Frequency and pattern of chromosomal abnormalities in acute myeloid leukemia from Western India: A retrospective study.

Context: Chromosomal abnormalities play an important role in diagnosis and prognosis of hematological diseases. Aims: The aim of the present study was to study the pattern and frequency of chromosomal aberrations in acute myeloid leukemia (AML) subgroups from western India. Settings and Design: A retrospective study was conducted through evaluating laboratory proforma which were filled during 2005 to 2014 for diagnosis and treatment of AML subjects. Methods and Material: We have studied chromosomal aberrations in 282 subjects with AML from western India. AML patients were sub-grouped according to FAB classification. Cytogenetic study using conventional cytogenetics (GTG-banding) and Fluorescence in situ hybridization (FISH) was carried out using FISH probes (AML1/ETO, PML/RARA, CBFB). Statistical analysis: Student's t test for continuous variables and Pearson's Chi-squared test for categorical variables were used to identify the relationship between variables. Results: Cytomorphological study revealed AML- M3 as most frequent (32.3%) group followed by AML-M2 (25.2%) and AML-M4 (19.9%). Chromosomal abnormalities were identified in 145 (51.42%) of the total AML cases. A high frequency (38.6%) of chromosomal abnormalities was identified in AML-M3 subgroup as compared to AML-M2 (31%) and AML-M4 (20.6%). Conclusions: Cytogenetic study is important for the diagnosis and management of the AML patients. Our study identified chromosomal abnormalities in AML subgroups with varied frequencies. It is important in diagnosis and monitoring of the disease. As younger AML patients were more affected in our study, etiological factors such as environmental factors need to be studied. Combination of conventional cytogenetics and FISH has an advantage of identifying high frequency of chromosomal aberrations in AML patients.

A Cross-Sectional Study to Correlate Disease Severity in Bullous Pemphigoid Patients with Serum Levels of Autoantibodies Against BP180 and BP230.

CONTEXT: Enzyme-linked immunosorbent assay (ELISA) for BP 180 and 230 antibodies is commonly done in patients with bullous pemphigoid. We could not find much data regarding the usefulness of this test to predict the disease severity in Indian population. AIMS: We studied the correlation of IgG anti BP180 and anti BP230 antibody titer with disease severity and clinical features in bullous pemphigoid. SETTINGS AND DESIGN: This cross-sectional study was conducted at a tertiary care center in western India. MATERIALS AND METHODS: Forty-two clinically diagnosed treatment-naive cases of bullous pemphigoid were enrolled and investigated with skin punch biopsy, IgG anti BP180, and anti BP230 ELISA, direct immunofluorescence, and indirect immunofluorescence tests. Disease severity was assessed by calculating modified Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) score. Thirty patients with a final diagnosis of bullous pemphigoid were included in the statistical analysis. Pearson's correlation coefficient (r) was used to study correlation. RESULTS: The mean ABSIS skin score was 32.81 when both tests were negative, 42.13 when only BP230 was positive, 76.28 when only BP180 was positive, and 78.16 when both were positive. Pearson's correlation coefficient (r) for BP180 and ABSIS skin score was 0.6 (P value: 0.0005), and for BP230 was -0.055 (P value: 0.600). CONCLUSIONS: BP antibody titers correlate partially with disease severity. Anti-BP180 antibody is associated with more severe disease. Anti-BP230 antibody titer does not correlate with disease severity.

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.

Partial trisomy 9 (9pter->9q22.1) and partial monosomy 14 (14pter- >14q11.2) due to paternal translocation t(9;14)(q22.1;q11.2) in a case of Dysmorphic features.

Trisomy 9 including mosaic and partial trisomy is less frequently seen chromosomal abnormality in live born children. The pure or partial trisomy 9 frequently been reported in prenatal diagnosis and product of conception. However few studies reported partial trisomy 9 in live born children. In addition data on genotype and phenotype correlation of partial trisomy is not well understood except few case reports. Here we report a case of partial trisomy 9 and monosomy 14 with a 46,XY,der(9)t(9;14)(q22.1;q11.2)pat,-14 karyotype in a 5-year old dysmorphic child. The proband was confirmed as trisomic for 9pter->9q22.1 and monosomic for 14pter->q11.2 due to paternal t(9;14)(q22.1;q11.2) balanced translocation using a combination of conventional and molecular cytogenetic (fluorescence in situ hybridization, array-comparative genomic hybridization) techniques. The clinical features similar to pure trisomy 9 is due to duplication of the large region of chromosome 9. However, the present report of partial trisomy 9 and monosomy 14 is a novel case report and showing comparatively longer survival which have not been previously reported in the literature. The parent of the proband was counseled for the future pregnancies.

Cytogenetic abnormalities and genomic copy number variations in EPO (7q22) and SEC-61(7p11) genes in primary myelodysplastic syndromes.

Myelodysplastic syndromes (MDSs) are heterogeneous clonal haematopoeitic stem cell disorders characterized by ineffective haematopoeisis, cytopenias and risk of progression to AML. We studied 150 MDS patients for cytogenetic aberrations and 60 patients with normal karyotype and 40 patients harboring cytogenetic abnormalities for copy number variations (CNVs). Cytogenetic abnormalities were detected in 46% of patients with a majority of patients harboring abnormalities of chromosome 7 and del (20q) at frequencies of 16% and 12% respectively. We explored the potential of quantitative multiplex PCR assay of short fluorescent fragments (QMPSF) to identify CNVs and correlated the findings with cytogenetic data and disease prognosis. CNVs (n=31) were detected in 28.3% of karyotypically normal and 23% patients with abnormal karyotype. Genetic losses or deletions (n=26) were more frequent than duplications (n=5). EPO (7q22) and SEC-61(7p11) emerged as new candidate genes susceptible to genetic losses with 57.7% deletions identified in regions on chromosome 7. The CNVs correlated with International Prognostic Scoring System (IPSS) intermediate disease risk group. Our integrative cytogenetic and copy number variation study suggests that abnormalities of chromosome 7 are predominant in Indian population and that they may play a secondary role in disease progression and should be evaluated further for asserting their clinical significance and influence on disease prognosis.

Association of XPD (Lys751Gln) and XRCC1 (Arg280His) gene polymorphisms in myelodysplastic syndrome.

Myelodysplastic syndromes (MDSs) are heterogeneous hematopoietic disease characterized by ineffective haematopoiesis that frequently transforms into acute leukaemia. Alterations in many individual biologic pathways have been reported in MDS pathophysiology. Disease progression along the MDS, acute myeloid leukemia (AML) continuum is believed to be a consequence of stepwise accumulation of DNA mutations which infers a defect in DNA repair. The present study investigated the association between DNA repair genes (XRCC1, XRCC3, OGG1, XPD and RAD51) and the risk of developing MDS. The study was carried out in 92 primary MDS patients. The genotyping study was carried out by PCR-RFLP technique. We have studied seven single-nucleotide polymorphisms (SNPs) of five DNA repair genes (XRCC1 (Arg194Trp, Arg280His, Arg399Gln), XRCC3, XPD, RAD51 and OGG1). Significantly, a high frequency of DNA repair gene XRCC1 (Arg280His) (p=0.05) and XPD (Lys751Gln) (p=0.01) polymorphism was observed in MDS patients compared to controls. The distribution of polymorphisms in MDS subgroups showed a significant association of XRCC1 with RAEB I compared to other subgroup. Though a high frequency of XRCC1 gene polymorphism was observed in farmers and tobacco chewers, it was not statistically significant. Our study suggests that XRCC1 (Arg280His) and XPD polymorphisms are associated with risk of MDS and XRCC1 polymorphism strongly associated with advanced MDS subgroup. Hence, these polymorphisms can be used as a prognostic marker in MDS.

Down-regulation of miR-199b associated with imatinib drug resistance in 9q34.1 deleted BCR/ABL positive CML patients.

Chronic myeloid leukemia (CML) occurs due to t(9,22) (q34;q11) and molecularly BCR/ABL gene fusion. About 15-18% Philadelphia positive CML patients have gene deletions around the translocation breakpoints on 9q34.1. The microRNAs (miRNAs), namely miR-219-2 and miR-199b, centromeric to the ABL1 gene are frequently lost in CML patients. We have designed a study to determine miR-219-2 and miR-199b expression levels which would help to understand the prognosis of imatinib therapy. A total of 150 CML patients were analyzed to identify 9q deletion. Fluorescent in-situ hybridization (FISH) was performed using BCR/ABL dual color, dual fusion probe to study the signal pattern and BAC probes for miR-199b and miR-219-2 (RP11-339B21 and RP11-395P17) to study the miRNA deletions. The expression level of miRNA was analyzed by real-time polymerase chain reaction (RT-PCR). FISH analysis revealed 9q34.1 deletion in 34 (23%) CML patients. The deletions were not detected using BAC probes for miRNAs in 9q deleted patients. The expression analysis showed down-regulation of miR-199b and miR-219-2 in the 9q deleted patients (34 CML) as compared to a pool of patients without deletion. However, miR-199b (9q34.11) was significantly (p=0.001) down-regulated compared to miR-219-2. The follow-up study showed that the miR-199b was found to be strongly associated with imatinib resistance, as 44.11% patients showed resistance to imatinib therapy. Hence, the deletion in 9q34.1 region (ABL) plays an important role in disease pathogenesis. Eventually, miRNAs can provide new therapeutic strategies and can be used as a prognostic indicator.

De novo origin of multiple small supernumerary marker chromosomes (sSMCs) in a child with intellectual disability and dysmorphic features.

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.

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.

A first case of primary amenorrhea with i(X)(qter---q10::---qter), rob(13;14)(q10;q10), inv(9)(p13q33) karyotype.

Primary amenorrhea (PA) refers to the absence of menarche by the age of 16-18 years although secondary sexual characters are developed. PA occurs in 1-3% of women in the reproductive age group. Various factors such as anatomical, genetic and hormonal factors reported to influence PA. We report triple chromosomal abnormalities of rob(13;14)(q10;q10),inv(9)(p13q33), i(Xq)(qter---q10::---qter) in a case of PA and short stature. Though proband has multiple chromosome aberrations, genotypic effect of only i(Xq) is evident as proband has PA and short stature. The rob(13;14) and inv(9), which are paternally derived may have role in later reproductive age. Therefore, chromosomal analysis is essential in such cases for the accurate diagnosis and management of the disease.

Chromosomal breakage study in children suspected with Fanconi anemia in the Indian population.

Chromosomal breakage investigation using diepoxybutane induction was carried out in 195 pediatric patients suspected with Fanconi anemia (FA). Chromosomal breakage evaluation showed 33 (17%) patients with classical FA, 9 (4%) with somatic mosaicism FA, (when at least 50% of the metaphases showed chromosomal breakage and radial figures), 25 (13%) with FA with high frequency of chromosomal breakage and without clinical features, and 128 (66%) with suspected FA but had no chromosomal breakage and clinical features of FA. Chromosomal breakage investigation is an important diagnostic tool for differentiating FA from idiopathic aplastic anemia.

Clinical, genetic and cytogenetic study of Fanconi anemia in an Indian population.

Fanconi anemia (FA) is a rare autosomal recessive genetic disease, associated with congenital anomalies and a predisposition to cancers. FA patients exhibit spontaneous chromosome breakage and FA cells are sensitive to DNA interstrand crosslink agents and expresses high frequency of chromosome breakage. Recently 13 genes have been shown to be involved with the FA phenotype. We have carried out a detailed study in clinically diagnosed FA patients in an Indian population. Thirty three patients were clinically diagnosed with FA and had aplastic anemia and bleeding abnormalities. The genetic analysis revealed a significantly (P<0.0001) high frequency (36.4%) of parental consanguinity in FA patients compared to controls (3.33%). Chromosomal analysis revealed spontaneous chromosome breakage in 63.64% FA patients. The mitomycin C and diepoxybutane induced cultures showed a significantly (P<0.001) high frequency of chromosome breakage and radial formation compared to controls. Among 33 patients, nine (27.27%) patients developed malignancies and chromosomal abnormalities were detected in five (55.5%) patients bone marrow cells including monosomy 5 and 7, trisomy 10, der(1q) and inv(7). Cytogenetic investigation is important in aplastic anemia to rule out FA. The clinical presentation and the associated high frequency of consanguinity in FA, and the molecular analysis are complementary in the study of an Indian population.

Familial small supernumerary marker chromosome (sSMC) (14)(:p11-q11:) [corrected] in a child with translocation Down syndrome.

We report a case of familial small supernumerary marker chromosome (sSMC)in a child with translocation Down syndrome (DS)and mother. The GTG-banded chromosomal analysis of DS child revealed 47,XY,+21,+mar and mother karyotype was 47,XX,+mar. The GTG-banded sSMC had a similar morphology of small acrocentric chromosomes. Fluorescence in situ hybridization (FISH)evaluation of sSMC using centromere probes(13/21,14/22,22)confirmed sSMC as derivative chromosome 14. The sSMC was not specifically stained with whole chromosome paint and arm-specific probes for chromosome 14;thus it has been described as der(14)(:p11-q11:).The phenotypic changes were not evident, may be due to trisomy condition in the child or the sSMC contain repetitive sequences.

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.