scholarly journals A Family Based Whole Exome Sequence Study to Indentify Modifier Genes for Phenotype Heterogeneity Between Severe and Non-Severe Thalassemia Patients

2021 ◽  
Author(s):  
ANUPAM BASU ◽  
Dipankar Saha ◽  
Prosanto Kumar Chowdhury ◽  
, Debashis Pal ◽  
Kaustav Nayek ◽  
...  
Keyword(s):  
Kegg Pathway ◽  
Pcr Amplification ◽  
Beta Thalassemia ◽  
Illumina Hiseq ◽  
Hormone Synthesis ◽  
Junction Protein ◽  
Index Sequence ◽  
Ppar Signaling ◽  
Phenotype Heterogeneity ◽  
Family Based

* Corresponding author: Prof. Anupam Basu, Department of Zoology, The University of Burdwan, PurboBarddhaman, West Bengal- 713104, India, Email- [email protected] & b are equal contribution Whole Expanded Exome Sequencing Study of two families father, mother and index cases (trio) was undertaken for two E/ beta thalassemia subjects with same HBB genotype. Approximately 200ng of DNA was taken from each individual and shared into 300-400bp fragment. Then the shared fragments are end repair. Klenow exonuclease was used to add an adapter. After adapter ligation 10 cycle PCR amplification was done for each sample. The targeted Exome was captured by the Agilent Sure select XT Human all Exome V6+ UTR kit as per the manufacturer’s protocol. Captured library was then amplified 10 cycles with 8 bp index sequence for each sample. Then the indexed capture library was pooled together. Pair end sequencing of the pooled library was performed in Illumina HiSeq2500 using Illumina HiSeq SBS kit. Finally, both the genes, inherited and denovo, from both the subjects were separately functionally annotated by DAVID online tools 6.8. Functionally annotation result shows that in case of subject-1, 6 KEGG pathway were involved. These are Adherent junction, Protein digestion and absorption, Inflamatory Bowel Disease, Amoebiasis, PPAR signaling pathway and glycolysis or gluconeogenesis. Interestingly in case of subject-2, only 2 KEGG pathway were found, Thyroid hormone synthesis and carbon metabolism.

scholarly journals Ultrahigh-Throughput Multiplexing and Sequencing of >500-Base-Pair Amplicon Regions on the Illumina HiSeq 2500 Platform

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

ABSTRACT Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.

X-Linked Gray Platelet Syndrome Due to a GATA1 Arg216Gln Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5-5 ◽  
Author(s):  
Venée N. Tubman ◽  
Jason E. Levine ◽  
Dean R. Campagna ◽  
Mark D. Fleming ◽  
Ellis J. Neufeld
Keyword(s):  
X Chromosome ◽  
Blood Smear ◽  
Pcr Amplification ◽  
Peripheral Blood Smear ◽  
Genetic Mutation ◽  
Beta Thalassemia ◽  
Common Haplotype ◽  
Obligate Carrier ◽  
Gray Platelet Syndrome ◽  
Chain Synthesis

Abstract Gray Platelet Syndrome (GPS) is characterized by a variable, mild bleeding diathesis, associated with thrombocytopenia and large, agranular, functionally abnormal platelets. While the disorder has been well-described both biochemically and pathologically, to our knowledge, no genetic mutation has been associated with the disease. Most cases are sporadic, with a few sibships and apparent autosomal kindreds reported. In our investigation, the proband is a healthy 1 year-old female whose father and uncle had recently been diagnosed with GPS. The child had a platelet count of 382 x103 platelets/μl, comprised of a dimorphic population of normal and large, agranular forms. Her CBC and peripheral blood smear were otherwise unremarkable. We subsequently analyzed four generations of this family. In addition to the proband’s father and uncle, several other members had known bleeding tendencies. The symptomatic individuals were invariably males, consistent with a sex-linked pattern of inheritance. Several symptomatic and asymptomatic family members were evaluated by CBC and blood smear. Large, agranular platelets were found in symptomatic males, while obligate carrier females exhibited both normal and abnormal forms. Using a set of fifteen microsatellite markers spanning the X chromosome, a common haplotype was identified in all affected men, their mothers, and their daughters. Aided by the published sequence of the X chromosome, we examined this region for candidate genes. Although the common haplotype between markers GATA144D04 and DXS6797 (Xp11.3-Xq22.3) contains hundreds of genes, only two, GATA1 and WAS, have known associations with thrombocytopenia. PCR amplification and sequencing of a 3′ segment of the GATA-1 promoter and the five coding exons of the GATA-1 gene revealed an G759A missense mutation resulting in an Arg216Gln substitution in exon 4 (NCBI RefSeq: NM_002049) that segregated with the phenotype and was present in all obligate carrier females. This mutation has been previously associated with X-linked thrombocytopenia and beta-thalassemia (XLTT), a syndrome characterized by splenomegaly, thrombocytopenia, and imbalanced globin chain synthesis (Balduini et al, Thromb Haemost. 2004; 91:129). Comparison of the ultrastructural characteristics of the platelets in both disorders and a review of literature on both diseases suggests that XLTT, and the associated mutation in GATA1, could represent one genetic origin for GPS. As carrier females generally display a less severe phenotype than affected males, without thrombocytopenia and with subtle dimorphism on smears, it is possible that this mutation may account for some previously reported “sporadic” cases of GPS.

Biased Paternal Transmission of TRH Variant to Female down Syndrome Probands: Possible Correlation with Low Breast Cancer Frequency

2015 ◽  
Vol 30 (1) ◽  
pp. 142-147
Author(s):  
Arpita Dey ◽  
Arpita Chatterjee ◽  
Atanu Maiti ◽  
Nupur Mukherjee ◽  
Swagata Sinha ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Down Syndrome ◽  
General Population ◽  
Pcr Amplification ◽  
Analysis Data ◽  
Protective Role ◽  
Thyroid Stimulating Hormone ◽  
Control Analysis ◽  
Functional Polymorphisms ◽  
Family Based

Thyroid malfunction is more common in individuals with Down syndrome (DS) than in the general population. It has been hypothesized that thyroid may influence cancer risk. Individuals with DS are at greater risk of developing leukemia than the general population, while solid tumors especially breast cancer (BC) are rare. BC patients have higher levels of circulating thyroid-stimulating hormone (TSH) and prolactin (PRL), both regulated by the thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide. This study was aimed at investigating the status of TRH functional polymorphisms in subjects with DS and BC. Unrelated families with DS probands (n=180), individuals with BC (n=99) and ethnically matched controls (n=216) were recruited. Genomic DNA isolated from peripheral blood was subjected to PCR amplification followed by DNA sequence analysis. Data obtained were analyzed by population- and family-based statistical analysis. Among 30 studied sites, only 2 (rs7645772 and rs13097335) were polymorphic. Case-control analysis showed a lack of any significant association with DS, while the rs13097335 GG and GT genotype frequency was significantly different in the BC samples. A paternal-biased transmission of the G allele was observed in female DS probands. It may be concluded that rs13097335 may have a protective role toward the development of BC.

scholarly journals Family-Based Haplotype Estimation and Allele Dosage Correction for Polyploids Using Short Sequence Reads

2018 ◽  
Author(s):  
Ehsan Motazedi ◽  
Richard Finkers ◽  
Chris Maliepaard ◽  
Dick de Ridder
Keyword(s):  
Sequence Data ◽  
Pedigree Information ◽  
Short Sequence ◽  
Haplotype Estimation ◽  
Illumina Hiseq ◽  
Single Chromosome ◽  
Sequencing Errors ◽  
Allele Dosage ◽  
Missing Genotypes ◽  
Family Based

AbstractDNA sequence reads contain information about the genomic variants located on a single chromosome. By extracting and extending this information (using the overlaps of the reads), the haplotypes of an individual can be obtained. Adding parent-offspring relationships to the read information in a population can considerably improve the quality of the haplotypes obtained from short reads, as pedigree information can compensate for spurious overlaps (due to sequencing errors) and insufficient overlaps (due to shallow coverage). This improvement is especially beneficial for polyploid organisms, which have more than two copies of each chromosome and are therefore more difficult to be haplotyped compared to diploids. We develop a novel method, PopPoly, to estimate polyploid haplotypes in an F1-population from short sequence data by considering the transmission of the haplotypes from the parents to the offspring. In addition, PopPoly employs this information to improve genotype dosage estimation and to call missing genotypes in the population. Through realistic simulations, we compare PopPoly to other haplotyping methods and show its better performance in terms of phasing accuracy and the accuracy of phased genotypes. We apply PopPoly to estimate the parental and offspring haplotypes for a tetraploid potato cross with 10 offspring, using Illumina HiSeq sequence data of 9 genomic regions involved in plant maturity and tuberisation.

scholarly journals Identification and differential expression of piRNAs in the gonads of Amur sturgeon (Acipenser schrenckii)

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6709
Author(s):  
Lihong Yuan ◽  
Linmiao Li ◽  
Xiujuan Zhang ◽  
Haiying Jiang ◽  
Jinping Chen
Keyword(s):  
Small Rna ◽  
Kegg Pathway ◽  
Technological Development ◽  
Sex Differentiation ◽  
Economic Value ◽  
Gonadal Development ◽  
Small Rna Sequencing ◽  
Illumina Hiseq ◽  
Specific Expression ◽  
Go Annotation

Objective Sturgeons are considered living fossils, and have a very high conservation and economic value. Studies on the molecular mechanism of sturgeon gonadal development and sex differentiation would not only aid in understanding vertebrate sex determination but also benefit sturgeon aquaculture. Piwi-interacting RNAs (piRNAs) have been shown to function in germline or gonadal development. In this study, we performed small RNA deep sequencing and microarray hybridization to identify potential sturgeon piRNAs. Methods Male and female sturgeon gonads were collected and used for small RNA sequencing on an Illumina HiSeq platform with the validation of piRNA expression by microarray chip. The program Bowtie and k-mer scheme were performed to filter small RNA reads and discover potential sturgeon piRNAs. A known piRNA database, the coding sequence (CDS), 5′ and 3′ untranslated region (UTR) database of the A. Schrenckii transcriptome, Gene Ontology (GO) database and KEGG pathway database were searched subsequently to analyze the potential bio-function of sturgeon piRNAs. Results A total of 875,679 putative sturgeon piRNAs were obtained, including 93 homologous to known piRNAs and hundreds showing sex-specific and sex-biased expression. Further analysis showed that they are predominant in both the ovaries and testes and those with a sex-specific expression pattern are nearly equally distribution between sexes. This may imply a relevant role in sturgeon gonadal development. KEGG pathway and GO annotation analyses indicated that they may be related to sturgeon reproductive processes. Conclusion Our study provides the first insights into the gonadal piRNAs in a sturgeon species and should serve as a useful resource for further elucidation of the gene regulation involved in the sex differentiation of vertebrates. These results should also facilitate the technological development of early sex identification in sturgeon aquaculture.

scholarly journals PSI-34 The Impact of Varying Protein Sources on Feedlot Goat Fecal Microbiome

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 256-256
Author(s):  
Allianna Mitchell ◽  
Cassandra K Jones
Keyword(s):  
Soybean Meal ◽  
Pcr Amplification ◽  
Alpha Diversity ◽  
Protein Source ◽  
Illumina Hiseq ◽  
Fecal Microbiome ◽  
Protein Sources ◽  
Β Diversity ◽  
Α Diversity ◽  
The Impact

Abstract The effect of protein sources with different RUP values on feedlot goat fecal microbiome has not been studied. This experiment evaluated the impact of varying protein sources on feedlot goat fecal microbiome. Fourty-five Boer-influenced goats (23.53 kg ± 1.07; approximately 75 d of age) were blocked by body weight and randomly allocated to 1 of 3 treatment diets (15 goats/treatment) with ad libitum feed and water for 42 d. Diets were formulated to be isocaloric and isonitrogenous, with varying primary protein source: 1) 18.7% solvent-extracted soybean meal (SBM), 2) 22.0% expelled soybean meal (SoyPlus), or 3) 34.4% distillers’ dried grains with solubles (DDGS). On d 42, fecal pellets were collected from each goat and stored at -80°C until microbiome sequencing. Samples underwent PCR amplification of the V4 region of the 16S gene and sequencing on Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) by a commercial lab (MR DNA, Shallowater, TX). Data were analyzed using the GLIMMIX procedure of SAS with Turkey’s test for post hoc comparisons. Beta and alpha diversity were analyzed using Qiime with Kruskal-Wallis pairwise comparisons and ANOSIM. Genera, with individual abundance greater than 1%, that were higher (P ≤ 0.05) in SBM-fed goats compared to goats fed DDGS and SoyPlus include Clostridium, Paludibacter, Tannerella, and Methanobrevibacter. At the phylum level, Bacteroidetes was greater in SBM-fed goats compared to DDGS-fed goats (P = 0.04) and Euryarchaeota was more abundant in goats fed SBM compared to DDGS (P = 0.003) and SoyPlus (P = 0.0001). Protein source did not affect β-diversity (r ≈ 0) or α-diversity (P > 0.10). In summary, altering protein source in finishing goat diets created shifts in some phyla and genera, however the diversity within and between microbial communities across treatments were uninfluenced.

scholarly journals Identification of biological processes and signaling pathways for the knockout of REV-ERB in mouse brain

2021 ◽  
Author(s):  
Jing Li ◽  
Wei Wang ◽  
Hanming Gu
Keyword(s):  
Signaling Pathways ◽  
Mouse Brain ◽  
Hippo Signaling ◽  
Biological Processes ◽  
Orphan Nuclear Receptor ◽  
Ppi Network ◽  
Illumina Hiseq ◽  
Number Of Genes ◽  
Ppar Signaling ◽  
The Brain

REV-ERB is an orphan nuclear receptor that is widely expressed in the brain and inhibits transcriptional activities. A variety of genes affect the activity and expression of REV-ERB. In this study, our objective is to identify significant signaling pathways and biological processes in the knockout of the REV-ERB mouse brain. The GSE152919 dataset was originally created by using the Illumina HiSeq 4000 (Mus musculus). The KEGG and GO analyses suggested that biological processes "PPAR signaling", "Hippo signaling", and "Hypertrophic cardiomyopathy (HCM)" are mostly affected in the knockout of REV-ERB. Furthermore, we identified a number of genes according to the PPI network including NPAS2, CRY2, BMAL1, and CRY1 which were involved in the lack of REV-ERB in the brain. Therefore, our study provides further insights into the study of circadian clocks.

scholarly journals Analyzing the gonadal transcriptome of the frog Hoplobatrachus rugulosus to identify genes involved in sex development

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yun Tang ◽  
Jing-Yi Chen ◽  
Guo-Hua Ding ◽  
Zhi-Hua Lin
Keyword(s):  
Gene Expression ◽  
Sex Differentiation ◽  
Gonadal Development ◽  
Future Research ◽  
Growth Stages ◽  
Illumina Hiseq ◽  
Hormone Synthesis ◽  
Sex Development ◽  
Vital Functions ◽  
Development And Differentiation

Abstract Background The tiger frog (Hoplobatrachus rugulosus) is listed as a national Class II protected species in China. In the context of global warming, the sex ratio of amphibians will be affected, and the development of the population will be limited. Therefore, considering the potential for a decrease in the number of amphibians, studying sex evolution and molecular regulation of gonadal development in H. rugulosus, phenomenon that are currently unclear, is of great significance. Results Here, H. rugulosus was used to explore the mechanisms regulating gonadal development in amphibians. Illumina HiSeq 3000 was used to sequence the gonadal transcriptome of male and female H. rugulosus at two growth stages to identify genes related to gonadal development and analyze expression differences in the gonads. This analysis indicated that cyp17α, hsd3β, hsd11β1, cyp19α, and hsd17β12 perform vital functions in sex development in amphibians. Specifically, the expression of cyp3α, cyp17α, hsd3β, hsd11β1, sox2, sox9, sox30, soat, cyp19α, hsd17β12, and hspα1s was correlated with gonadal development and differentiation in H. rugulosus, as determined using the quantitative reverse transcriptase-polymerase chain reaction. Conclusion Significant differences were found in the gonadal gene expression levels in H. rugulosus of both sexes, and we identified a steroid hormone synthesis pathway in this species and analyzed related gene expression, but the changes during sex differentiation were still unclear. To our knowledge, this report presents the first analysis of the H. rugulosus gonadal transcriptome and lays the foundation for future research.

Ultrastructural morphology of nontumorous lactotrophs in human pituitaries exposed to high prolactin levels

1987 ◽  
Vol 45 ◽  
pp. 896-897
Author(s):  
S. Jalalah ◽  
K. Kovacs ◽  
E. Horvath
Keyword(s):  
Anterior Pituitary ◽  
Secretory Activity ◽  
Pituitary Hormones ◽  
Feedback Mechanism ◽  
Endocrine Activity ◽  
Hormone Synthesis ◽  
Anterior Pituitary Hormones ◽  
Negative Feedback Mechanism ◽  
Ultrastructural Morphology ◽  
Transmission Electron

Lactotrophs, as many other endocrine cells, change their morphology in response to factors influencing their secretory activity. Secretion of prolactin (PRL) from lactotrophs, like that of other anterior pituitary hormones, is under the control of the hypothalamus. Unlike most anterior pituitary hormones, PRL has no apparent target gland which could modulate the endocrine activity of lactotrophs. It is generally agreed that PRL regulates its own release from lactotrophs via the short loop negative feedback mechanism exerted at the level of the hypothalamus or the pituitary. Accordingly, ultrastructural morphology of lactotrophs is not constant; it is changing in response to high PRL levels showing signs of suppressed hormone synthesis and secretion.By transmission electron microscopy and morphometry, we have studied the morphology of lactotrophs in nontumorous (NT) portions of 7 human pituitaries containing PRL-secreting adenoma; these lactotrophs were exposed to abnormally high PRL levels.

Melatonin reduces high levels of lipid peroxidation induced by potassium iodate in porcine thyroid

2019 ◽  
pp. 1-7 ◽  
Author(s):  
Paulina Iwan ◽  
Jan Stepniak ◽  
Malgorzata Karbownik-Lewinska
Keyword(s):  
Lipid Peroxidation ◽  
Oxidative Damage ◽  
Membrane Lipids ◽  
Chemical Properties ◽  
Iodine Concentration ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Protective Effects ◽  
Potassium Iodate ◽  
Hormone Synthesis

Abstract. Iodine is essential for thyroid hormone synthesis. Under normal iodine supply, calculated physiological iodine concentration in the thyroid is approx. 9 mM. Either potassium iodide (KI) or potassium iodate (KIO3) are used in iodine prophylaxis. KI is confirmed as absolutely safe. KIO3 possesses chemical properties suggesting its potential toxicity. Melatonin (N-acetyl-5-methoxytryptamine) is an effective antioxidant and free radical scavenger. Study aims: to evaluate potential protective effects of melatonin against oxidative damage to membrane lipids (lipid peroxidation, LPO) induced by KI or KIO3 in porcine thyroid. Homogenates of twenty four (24) thyroids were incubated in presence of either KI or KIO3 without/with melatonin (5 mM). As melatonin was not effective against KI-induced LPO, in the next step only KIO3 was used. Homogenates were incubated in presence of KIO3 (200; 100; 50; 25; 20; 15; 10; 7.5; 5.0; 2.5; 1.25 mM) without/with melatonin or 17ß-estradiol. Five experiments were performed with different concentrations of melatonin (5.0; 2.5; 1.25; 1.0; 0.625 mM) and one with 17ß-estradiol (1.0 mM). Malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) concentration (LPO index) was measured spectrophotometrically. KIO3 increased LPO with the strongest damaging effect (MDA + 4-HDA level: ≈1.28 nmol/mg protein, p < 0.05) revealed at concentrations of around 15 mM, thus corresponding to physiological iodine concentrations in the thyroid. Melatonin reduced LPO (MDA + 4-HDA levels: from ≈0.97 to ≈0,76 and from ≈0,64 to ≈0,49 nmol/mg protein, p < 0.05) induced by KIO3 at concentrations of 10 mM or 7.5 mM. Conclusion: Melatonin can reduce very strong oxidative damage to membrane lipids caused by KIO3 used in doses resulting in physiological iodine concentrations in the thyroid.

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