondary schools contained the same questions presented in the form suitable for the participants' age.Anthropometric measurements: body height, body weight, measured in all subjects.e height was measured using vertical stat meter, the values were expressed in centimetres (cm) and rounded at , cm.Body weight was measured using digital balance, the values were expressed in kilograms (kg) and rounded at , kg.The study was conducted by  teams which included  physicians and a certified nurse.The subjects participated in the study on voluntary basis.The data entered into database were anonymous.e statistical data processing was performed according to the age groups: I-IV and V-VIII grades of elementary school and I-IV grade of secondary school.BMI was used to estimate the nutritional status.It was calculated automatically according to the formula: BMI = body weight (kg) / height (m)    Nutritional status was derived automatically based on CDC criteria (): -BMI <  percentile indicates malnourishment.

Results
The total of  students ( students from  elementary schools and  from  secondary schools from Sarajevo Canton) filled in the questionnaire and were examined anthropometrically.

TABLE 1
. Overview of patients characteristics

TABLE 2 .
Prevalence of IVS14+1G> A DPYD mutation in persons from diff erent countries (12)  BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2010; 10 (2): 139-139 TIMUR CERIĆ ET AL.: INVESTIGATION OF IVS14+1G>A POLYMORPHISM OF DPYD GENE IN A GROUP OF BOSNIAN PATIENTS TREATED WITH 5FLUOROURACIL AND CAPECITABINE

Table 
presents an overview of the total number of subjects and breakdown according to school age and gender.

Table  .
Table  presents distribution of beverages consumption during the day among the student groups.

Table  .
Tablepresents the distribution of the degree of physical activity among students that participated in the study.Tablepresents the distribution of the time that students spend with computer or TV set (higher grades of elementary school and secondary school students).isis the fi rst study in the Federation of Bosnia and Herzegovina that presents data on the frequencies of obesity among students of elementary and second-

TABLE 1 .
Analysis of the examined student group According to school level, grade and gender TABLE 3. Quality of nourishment during school hours TABLE 4. Types of beverages consumed during school hours

TABLE 6 .
Distribution of the degree of physical activity among the students

TABLE 7 .
Distribution of the time spent with computer or TV set TABLE 5. Distribution of sweets consumption frequencies TABLE 2. BMI classifi cation of students according to the school grade and gender