The E368Q Mutant Allele of GJA8 is Associated with Congenital Cataracts with Intrafamilial Variation in a South Indian Family.

PURPOSE: To determine the basis of the autosomal dominant congenital cataracts in a three generation south Indian pedigree. METHODS: The proband and several family members underwent a complete ophthalmic examination. The coding regions of eight candidate genes (CRYAA, CRYBB2, CRYGC, CRYGD, GJA3, GJA8, AQP0, and PITX3) were amplified by PCR and directly sequenced. Wild type and mutant connexin50 (Cx50) were expressed by stable transfection of HeLa cells. Their cellular distributions and function were examined by immunofluorescence microscopy and by microinjection of gap junction permeant tracers, respectively. RESULTS: Congenital cataracts (with some variations in phenotype) segregated as an autosomal dominant trait within a three generation pedigree. Three affected individuals (proband, sibling and mother) showed a sequence variation in the candidate gene GJA8 encoding Cx50: a c.1102G>C transversion encoding a substitution of glutamate for glutamine at position 368 (E368Q). This substitution was absent from an unaffected family member (paternal aunt) and 100 healthy controls of the same ethnicity. In transfected HeLa cells, both wild type Cx50 and E368Q localized to gap junction plaques, and supported similar levels of intercellular transfer of Neurobiotin. CONCLUSIONS: The E368Q mutant allele of GJA8 is associated with autosomal dominant congenital cataracts with phenotypic variability. E368Q forms gap junction plaques and functional channels in transfected HeLa cells.

Translocation t(X;11)(q22;q25) in a woman with premature ovarian failure.

Genetic, autoimmune, environmental, iatrogenic, and idiopathic factors are known to cause premature ovarian failure (POF). This report describes an X;11 translocation, t(X;11)(q22;q25), in a woman diagnosed with POF. The FSH level was found to be elevated. Menstrual cycle was regular initially, and she had a spontaneous abortion at the 5th month of gestation at 16 years of age. Her mother was karyotypically normal while her father was not investigated. Male carriers of X;autosome translocations are mostly infertile, and hence the translocation is presumed to be of de novo origin. Fluorescence in situ hybridization using whole chromosome paint probes confirmed the rearrangement.

An MIP/AQP0 mutation with impaired trafficking and function underlies an autosomal dominant congenital lamellar cataract.

Autosomal dominant congenital cataracts have been associated with mutations of genes encoding several soluble and membrane proteins. By candidate gene screening, we identified a novel mutation in MIP (c.494 G > A) that segregates with a congenital lamellar cataract within a south Indian family and causes the replacement of a highly conserved glycine by aspartate (G165D) within aquaporin0 (AQP0). Unlike wild type AQP0, expression of AQP0-G165D in Xenopus oocytes did not facilitate swelling in hypotonic medium. In transfected HeLa cells, wild type AQP0 localized at the plasma membrane while AQP0-G165D was retained within the secretory pathway, and localized mainly within the endoplasmic reticulum. These results suggest that mutation of this conserved glycine residue leads to improper trafficking of AQP0-G165D and loss of water channel function. They emphasize the importance of AQP0 for maintenance of lens transparency and identify a critical residue that is conserved among aquaporins, but has not previously been associated with disease-associated replacement.

A novel human CRYGD mutation in a juvenile autosomal dominant cataract.

PURPOSE: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India. METHODS: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site. RESULTS: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein. CONCLUSIONS: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.

Genotoxic and antigenotoxic effects of Hemidesmus indicus R. Br. root extract in cultured lymphocytes.

AIM OF THE STUDY: The genotoxic and antigenotoxic potential of the ethanolic extract of Hemidesmus indicus roots were evaluated in cultured human lymphocytes using cisplatin as the positive mutagen. MATERIALS AND METHODS: Cytogenetic damage and cytotoxicity were determined in cells exposed to different doses of the extract, ranging from 2 to 32 microg/ml of culture medium, either alone or together with cisplatin. RESULTS: There was a significant reduction in cisplatin-induced frequencies of sister chromatid exchanges, chromosome aberrations and micronucleated binucleate cells at the lower concentrations of 4 and 8 microg/ml (P<0.05). However, the extract by itself reduced the proliferative rate index, mitotic index and cytokinesis-block proliferative index (P<0.05). Further, a significant increase in the percentage of chromosome aberrations was noticed at the higher concentrations. CONCLUSION: Hemidesmus indicus root extract possesses significant genoprotective effect at the lower concentrations although it is cytotoxic and probably genotoxic at higher doses.

Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice.

The genotoxic potential of anti-tumor drugs limits their efficacy in the treatment of cancers. Since ancient times, saffron (dried stigmas of Crocus sativus L.) has been used as a spice and medicinal herb. Saffron is a rich source of carotenoids and is known for its anti-cancer and anti-tumor properties. The present study was designed to ascertain the chemoprotective potential of saffron against the genotoxicity of three well-known anti-tumor drugs-cisplatin (CIS), cyclophosphamide (CPH) and mitomycin-C (MMC)--using comet assay. Three doses of saffron (20, 40 and 80 mg/kg b.w.) were orally administered to mice for five consecutive days prior to the administration of anti-tumor drugs under investigation. Pre-treatment with saffron significantly inhibited anti-tumor drugs induced cellular DNA damage (strand breaks) as revealed by decreased comet tail length, tail moment and percent DNA in the tail. These findings, together with our previous results, suggest a potential role for saffron as an anti-genotoxic, anti-oxidant and chemopreventive agent and could be used as an adjuvant in chemotherapeutic applications.

Protective effect of Spirulina fusiformis on chemical-induced genotoxicity in mice.

Spirulina fusiformis given by oral route to mice at doses of 250, 500 and 1000 mg kg(-1) significantly inhibit the genotoxicity induced by cisplatin and urethane. In addition, a significant reduction in the extent of lipid peroxidation with concomitant increase in the liver enzymatic (GPx, GST, SOD, CAT) and non-enzymatic (reduced glutathione) antioxidants were observed.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced in vivo clastogenicity: protective effects of aqueous neem leaf extract.

We evaluated the modifying effects of aqueous neem leaf extract on the in vivo clastogenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent gastric carcinogen by quantitation of micronuclei and chromosomal aberrations in metaphase cells from the bone marrow of male Wistar rats. Intraperitoneal injection of MNNG (40 mg/kg body weight) induced a significant increase in the frequency of micronuclei and chromosomal aberrations. Pretreatment with aqueous neem leaf extract (100 mg/kg body weight) significantly reduced MNNG-induced increase in micronuclei and chromosomal aberrations. These results reveal the chemoprotective potential of aqueous neem leaf extract against the clastogenic effects of MNNG.

Protective effects of saffron (Crocus sativus Linn.) on genotoxins-induced oxidative stress in Swiss albino mice.

The modifying effects of the aqueous extract of saffron (dried stigmas of Crocus sativus Linn.) on cisplatin (CIS), cyclophosphamide (CPH), mitomycin-C (MMC) and urethane (URE) induced alterations in lipid peroxidation and antioxidant status were investigated in Swiss albino mice. Three doses of saffron (20, 40 and 80 mg/kg body weight) were orally administered to mice for 5 consecutive days prior to administration of genotoxins. A significant reduction in the extent of lipid peroxidation with a concomitant increase in the liver enzymatic (SOD, CAT, GST, GPx) and non-enzymatic antioxidants (reduced glutathione) were observed in saffron pretreated animals compared with the genotoxins alone treated animals. However, the modulatory effects were not always dose dependent. Our data suggest that saffron may exert its chemopreventive effects by modulation of lipid peroxidation, antioxidants and detoxification systems.

Cytogenetic studies on spray painters in south India.

Studies on the frequencies of chromosomal aberrations were carried out on 104 spray painters working in automobile body reconditioning, steel furniture making and refrigerator repainting workshops in the metro city, Chennai, of south India. Randomly selected 50 male subjects not connected with this occupation were included as controls in the study. Chromosomal analysis was carried out in 48h lymphocyte (short duration) cultures representing the first mitotic division, on a subset of samples consisting of 50 spray painters, 20 controls and 72h (longer duration) cultures representing the second cell division, on all subjects. Baseline frequency of chromosomal aberrations was significantly higher among painters as compared to matched controls. Smoking and alcoholism as modulating factors had no added effect on the frequency of aberrant metaphases. Stepwise multiple linear regression analysis indicated that duration of service and age were significant factors that influence the frequency of chromosomal aberrations observed.

Effect of Spirulina fusiformis on cyclophosphamide and mitomycin-C induced genotoxicity and oxidative stress in mice.

Spirulina fusiformis was tested for its possible in vivo protective effects against cyclophosphamide (CP) and mitomycin-C (MMC) induced genotoxicity and oxidative stress in mice. Pre-treatment with S. fusiformis (250, 500 and 1000 mg kg(-1), p.o., daily for 5 days) significantly reduced the chromosomal damage and lipid peroxidation with concomitant changes in antioxidants and detoxification systems. All the three tested doses were effective in exerting a protective effect against CP and MMC.

Protection of N-methyl-N'-nitro-N-nitrosoguanidine-induced in vivo clastogenicity by aqueous garlic extract.

The modulatory effects of garlic extract on the in vivo clastogenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a carcinogenic nitrosamine, were evaluated by quantification of micronuclei and chromosomal aberrations in metaphase cells from the bone marrow of male Wistar rats. A single intraperitoneal injection of MNNG (40 mg/kg bodyweight) was found to be clastogenic as revealed by the increased frequency of micronucleated polychromatic erythrocytes and chromosomal aberrations. Pretreatment with aqueous garlic extract (250 mg/kg bodyweight) for 5 days significantly reduced the frequencies of MNNG-induced micronuclei and chromosomal aberrations. The results demonstrate that administration of garlic extract protects against the clastogenic effects of MNNG.

Inhibition of genotoxicity by saffron (Crocus sativus L.) in mice.

Experiments were carried out to ascertain whether or not saffron (dried stigmas of Crocus sativus L.), a commonly used agent for flavoring and coloring food can exert modulatory effects on the in vivo genotoxicity of cisplatin (CIS), cyclophosphamide (CPH), mitomycin C (MMC) and urethane (URE). For this purpose, Swiss albino mice were pretreated for five consecutive days with three doses (20, 40 and 80 mg/kg body weight) of the aqueous extract of saffron. Genotoxic effects were assessed in the mouse bone marrow micronucleus test. The results obtained suggest that pretreatment with saffron can significantly inhibit the genotoxicity of CIS, CPH, MMC and URE. This inhibitory effect was not always dose-dependent. In addition, the hepatic glutathione S-transferase (GST) activity was assessed in the control and treated animals. No significant change in GST activity was observed after pretreatment with saffron alone. Treatment with the genotoxins alone significantly inhibited GST activity. Saffron pretreatment attenuated the inhibitory effects of the genotoxins on GST activity.

Trisomy 4p and partial monosomy 18q due to paternal translocation t(4;18) (p11; q21.3).

A carrier status for balanced translocation in either of the parents increases the risk of congenital malformation in the offspring. A case of multiple congenital anomalies in a female newborn was found to be associated with trisomy 4p and partial monosomy 18q as a result of a reciprocal translocation, t(4; 18) (p11; q21.3) in the father. The clinical and cytogenetic findings are compared with characteristic features of trisomy 4p, monosomy 18q and two similar cases reported earlier.

Reduced levels of gamma-crystallin transcripts during embryonic development of murine Cat2nop mutant lenses.

BACKGROUND: From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma-crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma-crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. METHODS: The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. RESULTS: The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. CONCLUSION: The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice.

Cytogenetic damage in operation theatre personnel.

The study in a group of 24 (11 anaesthetists and 13 support staff) was planned to ascertain the cytogenetic risk in a group of theatre personnel who worked in various city hospitals. Their exposure in terms of duration of service vary from 3-30 years. The control group (n = 24) consisted of people with different occupations matched for possible confounding variables. Cytogenetic risk was assessed in terms of chromosome aberration and sister chromatid exchange in 72-hour lymphocyte cultures. A significant increase in the percentage of chromosome aberration was observed. The sister chromatid exchange was double that of the baseline value in 20% of the exposed individuals. These findings indicate the possible risk of cytogenetic damage for staff working in unscavenged rooms.