Towards solving the genetic diagnosis odyssey in Iranian patients with congenital anomalies.

Understanding the underlying causes of congenital anomalies (CAs) can be a complex diagnostic journey. We aimed to assess the efficiency of exome sequencing (ES) and chromosomal microarray analysis (CMA) in patients with CAs among a population with a high fraction of consanguineous marriage. Depending on the patient's symptoms and family history, karyotype/Quantitative Fluorescence- Polymerase Chain Reaction (QF-PCR) (n = 84), CMA (n = 81), ES (n = 79) or combined CMA and ES (n = 24) were performed on 168 probands (66 prenatal and 102 postnatal) with CAs. Twelve (14.28%) probands were diagnosed by karyotype/QF-PCR and seven (8.64%) others were diagnosed by CMA. ES findings were conclusive in 39 (49.36%) families, and 61.90% of them were novel variants. Also, 64.28% of these variants were identified in genes that follow recessive inheritance in CAs. The diagnostic rate (DR) of ES was significantly higher than that of CMA in children from consanguineous families (P = 0·0001). The highest DR by CMA was obtained in the non-consanguineous postnatal subgroup and by ES in the consanguineous prenatal subgroup. In a population that is highly consanguineous, our results suggest that ES may have a higher diagnostic yield than CMA and should be considered as the first-tier test in the evaluation of patients with congenital anomalies.

TERT Promoter Mutation in Benign and Malignant Salivary Gland Tumors; A Cross-Sectional Study.

Background & Objective: Telomere-related tumorigenesis mechanisms in the salivary gland, including mutation in the promoter region of TERT, have been rarely investigated. Therefore, the present study aimed to investigate the mutation in the promoter region of TERT in benign and malignant salivary gland tumors. Methods: This was a descriptive-analytical cross-sectional study. Tissue samples of 54 patients with primary salivary gland tumors sent to the pathology department of Rasool-e-Akram Hospital from September 2017 to September 2021 were examined. Fifteen samples including two groups of the most common benign tumors (n=5; 3 pleomorphic adenomas and 2 Warthin tumors) and four groups of the most common malignant tumors (n=10; 3 mucoepidermoid carcinomas, 3 adenoid cystic carcinomas, 2 acinic cell carcinoma, and 2 salivary duct carcinoma) were selected. The promoter region of TERT, including well-known hot spot regions, is sequenced using the Sanger sequencing method. Data were analyzed using statistical software R version 4.1.2. Results: Of 15 salivary gland tumor specimens, consisting of 5 benign tumors and 10 malignant tumors after DNA sequencing, TERT promoter region mutation was only seen in one of the adenoid cystic carcinoma samples, located at -146 bp upstream from ATG (chr5: 1,295,250 C>T). Conclusion: TERT promoter mutation was not different in malignant and benign salivary tumors. Nonetheless, there are a few studies that report TERT promoter mutation in adenoid cystic carcinoma of the salivary gland, necessitating the need for further investigations.

Low expression of isocitrate dehydrogenase 1 (IDH1) R132H is associated with advanced pathological features in laryngeal squamous cell carcinoma.

INTRODUCTION: Recent developments in genomic sequencing have led to the identification of somatic mutations in isocitrate dehydrogenase 1 (IDH1) in various malignancies. IDH1 R132H is the most common mutation of IDH1, which affects codon 132 and results in the conversion of amino acid residue arginine (R) to histidine (H). This study is designed to evaluate the association between the expression of IDH1 R132H and clinicopathological characteristics in laryngeal squamous cell carcinoma (LSCC). METHODS: The expression pattern and clinical significance of IDH1 R132H were investigated in tissue microarrays (TMAs) of 50 LSCC tumors as well as adjacent normal tissues using immunohistochemistry. Then the exons of the 12 tumor samples with negative/weak positive staining were sequenced by applying polymerase chain reaction (PCR). RESULTS: The results demonstrated that the cytoplasmic expression of IDH1 R132H was downregulated in tumor cells compared to adjacent normal tissues. A statistically significant association was found between a low level of cytoplasmic expression of IDH1 R132H protein and an increase in histological grade (p < 0.001), perineural invasion (p = 0.019), and lymph node involvement (p < 0.001). The exon4 sequencing results showed that only one sample was positive for IDH1 R132H mutation. IDH1 R132H expression was observed in 39 (78.0%) LSCC samples. CONCLUSION: These findings indicate that low cytoplasmic expression of IDH1 R132H may have clinical significance in LSCC patients and is associated with more aggressive tumor behavior and progression of the disease, which can help improve potential treatment in patients with LSCC. Further investigations are needed to understand the biological function of IDH1 R132H and larger sample size to confirm our findings.

Clinical whole-exome sequencing analysis reveals a novel missense COL11A1 mutation resulting in an 18-week Iranian male aborted fetus with Fibrochondrogenesis 1: A case report.

Fibrochondrogenesis 1, an autosomal recessive syndrome, is a rare disease that causes short-limbed skeletal dysplasia. Mutations in the gene encoding the α1 chain of type XI collagen (COL11A1) are seen to be the main cause of this disease. We present an 18-week Iranian male aborted fetus with Fibrochondrogenesis 1 from consanguineous parents. Whole-exome sequencing revealed a novel missense variant from G to A in exon 45 of 68 in the COL11A1 gene (NM_080629.2: c.3440G > A, [p.G1147E, g.103404625]). The mutation was confirmed by Sanger sequencing and further, MutationTaster predicted this variant to be disease-causing. Bioinformatic analysis suggests that this variant is highly conserved in both nucleotide and protein levels, suggesting that it has an important function in the proper role of COL11A1 protein. In silico analysis suggests that this mutation alters the COL11A1 protein structure through a Glycine to Glutamic acid substitution.

Improved microsatellite instability detection in colorectal cancer patients by a combination of fourteen markers especially DNMT3a, DCD, and MT1X.

BACKGROUND: Microsatellite instability (MSI) results from genetic and epigenetic changes. Studying Microsatellite instability can help in treatment and categorization of colorectal cancer (CRC) patients. OBJECTIVES: We aimed to investigate whether 14 genomic markers consisting of BAT-62, BAT-60, BAT-59a, BAT-56a, BAT-56b, DCD, RIOX, RNF, FOXP, ACVR, CASP2, HSP110, MT1X, and DNMT3a can increase the detection rate of MSI in CRC. METHODS: Samples were stratified by pentaplex panel (Promega) and 14 markers using multiplex PCR and fragment analysis. In MSI+ samples, to identify the pattern of BRAF V600E mutation and MLH1 promoter methylation, ARMS-scorpion, and Methylation-Specific High-Resolution Melting Curve analysis, were applied respectively. RESULTS: Totally, 35 MSI+ cases identified by 14 marker panel. Only 18 cases of them were detected by both panels which are pentaplex and 14 marker. On the other hand, 17 new MSI+ cases just were identified by 14 markers panel. The highest diagnostic value among 14 markers is related to three makers, namely DCD, MT1X, and DNMT3a. In MSI+ cases, the rate of MLH1 promoter methylation was insignificant, (P value = 0.3979) while the rate of observed BRAFV600E mutation was significantly higher (P value = 0.0002). CONCLUSION: Fourteen marker panel showed higher sensitivity in comparison with the pentaplex panel increasing the detection rate of MSI+ cases up to 1.94 fold. Three markers namely DNMT3a, DCD, and MT1X of 14 marker panel were the best among them showing excellent diagnostic value. A combination of these markers showed 100% sensitivity and specificity in the studied group. In contrary to the markers in the pentaplex panel, these markers had the ability to detect MSI without any bias for the clinicopathological features. These markers will help to identify more end-stage MSI+ tumors which are located distal colon.

Identification of candidate genes and miRNAs for sensitizing resistant colorectal cancer cells to oxaliplatin and irinotecan.

Drug resistance to irinotecan and oxaliplatin, two widely used chemotherapeutic, has become a common problem in cancerous patients. Despite numerous valuable studies, distinct molecular mechanisms involved in the acquisition of resistance to these anti-cancer drugs have remained a challenge. In this study, we studied the possible resistance mechanisms to irinotecan and oxaliplatin in three CRC cell lines (HCT116, HT29, and LoVo) via integration of microarray data with gene regulatory networks. After determination of hub genes, corresponding miRNAs were predicted using several databases and used in construction and subsequent analysis of miRNA-gene networks. Following to preparation of chemo-resistance CRC cells, a standard real-time PCR was conducted for validation of in silico findings. Topological and functional enrichment analyses of the resulted networks introduced several previously reported drug-resistance genes as well as novel biomarkers as hub genes which seem to be crucial in resistance of colon cancer cells to irinotecan and oxaliplatin. Furthermore, results of the functional annotation revealed the essential role of different signaling pathways like metabolic pathways in drug resistance of CRC cell lines to these drugs. A part of in silico findings was also validated in vitro using oxaliplatin-resistant cell lines. While FOXC1 and NFIC were upregulated in cell lines which were resistant to oxaliplatin, silencing FOXC1 decreased the resistance of SW480 cell line to oxaliplatin. In conclusion, our comparative in silico and in vitro study introduces several novel genes and miRNAs as the resistance-mediators which can be used for sensitizing resistant CRC cells to oxaliplatin and irinotecan.

Hypomethylation of NANOG promoter in colonic mucosal cells of obese patients: a possible role of NF-κB.

Obesity and particularly central obesity are the main risk factors of colon cancer. All intestinal cell populations including stem cells, their progenitors and differentiated colonocytes seem to be the origin of colorectal cancer. However, recent data support the role of differentiated cells as cancer origin especially during inflammation. Based on Yamanaka's seminal work, re-expression of few transcription factors in terminally differentiated cells creates stemness properti'es. Although these transcription factors are involved in tumorigenesis, they are epigenetically repressed in adult tissues. We proposed that obesity might regulate methylation of stemness genes in colonocytes via inflammatory signalling. Obesity-associated inflammation was analysed using Western blot analysis of phospho-IκB (inhibitor of NF-κB). Methylation-sensitive high-resolution melting analysis was performed on colonic mucosal samples of twenty obese and twenty normal-weight men to analyse promoter methylation of POU5F1 (OCT4), NANOG, MYC and CDKN2A. TNF-treated HT-29 cells were used to recapitulate the effect of NF-κB activation on stemness genes methylation. Our results showed that colonic phosphorylation of IκB, as a signal of NF-κB activation, was higher in obese subjects compared with their normal-weight counterparts. Moreover, promoter methylation of NANOG was likely to be lower in obese subjects and correlated with central obesity. HT-29 cells incubated by TNF-α showed hypomethylation of POU5F1 and MYC genes in addition to the NANOG. These results suggest that obesity-induced inflammation might be involved in the regulation of DNA methylation of oncogenic genes such as NANOG in differentiated colonocytes and thus predispose them to later oncogenic alterations.

Among autophagy genes, ATG16L1 but not IRGM is associated with Crohn's disease in Iranians.

BACKGROUND: The prevalence of inflammatory bowel diseases is uprising in countries like Iran. Genetic predisposing elements play prominent role in the pathogenesis of Crohn's disease. In this study we studied the role of autophagy genes like IRGM (Immunity related GTPase M) and ATG16L1 (Autophagy related 16 like 1) in the pathogenesis of Crohn's Disease in Iranian patients. METHODS: One hundred thirty-eight patients and 99 normal controls were recruited in this study. Polymorphisms in -1644 and -308 upstream of IRGM gene were studied by PCR-sequencing and 20 kb CNVdel/insertion was studied by specific PCR. Rs10065171, rs4958847 in IRGM gene and rs2241880 in ATG16L1 were studied by Taqman genotyping assays. RESULTS: None of the so-called predisposing alleles of IRGM gene predispose Iranians to Crohn's disease while the prevalence of some of them like CNV deletion was higher in normal controls. Surprisingly all the so-called predisposing alleles in IRGM were linked to each other (especially rs4958847 with rs10065172 and polymorphisms in -308 region with rs4958847). Patients harboring A allele in rs4958847 site showed higher ratio of fibrostenotic type of disease while in patients with C/T genotype in rs4958847, colonic involvement was seen more frequently. G allele in ATG16L1 was associated with Crohn's disease though it was not associated with any phenotypic manifestation. CONCLUSION: In our study the association of ATG16L1 to Crohn's disease in Iranian patients was confirmed while it was shown that the studied polymorphisms in IRGM was not associated with Crohn's disease. Therefore in order to have a better picture about the genetics of Crohn's disease in Iranian patients, it is recommended to study other clinically effective polymorphisms in IRGM and ATG16L1 in addition to other genes which are responsible for the process of autophagy.

Comparison of gut microbiota in adult patients with type 2 diabetes and healthy individuals.

Recent studies indicate that inflammatory reactions leading to the development of type 2 diabetes mellitus (T2DM) may also contribute to variations in the composition of the intestinal microbiota, suggesting a relation between T2DM and bacterial residents in the intestinal tract. This case-control study was designed to evaluate the composition of the gut microbiota dominant bacterial groups in patients with T2DM compared to the healthy people. A total of 36 adult subjects (18 patients diagnosed with T2DM and 18 healthy persons) were included in the study. The intestinal microbiota composition was investigated by quantitative real-time polymerase chain reaction (qPCR) method using bacterial 16S rRNA gene. The quantities of two groups of bacteria were meaningfully different among T2DM patients and healthy individuals. While, the level of Lactobacillus was significantly higher in the patients with T2DM (P value < 0.001), Bifidobacterium was significantly more frequent in the healthy people (P value < 0.001). The quantities of Prevotella (P value = 0.0.08) and Fusobacterium (P value = 0.99) genera in faecal samples were not significantly different between the two groups. The significant alterations in dominant faecal bacterial genera found in T2DM patients participating in the current study highlight the link between T2DM disease and compositional variation in intestinal flora. These findings may be valuable for developing approaches to control T2DM by modifying the gut microbiota. More investigations with focus on various taxonomic levels (family, genus and species) of bacteria are necessary to clarify the exact relevance of changes in the gut microbial communities with the progression of T2DM disorder.

RAB7L1 promoter polymorphism and risk of Parkinson's disease; a case-control study.

INTRODUCTION: Recent genome-wide association studies have explored some new loci in association with Parkinson's disease (PD). RAB7L1 is an important gene involved in one of the important neurological pathways, located in PARK16 locus. We performed a case-control study to examine the association between rs823144 SNP located in the promoter region of the RAB7L1 gene and PD risk in Iranian population. METHODS: A total of 960 samples including 480 PD patients and 480 healthy controls were collected for analysis of the RAB7L1 rs823144 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR - RFLP) method. RESULTS: We found significant differences in genotypic and allelic frequencies between patients and controls. Significant association was found between presence of minor allele (C) and decreased risk of PD development (p = 0.008, OR = 0.74 (0.605-0.924)). Also another significant association was observed between the CC genotype and PD (p = 0.004, OR = 0.441 (0.252-0.772)). CONCLUSION: Our data support the association between rs823144 and decreased risk of PD.

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition.

It is observed that upregulation of DNMT3B enzyme in some cancers, including colon cancer, could lead to silencing of tumor suppressor genes. MiR-339 and miR-766 have been predicted to target 3'UTR of DNMT3B gene. Luciferase reporter assay validated that individual and co-transfection of miR-766 and miR-339 into the HEK293T cell reduced luciferase activity to 26% ± 0.41%, 43% ± 0.42 and 64% ± 0.52%, respectively, compared to the control (P < 0.05). Furthermore, transduction of miR-339 and miR-766 expressing viruses into colon cancer cell lines (SW480 and HCT116) decreased DNMT3B expression (1.5, 3-fold) and (3, 4-fold), respectively. In addition, DNA methylation of some tumor suppressor genes decreased. Expression of these genes such as SFRP1 (2 and 1.6-fold), SFRP2 (0.07 and 4-fold), WIF1 (0.05 and 4-fold), and DKK2 (2 and 4-fold) increased in SW-339 and SW-766 cell lines; besides, expression increments for these genes in HCT-339 and HCT-766 cell lines were (2.8, 4-fold), (0.005, 1.5-fold), (1.7 and 3-fold) and (0.04, 1.7-fold), respectively. Also, while in SW-766, cell proliferation reduced to 2.8% and 21.7% after 24 and 48 hours, respectively, SW-339 showed no reduced proliferation. Meanwhile, HCT-766 and HCT-339 showed (3.5%, 12.8%) and (18.8%, 33.9%) reduced proliferation after 24 and 48 hours, respectively. Finally, targeting DNMT3B by these miRs, decreased methylation of tumor suppressor genes such as SFRP1, SFRP2, WIF1 and DKK2 in the mentioned cell lines, and returned the expression of these tumor suppressor genes which can contribute to lethal effect on colon cancer cells and reducing tumorigenicity of these cells.

TCF4 silencing sensitizes the colon cancer cell line to oxaliplatin as a common chemotherapeutic drug.

Colon cancer is among the most prevalent cancers worldwide. Although the main modality of treatment is surgery, resistance to chemoradiotherapy raises concerns. Hence, we aimed to determine the effect of RNA-mediated silencing of tcf4, the downstream effector of the wnt signaling pathway, on the response of the SW480 cell line to oxaliplatin, a common chemotherapeutic drug. For this, two different silencing sequences against TCF4 mRNA were selected and cloned into pSilencer neo2.1. The SW480 cell line was stably transfected with the silencing constructs (namely p1396, p1874, and p silencer containing a scrambled sequence) and labeled SW1396, SW1874, and SW-Sc, respectively. Subsequently, the effect of oxaliplatin (from 0 to 11.25 µmol/l) on these cells was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide proliferation assay. Suppression of tcf4 expression in stable transfected cells with p1396 and p1874 was confirmed by quantitative reverse transcription-PCR and western blot analysis. Although oxaliplatin was not toxic to SW480 and SW-sc in the range tested, in SW1396 and SW1874 cells, a toxic effect was evident at 3.75 and 4.375 µmol/l. Also, SW1396 and SW1874 cells appeared to have a round shape in comparison with SW480 and SW-Sc cells. Only for SW1396, the number of apoptotic cells was significantly different before and after the addition of oxaliplatin (LC50 of oxaliplatin). The proliferating cells in SW480, SW1874, and SW-Sc increased after treatment with oxaliplatin; however, this was not observed in SW1396. Although silencing the tcf4 gene would confer sensitivity to oxaliplatin in SW1874 and especially SW1396, in SW480 and SW-Sc, the lethal effect of oxaliplatin was compensated by its effect in increasing the proliferation of cells. This sensitization effect may be because of different mechanisms including TCF4 motifs in the ABCB1 promoter or defects in nucleotide excision repair or double-strand break repair systems after tcf4 silencing.