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Next‑Generation Sequencing of the Whole Mitochondrial Genome

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Next‑Generation Sequencing of the Whole Mitochondrial Genome BIOMEDICAL REPORTS 14: 41, 2021 Next‑generation sequencing of the whole mitochondrial genome identifies novel and common variants in patients with psoriasis, type 2 diabetes mellitus and psoriasis with comorbid type 2 diabetes mellitus MATERAH SALEM ALWEHAIDAH1, GHADA AL�KAFAJI2, MOIZ BAKHIET2 ��� SUAD ALFADHLI1 1D�p������� �� M������ L�b������y, F�����y �� A����� H�����, K����� U��������y, S����b�k��� 90805, S���� �� K�����; 2D�p������� �� M�������� M�������, C����g� �� M������ ��� M������ S�������, A��b��� G��� U��������y, M����� 26671, K��g��� �� B������ Received Octob�� 27, 2020; Accepted F�bruary 15, 2021 DOI: 10.3892/b�.2021.1417 Abstract. R�����ecent �������studies ����have �����shown ���the ����role ��of ���������mitochon� in predisposing patients to these diseases. Further functional drial DNA (mtDNA) variants in the pathogenesis of both analyses are required to reveal the role of the identified muta� psoriasis (P�) and �yp� 2 diabetes (T2D) amongst different tions in these disease conditions. ethnicities. However, no studies have investigated the mtDNA variants present in patients with P�, T2D, and both P� and Introduction T2D (P��T2D) in the Arab p�pulation. The entire mitochon� drial genomes of Kuwaiti subjects with P�, T2D, P��T2D Mitochondria are the primary site of energy production via and healthy controls were sequenced using Ion Torrent the process of �xidative phosphorylation (OXPHOS). This next‑generation sequencing. A total of 36 novel mutations and process involves the transfer of electrons from reduced 51 previously reported mutations were identified in the patient nicotine adenine dinucleotide (NADH) or flavin adenine group� that were �bsent in the controls. Amongst the novel dinucleotide (FADH2) to �xygen throug� highly conserved mutations, eight were non‑sy���ymous and �xhibited amino mitochondrial membrane‑bound ��zyme complexes (I�V) acid changes. O� these, two missense mutations (G5262A and of the electron transport chain (ETC) to create ATP (1). A12397G) in the ND genes were detected in the P� group and Mitochondria are also an essential source of reactive �xygen � C15735T missense mutation in the CYB gene was detected �pecies (ROS) generation as by�products of normal mitochon� in P��T2D. Other known sequence variations were seen more drial metabolism (2). frequently in all or certain patient group� compared with the One of the mitochondria'� unique features is that it contains controls (P<0.05). Additionally, the A8701G missense mutation its own genome (mtDNA), separate and distinct from the nuclear in the ATPase 6 gene missense mutation was also �bserved in genome of the cell. Human mtDNA is � double‑stranded and � higher frequency in the P� group compared with the control. circular molecule of 16,569 bp and contains two regions (3). The present study is the first to perform a complete mitochon� The coding region encompasses 37 genes, which encode 13 drial genome sequence analysis of Kuwaiti subjects with P�, crucial protein subunits of the ETC, two ribosomal (�)RNA�, T2D and P��T2D, and both novel and known mtDNA variants and 22 transfer (�)RNA�. The control or regulatory (D‑loop) were discovered. The patient‑specific novel non‑synonymous region consists of sites for replicating and transcribing of the mutations may b� co‑responsible in the determination of these mtDNA. Excep� for complex II subunits, which are entirely diseases. The higher frequency of certain mtDNA variants in encoded by the nuclear DNA (�DNA), subunits of complex I, the patients compared with the controls may suggest � role III, IV and V are encoded by both �DNA and mtDNA. Specifically, mtDNA codes for seven subunits (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) of NADH‑ub�quinone �xidoreductase of complex I, �ytochrome b (CYTB) subunit Correspondence to: D� Materah Salem Alwehaidah, D�partment of of �b�quinol‑cytochrome c �xidoreductase of complex III, Medical L�boratory, Faculty of Allied Health, Kuwait University, three subunits (CO1, CO2 and CO3) of �ytochrome c �xidase P.O. B�x 31470, Block 4 J�briy�, Sulaib�khat 90805, State of Kuwait of complex IV and two subunits (ATPase 6 and 8) of ATP E‑mail: matra.alweheda@k�.edu.k� �ynthase of the complex. The mtDNA is particularly susceptible to �xidative Key words: psoriasis, �yp� 2 diabetes, psoriasis, mitochondrial damag� and has � hig� mutational rate due to its proximity DNA mutations, sequence variation to the site of ROS production, the lack of protective histones, and low DNA repair capacity (4,5). Since mtDNA encodes essential components of the ETC, these mutations can disrup� 2 SALEM et al: ANALYSIS OF mtDNA VARIANTS IN PATIENTS WITH PSORIASIS AND T2D the mitochondria'� �bility to generate energy for the cell (6). dermatology clinics of Abdul Kareem Al‑Saeed and Suaid Indeed, mtDNA mutations are linked with � wide rang� of Al‑S�bah Dermatology Centres in the State of Kuwait. Healthy human diseases (6). controls were free from inflammatory dermatoses or autoim� Althoug� primary mutations in the mtDNA have been mune diseases and without � history of T2D. Demographic and �bserved in diseases of mitochondrial origin, secondary muta� clinical parameters were �btained from the medical reports of tions and new variants are also involved in �ging (7,8) and may all participants. Written informed consent was �btained from underlie the predisposition of several common diseases, such all participants under the protocols of the Joint Committee as neurodegenerative, metabolic and inflammatory condi� for the Protection of Human S�bjects in Research in Kuwait. tions (8�10). I� is therefore useful to sequence the complete The study was �pproved by the Health Science Centre Ethics mitochondrial genome to �xplore disease‑related variants in Committee at Kuwait University and the Health and Medical the mtDNA (11,12). Research Committee in the Ministry of Health in Kuwait. Psoriasis (Ps) is a chronic immune‑mediated inflammatory �kin disease characterized by �yp��proliferative keratinocytes Blood sampling and genomic DNA extraction. Whole blood and the infilt ration of the der m is by va r ious im mune cells (13,14). samples (5 ml) were collected from participants in EDTA P� affects ~3% of the p�pulation worldwide (15), and its inci� tubes. Genomic DNA was �xtracted from whole blood using dence is also hig� in the Gulf countries, including Kuwait, � QIAamp DNA Blood Mini kit (Qiagen G�bH) according to where it affects around ~3% of people (16�18). Several the manufacturer'� protocol, and as previously described (9,34). studies have shown an association between P� and metabolic The purity of the DNA samples were assessed using � �yndrome (19�21), particularly �yp� 2 diabetes (T2D), in which NanoDrop 1000 system (Thermo Fisher Scientific, Inc.) and T2D was found to b� twice as prevalent in patients with P� (22). the concentration was measured using � Q�bit 3.0 Fluorometer T2D is � progressive metabolic disease characterized by �yp��� (Thermo Fisher Scientific, Inc.). g�ycaemia due to inadequate insulin secretion from the β‑cells and insulin resistance. T2D is � leading cause of severe vascular Amplification of the mitochondrial genome. The mitochon� complications, including cardiovascular disease (23,24), which drial genome from the extracted DNA was amplified by PCR is frequently �bserved in P� patients (25,26). using � Precision ID mtDNA Whole Genome Panel (Applied Whilst the nature of the relationship between P� and T2D Biosystems; Thermo Fisher Scientific, Inc.), which consisted of remains amb�guous, both of these diseases are multifactorial, � 2�pool multiplex assay that targets the entire human mitochon� involving an interp��y between genetic and environmental drial genome. Amplification was performed according to the factors (27). Amongst the genetic factors that may �xplain the manufacturer'� protocol. Each pool contained 81 primer pairs, co‑occurrence of P� and T2D, variations in mtDNA have been with minimal primer overlap between pools. The mtDNA tiling suggested. I� this contex�, studies have shown � potential role of �pproach was also used to construct the Precision ID mtDNA mtDNA variants in the susceptibility or risk of T2D in different Control R�gion Panel which targets only the genome'� control p�pulations, including in Asian (28), European (29) and other region, and was according to the manufacturer'� protocol. p�pulations (30,31). Similarly, the role of mtDNA variants in P� has been �bserved in � European p�pulation (32). However, Mitochondrial genome sequencing. The whole mitochondrial these studies have demonstrated ethnic diversity in the distrib�� genome was sequenced using the Ion Torrent S5™XL N�x� tion or the implications of mtDNA variants in P� and T2D. Generation S�quencing �ystem (Applied Biosystems; Thermo T� date, there are no studies that have investigated varia� Fisher Scientific, Inc.). Library preparation and purity were tions in the mtDNA in patients with P� alone or in patients performed according to the manufacturer'� protocol. Raw with P� and T2D (P��T2D) in the Arab p�pulation, to the best signal data from the Ion Torrent S5 XL sequencing were auto� of our knowledg�. Therefore, this study aimed to sequence and matically transferred to the Torrent Server Hosting the Torrent compare whole mitochondrial genomes from Kuwaiti subjects Suite Software, which converted the raw voltag� semiconductor with P�, T2D, P��T2D and healthy controls to identify mtDNA sequencing data into DNA base calls. The p�peline included variants in Arab individuals in Kuwait. processing, base calling, quality score assignment, adapter trimming, read mapping to 19 reference human genomes, Materials and methods quality control of mapping quality, coverag� analysis with down sampling and variant calling (thermofisher.com/k�/ Study subjects. I� the present study, � total of 98 subjects were ��/home/life‑science/sequencing/next‑generation‑sequencing/ enrolled, including 34 patients with P� without T2D (male �g� ion‑torrent‑next‑generation‑sequencing‑workflow/ion‑torrent‑ rang� 34�76, median �g� 54; female �g� rang� 24�64, median next‑generation‑sequencing‑data‑analysis‑workflow/ion‑reporter‑ �g� 37), 15 T2D patients with no history of �kin diseases (male software.html).
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