Prenatal Diagnosis and Screening of Thalassemia Mutations in Bangladesh: Presence of Rare Mutations.

Thalassemia is the most common congenital monogenic disorder in Bangladesh. Prenatal diagnosis (PND) and pregnation termination of an affected child is one of the best options to reduce the burden of thalassemic children. This article reports the results of DNA analyses of chorionic villus sampling (CVS) and amniocentesis of fetuses of mothers who came to the thalassemia center of Dhaka Shishu (Children) Hospital, Dhaka, Bangladesh. DNA analysis was done by real-time polymerase chain reaction (qPCR) and Sanger sequencing. Maternal contamination was ruled out by variable number of tandem repeats (VNTRs). A total of 232 samples were analyzed. Hb E (HBB: c.79G>A)/ß-thalassemia (Hb E/ß-thal) was the most common type of thalassemia seen in 32 samples (13.79%) followed by ß-thal major (ß-TM) in 10 cases (4.31%). Molecular characterization of the most predominant mutation was IVS-I-5 (G>A) (HBB: c.92+5G>C). The analysis also revealed five rare mutations: IVS-II-654 (C>T) (HBB: c.316-197C>T), IVS-II-1 (G>A) (HBB: c.315+5G>A), codon 44 (-C) (HBB: c.135delC), -86 (C>A) (HBB: c.-136C>A) and codons 14/15 (+G) (HBB: c.45_46insG), which have not been reported previously in Bangladesh. This study provides important information for PND and will help in the development of similar diagnostic programs for other DNA centers in Bangladesh.

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

BACKGROUND: Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS: Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS: Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.

A Novel ß-Thalassemia Insertion/Frameshift Mutation Between Codons 77/78 (p.Leu78Profs*13 or HBB: c.235_236insC) Observed in a Family in Bangladesh.

ß-Thalassemia (ß-thal) is one of the most common inherited hemoglobin (Hb) disorders, worldwide. A 28-year-old female and her husband came to Dhaka Shishu (Children) Hospital, Bangladesh for prenatal diagnosis for thalassemia mutations. We identified and characterized a novel ß-thalassemia (ß-thal) mutation due to an insertion of cytosine between codons 77 and 78 (p.Leu78Profs*13) found in mother in a heterozygous state. This mutation caused an insertion in the normal reading frame of the ß-globin coding sequence and the new stop codon being the amino acid 90 (HBB: c.235_236insC) in exon 2 that leads to a ß0-thal phenotype.

Risk factors associated with injury to the inferior alveolar and lingual nerves following third molar surgery-revisited.

OBJECTIVE: Earlier reports, including a preliminary study within our unit, have shown that the surgeon's experience is one of the most influential factors in determining the likelihood of both permanent inferior alveolar nerve (IAN) and lingual nerve (LN) paresthesia, following third molar surgery. The effect of this and other factors influencing such prevalence are assessed in this study. STUDY DESIGN: This prospective study involved 3236 patients who underwent surgical removal of impacted third molars. Patients' demographics and radiological parameters were recorded along with the grade of the treating surgeon. The prevalence of inferior alveolar and lingual nerves paresthesia at 1 month, 6 months, and 18 to 24 months postoperatively were also traced. RESULTS: At 1 month postoperatively, the incidence of IAN paresthesia was 1.5% and the LN was 1.8%. These figures decreased over time and 18 to 24 months postoperatively, the incidence of permanent dysfunction of the IAN was 0.6% and LN was 1.1%. With regard to inferior alveolar nerve paresthesia, risk factors included the patient's age (26-30 years), horizontally impacted teeth, close radiographic proximity to the inferior alveolar canal (IAC), and treatment by trainee surgeons. With regard to the lingual nerve, risk factors included male patients, distoangular impactions, close radiographic proximity to the IAC, and treatment by trainee surgeons. CONCLUSION: One of the main risk factors of developing permanent sensory dysfunction in the distribution of these nerves is related to the surgical skills/experience of the operator. Other factors are associated with the type of impaction and the radiographic proximity of the tooth to the inferior alveolar nerve. Such long-term complications can affect the patient's quality of life; the impact on profession, education, and research is unknown.

Experience versus complication rate in third molar surgery.

OBJECTIVES: The records of 1087 patients who underwent surgical removal of third molar teeth were prospectively examined to analyse the possible relationship between postoperative complications and the surgeon's experience parameter. METHOD AND MATERIALS: Seven surgeons (three specialists in surgical dentistry [specialists SD] and four oral and maxillofacial Senior House Officers [OMFS residents]) carried out the surgical procedures. For each patient, several variables were recorded including age, gender, radiographic position of extracted teeth, treating surgeon, duration of surgery and postoperative complications. RESULTS: Analysis of the data revealed some differences in the incidence of complications produced by the specialists SD and OMFS residents. The main statistically relevant differences were increase the incidences of trismus, nerve paraesthesia, alveolar osteitis and infection in the resident-treated group, while the specialist-treated group showed higher rates of post-operative bleeding. CONCLUSION: The higher rate of postoperative complications in the resident-treated group suggests that at least some of the complications might be related to surgical experience. Further work needs to compare specialists of training programmes with different years of experience, using large cross - sectional studies.