Chromosomal Aberrations in Primary Amenorrhea: A Retrospective Study.

OBJECTIVES: The aim of this study was to estimate the frequency of chromosomal abnormalities and establish the association with clinical of factors such as secondary sexual characters and gonad development in primary amenorrhea (PA). STUDY DESIGN: The study was carried out in a large cohort of PA. The chromosomal aberrations were correlated with secondary sexual characters and anatomical abnormalities. MATERIALS AND METHODS: The data of 490 cases of PA were collected retrospectively. The chromosomal preparations were done from the peripheral blood and subjected to giemsa-trypsin-giemsa banding and karyotyped according to the International System of Human Cytogenetic Nomenclature 2013. The fluorescence in situ hybridization was carried out using centromeric and whole painting probes for X and Y chromosome. STATISTICAL ANALYSIS: Statistical analysis of the data was performed using online version of social science statistics software. RESULTS: A high frequency of abnormal uterus (81.9%) and ovaries (86.7%) were detected in our study. A total of 121 (24.7%) cases were identified with abnormal karyotype. The numerical chromosomal abnormalities were identified in 53 (43.8%) cases while structural abnormalities were identified in 32 (26.4%) cases. The XY karyotype was detected in 29.8% females with PA. The PA individuals with anatomical abnormalities (84.3%) had a high frequency (24.6%) of chromosomal aberrations. CONCLUSIONS: The present study concluded that cytogenetics plays an important role in precise diagnosis which helps in the management of PA. The cytogenetic analysis should be carried out to know the genetic basis of PA.

Centrosome Aberration Frequency and Disease Association in B-Acute Lymphoblastic Leukemia.

Recent developments in genome-wide genetic analysis in B-acute lymphoblastic leukemia (B-ALL) have provided insight into disease pathogenesis and prognosis. B-ALL cases usually carry a primary genetic event, often a chromosome translocation, and a constellation of secondary genetic alterations that are acquired and selected dynamically in a nonlinear fashion. As far as we are aware of, for the first time, we studied centrosome aberration in patients with B-ALL to understand the progression of the disease. A cytogenetic study was carried out by GTG-banded karyotyping and fluorescence in situ hybridization. DNA index study was carried out with flow cytometry. Indirect immunostaining of centrosomes was performed on mononuclear cells using primary and corresponding secondary antibodies for centrosome-specific protein γ-tubulin. Three primary and corresponding secondary antibodies to three different centrosome-specific proteins, namely α-tubulin, γ-tubulin and pericentrin, were used for indirect immunostaining. The study was carried out on 50 patients with B-ALL. Centrosomal abnormalities were detected in 36 (72%) patients and the remainder (28%) had normal centrosome structure and numbers. Out of these 36 patients with abnormal centrosome, structural abnormalities were detected in 12 (33.3%) and numerical abnormalities in six (16.6%). Both structural and numerical aberrations were detected in 18 (50%) patients. When correlated with the cytogenetic and DNA index findings, 26/27 (96.2%) patients had centrosome defects concomitant with both abnormal karyotype and aneuploidy. Out of 50 patients with B-ALL, 17 (34%) had normal karyotype detected by both karyotype and DNA index, among these, seven (41.17%) patients had centrosome aberration. The morphological and structural abnormalities of the centrosome present in B-ALL cells have a role in disease development and can be considered as prognostic markers.

Pattern of chromosome involvement in childhood hyperdiploid pre-B-cell acute lymhoblastic leukemia cases from India.

BACKGROUND: Hyperdiploid pre-B-cell acute lymhoblastic leukemia (pre-B-ALL) is a common form of childhood leukemia with very good prognosis with present day chemotherapy. However, the chromosomal composition of the hyperdiploidy has not been extensively studied and possible mechanism for this pathology remains so far conjectural. OBJECTIVE: To analyze the pattern of chromosome involvement in a cohort of childhood hyperdiploid pre-B-ALL from India and from this pattern to develop an understanding on the causation of such pathology. Whether such patients also carry translocations and FLT3 mutations in addition to their hyperdiploid karyotype. MATERIALS AND METHODS: One hundred and twenty-six childhood pre-B-ALL patients were studied. Bone marrow aspirate of these patients were evacuated for morphology, FAB classification, immunophenotyping and both conventional and molecular cytogenetics. RESULTS: Of 126 patients with pre-B-ALL (age 2-15 years), 90 patients with abnormal karyotype showed 50 with hyperdiploid karyotype (50/90 i.e. 55.5%). Chromosomes 9, 10, 14, 17, 18, 20 and 21 were more often involved in hyperdiploidy. Chromosome 21 duplication was present in 92% of the cases. Chromosomes 5, 15, 16, 17 and Y were less often involved (18-20%) in hyperdiploidy. About 44% of patients with hyperdiploidy had additional karyotypic abnormality of which TEL-AML1 was predominant (24%). Chromosome loss was rare and accounted for 20% of the cases only. We did not find any FLT3 mutation in our patients. CONCLUSION: In this study, the pattern of chromosome involvement in hyperdiploid karyotype of ALL patients is same as other studies except some chromosomes like 1, 6, 11, 12, 19 and 22 have some more frequent involvement than other studies. This study also showed the occurrence of TEL/AML1 fusion is more (19.8%) than other reports from India.

Chimeric fusion karyotypes in childhood B-cell acute lymphoblastic leukemia.

Cytogenetics study using combination of conventional cytogenetics and fluorescent insitu hybridization was carried out in 171 pediatric acute lymphoblastic leukemia patients subgrouped to B-ALL (n=126) and T-ALL (n=45) by bone marrow morphology and immunophenotype. The chromosomal aberration frequency in B-ALL and T-ALL was 79% and 71%, respectively. TEL/AML1 translocation was detected in 28% of patients.

Standard karyotyping concentrates microfilaria and can be a valid concentrating technique for their detection.

During karyotype preparation from the bone marrow aspirates of 209 haematological malignancy cases, microfilaria were detected in four samples, whereas routine marrow and peripheral blood smears of these four cases did not show any parasite. The patients were recalled, and their peripheral blood was processed by karyotyping and standard concentration techniques. Karyotype preparation from peripheral blood was performed with and without addition of colchicine. When the blood was processed for karyotyping with colchicine, microfilaria were detected in the peripheral blood of all four patients. In samples without added colchicine, no parasite was observed. The same samples were processed by Knott's concentration technique, which showed microfilariae only in one of the four patients. Routine thick and thin smears of these patients showed no parasite. It seems that the standard karyotype preparation technique with colchicine concentrates the microfilariae in samples where parasite load is small and not demonstrable with standard techniques. Serological tests are available for W. bancrofti and costly, whereas no regular serodiagnosis is available for B. malayi. In a country like India, both parasites are endemic and patients are treated on clinical suspicion when parasitaemia could be low. Low parasitaemia is common because of repeated infection and partial immunity. In such circumstances, a cost-effective concentration technique like this may be useful.

FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India.

Acute myeloid leukemia (AML) is an aggressive hematological disorder characterized by the loss of ability of the hematopoietic progenitor cells to differentiate and proliferate normally leading to an accumulation of immature myeloid cells in the bone marrow. Several novel molecular genetic aberrations in FLT3 and NPM1 have been shown to have a prognostic impact in AML, particularly in those having normal karyotype. Though there is substantial amount of data on these mutations from western literature, there is surprisingly little data from Indian subcontinent on the frequency of this mutation in AML patients from India. The present study screens a large cohort of non-acute promyelocytic leukemia (APL) AML patients (207 patients) for the presence of FLT3 and NPM1 mutations and further correlates with cytogenetics, immunophenotypic characteristics and with follow-up data wherever available. During the course of study, 56 APL patients were also studied. Briefly, both FLT3 (internal tandem duplication (ITD) in 19.4% and tyrosine kinase domain (TKD) in 9%) and NPM1 mutations were detected in 28.4% of the total non-APL AML patients screened showing distinct correlations with hematologic, immunophenotypic, cytogenetics characteristics and follow-up. With regards to adult APL patients, 22.2 and 32.6% of the patients showed FLT3 and NPM1 mutation, respectively. In the pediatrics age group (<15 years), 23 and 16% of patients with APL showed FLT3 and NPM1 mutation, respectively, while in non-APL patient is this age group, 23% of patients showed both FLT3 and NPM1 mutation. NPM1 mutation was distinctly uncommon in younger age group of patients. In contrast to report elsewhere, most of our FLT3 mutation was in exon 11 rather than in exon 12. FLT3 mutation due to ITD or TKD mutation was detected in 2:1 ratio in our patients and a new TKD mutation was also detected S840G in an M5 patient who did not go into remission and had a short survival of 3 months from diagnosis. Generally, patients with NPM1 mutation had a very high white cell count but they went into remission more often than those with wild (Wt)-type allele (written as NPM1- and FLT3-, respectively) and FLT3 mutation. These patients also tended to have significantly lower expression of CD34 antigen on flowcytometry. Distinct prognostic subclasses of adult AML patients were identified based on the presence of NPM1 and FLT3 mutations.

DNA copy number changes and immunophenotype pattern in karyotypically normal acute myeloid leukemia patients from an Indian population.

Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). The purpose of this study was to identify DNA copy number variations in karyotypically normal AML patients and their correlation with immunophenotypes. Conventional comparative genomic hybridization (CGH) and immunophenotyping were performed in 46 untreated AML patients aged 7-68 years. Among the 86 Indian patients who had AML, 40 (46.5%) showed an abnormal karyotype and 46 (53.4%) showed no chromosome aberrations. The karyotypically abnormal AML patients were excluded from the study. Out of the 46 patients without chromosomal aberrations, 24 (52.2%) showed DNA copy number variations including losses and gains. The DNA copy number variations involved chromosomes 1, 3, 6, 12, 15, 16, 17 (gains) and 1, 4, 2, 3, 5, 7, 8, 9, 10, 11, 13, 15, 18, 20, 21 (losses). The aberrant immunophenotype was noticed in 13 of these 24 (54%) cases. The hidden chromosome rearrangements in karyotypically normal AML, which could not be detected by conventional cytogenetics and fluorescence in situ hybridization, were detected by CGH. These genetic changes have an important role in the prognosis of the disease. The DNA copy number changes might also be involved in the aberrant immunophenotypes in our study.

Cytogenetic study of myelodysplastic syndrome from India.

BACKGROUND & OBJECTIVE: Myelodysplastic syndrome (MDS) represents a group of clonal haematological disorders characterized by progressive cytopenia reflecting defects in erythroid, myeloid and megakaryocytic maturation. The incidence of MDS is more in older age groups and frequent chromosome abnormalities reported to be monosomies 5 and 7. However, the data on cytogenetic changes in Indian MDS patients are scanty. The present study was therefore undertaken to study the aetiology and frequency of chromosomal changes in MDS patients, attending a tertiary care hospital in Maharashtra, India. METHODS: The study was carried out in 145 MDS patients for six years (2001-2006) at National Institute of Immunohaematology (ICMR), and KEM Hospital, Mumbai, India. The patients were diagnosed according to FAB and WHO classification. Cytogenetic study was carried out using GTG-banding and fluorescence in situ hybridization (FISH) methods. Statistical analysis was done with Chi(2) and Fisher's exact test. RESULTS: Chromosomal abnormalities, including novel chromosome aberrations were detected in 54.48 per cent MDS patients and frequency of chromosomal aberrations increased with increase in age (> or = 30 yr). Among occupational exposure factors, chromosomal aberrations significantly (P<0.05) associated with pesticides exposure. INTERPRETATION & CONCLUSION: Our findings showed 54.48 per cent chromosome abnormalities including novel chromosome aberrations in MDS patients and these chromosome aberrations were increased with advancing age. In our series a high frequency of younger population (53%) developed MDS, a detailed molecular genetics and aetiological factors need to be studied.

Chromosomal breakage in myelodysplatic syndrome.

The myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia reflecting defects in erythroid, myeloid and mega karyocytic maturation. The incidence of MDS is greter in older age groups. Detailed studies on MDS from India are not available. Cytogenetic study using GTG-banding and FISH revealed 54.5% clonal chromosomal abnormalities. We have carried out chromosomal breakage study from peripheral blood cultures induced with mitomycin C, in karyotypically normal MDS (49) and 15 (30.6%) showed significant (p < 0.001) increase in chromosome damage compared to controls. Among 22 occupationally exposed MDS, 6 (27.3%) showed a high frequency of chromosome breakage while in the non-exposure (n=27) group, high chromosome breakage was noted in 9 (33.3% ) MDS patients. Our results suggest that the high chromosome damage may be due to acquired Fanconi anemia which leads to multiple defects in chromosomes and clonal chromosome anomalies.

Dandy-Walker malformations in a case of partial trisomy 9p (p12.1→pter) due to maternal translocation t(9;12)(p12.1;p13.3).

We describe a five-year-old proband presented with Dandy-Walker malformations, right microopthalmia, hamstring contractures, undescended testis with absence of testis in right scrotum in addition to typical trisomy 9p clinical features. Routine cytogenetic studies with GTG - banding showed 46,XY,der(12)t(9;12) (p12;q13.3),mat karyotype (trisomy 9p). Chromosomal analysis of the father was normal and phenotypically normal mother had 46,XX,t(9;12)(p12;q13) karyotype. Fluorescence in situ hybridization analysis with single copy probes bA5OIA2 (9p11.2), bA562M8 (12p12.1) and centromere probes (9) showed break point at 9p12.1 region. The gene dosage effect of Chromosome 9p along with environmental factors might be associated with Dandy- Walker malformations in the patient.