Lily Kerketta

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.

Three way translocation in a new variant of t(8;21) acute myeloid leukemia involving Xp22.

The t(8;21)(q22;q22) is one of the most frequent chromosomal abnormality associated with acute myeloid leukemia (AML) M2 sub type. The additional chromosomal abnormalities including structural and numerical are frequently reported with the translocation, t (8;21)(q22;q22). We report a case of AML-M2 with t(X;8;21)(p22;q22;q22) associated with loss of Y chromosome. Using a dual color fluorescence in situ hybridization (FISH) analysis with ETO and AML1 probes, we demonstrated an ETO/AML1 fusion signal on the derivative chromosome 8 and one ETO signal on derivative Chromosome Xp22. The patient did not respond to therapy and follow-up of cytogenetics revealed same chromosome abnormality. Hence, this three way translocation involving X chromosome might be associated with poor prognosis.

Novel immunophenotypic and morphologic presentation in acute myeloid leukemia (AML) with JAK2 V617F mutation

To the Editor: JAK2 V617F mutation (V617F), although most commonly seen in chronic myeloproliferative disorder (CMPD), is a rare occurrence in de novo acute myeloid leukemia (AML) (1). It is responsible for excessive myeloid cell proliferation by aberrantly signaling JAK2-associated receptors. We tested a large cohort of de novo AML for the mutation and correlated with cytogenetics, immunophenotype, morphology, and other molecular markers. Bone marrow (BM) and ⁄or peripheral blood (PB) collected after informed consent of the 261 patients of de novo AML were included in this study. The BM ⁄PB smears were stained by standard techniques for morphologic analysis and classification according to French-American-British (FAB) criteria (2, 3). Immunophenotyping by multiparameter flowcytometry (4) and cytogenetic investigations (5) was performed as previously described. DNA extracted from these frozen samples were amplified by PCR techniques with appropriate set of primers to JAK2, FLT3, and NPM1 genes and further screened for mutation (6–8) followed by confirmation with DNA sequencing. JAK2 V617F mutational analysis showed its presence in six (2.3%) of the 261 de novo AML. Of these, four were from the AML-M2 subtype with t(8;21) and two from the AML-M4 subtype with normal karyotype. The results tabulated in Table 1 give an overview of the biologic and immunophenotypic characteristics of the V617F positive AML in comparison with the V617F negative AML. This study showed the positive cases had low peripheral white blood cell (WBC) counts and blast percentage at presentation. However, BM showed hypercellularity with increased blast percentages, and characteristic blast morphology with Swiss-cheese ⁄honeycomb like nucleus, prominent cytoplasmic vacuolation resembling Golgi complex and an increase in the dead cell population and presence of hypolobulated megakaryocyte with emperipoiesis (Fig. 1B–D). Flow cytometric immunophenotyping revealed significantly high frequency of CD19 and CD56 aberrancy, 83.3% and 66.7%, respectively in V617F positive cases as against 12% of the total V617F negative AML. Unique aberrant coexpression of CD19 and CD56 was seen in four of these six V617F positive cases (Fig. 1A). Three of the six positive cases were also positive for FLT3-ITD. Present study identified JAK2 V617F mutation as a rare occurrence in 2.3% of de novo AML patients, mainly confined to the AML-M2 subtype with t(8;21) and the AML-M4 subtype with normal karyotype. Few studies from the literature have shown the V617F mutation to be distributed mainly among the more immature AML-M1 and M2 subtypes (1, 9, 11) with Jelinek et al., 2005 and Steensma et al., 2006 (1, 10) showing this to be in AML-M6 and M7 subtypes. Although only 16 and five cases of AML-M6 and M7, respectively were studied, we did not detect the V617F mutation in any of them. V617F mutation alone results in CMPD where the total WBC counts are often increased (12) and same has been reported by Iwanaga et al. in AML. From literature mutations of the FLT3 gene are also strongly associated with increased peripheral WBC count and increased blast

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.

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.

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.

Translocation t(2;14)(p13;q32) in a case of Ph+ acute lymphoblastic leukemia.

Online at www.ijhg.com The copies of the journal to members of the association are sent by ordinary post. The editorial board, association or publisher will not be responsible for nonreceipt of copies. If any of the members wish to receive the copies by registered post or courier, kindly contact the journal’s / publisher’s offi ce. If a copy returns due to incomplete, incorrect or changed address of a member on two consecutive occasions, the names of such members will be deleted from the mailing list of the journal. Providing complete, correct and up-to-date address is the responsibility of the members. Copies are sent to subscribers and members directly from the publisher’s address; it is illegal to acquire copies from any other source. If a copy is received for personal use as a member of the association/society, one cannot resale or give-away the copy for commercial or library use. ISSN 0971-6866 Indian Journal of Human Genetics Volume 13, Issue 3, September-December 2007