Frontal Theta Asymmetry as a Biomarker of Depression.

INTRODUCTION: Electroencephalography (EEG) has been used extensively to study affective disorders. Quantitative spectral analysis of an EEG scan has been used to assess the biological basis of emotional disorders such as depression as well as to investigate biomarkers of affective disorders. Inter-hemispheric asymmetries in both baseline and stimulus-evoked frequencies (alpha, beta, theta, and delta) are potential biomarkers of depression. The role of frontal alpha asymmetry has been established, but other spectral frequencies such as frontal theta remain elusive. We compared the hemispheric differences in frontal theta power in depressed patients and controls before and during listening to music to study the correlation of frontal theta asymmetry with depression. METHODS: To determine whether stimulus-evoked frontal theta asymmetry is a biomarker of depression, we compared 23 patients with mild depression (based on the Hamilton Depression Rating Scale) with 17 age- and sex-matched controls by conducting EEG at rest and after listening to Indian classical music. RESULTS: In controls without depression, the mean frontal theta power of the left hemisphere and frontal theta asymmetry increased significantly during music listening. In depressed patients, frontal theta asymmetry was reversed during music listening. CONCLUSION: Frontal theta asymmetry is a potential biomarker of depression.

HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer patients from India.

OBJECTIVES: The HPV16/18 code for an oncoprotein-E6, which binds to p53 tumor suppressor protein and degrades the protein via ubiquitination. A common polymorphism of p53 in exon 4 codon 72, resulting in either proline (Pro) or arginine (Arg), affects HPV16/18 E6-mediated degradation of p53 protein in vivo. Hence, in the current study we investigated the prevalence of HPV16/18 in cervical lesions and the distribution of p53 genotypes in cervical cancers and normal healthy women. METHODS: DNA from 337 Indian women with invasive cervical cancers, 164 women with clinically normal cervix, 64 women with low-grade squamous intraepithelial lesions (LSIL), and 5 women with high-grade squamous intraepithelial lesions (HSIL) was examined for the presence of HPV16/18 using consensus primers in a polymerase chain reaction (PCR), and the specific HPV type was identified by Southern hybridization of the PCR product using HPV16/18 type-specific nucleotide sequences as probes. Further, 134 women with cervical cancers and 131 healthy women were used to determine the frequency of p53 genotypes, Pro/Pro, Arg/Arg, and Pro/Arg, using peripheral blood cell DNA to indicate the constitutional genotypes and allele-specific primers, in a PCR-based assay. RESULTS: We observed a prevalence of HPV16/18 in 77% (258/337) of cervical cancer patients, 38% (24/64) of LSILs, 4 of 5 HSILs, and 15.2% (25/164) of normal healthy women. The frequency of distribution of the three genotypes of p53 codon 72 in a subgroup of the HPV16/18-positive cervical cancer patients was Pro/Pro 0.18 and Arg/Arg 0.26, with the heterozygous Pro/Arg 0.56, differing significantly from the genotype frequency in the normal healthy women (chi(2) = 6.928, df = 2, P < 0.05). CONCLUSIONS: A high prevalence of HPV16/18 was observed in the cervical cancers. The prevalence in LSILs confirms HPV16/18 infection as an early event and further indicates a role in progression of lesions. The p53 genotype distribution indicated that women homozygous for Arg genotype were at a 2.4-fold higher risk for developing HPV16/18-associated cervical carcinomas, compared to those showing heterozygous Pro/Arg genotype (odds ratio 2.4, 95% confidence interval 1.89 to 3.04).

Determination of p53 genotypes in oral cancer patients from India.

The p53 tumour suppressor gene is inactivated in various types of human cancers, and has been implicated as an early event in several cancers. A p53 Pro/Arg polymorphism at exon 4 codon 72, has been suggested to be involved in susceptibility to cancers as well. Hence, in the current study, we investigated p53 exon 4 codon 72 polymorphism using Proline or Arginine specific primers from the peripheral blood cells (PBC) representing constitutional DNA from 72 oral cancer patients. PBC from 153 normal healthy individuals were used to determine the frequency of the p53 genotypes, Pro/Pro, Arg/Arg and Pro/Arg, in the Indian population. The frequency of distribution of genotypes in the normal healthy individuals was, Pro/Pro - 0.20 (31/153), Arg/Arg -- 0.14 (22/153) and Pro/Arg -- 0.65 (100/153); and in the oral cancer patients was, Pro/Pro -- 0.19 (14/72), Arg/Arg -- 0.08 (6/72) and Pro/Arg -- 0.72 (52/72). Thus, we observed an equidistribution of the genotypes in normal control and oral cancer patients (chi(2)= 1.77, df = 2, 0.3

Infrequent loss of heterozygosity at adenomatous polyposis coli gene locus in Indian oral cancers.

We have investigated loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) tumor suppressor gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) in 86 untreated oral cancer patients, using matched oral cancer tissue and corresponding peripheral blood cell (PBC) DNA samples. PBC from 70 normal healthy individuals, were also analyzed for allelic distribution of APC gene. A 133 bp fragment, spanning exon 11 of the APC gene was amplified, and RsaI digestion of the PCR product defined the alleles as either homozygous 133 bp (Rsa(-/-)) or 87 and 46 bp (Rsa(+/+)) fragments, and heterozygous (Rsa(+/-)) exhibiting the three fragments. Distribution of the three alleles, Rsa(-/-), Rsa(+/+), and Rsa(+/-) in the oral cancer patients was observed as 10.5, 51.1 and 38.4%; whereas normal healthy individuals showed 11.4, 37.1 and 51.4%, respectively. In the informative heterozygous (Rsa(+/-)) oral cancer patients, LOH was infrequent, demonstrated in two of 33 (6%) samples. Thus, the APC gene was infrequently altered by LOH at the polymorphic RsaI locus in exon 11 in the tobacco associated Indian oral cancer, unlike the smoking tobacco/alcohol associated oral cancers from Western countries.

Characterization of Ngef, a novel member of the Dbl family of genes expressed predominantly in the caudate nucleus.

We have identified Ngef as a novel member of the family of Dbl genes. Many members of this family have been shown to function as guanine nucleotide exchange factors for the Rho-type GTPases. Ngef is predominantly expressed in brain, with the strongest signal in the caudate nucleus, a region associated with the control of movement. Ngef contains a translated trinucleotide repeat, a polyglutamic acid stretch interrupted by a glycine. We have localized the Ngef gene to mouse chromosome 1 and the human homologue of Ngef to human chromosome 2q37. We have shown in preliminary experiments that Ngef has transforming potential in cell culture and is able to induce tumors in nude mice.

p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India.

The inactivation of p53 tumour suppressor gene vis-á-vis point mutation, overexpression and degradation due to Human Papilloma virus (HPV) 16/18 infection, was examined in chewing tobacco-associated oral cancers and oral leukoplakias from India. The analysis of mutations was assessed by polymerase chain reaction (PCR) with single strand conformation polymorphism (PCR-SSCP) of exons 5-9 on DNA from 83 oral cancer cases, and the mutations confirmed by direct nucleotide sequencing of the PCR products. p53 protein expression was evaluated by immunohistochemical analysis on paraffin-embedded sections of 62 representative oral cancer biopsies and 22 leukoplakias, using p53-specific monoclonal antibody DO-7. The presence of HPV16/18 was detected in the 83 oral cancer cases by PCR analysis using HPV L1 consensus sequences, followed by Southern hybridization with type-specific oligonucleotide probes. Forty-six per cent (38/83) of oral cancer tumours showed p53 alterations, with 17% (14/83) showing point mutations, 37% (23/62) with overexpression and 25% (21/83) with presence of HPV16 wherein the E6 HPV16 protein degrades p53. HPV18 was not detected in any of the samples. Ninety-two per cent concordance was observed between missense point mutations and overexpression of p53 protein. A significant correlation was not observed between p53 alterations in oral cancer and clinico-pathological profile of the patients. Twenty-seven per cent (6/22) of oral leukoplakias showed p53 overexpression. The overall p53 alterations in oral cancer tissues and oral lesions are comparable to data from the oral cancers reported in the Western countries with smoking and alcohol-associated oral cancers, and suggest a critical role for p53 gene in a significant proportion of oral cancers from India. The overexpression of p53 protein in leukoplakias may serve as a valuable biomarker for identifying individuals at high risk of transformation to malignant phenotype.

Loss of p53 gene as a biomarker of high risk oral leukoplakias.

We have investigated loss of heterozygosity of p53 tumor suppressor gene in Indian oral cancer patients, individuals with premalignant leukoplakia lesions, and corresponding normal mucosa, to study the status of p53 alleles in oral cancer pathogenesis. Fifty oral cancers, and 42 oral leukoplakia lesions and corresponding clinically normal oral mucosa from 18 individuals, were analysed. Peripheral blood cells (PBCs) from all the individuals and 47 normal healthy volunteers were also included in the study. Polymerase chain reaction(PCR) of p53 Exon4, followed by restriction enzyme digestion with AccII due to the enzyme polymorphic site at Exon4 codon72, was used to detect homozygosity/heterozygosity of p53 alleles, and compared with the allelic pattern in the corresponding PBC. The PCR product subjected to AccII digestion detected 259 bp, 160/99 bp fragments indicating heterozygosity of p53 alleles in 69% of the 139 individuals. On comparison of the p53 allelic distribution in the lesions or tumour tissues, and corresponding PBC, LOH was observed in 20.5% oral tumors and 22% leukoplakias. However, there was no evidence of LOH in the clinically normal mucosa available from 16 individuals with leukoplakia. Our studies demonstrated LOH of p53 allele in early and advanced stages of oral cancers, as well as leukoplakias, perhaps indicating p53 LOH as one of the early events in oral carcinogenesis. Thus, p53 LOH may be useful as a biomarker in defining a certain population of high risk leukoplakias that may progress to oral cancer.

Detection and cloning of potent transforming gene(s) from chewing tobacco-related human oral carcinomas.

High molecular weight DNA isolated from 14 primary tumour tissues of human oral carcinoma patients was analysed for transforming activity by NIH3T3 co-transfection assay using pSV2neo gene as a selectable marker, followed by nude mouse tumorigenicity assay. Ten of the patient tumour tissues demonstrated molecular lesions in myc, ras or/and EGF-R genes, whereas 4 patients did not show tumour associated aberrations in these oncogenes. The G418-resistant transfected cells from 12 of 14 individual patients demonstrated transforming potential by colony formation in soft agar and tumour induction in nude mice within 25-80 days. DNAs from the transfected cells, consequent nude mice tumours and corresponding cell lines, contained human Alu sequences. Southern blot hybridisation with ras, myc, EGF-R oncogenes demonstrated the presence of human H-ras oncogene in one of the 12 sets of nude mice tumours. In contrast, DNA from the other 11 sets of nude mice tumours indicated absence of c-myc, N-myc, L-myc, H-ras, K-ras, N-ras and EGF-R genes on Southern analysis. Further, DNAs from five first cycle tumorigenic transformants were subjected to a second cycle of transfection, and induced tumours in nude mice with a shorter latency period of 21-50 days. The secondary transformants contained discrete human Alu sequences; however, the DNA did not hybridise with myc/ras/EGF-R probes. A genomic library was constructed from a second cycle nude mice tumour, using EMBL-3 as the vector. Four human Alu sequence positive clones were isolated on screening 2 x 10(5) plaques, and one of the recombinant clones subjected to fine restriction mapping using 16 restriction enzymes. The lack of association of the nude mice tumour DNA with myc/ras/EGF-R showing aberrations in the primary human tumour, implies activation of an alternative potent transforming gene(s) in the chewing tobacco-related oral carcinomas in India.