Cytogenetic Analysis Of Patients Research Articles

Parallel Genomic Analysis from Paired Bone Marrow and Peripheral Blood Samples of 200 Cytopenic Patients

Background: Analysis of bone marrow is the gold standard in patients with unexplainable cytopenia for diagnosing (or excluding) myelodysplastic neoplasm (MDS). Besides morphology also molecular genetics and cytogenetics are currently assessed from BM as both are required for diagnosis and risk stratification. Substitution of BM analysis with peripheral blood (PB) analysis would be a substantial progress especially in the elderly to allow for a less invasive strategy. Aims: Validate, whether PB reliably reflects molecular and cytogenetic aberrations of BM by evaluating somatic mutations and copy number variations (CNVs) in concurrent PB and BM samples. Methods: We analyzed paired BM and PB samples (time difference of sampling: <15 days) of 200 cytopenic patients. All samples were subjected to targeted panel sequencing analyzing mutations (assessed as positive at variant allelic frequencies/VAFs >1%) in 40 genes associated with myeloid neoplasms (coverage: 1500x). If available, BM chromosome analysis (CA) and BM FISH analysis were compared to NGS CNV analysis from PB using a human CNV backbone hybridization panel (coverage: 50x). Results: In 69 patients (35%) no mutations (MUTs) were detected in either BM or PB (Figure 1A). For the remaining 131 patients at least one MUT was found in BM (median MUTs/patient: 2; range 1-10). In 130/131 patients MUTs were detected in both BM and PB (same number of MUTs: n=121; higher number of MUTs in BM than in PB: n=9) while in only one patient harboring a DNMT3A MUT as the sole MUT in BM (VAF: 9%) no MUT was detected in PB. This resulted in a negative predictive value of 98.6% (69/70). Overall, we detected 364 MUTs in 29 of the analyzed genes (BM MUTs: n=362; PB MUTs: n=349) and a concordance between BM and PB of 95% (347/364; Figure 1A). The median VAF was lower in PB compared to BM across all MUTs (median VAF: 20% vs. 12%; p<0.001). All MUTs detected in BM at VAF ≥13% (n=229) were also detected in corresponding PB. Next, we compared CNVs detected by PB NGS to BM karyotype (KT). For 119/200 patients PB NGS including a CNV-backbone panel was available. BM CA revealed a normal KT in 71 and an aberrant KT in 48 patients. In total, 126 aberrations were detected in BM CA including 7 balanced aberrations that can technically not be detected using the CNV backbone panel. CNV analysis from PB revealed 68 aberrations (of 119 detectable BM CNVs; 54%) with 64 being concordant with BM CA. In total, in 88/119 patients (74%) concordant results were found in BM and PB (70/71 with normal and 18/48 with aberrant BM KT). In 3 patients (2.5%) different CNVs were detected (1/3: no CNV in BM CA), while in 28 (23.5%) either fewer CNVs (n=8; all complex KTs) or no CNVs (n=20) were detected in PB compared to BM. The majority (76%) of BM CNVs not detected by PB NGS with available BM FISH data were small BM clones (clone size ≤30%) and therefore not reliably captured by NGS PB with a technical sensitivity of 30%. Interestingly, 4 of the discordant patients with aberrant BM KT did not harbor any somatic MUT in BM and PB. Regarding the IPSS-R cytogenetic risk group, 95 (80%) patients fell into the same category, while 22 (18%; all with aberrant BM KT) were assigned to a better risk category by NGS PB than based on BM CA data and 2 patients (2%) to a worse risk category by NGS PB (Figure 1B). Conclusion: In cytopenic patients mutations can be reliably detected from PB showing a concordance with BM of individual mutations of 95% and a consistency regarding overall mutation detection between PB and BM in 99.5% of patients. Cytogenetic risk group assignment based on NGS CNV analysis from PB compared to chromosome analysis from BM resulted in the same risk group in 80% of patients. However, to further decrease the number of discrepant cases, the technical limitations (sensitivity, inability to detect balanced CNVs) of the CNV backbone method might be overcome by increasing the sequencing depth and/or using CD34-enriched PB samples and adding probe sets. Alternatively, FISH on PB smears could be used for CNV detection. Overall, our data indicate a high degree of overlap between PB and BM regarding mutational and cytogenetic analysis in patients with unexplained cytopenia and suggest PB as reliable surrogate especially in patients without any genetic aberrations.

Cytogenetic Analysis of Patients with Hematological Malignancies

With this study, we aim to summarize and assess the activity and performance of the Cytogenetic sector of the Laboratory of Medical Genetics—Varna, regarding the conventional cytogenetic analysis of bone marrow samples from patients with (onco)hematological diagnoses. Another purpose is to evaluate the tendencies noticed over a period of eleven years to draw conclusions and share our experience. We have performed the analysis with the G-banding technique on 2653 samples from patients of age 0–93 yr by the current European recommendations and the International System for Human Cytogenomic Nomenclature. The greater part of these samples (90.9%) was with an indication of a hematological malignancy, most commonly Acute myeloid leukemia, Myelodysplastic syndrome, Acute lymphoid leukemia, Chronic myeloid leukemia, and Multiple myeloma. Analysis was successful in 2215 (83.5%)—from those normal karyotypes were found in 1492 (67.4%) and pathology in 723 (32.6%). Regarding the latter, the most common were complex karyotypes (30.6%), Philadelphian chromosome (21.3%), trisomy 8 (5.9%), and deletion in the long arm of chromosome 5 (4.3%). Cytogenetic analysis is a method with great impact on the evaluation of many hematological malignancies and for this reason, it remains an essential part of routine assessment of these diseases. The disadvantages of it mainly in the field of oncohematological diseases, recognized by the scientific society and confirmed in our own experience, suggest a need for an additional genetic method to overcome these limitations.

Cytogenetic culture failure and its causes in hematological disorders; a single centre perspective

ObjectiveTo highlight the reasons of culture failure in bone marrow aspirate samples sent for Cytogenetic analysis and to identify the associated parameters causing this impact.MethodologyThis is a retrospective cross-sectional study conducted in the Clinical and Molecular Cytogenetics Laboratory of NIBD Hospital, Karachi, Pakistan. The rates of culture failure are assessed from the year 2017–2020 along with their reasons. Bone Marrow aspirate samples of patients with hematological malignancies were cultured for chromosomal analysis, both at the time of diagnosis or relapse. Statistical analysis was performed using SPSS version 25.ResultsA total of 1061 bone marrow aspirate samples were assessed for cytogenetic culture failures from the duration of 2017 to 2020. Ratio of males was predominantly higher i.e. 62.7% than female 37.3% with Mean ± SD age was 36.78 ± 18.94. Frequency of culture failure in the year 2020 was relatively high 20% as compared to the preceding years i.e. 8% in 2017, 6% in 2018, 7% in 2019. However, the patients were diagnosed with the following hematological malignancies; ALL 23%, CML 17.1%, AML 16.5% and AA 12.5%. Among the reasons of culture failure, cytogenetic analysis of patients with on-going chemo resulted in significant culture failures with p-value < 0.001 and the hematological malignancy, Acute Promyelocytic Leukemia, significantly impacted the growth of bone marrow aspirate cultures, with p-value < 0.001.ConclusionSignificant findings were associated with causative factors of culture failure including on-going treatment and sample issues of clotted bone marrow as well as with the clinical diagnosis. These evaluations facilitated in overcoming the rise in culture failures. As per our knowledge, no such data, discussing the effects of various parameters such as sample quality, diagnosis, effects of treatment etc., has been documented previously.

Cytogenetic Analysis in Patients with Azoospermia

BACKGROUND&#x0D; Azoospermia (the absence of sperm in the semen) significantly amounts to infertility in men (15 % of infertile men). Since genetics plays a huge part in its origin, genetic testing needs to be considered as an important part of early diagnostics when evaluating azoospermic patients. The aim of our study was to determine the genetic cause of azoospermia, to analyze karyograms and the possible presence of Y chromosome microdeletion.&#x0D; METHODS&#x0D; The study included 20 men diagnosed with azoospermia. The oldest male was 50 and the youngest was 24 years old. They were sent for analysis of karyotype and microdeletions of the Y chromosome (AZF region) to the Center for Genetics of the Faculty of Medicine.&#x0D; RESULTS&#x0D; The results of the cytogenetic analysis showed 3/20 (15 %) of abnormal karyotypes. Three patients were identified with two different translocations that were not previously reported. Two brothers were identified with the same translocation between the short arm of chromosome 8 and the long arm of chromosome 17 [46, XY, t(8;17)(p23;q11)], after both of them had failed in-vitro fertilization (IVF). The third patient with chromosomal translocation between the short arm of chromosome 4 and the long arm of chromosome 11 [46, XY, t(4;11)(p16;q13)]. Molecular genetic analysis showed no Y chromosome microdeletion in any of these three cases. However, from the rest of the group, only one patient had Y chromosome microdeletion in the AZFc region (one of the most common causes of genetic azoospermia). His karyotype was normal.&#x0D; CONCLUSIONS&#x0D; Our study confirms that cytogenetic and molecular genetic analysis plays an important role in identifying the cause of azoospermia. Genetic counseling should be provided when these abnormalities are identified before making any future medical decisions.

Application of CD138 Immunomagnetic Bead Sorting Combined with Fluorescence in Situ Hybridization in Multiple Myeloma

To compare the effects of direct fluorescence in situ hybridization (D-FISH) detection without sorting and CD138 immunomagnetic bead sorting technology combined with FISH (MACS-FISH) on cytogenetic analysis of patients with multiple myeloma (MM). FISH test results of 229 patients with initial MM were retrospectively analyzed. The patients were divided into two groups, 140 patients were tested with D-FISH and 89 patients with MACS-FISH. The combination probe was designed as P53, D13S319, RB1, 1q21, and IgH. Cytogenetic detection results were compared between the two groups. The total detection rate of cytogenetic abnormalities in D-FISH group was 52.9%, and that in MACS-FISH group was 79.8%. There was a significant difference in the cytogenetic abnormality rate between the two groups (P=0.020). The abnormal genes with the highest detection rate in the two groups were 1q21 and IgH, respectively, while the lowest was P53. There was no significant difference in the percentage of P53 positive cells (positive rate) between the two groups, while D13S319, RB1, 1q21, and IgH showed significant difference in positive cell rate (P=0.0002, P<0.0001, P=0.0033, P=0.0032). There was no significant correlation between the proportion of plasma cells (PC) detected by bone marrow morphology and cytogenetic abnormality rate in the D-FISH group, while there was a correlation between the proportion of PC detected by flow cytometry and cytogenetic abnormality rate (r=0.364). The PC proportion detected by bone marrow morphology and flow cytometry in the MACS-FISH group had no correlation with the cytogenetic abnormality rate and positive cell rate of the 5 genes mentioned above. Additionally, the PC proportion detected by bone marrow morphology and flow cytometry showed significant difference (P<0.0001). CD138 immunomagnetic bead sorting combined with FISH technology can significantly improve the abnormality detection rate of MM cytogenetics.

Cytogenetic analysis of patients with primary amenorrhea in Eastern India

Amenorrhea is the absence of menstruation in women of reproductive age. Previous reports suggest that chromosomal abnormality is the second most common cause of amenorrhoea. Early referral for cytogenetic evaluation is recommended for the identification of underlying chromosomal aberrations in amenorrhoea patients. This was an observational study which was conducted in Vivekananda Institute of Medical sciences, Kolkata, during January 2013–December 2015. This study aims to estimate the frequency and types of chromosomal abnormalities in primary amenorrhoea (PA) patients in Eastern India and correlate their hormonal profile with chromosomal reports. Clinical features of 150 patients were recorded with clinical expertise. Peripheral venous blood was taken following informed consent, followed by karyotyping for chromosomal analysis. Results revealed 76.1% of PA with normal female karyotype (46, XX) and 23.9% with different abnormal karyotypes. Among the abnormal karyotype constituents, 50% numerical abnormalities, most frequent being Turner syndrome, pure (n = 12, 8%) and mosaic (n = 5, 3.3%). Three cases (2%) showed male (XY) karyotype. The other cases showed X structural abnormalities. This study emphasises the need for cytogenetic analysis as integral part of the diagnostic protocol in case of PA for precise identification of chromosomal abnormalities and for appropriate management and counselling of these patients.

Cytogenetic Studies of Rwandan Pediatric Patients Presenting with Global Developmental Delay, Intellectual Disability and/or Multiple Congenital Anomalies.

Global developmental delay (GDD) is defined as a significant delay in two or more developmental domains: gross or fine motor, speech/language, cognitive, social/personal and activities of daily living. Many of these children will go on to be diagnosed with intellectual disability (ID), which is most commonly defined as having an IQ <75 in addition to impairment in adaptive functioning. Cytogenetic studies have been performed in 664 Rwandan pediatric patients presenting GDD/ID and/or multiple congenital abnormalities (MCA). Karyotype analysis was performed in all patients and revealed 260 chromosomal abnormalities. The most frequent chromosomal abnormality was Down syndrome and then Edward syndrome and Patau syndrome. Other identified chromosomal abnormalities included 47,XX,+del(9)(q11), 46,XY,del(13)(q34) and 46,XX,der(22)t(10;22)(p10;p10)mat. In conclusion, our results highlight the high frequency of cytogenetically detectable abnormalities in this series, with implications for the burden on the healthcare. This study demonstrates the importance of cytogenetic analysis in patients with GDD/ID and MCA.

Are FSHR polymorphisms risk factors to premature ovarian insufficiency?

Premature ovarian insufficiency (POI) is an ovarian dysfunction characterized by increased FSH levels and amenorrhea before 40 years old. In recent years, the search for genetic causes of POI intensified and studies have been published relating the presence of mutations and polymorphisms in genes associated with development, recruitment and oocyte atresia. The aim of this study was to evaluate the presence of FSHR polymorphisms in our population and contribute with the elucidation of POI etiology. To achieve it, we have studied 100 patients with POI (G1), 60 patients with border line levels of FSH (G2) and 123 controls with regular menopause onset. Cytogenetic analysis of patients’ samples and genotyping of Asn680Ser and Ala307Thr polymorphisms were performed in cases and controls. Cytogenetic analysis showed that 92% of G1 patients had normal karyotype, 4% presented polymorphic variants, 3% presented mosaic karyotype involving X chromosome. In G2, 91.6% had normal karyotype results, 3.2% displayed polymorphic variants, and 3.3% presented a mosaic karyotype involving X chromosome. Statistical comparison showed that the polymorphic allele of Ala307Thr polymorphism is more frequent in patients than in controls (G1: p < 0.001 and G2: p = 0.0259). This association has not been previously reported. We concluded that Ala307Thr polymorphism in FSHR can be potentially associated to POI development and can be considered as a screening marker in patients with ovarian failure signals.

Cytogenetic Analysis in Patients with Newly Diagnosed Myelodysplastic Syndromes in Southern Italy

AbstractAbstract 50623 Background:Bone marrow karyotype in myelodysplastic syndromes (MDS) is essential to define the prognosis and to guide treatment decisions, including targeted therapies. Due to the lack of an extensive national MDS registry in Italy, epidemiological data on MDS, including cytogenetics, throughout the territory is unknown. Objective:We evaluated the incidence of cytogenetic abnormalities amongst newly diagnosed MDS patients in in 2 southern Italian regions (Calabria and Puglia). Methods:A pilot project, denominated ANDROMEDA (ANalysis of cytOgenetics alteRatiOn in the MyEloDysplAstic syndromes) was developed in 17 centers to offer a service of conventional cytogenetic analysis for all consecutive patients undergoing diagnostic evaluation for cytopenia and suspected MDS between January 1 and December 31, 2011. The study conformed to the ethical standards set out in the Declaration of Helsinki and was approved by institutional review boards at each participating center. Patients were required to provide their written informed consent. Clinical characteristics of patients and bone marrow morphology, iron staining and histology were registered. Bone marrow samples were centralized for standard cytogenetic studies and fluorescence in situ hybridization to two dedicated genetics laboratories (one for each region), blind to patients' data. Results:Two hundred and thirty-five patients were evaluated and MDS diagnosis was confirmed in 220 cases (88. 3%), according to WHO criteria. The overall incidence of clonal chromosome abnormalities detected by conventional analysis was 36. 9%. Single abnormalities included +8 (13 cases, 5. 8%), del(5q) (12 cases, 5. 4%), –Y (11 cases, 5. 0%) and del(7)/-7 (4 cases, 1. 8%). Complex karyotypes were detected in 18 (8. 1%) cases. Among all cases only 10 (4. 5%) bone marrow samples were not evaluable for cytogenetic analysis. FISH revealed additional abnormalities not identified by conventional analysis only in 3 (1. 3%) out of 72 cases. Patients were classified in WHO subtypes: 39. 2% refractory cytopenia with unilineage dysplasia (RCUD), 1. 5% refractory anemia with ring sideroblasts (RARS), 32. 5% refractory cytopenia with multilineage (RCMD), 10. 8% refractory anemia with excess of blast-1 (RAEB-1), 9. 3% refractory anemia with excess of blast-2 (RAEB-2), 4. 1% MDS with deletion 5q (MDS 5q-) and 2. 6% MDS unclassifiable (MDS-U). Conclusions:These preliminary results demonstrate that the incidence of abnormal karyotype patterns and WHO subgroups in MDS patients in Southern Italy is comparable with that described in other geographical areas. It is confirmed that conventional cytogenetic analysis is a standard in the diagnostic workup of MDS of patients with a suspected myeloid malignancy in order to identify primary abnormalities and prognostic models. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Analysis in Patients with Newly Diagnosed Myelodysplastic Syndromes in Southern Italy

AbstractAbstract 5200 Background:Bone marrow karyotype in myelodysplastic syndromes (MDS) is essential to define the prognosis and to guide treatment decisions, including targeted therapies. Due to the lack of an extensive national MDS registry in Italy, epidemiological data on MDS, including cytogenetics, throughout the territory is unknown. Objective:We evaluated the incidence of cytogenetic abnormalities amongst newly diagnosed MDS patients in in 2 southern Italian regions (Calabria and Puglia). Methods:A pilot project, denominated ANDROMEDA (ANalysis of cytOgenetics alteRatiOn in the MyEloDysplAstic syndromes) was developed in 17 centers to offer a service of conventional cytogenetic analysis for all consecutive patients undergoing diagnostic evaluation for cytopenia and suspected MDS between January 1 and December 31, 2011. The study conformed to the ethical standards set out in the Declaration of Helsinki and was approved by institutional review boards at each participating center. Patients were required to provide their written informed consent. Clinical characteristics of patients and bone marrow morphology, iron staining and histology were registered. Bone marrow samples were centralized for standard cytogenetic studies and fluorescence in situ hybridization to two dedicated genetics laboratories (one for each region), blind to patients' data. Results:Two hundred and thirty-five patients were evaluated and MDS diagnosis was confirmed in 220 cases (88. 3%), according to WHO criteria. The overall incidence of clonal chromosome abnormalities detected by conventional analysis was 36. 9%. Single abnormalities included +8 (13 cases, 5. 8%), del(5q) (12 cases, 5. 4%), –Y (11 cases, 5. 0%) and del(7)/-7 (4 cases, 1. 8%). Complex karyotypes were detected in 18 (8. 1%) cases. Among all cases only 10 (4. 5%) bone marrow samples were not evaluable for cytogenetic analysis. FISH revealed additional abnormalities not identified by conventional analysis only in 3 (1. 3%) out of 72 cases. Patients were classified in WHO subtypes: 39. 2% refractory cytopenia with unilineage dysplasia (RCUD), 1. 5% refractory anemia with ring sideroblasts (RARS), 32. 5% refractory cytopenia with multilineage (RCMD), 10. 8% refractory anemia with excess of blast-1 (RAEB-1), 9. 3% refractory anemia with excess of blast-2 (RAEB-2), 4. 1% MDS with deletion 5q (MDS 5q-) and 2. 6% MDS unclassifiable (MDS-U). Conclusions:These preliminary results demonstrate that the incidence of abnormal karyotype patterns and WHO subgroups in MDS patients in Southern Italy is comparable with that described in other geographical areas. It is confirmed that conventional cytogenetic analysis is a standard in the diagnostic workup of MDS of patients with a suspected myeloid malignancy in order to identify primary abnormalities and prognostic models. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Analysis of Patients with Primary Amenorrhea

Cytogenetic Analysis of Patients with Primary Amenorrhea

Cytogenetic Analysis of Patients with Primary Amenorrhea

Amenorrhea is a normal clinical feature in prepubertal, pregnant, and postmenopausal females. It also accounts for 20% of patients with infertility. The physiology of menstruation and reproduction ...

Use of Chromosome Engineering To Model a Leukemia-Associated 7q22 Deletion in the Mouse.

Identifying tumor suppressor genes from intervals that are deleted in human hematologic malignancies such as 5q, 7q, 9q, and 20q has proven extremely challenging, and several laboratories have implicated haploinsufficiency as a likely mechanism. Chromosome engineering, which involves performing sequential rounds of gene targeting to insert loxP sites at flanking loci in mouse embryonic stem (ES) cells and using Cre recombination to delete the intervening sequences, was recently used to successfully interrogate the 1p36 interval in human solid tumors (Cell 128(3):459–75, 2007). Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Cytogenetic analysis of patients who developed myeloid disorders with del(7q) uncovered a 2.5 Mb commonly deleted segment (CDS) within 7q22 (Blood 88(6):1930–5, 1996), suggesting that this region plays an important role in leukemogenesis. To investigate the in vivo consequences of somatic loss of this interval, we generated a 5A3flox mouse model that harbor loxP sites flanking a ∼2 Mb interval on mouse chromosome 5A3 that is syntenic to the human 7q22 CDS. We intercrossed these mice with the interferon inducible Mx1-Cre transgenic strain, and injected these mice with polyinosinic-polycytidylic acid (pIpC) to delete the region in the hematopoietic compartment. The desired recombination is relatively inefficient; however, hematopoietic cells with loss of this region persist in the stem/progenitor compartment for over 1 year and are transplantable. We neither observed a block in differentiation nor clonal outgrowth of mutant hematopoietic cells, suggesting that additional mutations are necessary to initiate leukemia. We initiated AML in these mice by introducing additional genetic lesions using retroviral insertional mutagenesis, and we are characterizing these leukemias to study the effects of the 5A3 deletion on leukemogenesis and to clone cooperating genes. Chromosome engineering is a robust strategy for modeling leukemia-associated deletions in vivo, and for interrogating how loss of a specific interval alters hematopoietic growth. We are using the 5A3 strain to analyze candidate myeloid tumor suppressor genes from chromosome 7q and to uncover genes and pathways that cooperate in leukemogenesis.

Multimodal genetic diagnosis of solid variant alveolar rhabdomyosarcoma

The most common types of rhabdomyosarcoma (RMS) are alveolar RMS (ARMS), which are characterized by the specific translocation t(2;13)(q35;q14) or its rarer variant, t(1;13)(p36;q14), producing the fusion genes PAX3-FKHR and PAX7-FKHR, respectively, and embryonal RMS (ERMS), which is characterized by multiple numeric chromosome changes. A solid variant of ARMS that is morphologically indistinguishable from ERMS has been described recently. We present two cases with an initial histopathologic diagnosis of ERMS in which the combined findings by cytogenetic, reverse-transcriptase polymerase chain reaction (RT-PCR), and comparative genomic hybridization (CGH) analyses demonstrate that both tumors were in fact the solid variant of ARMS. The cytogenetic analysis of patient 1 revealed a t(2;13)(q35;q14) and the RT-PCR study detected the corresponding PAX3-FKHR chimeric transcript. In patient 2, the cytogenetic finding of multiple trisomies was compatible with the initial histopathologic diagnosis of ERMS, but the finding of a PAX7-FKHR fusion transcript by RT-PCR pointed to the diagnosis of ARMS. Interestingly, the CGH findings of this case reconciled the molecular and cytogenetic data by detecting, in addition to the trisomies, amplification of chromosomal bands 1p36 and 13q14, where the PAX7 and FKHR genes are located, respectively. Our data indicate that this multimodal genetic analysis could be important for the differential diagnosis of these tumors. Furthermore, our findings and previous studies indicate that there are no apparent genetic differences between solid variant and typical ARMS.

Apparently unrelated cytogenetic abnormalities among 462 probands referred for the detection of del(22q) by FISH.

Laboratory-based reports of the cytogenetic abnormalities detected during the course of testing for deletion del(22q) are scant. We report our findings from the testing with FISH of 462 patients suspected to have del(22q) between 1994 and 2000. Of these, 447 had a normal karyotype. An apparently unrelated cytogenetic abnormality was detected in 15 (3.2%). Two of these abnormalities involved reciprocal translocation with chromosome 22q and one of these showed del(22q) with FISH. The other abnormalities included sex chromosome aneuploidies and unbalanced rearrangements of various chromosomal segments. There was no commonality among these abnormalities and no correlation with other reported cases. Among those with a normal karyotype, an unexpected deletion of the control arylsulphatase A (ARSA) probe was found, providing a definite frequency of 1/262 for ARSA deletions among patients suspected to have del(22q). Of the 462 referrals, 48 (10%) had one or more additional diagnoses, and in this group, 4 (8%) had del(22q) and 2 (4%) had an apparently unrelated cytogenetic abnormality. The data highlight the importance of initial cytogenetic analysis in patients suspected of del(22q) and negates the use of interphase FISH screening by itself for del(22q). The finding of 3.2% unrelated cytogenetic abnormalities is noteworthy. FISH should be used in any structural rearrangement to ascertain if the relevant locus is deleted or not. The continued reporting of patients diagnosed with del(22q) found to have an unrelated cytogenetic abnormality will expand the phenotypic spectrum and possible gene mapping may refine phenotypic specificity.

Pesquisa de sequências do cromossoma Y em indivíduos com síndroma de Turner.

The Turner syndrome (TS) has been described in association with different sex chromosome aberrations. Although most TS patients show no evidence of Y chromosome sequences, according to different authors some TS patients may have Y chromosome material present in a few cells that are not detected by standard cytogenetic analysis. The importance of identification of this low level Y mosaicism is of clinical relevance due to the patient's increased risk of developing gonadoblastoma. In the present study, standard chromosome analysis performed on peripheral blood lymphocytes from 22 TS patients showed 12 patients with 45,X karyotype, 7 patients were mosaics with or without structural abnormalities in one X chromosome and, the remaining three patients had the following karyotypes: 46,X,i(X)(q10); 46,X,+mar/47,X,idic(Y),+mar and 45,X/46,X,+r. Molecular studies were performed on genomic DNA extracted from peripheral blood lymphocytes and mouth epithelial cells, which derive from two different embryonic germ layers, mesoderm and ectoderm, respectively. The screening for low level Y mosaicism was carried out by simplex PCR and by nested PCR of the following Y specific loci: SRY (sex determining region Y), TSPY (testis specific protein Y encoded), DYZ3 (centromeric locus) and DAZ1 (deleted in azoospermia). In two TS patients a set of STSs of the Y long and short arms were used to characterize the idic(Y) and the ring chromosomes. The high sensitivity of the nested PCR (1 male cell/125,000 female cells) allowed for exclusion of the presence of low level Y mosaicism in 20 out of 22 TS patients. In the patient with the idic(Y), PCR analysis was positive for all Y loci tested excluding the heterochromatic region. This result identified the breakpoint between sY158 and sY159 on the long arm and, by fluorescence in situ hybridization (FISH) it was confirmed that the euchromatic part of the long arm, centromere and short arm of the Y chromosome were duplicated. The characterization of the ring chromosome, detected in one of the TS patients, was only possible to analyse by FISH and PCR. In this ring, derived from the Y chromosome, a deletion was identified including the pseudoautosomal region 1 (PARY1) and Y intervals 6 and 7. However, the ring Y was positive for SRY, RPS4Y, AMGY, TSPY loci on the short arm, DYZ3 (centromere) and, sY85, DFFRY, GY6, sY87, sY113, sY119, sY122, sY126 and RBMY1 on the long arm. This study excluded the presence of low level Y mosaicism in two tissues collected from 20 TS patients. FISH and molecular analysis allowed us to characterize, in 2 out of 22 patients, one idic(Y) and one ring chromosome. The nature of the latter had not been completely identified by standard cytogenetics. The potential increased risk of gonadoblastoma in TS patients carrying Y chromosome sequences justifies the application of FISH and PCR for the characterization of marker chromosomes and the application of nested PCR for the detection of low level Y mosaicisms when Y chromosome material is not detected by standard cytogenetic analysis in patients with a 45,X karyotype and/or with virilization.

Loss of heterozygosity on chromosome 5 in adults with acute lymphoblastic leukemia

Cytogenetic abnormalities are among the most important pretreatment predictors of outcome in patients with acute lymphoblastic leukemia (ALL). Deletions of genetic material can result in loss of tumor suppressor genes or other translation products that are crucial in maintaining an orderly cell cycle sequence or viability of the apoptotic cascade. Chromosome 5 contains many genes that are relevant in hematopoiesis. Deletions of chromosome 5 or parts thereof are found frequently in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) where they are associated with a poor prognosis. Although abnormalities of chromosome 5 are not commonly detected by cytogenetic analysis in patients with acute lymphoblastic leukemias, we hypothesized that loss of heterozygosity (LOH) of microsatellite markers on chromosome 5 may occur more frequently and likewise influence outcome in these patients. Therefore, we analyzed peripheral blood and bone marrow samples of 41 adults with a diagnosis of ALL for LOH by polymerase chain reaction (PCR) and correlated our findings with overall survival of patients with and without LOH. LOH for at least one microsatellite marker was found in seven of 41 patients (17%). All patients demonstrated LOH on the long arm of chromosome 5. In three patients, LOH was extended to 5p. A region of minimal deletion which overlapped in all seven patients could be localized between markers D5S410 and D5S436 corresponding to chromosomal location 5q31–33 which is similar to the area of minimal deletion seen in AML. None of these patients showed involvement of chromosome 5 by cytogenetic analysis. We conclude that patients with ALL have LOH for gene segments on chromosome 5, especially 5q, more frequently than expected from cytogenetic studies. Although, unlike AML, no significant impact on prognosis could be found between patients with and without LOH on chromosome 5: The current data suggest that 5q abnormalities are not specific for AML and can also occur in patients with ALL.

Effect of Conditioned Medium, Nutritive Elements and Mitotic Synchronization on the Accuracy of the Cytogenetic Analysis in Patients with Chronic Myeloid Leukemia at Diagnosis and During α-Interferon Therapy

To improve the yield of the cytogenetic analysis in patients with CML at presentation and during α-interferon therapy, three culture conditions for bone marrow or peripheral blood cells were tested in parallel. The effects of 5637 conditioned medium (CM), nutritive elements (NE), and methotrexate (MTX) cell synchronization were investigated in 10 Ph-positive (Ph +) CML patients at diagnosis (group 1), and in 13 Ph + CML patients receiving treatment with α-interferon (group 2). In the presence of 5637 CM and NE with or without MTX, the mitotic index values were significantly improved in both groups. In group 2, the morphological index was significantly increased when using 5637 NE, and percentages of abnormal cells did not differ in 5637 NE and 5637 NE MTX compared to the control condition. Although cessation of interferon administration before sampling may improve the yield of the technique, it does not seem necessary when using 5637 CM and NE. The variability of the response of leukemic cells to different culture conditions further supports the recommendation that, in addition to the control condition, supplementations with 5637 CM and NE with or without cell synchronization be used in parallel in all CML patients. Results suggest that, when the number of cells available is not sufficient for several cultures, 5637 NE with or without MTX should replace the control condition.

PCR detection of Y‐specific sequences in patients with Ullrich‐Turner syndrome: Clinical implications and limitations

Cytogenetic analysis of patients with Ullrich-Turner syndrome (UTS) may fail to detect low levels of Y chromosome mosaicism or Y-derived marker chromosomes. More sensitive polymerase chain reaction (PCR)-based tests have been developed; however, applicability of these data to prognosis of virilization and gonadoblastoma development has not been investigated adequately. We used a multiplex PCR-based method to detect two Y-specific sequences, SRY and AMGLY. Thirteen patients with UTS without cytogenetically detected Y chromosomes were studied. Y-specific sequences were detected in 5 patients by multiplex PCR. A cryptic translocation involving the Y chromosome was found in one patient with severe virilization of external genitalia and a male phenotype. Y chromosomal mosaicism was detected in peripheral blood and in both gonads of one patient, and only in the left gonad of another patient. Existence of a Y-derived marker was demonstrated in 2 patients, one of whom had no testicular tissue or virilization. Consistent with previous reports, we conclude that PCR is more sensitive than classical cytogenetic analysis and detects patients with Y-specific sequences in blood cells. However, the absence of Y-specific material in blood is not a sufficient reason to reject surgical treatment in case of virilization. Am. J. Med. Genet. 76:283–287, 1998. © 1998 Wiley-Liss, Inc.

PCR detection of Y-specific sequences in patients with Ullrich-Turner syndrome: Clinical implications and limitations

Cytogenetic analysis of patients with Ullrich-Turner syndrome (UTS) may fail to detect low levels of Y chromosome mosaicism or Y-derived marker chromosomes. More sensitive polymerase chain reaction (PCR)-based tests have been developed; however, applicability of these data to prognosis of virilization and gonadoblastoma development has not been investigated adequately. We used a multiplex PCR-based method to detect two Y-specific sequences, SRY and AMGLY. Thirteen patients with UTS without cytogenetically detected Y chromosomes were studied. Y-specific sequences were detected in 5 patients by multiplex PCR. A cryptic translocation involving the Y chromosome was found in one patient with severe virilization of external genitalia and a male phenotype. Y chromosomal mosaicism was detected in peripheral blood and in both gonads of one patient, and only in the left gonad of another patient. Existence of a Y-derived marker was demonstrated in 2 patients, one of whom had no testicular tissue or virilization. Consistent with previous reports, we conclude that PCR is more sensitive than classical cytogenetic analysis and detects patients with Y-specific sequences in blood cells. However, the absence of Y-specific material in blood is not a sufficient reason to reject surgical treatment in case of virilization.