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Anthocyanins are antioxidant compounds constitute the primary dyes of the pomegranate arils. Anthocyanins could protect the aged skin induced by oxidant exposure as a major role in aging processing and skin degeneration.The study aimed to evaluate the anti-aging activity of anthocyanins rich pomegranate (Anthocyanins were extracted from fresh pomegranate arils using acidified methanol and were purified by Sephadex LH-20 gel-column chromatography. Further, the fusion method was used to prepare cold cream containing pomegranate anthocyanins. The formulated cream was evaluated for their compatibility study, irritation, homogeneity, drug content, drug release, and stability tests. Furthermore, permeation study through abdominal rabbits, as well as Human application was performed.Compatibility study showed the absence of any interaction between anthocyanins and the used polymers. The formulated cream was nonirritant, homogenous and potentially reduced skin aging when applied to Human volunteers' skin. Furthermore, the skin permeation displayed a good permeation of 43.16% after 210 min.Pomegranate anthocyanins could be used as a safe, stable, homogeneous, nonirritant and effective topical anti-aging drug formulation for aged human people.

Background: Eczema is also known as atopic dermatitis is well-known for the skin disease globally. In Saudi Arabia, exome sequencing studies have not been documented. The purpose of this study was to scrutinize the disease causing mutations in children affected with eczema with exome sequencing in the Saudi population. Methods: We recruited randomly three sporadic cases of children diagnosed with eczema and simultaneously, three more cases were adopted for control samples. Exome sequencing was carried out by applying a pipeline that captures all the variants of concern related to the samples by using the Ion torrent. Results: In this study, we have documented 49 variants, among which 37 variants were confirmed through eczema children and remaining 30 variants through control children. However, from the analysis of the 6 samples, we have identified rs10192157 (1646C > T; Thr549Ile), rs2899642 (27C > G; Asn9Lys), chr1:152127950 (1625 G > A; Gly542Asp) and chr1:152128041 (1534C > G; Gly512Arg) variants which are rarely linked to the disease eczema. In the rs10192157, we have documented these mutations in all three eczema children and one in the control; the rs2899642 mutation appeared in only a couple of eczema children, whereas the mutation in the chr1:152127950 regions appeared in only one eczema patient. However, the chr1:152128041 mutations appeared in only one case of eczema and also in two control children. Conclusion: Our study revealed four mutations which had not previously been connected with eczema within the database. However, the rs10192157 and rs2899642 mutations were documented with asthma disease. The remaining mutations such as chr1:152127950 and chr1:152128041 have not been reported anywhere else. This study recommends screening these 4 mutations in eczema cases and their relevant controls to confirm the prevalence in the Saudi population. It is recommended that future studies examine the 4 mutations in detail. Keywords: Eczema, Exome-sequencing, Mutation, Variants

Background. Different common gene variants were related to coronary artery disease (CAD) in many studies. Yet, the relation of these loci to the severity of CAD is not completely elucidated. Methods. We enrolled 520 subjects (315 CAD cases and 205 controls). CAD presence and extension were assessed by coronary angiography (CAG). Genotyping of five SNPs (namely, rs2230806 (1051G > A) in ABCA1 on chromosome 9, rs2075291 (553G > T) in ApoA5 on chromosome 11, rs320 in LPL on chromosome 8 intron (T → G at position 481), rs10757278 (c.22114477A > G), and rs2383206 (c.22115026 A > G) on chromosome 9p21 locus) was performed by allele-specific PCR. The degree and site of arterial lesions were used to classify patients, tested for association with CAD severity, and related to allele dosage. Results. The polymorphisms rs2383206 and rs10757278 showed significant associations with 2- and 3-vessel coronary disease (p =0.003 and 0.006, respectively). The homozygous GG genotypes of rs10757278 was associated with higher frequency of left anterior descending (LAD), right coronary artery (RCA) and left circumflex (LCX) diseases (p =0.002, 0.016 and 0.002, respectively). The GG genotypes of rs2383206 were found in higher percentage in patients with left main (LM) trunk and left circumflex (LCX) diseases ( p = 0.013 and 0.002, respectively). Conclusion. SNPs rs10757278 and rs2383206 allele dosage could predict CAD severity in the Saudi Arab population.

Whole exome sequencing (WES) is an innovative, precise procedure established on the next generation sequencing and has rapidly become esteemed in medical genetics. We have used WES to study patient with suspected genetic conditions with no apparent recognizable familial hypercholesterolemia (FH). In the tested proband, multiple causative mutations were identified in LPL, EPHX2, PCSK9, APOB, ABCA1 and ABCG8 genes. The identified mutations in the LPL gene were heterozygous mutation [c.106G>A, p. (Asp36Asn)] in exon 2 and another heterozygous mutation [c.1421C>G, p.(Ser474*)] in exon 9. EPHX2 gene of the proband showed a novel heterozygous mutation [c.1644delC, p.(Pro549fs)] in exon 19. In addition, heterozygous mutations were identified in exon 1 of PCSK9 gene [c.158C>T, p.(Ala53Val)] and exon 4 of APOB [c.293C>T, p.(Thr98Ile)]. Likewise, homozygous mutations were found in exon 35 of ABCA1 gene [c.4760A>G, p.(Lys1587Arg)] and exon 13 of ABCG8 gene [c.1895T>C, p.(Val632Ala)], a common mutation highly associated with hypercholesterolemia based on the p-value by phenolyzer prioritization method. All causative mutations were validated by capillary sequencing method and confirmed the presence of homozygous and heterozygous mutations detected in the proband. All the causative mutations were functionally predicted to be deleterious including a novel frameshift and common missense mutation. Further investigations of the common mutation using Molecular dynamic (MD) simulation with solvated condition confirmed the functional and structural impact of the changes on the mutated proteins.

Cancer may be difficult to target, however, if cancer targeted this provides the chance for a better and more effective treatment. Quantum dots (Qdots) coated vapreotide (VAP) as a somatostatin receptors (SSTRs) agonist can be efficient targeting issue since may reduce side effects and increase drug delivery to the target tissue. This study highlights the active targeting of cancer cells by cells imaging with improving the therapeutic outcomes. VAP was conjugated to Qdots using amine-to-sulfhydryl crosslinker. The synthesized Qdots-VAP was characterized by determination of size, measuring the zeta-potential and UV fluorometer. The cellular uptake was studied using different cell lines. Finally, the Qdots-VAP was injected into a rat model. The results showed a size of 479.8 ± 15 and 604.88 ± 17 nm for unmodified Qdots and Qdots-VAP respectively, while the zeta potential of particles went from negative to positive charge which proved the conjugation of VAP to Qdots. The fluorometer recorded a redshift for Qdots-VAP compared with unmodified Qdots. Moreover, cellular uptake exhibited high specific binding with cells which express SSTRs using confocal microscopy and flow cytometry (17.3 MFU comparing to 3.1 MFU of control, P

Internal ribosome entry site (IRES) sequences have become a valuable tool in the construction of gene transfer and therapeutic vectors for multi-cistronic gene expression from a single mRNA transcript. The optimal conditions for effective use of this sequence to construct a functional expression vector are not precisely defined but it is generally assumed that the internal ribosome entry site dependent expression of the second gene in such as cassette is less efficient than the cap-dependent expression of the first gene. Mainly tailoring inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector further in construction of optimised cassette for gene therapy of familial hypercholesterolemia. We tailored the size of the inter-cistronic spacer sequence at the 5′ region of the internal ribosome entry site sequence using sequential deletions and demonstrated that the expression of the 3′ gene can be significantly increased to similar levels as the cap-dependent expression of the 5’ gene. Maximum expression efficiency of the downstream gene was obtained when the spacer is composed of 18–141 base pairs. In this case a single mRNA transcriptional unit containing both the first and the second Cistron was detected. Whilst constructs with spacer sequences of 216 bp or longer generate a single transcriptional unit containing only the first Cistron. This suggests that long spacers may affect transcription termination. When the spacer is 188 bp, both transcripts were produced simultaneously in most transfected cells, while a fraction of them expressed only the first but not the second gene. Expression analyses of vectors containing optimised cassettes clearly confirm that efficiency of gene transfer and biological activity of the expressed transgenic proteins in the transduced cells can be achieved. Furthermore, Computational analysis was carried out by molecular dynamics (MD) simulation to determine the most emerges as viable containing specific binding site and bridging of 5′ and 3′ ends involving direct RNA-RNA contacts and RNA-protein interactions. These results provide a mechanistic basis for translation stimulation and RNA resembling for the synergistic stimulation of cap-dependent translation. Keywords: Internal ribosome entry site, IRES, Gene therapy, Inter-cistronic sequences, MD simulation, Protein structure modeling, Familial hypercholesterolemia

Background: Multiple genes have been implicated to have a role in asthma predisposition by association studies. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition could play a more substantial role in this group. This study aims to identify the spectrum of rare and novel variation in known paediatric asthma susceptibility genes using whole exome sequencing-analysis in nine individual cases of childhood onset allergic asthma. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, identify and annotate variants where patient sequence differed from the reference sequence. Results: DNA samples from the nine children with a history of bronchial asthma diagnosis underwent targeted exome capture and sequencing. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued. The analysis showed 21 variants in the subjects, 13 had been previously identified and 8 were novel. Also, amongst of which, nineteen were non-synonymous and 2 were nonsense. With regard to the novel variants, the 2 non-synonymous variants in the PRKG1 gene (PRKG1: p.C519W and PRKG1: p.G520W) were presented in 4 cases, and a non-synonymous variant in the MAVS gene (MAVS: p.A45V) was identified in 3 cases. The variants we found in this study will enrich the variants spectrum and build up the database in the Saudi population. Novel eight variants were identified in the study which provides more evidence in the genetic susceptibility in asthma among Saudi children.Conclusion: Screening a cohort of Saudi children for molecular identification of polymorphisms associated with allergic asthmatic response. These, together with the clinical phenotypes, revealed genetic determinants for paediatric asthma and also we compared to the similar previous reports. Providing a genetic screening map for the molecular genetic determinants of allergic disease in Saudi children, with the goal of reducing the impact of chronic diseases on the health and the economy. We belief that the advanced specified statistical filtration/annotation programs used in this study succeeded to release such results in preliminary study, exploring the genetic map of that disease in Saudi children.

The current COVID-19 pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has raised significant economic, social, and psychological concerns. The rapid spread of the virus, coupled with the absence of vaccines and antiviral treatments for SARS-CoV-2, has galvanized a major global endeavor to develop effective vaccines. Within a matter of just a few months of the initial outbreak, research teams worldwide, adopting a range of different strategies, embarked on a quest to develop effective vaccine that could be effectively used to suppress this virulent pathogen. In this review, we describe conventional approaches to vaccine development, including strategies employing proteins, peptides, and attenuated or inactivated pathogens in combination with adjuvants (including genetic adjuvants). We also present details of the novel strategies that were adopted by different research groups to successfully transfer recombinantly expressed antigens while using viral vectors (adenoviral and retroviral) and non-viral delivery systems, and how recently developed methods have been applied in order to produce vaccines that are based on mRNA, self-amplifying RNA (saRNA), and trans-amplifying RNA (taRNA). Moreover, we discuss the methods that are being used to enhance mRNA stability and protein production, the advantages and disadvantages of different methods, and the challenges that are encountered during the development of effective vaccines.

Background Leukaemia is the most prevalent form of cancer-causing death in a large number of populations and needs prompt and effective treatment. Chemotherapeutics can be used to treat leukaemia, but their pronounced killing effects to other living cells is still an issue. Active targeting to certain specific receptors in leukaemic cells is the best way to avoid damage to other living cells. Leukaemic cells can be targeted using novel nanoparticles (NPs) coated with a specific ligand, such as octreotide (OCD), to target somatostatin receptor type 2 (SSTR2), which is expressed in leukaemic cells. Methods Amino-PEGylated quantum dots (QDs) were chosen as model NPs. The QDs were first succinylated using succinic anhydride and then coated with OCD. The reactivity and selectivity of the formulated QDs-OCD were studied in cell lines with well-expressed SSTR2, while fluorescence was detected using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). Conclusively, QD-OCD targeting to blood cells was studied in vivo in mice and detected using inductively coupled plasma mass spectrometry and CLSM in tissues. Results Highly stable QDs coated with OCD were prepared. FACS and CLSM showed highly definite interactions with overexpressed SSTR2 in the investigated cell lines. Moreover, the in vivo results revealed a higher concentration of QDs-OCD in blood cells. The fluorescence intensity of the QDs-OCD was highly accumulated in blood cells, while the unmodified QDs did not accumulate significantly in blood cells. Conclusion: The formulated novel QDs-OCD can target SSTR2 overexpressed in blood cells with great potential for treating blood cancer.

Background and aims Familial hypercholesterolemia (FH) is a predominantly autosomal dominant hereditary disorder with significant potential for expansion of coronary artery disease. Methods To identify candidate variant/s in FH phenotype implicated genes, next-generation sequencing was performed using a targeted customized gene panel. Results We recognized a 45-year-old Saudi female FH patient with double variants in the LDLR [c.1255 T > G, p.(Y419D)] and LDLRAP1 genes [c.604_605delTCinsA, p.(S202Tfs*2)]. The proband was found to be homozygous for the LDLR variant and heterozygous for the LDLRAP1 variant. Three of the proband's children were found to be double heterozygous for the LDLR/LDLRAP1 gene variant. While her other three children were heterozygous for the same single LDLR variant. Both variants were not previously reported. The variants segregation pattern correlated with the clinical picture and with the patient's lipid profile. FH severity was greater in the proband while her children did not show any clinical manifestations. The missense variant p.(Y419D) was found to be deleterious and clinically significant based on prediction identified by PolyPhen-2 and Proven. Molecular dynamics simulation was used to further analyze the effect of the variant p.(Y419D) on the structure and function of the LDLR protein. The secondary structure was investigated, as well as the solvent accessibility and stabilizing residues. The frameshift variant of the LDLRAP1 gene results in a truncated peptide that could affect the cellular internalization of LDLR/LDL complex. Conclusions The finding of the combined variants in LDLR/LDLRAP1 genes triggering a severe FH phenotype is essential to elaborate the spectrum of variants causing FH and to understand the genotype-phenotype correlation.

Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5&prime, LDLReGFP-3&prime, fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3&prime, and 5&prime, ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD&ndash, eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein&ndash, protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.

Systemic steroids are used to treat acute graft-versus-host disease (aGVHD) caused by allogenic bone marrow transplantation (allo-BMT), however, their prolonged use results in complications. Hence, new agents for treating aGVHD are required. Recently, a new compound A (CpdA), with anti-inflammatory activity and reduced side effects compared to steroids, has been identified. Here, we aimed to determine whether CpdA can improve the outcome of aGVHD when administered after transplantation in a mouse model (C57BL/6 in B6D2F1). After conditioning with 9Gy total body irradiation, mice were infused with bone marrow (BM) cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. The animals were subsequently treated (3 days/week) with 7.5 mg/kg CpdA from day +15 to day +28, the controls received 0.9% NaCl. Thereafter, the incidence and severity of aGVHD in aGVHD target organs were analyzed. Survival and clinical scores did not differ significantly, however, CpdA-treated animals showed high cell infiltration in the target organs. In bulk mixed lymphocyte reactions, CpdA treatment reduced the cell proliferation and expression of inflammatory cytokines and chemokines compared to controls, whereas levels of TNF, IL-23, chemokines, and chemokine receptors increased. CpdA significantly reduced proliferation in vitro but increased T cell infiltration in target organs.

To the editor: Allogeneic bone marrow (BM) transplantation (alloBMT) is a unique curative therapy for diverse diseases. However, its use is limited by the development of severe treatment-related complications, most importantly, the occurrence of acute graft-versus-host-disease (aGVHD). Despite the

A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD) phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C > T, p.(Arg1624Trp), c.5725C > T, p.(Arg1909Trp), c.1736C > T, p.(Thr579Met) and c.10628T > G, p.(Leu3543Trp)] among 12 out of 18 samples. However, one variant c.4870C > T, p.(Arg1624Trp) was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD) disease causing genes PKD1 and PKD2 such as c.12433G > A, p.(Val4145Ile) and c.1445T > G, p.(Phe482Cys), respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T > G, p.(Leu3543Trp) in a male proband. We have recently published the results of these studies (Edrees et al., 2016). Here we report for the first time the effect of the common mutation p.(Arg1624Trp) found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp) in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1, PKD1, and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1, PKD1, and PKD2 genes among various related and unrelated mammalian species will provide important insights into their evolutionary process and understanding for further disease characterization and management.

Background: Ependymomas are glial tumors derived from differentiated ependymal cells. In contrast to other types of brain tumors, histological grading is not a good prognostic marker for these tumors. In order to determine genomic changes in an anaplastic ependymoma, we analyzed its mutation patterns by next generation sequencing (NGS). Methods: Tumor DNA was sequenced using an Ion PI v3 chip on Ion Proton instrument and the data were analyzed by Ion Reporter 5.6. Results: NGS analysis identified 19 variants, of which four were previously reported missense variants; c.395G>A in IDH1, c.1173A>G in PIK3CA, c.1416A>T in KDR and c.215C>G in TP53. The frequencies of the three missense mutations (PIK3CA c.1173A>G, KDR c.1416A>T, TP53, c.215C>G) were high, suggesting that these are germline variants, whereas the IDH1 variant frequency was low (4.81%). However, based on its FATHMM score of 0.94, only the IDH1 variant is pathogenic; other variants TP53, PIK3CA and KDR had FATHMM scores of 0.22, 0.56 and 0.07, respectively. Eight synonymous mutations were found in FGFR3, PDGFRA, EGFR, RET, HRAS, FLT3, APC and SMAD4 genes. The mutation in FLT3 p.(Val592Val) was the only novel variant found. Additionally, two known intronic variants in KDR were found and intronic variants were also found in ERBB4 and PIK3CA. A known splice site mutation at an acceptor site in FLT3, a 3’-UTR variant in the CSF1R gene and a 5’_UTR variant in the SMARCB1 gene were also identified. The p-values were below 0.00001 for all variants and the average coverage for all variants was around 2000x. Conclusions: In this grade III ependymoma, one novel synonymous mutation and one deleterious missense mutation is reported. Many of the variants reported here have not been detected in ependymal tumors by NGS analysis previously and we therefore report these variants in brain tissue for the first time.

Choroid plexus papilloma (CPP) is a rare benign tumor of the central nervous system that is usually confined to the cerebral ventricles. According to the World Health Organization, CPP corresponds to a grade I atypical CPP (a-CPP); however, it can become more aggressive and reach grade II, which can rarely undergo malignant transformation into a choroid plexus carcinoma (grade III). To the best of our knowledge, identification of these tumors mutations by next generation DNA sequencing (NGS) has not been yet reported. In the present study, NGS analysis of an a-CPP case was performed. Data were analyzed using Advaita Bioinformatics i-VariantGuide and Ion Reporter 5.6 programs. The results from NGS identified 12 novel missense mutations in the following genes: NOTCH1, ATM, STK36, MAGI1, DST, RECQL4, NUMA1, THBS1, MYH11, MALT1, SMARCA4 and CDH20. The PolyPhen score of six variants viz., DST, RECQL4, NUMA1, THBS1, MYHI1 and SMARCA4 were high, which suggested these variants represents pathogenic variants. Two novel insertions that caused frameshift were also found. Furthermore, two novel nonsense mutations and 14 novel intronic variants were identified in this tumor. The novel missense mutation detected in ATM gene was situated in c.5808A>T; p. (Leu1936Phe) in exon 39, and a known ATM mutation was in c.5948A>G; p. (Asn1983Ser). These novel mutations had not been reported in previous database. Subsequently, the quality statistics of these variants, including allele coverage, allele ratio, P-value, Phred quality score, sequencing coverage, PolyPhen score and alleles frequency was performed. For all variants, P-value was highly significant and the Phred quality score was high. In addition, the results from sequencing coverage demonstrated that 97.02% reads were on target and that 97.88% amplicons had at least 500 reads. These findings may serve at determining new strategies to distinguish the types of choroid plexus tumor, and at developing novel targeted therapies. Development of NGS technologies in the Kingdom of Saudi Arabia may be used in molecular pathology laboratories.

Background: Familial hypercholesterolemia (FH) is an autosomal dominant inherited genetic disorder and results in the development of coronary artery disease (CAD). Clinical diagnosis of homozygous HH patients is usually straightforward because persistent hypercholesterolemia can produce xanthoma and corneal arcus. However, xanthoma may also be misdiagnosed as skin lesions and could therefore be mistreated. The aim of this case study report is to highlight the plight of patients with FH as means of raising awareness of the condition among dermatologists and health care practitioners, also to determine the genotype-phenotype correlation in severely affected homozygous FH proband patients. Methods: Genetic screening of FH associated genes was performed by Ion Torrent next-generation sequencing and cascade screening by capillary sequencing. Results: We present two clinical cases with prominent skin lesions seen in a dermatology clinic that were referred to plastic surgery for excision. Genetic testing was performed later, and confirmed common single nucleotide deletion variant (c.2027delG) in the 'LDLR' alleles consequent to a frameshift mutation p.(G676Afs*33). In addition to the 'LDLR' variant, two possibly damaging 'APOB' variants p.(L3313I) and p.(L1212M) and three damaging variants p.(R19*), p.(G83Q) and p.(S474*) in 'APOC3, PON2' and 'LPL' genes respectively were identified. The 'PON2' gene variant p.(G83Q) was found to be novel, while others have been previously reported. Both patients were refractory to pharmacological therapies and are currently on lipoprotein apheresis (LA). Conclusions: The present report indicates the need for increased awareness of FH, among the public and healthcare practitioners and supports the need for diagnostic screening and cascade genetic testing of this high-risk condition, which could ultimately lead to better prevention of CHD in this lethal condition.

Type Basic science research. Presentation Type Oral presentation. Introduction Coronary artery diseases (CAD) inflict heavy economical and social cost on most populations including Saudi’s and contribute significantly to their morbidity and mortality rates. Familial hypercholesterolemia (FH) is hereditary in an autosomal dominant disease and is a major risk factor for the development of CAD. FH is predominantly caused by variants in the low-density lipoprotein (LDL) receptor gene (LDLR). Methodology More than 140 FH samples including 44 probands were collected from 17 unrelated Saudi families who live in the central, northern, western and eastern regions of Saudi Arabia. Patient samples were screened using Next-generation sequencing (NGS) and Capillary sequencing. We described the genetic analysis of severely affected homozygous FH patients who were mostly resistant to statin therapy and were managed on an apheresis program. Results We identified a common frameshift mutation p. (G676Afs*33) in exon 14 of the LDLR gene in 17 probands and their first-degree blood relatives in apparently unrelated Saudi families. This founder mutation was found in about 40% Saudi FH population. We also describe a three dimensional homology model of the LDL receptor protein (LDLR) structure and examine the consequence of the frameshift mutation p. (G676Afs*33), as this could affect the LDLR structure in a region involved in dimer formation, and protein stability. Conclusion This finding of a recurrent mutation causing FH in the Saudi population could serve to develop a rapid genetic screening procedure for FH, and the 3D-structure analysis of the mutant LDLR, may provide tools to develop a mechanistic model of the LDLR function.

Background: Hemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulation. Methods: For genotyping, blood samples from Saudi Arabian patients were collected, and the genomic DNA was amplified, and then sequenced by Sanger method. For molecular simulations, we have used softwares such as CHARMM (Chemistry at Harvard Macromolecular Mechanics; http://www.charmm-gui.org) and GROMACS. In addition, the secondary structure was determined based on the solvent accessibility for the confirmation of the protein stability at the site of mutation. Results: Six mutations (three novel and three known) were identified in factor VIII gene, and six mutations (one novel and five known) were identified in factor IX gene. The factor VIII novel mutations identified were c.99G>T, p. (W33C) in exon 1, c.2138 DelA, p. (N713Tfs*9) in eon14, also a novel mutation at splicing acceptor site of exon 23 c.6430 - 1G>A. In factor IX, we found a novel mutation c.855G>C, p. (E285D) in exon 8. These novel mutations were not reported in any factor VIII or factor IX databases previously. The deleterious effects of these novel mutations were confirmed by PolyPhen2 and SIFT programs. Conclusion: The protein functional and structural studies and the models built in this work would be appropriate for predicting the effects of deleterious amino acid substitutions causing these genetic disorders. These findings are useful for genetic counseling in the case of consanguineous marriages which is more common in the Saudi Arabia. J Clin Med Res. 2017;9(4):317-331 doi: https://doi.org/10.14740/jocmr2876w

Hemophilia A is an X-linked recessive hemorrhagic disorder caused by mutations in the factor VIII gene. To find out known and novel causative mutations in Hemophilia A, we carried out genetic analysis among Saudi patients. Twenty six Patients who were negative for inv-1/inv-22 were selected for analysis with Multiplex Ligation-dependent Probe Amplification (MLPA) and Sanger sequencing. Furthermore the functional and structural effects of the novel mutations were predicted using Molecular dynamic simulation. The results showed three known large deletions in 6 samples (delE8,9,10,11,12,13,14; delE7,8,9,10; and delE4) and twelve mutations in 18 samples; four of them were novel. The novel mutations detected were two substitutions in exon 8 at position c.1021G>C, p.(Ala341Pro) and position c.1183A>C, p.(Lys395Gln), one substitution in exon13 at position c.1930T>A, p.(Leu644Met), and one substitution in exon22 at position c.6322G>C, p.(Ala2108Pro). Clinical data of Patients with novel mutations showed

Familial hypercholesterolemia (FH) is most commonly caused by mutations in the LDL receptor (LDLR), which is responsible for hepatic clearance of LDL from the blood circulation. We described a severely affected FH proband and their first-degree blood relatives; the proband was resistant to statin therapy and was managed on an LDL apheresis program. In order to find the causative genetic variant in this family, direct exon sequencing of the LDLR, APOB and PCSK9 genes was performed. We identified a compound heterozygous mutation in the proband with missense p.(W577C) and frameshift p.(G676Afs33) variants at exons 12 and 14 of the LDLR gene respectively. DNA sequencing of LDLR gene from the parents demonstrated that the missense variant was inherited from the mother and frameshift variant was inherited from the father. The frameshift variant resulted in a stop signal 33 codons downstream of the deletion, which most likely led to a truncated protein that lacks important functional domains, including the trans-membrane domain and the cytoplasmic tail domain. The missense variant is also predicted to be likely pathogenic and affect EGF-precursor homology domain of the LDLR protein. The segregation pattern of the variants was consistent with the lipid profile, suggesting a more severe FH phenotype when the variants are in the compound heterozygous state. The finding of a compound heterozygous mutation causing severe FH phenotype is important for the genotype-phenotype correlation and also enlarges the spectrum of FH-causative LDLR variants in the Arab population, including the Saudi population.

Autosomal recessive polycystic kidney disease (ARPKD) a rare genetic disorder, described by formation of cysts in the kidney. A targeted customized sequencing of genes implicated in ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified likely pathogenic disease causing variants during the validation process. Four potential pathogenic variants [c.4870C>T, p.(Arg1624Trp)], [c.5725C>T, p.(Arg1909Trp)], c.1736C>T, p.(Thr579Met)] and [(c.10628T>G), p.(Leu3543Trp)] were observed in PKHD1 gene among 12 out of 18 samples. The rest of the patient samples also showed few variants in ADPKD (Autosomal Dominant Polycystic Kidney Disease) disease causing genes PKD1 and PKD2 i.e. [c.12433G>A, p.(Val4145Ile)] and [c.1445T>G, p.(Phe482Cys)], respectively. All causative variants were validated by capillary sequencing, confirming the presence of a novel homozygous variants [c.10628T>G, p.(Leu3543Trp)] found in exon 61 of a male proband. All potentially deleterious variants identified in PKHD1, PKD1, and PKD2 gene, also exhibited pathologically or clinically significance based on the computational predictions involved in predicting the impact of non-synonymous SNPs (nsSNPs) on protein function such as Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen2). SIFT classified 50% of our nsSNPs as "deleterious", while PolyPhen2 identified 45% of our nsSNPs as "Probably damaged" and the results from both programs were largely complementary. Taken together, these results suggest that the NGS strategies provide a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in targeted genes sequence analysis.

Background: Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular testing for the factor VIII gene is difficult due to its large size. More than 1000 different mutations have been described in factor VIII gene. In this study we have investigated the factor VIII gene mutations in Saudi Arabian population. Methods: For genotyping factor VIII cohorts of 110 samples from Saudi Arabian patients undergoing treatment for hemophilia A were collected. All patients were tested for factor VIII coagulant activity on Behring Coagulation System. Genomic DNA was isolated from blood on MagNapure system. Screening for inv-1 was done by multiplex PCR method, and inv-22 was done by ligation (inverse) PCR method. DNA sequencing was performed by Sanger method for all 26 exons of factor VIII gene. PCR products were sequenced on ABI 3500 Genetic analyzer. For molecular simulations we have used softwares such as CHARMM and GROMACS v4.0.527. In order to predict the possible impact of a variation on the function of factor VIII gene the online tools Polyphen 2, and SIFT were used. Results: Out of 110 cases screened, 2 patients were positive (affected) for inv-1 and 15 patients were positive (12 affected and 3 carriers) for inv-22. Out of 32 cases sequenced for coding exons, 2 novel mutations were found, one novel missense mutation c.355G>C, p. (A119P) in exon 3, and another novel frame shift mutation c.6482delC, p.(P2161Lfs*25) in exon 23. Also known mutations such as, c.409 A>C, p. (T137P) in 2 individual patients in exon 4, another known mutation c.1804C>T, p.(R602*) in 1 patient in exon 12 were found. Genotype-phenotype correlations and computer prediction analysis on these novel mutations and the secondary structure analysis of the factor VIII protein were performed, and compared with the predicted native proteins. Conclusions: These novel mutations in factor VIII gene and molecular dynamic simulation results to appropriately predict the deleterious effects of these mutations are presented in this study. In addition, for the native and mutant proteins models, the amino acid residues and its secondary structures were determined. Our In-silico study suggests that these mutations have significant impact on the structure and function of the factor VIII protein.

Familial hypercholesterolemia (FH) is an autosomal dominant disease, predominantly caused by variants in the low-density lipoprotein (LDL) receptor gene (LDLR). Herein, we describe genetic analysis of severely affected homozygous FH patients who were mostly resistant to statin therapy and were managed on an apheresis program. We identified a recurrent frameshift mutation p.(G676Afs*33) in exon 14 of the LDLR gene in 9 probands and their relatives in an apparently unrelated Saudi families. We also describe a three dimensional homology model of the LDL receptor protein (LDLR) structure and examine the consequence of the frameshift mutation p.(G676Afs*33), as this could affect the LDLR structure in a region involved in dimer formation, and protein stability. This finding of a recurrent mutation causing FH in the Saudi population could serve to develop a rapid genetic screening procedure for FH, and the 3D-structure analysis of the mutant LDLR, may provide tools to develop a mechanistic model of the LDLR function.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease., The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome–mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro–IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.