OAR@UM Collection:

Preliminary genome mapping of the Maltese goat

2021-09-02T07:11:10Z

Title: Preliminary genome mapping of the Maltese goat Abstract: In this study two different strategies to amplify anonymous fragments from the genome of Maltese Goat were studied. The first strategy ultised "arms" generated by restriction digests of amplicons commonly available in this laboratory. Ligation of the "arms" to a fragment from the goat genome were amplified using the primers complimentary to "arms", by a standard PCR technique. The second strategy involved the utilisation of arbitrary primed PCR known as Random Amplification of Polymorphic DNA (RAPD) and utilised a single 10 bp primer. A total of 66 Maltese goats were studied usmg the RAPD methods and 11 polymorphic zones were identified. The proportional incidence of presence and Polymorphism Information Content (PlC) of these zones were calculated. Due to the fact that in RAPD PCR only a single primer is used, these zones could not be sequenced directly and thus were cloned in a pUC 18 vector. Eleven different clones were sequenced using both the forward (universal) and reverse primers specific for the pUC 18 vector. After sequencing a total of nine new markers for the goat genome were identified and their homologies with known nucleic acid and protein databases were described. Description: M.PHIL.

Clinical and molecular pathology of the β+ IVSI-6C thalassaemia in Malta

2020-11-25T12:19:47Z

Title: Clinical and molecular pathology of the β+ IVSI-6C thalassaemia in Malta Abstract: CLINICAL AND MOLECULAR PATHOLOGY OF THE β+ IVSI-6C THALASSAEMIA IN MALTA The main objectives of this study were to characterise the β -thalassaemia mutations present in Malta, evaluate treatment protocols, study the correlation between the genotypical and phenotypical pictures, evaluate the criteria for the proper identification of thalassaemia heterozygotes and investigate the causes for the bone disease in thalassaemia homozygotes. Data on the molecular defects leading to β -thalassaemia were obtained from 28 homozygotes out of the known 29 subjects. Four different mutations were encountered, with the β+IVSI-6(T->C) accounting for 71.4% of all β-thal alle1es [β+IVSI-110(G->A) = 12.5%; β °IVSII-1 (G->A) = 10.7%; β °Codon 39(C->T) = 5.4%]. The β+IVSI-6 (C) allele was present on both haplotype VI and VII while the β °Codon 39(T)and the β+IVSI-110(A) were associated with haplotypes I and IX respectively. The β °IVSII-1(A) mutation was found within haplotype III except in one case that had an unusual VI/Ill hybrid haplotype. The VI/Ill hybrid haplotype was characterised by a low HbF (7.7%) in contrast to the other 5 cases that had a high HbF (-60%). Data collected prospectively on the β+IVSI-6C homozygote children, indicated that the disease presented as a moderate to severe condition needing regular blood transfusions (mean = 70ml/kg/year) for normal growth and development. Splenectomy had little effect on the blood transfusion requirement of the β+IVSI-6C homozygotes. On the other hand, the adult 13+ rvSI-6C homozygous condition was characterised by a mild disease (mean Hb= 8.2g/dl) with only occasional transfusions. Intra-allelic heterogeneity in the level of HbF was observed among the β+IVSI-6C homozygotes and these could be divided into two groups, one with a relatively high HbF (mean=15.0%) and one with a low HbF (4.5%). Statistically significant (p<0.05) gender difference in the level of HbF was also observed with female patients having in general, a higher HbF level. The high incidence of a relatively mild mutation and the presence of iron deficiency amongst the population, posed problems in the proper identification of β-thalassaemia heterozygotes. Indeed 37% (N=19) of obligate β+IVSI-6C heterozygotes had an MCV<80fl and a HbA2 between 3.0 and 3.5%. In an attempt to improve in the identification of the β+IVSI-6C heterozygotes, while keeping the cost of population testing to a minimum, a new approach in the identification of individuals at risk was evaluated with a computed index (10 X RBC3 x HbA²1Hb3) The exclusion of iron deficiency was further considered with a revised discriminant/cut-off value for serum ferritin, which was quite higher than that employed so far. A definite diagnosis amongst those individuals deemed at risk would be obtained by DNA analysis for the prevalent mutation within the population. Osteopenia as documented by a low bone mineral density using DEXA, was present in the majority of the homozygote subjects and was apparent as early as 5 years of age, despite a seemingly adequate hypertransfusion regime. In an attempt to elucidate possible causes for the observed osteopenia, the level of bone biochemical markers for bone formation (serum procollagen I carboxyterminal propeptide and osteocalcin) and for bone turnover (urinary deoxypyridinoline crosslinks and serum tartrate resistant acid phosphatase) were measured amongst the thalassaemia patients. The low serum osteocalcin level accompanied by normal levels for urinary deoxypyridinoline crosslinks and serum procollagen I carboxyterminal propeptide indicated that lack of proper mineralisation could result in the osteopenia observed within this group of patients. Nutritional deficiencies associated with low body weight might be possible causes of the lack of mineralisation among the patients with "mild" alleles. Description: PH.D.

Molecular epidemiology of haemoglobin in the population of Libya and the molecular biology of normal and abnormal globin gene expression

2020-11-23T15:29:02Z

Title: Molecular epidemiology of haemoglobin in the population of Libya and the molecular biology of normal and abnormal globin gene expression Abstract: The aim of this study is to evaluate with modern techniques the prevalence of different types of abnormal haemoglobins and thalassaemia in the population of Tripoli, Western and Southern Regions of Libya. 985 newborn babies from Tripoli were tested. Abnormal Hb's were noted in 10 samples (= 1.0%). Some of these were Hb C, Hb S, Hb S-β+ thaI, Hb Setif The aim of this study is to evaluate with modern techniques the prevalence of different types of abnormal haemoglobins and thalassaemia in the population of Tripoli, Western and Southern Regions of Libya. 985 newborn babies from Tripoli were tested. Abnormal Hb's were noted in 10 samples (= 1.0%). Some of these were Hb C, Hb S, Hb S-β+ thaI, Hb Setif and Hb F Malta 1. Among 866 adult samples were pregnant women, 14 (= 1.6%) had an abnormal Hb, including Hb S, Hb C and others. The total of 2595 blood samples from Western and Southern Region of Libya were investigated for abnormal haemoglobins. Out of these samples were tested, Hb AS (= 1.6%), Hb AC (= 0.72%), HPFH (= 1.3%), Hb CC (= 0.08%), Hb SC (= 0.04%) and Hb AX (= 0.16%). None of the 985 newborn samples which were tested had any amount of Hb Bart's (Y4), indicating that a-thalassaemia was absent or extremely rare. Among adult samples from Tripoli, 10 had an elevated Hb A2 ( > 3.6%), giving an incidence of ~-thalassaemia of 1.2%. Eleven cases from Western and Southern Regions had elevated HbA2, giving a gene incidence of β-thalassaemia of 0.5%. Eighteen different haplotypes have been found with polymorphic sites associated with 88 β~A -globin gene bearing chromosomes. Fifteen per cent of these are found in the Mediterranean, 50% in Black Africans and 35% are rare ones. Ten different haplotypes were found, associated with 40 βs globin gene-bearing chromosomes. Four major haplotypes were found. Three were y- βS-globin gene related haplotypes (Sub Hap Mor, CAR and Cameroon) and one is y_ βA_ globin gene haplotype (Sub Hap 16) and six were minor haplotypes. The β-globin DNA mutations present in 25 subjects with β-thalassaemia major and minor, in 5 patients with Hb S β-thalassaemia were analyzed by RFLP, ASOH and DNA sequencing of amplified DNA. Five different known β-thalassaemia mutations were identified. The common three β-thalassaemia mutations found in Libya were βo CD39 (C -> T) N = 14, β+ IVS-I,6 (T -> C) N = 8 and β+ IVS-I, 110 (G -> A) N = 6 and these mutations accounted for more than 90% of all β-thalassaemia alleles studied. The two rare mutations found were ~o IVS-I,I(G ->A) N = 1 and IVS-I,-1 (CD30) (G ->A) N = 1. Also, the associated haplotypes with the most common β-thalassaemia genes had been studied. Haplotypes I and VII were associated with βo CD39, Haplotypes VI and VII with β+IVS-I, 6, Haplotype I with β+IVS- I,110 and haplotype V with β °IVS-I,1. The MCH- β ° A value was used to evaluate the index of thalassaemia severity of two β+ thalassaemia from this study and those of six others from the literature which may be useful for understanding the effect of mutations in globin genes on pathophysiology meaningful gene expression. Description: PH.D.

Characterisation of two naturally occuring coagulation factor VII variants (ALA244V AL AND PR0134THR)

2020-11-19T12:16:42Z

Title: Characterisation of two naturally occuring coagulation factor VII variants (ALA244V AL AND PR0134THR) Abstract: Pre-operative coagulation studies on a 4-year old girl indicated the presence of coagulation factor VII deficiency in a kindred from Malta. Factor VII assays with a panel of thromboplastins from four different species, and factor VII antigen assays, indicated phenotypic heterogeneity within the kindred. Sequence analysis of the factor VII coding region and 5' untranslated region demonstrated the presence of two new missense mutations. One of these was a cytosine to adenine transversion at position 8,906. This mutation, designated factor VII Malta I, leads to the substitution of the proline at position 134 by threonine. The other mutation was a cytosine to thymine transition at position 10,648. This mutation, designated factor VII Malta II, leads to the substitution of the alanine at position 244 by valine. Sequence analysis also showed that the factor VII alle1es of the kindred were of two different frameworks, framework 1 and framework 2. Framework 2 is known to be associated with decreased levels of factor VII coagulant activity, factor VII antigen and activated factor VII. The factor VII Malta I mutation occurred on a framework 1 allele, whereas the factor VII Malta II mutation occurred on a framework 2 allele. In all, four factor VII alle1es segregated in the kindred. These were: (i) a wild-type framework 1 allele, (ii) a wild- type framework 2 allele, (iii) a framework I-factor VII Malta I allele, and (iv) a framework 2-factor VII Malta II allele. These four alle1es resulted in a complex pattern of genotypic combinations, which accounted for the phenotypic heterogeneity among members of the kindred. Both the factor VII Malta I and the factor VII Malta II mutations were likely associated with a cross-reacting material reduced defect. In order to investigate the effect of the two amino acid substitutions on the framework 1 gene product, framework 1 factor VII cDNAs coding for factor VII Malta I and factor VII Malta II were constructed and expressed in Chinese hamster ovary cells. The specific activity of the factor VII Malta I recombinant variant did not differ significantly from that of recombinant wild-type factor VII expressed by the wild-type framework 1 factor VII cDNA. On the other hand, the factor VII Malta II recombinant variant had a specific activity slightly decreased as compared to that of the recombinant wild-type protein. Both variants were fully activated by activated factor X. Furthermore, the Kcat and KM for activation of factor X in the presence of tissue factor, by activated recombinant factor VII Malta I and by activated recombinant factor VII Malta II, did not differ significantly from those by activated wild-type factor. The data obtained excluded gross dysfunctional effects of the two ammo acid substitutions on the functional characteristics of the framework 1 gene product. However, the studies done to date suggest a complex defect perhaps due to a small decrease in activity, defective secretion, decreased stability, or sequestration in an inactive complex in plasma. Description: PH.D.