CDR grafting and site-directed mutagenesis approach for the generation and affinity maturation of Anti-CD20 nanobody.

BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment. METHODS AND RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20. CONCLUSION: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.

Novel and deleterious nucleotide variations in the HAND1 gene probably affect miRNA target sites and protein function in pediatric patients with congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the most prevalent developmental defect and principal cause of infant mortality and affects cardiac and large blood vessel structures in approximately 1% of live births worldwide. To date, numerous studies have related critical genetic dysfunctions to the pathogenesis of CHDs. However, the genetic basis underlying CHD remains largely unknown. In the present study, we investigated the association of nucleotide variations in coding and noncoding regions of the HAND1 gene with the risk of CHD. The HAND1 gene, encoding a helix-loop-helix transcription factor, is particularly relevant for mechanisms underlying CHD since it plays a significant role in heart development. METHODS AND RESULTS: The genomic DNA of 150 unrelated pediatric patients with CHD was screened by PCR-SSCP and direct sequencing. Four novel and heterozygous missense mutations were identified in the first exon, with three causing amino acid substitutions (p.Val149Met, p.Tyr142His, and p.Leu146Met). In-silico analysis also indicated their deleterious impact on protein structure and function. In addition, we identified five novel nucleotide variants in the 3'UTR region (c.*461, c.*342, c.*529, c.*448, c.*593), potentially altering the target sites of miRNAs. These changes include the loss of certain target sites and the acquisition of new ones. CONCLUSIONS: These findings confirm the phenotypic association between CHDs and HAND1 mutations and can pave the way for developing new preventive and therapeutic strategies.

Mutational screening through comprehensive bioinformatics analysis to detect novel germline mutations in the APC gene in patients with familial adenomatous polyposis (FAP).

BACKGROUND: Familial adenomatous polyposis (FAP) as a colon cancer predisposition syndrome is an autosomal-dominant inherited condition and is diagnosed by the progress of hundreds or thousands of adenomatous colonic polyps in the colon. This study aims at the nature and effect of Adenomatous Polyposis Coli (APC) gene mutations in FAP tumorigenesis. METHODS: The genetic screening of 59 FAP Iranian patients in 10 families was performed by polymerase chain reactions and the direct sequencing of the entire coding exons of the APC gene. To do linkage haplotype analysis and multiplex PCR-based microsatellite examination, six short tandem repeat loci were selected in this gene. To evaluate and predict the potentially deleterious effects, comprehensive bioinformatics pathogenicity assays were used. RESULTS: A total of 12 germline heterozygous and homozygous nucleotide variations were identified. They included two missense mutations, four nonsense mutations, which would lead to the truncated and nonfunctional protein products, four synonymous or silent variations, and two nucleotide deletions of 1 to 5 bp or frameshift mutations. In addition, three novel heterozygous nonsense mutations were found in exons 10, 14, and 15 of the gene. There was also p.Arg653Met as a novel heterozygote mutation in exon 14 of the gene. CONCLUSIONS: Bioinformatics analysis and three-dimensional structural modeling predicted that these missense and nonsense mutations generally are associated with the deleted or truncated domains of APC and have functional importance and mainly affected the APC protein. These findings may provide evidence for the progress of potential biomarkers and help to understand the role of the APC gene in FAP.

Novel Point Mutations in the NKX2.5 Gene in Pediatric Patients with Non-Familial Congenital Heart Disease.

Background and objective: Congenital heart disease (CHD) is the most common birth abnormality in the structure or function of the heart that affects approximately 1% of all newborns. Despite its prevalence and clinical importance, the etiology of CHD remains mainly unknown. Somatic and germline mutations in cardiac specific transcription factor genes have been identified as the factors responsible for various forms of CHD, particularly ventricular septal defects (VSDs), tetralogy of Fallot (TOF), and atrial septal defects (ASDs). p. NKX2.5 is a homeodomain protein that controls many of the physiological processes in cardiac development including specification and proliferation of cardiac precursors. The aim of our study was to evaluate the NKX2.5 gene mutations in sporadic pediatric patients with clinical diagnosis of congenital heart malformations. Materials and methods: In this study, we investigated mutations of the NKX2.5 gene's coding region in 105 Iranian pediatric patients with non-familial CHD by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) and direct sequencing. Results: We observed a total of four mutations, of which, two were novel DNA sequence variants in the coding region of exon 1 (c. 95 A > T and c. 93 A > T) and two others were previously reported as single-nucleotide polymorphisms (SNPs), namely rs72554028 (c. 2357 G > A) and rs3729753 (c. 606 G > C) in exon 2. Further, observed mutations are completely absent in normal healthy individuals (n = 92). Conclusion: These results suggest that NKX2.5 mutations are highly rare in CHD patients. However, in silico analysis proves that c.95 A > T missense mutation in NKX2.5 gene is probably pathogenic and may be contributing to the risk of sporadic CHD in the Iranian population.

Synthesis, structure characterization, DNA binding, and cleavage properties of mononuclear and tetranuclear cluster of copper(II) complexes.

Two copper(II) complexes, cluster 1, and mononuclear 2, have been synthesized by reacting acetylacetone and benzohydrazide (1:1 ratio for 1 and 1:2 ratio for 2) with CuCl(2) in a methanol solution. In 2, which is a new complex, the ligand acts as a tetradentate which binds the metal ion via two amide-O atoms and two imine-N atoms providing an N(2)O(2) square-planar around the copper(II) ion. The absorption spectra data evidence strongly suggested that the two copper(II) compounds could interact with CT-DNA (intrinsic binding constant, K(b) = 0.45×10(4) M-1 for 1 and K(b) = 2.39×10(4) M-1 for 2). The super coiled plasmid pBR322 DNA cleavage ability was studied with 1 and 2 in the presence and absence of H(2)O(2) as an oxidant. In both the absence and the presence of an oxidizing agent, complex 2 exhibited no nuclease activity. However, even in the absence of an oxidant, complex 1 exhibited significant DNA cleavage activity.

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: application in human genomic sample.

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome.

Design and Production of a Novel Anti-PD-1 Nanobody by CDR Grafting and Site-Directed Mutagenesis Approach.

Programmed cell death protein-1 (PD-1) is a membrane protein expressed on the surface of activated T-cells, B-cells, natural killer cells, dendritic cells, macrophages, and monocytes. Inhibition of the PD-1/PD-L1 interaction by monoclonal antibodies (mAbs) has many therapeutic benefits and has led to a major advance in the treatment of various types of tumors. Due to the large size and immunogenicity of the antibodies (Abs), using small molecules such as nanobodies (nanobodies or VHH) is more appropriate for this purpose. In this research, the complementarity determining regions (CDR) grafting method was used to produce anti-PD-1 nanobody. For producing the grafted anti-PD-1 nanobody, CDRs from the tislelizumab mAb were grafted into the frameworks of a nanobody whose sequence is similar to the tislelizumab mAb. Also, the site-directed mutagenesis method was used to produce two mutated anti-PD-1 nanobodies which increased the affinity of grafted anti-PD-1 nanobodies. Two amino acid substitutions (Tyr97Arg and Tyr102Arg) in the VHH-CDR3 were used to improve grafted nanobody affinity and the binding capacity of the mutated nanobodies. The binding of the anti-PD-1 nanobodies and PD-1 antigen (Ag) was confirmed by Dot blot, western blot, and indirect ELISA analysis. According to the results of these in silico and in vitro studies, the binding between grafted and mutated nanobodies with PD-1 was confirmed. Also, our findings show that site-directed mutagenesis can increase the affinity of nanobodies.

Novel nucleotide variations in the thrombomodulin (THBD) gene involved in coagulation pathways can increase the risk of recurrent pregnancy loss (RPL).

Recurrent pregnancy loss (RPL) is a common but complex complication in fertility conditions, affecting about 15-20% of couples. Although several causes have been proposed for RPL, it occurs in about 35-60% of cases without a known explanation. A strong assumption is that genetic factors play a role in the etiology and pathophysiology of PRL. Therefore, several genes are proposed as candidates in the pathogenesis of RPL. The current study aimed to investigate the effects of nucleotide changes in the THBD (thrombomodulin) gene as an RPL-related candidate gene. This gene encodes a cell receptor for thrombin and is involved in reproductive loss in RPL cases. Its involvement in the natural anticoagulant system has been extensively studied. By genetic screening of the entire coding and noncoding regions of the THBD gene, we found twenty-seven heterozygous and homozygous nucleotide changes. Ten of them led to amino acid substitutions, seven variants were identified in the promoter region, and eight of them occurred in 3'UTR. Potentially, the pathogenicity effects of these variations on THBD protein were evaluated by several prediction tools. The numerous genomic variations prompted noticeable modifications of the protein's structural and functional properties. Furthermore, in-silico scores were consistent with deleterious effects for these mutations. The results of this study provide genetic information that will be useful in the future for clinicians, scientists, and students to understand the unknown causes of RPL better. It may also pave the way for developing diagnostic/prognostic approaches to help treat PRL patients.

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle.

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH-ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.

Systemic lupus erythematosus: From non-coding RNAs to exosomal non-coding RNAs.

Systemic lupus erythematosus (SLE), as an immunological illness, frequently impacts young females. Both vulnerabilities to SLE and the course of the illness's clinical symptoms have been demonstrated to be affected by individual differences in non-coding RNA expression. Many non-coding RNAs (ncRNAs) are out of whack in patients with SLE. Because of the dysregulation of several ncRNAs in peripheral blood of patients suffering from SLE, these ncRNAs to be showed valuable as biomarkers for medication response, diagnosis, and activity. NcRNAs have also been demonstrated to influence immune cell activity and apoptosis. Altogether, these facts highlight the need of investigating the roles of both families of ncRNAs in the progress of SLE. Being aware of the significance of these transcripts perhaps elucidates the molecular pathogenesis of SLE and could open up promising avenues to create tailored treatments during this condition. In this review we summarized various non-coding RNAs and Exosomal non-coding RNAs in SLE.

Digestion of restriction enzyme for the detection of single-base mismatch in DNA.

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.

A nonsense mutation in MME gene associates with autosomal recessive late-onset Charcot-Marie-Tooth disease.

BACKGROUND: The genetic cause for the majority of patients with late-onset axonal form of neuropathies have remained unknown. In this study we aimed to identify the causal mutation in a family with multiple affected individuals manifesting a range of phenotypic features consistent with late-onset sensorimotor axonal polyneuropathy. METHODS: Whole exome sequencing (WES) followed by targeted variant screening and prioritization was performed to identify the candidate mutation. The co-segregation of the mutation with the phenotype was confirmed by Sanger sequencing. RESULTS: We identified a nonsense mutation (c.1564C>T; p.Q522*) in membrane metalloendopeptidase (MME) gene as the cause of the disease condition. The mutation has a combined annotation- dependent depletion (CADD) score 45 and predicted to be deleterious based on various algorithms. The mutation was inherited in an autosomal recessive mode and further confirmed to co-segregate with the disease phenotype in the family and showed to has the required criteria including rarity and deleteriousness to be considered as pathogenic. CONCLUSION: The MME gene encodes for the membrane bound endopeptidase neprilysin (NEP) which is involved in processing of various peptide substrates. The identified mutation causes a complete loss of carboxy-terminal region of the NEP protein which contains the zinc binding site and the catalytic domain and thus considered to be a loss-of-function mutation. The loss of NEP activity is likely associated with impaired myelination and axonal injury which is hallmark of CMT diseases.

Mutational and bioinformatics analysis of the NKX2.1 gene in a cohort of Iranian pediatric patients with congenital hypothyroidism (CH).

Congenital hypothyroidism (CH) occurs with a relatively alarming prevalence in infants, and if not diagnosed and treated in time, it can have devastating consequences for the development of the nervous system. CH is associated with genetic changes in several genes that encode transcription factors responsible for thyroid development, including mutations in the NK2 homeobox 1 (NKX2.1) gene, which encodes the thyroid transcription factor-1 (TTF-1). Although CH is frequently observed in pediatric populations, there is still a limited understanding of the genetic factors and molecular mechanisms contributing to this disease. The sequence of the NKX2.1 gene was investigated in 75 pediatric patients with CH by polymerase chain reaction (PCR), single-stranded conformation polymorphism (SSCP), and direct DNA sequencing. Four missense heterozygous variations were identified in exon 3 of the NKX2.1 gene, including three novel missense variations, namely c.708A>G, p.Gln202Arg; c.713T>G, p.Tyr204Asp; c.833T>G, p.Tyr244Asp, and a previously reported variant rs781133468 (c.772C>G, p.His223Gln). Importantly, these variations occur in highly conserved residues of the TTF-1 DNA-binding domain and were predicted by bioinformatics analysis to alter the protein structure, with a probable alteration in the protein function. These results indicate that nucleotide changes in the NKX2.1 gene may contribute to CH pathogenesis.

Mitochondrial mutations in protein coding genes of respiratory chain including complexes IV, V, and mt-tRNA genes are associated risk factors for congenital heart disease.

Most studies aiming at unraveling the molecular events associated with cardiac congenital heart disease (CHD) have focused on the effect of mutations occurring in the nuclear genome. In recent years, a significant role has been attributed to mitochondria for correct heart development and maturation of cardiomyocytes. Moreover, numerous heart defects have been associated with nucleotide variations occurring in the mitochondrial genome, affecting mitochondrial functions and cardiac energy metabolism, including genes encoding for subunits of respiratory chain complexes. Therefore, mutations in the mitochondrial genome may be a major cause of heart disease, including CHD, and their identification and characterization can shed light on pathological mechanisms occurring during heart development. Here, we have analyzed mitochondrial genetic variants in previously reported mutational genome hotspots and the flanking regions of mt-ND1, mt-ND2, mt-COXI, mt-COXII, mt-ATPase8, mt-ATPase6, mt-COXIII, and mt-tRNAs (Ile, Gln, Met, Trp, Ala, Asn, Cys, Tyr, Ser, Asp, and Lys) encoding genes by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) in 200 patients with CHD, undergoing cardiac surgery. A total of 23 mitochondrial variations (5 missense mutations, 8 synonymous variations, and 10 nucleotide changes in tRNA encoding genes) were identified and included 16 novel variants. Additionally, we showed that intracellular ATP was significantly reduced (P=0.002) in CHD patients compared with healthy controls, suggesting that the mutations have an impact on mitochondrial energy production. Functional and structural alterations caused by the mitochondrial nucleotide variations in the gene products were studied in-silico and predicted to convey a predisposing risk factor for CHD. Further studies are necessary to better understand the mechanisms by which the alterations identified in the present study contribute to the development of CHD in patients.

Novel Point Mutations in 3'-Untranslated Region of GATA4 Gene Are Associated with Sporadic Non-syndromic Atrial and Ventricular Septal Defects.

OBJECTIVE: Transcription factor GATA4 has significant roles in embryonic heart development. Mutations of GATA4 appear to be responsible for a wide variety of congenital heart defects (CHD). Despite the high prevalence of GATA4 mutations in CHD phenotypes, extensive studies have not been performed. The 3'-untranslated region (3'-UTR) of the GATA4 gene comprises regulatory motifs and microRNA binding sites that are critical for the appropriate gene expression, nuclear transportation, and regulation of translation, and stability of mRNA. This study aimed to evaluate the association between mutations in the 3'-UTR of the GATA4 gene and CHD risk among Iranian patients. METHODS: We analyzed the coding region of exon 6 and the whole 3'-UTR of GATA4 in DNA isolated from 175 blood samples of CHD patients and 115 unrelated healthy individuals. The functional importance of the observed GATA4 mutations was evaluated using a variety of bioinformatics algorithms for assessment of nonsynonymous mutations and those observed in miRNA binding sites of 3'-UTR. RESULTS: Twenty-one point mutations including one missense mutation (c.511A>G: p.Ser377Gly) in exon 6 and 20 nucleotide variations in 3'-UTR of GATA4 gene were identified in 65 of the 175 CHD patients. In our patients, we identified 12 novel sequence alterations and 8 single nucleotide polymorphisms in the 3'-UTR of GATA4. Most of them had statistically significant differences between CHD patients and controls. CONCLUSION: Our results suggest that 3'-UTR variations of the GATA4 gene probably change microRNA binding sites and present an additional molecular risk factor for the susceptibility of CHD.

Carbamylated erythropoietin increases frataxin independent from the erythropoietin receptor.

BACKGROUND: Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the mitochondrial protein frataxin. Recently we showed in a clinical pilot study in Friedreich's ataxia patients that recombinant human erythropoietin (rhuEPO) significantly increases frataxin-expression. In this in vitro study, we investigated the role of the erythropoietin receptor (EPO-R) in the frataxin increasing effect of rhuEPO and if nonerythropoietic carbamylated erythropoietin (CEPO), which cannot bind to the classical EPO-R increases frataxin expression. MATERIALS AND METHODS: In our experiments human erythroleukaemic K562 cells (+ EPO-R), human monocytic leukemia THP-1 cells (- EPO-R) and isolated primary lymphocytes from healthy control and FRDA patients were incubated with different concentrations of rhuEPO or CEPO. Frataxin-expression was detected by an electrochemical luminescence immunoassay (based on the principle of an ELISA). RESULTS: We show that rhuEPO increases frataxin-expression in K562 cells (expressing EPO-R) as well as in THP-1 cells (without EPO-R expression). These results were confirmed by the finding that CEPO, which cannot bind to the classical EPO-R increased frataxin expression in the same concentration range as rhuEPO. In addition, we show that both EPO derivatives significantly increase frataxin-expression in vitro in control and Friedreich's ataxia patients primary lymphocytes. CONCLUSION: Our results provide a scientific basis for further studies examining the effectiveness of nonerythropoietic derivatives of erythropoietin for the treatment of Friedreich's ataxia patients.

Relationship of hypomethylation CpG islands in interleukin-6 gene promoter with IL-6 mRNA levels in patients with coronary atherosclerosis.

Introduction: Atherosclerosis is the important cause of most cardiovascular diseases, with high prevalence and mortality. Atherosclerosis is not only a lipid metabolism disorder but also recently is defined as a chronic inflammatory disease. Several studies showed that interleukin-6 (IL-6) is involved in the pathogenesis of atherosclerosis. The aim of the present study is the examination of IL6 mRNA Levels and hypomethylation of IL6 promoter in atherosclerosis patients. Methods: In this assay, a total of 35 cases with atherosclerosis and 30 controls were enrolled. RNA and DNA were isolated from the peripheral blood of all samples. Mean IL6 gene expression was determined by RT-PCR and methylation status at six CpG motifs in IL6 promoter was determined using bisulfite genomic sequencing. Results: Real Time-PCR analysis results showed the mean IL6 RNA level in atherosclerosis patients candidate for CABG (coronary artery bypass grafting) was significantly higher than controls (P value = 0.01). Also, the upstream CpG motifs (-1038 to -952) in IL6 promoter were predominantly unmethylated in patients than in the controls (P value = 0.01). Conclusion: These findings suggest that an increase in IL-6 gene expression and its DNA hypomethylation promoter are associated with atherosclerosis patient's candidate for CABG surgery.

Novel Point Mutations of CITED2 Gene Are Associated with Non-familial Congenital Heart Disease (CHD) in Sporadic Pediatric Patients.

CITED2 is a cardiac transcription factor that plays a critical role in cardiac development. Gene mutations in CITED2 lead to a series of cardiac malformations and congenital heart defects (CHD). Congenital heart disease generally refers to defects in the heart's structure or function and often seen in many forms such as ventricular septal defects (VSDs), atrial septal defects (ASDs), and tetralogy of Fallot (TOF). However, the mechanisms involved in these mutations are poorly understood. The aim of the present study was to evaluate the mutations of the CITED2 gene in pediatric patients with congenital heart defects. We studied the potential impact of sequence variations of the CITED2 gene in a cohort of 150 patients with non-familial CHD and 98 control individuals by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and subsequently direct sequencing. We identified seven novel CITED2 nucleotide changes. Four of these alterations were found in the coding region (c.716insG, c.389A>G, c.450G>C and c.512-538del27) and were only seen in our patients, and not detected in the control group. These mutations are leading to changes in the amino acid sequence in the position of p.Gly236fs, p.Asn125Ser, p.Gln145His, and p.Ser170-Gly178del, respectively. Other variations are located in the 5'UTR region of the gene (c.-43C>T, c.-64C>T and c.-90A>G). CITED2 gene mutations in control subjects were not observed. Our Bioinformatics assay results showed that these novel mutations alter the RNA folding, protein structure, and, therefore, probable effect on the protein function and may play a significant role in the development of congenital heart diseases.

Association of Pathogenic Missense and Nonsense Mutations in Mitochondrial COII Gene with Familial Adenomatous Polyposis (FAP).

Nuclear genetic mutations have been extensively investigated in solid tumors. However, the role of the mitochondrial genome remains uncertain. Since the metabolism of solid tumors is associated with aerobic glycolysis and high lactate production, tumors may have mitochondrial dysfunctions. Familial adenomatous polyposis (FAP) is a rare form| of colorectal cancer and an autosomal dominant inherited condition that is characterized by the progress of numerous adenomatous polyps in the rectum and colon. The present study aimed at understanding the nature and effect of mitochondrial cytochrome c oxidase subunit 2 (COII) gene mutations in FAP tumorigenesis. Fifty-six (26 familial and 30 sporadic) FAP patients and 60 normal controls were enrolled in this study. COII point mutations were evaluated by PCR and direct sequencing methods, and a total of 7 mtDNA mutations were detected (3 missense, 1 nonsense, and 3 synonymous variations). Novel non-synonymous COII gene mutations were mostly in heteroplasmic state. These mutations change amino acid residues in the N-terminal and C-terminal regions of COXII. Bioinformatics analysis and three-dimensional structural modeling predicted that these missense and nonsense mutations have functional importance, and mainly affected on cytochrome c oxidase (complex IV). Also, FAP patients carried a meaningfully higher prevalence of mutations in the COII gene in comparison with healthy controls (P <0.001). Analysis of cancer-associated mtDNA mutation could be an invaluable tool for molecular assessment of FAP so that these findings can be helpful for the development of potential new biomarkers in the diagnosis of cancer for future clinical assessments.

Novel Point Mutations in Mitochondrial MT-CO2 Gene May Be Risk Factors for Coronary Artery Disease.

A wide range of genetic and environmental interactions are involved in the development of coronary artery disease (CAD). Considerable evidence suggests that mitochondrial DNA mutations are associated with heart failure. In this work, we examined the possible mutations in hotspot mitochondrial genes and their association with Iranian patients with coronary artery disease. In this case-control study, nucleotide variations were investigated in 109 patients with coronary atherosclerosis and 105 control subjects with no family history of cardiovascular disease. The molecular analysis of related mitochondrial genes was performed by polymerase chain reaction sequencing. Our results showed 25 nucleotide variations (10 missense mutations, 9 synonymous polymorphisms, and 6 variants in tRNA genes) that for the first time were presented in coronary artery disease. Our results suggest that novel heteroplasmic m.8231 C>A mutation is involved in CAD (p = 0.007). These nucleotide variations suggest the role of mitochondrial mutations as a predisposing factor which in combination with environmental risk factors may affect the pathogenesis of coronary atherosclerosis. So, further investigation is needed for a better understanding of the pathogenesis and predisposing effects of these variations on the disease.