Karyotype patterns, clinical features, and parental ages of three predominant live born autosomal trisomies of Northeast Malaysia.

Chromosomal abnormality is one of the causes of congenital disorders among newborns. Despite aneuploidy being the major cause of first trimester miscarriages, very few aneuploidies such as trisomies of chromosomes 13, 18 and 21 survive to birth. The results of 4,064 patients referred for cytogenetic analysis at Human Genome Centre, Universiti Sains Malaysia, Kelantan, Malaysia between 2008 and 2019 were reviewed. We retrospectively investigated the karyotype patterns, clinical features and parental ages of the three common live-born autosomal trisomies such as trisomy 13, trisomy 18 and trisomy 21. The relative frequency of cases with the total sample received and cultured was calculated in each group and compared with those reported elsewhere. Between 2008 and 2019, a total of 1034 live-born trisomic cases which accounted for 25.4% of the 4064 total referred cases and 73.7% of 1403 suspected trisomy cases, were identified, with age ranging from newborns to 57 years. Down syndrome was the commonest aneuploidy (857 cases; 21.1%) followed by Edwards syndrome (133 cases; 3.3%) and Patau syndrome (44 cases; 1.1%). The number of diagnosed cases for each of the trisomies was fairly stable from year to year. About two-thirds of both maternal and paternal ages were ≥ 35 years. This is the first cytogenetic report on the common live-born autosomal trisomies in the North-Eastern region of Malaysia. The prevalence of trisomies 21 was found to be higher compared to an earlier study in the North-Western region of Malaysia, wherein also, advanced maternal age was a significant risk factor.

Clinical implications of conventional cytogenetics, fluorescence in situ hybridization (FISH) and molecular testing in chronic myeloid leukaemia patients in the tyrosine kinase inhibitor era - A review.

Chronic myeloid leukaemia (CML) provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Imatinib mesylate (IM), a BCR-ABL1 targeted tyrosine kinase inhibitor (TKI) drug, is the first line gold standard drug for CML treatment. Conventional cytogenetic analysis (CCA) can identify the standard and variant Philadelphia (Ph) chromosome, and any additional complex chromosome abnormalities at diagnosis as well as during treatment course. Fluorescence in situ hybridization (FISH) is especially important for cells of CML patients with inadequate or inferior quality metaphases or those with variant Ph translocations. CCA in conjunction with FISH can serve as powerful tools in all phases of CML including the diagnosis, prognosis, risk stratification and monitoring of cytogenetic responses to treatment. Molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) is used for the detection of BCR-ABL1 transcripts at diagnosis whereas quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) is used at the time of diagnosis as well as during TKI therapy for the quantitation of BCR-ABL1 transcripts to evaluate the molecular response and minimal residual disease (MRD). Despite the excellent treatment results obtained after the introduction of TKI drugs, especially Imatinib mesylate (IM), resistance to TKIs develops in approximately 35% - 40% of CML patients on TKI therapy. Since point mutations in BCR-ABL1 are a common cause of IM resistance, mutation analysis is important in IM resistant patients. Mutations are reliably detected by nested PCR amplification of the translocated ABL1 kinase domain followed by direct sequencing of the entire amplified kinase domain. The objective of this review is to highlight the importance of regular and timely CCA, FISH analysis and molecular testing in the diagnosis, prognosis, assessment of therapeutic efficacy, evaluation of MRD and in the detection of BCR-ABL1 kinase mutations which cause therapeutic resistance in adult CML patients.

Gene therapy: An updated overview on the promising success stories.

Gene therapy is a method of treatment of disease aimed at its molecular level. The progress of gene therapy, however, was as promising as it was tardy mainly due to the limitations in the resources and financial part of its development as well as owing to the rarity of most diseases it can offer its benefits to. The methods of gene therapy can vary depending on factors such as the physiology of tissue of interest, affinity of vectors to a certain type of cells, depth and accessibility of the tissue of interest, and size of the gene to be replaced or edited. The concept behind gene therapy has inspired scientists and clinicians alike leading to a rapid expansion of its clinical utility that has become so widespread to not only include diseases of monogenic origin, but also polygenic diseases, albeit not so commonly. This article delves into notable success stories of gene therapy which has been regarded as the beacon of medical novelty expected to blossom in the near future to provide a holistic, targeted, precise, and individualistic personalised-medicine as well as laying out the future hopes of gene therapy in the treatment of debilitating diseases such as solid tumours, AIDS, Tuberculosis, Diabetes Mellitus, psychiatric illnesses, which are still at a standstill, from a gene therapy point of view.

Clinical impact of ABCC1 and ABCC2 genotypes and haplotypes in mediating imatinib resistance among chronic myeloid leukaemia patients.

WHAT IS KNOWN AND OBJECTIVE: The introduction and success of imatinib mesylate (IM) has brought about a paradigm shift in chronic myeloid leukaemia (CML) treatment. However, despite the high efficacy of IM, clinical resistance develops due to a heterogeneous array of mechanisms. Pharmacogenetic variability as a result of genetic polymorphisms could be one of the most important factors influencing resistance to IM. The aim of this study was to investigate the association between genetic variations in drug efflux transporter ABCC1 (MRP1) and ABCC2 (MRP2) genes and response to IM in patients with CML. METHODS: We genotyped 215 Malaysian patients with CML (comprising of two groups with 108 IM resistant and 107 IM responsive) for polymorphisms of ABCC1 (2012G>T and 2168G>A) and ABCC2 (-24C>T, 1249G>A and 3972C>T) genes. Genotype, allele and haplotype frequencies were compared between two groups of patients. Patients with CML were further stratified according to their clinical response to IM into those having cytogenetics and molecular responses, and the associations with genotypes were evaluated. RESULTS AND DISCUSSION: We observed no significant differences in the distribution of any of the tested genotypes between the investigated groups. However, on evaluating the risk association, ABCC2 T₋24 G1249 T3972 haplotype was found to be associated with IM resistance (P = 0·046). These results suggest that haplotype variants -24T and 3972T might be associated with lower expression of ABCC2 protein and reduced transport activity and hence might be contributing to development of IM resistance. WHAT IS NEW AND CONCLUSION: Our results suggest the ABCC2 T₋24 G1249 T3972 haplotype was associated with imatinib resistance. However, the evidence is as yet insufficient to establish this haplotype as a predictive biomarker for response to the drug.

The differential metabolite profiles of acute lymphoblastic leukaemic patients treated with 6-mercaptopurine using untargeted metabolomics approach.

BACKGROUND: Acute lymphoblastic leukaemia (ALL) has posed challenges to the clinician due to variable patients' responses and late diagnosis. With the advance in metabolomics, early detection and personalised treatment are possible. METHODS: Metabolomic profile of 21 ALL patients treated with 6-mercaptopurine and 10 healthy volunteers were analysed using liquid chromatography/mass spectrometry quadrupole-time of flight (LC/MS Q-TOF). Principal components analysis (PCA), recursive analysis, clustering and pathway analysis were performed using MassHunter Qualitative and Mass Profiler Professional (MPP) software. RESULTS: Several metabolites were found to be expressed differently in patients treated with 6-mercaptopurine. Interestingly, 13 metabolites were significantly differently expressed [p-value <0.01 (unpaired t-test) and 2-fold change] in 19% of the patients who had relapses in their treatment. Down-regulated metabolites in relapsed patients were 1-tetrahexanoyl-2-(8-[3]-ladderane-octanyl)-sn-GPEtn, GPEtn (18:1(9Z)/0:0), GPCho(O-6:0/O-6:0), GPCho(O-2:0/O-1:0), methyl 8-[2-(2-formyl-vinyl)-3-hydroxy-5-oxo-cyclopentyl]-octanoate and plasma free amino acids (PFAA). Characterizing the subjects according to their ITPA 94C>A genotypes reveal differential expression of metabolites. CONCLUSIONS: Our research contributes to identification of metabolites that could be used to monitor disease progress of patients and allow targeted therapy for ALL at different stages, especially in preventing complication of relapse.

Somatic copy-neutral loss of heterozygosity and copy number abnormalities in Malaysian sporadic colorectal carcinoma patients.

Colorectal cancer is one of the most common cancers in many countries, including Malaysia. The accumulation of genomic alterations is an important feature of colorectal carcinogenesis. A better understanding of the molecular events underlying the stages of colorectal carcinogenesis might be helpful in the detection and management of the disease. We used a commercially available single-nucleotide polymorphism genotyping array to detect both copy number abnormalities (CNAs) and copy-neutral loss of heterozygosity (LOH) in sporadic colorectal carcinomas. Matched tumor and normal tissues of 13 colorectal carcinomas (Dukes' stages A-D) were analyzed using a 250K single nucleotide polymorphism array. An additional assay was performed to determine the microsatellite instability status by using the National Cancer Institute-recommended BAT-26 panel. In general, copy number gain (92.3%) was most common, followed by copy number loss (53.8%) and copy-neutral LOH (46.2%). Frequent CNAs of gains and losses were observed on chromosomes 7p, 8, 13q, 17p, 18q, and 20q, and copy-neutral LOH was observed on chromosomes 2, 6, 12, 13q, 14q, 17, 20p, 19q, and 22q. Even though genomic alterations are associated with colorectal cancer progression, our results showed that DNA CNAs and copy-neutral LOH do not reflect disease progression in at least 50% tumors. Copy-neutral LOH was observed in both early and advanced tumors, which favors the involvement of these genomic alterations in the early stages of tumor development.

Chromosomal abnormalities and reproductive outcome in Malaysian couples with miscarriages.

INTRODUCTION: This study was done to determine the prevalence of chromosomal abnormalities and the subsequent reproductive outcome in couples who had two or more miscarriages. METHODS: 56 couples with a history of at least two previous miscarriages were evaluated for prevalence and types of chromosomal abnormalities from their karyotype records. The study was a retrospective one, and subsequent reproductive outcome after a period of 12-24 months from the time of karyotyping was obtained by telephone interviews and scrutiny of the case records. The comparison of reproductive outcome was done by chi-square statistics. RESULTS: Five couples (8.9 percent) had a chromosomal abnormality in one partner. Three cases of reciprocal translocations t(5;11), t(9;14), dup(9q); one Robertsonian D/D translocation 13/14; and one mosaic Down syndrome male karyotype were found. Among the 32 couples available for follow-up, there was a lower incidence of subsequent live healthy births among chromosomally-normal couples (35.7 percent) compared to chromosomally-abnormal ones (25 percent). However, the difference was not statistically significant (p-value is 1.0). There was a lower incidence of subsequent abortions in chromosomally-normal couples (42.8 percent) compared to chromosomally-abnormal ones (50 percent), but the difference was also not statistically significant (p-value is 1.0). CONCLUSION: Chromosomal abnormalities were seen in 8.9 percent of the couples, and translocations were the commonest abnormality found. The frequencies of subsequent live healthy births and subsequent abortions showed no significant difference between couples having normal karyotypes and those having chromosomal abnormality in one partner.

Clinical manifestations in trisomy 9.

Complete trisomy 9 is a lethal diagnosis and most fetuses diagnosed thus die prenatally or during the early postnatal period and majority of such cases have been known to end in spontaneous abortion in the first trimester itself. One such rare survival of fetus ending in normal delivery and surviving until 20 days is reported here detailing the clinical manifestations of the child during the period of survival. The salient clinical features observed were small face, wide fontanel, prominent occiput, micrognathia, low set ears, upslanting palpebral fissures, high arched palate, short sternum, overlapping fingers, limited hip abduction, rocker bottom feet, heart murmurs and also webbed neck, characteristic of this trisomy 9 syndrome.

Screening of dystrophin gene deletions in Malaysian patients with Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive genetic disorder characterized by rapidly progressive muscle weakness. The disease is caused by deletion, duplication or point mutation of the dystrophin gene, located on the X chromosome (Xp21). Deletion accounts for 60% of the mutations within the 79 exons of the dystrophin gene. Seven exons (43, 44, 45, 46, 49, 50, and 51) were found to be most commonly deleted among the Asian patients. To detect the frequency of deletion of these 7 exons in Malaysian DMD patients, we carried out a molecular genetic analysis in 20 Malaysian DMD patients. The mean age of initial presentation was 60 months (SD 32 months, range 5-120 months). Fourteen patients were found to have deletion of at least one of the seven exons. The remaining six patients did not show any deletion on the tested exons. Deletions of exons 49, 50 and 51 were the most frequent (71.43%) and appear to be the hot spots in our cohort of patients.

Two cases of deletion 5p syndrome: one with paternal involvement and another with atypical presentation.

We report two cases of deletion 5p or cri du chat syndrome (CdCS) with different presentations and risks of transmission: one case with paternal chromosome 5 involvement and another, a de novo case with atypical clinical presentation. Cytogenetic analysis was performed on the two cases and their parents. GTG-banded karyotype analysis of Cases 1 and 2 revealed abnormal 46,XY,del(5)(p13-15) male karyotypes. For Case 1, the mother showed normal female karyotype while the father showed an abnormal karyotype involving a balanced translocation 46,XY,t(5;10)(p13;p15). For Case 2, however, both parents showed a normal karyotype pattern. In Case 1, the clinical features, particularly the distinct facial phenotype in combination with a characteristic cat-like cry and hypotonia, aided in the diagnosis at birth and the karyotype analysis was resolutive. The boy in Case 2 presented with atypical clinical features. Even though this patient had multiple syndromic features, the typical high pitched cat-like cry was not prominent. Instead, the patient manifested persistent stridor (from day three of life), which might have prevented the clinician from suspecting CdCS at birth. However, when this patient was presented at seven months of age for cytogenetic analysis, a confirmatory diagnosis of CdCS was established. For children with congenital abnormalities, an early clinical diagnosis confirmed through cytogenetic and molecular investigations, is important for providing personalised diagnostic and prognostic evaluation, and also for genetic counselling on the reproductive risk, particularly for patients with parental chromosome translocation involvement.

Germline BRCA1 mutation and survival analysis in familial breast cancer patients in Kerala; South India.

Mutations in breast cancer susceptibility gene BRCA1 have been identified in breast or breast/ovarian cancer families from different ethnic background. We analyzed a total of 79 samples for BRCA1 mutation, using Conformation Sensitive Gel Electrophoresis (CSGE) followed by sequencing. The overall survival of BRCA1 mutation carriers was also investigated. BRCA1 mutation was detected in 11 out of the 29 (38%) patients. Four different alterations were detected of three which were novel. A missense mutation in exon 7, 465G>A was detected in 1 patient (9%). Another missense mutation 932 G>A was observed in three patients (27.3%) and a truncation mutation 1027delA, was observed in one patient (9%). The fourth type of mutation (185delAG) which also results in protein truncation was observed in 6 different patients (54.5%). Kaplan-Meier survival analysis revealed a median overall survival of 34 months for BRCA1 mutation positive breast cancer patients and 71 months for BRCA1 negative breast cancer patients. The median overall survival of BRCA1 truncation mutation carriers was 26 months. Our data showed high prevalence of BRCA1 gene mutation among breast or breast/ovarian cancer families in South India and breast cancer patients having BRCA1 mutations were associated with poor prognosis.

Cytogenetic and clinical profile of Down syndrome in Northeast Malaysia.

INTRODUCTION: This study was designed to evaluate the karyotype pattern, clinical features and other systemic anomalies of patients with Down syndrome in Malaysia. METHODS: Retrospective analysis was performed on the case records of 149 patients confirmed as Down syndrome by cytogenetic analysis at Human Genome Centre and Genetic Clinic at the Universiti Sains Malaysia. RESULTS: Among the 149 cases of Down syndrome presenting over a period of 4.2 years, free trisomy (non-disjunction) was present in 141 cases (94.6 percent). One case (0.7 percent) had translocation, and seven cases (4.7 percent) were mosaics. Average age at presentation was 10.6 months. Average maternal age at birth of the affected child was 32.3 years. The prominent craniofacial features noted were upslanting palpebral fissures (89.3 percent), flat facial profile (64.9 percent), low set ears (56.1 percent), epicanthic folds (17.5 percent) and protruding tongue (19.2 percent). A total of 52.6 percent of the cases had documented hypotonia. Characteristic limb and dermatoglyphic anomalies included short stubby fingers (24.5 percent), sandal gap (33.3 percent), unilateral or bilateral simian crease (36.8 percent) and clinodactyly (19.2 percent). Ophthalmological abnormalities, such as hypertelorism, were presented in 33.3 percent of the cases. Congenital heart disease was diagnosed in 35 out of 71 cases (49.3 percent) and gastrointestinal anomalies were noted in 18 out of 79 cases (22.7 percent) analysed. CONCLUSION: Efforts to establish early diagnosis and a proper screening for high association with systemic anomalies should be undertaken among the Down syndrome patients in this population.

Two cases of isochromosome 18q syndrome.

Patients with isochromosome 18q, a rare cytogenetic abnormality, also reported as Edwards syndrome, is the second most common autosomal trisomy. However, the phenotypic features and survival of these patients are not uniform and depend upon the portion of chromosomes getting duplicated or deleted. The survival of these children may be longer, hence a good cytogenetic diagnosis is a must. Morphological characteristics of isochromosome 18q are not yet fully delineated because of the rarity of the cases and as most cases are aborted medically or terminate spontaneously. We report two cases of isochromosome 18q, one male aged two years old and the other a male aged eight months old, and review the literature on this rare syndrome.

Expression of folate sensitive and aphidicolin induced chromosomal fragile sites in familial neuroblastoma.

The expression of folate sensitive and aphidicolin induced fragile sites in the blood lymphocyte chromosomes of affected and unaffected members from 2 neuroblastoma families were studied. The subjects included 4 neuroblastoma patients, and 9 of their clinically healthy first degree relatives and corresponding number of age and sex matched controls. Lymphocytes cultured in folate deprived culture medium showed rare fragile sites at band p13.1 of chromosome 1, in a frequency of 3%-5% in all the 4 neuroblastoma patients. In aphidicolin treated cultures, the patients and unaffected members in neuroblastoma families, showed hypersensitivity to aphidicolin, as evidenced by the significant increase in percentage of aberration/cell (ab/c) and damaged cells (dc), over that of controls (P < 0.01). Aphidicolin induced fragile sites were more pronounced in chromosomes 1 and 2. A larger number of subjects have to be studied to prove whether altered fragile site expression may be a cytogenetic evidence for an individual or familial cancer predisposing genetic constitution.

Deficient DNA repair capacity: a predisposing factor and high risk predictive marker in familial colorectal cancer.

Even though colorectal cancer tends to aggregate in families, there is paucity of information on the genetic determinism for familial colorectal cancer (FCRC) predisposition. Therefore, we investigated constitutional chromosome abnormalities and bleomycin induced chromosome sensitivity of 26 familial and 30 sporadic colorectal cancer (SCRC) patients, 60 unaffected family members (first/second degree relatives) and 30 normal healthy controls to determine whether these parameters could give any clues on genetic predisposing factors by which high risk members in CRC families could be identified. The test assay used bleomycin-induced chromatid breaks in short term microcultures of peripheral blood lymphocytes of the subjects. The CRC patients, the unaffected family members and the controls did not show any constitutional chromosomal abnormalities. However, with regard to bleomycin sensitivity, there was significant difference between the CRC patients, unaffected relatives and controls. The mean b/c values of 1.64+/-0.42 for the FCRC patients and 1.08+/-0.34 for the SCRC patients were significantly higher than the mean b/c values of 0.62+/-0.18 for the unaffected relatives and 0.52+/-0.12 for the controls (P<0.001). A noteworthy observation was that 6 unaffected members from 6 CRC families also showed bleomycin hypersensitivity, at the initiation of the study. Since they expressed mean b/c values greater than 1.0, which was as high a value as those of the patients, they were regularly followed up. Out of these 6 members, 2 developed CRC later. This clearly demonstrates that mutagen hypersensitivity among unaffected relatives in CRC families may be related to cancer predisposition. Hence, this cytogenetic assay could be utilised to identify the genetically high risk individuals in CRC families.

DNA repair proficiency: a potential marker for identification of high risk members in breast cancer families.

Breast cancer is the single largest cancer and causes the high rate of cancer mortality among women. A positive family history of breast cancer is recognized as one of the major risk factors for this disease. The present study evaluates bleomycin (BLM)-induced chromosome sensitivity analysis in breast cancer families which provides indirectly a measure of the DNA repair defect of each person. BLM sensitivity assay on cultured lymphocytes of 36 familial breast cancer patients, their 85 first or second degree female relatives, 36 sporadic breast cancer patients and 40 age- and sex-matched controls (without any family history of cancer) were carried out to measure interindividual variation in their DNA repair capacity through mutagen-induced chromosome sensitivity analysis. Fifty percent of familial breast cancer patients and seven unaffected relatives showed hypersensitivity. Compared to hyposensitive relatives these seven subjects may be considered as high risk individuals.

Increased levels of mutagen-induced chromosome breakage in Down syndrome children with malignancy.

Even though an association between Down syndrome (DS) and malignancies has been established, the mechanism behind this is still unclear. We therefore investigated constitutional chromosomal abnormalities and bleomycin-induced chromosome sensitivity in 12 DS children, 8 DS children with malignancies, and 10 normal controls to explore whether these factors play any role in cancer predisposition. Trisomy 21 was the only constitutional cytogenetic abnormality observed in all the DS children. But there was significant variation between the patients and controls with regard to bleomycin sensitivity. Compared to the normal controls, all the DS patients expressed significantly higher chromosomal breaks per cell (b/c) values indicating sensitivity to bleomycin. Furthermore, DS children with malignancies demonstrated significantly higher b/c values than DS children with malignancies. These results permit us to assume that DS children showing mutagen hypersensitivity may be having defective DNA repair competence and hence may be predisposed to malignancies.

The cytogenetic identification of high risk members in colorectal cancer families.

Family history of colorectal cancer is recognized as a risk factor for the disease and the development of colorectal cancer represents a suitable model for illustrating multistep tumor development. Bleomycin induced chromosome sensitivity studies were done in 7 colorectal cancer families consisting of 12 colorectal cancer patients and their 34 first degree relatives and 12 sporadic colorectal patients for comparison and identification of high risk family members with genetic instability. All patients and 4 unaffected relatives showed increased bleomycin sensitivity, which might be due to defective DNA repair system. These four relatives may be classified as high risk (without cancer at present) individuals. The study is being continued in more number of familial colorectal cancer patients and their relatives to arrive at definite conclusions.