Point mutation of p53 gene exon 7 as a new early diagnostic marker of hepatocellular carcinoma

Background: One of the characteristics of hepatocellular carcinoma [HCC] is the selective mutation resulting in serine substitution at codon 249 of the p53 tumor suppressor gene, and it has been identified as a "hotspot" mutation in hepatocellular carcinomas occurring in populations exposed to aflatoxins and with high prevalence of hepatitis B virus [HBV] and hepatitis C virus [HCV]. Objective: To evaluate whether this "hotspot" mutation could be detected in cell free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis, and tried to determine the significance of the detection of this molecular biomarker

Subjects and Methods: Blood samples were collected from 105 subjects. Fourty two patients with hepatocellular carcinoma, 35 cirrhotic patients and 28 healthy control. DNA was extracted from the patient's plasma. The 249ser p53 mutation was detected by restriction digestion analysis after PCR amplification

Results: A total of 34 of the 105 subjects [32.4%] had p53 mutation, 22 from 42 subjects [52.4%] with hepatocellular carcinoma, 10 from 35 subjects [28.6%] with cirrhosis, and 2 from 28 subjects [7.1%] were healthy controls. The adjusted odds ratio [OR] for having mutation was 7.33 [95% confidence intervals [CI] 1.87-28.75] for HCC cases compared to controls which was extremly significant statistically [P <0.0001]. Eight out of the 34 positive mutation were HbsAg+ [23.5%] "5 from HCC and 3 from cirrhotic patients" while 17 out of 34 positive mutation were positive Anti-HCV [50%] "13 from HCC and 4 from cirrhotic patients"

Conclusion: These data show that the 249ser p53 mutation in plasma is strongly associated with HCC. We found this mutation was also detected in plasma DNA of cirrhotics and healthy controls but at a much lower frequency. We consider that these findings, together with the conventional methods of HCC diagnosis, will give more information in ealry diagnosis of HCC, and 249ser p53 mutation may be a new early diagnostic marker for HCC

Variations and genotypes of interleukin-10 in diabetes mellitus complicating chronic hepatitis C virus infection

To study whether genetic polymorphism influence lnterleukin-10 [IL-10] production and immune derangement that may contribute to the development of Diabetes Mellitus [DM] in chronic Hepatitis C Virus [HCV] infection, Two groups of HCV positive patients with liver cirrhosis [23 diabetic and 29 non-diabetic] were studied in addition to 10 healthy subjects. IL-10 serum levels were assayed for all the studied groups using ELISA technique. Two single nucleotide polymorphisms [SNPs] in the promoter region of IL-10 gene namely -1082 [A/G] and-592 [A/C] were genotyped in the HCV groups using Polymerase Chain Reaction, restriction fragment length polymorphism [PCR-RFLP] Technique. Serum IL-10 levels were found to be significantly higher in each of HCV groups compared to healthy control group [P<0.01] with positive correlation to liver enzymes, Fasting Blood Sugar [FBS] and negative correlation to serum albumin. Significant differences of IL-10 levels were detected in diabetic compared to non-diabetic HCV patients [9.94 +/- 3.5 versus 7.68 +/- 2.0 pg/ml, P<0.05].The frequency of carriage of allele G of-1082 A/G and allele C of-592 A/C markers were found to be higher in diabetic HCV compared with non diabetic patients [p=0.024, OR=3.12[95% Cl, 1.16- 8.39] and [p=0.045, OR=2.64 [95% Cl, 1.02, 6.84]] respectively. Haplotype analyses of both markers revealed that the carriage of haplotype GC was significantly higher in diabetic HCV compared to that of non diabetic patients [p<0.0001 and OR=7.49] [95% Cl, 3.45, 15.87]] It was concluded that IL-10 gene polymorphism and subsequently high IL-10 levels is associated with chronic HCV infection and may be involved the pathogenesis of diabetes mellitus

pattern of common haemoglobinopathies among anaemic patients in eastern province of Saudi Arabia

Haemoglobinopathy is a collection of a number of diseases, including sickle-cell disease and thalassemia. Several reports indicate that haemo-globinopathies with or without G6PD are the most common genetic abnormalities in the population of the Arabian- Peninsula. However the exact frequencies of neglected cases of these abnormalities among anaemic patients have not yet been determined. 1372 patients were selected from those who attending the out patients from multicenter clinics in Eastern Province of Saudi Arabia.. No one of our patients had been diagnosed for haemoglobinopathies or other chronic diseases. The control groups were as following: 152 children who attended the pediatric clinics for general checkup; 84 adult females; and 92 adult males who attended for blood donation. All individuals were subjected for the following tests: [1] complete blood picture and blood smear, [2] HB electrophoresis, [3] Sickling test [4] G6PD screening, [5] G6PD assay for positive screening cases [6] anemia test panel including serum iron, serum ferritin, and total iron binding capacity. The highest prevalence of haemoglobinopathies is sickle cell diseases. The carriage of at least one of S allele was 41.9%. The second commonest haemoglobinopathies in this study was beta thalassemia represent about 23.46% of total haemoglobinopathies. The third commonest haemoglobinopathies were hereditary persistent fetal haemoglobin [HPFH] and G6PD representing about 15% and 10.26% respectively. Significant differences of haemtological parameters were observed among haemoglobinopathies compared with control groups. Our results suggest the importance of screening tests of Haemoglobinopathies in individuals in Eastern region of Saudi Arabia

Accuracy of different clinicalsamples for prenatal diagnosisof toxoplasmosis in pregnant women

To evaluate the diagnostic sensitivity and specificity of different clinical samples for detection of prenatal toxoplasmosis in pregnant women,paired samples of blood, amniotic fluid, urine and saliva were collectedfrom one hundred eighty six pregnant women who living in Northern Egypt. Total immunoglobulin G [IgG], Toxoplasma gondii-specific IgM, and avidity of T. gondii-specific IgG were quantified by enzyme-linked immunosorbent assay in serum samples. DNA was extracted from different clinical samples and amplified for detection of B1 gene sequence of T.gondii. All positive PCR serum samples were found to be positive by PCRin amniotic fluid samples. Compared to serum and amniotic fluid PCR, the overall sensitivity of IgM, IgG, and Avidity IgG in serum samples was34.1%, 63.5%, and 72.1, respectively, but the specificity was 69.1%,52.4%, and 76.8% for IgM, IgG, and Avidity IgG respectively. The PCR of urine and saliva samples was 100% specific for both sets of PCR primers. The sensitivity of the PCR urine was 65.9% and 62.5% for 1st and 2ndprimer sets respectively. The sensitivity of PCR saliva tended to be lower than PCR urine, representing 28.4% and 43.3% for 1st and 2nd primersets respectively. None of the PCR-negative amniotic and serum samples were positive by PCR of urine or saliva samples. These results suggest that PCR analysis of urine or saliva samples may be a valuable approach to the diagnosis of toxoplasmosis in pregnant women

Molecular diagnosis of duchennemuscular dystrophy by multiplexpolymerase chain reaction

Duchenne or Becker muscular dystrophy [DMD/BMD] is one of the most common X-linked lethal genetic diseases with a worldwide frequency of one in 3500 live male births. The Dmd locus is, the largest human gene known, span 2.4 Mb in Xp21. It consists of 79 exons that encode a14 Kb transcript. We have investigated the frequency of deletion in the Dmd gene in 37 unrelated Duchenne muscular dystrophy [DMD] Egyptian patients. All patients were screened for 21 exonic deletion which pro-posed by Multicenter Study Group. The screening of deletion was done using single strand conformation polymorphism [SSCP] after multiplex PCR. The most common exonic deletion in our study are exon 48 [46 %],exons-19, 45 [37.8 % of each] followed by exons-43 [ 29.7 %], PM, 44, 47,and 50 [27 %]. The overall deletion in our study about 83.8% in DMD. Deletions were clustered at two spots: 76 % at the hot spot in the distal region of the gene and 24 % at the hot spot in the proximal region of the gene. The multiplex PCR is simple, reliable and rapid technique for detection of carrier in families with DMD