Two rare autosomal recessive neurological disorders identified by combined genetic approaches in a single consanguineous family with multiple offspring.

INTRODUCTION: Neurodevelopmental disorders (NDDs) refer to a broad range of diseases including developmental delay, intellectual disability, epilepsy, autism spectrum disorders, and attention-deficit/hyperactivity disorder caused by dysfunctions in tightly controlled brain development. The genetic backgrounds of NDDs are quite heterogeneous; to date, recessive or dominant variations in numerous genes have been implicated. Herein, we present a large consanguineous family from Turkiye, who has been suffering from NDDs with two distinct clinical presentations. METHODS AND RESULTS: Combined in-depth genetic approaches led us to identify a homozygous frameshift variant in NALCN related to NDD and expansion of dodecamer repeat in CSTB related to Unverricht-Lundborg disease (ULD). Additionally, we sought to functionally analyze the NALCN variant in terms of mRNA expression level and current alteration. We have both detected a decrease in the level of premature stop codon-bearing mRNA possibly through nonsense-mediated mRNA decay mechanism and also an increased current in patch-clamp recordings for the expressed truncated protein. CONCLUSION: In conclusion, increased consanguinity may lead to the revealing of distinct rare neurogenetic diseases in a single family. Exome sequencing is generally considered the first-tier diagnostic test in individuals with NDD. Yet we underline the fact that customized approaches other than exome sequencing may be used as in the case of ULD to aid diagnosis and better genetic counseling.

Targeted resequencing reveals high-level mosaicism for a novel frameshift variant in WDR45 associated with beta-propeller protein-associated neurodegeneration.

OBJECTIVES: Beta-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant neurodegenerative disease, which is characterized by iron accumulation in the basal ganglia. BPAN is associated with pathogenic variation in WDR45, which has been reported almost exclusively in females most probably due to male lethality in the hemizygous state. METHODS: Whole exome sequencing (WES) and targeted deep sequencing were performed for a male with a clinical diagnosis of BPAN at the age of 37. RESULTS: The novel frameshift variant in WDR45 detected by WES was further analyzed with targeted resequencing to detect a mosaic variant with a level of 85.5% in the blood sample of the proband. DISCUSSION: Although the main role of WDR45 remains elusive, recent studies show that WDR45 may contribute to neurodegeneration through defects in autophagy, iron storage and ferritin metabolism, mitochondria organization, and endoplasmic reticulum homeostasis. The extend of spatiotemporal haploinsufficiency of WDR45 frameshifting variants caused by mosaicism in males may lead to variable clinical severity, which may be hard to elaborate clinically. Promising genetic analysis strategies using targeted deep sequencing may help determine the clinical outcome of somatic mosaicism in neurological disorders including BPAN. Additionally, we suggest that deep sequencing should be conducted in cerebrospinal fluid samples to provide more reliable results in terms of reflecting the mosaicism level in the brain for future studies.

Reanalysis of exome sequencing data reveals a treatable neurometabolic origin in two previously undiagnosed siblings with neurodevelopmental disorder.

Neurodevelopmental disorders (NDDs) have broad heterogeneity both clinically and genetically. Inborn errors of metabolism can be one of the reasons of neurodevelopmental disruption causing specific NDDs. Although there is tremendous advance in molecular identification via next-generation sequencing (NGS), there are still many unsolved patients with NDD. Reanalysis of NGS data with different pipelines can at least partially accomplish this challenge. Herein, we report clinic and genetic components of an adult sib-pair with an undiagnosed NDD condition, which has been solved through reanalysis of whole-exome sequencing (WES). Parallel analysis of SNP-based genotyping and WES was performed to focus on variants only in loci with positive logarithm of the odds scores. WES data was analyzed through three different pipelines with two distinct bed files. Reanalysis of WES data led us to detect a homozygous FOLR1 variant (ENST00000393676.5:c.610C > T, p.(Arg204Ter), rs952165627) in the affected sib-pair. Surprisingly, the variant could not be detected in the first analysis as the variant region is not included in the first bed file which may frequently be used. Biochemical tests of CSF have confirmed the genetic analysis, CSF folic acid levels were detected low in sib-pair, and intravenous folinic acid treatment improved the disease course for the first 6 months of follow-up even at late diagnosis age. Although combined analysis of SNP-based genotyping and WES is a powerful tool to reveal the genetic components of heterogeneous diseases, reanalysis of genome data still should be considered in unsolved patients. Also, biochemical screening helps us to decipher undiagnosed NDD that may be a treatable neurometabolic condition.

Effects of Methylprednisolone in the Treatment of Spinal Cord Injuries by Evaluation of microRNA-21: An Experimental Study.

BACKGROUND AND STUDY AIMS: Spinal cord injury (SCI) is one of the most complicated pathologies that affect active young males. miR-21 primarily regulates several cellular processes. We aimed to elucidate the regulatory role of miR-21 and test methylprednisolone as a disease-modifying agent on experimental SCI tissues. METHODS: A total of 36 8- to 10-week-old adult female Sprague-Dawley rats weighing 250 to 300 g were used. Animals were randomly divided into six groups. Except for groups 1 and 4, the spinal trauma model was applied to all animal groups using the clipping method. In groups 3 and 6, methylprednisolone was given. For real-time polymerase chain reaction (PCR) investigations, rats in groups 1, 2, and 3 were reoperated on after the first postoperative day, whereas those in groups 4, 5, and 6 were reoperated on after postoperative day 7 and spinal cord samples from the laminectomy area were removed for gene expression analysis. Relative gene expression of miR-21, Gfap, Vim, Stat3, Faslg, Pten, Bax, Bcl2, Cox2, and Il6 were determined with quantitative reverse transcription (qRT) PCR. RESULTS: In group 3, the miR-21 expression significantly increased compared with groups 1 and 2. When compared with group 3, a decrease in miR-21 expression was observed in group 6 (p < 0.05). When compared with group 4, group 6 had lower levels of Gfap, Pten, Stat3, and Bax (p < 0.05). CONCLUSIONS: miR-21 supports the beneficial aspects of the body's healing mechanisms following SCI. In the acute phase, the use of methylprednisolone increases miR-21 expression in the early period of trauma. Methylprednisolone increases some astrogliosis and inflammation biomarkers' levels; however, it did not affect the apoptotic biomarkers.

Hypomyelinating spastic dyskinesia and ichthyosis caused by a homozygous splice site mutation leading to exon skipping in ELOVL1.

INTRODUCTION: Next generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human. METHODS: We have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ELOVL1 protein structure was computationally modelled. RESULTS: The novel c.376-2A > G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC-MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides. DISCUSSION: It has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.

The utility of serum miRNA-93 and miRNA-191 levels for determining injury severity in adults with multiple blunt trauma.

BACKGROUND: Various scoring systems have been developed to determine the trauma severity and prognosis of patients following multiple blunt trauma (MBT). However, these scoring systems do not provide exactly the desired severity assessment. In recent years, serum concentration of many specific microRNAs (miRNAs), especially for head trauma, has been shown to play an important role in determining the diagnosis, severity, and prognosis of injury. To date, however, no studies have investigated serum miRNAs in patients with MBT. Thus, this study measured the expression of miRNA-93 and -191 in the serum of adults with MBT and examined the correlations of Injury Severity Score (ISS) and Revised Trauma Score values with serum miRNA-93 and -191 levels in these patients with the aim of predicting trauma severity based on the miRNA levels. METHODS: This prospective case-control study enrolled 50 consecutive adults with MBT and age- and sex-matched 60 healthy controls. The patients were divided into ISS >16 (Group 1, major or severe trauma) and ISS ≤16 (Group 2, minor or mild-moderate trauma) groups. Serum miRNA-93 and -191 levels were assessed using quantitative real-time reverse transcription-PCR. We evaluated whether the miRNAs were differentially expressed in major and minor MBT patients and determined their utility for assessing the severity of injury. RESULTS: The mean serum miRNA-93 and -191 levels were significantly elevated in the patients compared to the controls and were higher in patients with ISS >16 compared to those with ISS ≤16, although the difference was not significant. In the patients with multitrauma, ISS was significantly, negative and weak correlated with serum miRNA-191 level (rho=-0.320, p=0.023) but not with the serum miRNA-93 level. No optimal cutoff for the serum miRNA-93 level was found with respect to trauma severity (AUC 0.617, [0.455-0.779]). However, an optimal cutoff value for serum miRNA-191 was identified, with values <1.94 indicating severe trauma (AUC 0.668 [0.511-0.826]; 65.6% sensitivity, 77.8% specificity). CONCLUSION: miRNA-191 and -93 levels were significantly upregulated in multitrauma patients compared to controls. The level of miRNA-191 in conjunction with ISS, but not that of miRNA-93, may be a useful biomarker for determining injury severity in patients with multitrauma.

Gene Hunting Approaches through the Combination of Linkage Analysis with Whole-Exome Sequencing in Mendelian Diseases: From Darwin to the Present Day.

BACKGROUND: In the context of medical genetics, gene hunting is the process of identifying and functionally characterizing genes or genetic variations that contribute to disease phenotypes. In this review, we would like to summarize gene hunting process in terms of historical aspects from Darwin to now. For this purpose, different approaches and recent developments will be detailed. SUMMARY: Linkage analysis and association studies are the most common methods in use for explaining the genetic background of hereditary diseases and disorders. Although linkage analysis is a relatively old approach, it is still a powerful method to detect disease-causing rare variants using family-based data, particularly for consanguineous marriages. As is known that, consanguineous marriages or endogamy poses a social problem in developing countries, however, this same condition also provides a unique opportunity for scientists to identify and characterize pathogenic variants. The rapid advancements in sequencing technologies and their parallel implementation together with linkage analyses now allow us to identify the candidate variants related to diseases in a relatively short time. Furthermore, we can now go one step further and functionally characterize the causative variant through in vitro and in vivo studies and unveil the variant-phenotype relationships on a molecular level more robustly. Key Messages: Herein, we suggest that the combined analysis of linkage and exome analysis is a powerful and precise tool to diagnose clinically rare and recessively inherited conditions.

The rare rs769301934 variant in NHLRC1 is a common cause of Lafora disease in Turkey.

Lafora disease (LD) is a severe form of progressive myoclonus epilepsy inherited in an autosomal recessive fashion. It is associated with biallelic pathogenic variations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The disease usually starts with adolescent onset seizures followed by progressive dementia, refractory status epilepticus and eventually death within 10 years of onset. LD is generally accepted as having a homogenous clinical course with no considerable differences between EPM2A or NHLRC1 associated forms. Nevertheless, late-onset and slow progressing forms of the disease have also been reported. Herein, we have performed clinical and genetic analyses of 14 LD patients from 12 different families and identified 8 distinct biallelic variations in these patients. Five of these variations were novel and/or associated with the LD phenotype for the first time. Interestingly, almost half of the cases were homozygous for the rare rs769301934 (NM_198586.3(NHLRC1): c.436 G > A; p.(Asp146Asn)) allele in NHLRC1. A less severe phenotype with an onset at a later age may be the reason for the biased inflation of this variant, which is already present in the human gene pool and can hence arise in the homozygous form in populations with increased parental consanguinity.

[Investigating of Relation Between Fibromyalgia Syndrome and Intestinal Microbiota]. / Fibromiyalji Sendromu ile Bagirsak Mikrobiyotasi Arasindaki Iliskinin Arastirilmasi.

Fibromyalgia syndrome (FMS) is one of the most frequent forms of chronic widespread pain, with a reported prevalence of 3-10% in the adult population. Clinical presentation of the typical pain and the presence of associated somatic and psychological symptoms form the basis of the diagnosis. FMS is associated with nervous system dysfunction and neurotransmitters act as targets of a number of drugs approved for fibromyalgia. However, although the underlying mechanisms in FMS are not yet known precisely, many hypotheses have been put forward. Considering the relation between fibromyalgia and irritable bowel syndrome (IBS), altered gut microbiome could be associated with fibromyalgia. In this study, it was aimed to investigate the variation of intestinal microbiome levels in patients with FMS compared to healthy controls. For the investigation of the microbiome, fecal samples were collected from a cohort of 54 patients with FMS and 36 healthy individuals. Those with any mental and/or physical illness in the control group were excluded from the study. The FMS patient group was determined according to the "American College of Rheumatology (ACR)" 2010 diagnostic criteria. The fecal samples were stored at -80°C until use and were thawed on ice; for each extraction, 0.3 g of faeces were weighed. Extraction of DNA was carried out with commercial kit according to the manufacturer's recommendations. Samples were compared using 16S rRNA gene amplification with specific primers of Bacteroidetes, Firmicutes, Enterobacter, Lactobacillus, Streptococcus and Bifidobacterium by the real-time PCR method. According to our results, while the increase of Bacteroidetes and Bifidobacterium was statistically significant (p<0.05), Firmicutes decreased (p<0.001) in the patient group. No statistically significant results were found for Enterobacter, Streptococcus and Lactobacillus (p> 0.05). When the relationship between bacteria was evaluated, a high statistically significance and negative correlation was found between Bacteroidetes and the percentage of Firmicutes (r= -0.778, p<0.001),while a moderate statistical significance and positive correlation was observed between the percentage of Enterobacter and Bifidobacterium (r= 0.460, p= 0.005). The results suggest that the gut microbiota may play a role in fibromyalgia. The balance of Firmicutes and Bacteroidetes phyla in the gut is known to have important effects on intestinal homeostasis. In summary, it is clear that large-scale further research in larger cohorts will be effective in understanding the relationship between the gut microbiome and FMS and evaluating possible treatment options.

Calcium homeostasis in cisplatin resistant epithelial ovarian cancer.

Intracellular calcium concentration ([Ca2+]i) may have an important role in the development of chemoresistance, which is an essential problem in cancer chemotherapy. Cisplatin (DDP), which modulates the intracellular calcium concentration by different mechanisms, is an antineoplastic agent with high success rate in cancer therapies. We investigated the regulatory role of [Ca2+] in cisplatin resistance in epithelial ovarian cancer cell line, in MDAH-2774, and its chemoresistant subclone MDAH-2774/DDP. The measurement of [Ca2+]i using fluorescence microscope, and flow cytometry revealed that the amount of intracellular calcium decreased in cisplatin resistant cells compared to the amounts in parental cells. mRNA expression profiles of calcium homeostasis-associated major genes (IP3R1/2/3, RYR1/2, SERCA1/2/3, NCX1/2/3, PMCA1/2/3, and PMCA4) decreased in cisplatin resistant cell line in comparison to the expression profiles in parental cells. Owing to the changes in the expression of genes involved in calcium regulation, these results show, drug resistance may be prevented by introducing a new perspective on the use of inhibitors and activators of these genes, and thus of cytostatic treatment strategies, due to changes in the expression of genes involved in calcium regulation.