MOLECULAR CHARACTERIZATION OF MICRORNA
INTERFERENCE AND ARISTOLOCHIC ACID
INTOXICATION FOUND IN UPPER TRACT UROTHELIAL
CARCINOMA IN PATIENTS WITH BALKAN ENDEMIC
NEPHROPATHY: A SYSTEMATIC REVIEW
OF THE CURRENT LITERATURE
Bašić D1*, Ignjatović I1, Janković Veličković Lj2, Veljković A3
*Corresponding Author: *Corresponding Authors: Dragoslav Bašić, Urology Clinic, University Clinical Center Niš, Faculty of
Medicine, University of Niš, Niš, Serbia, Puškinova 2, 18000 Niš, Serbia, Email: basicdr@gmail.com
page: 8
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MATERIAL AND METHODS
Ethical compliance
In this study, the Department of Cardiology of The
First Hospital of Lanzhou University in Lanzhou, China
enrolled a Han Chinese man with HCM (Figure 1). The
ethics committee of the First Hospital of Lanzhou University,
Lanzhou, China approved the study according to the
recommendations of the Declaration of Helsinki. Written
informed consent was obtained from the proband and all
of his family members.
Clinical test details
Clinical examinations were performed for the proband
for cardiovascular disorders. Electrocardiography (ECG)
and Holter were used to evaluate electrophysiology status.
Transthoracic echocardiography (TTE) was performed using
a Phillips EPIQ7C machine (Phillips, United States).
Magnetic resonance imaging (MRI) was performed using
a 3.0T machine (SIMENS Skyro, Germany). The proband
has a positive history of cardiac disorder in his family. The
proband’s father was identified with HCM and one of his
uncles died at a young age with no detailed diagnosis.
Therefore, the clinical evaluation and genetic counselling
for his family members was recommended.
Karyotype and chromosomal microarray analyses
G-banding karyotyping and chromosome microarray
analysis (CMA) has been done to investigate the structure of
the chromosome and copy number variations (CNV) [24].
Whole exome sequencing
Whole exome sequencing was performed with the
proband’s genomic DNA with an Illumina HighSeq 4000.
First, we use Agilent SureSelect version 6 (Agilent Technologies,
United States) for capturing sequence and prepare
the sequence library. Secondly, a Illumina HighSeq
4000 (Illumina, United States) was used to sequence the
enriched library.
After sequencing, we aligned the reads with GRCh37.
p10 by Burrows-Wheeler Aligner software. We locally
aligned and recalibrated the aligned reads again by the
GATK Indel Realigner and the GATK Base Recalibrator,
respectively (broadinstitute.org). Next, we identified the
single-nucleotide variants (SNVs) and small insertions
or deletions (InDels) by using GATK Unified Genotyper
(broadinstitute.org). For the last step, we annotated the
identified variants with Consensus Coding Sequences Database
(20130630) at the National Center for Biotechnology
Information (NCBI).
Bioinformatics data analysis and interpretation
Identified variants with minor allele frequencies
(MAF) less than 0.01 in the databases (dbSNP, HapMap,
1000 Genomes Project and in-house database) were selected.
We classified and categorized the selected variants
based on the established guideline of the American College
of Medical Genetics and Genomics (ACMG) [25].
Lastly, selected variants were interpreted according to their
function and association with disease with the reference of
the OMIM database and previously published literature.
Sanger sequencing
Sanger sequencing was performed to validate the
identified variants by whole exome sequencing.
Primer sequences are as follows; F1 5’-GCGTGCGCATCGTGGCGCTGG-
3’, R1 5’-GCTGATTAGCGCTGGATCGGCG-
3’. The reference sequence NM_000256.3
of MYBPC3 was used.
Analysis of Relative expression
of MYBPC3 mRNA
Realtime-PCR (RT-PCR) was performed for the proband
and his parents. RNA was extracted from whole
blood sample (PrimeScript™ RT Master Mix, Takara,
Japan). Primer sequences were as follows; forward primer:
5’-GCGCAGCTAGCGGCTCGGG-3’ and reverse primer:
5’- GGCGATCGGCCCGTGGCGG-3’.
In silico Analysis
The Mutation Taster (http://mutationtaster.org) was
used to analyze the identified variant in the proband [26].
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