Association of eating habits with mitochondrial DNA copy number in eveningness chronotypes: Origin research.

OBJECTIVE: To determine the relationship between eating habits and mitochondrial deoxyribonucleic acid copy number in adult cases of eveningness chronotypes. Methods: The cross-sectional, analytical study was conducted from September 2022 to June 2023 at the Physiology Department of the Islamic International Medical College, Rawalpindi, in collaboration with the Genetic Resource Centre, Rawalpindi, Pakistan, and comprised adult subjects who were assessed using the Morningness-Eveningness Questionnaire. The participants' eating habits were assessed using the Healthy Eating Assessment Questionnaire, and on they were divided into those with healthy eating habits in group A and those with unhealthy eating habits in group B. Deoxyribonucleic acid was extracted using the Chelex method, the mitochondrial deoxyribonucleic acid copy number of all participants was quantified using quantitative polymerase chain reaction. Data was analysed using SPSS 27. RESULTS: Of the 80 subjects, 30(37.5%) were males and 50(62.5%) were females. The overall mean age was 24.27±6.91 years (range: 18-45 years). There were 40(50%) subjects in each group. The mean mitochondrial deoxyribonucleic acid copy number in group A was 2.74±0.14 compared to 2.26±0.25 in group B (p<0.001). Conclusion: Subjects with healthy eating habits exhibited higher mitochondrial deoxyribonucleic acid copy numbers, indicating reduced damage to mitochondrial deoxyribonucleic acid.

Densitometry of STR-PAGE for donor chimerism in acute leukemia's: A simple method for routine use.

Objective: To evaluate a PCR based method of polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias. Methods: The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR- PCR assay. Electrophoresis was carried out and 6% polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. Results: The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients. Conclusion: Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients.

Optimizing fault tolerance of RAM cell through MUX based modeling and design using symmetries of QCA cells.

Extensive research is now being conducted on the design and construction of logic circuits utilizing quantum-dot cellular automata (QCA) technology. This area of study is of great interest due to the inherent advantages it offers, such as its compact size, high speed, low power dissipation, and enhanced switching frequency in the nanoscale domain. This work presents a design of a highly efficient RAM cell in QCA, utilizing a combination of a 3-input and 5-input Majority Voter (MV) gate, together with a 2 × 1 Multiplexer (MUX). The proposed design is also investigated for various faults such as single cell deletion, single cell addition and single cell displacement or misalignment defects. The circuit under consideration has a high degree of fault tolerance. The functionality of the suggested design is showcased and verified through the utilization of the QCADesigner tool. Based on the observed performance correlation, it is evident that the proposed design demonstrates effectiveness in terms of cell count, area, and latency. Furthermore, it achieves a notable improvement of up to 76.72% compared to the present configuration in terms of quantum cost. The analysis of energy dissipation, conducted using the QCAPro tool, is also shown for various scenarios. It is seen that this design exhibits the lowest energy dispersion, hence enabling the development of ultra-low power designs for diverse microprocessors and microcontrollers.

Effect of Sleep Quality on Mitochondrial DNA Copy Number in Eveningness Chronotypes.

OBJECTIVE: To determine the effect of sleep quality on mitochondrial DNA copy number in eveningness chronotype. STUDY DESIGN: Cross-sectional study. Place and Duration of the Study: Department of Physiology, Islamic International Medical College, in collaboration with Genetic Resource Centre, from August 2022 to May 2023. METHODOLOGY: A total of 80 participants with eveningness chronotype based on the Morningness-Eveningness Questionnaire were recruited. The participants' sleep quality was assessed using Pittsburg Sleep Quality Index (PSQI). Accordingly, they were categorised into Group A (good sleep quality) and Group B (poor sleep quality) with 40 participants in each group. After extracting DNA using the Chelex method, mitochondrial DNA copy number of all participants was measured using the quantitative polymerase chain reaction. The mean values in both groups were analysed by Independent-samples t-test. RESULTS: Analysis of mitochondrial DNA copy number in both groups showed normal distribution after being transformed into logarithmic values. The mean value of Group A (2.65 ± 0.26) exhibited a significant increase in mitochondrial DNA copy number (95% CI: 0.18, 0.44; p <0.001) as compared to that in Group B (2.34 ± 0.29). CONCLUSION: People with good sleep quality have a higher mtDNA-CN than those with poor sleep quality. Good sleep quality may counteract negative effects of increased oxidative stress brought on by eveningness behaviour, thus leading to better mitochondrial function and increased mitochondrial biogenesis that was indicated by higher mtDNA-CN in individuals who experience good sleep quality. KEY WORDS: Mitochondria, Mitochondrial DNA copy number, Sleep quality, Chronotype.

Bilateral versus single internal mammary artery in diabetic patients: systematic review and meta-analysis.

OBJECTIVES: Data on bilateral internal mammary artery (BIMA) versus single internal mammary artery (SIMA) on diabetics were analyzed; This is the only meta-analysis, the last 7 years. METHODS: Medline through PubMed/EMBASE/CINHAL and the Cochrane Central Register of Controlled Trials; 179 articles were studied; 19 studies deemed suitable and were included in the analysis. RESULTS: The mortality was 2.41% for BIMA versus 1.71% for SIMA (odds ratio [OR] = 0.95; 95% confidence interval [CI]: 0.74-1.22). Postoperative reopening for bleeding was higher at 3.75% for BIMA versus 2.91% for SIMA (OR = 1.49; 95% CI: 1.15-1.93). The incidence of MI was 0.87% for BIMA versus 0.83% for SIMA (OR = 0.73; 95% CI: 0.37-1.44). Deep sternal wound infection was 3.02% for BIMA and 1.95% for SIMA (OR = 1.57; 95% CI: 1.26-1.95). When skeletonized, the incidence of DSWI was 2.5% for BIMA versus 2.41% for SIMA. There was a significant difference at 5-year survival favoring the BIMA, 85.15% BIMA versus 80.77% SIMA (OR = 1.79; 95% CI: 1.60-2.01). The 10-year overall survival was 74.04% BIMA versus 61.57% SIMA (OR = 1.79; 95% CI: 1.61-1.98). The 15-year survival was 47.08% for BIMA versus 37.06% for SIMA (OR = 1.69; 95% CI: 1.52-1.88). CONCLUSIONS: Postoperative bleeding was higher in BIMA group. Bilateral internal mammary artery in diabetic patients should be carried out in a skeletonize fashion, to reduce DSWI. There is a survival benefit of using BIMA in diabetics within 5 years of surgery; it remains significant up to 15 years.

BSEMS-A Blockchain-Based Smart Energy Measurement System.

The modern world's increasing reliance on automated systems for everyday tasks has resulted in a corresponding rise in power consumption. The demand is further augmented by increased sales of electric vehicles, smart cities, smart transportation, etc. This growing dependence underscores the critical necessity for a robust smart energy measurement and management system to ensure a continuous and efficient power supply. However, implementing such a system presents a set of challenges, particularly concerning the transparency, security, and trustworthiness of data storage and retrieval. Blockchain technology offers an innovative solution in the form of a distributed ledger, which guarantees secure and transparent transaction storage and retrieval. This research introduces a blockchain-based system, utilising Hyperledger Fabric and smart contracts, designed for the secure storage and retrieval of consumers' energy consumption data. Finally, a user-friendly web portal was designed and developed using the node.js framework, offering an accessible and intuitive interface to monitor and manage energy consumption effectively.

A basis for prenatal diagnosis of Haemophilia-A in Pakistani patients.

Objective: To screen Haemophilia-A patients for five known recurrent F8 gene variants and to analyze CA repeats in intron 13 of F8 gene in the mother and the affected child pairs. Methods: In this descriptive cross sectional study, 80 unrelated pairs of "mother and son" affected by Haemophilia-A were recruited as subjects. After collection of samples DNA was extracted using the Chelex (Bio-Rad, USA) method. Five known F8 gene variants were screened in the mothers and the affected sons by the allele specific PCR. The amplified products were separated on 2% agarose gels.CA repeats in intron 13 of the F8 gene in the mother and the affected child pairs were analyzed by Sanger sequencing method. The CA repeat alleles were used to look for the feasibility of linkage based diagnosis of Haemophilia-A in the affected families. The data were analyzed using Statistical Package for Social Sciences (SPSS) version 22.0. Results: In the 80 subject "mother and son" pairs a recurrent F8 gene variant was found in 32 pairs (40%). The most recurrent variant c.6869G>A was seen in 12 (15%). Linkage based analysis of the CA repeats in intron 13 was found to be informative in 29 (36.2%) mother-son pairs. Conclusion: The five known Haemophilia-A disease causing variants were found in 40% of the Pakistani Haemophilia-A patients. The five recurrent F8 gene variants and the CA repeats in intron 13 of F8 gene can provide a comprehensive basis for carrying out prenatal diagnosis and carrier screening in majority of the Pakistani Haemophilia-A families.

Noninvasive prenatal testing of beta-thalassemia for common Pakistani mutations: a comparative study using cell-free fetal DNA from maternal plasma and chorionic villus sampling.

BACKGROUND: The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders. OBJECTIVE: Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method. METHODS: An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR. RESULTS: Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT). CONCLUSION: NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT.

Molecular Monitoring of BCR-ABL Fusion Transcripts in Patients with Chronic Myeloid Leukemia During Treatment Using the Endpoint Fluorescence Method.

OBJECTIVE: The purpose of the study was to compare results and evaluate the agreement between the endpoint fluorescence (EPF) method and quantitative real-time polymerase chain reaction (QPCR) during molecular monitoring of patients with chronic myeloid leukemia (CML) receiving treatment. MATERIALS AND METHODS: The study was conducted at Molecular Lab of Riphah International University, Islamabad, Pakistan, from January 2017 to December 2018. A total of 150 blood specimens from 30 patients with CML were analyzed at regular intervals during therapy. The detection/quantification of transcript mRNA was done simultaneously using QPCR and the EPF method. RESULTS: Out of a total of 150 RNA specimens analyzed, 117 (78%) specimens were positive, whereas 33 (22%) were negative for the transcript using both methods at various stages of treatment. Strong linear negative correlations between the cycle threshold and relative fluorescence unit values were observed with P <.0001 at 0, 3, 6, 9, and 12 months of treatment. No significant difference (P >.05) between the means of the BCR-ABL percentage was observed in either method at all stages of treatment. The bias between the 2 methods was calculated as 0.069 ± 3.50, and 95% limits of agreement were 6.92% to -6.79%. CONCLUSION: We found that EPF is s simple method to detect/quantify BCR-ABL mRNA expression during treatment with comparable results to QPCR.

Donor Chimerism Study by Single Nucleotide Polymorphism using SYBR green based Real Time PCR.

OBJECTIVE: To optimize and evaluate a real time PCR of Single Nucleotide Polymorphism by SYBR Green method for detection of donor chimerism after haematopoietic stem cell transplantation. METHODS: This descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Oct 2017 - Dec 2019. A total of twenty patients of post haematopoietic stem cell transplant with various haematological disorders were studied to see the status of donor chimerism by using SNP real time PCR using SYBR Green method and short tandem repeat PCR. These patients had undergone allogeneic HSCT from HLA-matched sibling donors at Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. RESULTS: Real time PCR using SYBR Green was able to detect significant amount of chimerism in all 20 patients having undergone HSCT. Regarding precision of the real time PCR assay the mean value of donor chimerism was 94.1% (SD 3.96) and by STR PCR it was 95.1% (SD 1.41). The assay was found to be sensitive with a detection limit of <1%. CONCLUSION: Our results demonstrate that SNP analysis by SYBR Green real time PCR may be used for the evaluation of chimerism status in patients having undergone HSCT with a sensitivity of <1%. Hence donor chimerism by this sensitive method can be used in monitoring of chimerism in post-transplant patients with various haematological disorders.

Characteristics of BCR-ABL gene variants in patients of chronic myeloid leukemia.

BACKGROUND: Depending on breakpoints of rearrangement different types of BCR-ABL fusion protein can be generated in patients of chronic myeloid leukemia (CML). The aim of this study is to observe frequencies of major transcripts in CML patients by reverse transcriptase polymerase chain reaction (RT-PCR) and their hematological features at the time of presentation. MATERIALS AND METHODS: This cross sectional study was performed at Molecular Lab of Riphah International University, Islamabad from January to June 2019. Consecutive peripheral blood samples of 70 newly diagnosed CML patients in chronic phase were analyzed by RT-PCR to detect different BCR-ABL transcripts. Routine blood cell counts were assessed by an automated hematology analyzer. RESULTS: All samples expressed typical BCR-ABL rearrangement. Expression of either e14a2 or e13a2 transcript was detected in 38 (54%) and 30 (43%) patients, respectively. Coexpression of e13a2 + e14a2 was found in 2 (3%) patients. The mean total leukocyte count was higher in group expressing e13a2 (P = 0.01). Higher mean platelet count was noted in patients with e14a2 transcript, but this difference was statistically insignificant (P = 0.1). The association of male gender was observed with the group exhibiting e14a2 (P = 0.01). There was no statistically significant association between transcript type and different ranges of age, hemoglobin levels, and platelet and total leukocyte counts (P > 0.05). CONCLUSION: e14a2 transcript was most common transcript in CML patients. Patients exhibiting e13a2 subgroup presented with significantly higher mean white blood cell count at the time of presentation. Significantly higher proportion of male patients was found to express e14a2 transcript over e13a2.

A Survey on Applications of Artificial Intelligence for Pre-Parametric Project Cost and Soil Shear-Strength Estimation in Construction and Geotechnical Engineering.

Ensuring soil strength, as well as preliminary construction cost and duration prediction, is a very crucial and preliminary aspect of any construction project. Similarly, building strong structures is very important in geotechnical engineering to ensure the bearing capability of structures against external forces. Hence, in this first-of-its-kind state-of-the-art review, the capability of various artificial intelligence (AI)-based models toward accurate prediction and estimation of preliminary construction cost, duration, and shear strength is explored. Initially, background regarding the revolutionary AI technology along with its different models suited for geotechnical and construction engineering is presented. Various existing works in the literature on the usage of AI-based models for the abovementioned applications of construction and maintenance are presented along with their advantages, limitations, and future work. Through analysis, various crucial input parameters with great impact on the estimation of preliminary construction cost, duration, and soil shear strength are enumerated and presented. Lastly, various challenges in using AI-based models for accurate predictions in these applications, as well as factors contributing to the cost-overrun issues, are presented. This study can, thus, greatly assist civil engineers in efficiently using the capabilities of AI for solving complex and risk-sensitive tasks, and it can also be used in Internet of things (IoT) environments for automated applications such as smart structural health-monitoring systems.

Genotype frequency and use of single nucleotide polymorphisms for detection of informative allele by polymerase chain reaction.

OBJECTIVE: To determine genotype frequency of biallelic single nucleotide polymorphisms and its use in detection of informative allele in donor/recipient pairs (sibling pairs) having undergone haematopoietic stem cell transplantation with various haematological disorders using a PCR based method. METHODS: This descriptive study was conducted at GRC Lab Rawalpindi from Jan 2018- Oct 2019.A total of twenty donor/ recipient pairs (sibling pairs) were studied for genotype frequency and informativeness of single nucleotide polymorphisms. Genomic DNA was extracted from the peripheral blood and amplification of single nucleotide polymorphisms was done by PCR based method. The amplified DNA was seen by electrophoresis on 6% polyacrylamide gel. RESULTS: A sharp band of DNA on the polyacrylamide gel indicated a positive reaction. At least two or more informative SNP markers were found in every sibling pair. CONCLUSION: Our results demonstrate that PCR amplification of polyacrylamide gel electrophoresis using single nucleotide polymorphism has allowed the successful screening and detection of informative allele in all the donor/recipient pairs. (Sibling pairs). This PCR based assay using SNPs appears to be a quick, simple, reliable and technically feasible method for a use in a Pakistani setting.

Mutational Analysis Of Beta Thalassaemia By Multiplex Arms-Pcr In Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Beta thalassaemia is one of the commonest genetic conditions in the world. More than 200 different mutations have been reported in the beta globin chain genes. Notably, regional and ethnic variations in most common mutations in beta-thalassaemia have been identified. It is therefore imperative that region- and ethnicity- specific commonest mutations be identified for cost-effective molecular diagnosis of ß-thalassaemia mutations. The objective of this study was to determine the molecular mutations in ß-globin chain gene in patients with thalassemia in Khyber Pakhtunkhwa (KP) using multiplex- Amplification Refractory Mutation System (ARMS) PCR. METHODS: It was a cross sectional descriptive study. Blood samples from newly diagnosed ß thalassemia patients was collected and used as source for DNA isolation. ARMS PCR was performed for detection of mutations in ß-globin gene. SDS-PAGE was conducted for visualization of the amplicon. RESULTS: Prominent mutations were Fr 8-9 (+G), CD 5 (-CT) and Fr 41-42 (-TTCT). Congenital marriages and lack of awareness are largest contributing factor for increasing the disease burden. Organomegaly being a serious clinical complication which contributes to morbidity was proportional to age and disease progression. Fr 8-9 (+G) & CD 5 (- CT) were the most frequent mutation prevalent among different ethnic groups residing in KP. CONCLUSIONS: Multiplex-ARMS PCR is capable of assessing for multiple mutations in a single tube. Regional- and ethnic- variations in the commonest mutations in KP are noted. Any mutational diagnostic strategy should consider costs and genetic variations in a particular setting..

Clinical and Demographic Features of 76 Children with Autism Spectrum Disorders at a Centre in Pakistan.

In this cross-sectional study, 76 consecutive children with autism spectrum disorder (ASD) were studied for the clinical and demographic parameters at Autism Resource Centre in Pakistan. The median age at first consultation was 30 months, 36 months at diagnosis, and 42 months at referral to a specialised centre. Clinical psychologists, therapists and paediatricians were the most frequently involved people in diagnosis. There was an average delay of one year between the first consultation and referral to the specialised centre. The male to female ratio was 4.4:1. Consanguinity was observed in 33 (43.4%) children. Three children had another affected sibling. Half of the children were from the affluent class, while two-thirds of the parents were professionals having good education. The severity of ASD showed that 13 (17%) children had borderline features, 50 (66%) had mild to moderate ASD, while 13 (17%) had severe ASD.

Microwave-assisted synthesis and in vitro osteogenic analysis of novel bioactive glass fibers for biomedical and dental applications.

Glass fiber-based materials have gained interest for use in biomedical and dental applications. The aim of this study was to make E-glass fiber bioactive by a novel method using the microwave irradiation technique. Industrial E-glass fibers were used after surface activation with the hydrolysis method. The ratio of calcium and phosphorous precursors was set at 1.67. After maintaining the pH of the calcium solution, E-glass fibers in two ratios, i.e. 30% (nHA/E30) and 50% (nHA/E50) wt/wt, were added. The phosphorous precursor was added later and the solution was irradiated in a microwave to obtain nano-hydroxyapatite (nHA) particles on E-glass fibers. The structural, physical and in vitro biocompatibility analyses of the resulting materials were conducted. The expression of osteopontin (OPN) and collagen (Col) type 1 was measured by reverse transcription polymerase chain reaction (RT-PCR) and comparison was made between all the groups. Fourier transform infrared spectroscopy and x-ray diffraction showed characteristic peaks of nHA, and a change in the peak intensities was observed with an increase in the concentration of E-glass fibers. Scanning electron microscopic (SEM) images confirmed the homogenous adhesion of nHA spherical particles all over the fibers. Cell viability with mesenchymal stem cells showed growth, proliferation, and adhesion. All the materials were able to upregulate the expression of the OPN and Col, where gene expression was highest in nHA followed by nHA/E30 and nHA/E50. The bioactive glass fibers were synthesized in the shortest time and showed osteogenic properties. These materials have the potential for use in bone tissue engineering, dental prosthesis, and tooth restoration.

Detection of asymptomatic carriers of malaria in Kohat district of Pakistan.

BACKGROUND: Kohat district is one of the medium intensity malaria transmission areas in Pakistan where asymptomatic carriers are likely to form a reservoir of infection. This study was done to explore the possibility of using microscopy, rapid diagnostic testing (RDT), real time polymerase chain reaction (RT-PCR) and RT-PCR followed by endpoint fluorometry (EPF) for detection of malaria in asymptomatic immediate family members of patients of malaria (homestead) and in a sample from the general population of Kohat. METHODS: This cross-sectional study was done at Combined Military Hospital Kohat and Molecular Lab of Riphah International University, Islamabad from Jan to Dec 2015. A total of 1000 individuals including 200 microscopy positive patients of malaria, 400 asymptomatic immediate family members (homestead) of the active patients of malaria and 400 apparently healthy controls were tested by microscopy, RDT and RT-PCR. At the end of RT-PCR the result were read by EPF. RESULTS: In the 200 malaria microscopy positive patients, 190 (95%) were RDT positive and all were RT-PCR positive. In the 400 individuals from the homestead of malaria patients, 6 (1.5%) individuals were malaria microscopy positive while RDT failed to pick any positive and 32 (8%) were RT-PCR positive for malaria. EPF of all the RT-PCR positive results were positive and the negative results were negative. The difference in the frequency of malaria in the homestead versus general population was very significant (p = 0.0002) and the relative risk of malaria was 4.0 times higher (95% CI 1.87-8.57). CONCLUSION: The chances of detecting asymptomatic malaria carriers is significantly higher in the homestead of malaria patients than in the general population and for this purpose RT-PCR with EPF can be very useful in the diagnosis of malaria especially with low parasite density.

Delta-Beta Thalassaemia in a Pathan Family.

Delta-beta-thalassaemia (δß-thalassaemia) is a rare type of thalassaemia which mostly results from deletion of δ and ß genes with preservation of γ genes. δß-thalassaemia is classified into (δß)+ and (δß)0 types. The (δß)0-thalassemia is further divided into GγAγ(δß)0-thalassaemia and Gγ(Aγδß)0-thalassaemia. In heterozygous state, (δß)0mutations give rise to phenotype resembling ß-thalassaemia trait but with raised Hb-F, ranging from 5 to 20%, without a rise in Hb-A2. In homozygotes, the clinical picture is usually that of thalassaemia intermedia and the patients have 100% Hb-F. Workup of a 1-year child suffering from pallor, chronic ill health, and splenomegaly referred to our laboratory with the suspicion of ß-thalassaemia, ultimately resulted in a diagnosis on polymerase chain reaction as having homozygous inversion/deletion Gγ(Aγδß)0-thalassaemia. Her family members were also investigated.

Rare ß-Globin Gene Mutations in Pakistan.

The aim of this study was to analyze the rare ß-thalassemia (ß-thal) mutations in the Pakistani population. A total of 8716 unrelated Pakistani individuals having children with transfusion-dependent thalassemia were investigated by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) for the previously reported common and rare ß-thal mutations. Genomic sequencing of the ß-globin gene and its immediate 5' and 3' flanking regions was done where no known mutation was found. Out of the 8716 individuals studied, 88 (1.0%) were not characterized by ARMS-PCR. Genomic sequencing revealed that 67 (0.82%) individuals had 19 different ß-thal mutations including one novel mutation (HBB: c.136delT). The remaining 21 (0.26%) individuals did not show any mutation on the ß-globin gene and its immediate flanking regions. The characterized alleles included seven (0.09%) in the 5' untranslated region (5'UTR), 29 (0.35%) in the coding regions, and 31 (0.38%) in the splice junction regions. HBB: c.92+1G>A and HBB: c.113G>A were the most frequently seen rare mutations. The spectrum of ß-thal mutations in the Pakistani population is very diverse. In addition to the already reported mutations, another 19 different types of mutations were found. Interestingly, 21 individuals who had children with transfusion-dependent thalassemia and one known ß-thal mutation, did not show any mutation on the ß-globin gene. HBB: c.92+1G>A and HBB: c.113G>A are the most frequently seen rare mutations in Pakistan.

Effect of Iron Deficiency on the Phenotype of β-Thalassaemia Trait.

The objective of this study was to determine the effect of iron deficiency on Hb-A2 level in ß-thalassaemia trait and to determine the frequency of individuals with ß-thalassaemia trait who could be missed due to concomitant iron deficiency. A total of 120 patients were studied, out of which 23 were iron deficient (serum ferritin < 20 ng/ml). Mean Hb-A2 in the iron deficient individuals was 4.1 ± 0.47% as compared to 5.1 ± 0.58% in the remaining 97 individuals without iron deficiency (p < 0.001). In the 120 individuals with ß-thalassaemia trait, mean Hb-A2 was 5.8% with range 3 - 6.8% and confidence interval was 95%. In 2 individuals with ß-thalassaemia trait, Iron deficiency was observed and showed Hb-A2 less than 3.5%. These could have been missed while screening by Hb-A2 estimation alone. Co-existence of Iron deficiency and ß-thalassaemia trait may mask the diagnosis of beta thalassaemia trait and such individuals can be missed during screening by Hb-A2 estimation alone.