M. Seshadri

Telomere Length in Human Adults and High Level Natural Background Radiation

Background Telomere length is considered as a biomarker of aging, stress, cancer. It has been associated with many chronic diseases such as hypertension and diabetes. Although, telomere shortening due to ionizing radiation has been reported in vitro, no in vivo data is available on natural background radiation and its effect on telomere length. Methodology/Principal Findings The present investigation is an attempt to determine the telomere length among human adults residing in high level natural radiation areas (HLNRA) and the adjacent normal level radiation areas (NLNRA) of Kerala coast in Southwest India. Genomic DNA was isolated from the peripheral blood mononuclear cells of 310 individuals (HLNRA: N = 233 and NLNRA: N = 77). Telomere length was determined using real time q-PCR. Both telomere (T) and single copy gene (S) specific primers were used to calculate the relative T/S and expressed as the relative telomere length. The telomere length was determined to be 1.22±0.15, 1.12±0.15, 1.08±0.08, 1.12±0.11, respectively, among the four dose groups (≤1.50, 1.51–3.00, 3.01–5.00 and >5.00 mGy per year), which did not show any dose response. The results suggested that the high level natural chronic radiation did not have significant effect on telomere length among young adult population living in HLNRA, which is indicative of better repair of telomeric ends. No significant difference in telomere length was observed between male and female individuals. In the present investigation, although the determination of telomere length was studied among the adults with an age group between 18 to 40 years (mean maternal age: 26.10±4.49), a negative correlation was observed with respect to age. However, inter-individual variation was (0.81–1.68) was clearly observed. Conclusions/Significance In this preliminary investigation, we conclude that elevated level of natural background radiation has no significant effect on telomere length among the adult population residing in HLNRAs of Kerala coast. To our knowledge, this is the first report from HLNRAs of the world where telomere length was determined on human adults. However, more samples from each background dose group and samples from older population need to be studied to derive firm conclusions.

Genetic association study of selected candidate genes (ApoB, LPL, Leptin) and telomere length in obese and hypertensive individuals

BackgroundA genetic study was carried out among obese and hypertensive individuals from India to assess allelic association, if any, at three candidate loci: Apolipoprotein B (ApoB) minisatellite and two tetranucleotide repeat loci; LPL (Lipoprotein lipase) and Leptin. Attempt has also been made to find out whether telomere length attrition is associated with hypertension and obese individuals.MethodsVenous blood samples were collected from 37 normal, 35 obese and 47 hypertensive individuals. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) and PCR amplifications were achieved using locus specific primers. Genotyping of ApoB minisatellite was performed using 4% polyacrylamide gel electrophoresis (PAGE) followed by silver staining, whereas LPL and Leptin loci were genotyped using ALF Express™ DNA sequencer. Telomere length was determined using a recently developed real time based quantitative PCR, where the relative telomere length was determined by calculating the relative ratio of telomere (T) and single copy gene (S) PCR products which is expressed as T/S ratio.ResultsAll the three loci are highly polymorphic, display high heterozygosity and conform to Hardy-Weinbergs equilibrium expectations. ApoB minisatellite displayed 14 alleles, whereas LPL and Leptin tetranucleotide loci were having 9 and 17 alleles, respectively. Interestingly two new alleles (9 and 11 repeats) were detected at ApoB locus for the first time. The alleles at Leptin locus were classified as Class I (lower alleles: 149-200 bp) and Class II alleles (higher alleles: >217 bp). Higher alleles at ApoB (>39 repeats), predominant allele 9 at LPL and alleles 164 bp and 224 bp at Leptin loci have shown allelic association with hypertensive individuals. After adjusting the influence of age and gender, the analysis of co-variance (ANCOVA) revealed the relative telomere length (T/S ratio) in hypertensive individuals to be (1.01 ± 0.021), which was significantly different (P < 0.001) from obese (1.20 ± 0.023) and normal (1.22 ± 0.014) individuals. However, no significant difference in the relative telomere length was observed among male and female individuals, although age related decrease in telomere length was observed in these limited sample size.ConclusionThe present study revealed that allelic association at ApoB, LPL, Leptin loci and loss of telomere length may have strong genetic association with hypertensive individuals. However, further study on larger sample size is needed to draw firm conclusions.

No evidence of telomere length attrition in newborns from high level natural background radiation areas in Kerala coast, south west India

Abstract Purpose: The tandemly repeated hexamers (TTAGGG)n present at the telomeric ends protect the human genome from a variety of environmental exposures including ionizing radiation. In order to find out the effect of chronic low dose radiation exposure, we have determined telomere length among newborns from high level natural radiation areas (HLNRA) of the Kerala coast in South west India. Methodology: Umbilical cord blood samples were collected from 128 newborns from HLNRA and 43 newborns from normal level natural radiation areas (NLNRA) and genomic DNA was isolated using a salt precipitation method. The mean telomere length was determined using SYBR green-based real time quantitative Polymerase chain reaction (qPCR), where telomere gene specific (T) and single copy gene specific (S) primers were used. The average of telomere versus single copy gene (T/S) ratio was calculated which was proportional to telomere length of each individual. Results: The mean relative telomere length was found to be 1.03 ± 0.01 (95% CI, 0.99–1.05) and 1.10 ± 0.03 (95% CI, 1.04–1.17) in HLNRA and NLNRA newborns, respectively (P > 0.05). Based on the level of background radiation dose, samples were divided into four groups, i.e., NLNRA: < 1.50 mGy/year and three HNLRA groups: 1.51–3.00 mGy/year, 3.01–5.00 mGy/year, and > 5.00 mGy/year. The mean relative telomere length in these groups were found to be 1.10 ± 0.03 (95% CI, 1.03–1.17), 0.98 ± 0.01 (95% CI, 0.95–1.01), 1.05 ± 0.02 (95% CI, 1.01–1.10) and 1.07 ± 0.03 (95% CI, 1.04–1.10), respectively. No significant difference was observed between the mean telomere length of male and female newborns. Conclusions: The elevated level of natural chronic background radiation prevailing in Kerala coast did not show any significant effect on telomere length of newborns. To our knowledge, this is the first report on newborn telomere length from a high level natural radiation area.

Evaluation of Spontaneous DNA Damage in Lymphocytes of Healthy Adult Individuals from High-Level Natural Radiation Areas of Kerala in India

Inhabitants of the high-level natural radiation areas (>1 mSv year−1) of Kerala in southwest India were evaluated for basal damage (spontaneous DNA strand breaks and alkali-labile sites) by the alkaline comet assay and oxidative DNA damage (ENDO III- and hOGG1-sensitive sites) by the enzyme-modified comet assay. Of the 67 adult male subjects studied, 45 were from high-level natural radiation areas and 22 subjects were from a nearby normal-level natural radiation area (≤1 mSv year−1). Basal damage due to the age and residential area (normal-level natural radiation area/high-level natural radiation areas) of the donors showed significant interaction (P < 0.001) when all subjects were analyzed using a general linear model (GLM). In subgroup analysis, basal damage increased with age in subjects from the normal-level natural radiation area (P = 0.02), while a significant negative correlation (P = 0.002) was observed in subjects from high-level natural radiation areas. Further, basal damage in elderly subjects from high-level natural radiation areas was significantly (P < 0.001) lower compared to the subjects from the normal-level natural radiation area. Oxidative DNA damage was not influenced by age, smoking habit or residential area in the entire sample. However, in a subgroup analysis, hOGG1-sensitive sites showed a significant increase with age in subjects from high-level natural radiation areas (P = 0.005). ENDO III-sensitive sites increased with natural radiation exposure in subjects from high-level natural radiation areas (P = 0.02), but when stratified according to smoking, a significant increase was observed only in smokers (P = 0.01). To the best of our knowledge, this is the first study on basal and oxidative DNA damage in healthy adults of this population. However, our findings need more validation in a larger study population.

Effect of chronic low dose natural radiation in human peripheral blood mononuclear cells: Evaluation of DNA damage and repair using the alkaline comet assay.

This study investigates whether peripheral blood mononuclear cells (PBMCs) from inhabitants of Kerala in southwest India, exposed to chronic low dose natural radiation in vivo (>1 mSv year(-1)), respond with a radioadaptive response to a challenging dose of gamma radiation. Toward this goal, PBMCs isolated from 77 subjects from high-level natural radiation areas (HLNRA) and 37 subjects from a nearby normal level natural radiation area (NLNRA) were challenged with 2 Gy and 4 Gy gamma radiation. Subjects from HLNRA were classified based on the mean annual effective dose received, into low dose group (LDG) and high dose group (HDG) with mean annual effective doses of 2.69 mSv (N=43, range 1.07 mSv year(-1) to 5.55 mSv year(-1)) and 9.62 mSv (N = 34, range 6.07 mSv year(-1) to 17.41 mSv year(-1)), respectively. DNA strand breaks and repair kinetics (at 7 min, 15 min and 30 min after 4 Gy) were evaluated using the alkaline single cell gel electrophoresis (comet) assay. Initial levels of DNA strand breaks observed after either a 2 Gy or a 4 Gy challenging dose were significantly lower in subjects of the HDG from HLNRA compared to subjects of NLNRA (2 Gy, P = 0.01; 4 Gy, P = 0.02) and LDG (2 Gy P = 0.01; 4 Gy, P=0.05). Subjects of HDG from HLNRA showed enhanced rejoining of DNA strand breaks (HDG/NLNRA, P = 0.06) during the early stage of repair (within 7 min). However at later times a similar rate of rejoining of strand breaks was observed across the groups (HDG, LDG and NLNRA). Preliminary results from our study suggest in vivo chronic low-level natural radiation provides an initial exposure that allows an adaptation to a subsequent higher radiation exposure, perhaps through improving DNA repair via an unknown mechanism. Therefore, further investigations would be necessary in this population to understand the biological and health effects of chronic low-level natural radiation exposures.

Cytogenetic studies on newborns from high and normal level natural radiation areas of Kerala in southwest coast of India

Abstract Purpose: To study, characterize and compare chromosome aberrations and karyotype anomalies among newborns from high (> 1.5 mGy/y) and normal (≤ 1.5 mGy/y) level natural radiation areas of monazite-sand bearing southwest coast of Kerala in India. Materials and methods: Cord blood samples from newborns were collected from selected Government hospitals in heparinized vials and cultures were set up employing standard microculture techniques, slides were prepared, coded and stained with giemsa. Well spread metaphases were analyzed for chromosome aberrations and karyotype anomalies. Results: A total of 1,267,788 metaphases from 27,295 newborns of mothers aged 17–45 years (17,298 from high and 9,997 from normal level radiation areas) were analyzed during 1986–2007. Frequencies of dicentrics in high and normal level radiation areas were 1.90 ± 0.14 and 2.01 ± 0.26 per 10,000 cells, respectively (Relative frequency [RF] = 0.94; 95% CI: 0.71–1.26). Karyotype anomalies had a frequency of 5.49‰ and 6.7‰, respectively (RF = 0.82; 95% CI: 0.60–1.12). No dose-related trend was observed in chromosome aberrations or karyotype anomalies. Conclusion: Frequencies of chromosomal aberration and karyotype anomalies between the newborns from the high level natural radiation area (HLNRA) and normal level natural radiation areas (NLNRA) were very similar.

Transcriptional expression of H2B, CTP synthase and PLK3 genes in whole blood exposed to 60 Co gamma radiation

Ionising radiation induces complex molecular responses in human cells resulting in changes at mRNA and protein expression. Limited data is available on the transcriptional status of functional genes in response to ionising radiation using peripheral blood mononuclear cells (PBMCs). In the present study, transcriptional profiles of Histone 2B , CTP synthase and PLK3 were studied. Blood samples were collected from ten random healthy males with informed consent. Whole blood irradiation was done at four different dose groups (0.3, 0.6, 1.0 and 2.0 Gy) at a dose rate of 0.68 Gy/minute. PBMCs were separated immediately as well as 4 hours post-irradiation. Total RNA was isolated, transcribed to cDNA and real-time quantitative PCR was performed. Our results revealed a dose-dependent significant upregulation at H2B and CTP synthase at 4 hours post-irradiation. At PLK3 significant upregulation was observed at 2.0 Gy ( P = 0.007). In conclusion, these genes can be used for population monitoring programme.

Indian ethnic populations characterized by dopamine (D4) receptor VNTR polymorphism.

Background: The human dopamine receptor D4 gene (DRD4) contains a 48-bp tandem repeat in exon 3 and shows alleles varying between repeats 2 and 11. The gene shows a high level of expression in the prefrontal cortex of the brain and association of particular alleles of this locus with various neuropsychiatric and personality disorders have been reported. Objective: The present study reports allele frequency distribution at the DRD4 variable number tandem repeat (VNTR) locus among five ethnic populations of India. This background information is fundamental to the field of pharmacogenetics for disease susceptibility and association studies. Subjects and methods: Three hundred and thirty two healthy unrelated adult individuals belonging to five ethnic groups: Konkanastha Brahmins, Marathas, Ezhavas, Nairs and Muslims, have been typed. Genomic DNA, extracted from peripheral blood, was PCR amplified using a two-enzyme system. The use of ALF™Express DNA sequencer was found to be helpful for large-scale population genotyping. Statistical analysis was performed using the POPGENE and DISPAN programs. Results: A total of eight alleles ranging from repeat 2 to repeat 9 were observed. Allele 4 was the predominant allele among all the five populations, consistent with the data on other world populations. A rare allele 9 was detected exclusively among Marathas. The observed heterozygosity was low, ranging from 0.38 to 0.54 while other parameters like Polymorphism Information Content (PIC) and Power of Discrimination (PD) showed moderate values. The populations were in genetic equilibrium when tested under Hardy–Weinberg expectations. Conclusion: The allele frequency estimates for DRD4 provided here will contribute towards developing an informative database for this functionally relevant locus. This will prove useful when studying the association between genetic factors and pathogenesis of disease in Indian populations and will address the concern of biased results of association due to population admixtures. Résumé. Le gène D4 récepteur de la dopamine humaine (DRD4) contient un tandem répété de 48-bp en exon 3 et présente des allèles variant entre les répétitions 2 et 11. Le gène présente un niveau élevé d’expression dans le cortex frontal du cerveau et on a fait état de diverses affections neuropsychiatriques et de troubles de personnalité en association avec les gènes de ce locus. Objectif: Cette étude concerne la distribution de fréquence allélique du nombre variable de répétitions de tandem (VNTR) au locus DRD4 dans cinq populations ethniques de l’Inde. Cette information d’arrière plan est fondamentale pour les études de susceptibilité et d’associations pathologiques en pharmacogénétique. Sujets et méthodes: On a typé 332 adultes sains et non apparentés, appartenant à cinq groupes ethniques : Konkanastha Brahmin, Maratha, Ezhava, Nair et des musulmans. L’ADN génomique extrait du sang périphérique, a été amplifié par PCR au moyen d’un système de deux enzymes. L’utilisation du séquenceur d’ADN ALFTM a été efficace pour le génotypage à grande échelle. Les analyses statistiques ont été effectuées à l’aide des programmes POPGENE et DISPAN. Résultats: On a observé au total huit allèles, variant de la répétition 2 à la répétition 9, l’allèle 4 étant prédominant dans les cinq populations, en accord avec les données mondiales. Un rare allèle 9 a été trouvé chez Maratha exclusivement. L’hétérozygosité observée est faible, variant de 0,38 à 0,54, tandis que d’autres paramètres tels le Contenu d’Information Polymorphique et le Pouvoir de Discrimination présentent des valeurs modérées. Les populations testées par rapport aux proportions de Hardy-Weinberg apparaissent en équilibre génétique. Conclusion: Ces estimations de fréquences alléliques pour DRD4 contribueront au développement d’une base de données sur ce locus. Ceci sera utile pour l’étude de l’association entre facteurs génétiques et pathogenèse dans les populations indiennes et nourrira le problème des biais de résultats d’association dus aux mélanges de populations. Zusammenfassung.Hintergrund: Das menschliche Dopaminrezeptor-D4-Gen (DRD4) enthält ein 48-bp-Tandem-Repeat in Exon 3 und zeigt Allele, die zwischen den Repeats 2 und 11 variieren. Das Gen zeigt eine starke Ausprägung im präfrontalen Cortex des Gehirns, und es wurden Beziehungen zwischen besonderen Allelen dieses locus und verschiedenen neuropsychiatrischen und Persönlichkeitsstörungen berichtet. Vorhaben: Die vorliegende Untersuchung berichtet über die Verteilung der Allelhäufigkeiten am DRD4-VNTR-locus (variable number tandem repeat, VNTR, verschiedene Anzahl von Tandem-Repeats) bei fünf ethnischen Bevölkerungsgruppen in Indien. Diese Hintergrundinformation ist wesentlich auf dem Gebiet der Pharmakogenetik für Krankheitsanfälligkeit und Assoziationsstudien. Probanden und Methoden: 332 gesunde, nicht verwandte Personen aus fünf ethnischen Gruppen, Konkanastha Brahmins, Marathas, Ezhavas, Nairs und Moslems, wurden typisiert. Genomische DNS aus peripherem Blut wurde PCR-amplifiziert unter Verwendung eines 2-Enzymsystems. Die Verwendung von ALF™Express DNA Sequencer wurde als hilfreich bei der genetischen Typisierung größerer Bevölkerungsgruppen angesehen. Statistische Analysen wurden mit POPGENE- und DISPAN-Programmen durchgeführt. Ergebnisse: Insgesamt wurden acht Allele mit Repeats zwischen 2 und 9 beobachtet. Das Allel 4 war unter allen fünf Populationen vorherrschend, passend zu Daten von anderen Völkern dieser Erde. Ein seltenes Allele 9 wurde ausschließlich bei den Marathas gefunden. Die beobachtete Heterozygotie war niedrig und reichte von 0,38 bis 0,54, während andere Parameter wie der Gehalt an Information betreffend Polymorphismus (Polymorphic Information Content) und die Unterscheidbarkeit (Power of Discrimination) mäßige Ausprägungen zeigten. Die Populationen waren bei Prüfung unter Hardy–Weinberg-Erwartungen im genetischen Äquilibrium. Zusammenfassung: Die hier vorgenommene Schätzung der Allelhäufigkeit für DRD4 wird zur Entwicklung einer informativen Datenbasis für diesen funktionell bedeutsamen locus beitragen. Dies wird sich als hilfreich für die Untersuchung der Beziehung zwischen genetischen Faktoren und der Pathogenese von Krankheiten in der Indischen Bevölkerung erweisen und wird Bedenken berücksichtigen hinsichtlich möglicher Beeinflussung von Ergebnissen aufgrund ethnischer Durchmischung. Resumen. Antecedentes: El gen D4 del receptor de la dopamina humana (DRD4) contiene una repetición en tándem de 48 pares de bases (pb) en el tercer exón y muestra alelos que varían entre 2 y 11 repeticiones. El gen muestra un elevado nivel de expresión en el córtex pre-frontal del cerebro y se ha señalado la asociación de alelos particulares de este locus con varios trastornos neuro-psiquiátricos y de la personalidad. Objetivo: El presente estudio informa sobre la distribución de frecuencias alélicas en el polimorfismo de repetición en tándem de número variable (VNTR) del locus DRD4, en cinco poblaciones étnicas de la India. Esta información es fundamental en el campo de la farmacogenética para los estudios sobre susceptibilidad a enfermedades y de asociación. Sujetos y métodos: Se tiparon 332 individuos adultos sanos, no emparentados, procedentes de cinco grupos étnicos, los Konkanastha, Brahmins, Marathas, Ezhavas, Nairs y Musulmanes. El ADN genómico, extraído de sangre periférica, se amplificó mediante PCR utilizando un sistema bi-enzimático. Se comprobó que el uso del secuenciador de ADN ALF™Express era útil para genotipar poblaciones a gran escala. Los análisis estadísticos se realizaron utilizando los programas POPGENE y DISPAN. Resultados: Se observó un total de ocho alelos que oscilaban entre 2 y 9 repeticiones. El alelo 4 fue el predominante en las cinco poblaciones, lo que era consistente con los datos de otras poblaciones del mundo. Se detectó un alelo 9 raro exclusivamente entre los Marathas. La heterozigosidad observada era baja, oscilando entre 0,38 y 0,54, mientras otros parámetros como el Contenido de Información Polimórfica (Polymorphic Information Content) y el Poder de Discriminación (Power of Discrimination) mostraron valores moderados. Las poblaciones estaban en equilibrio genético cuando se las testó bajo las condiciones de Hardy-Weinberg. Conclusión: Las estimaciones de las frecuencias alélicas para el gen DRD4 proporcionadas en este estudio, contribuirán al desarrollo de una base de datos informativa sobre este locus funcionalmente relevante. Esto será útil cuando se estudie la asociación entre los factores genéticos y la patogénesis de la enfermedad en poblaciones indias, y para tratar la inquietud por los resultados sesgados de asociación debidos a las mezclas de población.

Minimal Sharing of Y-Chromosome STR Haplotypes Among Five Endogamous Population Groups from Western and Southwestern India

We attempt to address the issue of genetic variation and the pattern of male gene flow among and between five Indian population groups of two different geographic and linguistic affiliations using Y-chromosome markers. We studied 221 males at three Y-chromosome biallelic loci and 184 males for the five Y-chromosome STRs. We observed 111 Y-chromosome STR haplotypes. An analysis of molecular variance (AMOVA) based on Y-chromosome STRs showed that the variation observed between the population groups belonging to two major regions (western and southwestern India) was 0.17%, which was significantly lower than the level of genetic variance among the five populations (0.59%) considered as a single group. Combined haplotype analysis of the five STRs and the biallelic locus 92R7 revealed minimal sharing of haplotypes among these five ethnic groups, irrespective of the similar origin of the linguistic and geographic affiliations; this minimal sharing indicates restricted male gene flow. As a consequence, most of the haplotypes were population specific. Network analysis showed that the haplotypes, which were shared between the populations, seem to have originated from different mutational pathways at different loci. Biallelic markers showed that all five ethnic groups have a similar ancestral origin despite their geographic and linguistic diversity.

Population Genetic Analysis among Five Indian Population Groups Using Six Microsatellite Markers

Genetic variation at six tetranucleotide microsatellites (HUMTHO1, HUMVWA, F13A01, D3S1359, D12S66, and D12S67) has been determined in five endogamous ethnic population groups of India belonging to two major linguistic families. The populations analyzed were Konkanastha Brahmins and Marathas (Maharashtra state) from the Indo-Aryan linguistic family and Nairs, Ezhavas, and Muslims (Kerala state) from the Dravidian family. All six loci show high gene diversity, ranging from 0.63 ± 0.04 to 0.84 ± 0.02. The average GST value observed was 1.7%, indicating that the differences between the populations account for less than 2% of the diversity, while the genetic variation is high within the five population groups studied (>98%). The phylogenetic tree fails to show any clear cluster. The absence of any cluster along with low average GST is suggestive of substantial genetic similarity among the studied populations, in spite of clear geographical, linguistic, and cultural barriers. This similarity indicates either a greater gene flow between these groups or, alternatively, may reflect a recent evolution for them, considering that the Indian caste system evolved only about 3000 years ago.