Biomedical subjects

P H Rao

Publications and source records attributed to P H Rao.

At least 19 recordsLinked to original sources

PubMed2002

The utility of spectral karyotyping in the cytogenetic analysis of newly diagnosed pediatric acute lymphoblastic leukemia.

We applied multicolor spectral karyotyping (SKY) to a panel of 29 newly diagnosed pediatric pre B-cell ALLs with normal and abnormal G-banded karyotypes to identify cryptic translocations and define complex chromosomal rearrangements. By this method, it was possible to define all add chromosomes in six cases, a cryptic t(12;21)(p13;q11) translocation in six cases, marker chromosomes in two cases and refine the misidentified aberrations by G-banding in two cases. In addition, we identified five novel non-recurrent translocations - t(2;9)(p11.2;p13), t(2;22) (p11.2;q11.2), t(6;8)(p12;p11), t(12;14)(p13;q32) and t(X;8)(p22.3;q?). Of these translocations, t(2;9), t(2;22) and t(12;14) were identified by G-banding analysis and confirmed by SKY. We characterized a t(12;14)( p13;q32) translocation by FISH, and identified a fusion of TEL with IGH for the first time in ALL. We identified a rearrangement of PAX5 locus in a case with t(2;9)(p11.2;p13) by FISH and defined the breakpoint telomeric to PAX5 in der(9)t(3;9)(?;p13). These studies demonstrate the utility of using SKY in combination with G-banding and FISH to augment the precision with which chromosomal aberrations may be identified in tumor cells.

Acute Disease↗

PubMed2001

Comparative genomic hybridization and amplotyping by arbitrarily primed PCR in stage A B-CLL.

Cytogenetic analysis is useful in the diagnosis and to assess prognosis of B-cell chronic lymphocytic leukemia (B-CLL). However, successful cytogenetics by standard techniques has been hindered by the low in vitro mitotic activity of the malignant B-cell population. Fluorescence in situ hybridization (FISH) has become a useful tool, but it does not provide an overall view of the aberrations. To overcome this hurdle, two DNA-based techniques have been tested in the present study: comparative genomic hybridization (CGH) and amplotyping by arbitrarily primed PCR (AP-PCR). Comparative genomic hybridization resolution depends upon the 400-bands of the human standard karyotype. AP-PCR allows detection of allelic losses and gains in tumor cells by PCR fingerprinting, thus its resolution is at the molecular level. Both techniques were performed in 23 patients with stage A B-CLL at diagnosis. The results were compared with FISH. The sensitivity of AP-PCR was greater than CGH (62% vs. 43%). The use of CGH combined with AP-PCR allowed to detect genetic abnormalities in 79% (15/19) of patients in whom G-banding was not informative, providing a global view of the aberrations in a sole experiment. This study shows that combining these two methods with FISH, makes possible a more precise genetic characterization of patients with B-CLL.

Adult↗

PubMed2001

Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification.

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization, to characterize the genetic aberrations in a panel of 11 cell lines derived from head and neck squamous cell carcinoma and 1 cell line derived from premalignant oral epithelium. CGH identified recurrent chromosomal losses at 1p, 3p, 4, 8p, 10p, and 18q; gains at 3q, 5p, 8q, 9q, and 14q; and high-level amplification at 3q13, 3q25-q26, 5q22-q23, 7q21, 8q24, 11q13-q14, 12p13, 14q24, and 20q13.1. Several recurrent translocations including t(1;13)(q10;q10), t(13;13)(q10;q10), t(14;14)(q10;q10), i(8)(q10), and i(9)(q10) and breakpoint clusters at 1p11, 1q21, 3p11, 5q11, 5q13, 6q23, 8p11, 8q11, 9p13, 9q13, 10q11, 11q13, 13q10, 14q10, and 15q10 were identified by SKY. There was a good correlation between the number of aberrations identified by CGH and SKY (r = 0.69), and the analyses were both confirmatory and complementary in their assessment of genetic aberrations. Amplification at 3q26-q27 was identified in 42% of cases. Although SKY defined the derivation of 3q gain, the precise breakpoint remained unassigned. Positional cloning efforts directed at the amplified region at 3q26-q27 identified three highly overlapping nonchimeric yeast artificial chromosome clones containing the apex of amplification. The use of these yeast artificial chromosome clones as a probe for fluorescence in situ hybridization analysis allowed a detailed characterization and quantification of the 3q amplification and refinement of unassigned SKY breakpoints.

Carcinoma, Squamous Cell↗

PubMed2001

IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy.

Abnormalities of chromosome 1q21 are common in B cell malignancies, but their target genes are largely unknown. By cloning the breakpoints of a (1;14) (q21;q32) chromosomal translocation in a myeloma cell line, we have identified two novel genes, IRTA1 and IRTA2, encoding cell surface receptors homologous to the Fc and inhibitory receptor families. Both genes are selectively expressed in mature B cells: IRTA1 in marginal zone B cells and IRTA2 in centrocytes, marginal zone B cells, and immunoblasts. As a result of the t(1;14), IRTA1 is fused to the immunoglobulin Calpha domain to produce a chimeric IRTA1/Calpha fusion protein. In tumor cell lines with 1q21 abnormalities, IRTA2 expression is deregulated. Thus, IRTA1 and IRTA2 are novel immunoreceptors implicated in B cell development and lymphomagenesis.

Amino Acid Sequence↗

PubMed2001

Multicolor spectral karyotyping identifies novel translocations in childhood acute lymphoblastic leukemia.

We used a recently described molecular cytogenetic method, spectral karyotyping (SKY), to analyze metaphase chromosomes from 30 pediatric patients with acute lymphoblastic leukemia (ALL). This group included 20 patients whose leukemic blast cells lacked chromosomal abnormalities detected by conventional cytogenetics and 10 patients whose blast cells had multiple chromosomal abnormalities that could not be completely identified by G-banding analysis. In two of the 20 patients (10%) with apparently normal karyotypes, SKY identified three cryptic translocations: a t(7;8)(q34-35;q24.1) in one patient and a t(13;17)(q22;q21) and a der(19)t(17;19)(q22;p13) in another. Fluorescence in situ hybridization using subtelomeric probes proved the latter translocation to be a t(17;19). SKY analysis was also successful in defining the nature of the chromosomal abnormalities in four of the 10 patients with marker and derivative chromosomes. The identified abnormalities in the latter group included three novel translocations: a der(X)t(X;5)(p11.4;q31), a der(21)t(X;21)(p11.4;p11.2) and a t(X;9)(p11.4;p13). The presence of the t(X;9) was suggested by conventional cytogenetics. The application of fluorescence in situ hybridization using chromosome-specific painting probes and locus-specific probes complemented the SKY analysis by confirming the nature of the chromosome rearrangements defined by SKY and by identifying the amplification of the AML1/CBFA2 gene in one patient with a duplicated 21q. Our study demonstrates the utility of SKY in identifying novel translocations and in refining the identity of chromosomal abnormalities in leukemias.

Adolescent↗

PubMed2000

An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.

Alleles↗

PubMed2000

Trisomy 1q, 2, and 20 in a case of hepatoblastoma: possible significance of 2q35-q37 and 1q12-q21 rearrangements.

Combined cytogenetic, chromosome painting, and spectral karyotyping (SKY) analyses in a case of hepatoblastoma revealed a karyotype of 49,XY,+Y,+der(2)t(2;3)(q35;q25),der(3)t(1;3)(q12; q25),+20. Trisomy 1q, 2, and 20 identified in the present case are consistent with the previously reported cytogenetic alterations in hepatoblastoma. The breakpoints at 1q12 and 2q35 identified in this case have also been reported previously as nonrandom changes. The frequent occurrence of these rearrangements in hepatoblastoma suggests that they may be of pathogenic significance.

Chromosome Painting↗

PubMed2000

Screening for genetic aberrations in papillary thyroid cancer by using comparative genomic hybridization.

BACKGROUND: Determination of the genetic composition of papillary thyroid cancers may help explain differences in observed clinical behavior. Comparative genomic hybridization (CGH) is a novel molecular cytogenetic assay that allows simultaneous detection of gains, losses, and amplification of genetic information, making it an ideal screening tool. The aim of this study was to identify genetic aberrations occurring in papillary thyroid cancers by using CGH analysis. METHODS: CGH analysis was performed on 21 individual cases of papillary thyroid cancers. Nonparametric statistical comparisons were performed with the Fisher exact test. RESULTS: Genetic abnormalities were identified by CGH in 10 of 21 cases (48%). A recurrent pattern of aberrations was seen in cases where genetic changes were detected, involving losses at chromosome arms 1p and 9q and chromosomes 17, 19, and 22, and gains at chromosome 4 and chromosome arms 5q, 6q, 9q, and 13q. The loss of chromosome 22 was unique to younger patients (P =.05) and was associated with a higher rate of regional lymphatic metastasis (19% vs 80%, P =.02). CONCLUSIONS: Two genetically unique groups of patients were identified by using CGH analysis. One group had no detectable aberrations; the other had a recurrent pattern of aberrations, localizing to the identical chromosomal loci. This pattern of aberrations suggests that the involved loci may contain genes important in thyroid carcinogenesis. The clinical significance of the presence of copy number changes detected by CGH needs to be determined. In addition, molecular cloning of involved genes in each of the aberrations is warranted.

Adolescent↗

PubMed2000

Genome-wide screening for radiation response factors in head and neck cancer.

INTRODUCTION: Radiation therapy is an integral part of the treatment of head and neck cancer. Factors predicting radiation response are ill defined. The aim of this study was to identify genetic aberrations associated with radiation response in cell lines derived from head and neck squamous cell carcinomas (HNSCC) using comparative genomic hybridization (CGH) for genome-wide screening. METHODS: Five cell lines derived from HNSCC were subjected to a single course of radiation (400 cGy) in parallel with a similarly handled, untreated control. Cellular response to radiation was determined on posttreatment days 1, 2, 3, 4, and 5 using a cell viability assay (MTT assay). Radiation response was defined as 35% or greater decrease in cell survival relative to control. Tumor doubling time was determined by cell counts performed at day 0 and 1 for each cell line. All experiments were done in quadruplicate. CGH analysis was performed by differentially labeling DNA from tumor and normal tissue with fluorescent agents. The labeled DNAs were simultaneously hybridized to normal metaphase chromosomes. Image analysis for fluorescence intensity along the entire length of each metaphase chromosome allowed generation of a color ratio, which was used to detect copy number changes. RESULTS: Radioresistance was identified in two of five cell lines. The tumor doubling time was not a predictor of radiation response. CGH identified a complex pattern of aberrations, with gain of 3q common to all cell lines. The number of genetic aberration was higher in radiation-sensitive cell lines than in radiation-resistant ones. No recurrent aberrations were unique to the radiation-resistant cell lines. Recurrent gains at 7p and 17q and losses at 5q, 7q, and 18q were unique to the radiation-sensitive cell lines. CONCLUSIONS: The number of aberrations identified by CGH analysis may be a predictor of radiation response. A large study of primary tumors is warranted to confirm this association and identify specific genetic aberrations associated with radiation response.

Carcinoma, Squamous Cell↗

PubMed2000

Comparative genomic hybridization study of de novo myeloid neoplasia.

Comparative genomic hybridization (CGH) was used to detect chromosomal imbalances in 20 patients with a diagnosis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). The results obtained were compared with G-banding analysis. This last methodology showed 50% of cases with clonal abnormalities whereas CGH detected 70% of cases with copy number changes. Gains were more frequent than losses and constituted 66% of total changes detected. The most common gains included chromosomes 21 and chromosome region 18p for AML and chromosome 17 and region 1p33p35 for MDS. Losses represent 34% of changes and the regions involved were 5q31q32, 7q22, 7p12 and 13q21q22. CGH revealed additional chromosome imbalances in 12 of 20 cases (60%) which were not detected by traditional cytogenetic studies, demonstrating complex karyotype in 50% (6/12). Combination of CGH and G-banding provides an efficient method to identify critical regions present in the malignant clone, which is of great value in the prognosis and outcome of myeloid neoplasias.

Acute Disease↗

PubMed2000

Improving degenerate oligonucleotide primed PCR-comparative genomic hybridization for analysis of DNA copy number changes in tumors.

Combining degenerate oligonucleotide-primed PCR (DOP-PCR) with comparative genomic hybridization (CGH) has made it possible to analyze genomic changes in single cells. Although DOP-PCR-CGH methodology has been reported, the reproducibility of the method has been uncertain. We have developed a reproducible DOP-PCR-CGH protocol by systematically evaluating different labeling methods (including nick translation, PCR incorporation, and random-primed labeling) and different hybridization mixtures (including amplified test DNA vs. amplified reference DNA, termed homo-hybridization; and amplified test DNA vs. unamplified reference DNA or vice versa, termed hetero-hybridization). We have analyzed DNA samples obtained from 16 tissue sources including fresh/frozen normal and tumor samples, formalin fixed and paraffin embedded tumor tissue, and tumor cell lines by using differently labeled probes and hybridization combinations, and we calculated the corresponding rate (CR) of DOP-PCR-CGH with standard CGH. We found that homo-hybridization produced reproducible results with high CRs as compared to standard CGH (91-100% CR, mean 97%); In contrast, hetero-hybridization failed to generate reproducible hybridization with low CRs (57-97% CR, mean 80%; chi(2) = 1245.8, P<0.0001), high background, uneven hybridization, and false deletions or amplifications. In addition, our improved DOP-PCR protocol raised the amplification efficiency at least five times as compared to previously reported protocols, allowing for the detection of genomic imbalances in as little as 12.5 pg of starting DNA. In conclusion, the DOP-PCR-CHG homo-hybridization method, especially when combined with labeling by nick translation, is reliable and reproducible. The method can be used in screening for genomic imbalances using minute amounts of tumor DNA, thereby facilitating CGH application. Genes Chromosomes Cancer 28:395-403, 2000.

Breast Neoplasms↗

PubMed1999

Translocation (4;15)(p16;q24): a novel reciprocal translocation in a patient with BCR/ABL negative myeloproliferative syndrome progressing to blastic phase.

A patient with BCR/ABL negative myeloproliferative syndrome with a 46,XY,del(3)(q21), t(4;15)(p16;q24) karyotype is described. Fluorescence in situ hybridization performed with chromosomes 4 and 15 painting probes confirmed a novel reciprocal (4;15) translocation. The absence of crkl tyrosine phosphorylation, no activation of the abl kinase as measured by autophosphorylation, and a normal-size abl transcript suggest an alternative mechanism for leukemogenesis to that operative in Ph positive BCR/ABL positive chronic myeloid leukemia. A number of genes potentially relevant to tumorigenesis, some involving the ras signaling pathway, map to the 4p16 and 15q24 chromosome regions.

Acute-Phase Reaction↗

PubMed1999

Effect of Detarium microcarpum (Dm) and Mucuna flagellipes (Mf) gums on the quality of white bread.

Incorporation of Detarium Microcarpum (Dm) and Mucuna flagellipes (Mf) water soluble polysaccharides (gums) at 0.0 to 0.5% levels in wheat flour was studied to evaluate their effect on the rheological properties of wheat flour dough and white bread quality. At all levels of incorporation, there were increases (p < or = 0.05) in water absorption of the dough. Doughs containing gums had higher (p < or = 0.05) mixing tolerance index than the control. Set back viscosities decreased by 4.0 RVU and 9.0 RVU with increased levels of Dm and Mf gum incorporation, respectively. Significantly (p < or = 0.05) higher oven spring occurred in all the gum substituted white bread when compared to the control. The 0.5% gum substituted breads had a significantly (p < or = 0.05) higher sensory score for crumb grain, texture but lower (p < 0.05) crumb firmness than the control as determined instrumentally. Textural analysis after 5 days storage revealed that Dm and Mf gums improved moisture retention properties of the bread and reduced crumb firming tendency.

Bread↗

PubMed1999

Chromosomal rearrangement of the PAX-5 locus in lymphoplasmacytic lymphoma with t(9;14)(p13;q32).

B-cell non-Hodgkin's lymphoma (NHL) consists of heterogeneous subtypes based on histologic, immunophenotypic, and clinical findings. Recent advances in molecular biology have provided us new insights into the pathogenesis of this neoplasm at the genetic level, such as the deregulation of the protooncogenes adjoining the immunoglobulin gene (Ig) loci, which is a specific event in mature B-cell tumors. Moreover, involvement of certain protooncogenes corresponds to certain subtypes of NHL. Recently, we found that t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytic lymphoma (LPL) juxtaposes PAX-5 gene encoding for an essential transcription factor (BSAP: B-cell specific activator protein) for B-cell proliferation and differentiation to the Ig heavy chain gene (IgH) locus. This results in deregulated expression of the PAX-5 mRNA. We also developed a diagnostic FISH (fluorescence in situ hybridization) procedure which is able to detect 80% of the widely scattering 9p13 breakpoints involved in this translocation. Thus, an understanding of the PAX-5 gene's physiological role in B-cell development and the pathological role in tumorigenesis may lead to the optimal clinical treatment strategy for LPL and LPL-derived diffuse large cell lymphoma (DLCL).

Animals↗

PubMed1999

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC.

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820-kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21.

Chromosomes, Human, Pair 11↗

PubMed1998

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability.

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.

Amino Acid Sequence↗

PubMed1998

The t(2;3)(q21;q27) translocation in non-Hodgkin's lymphoma displays BCL6 mutations in the 5' regulatory region and chromosomal breakpoints distant from the gene.

The BCL6 gene, mapped at the chromosomal band 3q27, encodes a POZ/Zinc finger transcription repressor protein. It is frequently activated in Non-Hodgkin's lymphomas (NHL) by translocations with breakpoints clustering in the 5' major breakpoint region (MBR) as well as by mutations in the same region. The translocations lead to BCL6 activation by substitution of promoters of rearranging genes derived from the reciprocal chromosomal partners such as IG. We report the molecular genetic analysis of a novel t(2;3)(q21;q27) translocation subset in NHL comprising three cases without apparent BCL6 involvement in the translocation. Southern blot analysis of tumor DNAs utilizing BCL6 MBR probes revealed no rearrangement in two cases. Two rearranged bands in the third case resulted from a deletion in one allele and a mutation in the other allele. Southern blot analysis of DNA from one of the two tumors without BCL6 rearrangement, using a probe derived from the recently identified alternative breakpoint region (ABR), showed a rearrangement. The ABR is located 200-270 kb telomeric to MBR. Mutations were identified in the previously reported hypermutable region of BCL6 in all three tumors. In one, the mutant allele alone was found to be expressed by RT-PCR analysis of RNA. These results demonstrate the presence of 3q27 translocation breakpoints at a distance from BCL6 suggesting distant breaks that deregulate the gene or involvement of other genes that may be subject to rearrangement.

Alleles↗

PubMed1998

Chromosomal amplification is associated with cisplatin resistance of human male germ cell tumors.

Chemotherapy resistance of tumors is an important biological and clinical problem. Studies from many tumor types have indicated that resistance may be based on multiple genetic pathways. Human male germa cell tumors (GCTs) are an especially good model system to study the genetic basis of tumor sensitivity and resistance to chemotherapy. GCTs are exquisitely sensitive to treatment with DNA-damaging drugs such as cisplatin, rarely exhibit TP53 gene mutations, express normal p53 protein, and undergo p53-mediated apoptosis upon drug treatment. A small proportion of tumors (20-30% of metastatic lesions) escape the apoptotic response and result in treatment resistance. We have recently shown (J. Houldsworth, et al., Oncogene, 16: 2345-2359, 1998) that in a subset of such tumors, resistance is linked to TP53 gene mutations. In a further search for genetic mechanisms underlying resistance, we subjected a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemotherapy-resistant tumors to comparative genomic hybridization analysis to identify possible amplified regions (implying amplified/overexpressed genes) associated with resistance. With the exception of 12p11.2-12, high level amplification was not detected in any of the sensitive tumors. We have identified eight amplified regions (1q31-32, 2p23-24, 7q21, 7q31, 9q22, 9q32-34, 15q23-24, and 20q11.2-12) in five resistant tumors, which suggests that chromosomal and, hence, gene amplification may comprise a pathway to drug resistance. Identification of amplified/overexpressed genes at these sites may elucidate new genetic pathways of chemotherapy resistance in GCTs and possibly also in other tumors.

Chromosome Mapping↗