WITHDRAWN: Mutational analysis of the F8 gene and phenotypic characterization of Hemophilia A.

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Total Reflection X-ray Fluorescence Analysis of Plasma Elements in Autistic Children from India.

Trace elements are essential for the human body's various physiological processes but if they are present in higher concentration, these elements turn to be toxic and cause adverse effect on physiological processes. Similarly, deficiency of these essential elements also affects physiological processes and leads to abnormal metabolic activities. There is a lot of interest in recent years to know the mystery behind the involvement of trace elements in the metabolic activities of autistic children suspecting that it may be a risk factor in the aetiology of autism. The present study aims to analyse the plasma trace elements in autistic children using the total reflection X-ray fluorescence (TXRF) technique. Plasma samples from 70 autistic children (mean age: 11.5 ± 3.1) were analysed with 70 age- and sex-matched healthy children as controls (mean age: 12 ± 2.5). TXRF analysis revealed the higher concentration of copper (1227.8 ± 17.8), chromium (7.1 ± 2.5), bromine (2695.1 ± 24) and arsenic (126.3 ± 10) and lower concentration of potassium (440.1 ± 25), iron (1039.6 ± 28), zinc (635.7 ± 21), selenium (52.3 ± 8.5), rubidium (1528.9 ± 28) and molybdenum (162,800.8 ± 14) elements in the plasma of autistic children in comparison to healthy controls. Findings of the first study from India suggest these altered concentrations in elements in autistic children over normal healthy children affect the physiological processes and metabolism. Further studies are needed to clarify the association between the altered element concentration and physiology of autism in the North Karnataka population in India.

Clinical profile of hemophilia B patients from Karnataka.

Background: The most prevalent severe inherited hemorrhagic condition is hemophilia, which means "love of blood." Hemophilia A and B are caused by a lack or malfunction of the factor VIII and factor IX proteins. Objective: The present study is to determine the prevalence and clinical profile of hereditary coagulation disorder, particularly hemophilia B, in Karnataka. Methods: The study comprised 150 HB patients with a mean age of 25, nmale = 148 and nfemale = 2. The samples were collected from hemophilia societies across Karnataka. The detailed history of HB patients was recorded in a predesigned Performa regarding family history, age, time of first bleed, site of the bleed, and bleeding history. Result: In our study cohort, the majority of the 58 (38.7%) cases belong to 21-30 years of age. The mean age of onset was 2.0 ± 1.0 years in severe, 7.5 ± 2.8 0 years in moderate, and 10.0 ± 3.5 years in mild HB patients. Out of 150 HB cases, 102 (68%) cases were diagnosed as severe, 30 (20%) as moderate, and 18 (12%) as mild. Mean factor IX levels were 0.6 ± 0.2, 2.5 ± 1.3, and 8.0 ± 2.6 in the severe, moderate, and mild group, respectively. A family history of bleeding was observed in 97 [64.7%] HB patients. Forty-seven (32.3%) HB patients had a history of consanguinity. The most common initial site of bleed was in joints in 86 [57.3%]. Conclusion: The present study is one of the fewer studies from Karnataka studying the demographic and clinicopathological features of hemophilia B. Early diagnosis can be only helpful with knowledge of spectral presentation of hemophilia B in a local population.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva.

BACKGROUND: The isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages - volume, time, cost, and yield are a few notable ones. OBJECTIVE: We aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. METHODS: Five DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. RESULTS: Out of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. CONCLUSION: In the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Novel and PCR ready rapid DNA isolation from Drosophila.

INTRODUCTION: Isolation of genomic DNA is an initial step in molecular biology techniques. The quality of isolated DNA depends on procedures and chemicals, as well as source and types of the sample used. Several existing procedures are expensive and time consuming. In this study, we isolated high quality genomic DNA with an inexpensive and least time consuming procedure using Drosophila melanogaster flies, larvae, and pupae. METHODS: Drosophila melanogaster samples were collected from pre-cultured bottles, and genomic DNA was extracted using a proposed novel and PCR-ready method from three different pools of flies [PF1, PF2, and PF3], similarly from larvae and pupae [PL1, PL2, PL3, PP1, PP2, and PP3, respectively]. Isolated genomic DNA was subjected to PCR amplification with different dilutions using the COI gene and further amplicons were used for RAPD and DNA sequencing. RESULTS: The high quality of isolated genomic DNA was confirmed by 0.8% agarose gel electrophoresis and the purity and quantity of the DNA isolated from single fly, larva and pupa was similar to the purity and quantity of the DNA isolated using the NucleoSpinR Tissue kit method. Isolated genomic DNA was successfully amplified when the template was diluted in the ratio of 1:10. Further successful RAPD amplification and sequencing analysis of the COI gene confirms the efficiency of the downstream application of the proposed novel method. CONCLUSION: The present Novel and PCR ready rapid DNA isolation method will be potentially beneficial, and it can be successfully used for quick isolation of high molecular weight DNA from Drosophila flies larvae and pupae for DNA barcoding, identification of new species, genotyping, RAPD analysis, etc. Moreover, it can also be easily scaled up for bulk preparations.

Analysis of Genetic Variations in Connexin 26 ( GJB2 ) Gene among Nonsyndromic Hearing Impairment: Familial Study.

Objective The goal of this research was to investigate the gap junction beta 2 ( GJB2 ) gene mutations associated with nonsyndromic hearing loss individuals in North Karnataka, India. Materials and Methods For this study, patients with sensorineural genetic hearing abnormalities and a family history of deafness were included. A total of 35 patients from 20 families have been included in the study. The patient's DNA was isolated from peripheral blood samples. The GJB2 gene coding region was analyzed through Sanger sequencing. Results There is no changes in the first exon of the GJB2 gene. Nine different variants were recorded in second exon of the targeted gene. W24X and W77X are two nonsense mutations and three polymorphisms viz. R127H, V153I, and I33T were reported along with four 3'-UTR variants. A total (9/20) of 45% of families have been identified with mutations in the targeted gene. Conclusion GJB2 mutations were identified in 19 deaf-mute patients (19/35), and 13 patients were homozygous for the mutations identified in our study cohort. In our study, W24X mutation was found to be the pathogenic with a high percentage, prompting further evaluation of the other genes, along with the study of additional genetic or external causes in the families, which is essential.

Genetic Analysis of Neuroligin 4Y Gene in Autism Population of India.

Background Autism is one of the most complex, heterogeneous neurological disorders. It is characterized mainly by abnormal communication, impaired social interaction, and restricted behaviors. Prevalence of autism is not clear in Indian population. Aim The present study hypothesized that Y chromosome plays role in sex bias of autism in Indian autistic population. To investigate our hypothesis, we underwent genetic analysis of neuroligin 4Y [ NLGN4Y ] gene by sequencing 85 male autistic children after screening large population of 1,870 mentally ill children from North Karnataka region of India. Result Detailed sequencing of the single targeted gene revealed nine variants including, one novel missense mutation and eight synonymous variants; this accounts for 88.9% of synonymous variants. A single novel missense mutation is predicted to be nonpathogenic on the functions of neuroligin4Y protein but it slightly affects the local configuration by altering the original structure of a protein by changing charge and size of amino acid. Conclusion Probably NLGN4Y gene may not be the risk factor for autism in male children in Indian autistic population. Functional analysis was an important limitation of our study. Therefore, detailed functional analysis is necessary to determine the exact role of novel missense mutation of neuroligin 4Y [ NLGN4Y ] gene especially in the male predominance of autism in Indian autistic population.

Mutation analysis and characterisation of F9 gene in haemophilia- B population of India.

BACKGROUND: Hemophilia B (HB) is an X-linked bleeding disorder resulting from coagulation factor IX defects. Over 3,000 pathogenic, HB-associated mutations in the F9 gene have been identified. We aimed to investigate the role of F9 variants in 150 HB patients using sequencing technology. METHODS: F9 gene sequences were amplified from peripheral blood-derived DNA and sequenced on an Applied Biosystems (ABI) 3500 Sanger sequencing platform. Functional and structural predictions of mutant FIX were analyzed. RESULTS: Among 150 HB patients, 102 (68%), 30 (20%), and 18 (12%) suffered from severe, moderate, and mild HB, respectively. Genetic analysis identified 16 mutations, including 3 novel mutations. Nine mutations (7 missense and 2 stop-gain) were found to be pathogenic. Only 3 mutations (c.127C＞T, c.470G＞A, and c.1070G＞A) were associated with different severities. While 2 mutations were associated with mild HB cases (c.304C＞T and c.580A＞G), 2 (c.195G＞A and c.1385A＞G) and 3 mutations (c.223C＞T, c.1187G＞A, and c.1232G＞A) resulted in moderate and severe disease, respectively. Additionally, 1 mutation each was associated with mild-moderate (c.*1110A＞G) and mild-severe HB disease (c.197A＞T), 4 mutations were associated with moderate-severe HB cases (c.314A＞G, c.198A＞T, c.676C＞T, and c.1094C＞A). FIX concentrations were lower in the mutated group (5.5±2.5% vs. 8.0±2.5%). Novel p.E66D and p.S365 mutations were predicted to be pathogenic based on changes in FIX structure and function. CONCLUSION: Novel single nucleotide polymorphisms (SNPs) largely contributed to the pathogenesis of HB. Our study strongly suggests that population-based genetic screening will be particularly helpful to identify risk prediction and carrier detection tools for Indian HB patients.

Loss of GSK-3ß mediated phosphorylation in HtrA2 contributes to uncontrolled cell death with Parkinsonian phenotype.

HtrA2, a proapoptotic mitochondrial serine protease, promotes cellular protection against oxidative damage. Literature reports show positive correlation between loss of HtrA2 protease activity and Parkinson's Disease (PD) susceptibility. Homozygous loss-of-function mutations in murine-HtrA2, and when they rarely occur in humans result in severe neurodegeneration and infantile death. Here, we report a novel heterozygous pathogenic HTRA2 variant, c.725C > T (p.T242M) in Indian PD patients. Although, this mutation exhibits no significant conformational changes compared to the wild-type, functional studies with HtrA2-T242M transfected neurons reveal common features of PD pathogenesis such as dysfunction, altered morphology and mitochondrial membrane depolarization. Despite exhibiting two-fold decrease in enzyme activity, observation of excessive cell-death due to over-expression of the mutant has been correlated with it being constitutively active. This interesting behavioral anomaly has been attributed to the loss of phosphorylation-mediated regulatory checkpoint at the T242M mutation site that is otherwise controlled by glycogen synthase kinase-3ß (GSK-3ß). This study, with seamless amalgamation of biophysical and biomedical research unravels a mechanistic pathway of HtrA2 regulation and delineates its biological role in PD. Therefore, this investigation will not only prove beneficial toward devising therapeutic strategies against HtrA2-associated diseases mediated by GSK-3ß but also suggest new avenues for treatment of Parkinsonian phenotype.

Molecular alteration in the Gap Junction Beta 2 (GJB2) gene associated with non-syndromic sensorineural hearing impairment.

Non-syndromic sensory neural hearing defect is one of the genetic diseases inherited from parents to offerings. The autosomal recessive form affects a large population worldwide and has become a major concern in the social and professional lives of many people. There are many factors and genes which are involved in hearing loss but the Gap Junction Beta 2 (GJB2) gene which encodes the connexin 26 protein, is a major cause of non-syndromic recessive deafness (NSRD). This study aims to record and analyze GJB2 gene mutations in the hearing-impaired population of North Karnataka, India. In this study, we included 368 congenitally hearing-impaired children from North Karnataka, India, under 18 years of age. After thorough clinical examinations, patient's history and proper audiological results, peripheral blood samples were collected and subjected to genetic analysis. We recorded that 54.8% of the NSRD cases have an autosomal recessive mutation in the coding region of the GJB2 gene. The frequency of W24X (25%) mutation was found to be high in the present study population. From this study we can suggest that, identifying this mutation in new-borns definitely helps in the early diagnosis of hearing loss.

Genetic analysis of the postsynaptic transmembrane X-linked neuroligin 3 gene in autism.

Autism is a complex neurodevelopmental disorder, the prevalence of which has increased drastically in India in recent years. Neuroligin is a type I transmembrane protein that plays a crucial role in synaptogenesis. Alterations in synaptic genes are most commonly implicated in autism and other cognitive disorders. The present study investigated the neuroligin 3 gene in the Indian autistic population by sequencing and in silico pathogenicity prediction of molecular changes. In total, 108 clinically described individuals with autism were included from the North Karnataka region of India, along with 150 age-, sex-, and ethnicity-matched healthy controls. Genomic DNA was extracted from peripheral blood, and exonic regions were sequenced. The functional and structural effects of variants of the neuroligin 3 protein were predicted. One coding sequence variant (a missense variant) and four non-coding variants (two 5'-untranslated region [UTR] variants and two 3'-UTR variants) were recorded. The novel missense variant was found in 25% of the autistic population. The C/C genotype of c.551T>C was significantly more common in autistic children than in controls (p = 0.001), and a significantly increased risk of autism (24.7-fold) was associated with this genotype (p = 0.001). The missense variant showed pathogenic effects and high evolutionary conservation over the functions of the neuroligin 3 protein. In the present study, we reported a novel missense variant, V184A, which causes abnormal neuroligin 3 and was found with high frequency in the Indian autistic population. Therefore, neuroligin is a candidate gene for future molecular investigations and functional analysis in the Indian autistic population.

Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells.

Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30 kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

Molecular analysis of α-synuclein gene in Parkinson's disease in North Karnataka, India.

BACKGROUND: Parkinson's disease (PD) is a disabling neurological disorder characterized by progressive degeneration of dopaminergic neurons. Mutations analysis within the α-synuclein gene (SNCA) on chromosome 4 has been reported in the last decade. OBJECTIVE: To elucidate the possible role of SNCA gene in the pathogenesis of PD in Indian population specifically in north Karnataka. MATERIALS AND METHODS: The study subjects included 100 clinically diagnosed PD patients and 100 ethnically matched healthy controls. Isolated deoxyribonucleic acid (DNA) samples from both were subjected to exon-specific polymerase chain reaction (PCR) amplification and amplicons were subjected to capillary-based direct DNA sequencing. RESULT: No mutations were observed in SNCA gene of PD samples in comparison with control samples. CONCLUSION: These findings support the hypothesis that the SNCA gene mutations might be population specific and may not be playing role in causing PD in all the populations.

Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences.

The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3' long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7 kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

Molecular basis of ß-thalassemia in Karnataka, India.

In ß-thalassemia, point mutations in the ß-globin gene are largely responsible for either decreased or no ß-globin synthesis. The ß-globin gene has three exons and two introns. The molecular characterization of ß-thalassemia is absolutely necessary for carrier screening, for genetic counseling, and to offer prenatal diagnosis. The objective of the present study was to identify the rare mutations in ß-globin gene of ß-thalassemia patients. We have sequenced the entire ß-globin gene in 36 clinically identified thalassemia patients from the Karnataka region using polymerase chain reaction and sequencing. Our analysis revealed 11 ß-thalassemia variants. The most common being IVSII-16 G>C, IVSI-5G>C, IVSII-74 T>G, codon 3 (T>C), and Poly A site (T>C). In addition, we have also documented a novel deletion at codon 6 (-CT) (HBB:c.16delCT). These data are useful in future molecular screening of the population for implementing a thalassemia prevention and control program. Further it is found that family studies and comprehensive hematological analyses would provide useful insights for accurate molecular diagnosis of thalassemia phenotype and offers an interesting subject for further investigations in the Indian populations.

Indian Siddis: African descendants with Indian admixture.

The Siddis (Afro-Indians) are a tribal population whose members live in coastal Karnataka, Gujarat, and in some parts of Andhra Pradesh. Historical records indicate that the Portuguese brought the Siddis to India from Africa about 300-500 years ago; however, there is little information about their more precise ancestral origins. Here, we perform a genome-wide survey to understand the population history of the Siddis. Using hundreds of thousands of autosomal markers, we show that they have inherited ancestry from Africans, Indians, and possibly Europeans (Portuguese). Additionally, analyses of the uniparental (Y-chromosomal and mitochondrial DNA) markers indicate that the Siddis trace their ancestry to Bantu speakers from sub-Saharan Africa. We estimate that the admixture between the African ancestors of the Siddis and neighboring South Asian groups probably occurred in the past eight generations (∼200 years ago), consistent with historical records.

Prevalence and distribution of high risk human papillomavirus (HPV) Types 16 and 18 in Carcinoma of cervix, saliva of patients with oral squamous cell carcinoma and in the general population in Karnataka, India.

BACKGROUND: In view of conducting HPV vaccination in India it is most important to understand the prevalence of HPV genotypes in this population, not only in squamous cell carcinoma of cervix and oral cavity but also in the general population. In this study we explored the prevalence and distribution of high-risk HPV types 16 and 18 in carcinoma of cervix, saliva of patients with oral squamous cell carcinoma and in general population in Karnataka. METHODS: Cervical cancer specimens after punch biopsy (n=60) were obtained from women attending Karnataka Institute of Medical Sciences and Karnataka Cancer Therapy and Research Institute, Hubli (KCTRI). Saliva rinse of (n=34) OSCC patients from KCTRI and (n=396) normal individuals from different regions of North Karnataka, were collected and PCR based high-risk HPV genotyping was carried out. RESULTS: Using consensus PCR primers it was observed that 96.7% patients were infected with HPV irrespective of specific type in cervical cancer. Among them, HPV 16 was observed in 89.7%, HPV 18 in 86.2% and both HPV 16 and 18 in 79.3% patients. In OSCC, 70.6% were positive for HPV, among which HPV 16 prevalence was observed in 45.8%, HPV 18 in 54.2%, and HPV 16 and 18 multiple infection in 4.18%. In general population, HPV prevalence was observed in 84.4%. Among them, HPV 16 was observed in 2.75% and HPV 18 in 22.0% patients. In general population, multiple infection with HPV 16 and 18 was not observed but 75.3% were found to be infected by HPV genotypes other than HPV 16 and 18. CONCLUSIONS: Our study reveals that multiple infection of HPV 16 and 18 is quite high in cervical cancer and in case of OSCC, it was in conformity with the other studies. In general population HPV 18 prevalence was observed to be high. With this, we can conclude that both HPV 16 and 18 vaccinations will reduce the burden of cervical cancer and OSCC in Karnataka.

Prevalence and risk of leukemia in the multi-ethnic population of North Karnataka.

BACKGROUND: The aim of this study was to analyze the trends in the incidence rates of four major types of leukemia in the population of North Karnataka, which accounts for the 2.5% of the whole population of India. Due to the lack of any nationwide leukemia screening program, the majority of the people are not aware of the disease. Epidemiological study can play a vital role in understanding the occurrence and outcome of the disease. PATIENTS AND METHODS: Focusing on variables like age, sex, race, blood group and lifestyle habits, detailed reports of 417 males and 230 females (M:F ratio 1.8:1) were collected from different hospitals of North Karnataka and analyzed for their risk of leukemia. RESULTS: Compared to female patients, Hindu males were found to have greater risk of occurrence of leukemia (p=0.0333). The males of scheduled caste (SC) and Lingayat communities showed a high risk than other communities (p=0.000). The occurrence of AML showed a significant relationship with ABO blood groups (p=0.0090). The frequency of leukemia is quite high in Belgaum district when compared to others districts of North Karnataka and totally absent in Bidar district. The reasons need precise molecular and genetical studies of the population. CONCLUSIONS: The localized communities of Lingayat and SCs needs to be further studied to get a better understanding of the higher risk of occurrence of leukemia in males. Moreover, since the spectrum of cancer epidemiology seen in India is different from that in developed countries more emphasis should be placed on better development of regional and national registries.