For many years counting cells and identifying them under the microscope has been the conventional method to determine the number of abnormal and normal cells in cancers. During the last decade, studies have shown that the detection and quantification of residual tumor cells is important in predicting the clinical outcome of several types of hematological malignancies. Detection of
minimal residual disease (MRD) is now becoming routinely implemented in treatment protocols and is increasingly used for guiding therapy and for evaluation of new treatment modalities (Raanani & Hashomer, 2004). A wide variety of techniques have been developed to detect residual malignant cells beyond the sensitivity of conventional approaches by cell morphology. One of these technology is by real time quantitative (RQ) polymerase chain reaction (PCR) using the Taqman and LightCycler systems.
In this report we demonstrate the role of fluorescence in situ hybridisation (FISH) and conventional cytogenetic methods in clinically and cytogenetically confirmed cases of microdeletion syndromes. A total of nine cases were referred to the Cytopathology and Cytogenetic Unit, Hospital Universiti Kebangsaan Malaysia (HUKM) from 2002 to 2004. They include three Prader-Willi syndrome, three DiGeorge syndrome, one Williams syndrome, one Miller-Dieker syndrome and one Kallmann syndrome. Blood samples from the patients were cultured and harvested following standard procedures. Twenty metaphases were analysed for each of the cases. FISH analysis was carried out for all the cases using commercial probes (Vysis, USA): SNRPN and D15S10 for Prader-Willi syndrome, LIS1 for Miller Dieker syndrome, ELN for Williams syndrome, KAL for Kallmann syndrome, TUPLE 1 and D22S75 for DiGeorge syndrome. Conventional cytogenetic analysis revealed normal karyotypes in all but one case with structural abnormality involving chromosomes 9 and 22. FISH analysis showed microdeletions in all of the nine cases studied. This study has accomplished two important findings ie. while the FISH method is mandatory in ruling out microdeletion syndromes, conventional cytogenetics acts as a screening tool in revealing other chromosomal abnormalities that may be involved with the disease.
Thalassaemia screening programme was conducted to reduce the burden of the disease [1]. Here, we describe one unexpected discovery in a 33-year-old gentleman and also the importance of DNA analysis in detecting the globin gene mutation.
The aims of this study are to identify and characterize the Haemoglobin G Makassar. Haemoglobin G Makassar was identified in Makassar, Sulawesi (Celebes), Indonesia in 1969 and has been reported in a family of Thai origin in 2002. Haemoglobin G Makassar was found to share identical properties with haemoglobin S in routine haemoglobin separation by cation-exchange HPLC. It is therefore, patients with Haemoglobin G Makassar and Haemoglobin S may sometimes be mistakenly identified for each other.
There were four cases identified from year 2015 to 2016 in Peninsular Malaysia by Molecular Genetics Laboratory, Institute for Medical Research. All patients were asymptomatic with mild hypochromic microcytic anaemia. All patients were analysis with Haemoglobin S trait. Analysis by Capillary Electrophoresis showed that these patients had 39.9 to 44.0% of haemoglobin variant in zone S. Alpha and Beta globin gene analysis were performed on these samples.
DNA sequence analysis, revealed a single nucleotide substitution GAG to GCG at codon 6 of the beta-globin gene (Glu>Ala), indicating of Haemoglobin G Makassar for all the patients (Fig. 1). All patients were positive with Haemoglobin S trait. Multiple Amplification Refractory Mutation System (MARMS) PCR for Haemoglobin S was negative in all cases. However alpha-globin gene analysis showed that two of them had single alpha deletion (α3.7). The mean reading for HGB is 11.95 g/dL, for MCV is 72.1 fL and for MCH is 23.65 pg which all are lower than normal peoples.
The screening method may mistakenly identify Haemoglobin G Makassar as Haemoglobin S. Therefore identification and characterization of Haemoglobin G Makassar by several molecular methods such as polymerase chain reaction (PCR) and sequence analysis are necessary for confirmation of the diagnosis.
The identification of chromosomal aberrations in prostate cancer has been widely studied with several known oncogenes and tumor suppressor genes have successfully been discovered. The most frequent aberrations detected in western population were losses in chromosome 5q, 6q, 8p, 13q, 16q, 17p, 18q and gains of 7p/q and 8q. The purpose of this study was to determine the chromosomal aberrations among Malaysian men of Southeast Asia population and discover those potential genes within that chromosomal aberrant region. Thirty-six formalin-fixed paraffin embedded specimens consist of eight organ-confined prostate cancer cases, five with capsular invasion, 14 showed metastasis and nine cases had no tumor stage recorded, were analyzed by array CGH technique. Chromosomal losses were frequently detected at 4q, 6q, 8p, 13q, 18q while gains at 7q, 11q, 12p, 16q and 17q. Gain of 16q24.3 was statistically significant with tumor size. Gains of 6q25.1 and Xq12 as well as losses of 3p13-p1.2 and 13q33.1-q33.3 were significantly correlated with Gleason grade whereas 12p13.31 gain was associated with bone metastasis. Several potential genes have also been found within that aberrant region which is myopodin (4q26-q27), ROBO1 (3p13-p11.2), ERCC5 (13q33.1-q33.3) and CD9 (12p13.31), suggesting that these genes may play a role in prostate cancer progression. The chromosomal aberrations identified by array CGH analysis could provide important clues to discover potential genes associated with prostate tumorigenesis of Malaysian men.
Turner syndrome is one of the most common chromosomal abnormalities affecting newborn females. More than half of patients with Turner syndrome have a 45X karyotype The rest of the patients may have structurally abnormal sex chromosomes or are mosaics with normal or abnormal sex chromosomes. Mosaicism with a second X sex chromosome is not usually of clinical significance. However, Turner syndrome patients having a second Y chromosome or Y chromosomal material are at risk of developing gonadoblastoma later in life. The aim of this study is to compare the results of conventional (karyotyping) and molecular cytogenetics (FISH), and discuss the advantages and limitations in the diagnosis of Turner syndrome. We also aim to compare the degree of mosaicism identified using conventional cytogenetics and FISH techniques. Conventional cytogenetics and FISH analyses were performed on eight peripheral blood samples of patients with Turner syndrome collected between 2004 and 2006. From this study, two out of eight patients with Turner syndrome were found to have the sex determining region on the Y chromosome (SRY) gene by FISH analysis. Our results showed that the rate of detection of mosaic cases in Turner syndrome was also increased to 88% after using the FISH technique. We concluded that FISH is more superior to conventional cytogenetics in the detection of the Y chromosomal material. FISH is also a quick and cost effective method in diagnosing Turner syndrome and assessing the degree of mosaicism.
Mutations in the δ globin gene are not pathologically significant [1]. However, coinheritance of β and δ thalassaemia can mask the diagnosis of β thalassaemia trait as it causes HbA2 level to be lowered [2,3]. Here, we reported 5 unrelated cases of compound heterozygous β0 Filipino ~ 45 kb deletion and codon 67 (GTG>ATG) HbA2 Deventer in Sabahan population.
Cases of β°-thalassemia traits with unusual low HbA2 were reviewed. These cases were initially referred to our laboratory for definitive diagnosis of β-thalassemia trait. Haematological parameters and Hb analysis were carried out at the referral hospital. Genomic DNA was extracted from the peripheral blood. Multiplex ARMS and Gap PCR were done to detect common point mutations and deletions for both alpha and beta globin genes. Sanger sequencing was performed to detect mutations in delta globin gene.
Patients’ consist of 4 males and 1 female aged between 25-38 years old. All of them are indigenous Sabahan (2 Kadazans, 1 Murut, 1 Dusun and 1 Sungai). Their haemoglobin level ranges between 10.8 – 12.8g/dl. Hb analysis findings of HbA2 and HbF level ranges between 2.9 – 4.0 and 2.2 – 9.4g/dl respectively. Molecular findings revealed heterozygous state of (β)º-thal, Filipino ~45Kb deletion, NG_000007.3:g.[66258_184734del];[66258_184734=] and heterozygous state of Codon 67 [GTG>ATG] Hb A2-Deventer mutation, NG_000007.3:g.[63512G>A];[63512G=] (Figure 1 and 2).
Detection of 5 unrelated cases of HbA2 Deventer may suggest that this delta variant is common among indigenous Sabahan. Since beta thalassaemia is also common in the population, more attention should be paid during diagnosis. Identification of delta variant in beta thalassaemia carrier is important because coinheritance of beta and delta thalassaemia results in a less elevated HbA2 level. Therefore, molecular testing of thalassemia carrier state in the case of borderline HbA2 is warranted to avoid misdiagnosis of beta thalassaemia carriers.
Chromosomal abnormalities (CA) can affect numerical or structural compositions of chromosomosal DNA leading to a diversity of clinical phenotypic presentations. Awareness of prenatal diagnosis and genetic counselling have improved with advancing medical research but CA remain prevalent as its aetiology is unknown. The objective of this study is to determine the frequencies of various CA in the principle region of north-western Malaysia and compare this data to previous reports to ascertain if statistical differences exist. Karyotype analyses performed at the Genetics Laboratory, Advanced Diagnostic Laboratory (ADL) during the first 5-years of cytogenetic services, totalling 1461 cases, were assessed in this report. Cases suspected of CA were initially diagnosed by clinicians and detailed clinical and family histories were recorded. Peripheral blood lymphocytes of patients were collected and cultured in vitro for acquisition of karyotype by standardized G-banding technique. Fluorescence in situ hybridization (FISH) was conducted in cases suspected of to be DiGeorge, Prader-Willi, Angelman and Williams syndrome. Of the total samples (1805) received and cultured, 1669 (92.46%) successfully yielded results. Abnormal outcomes were observed in 495 cases (29.66%) whereby pronounced majority of cases 299 (68.42%) were Down syndrome. This is followed by Edward, Turner and Patau syndrome, in order of frequency. Numerical CA appears to be prevalent accounting for 85.86% of cases. Structural CA accounted for 14.14% of total positive cases whereby the most common was deletions (34.29%) followed by translocations (20%), ring chromosomes (5.71%), Fragile X syndrome (4.29%), duplications (5.71%) and marker chromosomes (7.14%). The remainder of cases (22.86%) consisted of derivative chromosomes and other complex aberrations. The number of polymorphic variant cases were 27 (1.62%). The number of peripheral blood samples received has significantly increased from 14.3 per month in 2006 to 32.17 per month in 2011. Comparative analysis of our study to previous reports reveal statistical differences in the occurrence of several CA including Edward, Patau, Klinefelter and Fragile-X syndrome. Our experience with peripheral blood samples for cytogenetic analysis demonstrated a success rate of 92.46%. This showed an increase in clinicians validating patients’ diagnoses with karyotyping which is essential in confirming genetic anomalies with the goal to substantiate genetic counselling.
The most common inherited monogenic disorders in the world are the haemoglobinopathies and thalassaemia. Thalassaemia is a heterogeneous group of genetic disorders of haemoglobin synthesis, characterised by a reduction in the production of one or more of the subunits of haemoglobin chains [1]. Haemoglobin A2 (HbA2) level is an important parameter in thalassaemia diagnosis. High HbA2 level (≥4.0) detected in Hb analysis, points to the diagnosis of beta thalassaemia and other haemoglobinopathies. However, in some cases, the HbA2 levels are apparently normal or borderline high despite abnormal haematological profile. In these cases, further testing is required to confirm the diagnosis. The aim of this study is to examine any abnormality at molecular level in cases of Hb analysis results with normal or borderline high HbA2 level.
At the present time, the differentiation between follicular thyroid carcinoma (FTC) and adenoma can be made only postoperatively and is based on the presence of capsular or vascular invasion. The ability to differentiate preoperatively between the malignant and benign forms of follicular thyroid tumors assumes greater importance in any clinical setting. The PAX8-PPARG translocation has been reported to occur in the majority of FTC. In this study, a group of 60 follicular thyroid neoplasms [18 FTC, 1 Hurthle cell carcinoma (HCC), 24 follicular thyroid adenomas (FTA), 5 Hurthle cell adenomas (HCA), and 12 follicular variants of papillary thyroid carcinomas (FV-PTC)] were analyzed to determine the prevalence of the PAX8-PPARG translocation by fluorescence in situ hybridization. The PAX8-PPARG translocation was detected in 2/18 FTC (11.1%). In addition, 2/18 (11.1%) FTC and 1/5 (20%) HCA showed 3p25 aneusomy only. The frequency of the translocation detected in the study was lower compared to the earlier studies conducted in Western countries. This might be attributed to the ethnic background and geographic location. Detection of either the PAX8-PPARG translocation or the 3p25 aneusomy in FTC indicates that these are independent genetic events. It is hereby concluded that 3p25 aneusomy or PAX8-PPARG translocation may play an important role in the molecular pathogenesis of follicular thyroid tumors.