OAR@UM Collection:

Does summation of HMIP SNPs differentiate between the Hb F of the β°Codon39 and the β+IVS-I-6C thalassaemia heterozygotes?

Tue, 01 Jan 2013 00:00:00 GMT

Title: Does summation of HMIP SNPs differentiate between the Hb F of the β°Codon39 and the β+IVS-I-6C thalassaemia heterozygotes? Abstract: This research was intended to confirm the observations of Daw (2012) regarding the differential effect of summation of SNPs at the HMIP locus on (absolute) Hb F levels between β thalassemia (thal) heterozygotes with, either the β°Codon39T, or the β+IVS-I-6C mutations. The β°Codon39T mutation has been known to result in higher Hb F among heterozygotes and homozygotes. However, with the exception of the confounding effect of the conditional dimorphism (C/T) at -158 in the Gγ promoter, known as the XmnI site, an adequate explanation has not been available. The total dataset was increased from 167 of Daw, to 277 in this study. Furthermore, the thalassaemia heterozygotes with mild anaemia were separated from the rest with normal Hb level. Two cis regulatory loci, the Xmnl site and the (AT)xTy polymorphism as well as two trans regulatory sites, BCL11A and HMIP polymorphism were sequenced. Neither one of the trans or cis- regulatory sites nor the BCL11A were found to have significant effect on Hb F level. However, the Hb F of the 35 β°Codon39T heterozygotes with total Hb ≥ 10 g/dl was significantly different from that of β+IVS-I-6C hetetrozygotes (P < 0.005). In particular, those with HMIP (++++) haplotype had Hb F of (24 ± 9 N=8) in the β°Codon39 thalassaemia and (9 ± 2 N=10) in the β+IVS-I-6C thalassaemia even when all cis and trans heterogeneities tested were accounted for. Any explanation appeared inadequate at this stage, but functional experiments could serve to distinguish between alternative mechanisms including the possibility of a trans-regulator that bound both the HMIP locus and the p globin gene around β°Codon39T competitively or cooperatively. A much larger data set is being sought together with the direct quantification of the absolute HbF by immuno-assay and the enumeration of F-erythrocytes by flow cytometry, in order to put the observation on more solid ground and explore possiable mechanisms. Description: M.SC.BIOMED.SCI.

Deregulation of the phosphatase PP2A in haematopoietic cell lines

Tue, 01 Jan 2013 00:00:00 GMT

Title: Deregulation of the phosphatase PP2A in haematopoietic cell lines Abstract: Haematopoieisis is a long life process, providing different mature functional cells required by physiological demand. In addition to lineage commitment, expansion and differentiation of a specific progenitor pool has to be maintained. This complex process is under the control of transcription factors, growth factors and extrinsic factors. Deregulation of genes results in an imbalance between cell proliferation and differentiation, hence the malignant phenotype. Previous studies using cellular models show the importance of suppressed feedback mechanisms, in particular the regulation of the phosphatase PP2A. The aim of this study was to (1) characterise variations in PP2A transcripts; (2) correlate the PP2A activity with the presence of the mutants using haematopoietic cell lines; and (3) investigate the presence of the resulting mutants/isoforms in a panel of Chronic Myeloid Leukaemia patient samples using cDNA. 6 leukaemia cell lines were used to scan the PPP2CA coding sequence for variations. All nucleotide substitutions identified were silent mutations. A novel splice variant was identified in the PPP2CA as a result of exon 2 skipping, hence called PPP2CAL12 variant. This indicates that interfering with PPP2CA function is not a common event. Interestingly, the BCR-ABL positive cell line, BV173 expressed the PPP2CAL12 variant only. In addition, PPP2CAL12 splice variant was detected at low expression levels in chronic myeloid leukaemia (CML) patient samples. Description: M.SC.PATHOLOGY

Non-syndromic oligodontia

Tue, 01 Jan 2013 00:00:00 GMT

Title: Non-syndromic oligodontia Abstract: Introduction. Oligodontia is defined as the developmental absence of more than six permanent teeth, not including third molars. The aetiology is genetic, a number of candidate genes for this condition have been identified, for instance Muscle segment homeobox 1 (MSXl), and Paired box 9 (P AX9). Mutations in these genes are associated mainly with the absence of premolar and molar teeth respectively. The reported prevalence of oligodontia is 0.08-0.16 %, however the clinical impression is that the prevalence in Malta may be higher. Materials and Methods. A survey of 1000 Dental Panoramic Tomograms from the archives of the Dental Department, Mater Dei Hospital, Malta revealed a prevalence of oligodontia of 0.8%. Two unrelated nuclear families and a further two unrelated individuals with oligodontia were tested at a genetic level. Saliva samples were collected and DNA extracted. Primers were designed to span the introns and intron-exon junctions of MSXl and P AX9, The primers were optimised using gradient PCR, and the samples genotyped by Real Time PCR followed by High Resolution Melting Analysis. DNA sequencing of all samples exhibiting a shift of the melting curve was carried out. Results. A misscnsc mutation (A40G) in lY1SXl (rs36059701), was found to segregate with the phenotype in both nuclear families. A novel missense mutation in P AX9 (A99P) was also found in two severely affected members of one family. Discussion and Conclusion. The MSXI A40G SNP is relatively common with a Minor Allele Frequency (MAF) of 0.20 in European populations, but has been associated with both Oligodontia and Cleft Palate. The PAX9 mutation is in the DNA binding domain (homeobox) and is predicted to be pathogenic. It is possible that the two genes act synergistically to produce the oligodontia phenotype.

Anomalous urine drug testing results in Maltese heroin addicts

Tue, 01 Jan 2013 00:00:00 GMT

Title: Anomalous urine drug testing results in Maltese heroin addicts Abstract: The main aim in a urine drug testing programme for opiates is to detect and confirm heroin intake. The prevalence of atypical samples present amongst urine specimens obtained from the local population of patients undergoing methadone maintenance treatment (DETOX) was studied. For the purpose of this project, atypical samples would yield a negative total opiate immunoassay screen (cut-off 300 µg/L) and a positive 6-monoacetylmorphine (6 MAM) immunoassay screen (cut-off 1 0 µg/L). The strategy of utilizing the total opiate screen as the sole analytical tool may result in a number of individuals escaping detection. This phenomenon which was never studied locally cannot be satisfactorily explained with the current understanding of heroin metabolism. Possible xenobiotic inhibition in the heroin metabolic pathway and also the presence of enzymatic defects caused by known single-nucleotide polymorphism (SNP's) mutations were investigated. The results of the commercial 6 MAM immunoassay (CEDIA®) were compared with an HPLC and a GC-MS method to confirm authenticity and also assess the sensitivity of the assay. This study showed that 1.23% of the negative results issued for total opiates (cut-off 300 µg/L) are actually 'pseudo false negative'. Such results are analytically correct but might not be reflecting the actual patient situation. There seems to be no xenobiotic interactions with the heroin metabolic pathway, and the association with the presence of known SNP's was also excluded in 28.6% of the cases. By exclusion, it seems that urine collection timing in relation to heroin intake and to a much lesser extent heroin contamination of the urine sample are the most likely explanations for this phenomenon. With regards to the 6 MAM immunoassay kit currently being utilized at the Maltese Toxicology Lab (Mater Dei Hospital), the results indicate that it possesses excellent sensitivity since all preliminary positive 6 MAM results were confirmed by HPLC and GC-MS techniques. An additional 6 MAM immunoassay screen is not being recommended in cases of routine drugs of abuse screening since the prevalence of atypical samples may not be high enough to justify the additional expense. However it might be useful in cases of suspected opioid overdose that turn out to be total opiates negative. Description: M.SC.BIOMED.SCI.