Ajit Kumar Saxena

Clinical spectrum of neural tube defects with special reference to karyotyping study.

Background: Neural tube defects are common congenital malformations of the central nervous system. Despite years of intensive epidemiological, clinical, and experimental research, the exact etiology of NTD remains rather complex and poorly understood. The present study attempted to look into the association of occurrence of NTD with reference to folic acid levels, along with karyotyping status. Materials and Methods: Detailed history was taken with emphasis on age of the baby and mother, parity, antenatal folic acid intake. Five milliliters of blood was drawn from all the babies and their mothers and divided equally in preheparinized vials (for karyotyping) and plain vials (for folic acid estimation). The total duration was 2 years. Results: The total number (n) in the study group was 75. The folic acid level was less in affected babies and their mother when compared to matched controls. Chromosomal defect was observed in nine of the 75 patients. Karyotyping defects were higher in children born to mothers of the age group 31-40 years and when their birth order was second. Conclusion: Folic acid supplementation needs to be continued to prevent the occurrence of NTD, and the perinatal identification of NTD should alert one to the possibility of chromosomal abnormalities and prompt a thorough cytogenetic investigation and genetic counseling.

CYCLOPHOSPHAMIDE MODULATES GENE EXPRESSION IN NEONATAL RAT TESTIS FOLLOWING ANTENATAL EXPOSURE TO FETUSES DURING TESTICULAR DIFFERENTIATION

The present study was designed to evaluate the altered gene expression in neonatal rat testis after antenatal exposure of cyclophosphamide (one time single dose of either 2, 10, or 20 mg/kg body weight) to the developing fetuses, especially at the time of male sex differentiation. The rationale behind these experiments is to know about the involvement of Y-chromosome gene-dependent product(s) associated with gonadal dysfunction. Using SDS-polyacrylamide gel electrophoresis and photosensitive silver staining technique, the study shows that the variety of proteins of different molecular weight ranges 40,000 to 127,000 Da are modulated by cyclophosphamide exposure to the developing testis. Interestingly, the overexpression of one protein of 74,500 Da was observed both in supernatant as well as in pallet fractions. The qualitative and quantitative regulation of newly synthesized protein appearance or disappearance is observed in a dose-dependent manner.

Cyclophosphamide-induced chromosomal aberrations and associated congenital malformations in rats

Dear Editor: Cyclophosphamide (CP) is a known anticancer drug, metabolically activated in the presence of the cytochrome P-450 monooxygenase enzyme system to form highly reactive intermediates such as phosphoramide mustard and acrolein (3). These metabolites can induce DNA damage, cross-linking, and single strand breakage, and disturb the cell cycle at particular concentrations of teratogen (4). CP has been frequently used as a model teratogen, although little is known about the mechanism of action as a whole drug or its reactive metabolites on genotoxicity during early embryogenesis. Since the introduction of CP in the treatment of malignancies, its mutagenic and teratological effects have been studies by various workers (1,2,5,6). In vitro, studies indicate that both phosphoramide mustard and acrolein are teratogenic, may retard growth, and cause other congenital anomalies in rat and mouse embryos during organogenesis. Action of cyclophosphamide on the various stages of cell division from a cytogenetic point of view has been poorly understood, although CP is a S-dependent clastogen. Thus, the present study was designed to evaluate its genotoxicity in terms of chromosomal features between normal and abnormal (malformed)-looking pups when CP was given in utero. In the present study, rats (C. F. inbred strain) were used (n = 30) in the experiments. Sperm-positive d was designated as Day 0 of the pregnancy. Cyclophosphamide (Khandelwal Lab., Bombay, India) solution in water was given (i.p.) to pregnant rats on the 12th d of gestation (20 mg/kg body weight). Control rats received normal saline parallel to the experiment at the same site. Pregnancy was allowed to continue until delivery. The pups were classified as phenotypically normal or abnormal. Colchicine solution (8 ktg/kg body weight) was given to arrest mitosis 2 h before the pups were sacrificed. Liver was dissected out from control, normal, and abnormal pups for cytogenetic studies. A single-cell suspension formed and was centrifuged for 10 rain at 800 rpm; the supernatant was decanted. The pellet was then suspended in prewarmed 0.56% KC1 (British Drug House, India) solution, and after hypotonic treatment the cells were dispersed by gentle pipetting and again centrifuged for 10 rain at 800 rpm. The pellet was fixed in a freshly prepared methanol-acetic acid solution (3:1 ratio) and changed three times before the slides were prepared. The slides were flame-dried and stained with 5% Giemsa stain for 5 rain as in previous studies (9). Metaphase plates were observed under 1000 × magnification and photographed. Statistical analysis with a chi-square test was applied to compare differences between control and experimental groups with respect to total and individual chromosomal features. In the experimental group, two types of pups were collected after antenatal exposure of CP, either phenotypically normal looking or abnormal (malformed) with clinical features such as hydrocephalus, micrognathia, reductive malformation of limbs, edema, protruded tongue, kinking of the tail, swollen umbilical cord, and growth retardation. For cytogenetic analysis, dispersed metaphase plates were examined and showed a variety of abnormal chromosomal features such as gaps, chromatid and chromosomal breaks, acentric fragments, dicentrics, centromeric breakage, chromatin bodies (Cbs) and aneuploidy. After we split the data between total chromosomal abnormalities and Cbs alone, we observed that the frequency of Cbs was very high in malformed fetuses (29.60%), when compared with normal-looking fetuses (19.31%). The Cbs originate after extreme fragmentation of chromosomes, leading to the formation of a large number of rounded and oval structures with or without chromosomal complements as mentioned in earlier studies (8). The total number of ceils examined and the frequency of chromosomal aberrations is noted in Table 1. The results showed that the incidence of total chromosomal abnormalities in normal-looking fetuses (33.79%) was lower than that in abnormal-looking fetuses (45.81%). The statistical

Penetrance of MTHFR, MTRR and SHMT Gene Polymorphism Modulate Folate Metabolism in Maternal Blood and Increases “Risk Factor” in Neural Tube Defects in Eastern India.

Background: Neural Tube Defects (NTDs) are a multifactorial disorder that arises during first month after conception due to complex interactions between genetic and environmental factors. Role of folate metabolism plays a significant role in determining genetic predisposition of NTDs. Materials and Methods: Present study was conducted to evaluate the allele frequency of folate regulatory candidate genes methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and serine hydroxymethyltransferase (SHMT) as “risk factors” in NTD cases in Indian population. Results: Genomic DNA was isolated from NTD cases, NTD mothers and respective controls. PCR-RFLP analysis was performed using specific set of primers to determine the frequency of genotypes and their alleles after using restriction enzymes- Hinf, MboII, Nde I & EarI. The DNA fragments were separated on agarose gel and visualized by Gel documentation system. MTHFR 667CT genotype reveals variable frequency between homozygous (CC genotype, wild type) 64.00% and heterozygous (CT) condition (32.00%) in NTDs cases. MTHFR 1298AC genotype showed a frequency 35.78% in heterozygous (AC) and 5.54% in homozygous (CC) conditions. Statistical analysis was performed by calculating CC/TT genotype O.R (0.113) and C.I. at 95% (0.0054-2.367) of and of AA/AC genotype O.R. (3.24) at 95% C.I (0.690-15.205) that showed significant (p<0.05) differences between NTD mothers and their respective controls in MTHFR gene. Data was further analyzed by adding “T/C” alleles in MTHFR gene to increase statistical power which further showed significant (p<0.001) differences between NTD cases with respect to controls. MTRR 66A→G gene showed significant (p<0.05) difference between NTDs cases and NTD mothers after combining the genotypes (AA vs. AG+GG). SHMT 1420CT gene showed lack of significant differences between homozygous and heterozygous conditions in NTD cases and NTD mother with their respective control groups. Conclusion: Present study suggests that the variations in the genotype frequency are due to the penetrance of defective allele into maternal gene pool, affecting DNA synthesis during organogenesis leading to the onset of NTDs.