We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.
Generally, the karyotype profile of Down Syndrome has been reported to be full trisomy 21 in 92% of patients, mosaic trisomy 21 in 4% of patients and translocation involving chromosome 21 in 4% of patients in most of the population groups worldwide. But, karyotype analysis of 149 DS patients at the Human Genome Center, USM, during the past five years revealed that free trisomy accounted for 94.6%, mosaic trisomy 21 for 4.7% and translocation involving chromosome 21 in 0.7% of the Down Syndrome etiology in North East Malaysian population, indicating a low frequency of translocation DS in this region. Here, we report one case of translocation Down Syndrome encountered during karyotype analysis of 149 DS cases. Karyotype showed a robertsonian translocation where an entire extra chromosome 21 was attached to the centromere of one of the chromosome 14, resulting in a derivative chromosome 14 with attached chromosome 21. Karyotype analysis of the parents revealed a normal 46,XY pattern for father and 46,XX pattern for mother indicating that this robertsonian translocation had arisen de novo either prior to or at conception.
Fragile X syndrome is a result of an unstable expansion of (CGG)n trinucleotide sequences in the FMR-1 (Fragile X Mental Retardation 1) gene site at Xq27. In a normal person, n ranges from 6 to 40 repeats with an average of 30 repeats, whereas in a mutated FMR1 gene the sequence is repeated several times over (stuttering gene). Full mutation occurs when n equals 200 repeats or more. Where n equals 50 to 200 repeats, it is a premutation. Fragile X occurs when the FMR-1 gene is unable to make normal amounts of usable Fragile X Mental Retardation Protein, or FMRP. The amount of FMRP in the body is one factor that determines the severity of the Fragile X syndrome. A person with nearly normal levels of FMRP usually has mild or no symptoms, while a person with very little or no normal FMRP has more severe symptoms. The mechanism for the role of the FMRP gene is still being researched upon. However, it has been observed that large numbers of repeats (more than 200) inactivates the gene through a process of methylation and when the gene is inactivated, the cell may make little or none of the needed FMRP. Inheritance is X-linked with reduced penetrance and the frequency of occurrence goes up through generations. The phenotypic manifestations of fragile-X syndrome vary and are largely dependent on the size of the mutation or premutation. The identification of the fragile site on G banded metaphases is a time consuming and delicate process requiring experience and skill, however, molecular diagnosis using DNA analysis and Southern blotting, even though expensive, is more specific in determining the presence or absence of the gene. This study was aimed to establish a rapid polymerase chain reaction (PCR) based - touch down PCR, as a screening method for fragile X syndrome. A total of six cases were analysed. Of these, one was a known case of Fragile X (T1) diagnosed by conventional cytogenetics, two were from the latter’s family members namely, his mother (T2) and father (T3), and the other two (T4 and T5) were randomly selected from patients presenting with dysmorphic features and delayed development respectively. One normal control (TC) was included. Cytogenetic analyses for detection of the fragile site was carried out in all cases. Two culture systems were used, namely the synchronised lymphocyte culture and the folate - thymidine deficient culture. Stained metaphases from the fragile X cultures were screened for the presence of the fragile site on the X chromosome. G-banded karyotyping was done using an image analyser to exclude presence of chromosomal abnormalities. DNA was extracted from these samples and amplified by touch-down PCR. Cytogenetic analysis revealed a folate-sensitive fragile site in the affected male, but none in the other five samples. G-banded karyotyping exhibited no additional chromosomal abnormalities. All extracted DNA samples were successfully amplified. Five of the samples showed presence of the product at the expected band at 552bp, excluding the presence of an expansion of CGG segment of the FMR-1 gene. The absence of a band in an affected individual, suggested a fully mutated allele of FRAXA (Folate Sensitive Fragile Site at Xq28). We succeeded in establishing a slightly modified touch-down PCR analysis. Our study indicates that PCR testing offers a rapid and specific method for screening of normal allele and full mutation of the fragile X gene. We suggest this technique to be applied as a complementary tool for cytogenetic analysis to detect the FRAXA gene.
Down Syndrome (DS), is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. It is the most common autosomal abnormality among live births and the most commonly recognized genetic cause of mental retardation. The only well established risk factor for DS is advanced maternal age. The Human Genome Center , University Sains Malaysia, Kelantan has been carrying out cytogenetic studies in DS patients. Here we, report the karyotype pattern of Down Syndrome patients in correlation with maternal age, among referral cases to our Center.
The reciprocal translocation t(9;22)(q34;q11) which gives rise to the Philadelphia (Ph1) chromosome and BCR/ABL fusion gene, plays a pivotal role in the diagnosis and pathogenesis of chronic myeloid leukemia (CML). In this study, we evaluated the role of fluorescence in situ hybridisation (FISH) in detecting the BCR/ABL rearrangement in CML patients. The sensitivity, specificity and detection rate of BCR/ABL gene using FISH, PCR and conventional cytogenetics (karyotyping) methods were also compared. 18 bone marrow samples of patients with clinically diagnosed CML and suspected of CML were collected. The sensitivity, specificity and positive predictive values of FISH were altogether 100% while the sensitivity, specificity and positive predictive values for conventional cytogenetics (karyotyping) were 85%, 100% and 100% respectively. Convetional cytogenetics (karyotyping) detected an additional chromosomal aberration in addition to the Ph1 chromosome. In conclusion, FISH is a highly sensitive method in detecting the BCR/ABL gene. Conventional cytogenetics (karyotyping) remains an important investigation in the work up of suspected CML patients since there is a possibility of detecting chromosomal aberrations in addition to the Ph1 translocation. Therefore, conventional cytogenetics (karyotyping) and FISH are complementary techniques and their results should be interpreted together with clinical information.
A 25-year-old man was referred to Hospital UKM with a 2-week history of fever, productive cough and loss of appetite. Physical examination revealed an ill-looking, tachypnoeic young man. No obvious lymphadenopathy or organomegaly was noted. Examination of the respiratory system revealed right pleural effusion. Full blood picture demonstrated leukocytosis with 90% blasts, and bone marrow examination confirmed the diagnosis of acute myeloid leukemia (AML) French-American-British (FAB) classification of M4 with eosinophilia. His chromosome karyotyping showed complex karyotypic abnormalities. Cytological examination of the pleural fluid demonstrated numerous blast cells indicating leukemic infiltration of the lungs, which is a rare presentation in AML. He was then started on induction chemotherapy with intravenous daunorubicin and cytarabine. In the midst of treatment, he developed an episode of seizure and cerebro-spinal fluid cytology confirmed central nervous system (CNS) leukaemic infiltration. Additional intrathecal methotraxate was given. Repeat bone marrow examination done on day 15 of chemotherapy showed persistence of excess blasts indicating refractory AML. He was then reinduced with high dose cytarabine but to no avail. The disease progressed and he succumbed about 8 weeks after the initial diagnosis was made. We highlight here a case of AML-M4Eo with complex karyoyptic abnormalities presenting with leukaemic infiltration of the lungs and CNS which had imparted a bad prognosis for this subtype of AML, AML-M4Eo.
The present study is aimed at finding the mutagenicity and cytotoxicity of dense form of synthetic hydroxyapatite (Source: School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia) in the blood of sheep. The biomaterial was implanted in the tibia of Malin, an indigenous sheep breed of Malaysia. Blood was collected from the sheep before implantation of the biomaterial, cultured and a karyological study was made. Six weeks after implantation, blood was collected from the same animal, cultured and screened for chromosome aberrations. The mitotic indices and karyological analysis indicated that the implantation of synthetic hydroxyapatite (dense form) did not produce any cytotoxicity or chromosome aberrations in the blood of sheep.
Cytogenetic analysis performed at diagnosis is considered to be the most valuable prognostic factor in acute myeloid leukemia (AML). Large systematic studies of cytogenetic abnormalities in AML patients from Southeast Asia are not available. The karyotypic patterns in AML patients from a single center in Singapore were studied and compared with reports from other regions of the world to identify possible geographic heterogeneity.
Tumor cytogenetic analysis from 27 patients with breast cancer diagnosed at the Singapore General Hospital revealed complex karyotypic aberrations in 12 cases. The study group comprised 25 women and 2 men, ranging in age from 33 to 78 years (median 52 years). Ethnic distribution consisted of 22 Chinese, 3 Malaysian, and 2 Indian patients. Pathologic assessment disclosed 24 invasive ductal, 2 invasive mucinous, and 1 mixed invasive mucinous and ductal carcinomas. Histologic grading showed 3 grade 1, 10 grade 2, and 12 grade 3 tumors; 2 cancers were not graded, because they had been subjected to prior chemotherapy. Tumor sizes ranged from 1.5 to 10 cm (median 3 cm). Eleven cases were axillary node negative, whereas the remaining 16 node-positive cancers affected as many as 3 nodes in 8 cases and 4 or more nodes in another 8. Twenty cases demonstrated estrogen-receptor positivity, and 8 cases progesterone-receptor positivity. The spectrum of cytogenetic abnormalities involved chromosomes 1, 3, 6, 7, 8, 11, 16, and 17 and ranged from gains and deletions of both long and short arms, trisomy, monosomy, and other rearrangements. There was a trend toward the presence of karyotypic abnormalities in tumors of higher grade.
There has been no recent report on the dermatoglyphics of the Malays (normal population as well as patients with Down's syndrome). A study on the frequencies of the dermal patterns (dermatoglyphics) of the digits, palms and hallucal areas was done therefore in 40 Malay patients with Down's syndrome and 200 unrelated normal controls. Only the patients with the standard 21 trisomy karyotype were included in the study. Comparison was made with the published data on studies done in various racial groups. Significant differences of the dermal patterns were found not only between the controls but also among patients of different races.
A high-resolution G-banding technique was used to identify five metacentrics that characterize Suncus murinus from Sri Lanka. These metacentrics were shown to be the product of Robertsonian fusion of acrocentric chromosomes identical to those in the standard karyotype defined by M.B. Rogatcheva et al. Two of the metacentrics in the Sri Lankan shrews (Rb(10.12) and Rb(14.15)) were the same as those reported by C.H. Sam et al. in Malayan populations of S. murinus. This finding provides strong support for the suggestion of T.H. Yosida that metacentric-carrying shrews colonized Malaya from Sri Lanka and hybridized with individuals of standard karyotype, generating the Robertsonian polymorphism now observed. In addition to the Robertsonian variation in S. murinus, we have used our high resolution technique (G- and C-banding) to characterize variants on chromosome 7, the X chromosome, and the Y chromosome.
The finding of a supernumerary or marker chromosome in a karyotype poses difficulty in genetic counselling. The true incidence and significance of this chromosomal aberration is unknown in Malaysia. We report two patients who presented with supernumerary chromosomes in mosaic Turner syndrome.
A case report of a male true hermaphrodite with 46XX/46XY karyotype is presented. He was first diagnosed at the age of 9 years when he presented with hypospadias and a left undescended testis. He was lost to follow-up until he presented at the age of 23 years with bilateral gynaecomastia. A hormonal profile showed a low testosterone level, while a seminal assay showed very few sperms. However he claimed to be sexually active. A year later, after he got married, he began to complain of impotence. A review of the condition is presented.
The haematological findings and case history of 3 patients with the association of acute myeloid leukemia and translocation involving the long arm of chromosome no. 11 are presented. The recipient chromosome for the translocated material from chromosome 11 differs in all the three cases being namely chromosomes 1, 10 and 17.
We report here a rare case of bilateral pure gonadoblastoma which accounts for only 0.2% of all ovarian tumours seen at Universiti Kebangsaan Malaysia from 1980 to 1987. This tumour occurred in an 18 year old Chinese "female" who presented with primary amenorrhoea. Examination showed a phenotypic female with poorly developed external gentalia. Exploratory laparotomy revealed a hypoplastic uterus, rudimentary fallopian tubes and streak gonads. Histological examination of the gonads showed a mixed tumour comprising large germ cells and smaller sex cord derivatives arranged in characteristic nests or islands containing hyaline material.
Cytogenetic investigations were carried out on 117 women with primary amenorrhea who had been referred to our Genetics Laboratory by clinicians throughout Malaysia, after exclusion of other causes of the disorder. Thirty-six cases (31%) showed numerical or structural abnormalities of the sex chromosomes. These can be broadly classified into 4 main types, namely, presence of a Y chromosome (14%), X-chromosome aneuploidies (8%), structural anomalies of the X-chromosome (7%) and lastly, presence of a marker chromosome (2%). Mosaics constituted 17% of the abnormalities observed, always in association with a 45,X cell line. There was no observable correlation between the phenotype of the patients and their respective abnormal karyotypes. The aetiological role of sex chromosomal abnormalities in these amenorrheic women is discussed.
Chromosome analysis on different breed types of water buffaloes (Bubalus bubalis) was undertaken to identify their karyotypes and to determine the pattern of chromosome segregation in crossbred water buffaloes. Altogether, 75 purebred and 198 crossbred buffaloes including 118 from Malaysia and 80 from the Philippines, were analyzed in this study. The diploid chromosome number of the swamp buffalo from both countries was 48 and that of the river buffalo was 50, while all F1 hybrids exhibited 49 chromosomes. The F2 hybrids consisted of three different karyotype categories (2n = 48, 2n = 49, and 2n = 50), whereas the backcrosses included two different karyotype categories each, with 2n = 48 and 2n = 49 in the three quarters swamp types and 2n = 49 and 2n = 50 in the three quarters river types. Chi-square tests on pooled data from Malaysia and the Philippines indicated that the distribution of different karyotype categories of F2 animals did not deviate significantly from the 1:2:1 ratio expected if only balanced gametes with 24 and 25 chromosomes were produced by the F1 hybrids. In the three quarters swamp and three quarters river types, the respective karyotypic categories were in ratios approximating 1:1. The distribution of chromosome categories among the F2 hybrids and backcrosses suggests that only genetically balanced gametes of the F1 hybrids are capable of producing viable F2 and backcross generations.
The current information on isoenzyme studies of nematode parasites was reviewed. The genetic heterogeneity as reviewed by these studies was highlighted. Application of isoenzyme studies and the role of biotechnological techniques in isoenzyme studies was discussed, and the status of cytogenetic studies on nematode parasites was presented.
Karyotypes and crossing relationships were investigated for three allopatric populations of Anopheles leucosphyrus in Southeast Asia: South Kalimantan, Sumatra and Thailand. The mitotic karyotypes of these populations were similar to those previously observed in other species of the An. leucosphyrus group. Populations from Thailand and South Kalimantan exhibited telocentric and subtelocentric sex chromosomes, respectively, with a distinctive band of intercalary heterochromatin in the X chromosome. Strikingly different submetacentric X and Y chromosomes were observed in the population from Sumatra, and it seems likely that the evolution of these chromosomes occurred through the acquisition of constitutive heterochromatin. Sterile F1 males were observed in crosses between the Sumatra population and the populations from South Kalimantan and Thailand. No genetic incompatibility was observed in crosses between the latter two populations. We believe that the present concept of An. leucosphyrus includes two allopatric species, one inhabiting Borneo, West Malaysia and southern Thailand and one confined to Sumatra.