Diagnostic role of conventional cytogenetics and fluorescence in situ hybridization (FISH) in chronic myeloid leukemia patients.

INTRODUCTION: The limitation of cytogenetic analysis is that the Ph chromosome cannot be detected in clumped metaphase or in interphase cells. Fluorescence in situ hybridization (FISH) is a highly sensitive molecular genetic technique, which enables to detect break point cluster region--Abelson (BCR-ABL) complex and minimal residual disease in all Ph positive CML patients not only in metaphase but also in interphase cells. AIMS: To detect Ph chromosome in CML patients by the use of conventional cytogenetics and FISH. MATERIAL AND METHODS: The bone marrow samples were collected in heparinised syringe from 35 diagnosed CML patients and transported to cytogenetic laboratory for chromosomal analysis. Conventional karyotype was prepared by direct harvesting and short-term culture. The FISH analysis was carried out on interphase cells of two patients to confirm the cytogenetic diagnosis. RESULTS: Out of 35 CML patients, 17 (49.9%) were 100% Philadelphia positive, 10(28.5%) were 50-70% Ph+ mosaics and 3(9%) were 100% Ph negative. In 5 patients (14.25%) cytogenetic analysis failed to confirm the presence or absence of Ph chromosome. FISH was carried out in interphase cells from bone marrow preparations of two patients. The signals for BCR-ABL fusion gene was absent in Ph- negative CML patients. In Ph positive patients, the FISH analysis detected BCR-ABL fusion gene seen as a yellow signal on interphase cells. CONCLUSION: Conventional cytogenetics is a useful method for detection of Ph chromosome in metaphase stage of cell division. FISH can be used in interphase stage of cell division for the same purpose.

Molecular screening for Yq microdeletion in men with idiopathic oligozoospermia and azoospermia.

Infertility affects 15% couples attempting pregnancy and in 40-50% of these cases the male partner has qualitative or quantitative abnormalities of sperm production. Microdeletions in the azoospermia factor (AZF) region on the long arm of the Y chromosome are known to be associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb and AZFc) which are critical for spermatogenesis and are recurrently deleted in infertile males. Semen analysis was carried out on one hundred and twenty five infertile males with oligozoospermia and azoospermia. Cytogenetic analysis was done for all the cases and in all cytogenetically normal cases (n = 83) microdeletion analysis was carried out on DNA extracted from peripheral blood using PCR. The sequence tagged sites (STS) primers sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc) were used for each case. Eight of the eighty three cases (9.63%) showed deletion of at least one of the STS markers. Correlation of phenotype with microdeletion was done in each case to determine any phenotype association with deletion of particular AZF locus. Based on the present study, the frequency of microdeletion in the Indian population is 9.63%. This study emphasizes the need for PCR analysis for determining genetic aetiology in cases with idiopathic severe testiculopathy.

Spermatogenic alterations in men with high testiculo epididymal temperatures.

Sperms are produced by a highly complex and poorly understood differentiation process known as spermatogenesis. Occupational exposure to high temperatures adversely affect testicular function causing partial or complete spermatogenic arrest. Dyers, cooks, blast furnace workers and men with varicocele are known to develop testicular hyperthermia, which leads to oligoasthenoteratozoospermia (OAT) and azoospermia. Semen analysis of 125 infertile men (and 25 fertile controls following the WHO guidelines, 1999 showed azoospermia in 109 men and oligozoospermia in 16 men. Twenty azoospermic and 14 oligozoospermic men had high testiculoepididymal temperatures either due to occupational exposure to high temperature or varicocele. All the 14 oligozoospermic men showed a very high percentage of sperms with abnormal morphology, impaired motility and they were subclassified as OAT group. Observations made in this study reiterates that high intratesticular temperature causes partial or complete spermatogenic arrest and may lead to increased production of morphologically abnormal sperms with impaired motility. This inverse relationship of sperm function with elevated temperature has implications in clinical medicine both in understanding pathological states and for therapeutic measures.

Prenatal diagnosis of chromosomal abnormalities in women with high risk pregnancies.

BACKGROUND & OBJECTIVES: Prenatal diagnosis helps in averting the birth of infants with chromosomal abnormalities. Fluorescence in situ hybridization (FISH) has been introduced as a potentially powerful tool in clinical cytogenetics. Several studies have reported successful prenatal diagnosis of chromosomal abnormalities in high risk pregnancies using FISH, however there are no reported studies from an Indian set up. Prenatal diagnosis for the detection of chromosomal abnormalities was carried out by conventional cytogenetics in the present study in the foetuses of high risk pregnancies in women attending a tertiary care facility in north India. These cases were further analyzed using FISH, to test the efficiency and utility of this technique for prenatal detection of common aneuploidies. METHODS: A total of 82 women with high risk pregnancies (81 singleton, 1 twin) were included in the study. Prenatal diagnosis was performed in these women using conventional chromosomal analysis (CCA) and interphase or metaphase FISH on chorionic villus or amniotic fluid or cord blood samples. RESULTS: Chromosomal analysis was successful in 80 instances and uninformative in 2. Abnormal karyotypes were detected in five (6.09%) of these women. FISH was successful in all the subjects and the results were in conformity to the cytogenetic results. In the 2 women where cytogenetic analysis was uninformative, results were given on the basis of interphase FISH. INTERPRETATION & CONCLUSION: The study clearly demonstrates that prenatal diagnosis is useful in the detection of chromosomal abnormalities in foetuses of women with high risk pregnancies. FISH is a powerful sensitive molecular cytogenetic technique, through which specific chromosomal abnormalities can be diagnosed/identified rapidly and accurately and may be used as an adjunct to conventional cytogenetic analysis.

Detection of chromosomal abnormalities using fluorescence in situ hybridization (FISH).

BACKGROUND: A number of studies have demonstrated the use of molecular cytogenetic techniques for clinical diagnosis. We compared the results of FISH analysis and conventional cytogenetics on different tissue samples for detection of chromosomal aberrations and to assess the utility of FISH assay for clinical diagnosis. METHODS: Karyotypic analysis was carried out on 50 samples--20 peripheral blood samples, 20 bone marrow samples and 10 prenatal (chorionic villi/amniotic fluid) samples. The same chromosome preparations were further subjected to FISH analysis using probes specific for chromosome X, Y, 21 or bcr-abl gene. RESULTS: The results of FISH analysis were in conformity with the cytogenetic results in all the samples except one. FISH analysis could reveal hybridization signals even on poorly spread metaphase chromosomes and interphase nuclei. It was also possible to detect subtle chromosomal aberrations which were not detected using conventional chromosomal analysis. CONCLUSION: FISH is a powerful, sensitive molecular cytogenetic technique which can be used as an adjunct to conventional chromosomal analysis for prenatal diagnosis and the diagnosis and management of cancer patients. FISH analysis should be used as a supplement to conventional cytogenetics.

Confirmation Of Cytogenetic Diagnosis In Ph' Positive And Negative Cases By Fluorescence In Situ Hybridization.

Chronic myelogenous leukemia (CML) is a clonal bone marrow disease characteristics of CML is the presence of the Philadelphia (Ph1) chromosome which involves rearrangement of BCR-ABL genes as a result of reciprocal translocation between chromosomes 9 and 22. Cytogenetic analysis requires sufficient numbers of well-spread metaPhases, but, the recently described fluorescent in situ hybridization (FISH) technique can also be used on poorly spread metaPhases and on interPhase cells to identify the Ph1 chromosome. We have performed cytogenetic as well as FISH analyses using ber-abl probe to determine if 1) the two methods of analyses complement each other, and 2) FISH analysis is more sensitive in detecting the Ph1. Cytogenetic analysis on 23 patients with clinical diagnosis of CML, showed the presence of Ph1 chromosome in 15 patients, whereas 8 patients were Ph1 negative. Specimens from all the 23 patients were independently studied for the presence of Ph1 chromosome using FISH. A reliable correlation was seen between patients with Ph1 chromosome and hybrid signal in all the patients studied. In addition, 3 cytogenetically Ph1 negative patients showed significant numbers of cells with hybrid signal by FISH analysis. ber-abl hybrid was also seen in all the patients with Ph' + ve cells. These results underscore the significance of the FISH technique in identifying the ber/abl hybrids in cells from patients with normal karyotype and, therefore, has tremendous application in detecting minimal residual disease following chemotherapy or monitoring the persistence of leukemic cells after bone marrow transplantation.

PCR-Based Detection Of Occult Y Chromosome Sequences In Turner Subjects.

Two hundred and sixty three patients with clinically suspected gonadal dysgenesis were analyzed cytogenetically using banded metaphase chromosomes. There were 61 cases exhibiting karyotypic changes; of these 28 showed 45, X and the rest were largely mosaics. The employment of FISH proved helpful to detect some of the unrecognizable chromosomal changes in selected cases. PCR analysis conducted with eleven sets of primers from the Y chromosome of 36 patients and 35 normal healthy individuals revealed the usefulness of the molecular investigations in conjunction with cytogenetic analysis. In conclusion, the application of molecular techniques to detect low level mosaicism allowed better management of the patients with dysgenesis of the gonads.

Familial male pseudohermaphroditism.

Familial male pseudohermaphroditism (MPH) due to 17,20-desmolase deficiency is rare. Here we present two siblings with MPH possibly due to 17,20-desmolase deficiency. The first patient presented with unambiguous female external genitalia and hypergonadotrophic hypogonadism. Chromosomal analysis revealed 46 XY. Ultrasound evaluation of pelvis revealed gonads in the inguinal canal, and no uterus. These findings were confirmed on laparotomy. Histology revealed the gonads to be testes. The second patient had ambiguous genitalia (perineoscrotal hypospadias, bifid scrotum with palpable gonads) with a 46 XY chromosomal pattern. Both patients had high plasma 17-hydroxy progestrone (17 OHP), low normal dehydro epiandrosterone sulphate (DHEAS) and low plasma testosterone. Plasma testosterone and DHEAS showed no response to ACTH or HCG. These features are compatible with the diagnosis of 17,20-desmolase deficiency.

Marker Chromosome in a Turner Patient.

Out of the twenty seven suspected cases of Turner Syndrome analysed during 1993-1995, eleven had the chromosome pattern of 45,X; fifteen had 45,X / 46,XX and one had mosaic pattern of 45,X / 46,X, +mar. The paper discusses a patient with the mosaic chromosome pattern of 45,X / 46,X, +mar showing most of the Turner stigmata. The marker chromosome was seen in the 76% of the metaphases analysed. It was too small and G, C, Q banding could not confirm the origin. The position of the marker chromosome was random and no significant centromeric association was seen. It is well documented that the patients with the dysgenetic gonads and the presence of Y material has increased risk of malignancy. The origin of the marker chromosome is discussed in relation to phenotypic features.

Molecular Analysis Of The Additional X-Chromosome In Klinefelter's Syndrome Patients.

The parental origin of the extra X chromosome in five families with Klinefelter's syndrome (47,XXY) was studied DNA restriction fragment length polymorphisms (RFLPs). In four, the extra X chromosome was maternal in origin and one it was paternal. Four X-linked marker loci were used and we were able to specify the origin of the extra X chromosome in all cases. The parental origin of the extra X chromosome in five families with Klinefelter's syndrome (47,XXY) was studied DNA restriction fragment length polymorphisms (RFLPs). In four, the extra X chromosome was maternal in origin and one it was paternal. Four X-linked marker loci were used and we were able to specify the origin of the extra X chromosome in all cases. The parental origin of the extra X chromosome in five families with Klinefelter's syndrome (47,XXY) was studied DNA restriction fragment length polymorphisms (RFLPs). In four, the extra X chromosome was maternal in origin and one it was paternal. Four X-linked marker loci were used and we were able to specify the origin of the extra X chromosome in all cases. The parental origin of the extra X chromosome in five families with Klinefelter's syndrome (47,XXY) was studied DNA restriction fragment length polymorphisms (RFLPs). In four, the extra X chromosome was maternal in origin and one it was paternal. Four X-linked marker loci were used and we were able to specify the origin of the extra X chromosome in all cases.

Induction of sister chromatid exchanges in phenytoin treated & untreated patients with epilepsy.

The mutagenic potential of phenytoin (PHT) was studied using the sister chromatid exchange (SCE) assay. Twenty nine PHT treated epileptics, 32 untreated and 32 normal healthy controls were analysed. Similar SCE frequencies were observed in untreated patients and patients on PHT monotherapy. Both groups had significantly increased SCE frequency as compared to controls. No positive correlation of SCE frequency with sex and duration of therapy was observed. The results of the present study suggest the role of the disease condition in inducing genetic damage as assessed by increased SCE frequencies.