Prospective Case-Control Study to Evaluate the Role of Glutathione S Transferases (GSTT1 and GSTM1) Gene Deletion in Breast Carcinoma and Its Prognostic Significance.

Breast cancer is the most common cause of cancer death in women with the incidence rising in young women. GST gene polymorphisms are significant because of their role in the detoxification of both environmental carcinogens and also cytotoxic drugs used in therapy for breast cancer. The present study has been designed to identify the role of polymorphisms in GSTT1 and GSTM1 genes in the risk of development of breast cancer, in the prognostication of breast cancer, and in the prediction of response towards chemotherapy. Ninety-nine patients with breast cancer and 100 healthy controls with no history of cancer were taken from blood donors after informed consent. Epidemiological and clinical data was collected from participants and 5 ml of peripheral venous blood was collected for genotype analysis. Null genotype of GSTT1 was detected in 51.04 % of the controls in comparison to 20.2 % of patients with carcinoma breast, which was found to be statistically significant (OR 4.18; 95 % CI 2.01-8.75; P = 0.0001). GSTM1 gene deletion was also significantly more common among controls (60 %) than in patients with breast cancer (33 %) (OR 4.57; 95 % CI 2.20-9.51; P = 0.0001). Tumors more than 5 cm in size had greater tendency for GSTM1 gene expression (P value = 0.019), but other clinicopathological parameters did not show any correlation. GSTT1 and GSTM1 genes status did not show any association with response to chemotherapy. The results indicated the null genotype of both GSTT1 and GSTM1 to be protective for the development of carcinoma breast. None of the known etiological factors have any correlation with GSTT1 and GSTM1 gene deletion. Patients with small tumor size expressed GSTM1 gene deletion. Other tumor characteristics and clinicopathological parameters did not have any correlation with gene deletion.

Fertility among women with classical congenital adrenal hyperplasia: report of seven cases where treatment was started after 9 years of age.

BACKGROUND AND AIM: Androgen excess is believed to be one of the major factors responsible for poor fertility outcomes in females with congenital adrenal hyperplasia (CAH). Some believe that the adverse effect of androgens on fertility could have its origins as early as the antenatal years. To assess the impact of prolonged androgen exposure on fertility in CAH patients, we compiled the data of females with CAH followed in our clinic during the last 25 years who were sexually active and had not been initiated on steroids until age 9 years. STUDY DESIGN AND PATIENTS: This was an observational case study on seven patients with classical CAH who fulfilled the inclusion criteria. The age at initiation of therapy in these females ranged from 9 years to 29 years. RESULTS: All patients had varying degrees of genital ambiguity. The most common presenting complaints were genital ambiguity, non-development of secondary sexual characteristics, hirsutism and primary amenorrhea. Genital surgery was performed in all patients at ages ranging from 12 to 29 years, except for one patient who underwent surgery at age 5 years without a diagnosis of CAH being made. Breast development ensued within 2 to 12 months and periods started in all patients within 2-24 months of steroid initiation. There were 13 pregnancies (seven normal vaginal deliveries, two spontaneous abortions and four pregnancies were medically terminated). CONCLUSIONS: Late initiation of steroid therapy did not affect fertility in our cohort of CAH women. Androgen excess in situations of subnormal cortisol may not adversely affect fertility in females with CAH.

Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR.

BACKGROUND & OBJECTIVE: Analysis of the microdeletions in the azoospermia factor (AZF) region of Y chromosome by PCR is an important screening tool in the work-up of infertile males opting for assisted reproductive techniques. In the present study, the Y chromosome microdeletions were analyzed by PCR using primers corresponding to 16 sequence tagged sites (STS) and three genes of the AZF region in infertile Indian men. Feasibility of developing a simplified multiplex PCR for screening of the Y chromosome microdeletions has been explored. METHODS: A total of 271 male subjects were analyzed, of which, 170 were infertile patients (51 oligospermic and 119 azoospermic) and 101 were fertile controls. Subjects showing normal karyotype only were included in the study. The semen analysis was done and plasma follicle stimulating hormone (FSH) concentrations were determined by radioimmunoassay. Testicular histopathology was analyzed by fine needle aspiration cytology (FNAC). RESULTS: Y chromosome microdeletions were observed in nine out of 170 (5.29%) infertile males all of whom were azoospermic. Of the nine subjects, two had deletions in AZFa, one in AZFb, three in AZFc and three in AZFb+c regions. No deletions were observed in the infertile severe oligospermic men (< 5 million sperm/ml semen) and fertile controls. No difference in the FSH concentrations of infertile patients with and without deletions (18.36 and 18.10 mIU/ml respectively) was observed. A clear relationship between Y chromosome microdeletions and testicular phenotypes could not be established. Two multiplex PCRs were designed using 7 STSs markers, which could detect Y chromosome microdeletions in infertile male subjects as efficiently as PCR based on larger number of PCR reactions. INTERPRETATION & CONCLUSION: The multiplex PCRs described in the present study may be a suitable, cost-effective and less time consuming method for screening the Y chromosome deletions in infertile males in routine clinical diagnosis and counselling prior to assisted reproduction.

Gender identity of children and young adults with 5alpha-reductase deficiency.

Male pseudohermaphroditism (46,XY DSD) due to 5alpha-reductase deficiency has been recognized for the last few decades. There is scant literature on this entity in India. We compiled data on five patients with this disorder. Four of our five patients were reared as females. Our assessment of these children reveals that they had male gender identity from childhood. Three of the four reared as females chose to change gender role at adolescence, while the fourth is still prepubertal. We conclude that all these patients had male gender identity from early childhood. The parents took note of this only after the appearance of male secondary sexual characteristics at puberty, thereby giving an impression of change in gender identity and gender role.

Molecular diagnosis of 5alpha-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism.

AIM: To identify the genotype of two Indians with male pseudohermaphroditism. METHODS: Standard radioimmunoassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5alpha-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. RESULTS: We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. CONCLUSION: Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.

Higher frequency of Yq microdeletions in sperm DNA as compared to DNA isolated from blood.

AIM: To determine if Yq microdeletion frequency and loci of deletion are similar in two tissues (blood and sperm) of different embryological origin. METHODS: The present study included 52 infertile oligozoospermic cases. In each case, DNA was isolated from blood and sperms and polymerase chain reaction (PCR) microdeletion analysis was done from genomic DNA isolated from both the tissues. The PCR products were analyzed on a 1.8% agarose gel. PCR amplifications found to be negative were repeated at least three times to confirm the deletion of a given marker. RESULTS: Only 1 case harbored microdeletion in blood DNA, whereas 4 cases harbored microdeletion in sperm DNA. CONCLUSION: The frequency of Yq microdeletions is higher in germ cells as compared to blood. As the majority of infertile couples opt for assisted reproduction procreation techniques (ART), Yq microdeletion screening from germ cells is important to understand the genetic basis of infertility, to provide comprehensive counseling and most adapted therapeutics to the infertile couple.

Role of testicular fine-needle aspiration cytology in infertile men with clinically obstructive azoospermia.

BACKGROUND: Azoospermia due to obstruction of the vaso-epididymal junction is one of the few surgically correctable causes of male infertility. In patients where all clinical and laboratory parameters suggest a vaso-epididymal junction block amenable to surgery, failure to find normal spermatogenesis on fine-needle aspiration cytology (FNAC) of the testis may necessitate a change in treatment modality to the more expensive intracytoplasmic sperm injection. We evaluated the validity of FNAC findings in predicting failure of surgical exploration when clinical parameters suggest otherwise. METHODS: Infertile, azoospermic men in whom the semen volume and fructose content, testis size, follicle-stimulating hormone level were normal and the vas deferens was palpable with no evident cause for obstruction, underwent FNAC of the testis to confirm the presence of normal spermatogenesis before surgical exploration. Men with hypospermatogenesis or maturation arrest on FNAC and a normal karyotype with absence of Y chromosome microdeletion were offered assisted reproduction or surgical exploration to identify a reconstructable obstruction. Men who chose surgery were included in the study and the findings on exploration were compared with the FNAC reports. RESULTS: Of the 10 men who satisfied the inclusion criteria, 6 had hypospermatogenesis and in 4 FNAC showed maturation arrest. On surgical exploration, none had sperm in the epididymis. A biopsy of the testis taken at the time of exploration confirmed the FNAC findings. CONCLUSION: Clinical parameters are insufficient for diagnosing obstructive azoospermia. FNAC can accurately evaluate the testicular pathology and predict whether or not surgical exploration should be undertaken.

Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome.

AIM: To study the occurrence of Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome (KFS). METHODS: Blood and semen samples were collected from azoospermic patients with KFS (n = 14) and a control group of men of proven fertility (n = 13). Semen analysis was done according to World Health Organization (WHO) guidelines. Blood samples were processed for karyotyping, fluorescent in situ hybridization (FISH) and measurement of plasma follicle stimulating hormone (FSH) by radioimmunoassay. To determine Y chromosome microdeletions, polymerase chain reaction (PCR) of 16 sequence tagged sites (STS) and three genes (DFFRY, XKRY and RBM1Y) was performed on isolated genomic DNA. Testicular fine needle aspiration cytology (FNAC) was done in selected cases. RESULTS: Y chromosome microdeletions spanning the azoospermia factor (AZF)a and AZFb loci were found in four of the 14 azoospermic patients with KFS. Karyotype and FISH analysis revealed that, of the four cases showing Y chromosome microdeletion, three cases had a 47,XXY/46,XY chromosomal pattern and one case had a 46,XY/47,XXY/48,XXXY/48,XXYY chromosomal pattern. The testicular FNAC of one sample with Y chromosome microdeletion revealed Sertoli cell-only type of morphology. However, no Y chromosome microdeletions were observed in any of the 13 fertile men. All patients with KFS had elevated plasma FSH levels. CONCLUSION: Patients with KFS may harbor Y chromosome microdeletions and screening for these should be a part of their diagnostic work-up, particularly in those considering assisted reproductive techniques.

Conservation of the RB1 gene in human and primates.

Mutations in the RB1 gene are associated with retinoblastoma, which has served as an important model for understanding hereditary predisposition to cancer. Despite the great scrutiny that RB1 has enjoyed as the prototypical tumor suppressor gene, it has never been the object of a comprehensive survey of sequence variation in diverse human populations and primates. Therefore, we analyzed the coding (2,787 bp) and adjacent intronic and untranslated (7,313 bp) sequences of RB1 in 137 individuals from a wide range of ethnicities, including 19 Asian Indian hereditary retinoblastoma cases, and five primate species. Aside from nine apparently disease-associated mutations, 52 variants were identified. They included six singleton, coding variants that comprised five amino acid replacements and one silent site. Nucleotide diversity of the coding region (pi=0.0763+/-1.35 x 10(-4)) was 52 times lower than that of the noncoding regions (pi=3.93+/-5.26 x 10(-4)), indicative of significant sequence conservation. The occurrence of purifying selection was corroborated by phylogeny-based maximum likelihood analysis of the RB1 sequences of human and five primates, which yielded an estimated ratio of replacement to silent substitutions (omega) of 0.095 across all lineages. RB1 displayed extensive linkage disequilibrium over 174 kb, and only four unique recombination events, two in Africa and one each in Europe and Southwest Asia, were observed. Using a parsimony approach, 15 haplotypes could be inferred. Ten were found in Africa, though only 12.4% of the 274 chromosomes screened were of African origin. In non-Africans, a single haplotype accounted for from 63 to 84% of all chromosomes, most likely the consequence of natural selection and a significant bottleneck in effective population size during the colonization of the non-African continents.

Imaging in intersex disorders.

BACKGROUND: Evaluation of the gonads and internal genital structures is an essential component for evaluation of patients presenting with ambiguous genitalia. Ultrasonography (US) and magnetic resonance imaging (MRI) are the two preferred modalities. OBJECTIVE: To compare US and MRI in patients with intersex for localization of gonads and internal genitalia. PATIENTS AND METHODS: Ten patients with proven intersex disorders were included in the study. Findings from US and MRI were corroborated by those from surgery/laparoscopy. RESULTS: For evaluation of the gonads, MRI was found to be marginally more sensitive than US. For internal genital structures, both modalities were found to be equally sensitive and specific with no false positive results. CONCLUSION: US still remains the modality of choice for screening patients with intersex disorders. MRI is helpful in cases with equivocal US findings.

Differentiation of Fanconi anemia from idiopathic aplastic anemia by induced chromosomal breakage study using mitomycin-C (MMC).

This study was conducted to differentiate between Fanconi anemia (FA) and "idiopathic" aplastic anemia on the basis of induced chromosomal breakage study with mitomycin C (MMC). MMC-stress test was conducted on peripheral blood lymphocytes from 29 patients with aplastic anemia. Ten patients with very high percentage of chromosomal breakage and four patients exhibiting somatic mosaicism were diagnosed as FA on the basis of chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation while another eight lacked such anomalies or had minor physical problems. The present study illustrates that MMC stress test provides an unequivocal means of differentiation between Fanconi anemia and 'idiopathic' aplastic anemia. Further, the study, first of its kind from India, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of Fanconi anemia to implement appropriate therapy.

14q(22) deletion in a familial case of anophthalmia with polydactyly.

We report a family of anophthalmia with ocular and extraocular manifestations. The proband, his three sisters, and two sons had anophthalmia and preaxial polydactyly in the right hand. Cytogenetic analysis was done for the proband and two of his sons, one of whom was affected. Another male child was affected but was not available for cytogenetic analysis. Karyotypes of both affected individuals showed deletion on long arm of 14q22q23. Literature review shows four cases of anophthalmia with extra ocular anomalies associated with 14q (q22q23) deletion. Recently it has been suggested that the human homeobox gene, SIX6, and the BMP-4 gene are responsible for eye development. Both are located in the chromosome 14q22.3-q23 region. Deletion in this region has been known to be associated with anophthalmia and pituitary anomalies. This is the first family of anophthalmia, which showed polydactyly with a chromosomal deletion in the 14q22-q23 region and its familial transmission in two generations with a total of six affected individuals.

A case of chronic myeloid leukemia complicated with minimal change nephrotic syndrome.

We present a 28-year-old patient with chronic myeloid leukemia (CML) in chronic phase complicated with nephrotic syndrome. The bone marrow cells revealed the presence of Philadelphia chromosome, the cytogenetic hallmark of CML, that results from a balanced, reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11). This reciprocal translocation leads to the formation of the BCR/ABL fusion gene, the presence of which was confirmed using the highly sensitive fluorescence in situ hybridization technique. The renal biopsy was compatible with minimal change nephrotic syndrome. To the best of our knowledge, this is the first case of minimal change nephrotic syndrome associated with CML before the administration of any therapy.

Human molecular cytogenetics: diagnosis, prognosis, and disease management.

The year 2001 witnessed the sequencing of 90% of the euchromatic region in the human genome but the ultimate goal to delineate the positions of all genes is yet to be achieved. Fluorescence In Situ Hybridization (FISH) is one of the methods for localizing genes on chromosomes. In the present study, diagnostic utility of single-, dual-, and multicolor FISH was evaluated for prenatal diagnosis, cancer genetics, and screening of various congenital anomalies (sex chromosomal and autosomal). Centromeric probes for chromosomes X and Y were used for screening minor aneuploid cell lines (XXY, XO, and XXX) in the cases of primary amenorrhea and suspected Klinefelter syndrome. The cases with ambiguous genitalia were analyzed using a probe specific for the sex-determining region (SRY). Suspected cases of Down syndrome were subjected to FISH using probe specific for chromosome 21. FISH was also used to study gene alterations in retinoblastoma and myeloid leukemias. Prenatal diagnosis was done to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y using FISH on uncultured cells from amniotic fluid and chorionic villi sampling. The screening for common aneuploidies was extended to abortuses from spontaneous abortions. Using FISH, low-level mosaicism could be identified in some cases of primary amenorrhea and suspected Klinefelter syndrome. Submicroscopic gene rearrangements could be detected using FISH in cases of ambiguous genitalia and cancers. Further interphase FISH could provide results within 24 hours. To conclude, FISH adds to the diagnostic utility of routine cytogenetics and its use on interphase nuclei overcomes the difficulty of conventional cytogenetics, thereby reducing the time between sampling and diagnosis to 24 hr.

Spermatogenic arrest in men with testicular hyperthermia.

Sperm is 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 122 infertile men (and 25 fertile controls), following the WHO guidelines, 1999, showed azoospermia in 106 men and oligozoospermia in 16 men. Twenty azoospermic and fourteen 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 sperm 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 sperm 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.

Evaluation of minimal residual disease in patients with chronic myeloid leukaemia on IFN-alpha 2b therapy using conventional cytogenetics and fluorescence in situ hybridization.

BACKGROUND: Chronic myeloid leukaemia (CML) is a haematopoietic malignancy characterized by the presence of the Philadelphia (Ph) chromosome that results from balanced reciprocal translocation between chromosomes 9 and 22 leading to the formation of the bcr/abl fusion gene. Studies have shown that interferon-alpha (IFN-alpha) therapy induces both cytogenetic (reduction in Ph+ cells) and molecular response (reduction in the bcr/abl positive cells) in a large proportion of patients, thereby improving their prognosis and survival. There are no reports available from India on the clinical management of CML patients using IFN-alpha therapy and molecular methods for the evaluation of residual disease. We evaluated the efficacy of IFN-alpha 2b therapy bysequential cytogenetic and molecularanalysis. METHODS: Karyotypingwas done from G-banded metaphases obtained from 24-hour culture of bone marrow aspirates of 45 patients. Cytogenetic analysis was repeated at intervals of 4-6 months during the course of IFN-alpha therapy. Dual-colour fluorescence in situ hybridization (FISH) analysis using specific probes for bcr and abl genes was done to assess the molecular response. RESULTS: Eight patients achieved complete cytogenetic response with no Ph+ cells. Using FISH analysis, 4 of these patients were negative for the fusion gene implying a complete response, while the remaining 4 patients showed bcr/abl fusion signals that represent residual disease. CONCLUSION: Our study emphasizes the need for sequential cytogenetic and molecular analysis in the management of patients with CML and for the evaluation of minimal residual disease in patients on IFN-alpha therapy.

Prenatal detection of aneuploidies using fluorescence in situ hybridization: a preliminary experience in an Indian set up.

Fluorescence in situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics.