scholarly journals Uncontrolled Diabetes Mellitus Has No Major Influence on the Platelet Transcriptome

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Thomas G. Nührenberg ◽  
Marco Cederqvist ◽  
Federico Marini ◽  
Christian Stratz ◽  
Björn A. Grüning ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Next Generation Sequencing ◽  
Platelet Reactivity ◽  
Platelet Rich Plasma ◽  
Expression Profiles ◽  
Bioinformatic Analysis ◽  
Differentially Expressed ◽  
Major Influence ◽  
Next Generation ◽  
Generation Sequencing

Background. Diabetes mellitus (DM) has been associated with increased platelet reactivity as well as increased levels of platelet RNAs in plasma. Here, we sought to evaluate whether the platelet transcriptome is altered in the presence of uncontrolled DM. Methods. Next-generation sequencing (NGS) was performed on platelet RNA for 5 patients with uncontrolled DM (HbA1c 9.0%) and 5 control patients (HbA1c 5.5%) with otherwise similar clinical characteristics. RNA was isolated from leucocyte-depleted platelet-rich plasma. Libraries of platelet RNAs were created separately for long RNAs after ribosomal depletion and for small RNAs from total RNA, followed by next-generation sequencing. Results. Platelets in both groups demonstrated RNA expression profiles characterized by absence of leukocyte-specific transcripts, high expression of well-known platelet transcripts, and in total 6,343 consistently detectable transcripts. Extensive statistical bioinformatic analysis yielded 12 genes with consistently differential expression at a lenient FDR < 0.1, thereof 8 protein-coding genes and 2 genes with known expression in platelets (MACF1 and ITGB3BP). Three of the four differentially expressed noncoding genes were YRNAs (RNY1, RNY3, and RNY4) which were all downregulated in DM. 23 miRNAs were differentially expressed between the two groups. Of the 13 miRNAs with decreased expression in the diabetic group, 8 belonged to the DLK1–DIO3 gene region on chromosome 14q32.2. Conclusions. In this study, uncontrolled DM had a remote impact on different components of the platelet transcriptome. Increased expression of MACF1, together with supporting predicted mRNA-miRNA interactions as well as reduced expression of RNYs in platelets, may reflect subclinical platelet activation in uncontrolled DM.

32 NEXT-GENERATION SEQUENCING DISCLOSES DIFFERENCES IN microRNA EXPRESSION PROFILES OF BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY HAND-MADE CLONING AND IN VITRO FERTILIZATION

2017 ◽  
Vol 29 (1) ◽  
pp. 123 ◽  
Author(s):  
S. Lagah ◽  
T. J. Sood ◽  
P. Palta ◽  
M. Mukesh ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Success Rate ◽  
Embryo Development ◽  
Birth Rate ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Next Generation ◽  
Mammalian Embryo ◽  
Differentially Expressed Mirnas ◽  
Generation Sequencing

Mammalian embryo development is a complex process with a series of critical events taking place at every stage of development. It is an established fact that the birth rate of animals produced by nuclear transfer (NT) is far less (<2%) than that of IVF embryos (40%) after successful embryo transfers in different farm animal species. Micro(mi)RNAs are small non-coding RNAs of 17 to 25 nucleotides that alter the function of their target genes by either degrading them or inhibiting their expression. MiRNAs play a vital role during mammalian embryo development and may be adding to the extremely low birth rate and abnormalities in cloned animals. The present study was done with an objective of comparing the miRNA expression profiles of pre-implantation buffalo blastocysts produced by handmade cloning (HMC) and IVF. We hypothesised that there may be differences in the profiles of miRNAs expressed between the 2 groups that contribute to higher success rate in IVF group compared with HMC. Next-generation sequencing (NGS) was done to generate and compare the miRNA profiles and further discern the differentially expressed miRNAs between the 2 groups of blastocysts. For this study, NT blastocysts were produced using fibroblast donor cells isolated from ear skin of a buffalo bull. To produce genetically half-identical IVF blastocysts, the semen of the same bull was used. The oocytes used for generation of both HMC and IVF blastocysts were aspirated from buffalo ovaries obtained from abattoir. Total RNA was isolated from HMC and IVF blastocysts in 4 pools of each group. Each pool consisted of 40 blastocysts. A MiRNA cDNA library was prepared which was then subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). Bos taurus genome was taken as reference to align the reads generated. The data from NGS was validated by RT-qPCR, taking 10 miRNAs (mir-15a, mir-23a, mir-128, mir-130a, mir-133a, mir-194, mir-196b, mir-200b, mir-431 and mir-451). The results positively validated the NGS data. Differential expression analysis of miRNAs between the 2 types of blastocysts revealed that the number of differentially expressed miRNAs with fold change of ≥ 2.0 were 74, with 52 miRNAs up-regulated in HMC and 22 miRNAs up-regulated in IVF. At significance level of P < 0.2, there were 2 miRNAs (mir-202 and mir-133a) that were uniquely expressed in IVF blastocysts and 8 miRNAs (mir-219, mir-451, mir-497, mir-33a, mir-2448, mir-592, mir-187, and mir-502a) that were uniquely expressed in HMC blastocysts. According to the gene ontology analysis, mir-202 is involved in negative regulation of apoptosis and positive regulator of cell growth, whereas mir-133a is involved in generating immunity. Absence of mir-202 and mir-133a expression from HMC blastocysts may be contributing to high apoptosis and other abnormalities in them compared with IVF counterparts. The NGS results indicate that the miRNA profiles of HMC and IVF blastocysts show huge differences. Further analysis of these differentially expressed miRNAs may open the door to miRNA therapies for treating the HMC blastocysts by regulating the expression of critical miRNAs in HMC blastocysts, thereby improving the success rate of cloning.

Next generation sequencing reveals ABCC8 (MODY 12) variants in two families with diabetes mellitus (DM)

2019 ◽  
Author(s):  
Athina Markou ◽  
Amalia Sertedaki ◽  
Elizabeth Tatsi ◽  
George Piaditis ◽  
Theodora Kounadi ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1047 ◽  
Author(s):  
Lama Jaffal ◽  
Wissam H Joumaa ◽  
Alexandre Assi ◽  
Charles Helou ◽  
George Cherfan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Usher Syndrome ◽  
Bioinformatic Analysis ◽  
Next Generation ◽  
Novel Mutations ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

Identification of differentially expressed microRNAs in the skin of experimentally sensitized naturally affected atopic beagles by next-generation sequencing

2020 ◽  
Vol 72 (4) ◽  
pp. 241-250
Author(s):  
Domenico Santoro ◽  
Antonio Di Loria ◽  
Teresa Mirante ◽  
Duarte Mendes Oliveira ◽  
Carmelo Laudanna ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Differentially Expressed ◽  
Next Generation ◽  
Generation Sequencing
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