Novel mutation in AIRE gene with autoimmune polyendocrine syndrome type 1.

PURPOSE: Autoimmune polyendocrine type 1 (APS-1) is a complex inherited autosomal recessive disorder. Classically, it appears within the first decade of life followed by adrenocortical insufficiency, mucocutaneous candidiasis, Addison's disease, and hypoparathyroidism. The clinical phenotype of APS-1 varies depending upon mutations in the autoimmune regulator gene (AIRE) on chromosome 21q22.3. METHODS: In this study, we performed Sanger sequencing ofAIRE in Iranian patients to identify different variants and probable new mutations corresponding to a clinical diagnosis of APS-1. RESULTS: After analyzing 14AIRE exons, we detected a novel insertion mutation in exon 2 in a patient who presented with severe APS-1, Lys50AsnfsX168. Furthermore, the known mutations in AIRE, including Arg139X, Arg257X, and Leu323SerfsX51, were detected in enrolled patients. DISCUSSION: According to our results, sequencing analysis ofAIRE provides a useful screening method to diagnose patients with incomplete or unusual clinical presentations of APS-1.

Novel DNA variation of GPR54 gene in familial central precocious puberty.

BACKGROUND: Puberty can be considered the end point of a maturation process which is defined by the dynamic interactions of genes and environmental factors during prenatal and postnatal development. Kisspeptin/G protein-coupled receptor-54, is as an essential gatekeeper and regulator of GnRH neurons, and a key factor in initiation of puberty. Loss and gain of functional mutations in the GPR54 gene are associated with hypogonadotropic hypogonadism and precocious puberty, respectively. This study was designed to evaluate variations of GPR54 in familial precocious puberty. METHODS: Genomic DNA was extracted from peripheral whole blood of 25 subjects with familial precocious puberty. Coding exons 1-5 of the GPR54 gene were amplified by polymerase chain reaction (PCR) and the PCR products were purified and sequenced. DNA sequences were compared to the human GenBank GPR54 sequence using Sequencher sequence alignment software. RESULTS: We detected three different Single Nucleotide Polymorphisms (SNPs) in GPR54: rs10407968 (24A > T) in 13 subjects (52%); rs3050132 (1091 T > A) in 16 subjects (64%), and a novel polymorphism (492C > G) in one subject (4%), while three subjects (12%) had no SNPs. No mutations were found in the GPR54 gene. CONCLUSIONS: Regarding the presence of SNPs in 88% of the subjects in this study, it is likely a relationship exists between the SNPs of the GPR54 gene and familial precocious puberty. Further research is needed to investigate this possibility, and potential functional effects of these polymorphisms.

Mutations in HNF1A Gene are not a Common Cause of Familial Young-Onset Diabetes in Iran.

Mutations in hepatocyte nuclear factor-1 alpha (HNF1A) as a homeodomain transcription factor which regulates variety of genes, are the most common cause of maturity-onset diabetes of the young (MODY). Detection of HNF1A mutations not only classifies the subtype, but also predicts the likely clinical course and may alters the method of treatment from insulin to the oral sulphonylureas, which is shown to improve glycemic control. The coding and promoter regions of HNF1A gene were screened for mutations in 34 unrelated Iranian MODY patients. We identified one novel missense mutation (C49G) and two novel polymorphisms and 8 recently identified SNPs in the HNF1A gene. It is possible that in Iran, other yet to be identified genes are responsible for the familial young onset diabetes. Hence, there is a need for more extensive genetic analyses in Iranian patients with familial young onset diabetes.

Novel mutations and their genotype-phenotype correlations in patients with Noonan syndrome, using next-generation sequencing.

PURPOSE: Noonan Syndrome (NS) is an autosomal dominant disorder with many variable and heterogeneous conditions. The genetic basis for 20-30% of cases is still unknown. This study evaluates Iranian Noonan patients both clinically and genetically for the first time. MATERIALS/METHODS: Mutational analysis of PTPN11 gene was performed in 15 Iranian patients, using PCR and Sanger sequencing at phase one. Then, as phase two, Next Generation Sequencing (NGS) in the form of targeted resequencing was utilized for analysis of exons from other related genes. Homology modelling for the novel founded mutations was performed as well. The genotype, phenotype correlation was done according to the molecular findings and clinical features. RESULTS: Previously reported mutation (p.N308D) in some patients and a novel mutation (p.D155N) in one of the patients were identified in phase one. After applying NGS methods, known and new variants were found in four patients in other genes, including: CBL (p. V904I), KRAS (p. L53W), SOS1 (p. I1302V), and SOS1 (p. R552G). Structural studies of two deduced novel mutations in related genes revealed deficiencies in the mutated proteins. Following genotype, phenotype correlation, a new pattern of the presence of intellectual disability in two patients was registered. CONCLUSIONS: NS shows strong variable expressivity along the high genetic heterogeneity especially in distinct populations and ethnic groups. Also possibly unknown other causative genes may be exist. Obviously, more comprehensive and new technologies like NGS methods are the best choice for detection of molecular defects in patients for genotype, phenotype correlation and disease management.

Loss of heterozygosity and microsatellite instability as predictive markers among Iranian esophageal cancer patients.

OBJECTIVES: Variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer. MATERIALS AND METHODS: DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of heterozygosity (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12.3), D13S267 (13q12.3), D9S171 (9p21), D2S123 (2p), D5S2501 (5q21) and TP53 (17p13.1) analyzed on 6% denaturing polyacrylamide gel electrophoresis. RESULTS: Statistical analysis indicated a near significant reverse correlation between grade and LOH (P= 0.068, correlation coefficient= -0.272). Specifically, increased LOH in tumor DNA has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors (P= 0.002 and P= 0.016 respectively). In addition, higher number of chromosomal loci with LOH showed a reverse correlation with lymph node metastasis (P= 0.026, correlation coefficient= -0.485). Furthermore, there was a positive correlation between addiction and MSI (P= 0.026, correlation coefficient= 0.465). CONCLUSION: Microsatellite DNA alterations may be a prognostic tool for detection and the evolution of prognosis in patients with SCC of esophagus. It can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium, cigarette, water pipe or alcohol can be a susceptibility factor(s) for MSI.

Identification of four novel mutations of the WFS1 gene in Iranian Wolfram syndrome pedigrees.

AIMS: Wolfram syndrome is a rare neurodegenerative disorder with an autosomal recessive pattern of inheritance characterized by various clinical manifestations. The related gene, WFS1, encodes a transmembrane glycoprotein, named wolframin. Genetic analyses demonstrated that mutations in this gene are associated with WS type 1. Our aim in this study was to sequence WFS1 coding region in Iranian Wolfram syndrome pedigrees. METHODS: Genomic DNA was extracted from peripheral blood of 12 WS patients and their healthy parents. Exons 2-8 and the exon-intron junctions of WFS1 were sequenced. DNA sequences were compared to the reference using Sequencher software. RESULTS: Molecular analysis of WFS1 revealed six different mutations. Four novel and two previously reported mutations were identified. One novel mutation, c.1379_1381del, is predicted to produce an aberrant protein. A second novel mutation, c.1384G > T, encodes a truncated protein. Novel mutation, c.1097-1107dup (11 bp), causes a frameshift which results in a premature stop codon. We screened for the novel missense mutation, c.1010C > T, in 100 control alleles. This mutation was not found in any of the healthy controls. CONCLUSION: Our study increased the spectrum of WFS1 mutations and supported the role of WFS1 in susceptibility to WS. We hope that these findings open new horizons to future molecular investigations which may help to prevent and treat this devastating disease.

Neoantigen in esophageal squamous cell carcinoma for dendritic cell-based cancer vaccine development.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant tumor which usually is diagnosed in advanced stages due to its asymptomatic course of tumorigenesis. Current therapeutic modalities are not effective enough and the 5-year survival rate of the disease is still very low which prompts the urgent need for finding novel efficient therapeutic methods. In this study, we evaluated ex vivo immune response of ESCC patients against our newly designed chimeric construct consisting of highly immunogenic cancer-testis antigens. After confirming effective expression of the in vitro transcribed chimeric mRNA in ex vivo electroporated dendritic cells (DCs) of the ESCC patients, the patients' CTLs were primed by DCs and cytotoxicity assay was performed to evaluate how the primed CTLs can recognize and target the chimeric mRNA-loaded cells. The chimeric protein was strongly expressed relative to the housekeeping gene expression in electroporated cells. The cytotoxicity of the CTLs was significantly higher in DCs loaded with chimeric mRNAs compared to mock DCs (p<0.05) in all of the tested ESCC patients. We are introducing a novel construct that our functional study showed can stimulate and induce an effective immune response in ESCC patients. The designed chimeric mRNA-loaded DCs are capable of priming CTLs effectively and induce cytotoxicity against tumor. Therefore, loading DCs with chimeric epitopes of highly immunogenic antigens, such as cancer-testis antigens, are potentially interesting and effective therapeutic modalities for immunotherapy of ESCC.

Genetic properties of blaCTX-M and blaPER ß-lactamase genes in clinical isolates of Enterobacteriaceae by polymerase chain reaction.

OBJECTIVES: blaCTX-M and blaPER are two genes that encode class A extended-spectrum ß-lactamases (ESBLs) and can be responsible for therapeutic problems. This study was carried out to evaluate the molecular properties of these genes in clinical isolates of Enterobacteriaceae by polymerase chain reaction (PCR), restriction digestion and sequencing. MATERIALS AND METHODS: During six months, starting from January 2012, one hundred clinical isolates of Enterobacteriaceae were collected from urinary samples. The ESBL-producing isolates were detected by phenotypic confirmation test. After plasmid extraction, blaPER and blaCTX-M genes were detected using PCR by specific primers. The blaCTX-M PCR products were digested with Taq1, and two of the blaCTX-M genes were sequenced. RESULTS: Phenotypic tests showed that 27 (27%) isolates were ESBL producers with the highest frequency for Klebsiella pneumoniae (47.4%) and Escherichia coli (17.9%). Twenty six (26%) of Enterobacteriaceae isolates harbored the blaCTX-M gene, and none of them had blaPER . The restriction analysis of PCR products showed that all blaCTX-M amplified products had the same patterns. Both sequenced bacteria were CTX-M-15 type ESBL carriers. CONCLUSION: The results of this study showed the blaCTX-M-15 gene in Enterobacteriaceae isolates for the first time in Mashhad, Iran. High degrees of associated resistance to co-trimoxazole and gentamicin were found in ESBL producers. Therefore, an integrated and regular management of antibiotic prescription need to be trained in our society.

Novel mutation in the SLC19A2 gene in an Iranian family with thiamine-responsive megaloblastic anemia: a series of three cases.

Thiamine-responsive megaloblastic anemia (TRMA) is a clinical triad characterized by megaloblastic anemia, non-autoimmune diabetes mellitus, and sensory-neural hearing loss. Mutations in the thiamine transporter gene, solute carrier family 19, member 2 (SLC19A2), have been associated with TRMA. Three pediatric patients from a large consanguineous Iranian family with hyperglycemia, anemia, and hearing loss were clinically diagnosed with TRMA. In all three patients, TRMA was confirmed by direct sequencing of the SLC19A2 gene that revealed a novel missense homozygous mutation c.382 G>A (p.E128K). This mutation results in the substitution of glutamic acid to lysine at position 128 in exon 2 and was not detected in 200 control chromosomes. Thiamine therapy reversed the anemia and alleviated the hyperglycemia in all three patients. We recommend sequence analysis of the SLC19A2 gene in individuals with a clinical triad of diabetes mellitus, hearing loss, and anemia. The administration of thiamine ameliorates the megaloblastic anemia and the hyperglycemia in patients with TRMA.