Conventional G-banding Technique Research Articles

Cytogenetic abnormalities in patients with hematological malignancies in Lahore city, Pakistan.

Hematological and hematopoietic cells malignancies of the genes and hematopoietic cells are associated with the genetic mutation, often at the chromosomal level. The standard cytogenetic study is widely accepted as one of the main diagnostics and prognostic determinants in patients. Therefore, the current descriptive and cross-sectional study sought to determine the cytogenetic analysis of frequent hematological malignancies in Pakistan. A total of 202 peripheral bone marrow or blood samples from patients with benign and malignant hematological malignancy were taken using a conventional G-banding technique. Among enrolled patients, the mean age was 21.5 years ± 23.4, and gender-wise distribution showed a marked predominance of the male 147 (73%) population compared to the female 55 (27%). Patients in the age group (2-10 years) had the highest frequency, 48 (24%), of hematological neoplasms, followed by age (11-20 years) with 40 (20%). Normal karyotypes (46, XX/46, XY) was found in 51% (n=103) patients. Furthermore, the frequency of complex karyotype was 30 (15%), while normal was seen in 171 (85%) patients. Pre-B Acute Lymphoblastic Leukemia (Pre-B ALL) was the most prevalent malignancy of 66 (33%), followed by Chronic Myelogenous Leukemia (CML) of 41 (20%) and Acute Lymphocytic Leukemia of 29 (14%). Translocation was the most prevalent 50 (25%), followed by hypotriploidy 14 (7%) and monosomy 8 (4%) on chromosome aberration analysis. In addition, t(9:22) translocation was found to be 20 (10%) in CML, with the majority in the age group (31-40 years). This study recommends that karyotyping should be tested frequently in hematological conditions because it may provide insight into the relative chromosomal changes associated with particular malignancies.

CYTOGENETIC ANALYSIS OF PATIENTS WITH AMBIGUOUS GENITALIA

Objective: To determine the magnitude and classification of cases of ambiguous genitalia presenting to our setup.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Department of Haematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Aug 2018 to Feb 2019.
 Methodology: All the patients with ambiguous genitalia referred for cytogenetic analysis, were included in the study. The patients were subjected to a detailed history and physical examination. The record of radiological investigations was were obtained. Cytogenetic analysis was performed using the conventional G-banding technique. Hormonal testing included 17- hydroxyprogesterone (17-OHP) levels was also performed.
 Results: Fifty-one cases of ambiguous genitalia were studied. The median age was 15 months. Thirty-three patients (64.7%) had a 46XY karyotype, 17 (33.3%) had a 46XX karyotype while 1 (1.9%) had 45X/46, XY mosaic karyotype. Thirty patients (58.8%) were products of consanguineous marriage. Congenital adrenal hyperplasia was diagnosed in 12 cases (70.5%) of 46 XX karyotype and in 3 cases (9%) of 46XY karyotype.
 Conclusion: Ambiguous genitalia, currently categorized as disorders of sex development, are not uncommon in our populartion. Increased awareness and early diagnosis are crucial to prevent life threatening complications of congenital adrenal hyperplasia, to determine sex of rearing, and to counsel the parents or patients.

Prenatal diagnosis of a 4.5-Mb deletion at chromosome 4q35.1q35.2: Case report and literature review

ObjectiveWe present a genetic analysis of an asymptomatic family with a 4q terminal deletion; we also review other similar published studies and discuss the genotype–phenotype correlation.MethodsA karyotype analysis was performed on the amniotic fluid cells of a woman at 24 weeks of pregnancy and peripheral blood lymphocytes from both parents and their older son with the conventional G-banding technique. Chromosomal microarray analysis (CMA) testing was carried out for both parents and the fetus to analyze copy number variation (CNV) in the whole genome.ResultsThe results showed no abnormalities in the karyotypes of the father and older son, and the karyotypes of the mother and fetus were 46,XX,del(4)(q35.1) and 46,XY,del(4)(q35.1), respectively. CMA results showed a partial deletion at the 4q terminus in both the fetus and mother. The deletion region of the fetus was arr[GRCh37] 4q35.1q35.2(186,431,008_190,957,460) × 1; the loss size of the CNV was approximately 4.5 Mb and involved 14 protein-coding genes, namely, CYP4V2, F11, FAM149A, FAT1, FRG1, FRG2, KLKB1, MTNR1A, PDLIM3, SORBS2, TLR3, TRIML1, TRIML2, and ZFP42. No variation on chromosome 4 was detected in the father’s CMA results.ConclusionDeletion of the 4q subtelomeric region is a familial variation. The arr[GRCh37] 4q35.1q35.2(186,431,008_190,957,460) region single-copy deletion did not cause obvious congenital defects or mental retardation. The application of high-resolution genetic testing technology combined with the analysis of public genetic database information can more clearly elucidate the genotype–phenotype correlation of the disease and provide support for both prenatal and postnatal genetic counseling.

Relation between uterine natural killer cells and unexplained recurrent miscarriage

Objective: To evaluate the relation between uK (uterine Killer) cells and unexplained repeated miscarriage (RM). Patients and Methods: Eighty (80) women with unexplained repeated miscarriage and missed miscarriage of current pregnancy were studied. Fetal viability and gestational age of current pregnancy were confirmed by ultrasound, followed by suction evacuation to collect abortus specimens and uterine wall curettage to collect decidua specimens. Abortus specimens were collected for long-term monolayer cell culture and subsequent chromosome analysis using conventional G-banding technique. Decidua specimens were subjected to IHC (Immunohistochemical) staining using monoclonal antibodies specific to CD56+ and CD16+ expressed by uK cells. Results: CD56+ CD16+ uK cells was found in 85% (68/80) of studied decidua specimens of women with unexplained repeated miscarriage, 88.5% (54/61) had normal abortus karyotyping and 73.7% (14/19) had abnormal abortus karyotyping. 73.75% (59/80) of studied women with past history of early miscarriage had CD56+ CD16+ uK cells in their decidua specimens and 66.25% (53/80) of studied women with past history of late miscarriage had CD56+ CD16+ uK cells in their decidua specimens, the association between early and late miscarriage and CD56+ CD16+ uK cells in deciduas specimen was significant. Conclusion: CD56+ CD16+ uK cells were predominant in decidua specimens of studied women with repeated miscarriage. Significant association was found between presence of CD56+ CD16+ uK cells in studied decidua specimens and unexplained repeated miscarriage.

Karyotyping analysis of 396 newborns with congenital malformations and chromosomal abnormalities and the associated phenotypes

Objective To reveal the chromosome abnormalities and their relationship with the clinical phenotype of neonates with congenital malformation. Methods Karyotype analysis of peripheral blood lymphocytes was performed on 396 newborns with congenital malformation, who were recruited at the Children's Hospital Affiliated to Soochow University from Jan.2006 to May 2012, chromosome karyotypes were prepared with neonatal peripheral lymphocytes by conventional G-banding technique. Results 1.Of 396 newborns, 159(40.2%) cases were detected to have chromosomal abnormalities, including karyotype first reported domestically and internationally in 3 cases.2.Trisomy-21(Down's syndrome), which was the most common abnormal karyotype, was seen in 130 cases, accounting for 81.8%, of whom 119 cases show the standard type, 10 cases accompanied by the Robertsonian translocation involving group D or group G, and 1 case accompanied by sexual chromosomal abnormality: inv(Y)(p11q11), + 21.3.Other common kar-yotype abnormalities were as follows: del(5)(p12-14)(cats cry syndrome) in 4 cases, trisomy-18(Edwards syndrome) in 4 cases, 45, XO(Turner's syndrome) in 4 cases, inv(9)(p11q12-21) in 4 cases, trisomy-X(super female syndrome) in 1 case, rob(13; 14) in 1 case, trisomy-8 in 1 case and del(18)(q22) in 1 case.4.Special faces were seen in 147 cases(92.5%), congenital heart disease in 97 cases(61.0%), low birth weight in 72 cases(45.3%), congenital anal atresia in 13 cases(8.1%), multiple malformations in 11 cases(6.8%), intestinal malformations in 10 cases(6.2%), extrinsic genital abnormalities in 9 cases(5.7%), meow-like cry in 4 cases(2.5%), limb edema in 4 cases(2.5%), fingers and toe abnormalities in 6 cases(3.6%), congenital brain dysplasia in 6 cases(3.6%), webbed neck in 5 cases(3.1%) and cleft lip and palate in 3 cases(1.8%). Conclusions Chromosomal abnormality is an important factor leading to neonatal birth defects, of which special face, congenital heart disease, low birth weight, and multiple malformations are the main clinical manifestations of chromosomal diseases. Key words: Karyotype; Chromosomal abnormalities; Clinical phenotype; Infant, newborn

NUP98-NSD1 Fusion Gene Is Strongly Associated with a Poor Prognosis in Pediatric Acute Myeloid Leukemia: A Study of the Japanese Childhood AML99 Cooperative Study Group

Abstract 1475 Background:Acute myeloid leukemia (AML) is a complex disease caused by mutations, deregulated gene expression, and epigenetic modifications of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. In hematological malignancies with 11p15 translocations, the nucleoporin 98-kDa (NUP98) protein regulates nucleocytoplasmic transport of protein and RNA. More than 20 different chromosomal rearrangements involving NUP98 have been identified. The cryptic translocation t(5;11)(q35;p15.5), which creates a fusion gene between the NSD1 and the NUP98. The NUP98-NSD1 fusion gene has been associated with a deletion of the long arm of chromosome 5, del(5q), in pediatric AML patients. Recently, NUP98-NSD1 was identified in childhood AML by fluorescence in situ hybridization (FISH) with subtelomeric probes. However, the frequency and clinical impact of NUP98-NSD1 in pediatric AML remain uncertain. Methods:To estimate the frequency and clinical impact of the NUP98-NSD1 in pediatric AML, we examined the NUP98-NSD1 fusion transcript in a consecutive series of 157 newly diagnosed AML patients enrolled in the AML99 protocol in Japan clinical trial. Total RNA extracted from the leukemia cells of the patient at diagnosis was reverse transcribed to cDNA. The PCR was performed using the primer sets for detection of NUP98-NSD1. The PCR amplification was performed with a DNA thermal cycler followed by direct sequencing with an ABI PRISM 310 Genetic Analyzer. We also examined somatic mutations of the FLT3, KIT, WT1, NPM1, NRAS, KRAS and MLL genes, which are prevalent in AML, in these AML patients with NUP98-NSD1. Results:In the current study, we identified the NUP98-NSD1 by RT-PCR followed by direct sequencing in 6 (3.8%) out of 157 AML patients, and clustered significantly in the cytogenetically normal AML subgroup (6/33, 18.2%). Significant differences between patients with and without NUP98-NSD1 were observed in 4-year OS (33.3% versus 80.0%), and EFS (33.3% versus 69.2%). NUP98-NSD1 was highly seen in males (5 males and 1 female). The statistical differences between patients with and without NUP98-NSD1 was not significant in age (median: 7 years [range: 2–15 years] versus 6 years [range: 0–15 years]), and the initial WBC count (median: 115.9 × 109/L [range: 9.0–329 × 109/L] versus 51.8 × 109/L [range: 1.0–621 × 109/L]). Interestingly, NUP98-NSD1 was observed in 4 patients with normal karyotype, and was observed in 2 patients with del(9q). NUP98-NSD1 could not be identified by conventional G-banding technique in any cases. Patients with NUP98-NSD1 had any mutation of FLT3 -ITD (66.7%), WT1 (50%), KIT (16.7%) or RAS (33.3%). Furthermore, NUP98-NSD1 was frequently found in M4 or M5 patients according to FAB classification. Conclusions:We identified the NUP98-NSD1 in 6 out of 157 AML patients, and clustered significantly in the cytogenetically normal AML subgroup. Patients with NUP98-NSD1 had any FLT3 -ITD, WT1, KIT or RAS mutations. These results suggest that simultaneous occurrence of FLT3 -ITD, WT1, KIT mutations and RAS aberrations in NUP98 -related leukemia may be associated with poor prognosis. The NUP98-NSD1 was not identified by conventional G-banding technique, confirming that this translocation can exist as the only cytogenetic alteration. Therefore, FISH and RT-PCR analyses for NUP98-NSD1 fusion gene is recommended at the onset of disease in larger series of AML patients presenting an apparently normal karyotype, del(9q) or del(5q) to gain insight into the actual incidence and clinical features of patients with t(5;11)(q35;p15.5). Disclosures:No relevant conflicts of interest to declare.

Genome-wide discovery of chromosomal copy number variants in human amniotic cell using array-based comparative genomic hybridization

Chromosome aberrations (CA) are associated with several genetic heredity diseases. Of concerns, prenatal diagnosis or cytogenetic screening was administered to determine if there are chromosome abnormalities, genetic diseases, or birth defects in a fetus or embryo. It is very useful to diagnose high-risk pregnancies so that delivery can be scheduled. However, conventional G-banding technique which is generally applied in CA detection for prenatal diagnosis has limit on exploit of copy number variants (CNVs) due to low resolution and tedious multiple processes. Instead, array comparative genomic hybridization (aCGH) technology enabling incremented high-resolution, even in microscale segmental CNVs, can be considered as an alternative approach for improving prenatal diagnosis. In this study, our aCGH (Illumina Infinium Human 370Quad-Duo; coverage of SNP tag and CNV regions) was used to identify copy number variations from genomic DNA in human amniotic cell specimens. The aCGH results revealed various karyotypes of CNVs including loss, homozygous loss, gain, high copy gain, and copy neutral LOH whereas using conventional Gbanding only one benign cytogenetic CNV was observed in one case study. The aCGH was compatible to define small-scale chromosomal imbalances segment that were undetectable risk segment by G-banding. In contrast, abnormal G-banded karyotypes as balanced rearrangements were hidden from detection by aCGH analysis. This indicates that even though the aCGH enables us more advantage in detection of unbalanced CNVs at small-size regions with relatively high resolution, combination of both techniques is still required for efficient evaluation of various CNVs (both balanced and unbalanced ones). The aCGH also provides detailed database of copy number variant regions (CNVRs) in each chromosome. Interestingly, CNVRs containing important genes (ACADM, PPM1B, UGT2B17 and ZDHHC11) were discovered from our data, in which their defects or mutations have been reportedly to be involved in certain genetic heritable diseases and/or syndromes. Their relationships were analyzed using Pathway Studio 7.1 software to demonstrate key pathways concerning prenatal genetic defects. Respective components including protein kinases and phosphatases, transcription factors, hormones and receptors were participated in our analyzed pathway. Gathering together, our detailed CNVs and CNVR via aCGH of amniotic cells might be meaningful database to improve strategy in genetic disease-specific researches and follow-up after medication during pregnancy.

Chromosome 12q24.31-q24.33 deletion causes multiple dysmorphic features and developmental delay: First mosaic patient and overview of the phenotype related to 12q24qter defects

BackgroundGenomic imbalances of the 12q telomere are rare; only a few patients having 12q24.31-q24.33 deletions were reported. Interestingly none of these were mosaic. Although some attempts have been made to establish phenotype/genotype interaction for the deletions in this region, no clear relationship has been established to date.ResultsWe have clinically screened more than 100 patients with dysmorphic features, mental retardation and normal karyotype using high density oligo array-CGH (aCGH) and identified a ~9.2 Mb hemizygous interstitial deletion at the 12q telomere (Chromosome 12: 46,XY,del(12)(q24.31q24.33) in a severely developmentally retarded patient having dysmorphic features such as low set ears, microcephaly, undescended testicles, bent elbow, kyphoscoliosis, and micropenis. Parents were found to be not carriers. MLPA experiments confirmed the aCGH result. Interphase FISH revealed mosaicism in cultured peripheral blood lymphocytes.ConclusionsSince conventional G-Banding technique missed the abnormality; this work re-confirms that any child with unexplained developmental delay and systemic involvement should be studied by aCGH techniques. The FISH technique, however, would still be useful to further delineate the research work and identify such rare mosaicism. Among the 52 deleted genes, P2RX2, ULK1, FZD10, RAN, NCOR2 STX2, TESC, FBXW8, and TBX3 are noteworthy since they may have a role in observed phenotype.

Amplification of RUNX1 gene in two new cases of childhood B-cell precursor acute lymphoblastic leukemia: A case report

e21000 Background: Chromosome abnormalities of leukemia cells have important prognostic significance in childhood acute lymphoblastic leukemia (ALL). B-cell precursor acute lymphoblastic leukemia (BCP-ALL) ETV6/RUNX1 (alias TEL/AML1) is most frequent i.e. 15 - 35% in the children with 2 - 18 age group. We report two new cases with Pre B- cell ALL without ETV6/RUNX1 rearrangement, showing amplification of AML1 gene detected by FISH analysis. Methods: Bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding techniques and interphase fluorescence in situ hybridization (FISH) using probes to detect BCR/ABL t(9;22)(q34-q11) fusion, cryptic TEL/AML1 t(12:21)(p13-q22) and MLL rearrangement for del 11q23. Results: In first case a 3-year girl with four copies of AML (RUNX1) gene were observed in 95% of the cell with normal two copies of TEL (ETV6) gene in both interphase and metaphase FISH. We observed BCR-ABL negative translocation and no MLL gene rearrangement in all the interphase cells after doing FISH. We got a normal 46XX karyotype from bone marrow with conventional cytogenetics (CC) in the same patient. In second case, a 4-year male we observed four copies of AML and two copies of TEL gene in more than 80% of cells. In this patient, we got BCR-ABL negative translocation and three copies of MLL gene without any rearrangement through FISH. We got normal 46XY karyotype in the same patient through CC. Conclusions: In both the patients, we observed hyperdiploidy detected with four copies of RUNX1 gene showing tetrasomy of chromosome 21 detected with metaphase FISH analysis whereas G-banding shows normal diploidy. Bone marrow karyotype in combination with molecular cytogenetic techniques like FISH should be done for improvement in sensitivity and accurate cytogenetic analysis in childhood ALL patients for proper identification of prognostic group for optimum treatment. This is one of the few reported studies worldwide for amplification of RUNX1 gene from Indian subcontinent in childhood BCP-ALL. No significant financial relationships to disclose.

Marker chromosomes are a significant mechanism of high-level RUNX1 gene amplification in hematologic malignancies

To understand the cytogenetic mechanisms responsible for multiple RUNX1 gene copy numbers in hematologic malignancies, we analyzed the chromosomal and molecular cytogenetic findings in bone marrow or peripheral blood samples of individuals who were diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL). Included in the analysis were 113 consecutive samples received in our laboratory between January 2005 and June 2007. Bone marrow and/or peripheral blood samples were characterized using conventional G-banding techniques and fluorescent in situ hybridization (FISH) techniques with commercially available RUNX1/RUNX1T1 or ETV6/RUNX1 dual-color fusion probes. Eighty-one (72%) of the 113 samples showed an abnormal karyotype and/or abnormal FISH results. Eight of these had, by interphase FISH, RUNX1/RUNX1T1 or RUNX1/ETV6 fusion, and 19 had three or more RUNX1 signals not related to fusion with RUNX1T1 or ETV6 gene. Of the 19 cases with multiple RUNX1 gene signals, 5 had high-level RUNX1 amplification — defined as 5 or more RUNX1 signals in interphase cells — whereas the remaining 14 had 3–4 RUNX1 signals. Four of the five tumors with high-level RUNX1 amplification were myeloid disorders — three cases of AML and one case of MDS. The karyotypes of tumors with high-level amplification of RUNX1 were significantly characterized by the presence of marker chromosomes that harbored extra copies of the RUNX1 gene compared with tumors that had three to four RUNX1 gene signals ( P = 0.026, Fisher's exact test). Our findings show that high-level RUNX1 amplification, especially in myeloid disorders, often results from marker chromosomes harboring extra copies of the RUNX1 gene . This suggests that amplification of RUNX1 in these tumors may be secondary to a previous rearrangement of 21q22, which later evolved into a complex marker chromosome as part of tumor progression.

Characterization of a prenatally assessed de novo supernumerary minute ring chromosome 20 in a phenotypically normal male

BackgroundThe heterogeneous group of small supernumerary marker chromosomes (sSMCs) presents serious counseling problems, especially if they are present de novo and diagnosed prenatally. The incidence has been estimated at 1 in 1000 prenatal samples. We present a case of mosaic sSMC diagnosed prenatally after amniocentesis. The sSMC was characterized by various molecular cytogenetic techniques and determined to be a r(20) chromosome. After genetic counseling, the parents decided to continue the pregnancy, and a boy with minor phenotypic variants was born after 39 weeks of pregnancy. The case is compared with four other cases of prenatally detected r(20) mosaicism.ResultsHere we describe a 3 months old male child with normal pre- and postnatal development and with a de novo ring supernumerary marker chromosome in amniocytes cultures. Using new fluorescence in situ hybridization (FISH) techniques, three distinguishable sSMCs (cryptic mosaicism), all derived from chromosome 20, were observed, including ring and minute chromosomes. This heterogeneity was impossible to detect by the conventional G-banding technique or conventional FISH technique that were used before the application of new FISH techniques (subcentromere-specific multicolor-FISH [subcenM-FISH]) and a probe, specific for the 20p12.2 band. The sSMC present in 25% of the cells was present as r(20)(::p12.2~12.3->q11.1::)[5]/r(20;20)(::p12.1->q11.1::q11.1 >p12.1::)[2]/min(20;20)(:p12.1->q11.1::q11.1->p12.1:)[1]. The final karyotype was 47,XY,+r(20)[25%]/46,XY[75%].ConclusionWe emphasize the importance of application of molecular cytogenetics in a prenatally diagnostic laboratory and description of more cases to enable a better genetic counseling and risk evaluation.

Additional chromosomal abnormalities in Philadelphia-positive chronic myeloid leukemia

The emergence of non-random chromosomal abnormalities is a well-recognized occurrence in chronic myeloid leukemia (CML) and detection of these abnormalities is important in prognostic stratification. The frequency and types of additional chromosomal abnormalities in CML patients has not been determined in our region. We conducted a descriptive, prospective study of additional chromosomal abnormalities in patients with an established diagnosis of Philadelphia-positive CML from May 2001 to June 2007. Cytogenetic studies were repeated every three months with the conventional G-banding technique and described according to the international system for Human Cytogenetic Nomenclature. All patients received imatinib mesylate. In 219 patients with Philadelphia-positive CML, 34 (15.5%) (median age, 38 years) developed 51 additional chromosomal abnormalities. Five cases had variant translocations prior to starting imatinib; the remaining 29 cases acquired chromosomal abnormalities after starting imatinib, including 8 cases that received prior interferon-alfa. Twenty-one patients were in chronic phase, 10 in accelerated phase and 3 were in blast crisis. Trisomy 8 was the most frequent abnormality followed by random chromosomal abnormalities and variants of the Philadelphia chromosome. The overall frequency of additional chromosomal abnormalities was similar to that in previous reports. Early identification of these abnormalities may help in adapting to a more appropriate therapeutic approach.

Multiplex PCR/Liquid Chromatography Assay for Screening of Subtelomeric Rearrangements

In idiopathic or nonspecific mental retardation, the overall rate of cryptic subtelomeric rearrangements is estimated to be about 5%. Development of cost-effective screening for subtelomeric deletions would help clinical geneticists to make specific diagnoses in children with idiopathic mental retardation. Current screening modalities include fluorescence in situ hybridization (FISH) using subtelomeric probes and PCR-based quantitative analyses. Reductions in the cost and turnaround time will make the complete screening of subtelomeric rearrangements more widely used in clinical settings. Recently, a versatile method, called the multiplex PCR/liquid chromatography assay (MP/LC), was developed to assess copy numbers in this assay. Multiple genomic regions are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography. In the present study, we developed an MP/LC-based subtelomeric screening system that involves 21 multiple reactions and validated the protocol by analyzing 16 publicly available cell lines with known cytogenetic abnormalities involving at least one subtelomere per patient. To confirm the validity of the MP/LC method, we analyzed these cell lines concurrently with array-based comparative genomic hybridization (array-CGH), which gives higher resolution than the conventional G-banding technique. Among those 16 samples, the results from MP/LC and array-CGH agreed with each other perfectly. In 2 of the 16 samples, MP/LC correctly revealed subtelomeric duplications that were detected by array-CGH but were undetected by conventional cytogenetics, demonstrating the sensitivity of the MP/LC assay. This system is expected to be useful for making specific diagnoses and in genetic counseling for children with idiopathic mental retardation, a sizable fraction of whom have subtelomeric rearrangements.

Identification of a “cryptic mosaicism” involving at least four different small supernumerary marker chromosomes derived from chromosome 9 in a woman without reproductive success

To characterize the small supernumerary marker chromosomes (sSMCs) present in the female member of an infertile couple who has no further clinical symptoms. Case report. Faculty of medicine and institute of human genetics and anthropology. A young, healthy, nonconsanguineous couple asked for genetic evaluation for infertility. Intracytoplasmic sperm injection, conventional and molecular cytogenetic analyses. We characterized the sSMCs present in a woman, who was a member of an infertile couple, by molecular cytogenetic techniques. The G-banding technique showed that a marker chromosome was present in some of the examined cells describing the 47,XX,+mar[30]/46,XX[70] karyotype. Subsequently, using new fluorescence in situ hybridization (FISH) techniques, four distinguishable sSMCs (cryptic mosaicism), all derived from chromosome 9, were observed, including minute and ring chromosomes. This heterogeneity was impossible to detect by the conventional G-banding technique or conventional FISH technique that were used before the new FISH techniques (subcentromere-specific multicolor-FISH [subcenM-FISH]) and specific probe for the 9q12 band. In each metaphase with sSMCs, only one or two markers were observed. On the basis of the FISH analyses, the patient's karyotype was defined as 47,XX,+min(9)(:p12-->q12:)/47,XX,+min(9)(:p12-->q12::q12-->p12:)/47,XX,+r(9)(::p12-->q12::)/47,XX,+r(9)(::p12-->q12::p12-->q12::)x2/46,XX. The presence of sSMCs derived from chromosome 9 could influence the couple's infertility. The new subcenM-FISH techniques are very useful in the characterization of cryptic mosaicisms of marker chromosomes. Additionally, the hypothesis that the 9p12 chromosomal band is an euchromatic variant region without any phenotypic impact other than possible infertility is supported by this case study since the woman shows a normal phenotype.

Molecular Cytogenetic Study of Immunofluorescently Labeled Plasma Cells and Prognostic Significance of Clonal Chromosomal Aberrations in Multiple Myeloma.

Finding of clonal chromosomal aberrations in plasma cells is considered as one of the most important and independent prognostic factor in patients with multiple myeloma (MM). The most frequent and reliable cytogenetic indicators are chromosome 13q deletions (intermediate or moderately adverse prognosis), and translocations involving the immunoglobulin heavy chain gene (IgH) at 14q32 region. IgH gene is involved in translocations with different partner genes and these rearrangements are related to a very poor prognosis. The only exception is translocation t(11;14)(q13;q32) which is often connected with longer overall survival and therefore considered to be a favorable prognostic factor. The aim of the study was to assess the frequency of the most important chromosomal aberrations in immunofluorescently labeled plasma cells of patients with MM by interphase fluorescence in situ hybridization (I-FISH), and to evaluate their prognostic significance.During the last two years we examined 128 newly diagnosed patients with MM by conventional G-banding technique and by I-FISH. We focused on detection of aberrations of 13q, IgH gene rearrangements, and t(11;14)(q13;q32) translocation. I-FISH was done by locus-specific DNA probes (Abbott-Vysis, Des Plaines, Illinois, USA). Fifteen patients with complex karyotype were re-examined by multicolor FISH (mFISH, MetaSystems GmbH, Altlussheim, Germany). G-banding revealed abnormal karyotypes in 25% of patients, I-FISH detected chromosomal abberations in 82.8% cases. Abberations of chromosome 13 were found in plasma cells of 84 patients (65,6%), the incidence was: del(13)(q14) in 11%, monosomy 13 in 40%, combination of del(13)(q14)/monosomy 13 in 8%, and other aberrations of chromosome 13 in 7% of cases. Aberrations of IgH gene were proved in 82 patients (64%). t(11;14)(q13;q32) was seen in fourteen patients (11%) and other translocations affecting 14q32 region in 19 patients (15%). Besides translocations, different variations of total and/or partial deletions of IgH gene were detected in 28 cases (22%). In nine patients (7%) we found clones with translocation and deletion of 14q32 simultaneously, twelve patients (9%) had other IgH aberrations. Univariate statistical testing showed aberrations of 13q (p=0.042), and IgH gene rearrangments (translocations except t(11;14)(q13;q32) or deletions; p=0.007) to be associated with significantly shorter progression-free survival. The worst prognosis in our cohort was for 18 patients with combination of chromosome 13 anomalies together with rearrangements of IgH gene (p=0.049). Method of immunofluorescent labeling of plasma cells allows higher detection of chromosomal changes by I-FISH technique and therefore correct prognosis of the disease can be done.

Combined spectral karyotyping, comparative genomic hybridization, and in vitro apoptyping of a panel of Burkitt's lymphoma-derived B cell lines reveals an unexpected complexity of chromosomal aberrations and a recurrence of specific abnormalities in chemoresistant cell lines

The comprehensive cytogenetic profiles of a panel of 10 Burkitt's lymphoma (BL)-derived B cell lines, designated Akata, BL-28, BL-41, Daudi, DG-75, Mutu I, Mutu III, Namalwa, Rael, and Ramos, respectively, are reported herein. The unique origin of each cell line was established using multiplex quantitative fluorescence polymerase chain reaction (QF-PCR). Spectral karyotyping (SKY) revealed a large number of structural and numerical chromosomal aberrations, many of which had not been previously identified or resolved by conventional G-banding techniques. Notably, whereas all 10 cell lines harbored the hallmark translocation t(8;14)(q24;q32), no other common structural aberrations were identified, although translocations involving chromosomes 3, 13, and 17 were frequently seen. Moreover, analysis of chromosomal breakpoints by comparative genomic hybridization (CGH) revealed a number of recurring aberrations, such as gain of chromosomes 7 and 20, gains of regions at 2p, 3q, 13q and 16q, and losses at 3p, 4q and 17p. In addition, apoptyping (i.e. determination of in vitro responses to apoptosis stimulation) of the cell lines suggested specific association patterns between karyotypic changes (e.g. translocations involving 17p, and gains of portions of chromosomes 7 and 20) and resistance to the chemotherapeutic agent, etoposide. The current molecular cytogenetic characterization of 10 BL cell lines has thus identified several novel sites of rearrangements; moreover, the combined karyotyping and functional assessment (apoptyping) of these cell lines serves to enhance their utility in future studies aimed at gene discovery and gene function.

Preliminary cytogenetic characterization of an AZT-resistant subclone, clone 5, of the human colon tumor cell line, HCT-15

Stage II human trials have indicated that the combination of 3′-azido-3′-deoxythymidine (AZT) with 5-fluorouracil and leucovorin has improved efficacy in the adjuvant chemotherapy of colorectal cancer. The development of resistance to AZT has been well documented in viral metabolism, but has not been fully described in human cells. This renders the drug clinically unpredictable, limiting its chemotherapeutic utility. In order to better study the mechanism of AZT-resistance, we have developed an AZT-resistant colon cancer cell line in culture to serve as a model system.The human colon adenocarcinoma cell line, HCT-15 (American Type Culture Collection #CCL225), has been previously characterized by conventional cytogenetic studies and by fluorescent in situ hybridization (FISH). We have measured the concentration of AZT necessary to inhibit growth by 50% over 5 days (IC50) to be approximately 30 μM. AZT-resistant cell lines were derived by serial passage of HCT-15 cells in increasing concentrations of AZT until stable growth was achieved in 1000 μM AZT. The surviving cell colonies were then subcloned in soft agar. One such cell line, clone 5, was found to have eighty-fold increased resistance to AZT, with an IC50 of approximately 2500 μM AZT. We now report a preliminary characterization of the clone 5 cell line in comparison to its parent HCT-15 cell line, using conventional G-banding techniques.

FISH mapping and identification of canine chromosomes.

The karyotype of the domestic dog (Canis familiaris) is widely accepted as one of the most difficult mammalian karyotypes to work with. The dog has a total of 78 chromosomes; all 76 autosomes are acrocentric in morphology and show only a gradual decrease in length. Standardization of the canine karyotype has been performed in two stages. The first stage dealt only with chromosomes 1-21 which can be readily identified by conventional G-banding techniques. The remaining 17 autosomal pairs have proven to be very difficult to reliably identify by banding alone. To facilitate the identification of all canine chromosomes, chromosome-specific paint probes have been produced by DOP-PCR from flow-sorted dog chromosomes. Each paint probe has been used for FISH to identify the corresponding chromosome(s), allowing precise identification of all 78 canine chromosomes. The identification of the undesignated 17 autosomal pairs has been agreed upon by the standardization committee during the second stage of their role. Cosmid clones containing microsatellite markers may now be conclusively assigned to their chromosomal origin by simultaneous dual-color FISH with the corresponding paint probe. In this way a collection of chromosome-specific cosmid clones is being constructed, comprising at least one marker per chromosome, which will allow anchoring of existing and future linkage groups to the physical map.

Deletion and translocation involving chromosome 3 (p14) in two tumorigenic Kaposi's sarcoma cell lines.

Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have shown to have normal diploid characteristics are hyperplastic, and depends on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. We investigated whether the chromosomal changes that occurred in these KS cell lines were random contribute to the pathogenesis of KS. We used the conventional G-banding technique and fluorescence in sti hybridization to identify structural and numerical chromosomal changes in the KS cell lines. We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibits loss of heterozygosity of loci at region 3p14-ter. This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. Future research should focus on the identification cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process.