Seema Korgaonkar

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.

Pericentric inversion of chromosome 9[inv(9)(p12q13)]: Its association with genetic diseases

Background: The chromosomal polymorphism of short arms of acrocentric chromosomes and heterochromatin variation of Chromosomes 1, 9, 16 and Y have been reported in humans. The pericentric inversion of Chromosome 9 is commonly seen in normal humans and the frequency estimated to be 1 to 3% in general population and inherited in mendalian fashion or might occur spontaneously without any clinical significance. Aim: The aim of the study was to study the frequency of inv(9) and its clinical correlation with human genetic diseases. Materials and Methods:0 The chromosomal analysis using GTG-banding was carried out in 3,392 cases suspected with genetic diseases. Results: The pericentric inversion frequency of different chromosomes in our study was 1.24% and frequency of inv(9)(p12q13) was high (64.29%) compared to other pericentric inversions in our study. A high frequency (9.33%) of inv(9)(p12q13) was detected in children with dysmorphic features and congenital anomalies. Conclusion: As a high frequency of inv(9)(p12q13) detected in children with dysmorphic features, the inv(9) definitely have a role in the abnormal phenotype development. During inversion event there might be loss or suppression of euchromatin chromosome region and hence detailed chromosomal break point study is important to understand the clinical significance of the pericentric inversion of Chromosome 9.

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

Abstract 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.

Incidence of malignancy and clonal chromosomal abnormalities in Fanconi anemia

BACKGROUND Fanconi anemia (FA) is an autosomal recessive, cancer susceptibility disorder characterized by diverse clinical features, such as short stature, skeletal or skin abnormalities, progressive bone marrow (BM) failure, and increased risk of malignancies. Clonal chromosomal abnormalities are frequently reported in FA patients transformed to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). AIM To study the incidence of malignancy and clonal chromosomal abnormalities in FA patients. MATERIALS AND METHODS Thirty-eight clinically diagnosed FA patients were studied at the time of diagnosis and the patients were followed-up for a maximum of 28 months at 3-month intervals. The median duration of follow-up of these patients was 19.8 months. Chromosomal breakage investigation using mitomycin C (MMC)- and diepoxybutane (DEB)-induced peripheral blood cultures were stimulated with phytohemagglutinin. Cytogenetic study was done on the BM cells to detect clonal chromosomal aberrations. RESULTS Eleven (28.95%) out of 38 patients developed malignancies, including 6 (54.54%) MDS, 4 (36.36%) AML, and 1 (2.63%) squamous cell carcinoma. The clonal chromosomal abnormalities were detected in 5 (45.45%) FA patients who developed malignancies and the type of chromosomal abnormality detected were monosomies 5, 7, trisomy 10, dup(1)(q12-q24), and inv(7)(p11pter). CONCLUSION The FA patients have a high risk of developing malignancies, and clonal chromosomal abnormalities play an important role in the prognosis of the disease. Therefore, FA patients need to be followed-up at regular intervals for early diagnosis and optimal management of the disease.

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.

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.

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.

18p deletion syndrome with a 45, XY, t (14; 18) (p11;q11.2), -18, karyotype

A dysmorphic male child of 8 months age presented with microphthalmia, micrognathia, hypertelorism, wide anterior fontanelles, large forehead, short neck, prominent ears, macrotestis and delayed developmental milestones. The patient presented with generalised seizures hydrocephalaus and Coarctation of aorta (Pre subclavian). He also had mild hypocalcaemia with normal renal function. Cytogenetic study revealed 18p(-) picture due to translocation between 14 p & 18q. Since the spectrum of clinical expression is similar to that is seen in 18p(-) syndrome it is suggested that not only whole of 18p but part of chromosome no. 18 proximal to 18 q 11.2 may also be involved in this phenotype.

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.