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.

The c.1243T>C mutation in the PROC gene is linked with inherited protein C deficiency and severe purpura fulminans.

Purpura fulminans is a severe coagulation disorder that often leads to death in neonates. Mutations in the protein C (PROC) gene can cause protein C deficiency, leading to this disorder. This study aimed to investigate a family with a history of coagulopathies, particularly those related to protein C deficiency. The primary objective was to identify any genetic mutations in the PROC gene responsible for the coagulopathies. The study focused on a male neonate with purpura fulminans who ultimately died at 2 months of age. The patient had low protein C activity levels (6%). The entire PROC gene of the patient and his family was analyzed using next-generation sequencing to identify any genetic mutations. Segregation analysis was conducted to determine if the mutation followed an autosomal dominant inheritance pattern. In silico analysis was also conducted to evaluate the pathogenicity of the identified mutation. Analysis revealed a novel homozygous c.1243T>G variant PROC gene. The mutation resulted in a Phe415Val substitution. The mutation was found in at least three generations of the family. Carrier family members had lower protein C activity levels than wild-type homozygotes. Additionally, the mutation may account for the observed reduction in protein C enzyme activity.

Regulatory Role of Insulin on Endogenous L1 ORF1 and NEFM Gene Expression through PI3K Signaling Pathway Specifically in Neuroblastoma Cell Line.

Background: One of the most important endogenous factors causing genomic instability in human cells is L1s retrotransposons. In this study, we assume that increased activity of L1 retrotransposons (specifically L1 expression) might be induced by hyperglycemia and hyperinsulinemia in neuroblastoma cell line. Methods: Two different cell lines (BE (2)-M17 and HEK293) were treated with insulin and its PI3K signaling pathway inhibitor under three conditional media including hyperglycemic and retinoic acid treatment in the department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran in 2018. The expression of L1 ORF1, as well as genes involved in insulin signaling pathway and neuronal stress and structure were measured at RNA level. Results: Insulin could significantly down regulate the expression of L1 ORF1 and NEFM genes. Hyperglycemia result in severe decrease in expression of all candidate genes in control neuroblastoma but not HEK293 cells. Retinoic acid as the concentration used in this study cause increase stemness in neuroblastoma but not HEK293 cells. We could not find significant correlation between expression pattern of other genes tested in our study and L1 ORF1 expression. Conclusion: Total regulatory effect of insulin on L1 ORF1 RNA expression as well as NEFM markedly in BE (2)-M17 cell line. Although these results could not be interpreted as L1 retrotransposition, expression of L1 RNA during stress conditions might be considered following inhibition of the insulin pathway. The result of this study also confirms the impotence of insulin on human evolution.

CEP104 gene may involve in the pathogenesis of a new developmental disorder other than joubert syndrome.

BACKGROUND: The CEP104 gene (OMIM: 616,690) encodes the centrosome protein 104 (CEP104) that is involved in cilia function. Pathogenic variants in this gene have been described in four patients diagnosed with Joubert syndrome (JBTS) 25. Here, we challenged the concept that pathogenic variants in CEP104 gene are only involved in the development of JBTS 25. METHODS AND RESULTS: In a clinical setting, whole-exome sequencing (WES) was applied to investigate pathogenic variants in patients with unexplained developmental delay or intellectual disability (DD/ID).WES revealed a novel homozygous nonsense variant (c.643C > T) in CEP104 (NM _014704.3) in a girl with mild intellectual disability, hypotonia, and imbalanced gait. Her brain MRI data did not show molar tooth sign (MTS) or any other brain anomalies. CONCLUSION: Our study introduced a novel variant in the CEP104 gene that results in an ID phenotype other than JBTS25. Comparison of her phenotype with that of eight previously published DD/ID patients harboring pathogenic variants in CEP104 gene revealed that more than half of them did not show JBTS related symptoms. Therefore, we suggest that the CEP104 gene might also be involved in a disorder other than JBTS 25, a point that deserves to be emerged in the OMIM database.

Cephalometric Evaluation of the Upper Airway in Different Skeletal Classifications of Jaws.

BACKGROUND: Respiratory system is an important section in development of maxillofacial components and many studies indicated its effect on normal growth of the jaws. The aim of this study is to evaluate upper airway in different skeletal classifications of jaws in lateral cephalogram and its relation to age and gender. MATERIALS AND METHODS: Study samples were 105 digital cephalometric radiographies, 72 females and 33 males. Lateral cephalograms were hand traced and based on Stainer analysis, there were 30 samples in Class I, 30 samples in Class II and 45 samples in Class III subgroup. Vertical linear measurements, horizontal linear measurements, and angular measurement, proportions and space measurements of the airway in the Cephalograms were analyzed by AutoCAD software. RESULTS: Data were analyzed using SPSS software version 20. Two horizontal linear measurement (the hypo pharyngeal airway depth, the nasopharyngeal airway depth) and one space measurement (soft palate space) were significantly different in skeletal classes. Vertical and horizontal linear measurements in the 3 groups were increased significantly in men rather than women. The developmental age of groups showed some significant differences. CONCLUSION: Upper airway dimension is different in different skeletal classes, developmental ages, and gender.

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution.

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible.

Molecular changes in obese and depressive patients are similar to neurodegenerative disorders.

Background: Neurodegenerative disorders (NDs) are categorized as multifactorial conditions with different molecular and environmental causes. Disturbance of important signaling pathways, such as energy metabolism and inflammation induced by environmental agents, is involved in the pathophysiology of NDs. It has been proposed that changes in the lifestyle and nutrition (metabolism) during mid-life could trigger and accumulate cellular and molecular damages resulting in NDs during aging. Methods: In order to test the hypothesis, we investigated the expression level of two energy metabolism-related [forkhead box O1 (FOXO1) and forkhead box O3 (FOXO3A)] and two pro-inflammatory cytokines [interleukin 1ß (IL-1ß) and IL-6] genes, using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, changes in the ionic concentration of three essential heavy metals [iron (Fe), copper (Cu), and zinc (Zn)] by atomic absorption spectroscopy in patients with NDs, depression, obesity, and diabetes type II, were evaluated and compared with the results of normal individuals. Results: More than half of the participants in obesity, depression, and ND groups had significant up-regulation of FOXO1 and FOXO3A, down-regulation of IL-1ß and IL-6, and higher levels of Fe and Cu in their blood. This pattern of gene expression was not repeated in diabetic patients. Conclusion: It could be concluded that individuals affected with different levels of obesity and depression have increased the risk of developing NDs later in life, probably through changes in energy metabolism, inflammatory pathways, and ionic concentrations.

Mobile DNA Elements: The Seeds of Organic Complexity on Earth.

Mobile DNA or transposable elements (TEs) are genomic sequences capable of moving themselves independently into different parts of the genome. Viral invasion of eukaryotic genomes is assumed to be the main source of TEs. Selfish transposition of these elements could be a serious threat to the host cell, as they can insert themselves into the middle of coding genes and/or induce genomic instability. In response, through millions of years of evolution, cells have come up with various mechanisms such as genomic imprinting, DNA methylation, heterochromatin formation, and RNA interference to deactivate them. Interestingly, these processes have also greatly contributed to important cellular functions involved in cell differentiation, development, and differential gene expression. Propagation of TE copies during the course of evolution have resulted in increasing the genome size and providing proper space and flexibility in shaping the genome by creating new genes and establishing essential cellular structures such as heterochromatin, centromere, and telomeres. Yet, these elements are mostly labeled for playing a role in pathogenesis of human diseases. Here, we attempt to introduce TEs as factors necessary for making us human rather than just selfish sequences or obligatory guests invading our DNA.

Epileptic syndromes: From clinic to genetic.

Epilepsy is one of the most common neurological disorders. Studies have demonstrated that genetic factors have a strong role in etiology of epilepsy. Mutations in genes encoding ion channels, neurotransmitters and other proteins involved in the neuronal biology have been recognized in different types of this disease. Moreover, some chromosomal aberration including ring chromosomes will result in epilepsy. In this review, we intend to highlight the role of molecular genetic in etiology of epilepsy syndromes, inspect the most recent classification of International League against Epilepsy and discuss the role of genetic counseling and genetic testing in management of epilepsy syndromes. Furthermore, we emphasize on collaboration of neurologists and geneticists to improve diagnosis and management.

Effect of Copper Sulfate on Expression of Endogenous L1 Retrotransposons in HepG2 Cells (Hepatocellular Carcinoma).

The long interspersed element-1 (LINE-1 or L1) constitutes approximately 17% of human genome. The expression of these elements is deregulated upon exposure to environmental exposures resulting to genomic instability and cancer promotion. The effect of copper as essential elements in regulation of L1 expression remained to be elucidated. Using non-cytotoxic concentrations of the copper, the expression of endogenous L1 was analyzed by qPCR after 6 days of copper pretreatment in human hepatocellular carcinoma cells (HepG2). The results indicated that the expression of active L1 elements are significantly downregulated at concentrations of 12.5, 25, and 50 µM (p < 0.005). Our data imply that low-level copper exposure may have a protective effect to suppress the induction of L1 activity and decrease incidence of cancer-associated L1 mutagenesis. If this achievement is confirmed by further studies, it can be applied in the long-term goals of cancer prevention.

Novel trends in genetics: transposable elements and their application in medicine.

Forty-five percent of the human genome is composed of Transposable Elements (TEs); therefore, TEs have had an undisputed impact on evolution of the most evolved creature by a very simple mechanism of action.  Scientists have been studying this simple mechanism of action and are currently using it to develop efficient and safe gene delivery systems especially for treatment of diseases. TEs have also been used safely in generating induced Pluripotent Stem Cells (iPSC) for regenerative medicine, which opens the door to a world of possibilities in our approach in trying to wrestle with many challenges in medicine. The PiggyBac (PB) system has yielded more success in generation of induced pluripotent stem cells in regenerative medicine, and the Sleeping Beauty (SB) has been more successful in Gene Therapy. Recent advances are indicative of more good news to come regarding the potential heights of successes achievable by the use of the TE-based systems.

Exposure of hepatocellular carcinoma cells to low-level As2O3 causes an extra toxicity pathway via L1 retrotransposition induction.

Various mechanisms have been proposed for toxicity and carcinogenesis pattern of arsenic, a naturally occurring metalloid. The extent to which the long interspersed element-1 (LINE-1) retrotransposon, an ubiquitous retroelement with autonomous mobility, can be influenced upon exposure to low-level arsenic remains to be elucidated. The aim of this study was to evaluate the possible effect of low-level As2O3 on L1 retrotransposition alteration in human hepatocellular carcinoma cells (HepG2). L1 retrotransposition in HepG2 cells was performed by the in vitro retrotransposition assay using an EGFP-tagged L1RP. Following determination of non-cytotoxic concentrations of arsenic by a MTT assay, the cells were transfected with pL1RP-EGFP and then exposed to 0.25, 0.50 and 0.75 µM of As2O3. The amount of EGFP and its copy number in retrotransposed cells were evaluated by FACS and qPCR analysis in treated vs. control cells, respectively. Significant increase in retrotransposition frequency was found after 12 days exposure to 0.50 and 0.75 µM of As2O3 by FACS analysis (P<0.05). Obtained results were further confirmed by real time PCR, which showed significant induction of retrotransposition in all mentioned concentrations. Our findings indicate that low-level long-term As2O3 exposure may pave activation of L1 retrotransposon.

Evaluating the extent of LINE-1 mobility following exposure to heavy metals in HepG2 cells.

The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17% of the human genome and retain mobility properties within the genome. At present, 80-100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.

In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice.

BACKGROUND: Numerous in vitro reports suggest that Low Level Laser Therapy (LLLT) affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light (which is more useful in clinic because of its higher penetration) on secretion of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF), as important growth factors in wound healing. METHODS: Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT (energy density of 1 J/cm (2)) using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time poly-merase chain reaction. RESULTS: Diabetic irradiated group showed significant increase in FGF (p = 0.017) expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. CONCLUSION: These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts.

Is amyloid-ß an innocent bystander and marker in Alzheimer's disease? Is the liability of multivalent cation homeostasis and its influence on amyloid-ß function the real mechanism?

Two decades of the amyloid-ß (Aß) hypothesis in Alzheimer's disease (AD) and the prominence of Aß-targeting strategies have yet to meet the levels of original expectation. Disappointing results in numerous Phase II/III studies have called for a re-examination of the validity of the Aß-targeting approaches as an intervention strategy in AD. The mid-life onset of chronic conditions (e.g., hypertension, diabetes, insulin intolerance, and depression nominated as risk factors for the later development of AD) points to the possibility that each condition could involve mechanisms, which while relatively modest over a short-term, could have significant accumulative effects. What may also not be fully appreciated is that a number of these conditions involve potential disturbances to multivalent cations (MC) levels through various mechanisms such as autophagy, oxidative stress, and apoptosis. Furthermore, some MCs have intimate associations with the mechanisms by which Aß pathology manifests. Considering various lines of evidence and incorporating statistical analysis on Disability-Adjusted Life Years (DALYs) data of both causes of and prevalence of multifactorial risk factors in different world regions, we propose an MC hypothesis for AD. More specifically, we suggest that MC imbalance marks many chronic conditions and because of their involvement with Aß pathology, could reflect that Aß may be a vital manifestation and marker of underlying MC imbalance. Thus, careful targeting of MC imbalance may provide an alternative or complementary interventional approach to current Aß treatment strategies.

Global warming and neurodegenerative disorders: speculations on their linkage.

Climate change is having considerable impact on biological systems. Eras of ice ages and warming shaped the contemporary earth and origin of creatures including humans. Warming forces stress conditions on cells. Therefore, cells evolved elaborate defense mechanisms, such as creation of heat shock proteins, to combat heat stress. Global warming is becoming a crisis and this process would yield an undefined increasing rate of neurodegenerative disorders in future decades. Since heat stress is known to have a degenerative effects on neurons and, conversely, cold conditions have protective effect on these cells, we hypothesize that persistent heat stress forced by global warming might play a crucial role in increasing neurodegenerative disorders.

Retrotransposons and pediatric genetic disorders: Importance and implications.

Pediatric disorders are generally observed to have a greater genetic load than diseases occurring during adulthood. Clinical manifestations of many genetic defects including chromosomal abnormalities and mutations in specific genes appear during childhood. One of the notable mutagens in human cells is mobile DNA element. They possess the ability to move and insert themselves in new genomic locations including critical disease-causing genes. Although our cells inhibit their transport by different mechanisms, factors such as aging and environmental heavy metals have effect on increasing their activities. In this article, we try to go over the features of active human retroelements and highlight their role in the pathology of pediatric genetic disorders. We also propose two mechanisms in which aged parental gametes and embryonic exposure to environmental stresses followed by mobile elements insertion may result in de novo pediatric diseases.

Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

Effect of heavy metals on silencing of engineered long interspersed element-1 retrotransposon in nondividing neuroblastoma cell line.

BACKGROUND: L1 retrotransposons are the most active mobile DNA elements in human genome. Unregulated L1 retrotransposition may have deleterious effect by disrupting vital genes and inducing genomic instabilities. Therefore, human cells control L1 elements by silencing their activities through epigenetic mechanisms. It has been shown that cell division and heavy metals stimulate the frequency of L1 activities. Removal of silencing by L1 motivators may restart L1 element functions. Here, we have proposed that weather neurotoxic environmental heavy metals (as L1 stimulating factors) have a role in removing L1 silencing and restating its activities in nondividing neuronal cells. METHODS: L1-RP green fluorescent protein (GFP)-tagged knock-in human neuroblastoma clones were prepared. Single-cell clone was treated with mitomycin-c combined with nontoxic and toxic concentrations of iron (Fe), copper (Cu), and mercury (Hg). Silencing status of engineered L1 elements in dividing and nondividing cells was determined through measuring the amount of GFP expressing cells with flow cytometry. The cytotoxic effect of mitomycin-c combined with metals was measured by MTT assay. RESULTS: Hg in nondividing cells and Fe, Cu, and Hg in dividing neuroblastoma cells could significantly remove L1 silencing. Also, mitomycin-c treatment did not have any effect on metal toxicity status in neuroblastoma cells. CONCLUSION: Totally, our findings have shown that cell division has a role in removing L1 silencing as well as L1 retrotransposition induced by environmental heavy metals. It has been also indicated that Hg at all concentrations could remove silencing of engineered L1 element regardless of cell cycle state.

Afebrile Neuroleptic Malignant Syndrome associated with Fluphenazine decanoate: A case report.

Neuroleptic Malignant Syndrome (NMS) is unusual but could be a lethal reaction associated with neuroleptic drugs. It occurs in almost 0.07-2.2% of patients under treatment with neuroleptics. There are some medical treatments that may also be helpful for its treatment, including dopamine agonists, muscle relaxants, and electroconvulsive therapy (ECT). We present this case to alert the clinicians to the potential for inducing afebrile NMS.Our case is a 41-year-old man with a history of schizophrenia showing signs and symptoms in accordance with NMS, 2 weeks after receiving one dose of 12.5 mg fluphenazine decanoate, abruptly following the 3rdsession of ECT. The patient presented with decreased level of consciousness, muscular rigidity, waxy flexibility, mutism, generalized tremor, sever diaphoresis and tachycardia which progressed during the previous 24 h. Laboratory data indicated primarily leukocytosis, an increasing level of creatinine phosphokinase and hypokalemia during the next 72h.In patients receiving antipsychotics, any feature of NMS should carefully be evaluated whether it is usual or unusual particularly in patients receiving long acting neuroleptics.