Prevalence of Chromosomal Abnormalities in Iranian Patients with Infertility.

BACKGROUND: The numerical and structural abnormalities of chromosomes are the most common cause of infertility. Here, we evaluated the prevalence and types of chromosomal abnormalities in Iranian infertile patients. METHODS: We enrolled 1750 couples of reproductive age with infertility, who referred to infertility clinics in Tehran during 2014- 2019, in order to perform chromosomal analysis. Peripheral blood samples were obtained from all couples and chromosomal abnormalities were evaluated by G-banded metaphase karyotyping. In some cases, the detected abnormalities were confirmed using fluorescence in-situ hybridization (FISH). RESULTS: We detected various chromosomal abnormalities in 114/3500 (3.257%) patients with infertility. The prevalence of chromosomal abnormalities was 44/114 (38.596%) among infertile females and 70/114 (61.403%) among infertile males. Structural chromosomal abnormalities were found in 27/1750 infertile females and 35/1750 infertile males. Numerical chromosomal abnormalities were found in 17/1750 of females and 35/1750 of males. The 45, XY, rob (13;14) (p10q10) translocation and Klinefelter syndrome (47, XXY) were the most common structural and numerical chromosomal abnormalities in the Iranian infertile patients, respectively. CONCLUSION: In general, we found a high prevalence of chromosomal abnormalities in Iranian patients with reproductive problems. Our study highlights the importance of cytogenetic studies in infertile patients before starting infertility treatments approaches.

Identification of a novel homozygous mutation in the DDR2 gene from a patient with spondylo-meta-epiphyseal dysplasia by whole exome sequencing.

OBJECTIVES: The spondylo-meta-epiphyseal dysplasia (SMED) short limbs-hand type is a rare autosomal recessive disease, which is characterized by premature calcification leading to severe disproportionate short stature and various skeletal changes. Defective function of a conserved region encoding discoidin domain receptor tyrosine kinase 2 (DDR2 protein) by the discoidin domain-containing receptor 2 (DDR2 gene) is cause of this disease. The purpose of present study was to investigate disease-causing mutations on DDR2 gene in an Iranian family with SMED, and predict the DDR2 protein molecular mechanism in development of SMED. MATERIALS AND METHODS: In the present study, we evaluated a 2-year-old male with SMED. Detection of genetic changes in the studied patient was performed using Whole-Exome Sequencing (WES). PCR direct sequencing was performed for analysis of co-segregation of variants with the disease in family. Finally, in silico study was performed for further identification of molecular function of the identified genetic variant. RESULTS: We detected a novel splice-site mutation (NM_001014796: exon9: c.855+1G>A; NM_006182: exon8: c.855+1G>A) in DDR2 gene of the studied patient using WES. This mutation was exclusively detected in patients with homozygous SMED, not in healthy people. The effects of detected mutation on functions of DDR2 protein was predicted using in silico study. CONCLUSION: The causative mutation in studied patient with SMED was identified using Next-generation sequencing (NGS), successfully. The identified novel mutation in DDR2 gene can be useful in prenatal diagnosis (PND) of SMED, preimplantation genetic diagnosis (PGD), and genetic counseling.

Identification of Two Novel Mutations in PKHD1 Gene from Two Families with Polycystic Kidney Disease by Whole Exome Sequencing.

BACKGROUND: Polycystic kidney disease (PKD) is an autosomal recessive disorder resulting from mutations in the PKHD1 gene on chromosome 6 (6p12), a large gene spanning 470 kb of genomic DNA. OBJECTIVE: The aim of the present study was to report newly identified mutations in the PKHD1 gene in two Iranian families with PKD. MATERIALS AND METHODS: Genetic alterations of a 3-month-old boy and a 27-year-old girl with PKD were evaluated using whole-exome sequencing. The PCR direct sequencing was performed to analyse the co-segregation of the variants with the disease in the family. Finally, the molecular function of the identified novel mutations was evaluated by in silico study. RESULTS: In the 3 month-old boy, a novel homozygous frameshift mutation was detected in the PKHD1 gene, which can cause PKD. Moreover, we identified three novel heterozygous missense mutations in ATIC, VPS13B, and TP53RK genes. In the 27-year-old woman, with two recurrent abortions history and two infant mortalities at early weeks due to metabolic and/or renal disease, we detected a novel missense mutation on PKHD1 gene and a novel mutation in ETFDH gene. CONCLUSION: In general, we have identified two novel mutations in the PKHD1 gene. These molecular findings can help accurately correlate genotype and phenotype in families with such disease in order to reduce patient births through preoperative genetic diagnosis or better management of disorders.

Association of a novel homozygous mutation in the HMGCS2 gene with an HMGCSD in an Iranian patient.

BACKGROUND: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 gene (HMGCS2) encodes a mitochondrial enzyme catalyzing the first reaction of ketogenesis metabolic pathway which provides lipid-derived energy for various organs during times of carbohydrate deprivation, such as fasting. Mutations in this gene are responsible for HMG-CoA synthase deficiency (HMGCSD). The aim of present study was to investigate the association of mutation in the HMGCS2 gene with HMGCSD in a patient with atypical symptoms. METHODS: The clinical and genetic features of an 8-months-old girl with HMGCSD were evaluated. Molecular genetic testing was conducted using whole-exome sequencing (WES) in order to identify potential disease-causing mutation. The WES finding was confirmed by the polymerase chain reaction (PCR) amplification of the target sequence carried out for the patient and her parents. The PCR products were subjected to direct sequencing using forward and reverse specific primers corresponding to the HMGCS2 gene. RESULTS: A novel homozygous missense mutation (c.266G>A p.Gly89Asp) was detected in the HMGCS2 gene. Sanger sequencing along with co-segregation analysis of all family members confirmed this novel pathogenic germline mutation. The mutant gene was found to be pathogenic by bioinformatics analysis. CONCLUSION: To our best knowledge, this is the first report of HMGCSD in Iran which would expand our knowledge about the mutational spectrum of the HMGCS2 gene and the phenotype variations of the disease.

Identification of Two Novel Mutations in the ATM Gene from Patients with Ataxia-Telangiectasia by Whole Exome Sequencing.

BACKGROUND: Ataxia telangiectasia (AT) is one of the most common autosomal recessive hereditary ataxia presenting in childhood. The responsible gene for AT designated ATM (AT, mutated) encodes a protein which is involved in cell cycle checkpoints and other responses to genotoxicity. We describe two novel disease-causing mutations in two unrelated Iranian families with Ataxia-telangiectasia. METHODS: The probands including a 6-year-old female and an 18-year-old boy were diagnosed with Ataxia-telangiectasia among two different Iranian families. In this study, Whole-Exome Sequencing (WES) was employed for the detection of genetic changes in probands. The analysis of the co-segregation of the variants with the disease in families was conducted using PCR direct sequencing. RESULTS: Two novel frameshift mutations, (c.4236_4236del p. Pro1412fs) and (c.8907T>G p. Tyr2969Ter) in the ataxia telangiectasia mutated ATM gene were detected using Whole-Exome Sequencing (WES) in the probands. These mutations were observed in two separate A-T families. CONCLUSION: Next-generation sequencing successfully identified the causative mutation in families with ataxia-telangiectasia. These novel mutations in the ATM gene reported in the present study could assist genetic counseling, Preimplantation Genetic Diagnosis (PGD) and prenatal diagnosis (PND) of AT.

In vivo identification of novel TGIF2LX target genes in colorectal adenocarcinoma using the cDNA-AFLP method.

BACKGROUND AND STUDY AIMS: Homeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene(s) of TGIF2LX in colorectal adenocarcinoma. METHODS: A human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice (n = 6 per group). The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. RESULTS: The real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 (Nir1) gene was suppressed whereas Nir2 and fragile histidine triad (FHIT) genes were upregulated followed by the overexpression of TGIF2LX gene. CONCLUSION: Downregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma.

Transforming Growth Factor Beta-Induced Factor 2-Linked X (TGIF2LX) Regulates Two Morphogenesis Genes, Nir1 and Nir2 in Human Colorectal.

A member of homeodomain protein namely TGIF2LX has been implicated as a tumor suppressor gene in human malignancy as well as in spermatogenesis. However, to our knowledge, dynamic functional evidence of the TGIF2LX has not yet been provided. The aim of the present study was to investigate the human TGIF2LX target gene(s) using a cDNA-AFLP as a differential display method. A pEGFP-TGIF2LX construct containing the wild-type TGIF2LX cDNA was stably transfected into SW48 cells. UV microscopic analysis and Real-time RT-PCR were used to confirm TGIF2LX expression. The mRNA expressions of TGIF2LX in transfected SW48 cells, the cells containing empty vector (pEGFP-N), and untransfected cells were compared. Also, a Real-time PCR technique was applied to validate cDNA-AFLP results. The results revealed a significant down-regulation and up-regulationby TGIF2LX of Nir1 and Nir2 genes, respectively. The genes are engaged in the cell morphogenesis process. Our findings may provide new insight into the complex molecular pathways underlying colorectal cancer development.

Evaluation of SD-208, a TGF-ß-RI Kinase Inhibitor, as an Anticancer Agent in Retinoblastoma.

Retinoblastoma is the most common intraocular tumor in children resulting from genetic alterations and transformation of mature retinal cells. The objective of this study was to investigate the effects of SD-208, TGF-ß-RI kinase inhibitor, on the expression of some miRNAs including a miR-17/92 cluster in retinoblastoma cells. Prior to initiate this work, the cell proliferation was studied by Methyl Thiazolyl Tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays. Then, the expression patterns of four miRNAs (18a, 20a, 22, and 34a) were investigated in the treated SD-208 (0.0, 1, 2 and 3 µM) and untreated Y-79 cells. A remarkable inhibition of the cell proliferation was found in Y-79 cells treated with SD-208 versus untreated cells. Also, the expression changes were observed in miRNAs 18a, 20a, 22 and 34a in response to SD-208 treatment (P<0.05). The findings of the present study suggest that the anti-cancer effect of SD-208 may be exerted due to the regulation of specific miRNAs, at least in this particular retinoblastoma cell line. To the best of the researchers' knowledge, this is the first report demonstrating that the SD-208 could alter the expression of tumor suppressive miRNAs as well as oncomiRs in vitro. In conclusion, the present data suggest that SD-208 could be an alternative agent in retinoblastoma treatment.

Down-regulation of miR-135b in colon adenocarcinoma induced by a TGF-ß receptor I kinase inhibitor (SD-208).

OBJECTIVES: Transforming growth factor-ß (TGF-ß) is involved in colorectal cancer (CRC). The SD-208 acts as an anti-cancer agent in different malignancies via TGF-ß signaling. This work aims to show the effect of manipulation of TGF-ß signaling on some miRNAs implicated in CRC. MATERIALS AND METHODS: We investigated the effects of SD-208 on SW-48, a colon adenocarcinoma cell line. The cell line was treated with 0.5, 1 and 2 µM concentrations of SD-208. Then, the xenograft model of colon cancer was established by subcutaneous inoculation of SW-48 cell line into the nude mice. The animals were treated with SD-208 for three weeks. A quantitative real-time PCR was carried out for expression level analysis of selected oncogenic (miR-21, 31, 20a and 135b) and suppressor-miRNAs (let7-g, miR-133b, 145 and 200c). Data were analyzed using the 2-∆∆CT method through student's t-test via the GraphPad Prism software. RESULTS: Our results revealed that SD-208 could significantly down-regulate the expression of one key onco-miRNA, miR-135b, in either SW-48 colon cells (P=0.006) or tumors orthotopically implanted in nude mice (P=0.018). Our in silico study also predicted that SD-208 could modulate the expression of potential downstream tumor suppressor targets of the miR135b. CONCLUSION: Our data provide novel evidence that anticancer effects of SD-208 (and likely other TGF-ß inhibitors) may be owing to their ability to regulate miRNAs expression.

Identification and Sequencing of Candida krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis.

BACKGROUND: The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell. OBJECTIVES: The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition. MATERIALS AND METHODS: Candida krusei (ATCC: 6258) aconitase gene was determined by 5'Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares. RESULTS: One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues. CONCLUSIONS: Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.