CYP21A2 Gene Analysis in Southern Iranian CAH Patients and a Brief Review of the Mutation Spectrum.

Background: CYP21A2 gene mutations are responsible for more than 95% of Congenital Adrenal Hyperplasia (CAH) disorders with autosomal recessive inheritance. Most of these pathogenic mutations originate from the CYP21A1P, a neighboring pseudogene with 98% homology, due to unequal crossing over or gene conversion events. Mutation identification of the gene could be beneficial for accurate diagnosis and outcome prediction. Methods: Twelve unrelated patients with CAH diagnosis were recruited for genetic counseling. To ensure distinct amplification of the CYP21A2 gene rather than its pseudogene, the complete sequence of the gene was amplified through two overlapping fragments by specific primers. The entire sequences were screened by direct Sanger sequencing using new sequencing primers. Results: Only two pathogenic point mutations were identified. The c.293-13C>G, also known as In2G, and the c.955C>T mutations were found in 37.5 and 33.3% of alleles, respectively. One patient showed homozygous gene deletion. We also reviewed recent reports on CYP21A2 gene mutations in Iran. Conclusion: Evaluating the ethnicity-specific gene mutation data is significant for populations with diverse ethnic groups including the Iranian population. Although several common mutations have been reported as causative mutations among CAH patients, identifying only two common point mutations in Fars province would help prioritize exon sequencing and reduce the cost and time of genotyping.

Concise review: The heterogenous roles of BATF3 in cancer oncogenesis and dendritic cells and T cells differentiation and function considering the importance of BATF3-dependent dendritic cells.

The transcription factor, known as basic leucine zipper ATF-like 3 (BATF3), is a crucial contributor to the development of conventional type 1 dendritic cells (cDC1), which is definitely required for priming CD8 + T cell-mediated immunity against intracellular pathogens and malignancies. In this respect, BATF3-dependent cDC1 can bring about immunological tolerance, an autoimmune response, graft immunity, and defense against infectious agents such as viruses, microbes, parasites, and fungi. Moreover, the important function of cDC1 in stimulating CD8 + T cells creates an excellent opportunity to develop a highly effective target for vaccination against intracellular pathogens and diseases. BATF3 has been clarified to control the development of CD8α+ and CD103+ DCs. The presence of BATF3-dependent cDC1 in the tumor microenvironment (TME) reinforces immunosurveillance and improves immunotherapy approaches, which can be beneficial for cancer immunotherapy. Additionally, BATF3 acts as a transcriptional inhibitor of Treg development by decreasing the expression of the transcription factor FOXP3. However, when overexpressed in CD8 + T cells, it can enhance their survival and facilitate their transition to a memory state. BATF3 induces Th9 cell differentiation by binding to the IL-9 promoter through a BATF3/IRF4 complex. One of the latest research findings is the oncogenic function of BATF3, which has been approved and illustrated in several biological processes of proliferation and invasion.

siRNA-mediated downregulation of BATF3 diminished proliferation and induced apoptosis through downregulating c-Myc expression in chronic myelogenous leukemia cells.

OBJECTIVE: Despite considerable improvement in therapeutic approaches to chronic myeloid leukemia (CML) treatment, this malignancy is considered incurable due to resistance. However, investigating the molecular mechanism of CML may give rise to the development of extremely efficient targeted therapies that improve the prognosis of patients. Basic leucine zipper transcription factor ATF-like3 (BATF3), as transcription factor, is considered a key regulator of cellular activities and its function has been evaluated in tumor development and growth in several cancer types. This study aimed to evaluate the potential of the cellular impact of siRNA-mediated downregulation of BATF3 on CML cancer cells through cell proliferation, induction of apoptosis, and cell cycle distribution. MATERIALS AND METHODS: The transfection of BATF3 siRNA to K562 CML cells was performed by electroporation device. To measure cellular viability and apoptosis, MTT assay and Annexin V/PI staining were carried out, respectively. Also, cell cycle assay and flow cytometry instrument were applied to assess cell cycle distribution of K562 cells. For more validation, mRNA expression of correlated genes was relatively evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The data indicated that siRNA-mediated BATF3 inactivating severely promoted the cell apoptosis. Also, the targeted therapy led to high expression of Caspase-3 gene and Bax/Bcl-2 ratio. Silenced BATF3 also induced cell cycle arrest in phase sub-G1 compared to control. Finally, a noticeable decrement was obtained in c-Myc gene expression through suppression of BATF3 in CML cells. CONCLUSION: The findings of this research illustrated the suppression of BATF3 as an effective targeted therapy strategy for CML.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions.

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify -α3.7, -α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Regulatory Effects of Long Non-coding RNAs on Th17/Treg Differentiation and Imbalance.

Scientific research over the past decades has proven the pivotal role of long non-coding RNAs (LncRNAs) in regulating gene expression. The immune responses are controlled through the interaction of pro-inflammatory (predominance of T helper 17 cells (Th17)) and anti-inflammatory cytokines excretion (predominance of Regulatory T cells (Treg)). Recent studies have marked the impact of many diverse LncRNAs on Treg/Th17 imbalances. Moreover, some of the roots and causes of human diseases can be associated with the alterations in the Th17/Treg ratio. In this review study, we overviewed the association between LncRNAs and Th17/Treg, with the potential of providing novel prognostic and diagnostic biomarkers and promising therapeutic targets in various diseases, particularly cancer.

Prediction of potential deleterious nonsynonymous single nucleotide polymorphisms of HIF1A gene: A computational approach.

Hypoxia-inducible factor-1α (HIF-1α) is the oxygen sensitive subunit of HIF1 transcription factor. Its variations is associated with several diseases including different type of cancer, cardiovascular diseases, and liver and kidney failure. Despite all the investigations carried out on the single nucleotide polymorphisms (SNPs) of HIF1A gene and diseases, there are many uncharacterized nonsynonymous SNPs of this gene, which might have damaging effect on the protein function. Therefore, it is worthwhile to analyze these potential damaging nsSNPs, using different bioinformatics tools before launching large population studies. The objective of the present study was to predict the possible deleterious nsSNPs of HIF1A gene and their effects on the function and structure of HIF-1alpha protein, using different bioinformatics tools. Various prediction servers were used including SIFT, PROVEAN, PolyPhen-2, PANTHER, phD-SNP, SNP-GO, I-Mutant 2.0, Fathmm, SNPeffect 4.0, Mutation taster, CADD and RAMPAGE in a stepwise approach. After analyzing all 454 missense variants of the HIF1A gene using the abovementioned tools, we reported 11 variants with a significant impact on the function or structure of HIF-1α protein. Furthermore, among these variants only S274 P was predicted as stability enhancing variant with effect on protein function by increasing its stability. Although there are many advantages for computational analysis, the results has to be confirmed by experimental investigations.

CRB1-Related Leber Congenital Amaurosis: Reporting Novel Pathogenic Variants and a Brief Review on Mutations Spectrum

Background: LLeber congenital amaurosis (LCA) is a rare inherited retinal disease causing severe visual impairment in infancy. It has been reported that 9-15% of LCA cases have mutations in CRB1 gene. The complex of CRB1 protein with other associated proteins affects the determination of cell polarity, orientation, and morphogenesis of photoreceptors. Here, we report three novel pathogenic variants in CRB1 gene and then briefly review the types, prevalence, and correlation of reported mutations in CRB1 gene. Methods: Whole exome sequencing and targeted gene panel were employed. Then validation in the patient and segregation analysis in affected and unaffected members was performed. Results: Our detected novel pathogenic variants (p.Glu703*, c.2128+1G>A and p.Ser758SerfsX33) in CRB1 gene were validated by Sanger sequencing. Segregation analysis confirmed the inheritance pattern of the pathogenic variants. Conclusion: Our findings show that emerging the next-generation sequencing-based techniques is very efficient in identifying causative variants in disorders with locus heterogeneity.

Multi affected pedigree with congenital microcephaly: WES revealed PNKP gene mutation.

Microcephaly is a rare neurological disorder, occurs in both isolated and syndromic forms. This classification could be confusing in rare disorders with variable phenotypic characteristics. However, identification of the causative gene through genetic study would allow determining the definite diagnosis. Here we reported a novel missense variant c.1133A>C (p.Lys378Thr) on the 13th exon of PNKP gene identified by whole exome sequencing (WES) in an Iranian multi-affected family with microcephaly, seizures and developmental delay (MCSZ) disorder. Data analysis suggested this variant as a pathogenic mutation which is co-segregate with the disease in the pedigree. PNKP gene mutation is consistent with the clinical features of the affected family members. Regarding both genetic findings and clinical examinations, the reported pedigree can be considered as another affected family with MCSZ syndrome, which has been reported about 10 cases worldwide. This study proves the application of WES for determining the final diagnosis in complicated neurodevelopmental disorders.

Genetic study of the PAH locus in the Iranian population: familial gene mutations and minihaplotypes.

Phenylketonuria (PKU), one of the most common inborn errors of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene (PAH). PKU has wide allelic heterogeneity, and over 600 different disease-causing mutations in PAH have been detected to date. Up to now, there have been no reports on the minihaplotype (VNTR/STR) analysis of PAH locus in the Iranian population. The aims of the present study were to determine PAH mutations and minihaplotypes in Iranian families with PAH deficiency and to investigate the correlation between them. A total of 81 Iranian families with PAH deficiency were examined using PCR-sequencing of all 13 PAH exons and their flanking intron regions to identify sequence variations. Fragment analysis of the PAH minihaplotypes was performed by capillary electrophoresis for 59 families. In our study, 33 different mutations were found accounting for 95% of the total mutant alleles. The majority of these mutations (72%) were distributed across exons 7, 11, 2 and their flanking intronic regions. Mutation c.1066-11G > A was the most common with a frequency of 20.37%. The less frequent mutations, p.Arg261Gln (8%), p.Arg243Ter (7.4%), p.Leu48Ser (7.4%), p.Lys363Asnfs*37 (6.79%), c.969 + 5G > A (6.17%), p.Pro281Leu (5.56), c.168 + 5G > C (5.56), and p.Arg261Ter (4.94) together comprised about 52% of all mutant alleles. In this study, a total of seventeen PAH gene minihaplotypes were detected, six of which associated exclusively with particular mutations. Our findings indicate a broad PAH mutation spectrum in the Iranian population, which is consistent with previous studies reporting a wide range of PAH mutations, most likely due to ethnic heterogeneity. High prevalence of c.1066-11G > A mutation linked to minihaplotype 7/250 among both Iranian and Mediterranean populations is indicative of historical and geographical links between them. Also, strong association between particular mutations and minihaplotypes could be useful for prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) in affected families.

A Novel Variant in the PAH Gene Causing Phenylketonuria in an Iranian Pedigree.

BACKGROUND: Phenylalanine hydroxylase (PAH) gene is the well-known causative gene for classic Phenylketonuria (PKU) (OMIM#261600) disease, with more than 500 reported mutations. Through this study, a novel mutation in the PAH gene in an Iranian pedigree with phenylketonuria was introduced. METHODS: A consanguineous family with a 10-year old affected girl was referred for genetic analysis. Mutation screening of all exons and exon-intron boundaries was performed by Sanger sequencing, and mini haplotype analysis was carried out by genotyping of Short Tandem Repeat (STR) and Variable Number Tandem Repeat (VNTR) alleles. RESULTS: Mutation analysis revealed a novel homozygous insertion of a single adenine nucleotide at position 335 in exon 3 of the PAH gene. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the change is interpreted as a pathogenic mutation which produces a premature termination signal (TAA) at codon 113 according to in silico assessments. The mini haplotype analysis showed that this mutation was linked to STR (15) -VNTR (3). CONCLUSION: In this study, a novel mutation was reported in a patient who had PKU symptoms without any previously reported mutations in the PAH gene (NM_000277.1:p.Asp112Glufs*2) that can be responsible for the classical PKU phenotype in the Iranian population. Detection of novel mutations indicates notable allelic heterogeneity of the PAH locus among this population.

Comparison of Two Different PCR-based Methods for Detection of GAA Expansions in Frataxin Gene.

BACKGROUND: Expansion of GAA trinucleotide repeats is the molecular basis of Friedreich's ataxia (FRDA). Precise detection of the GAA expansion repeat in frataxin gene has always been a challenge. Different molecular methods have been suggested for detection of GAA expansion, including; short-PCR, long-PCR, Triplet repeat primed-PCR (TP-PCR) and southern blotting. The aim of study was to evaluate two PCR-based methods, TP-PCR and long-PCR, and to explore the use of TP-PCR accompanying with long-PCR for accurate genotyping of FRDA patients. METHODS: Blood samples were collected from six Iranian patients suspected to FRDA, who referred to the Department of Medical Genetics at Tehran University of Medical Sciences during the year 2014. For one of these patients' four asymptomatic members of the family were also recruited for the analysis. DNA extraction was performed by two different methods. TP-PCR and long-PCR were carried out in all samples. The type of this study is assessment / investigation of methods. RESULTS: Using a combination of the above methods, the genotypes of all samples were confirmed as five homozygous mutants (expanded GAA repeats), two heterozygous and three homozygous normal (normal repeat size). The results obtained by TP-PCR are consistent with long-PCR results. CONCLUSION: The presence or absence of expanded alleles can be identified correctly by TP-PCR. Performing long-PCR and Fluorescent-long-PCR enables accurate genotyping in all samples. This approach is highly reliable. It could be successfully used for detection of GAA expansion repeats.

An Efficient Trio-Based Mini-Haplotyping Method for Genetic Diagnosis of Phenylketonuria.

OBJECTIVE: The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping. MATERIALS AND METHODS: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N',N'-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software. RESULTS: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001). CONCLUSION: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems.

PKD2 mutation in an Iranian autosomal dominant polycystic kidney disease family with misleading linkage analysis data.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder caused by mutation in 2 genes PKD1 and PKD2. Thus far, no mutation is identified in approximately 10% of ADPKD families, which can suggest further locus heterogeneity. Owing to the complexity of direct mutation detection, linkage analysis can initially identify the responsible gene in appropriate affected families. Here, we evaluated an Iranian ADPKD family apparently unlinked to both PKD1 and PKD2 genes. This is one of the pioneer studies in genetic analysis of ADPKD in Iranian population. METHODS: Linkage reanalysis was performed by regenotyping of flanking microsatellite markers in 8 individuals of the ADPKD family. Direct mutation analysis was performed by Sanger sequencing. RESULTS: Mutation analysis revealed a pathogenic mutation (c.1094+1G>A) in the PKD2 gene in the proband. Analyzing 2 healthy and 4 clinically affected members confirmed the correct segregation of the mutation within the family and also ruled out the disease in 1 suspected individual. Misinterpretation of the linkage data was due to the occurrence of 1 crossing over between the PKD2 intragenic and the nearest downstream marker (D4S2929). Homozygosity of upstream markers caused the recombination indistinguishable. CONCLUSION: Although analysis of additive informative polymorphic markers can overcome the misleading haplotype data, it is limited because of the lack of other highly polymorphic microsatellite markers closer to the gene. Direct mutation screening can identify the causative mutation in the apparently unlinked pedigree; moreover, it is the only approach to achieve the confirmed diagnosis in individuals with equivocal imaging results.

Human herpesvirus-6 viral load and antibody titer in serum samples of patients with multiple sclerosis.

BACKGROUND: Despite the number of cases with definite diagnosis of multiple sclerosis (MS) being on increase, the role of human herpesvirus-6 (HHV-6) infection as a trigger for MS disease still is deliberated. Based on antibody detection and quantitative HHV-6 polymerase chain reaction assay, this study was achieved to find out the possible association between infection with HHV-6 and clinical progression of MS disease. METHODS: A total of 108 serum samples were obtained from 30 MS patients followed prospectively for a 6-month period. These samples were analyzed for the presence of HHV-6 DNA by nested polymerase chain reaction enzyme-linked immunosorbent assay and for anti-HHV-6 IgG titer. Activation of the disease was determined by either magnetic resonance imaging or by clinical status of the patients. Control groups were also included. RESULTS: The average antibody index for the MS patients in the first sample collection was higher than both control groups (p = 0.001). HHV-6 DNA was detected in the serum samples of 10 of 30 MS patients. The mean HHV-6 viral load in patients with relapsing-remitting multiple sclerosis (RRMS) with and without relapse was 973 and 714, respectively. Seven patients showed an exacerbation during the study period. Of those, four patients had HHV-6 DNA in their collected samples. The prevalence of HHV-6 DNA was significantly higher in patients with MS as compared with control groups (p = 0.001). CONCLUSIONS: The results indicate that HHV-6 is implicated somehow in MS disease. Over time, rising HHV-6 IgG antibody titers together with an exacerbation and detection of HHV-6 DNA in serum samples of some MS patients suggests possible association between the reactivation of the virus and disease progression.

Association between HIC1 and RASSF1A promoter hypermethylation with MTHFD1 G1958A polymorphism and clinicopathological features of breast cancer in Iranian patients.

BACKGROUND: Ras-associated domain family 1 (RASSF1A) and hypermethylated in cancer (HIC1) genes are methylated more frequently in breast cancer. Genetic factors that alter the DNA methylation levels in normal and tumor tissues could therefore influence the susceptibility to this tumor phenotype. We determined the frequency of aberrant methylation of HIC1 and RASSF1A gene promoters and their association with methylene tetrahydrofolate dehydrogenase (MTHFD1) G1958A polymorphism and major clinical and pathological features of breast cancer in Iranian women. METHODS: DNA was extracted from 81 primary breast tumors and 100 control blood samples. Gene promoter methylation was analyzed by methylation-specific polymerase chain reaction. RESULTS: Eighty four percent of the breast cancer samples showed total methylation in at least one of two tested loci. We detected HIC1 hypermethylation in 79% of invasive and metastasis tumors and RASSF1A gene hypermethylation in 51% of them. We found no association between HIC1 and RASSF1A gene hypermethylation and MTHFD1 G1958A polymorphism, but a significant correlation between methylation of HIC1 and RASSF1A promoters was indicated (r = 0.24, P = 0.02). There was a combination between hypermethylation of HIC1 locus and nodal involvement in the studied population (p=0.03). We found a significant association between total methylation and nodal involvement (P = 0.01) as well as tumor size more than 2 cm in all cases (P = 0.02). CONCLUSION: Methylation of HIC1 and RASSF1A promoters can be used as epigenetic markers to detect the malignant progression of breast carcinoma in Iranian women patients.