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Using exome sequencing, we analyzed 196 participants of the Cretan Aging Cohort (CAC; 95 with Alzheimer's disease [AD], 20 with mild cognitive impairment [MCI], and 81 cognitively normal controls). The APOE ε4 allele was more common in AD patients (23.2%) than in controls (7.4%; p0.01) and the PSEN2 p.Arg29His and p.Cys391Arg variants were found in 3 AD and 1 MCI patient, respectively. Also, we found the frontotemporal dementia (FTD)-associated TARDBP gene p.Ile383Val variant in 2 elderly patients diagnosed with AD and in 2 patients, non CAC members, with the amyotrophic lateral sclerosis/FTD phenotype. Furthermore, the p.Ser498Ala variant in the positively selected GLUD2 gene was less frequent in AD patients (2.11%) than in controls (16%; p0.01), suggesting a possible protective effect. While the same trend was found in another local replication cohort (n = 406) and in section of the ADNI cohort (n = 808), this finding did not reach statistical significance and therefore it should be considered preliminary. Our results attest to the value of genetic testing to study aged adults with AD phenotype.

Characterization of the novel HLA-DRB1*04:311 and HLA-DRB1*11:277 alleles in two Greek individuals of Cretan origin.

Characterization of the novel HLA-C*15:228 and HLA-C*04:434 alleles in two Greek individuals of Cretan origin.

Characterization of HLA-DQB1*03:439 allele in a Greek individual of Cretan origin.

Background aims The high genetic diversity of HLA across populations significantly confines the effectiveness of a donor or umbilical cord blood search for allogeneic hematopoietic stem cell transplantation (HSCT). This study aims to probe the HLA immunogenetic profile of the population of Crete, a Greek region with specific geographic and historical characteristics, and to investigate potential patterns in HLA distribution following comparison with the Deutsche Knochenmarkspenderdatei (DKMS) donor registry. It also aims to highlight the importance of regional public cord blood banks (PCBBs) in fulfilling HSCT needs, especially in countries with significant genetic diversity. Methods A cohort of 1835 samples representative of the Cretan population was typed for HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) loci by high-resolution second field next-generation sequencing. Data were compared with the respective HLA profiles of 12 DKMS populations (n = 20 032). Advanced statistical and bioinformatics methods were employed to assess specific intra- and inter-population genetic indexes associated with the regional and geographic distribution of HLA alleles and haplotypes. Results A considerable HLA allelic and haplotypic diversity was identified among the Cretan samples and between the latter and the pooled DKMS cohort. Even though the HLA allele and haplotype frequency distribution was similar to regions of close geographic proximity to Crete, a clinal distribution pattern from the northern to southern regions was identified. Significant differences were also observed between Crete and the Greek population of DKMS. Conclusions This study provides an in-depth characterization of the HLA immunogenetic profile in Crete and reveals the importance of demographic history in HLA heterogeneity and donor selection. The novel HLA allele and haplotype frequency comparative data between the Cretan and other European populations signify the importance of regional PCBBs in prioritizing HLA diversity to efficiently promote the HSCT program at the national level and beyond.

Background Dent disease is an X-linked disorder characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and chronic kidney disease (CKD). It is caused by mutations in the chloride voltage-gated channel 5 (CLCN5) gene (Dent disease-1), or in the OCRL gene (Dent disease-2). It is associated with chronic metabolic acidosis; however metabolic alkalosis has rarely been reported. Case presentation We present a family with Dent-2 disease and a Bartter-like phenotype. The main clinical problems observed in the proband included a) primary phosphaturia leading to osteomalacia and stunted growth; b) elevated serum calcitriol levels, leading to hypercalcemia, hypercalciuria, nephrolithiasis and nephrocalcinosis; c) severe salt wasting causing hypotension, hyperaldosteronism, hypokalemia and metabolic alkalosis; d) partial nephrogenic diabetes insipidus attributed to hypercalcemia, hypokalemia and nephrocalcinosis; e) albuminuria, LMWP. Phosphorous repletion resulted in abrupt cessation of hypercalciuria and significant improvement of hypophosphatemia, physical stamina and bone histology. Years later, he presented progressive CKD with nephrotic range proteinuria attributed to focal segmental glomerulosclerosis (FSGS). Targeted genetic analysis for several phosphaturic diseases was unsuccessful. Whole Exome Sequencing (WES) revealed a c.1893C > A variant (Asp631Glu) in the OCRL gene which was co-segregated with the disease in male family members. Conclusions We present the clinical characteristics of the Asp631Glu mutation in the OCRL gene, presenting as Dent-2 disease with Bartter-like features. Phosphorous repletion resulted in significant improvement of all clinical features except for progressive CKD. Angiotensin blockade improved proteinuria and stabilized kidney function for several years. Graphical abstract

Characterization of two novel HLA-A alleles in two Greek individuals of Cretan origin.

Characterization of the HLA-B*51:232:02 allele in a Greek individual of Cretan origin.

Inherited muscle disorders are caused by pathogenic changes in numerous genes. Herein, we aimed to investigate the etiology of muscle disease in 24 consecutive Greek patients with myopathy suspected to be genetic in origin, based on clinical presentation and laboratory and electrophysiological findings and absence of known acquired causes of myopathy. Of these, 16 patients (8 females, median 24 years-old, range 7 to 67 years-old) were diagnosed by Whole Exome Sequencing as suffering from a specific type of inherited muscle disorder. Specifically, we have identified causative variants in 6 limb-girdle muscular dystrophy genes (6 patients; ANO5, CAPN3, DYSF, ISPD, LAMA2, SGCA), 3 metabolic myopathy genes (4 patients; CPT2, ETFDH, GAA), 1 congenital myotonia gene (1 patient; CLCN1), 1 mitochondrial myopathy gene (1 patient; MT-TE) and 3 other myopathy-associated genes (4 patients; CAV3, LMNA, MYOT). In 6 additional family members affected by myopathy, we reached genetic diagnosis following identification of a causative variant in an index patient. In our patients, genetic diagnosis ended a lengthy diagnostic process and, in the case of Multiple acyl-CoA dehydrogenase deficiency and Pompe’s disease, it enabled specific treatment to be initiated. These results further expand the genotypic and phenotypic spectrum of inherited myopathies., Highlights • 24 consecutive patients with myopathy were investigated via WES. • 16 patients and 6 family members with causative variants in 14 different genes • Causative variants in 6 LGMD genes (ANO5, CAPN3, DYSF, ISPD, LAMA2, SGCA) • Other genes involved were CPT2, ETFDH, GAA, CLCN1, LMNA, MT-TE, MYOT, CAV3. • These results expand the genotypic and phenotypic spectrum of inherited myopathies.

Over the last 10 years, Next-Generation Sequencing (NGS) has become a powerful tool in clinical genetics and precision medicine. Techniques like Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES) and Target Sequencing are commonly used for the elucidation of common and rare variants in mendelian and complex diseases. One of the most vital parts of NGS pipelines is the prioritization of annotated variants according to their clinical significance. During this process, a clinical geneticist is presented with annotation information from external databases for each of the thousands of potential variants. The vast amounts of data and the vague nature of existing guidelines for variant reporting, like ACMG (American College of Medical Genetics) can make this procedure very cumbersome and time consuming. Here we present the main computational challenges and existing solutions for this task. We also present Zazz, an online environment for variant annotation, query and exploration. Zazz can efficiently support the submission of complex and dynamically generated queries to hundreds of millions of variants each having hundreds of annotation fields. Zazz also leverages the capabilities of modern browsers to dynamically filter, explore and visualize multidimensional data.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Evaluation of: Sidransky E, Nalls MA, Aasly JO et al.: Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N. Engl. J. Med. 361, 1651–1661 (2009). This study aimed to substantiate the role of glucocerebrosidase (GBA) mutations in Parkinson’s disease (PD) by pooling clinical and genotypic data for 5691 PD patients and 4898 unaffected individuals from 16 international centers. Genotyping was performed for either two mutations (N370S and L444P) or for three to eight GBA mutations. One of the N370S and L444P mutations was found in 15 and 3% of PD patients of Ashkenazi and non-Ashkenazi Jewish origin, respectively. Whole gene sequencing performed in a subgroup of these patients revealed a combined odds ratio of carrying any GBA mutation of 5.43. Genotype–phenotype correlations verified that GBA mutation carriers were more likely to develop PD at an earlier age, have affected relatives and show increased incidence of cognitive changes compared with PD patients without mutations. This study provides evidence that heterozygosity for GBA mutations is the most common susceptibility factor for the onset of PD symptoms to date. It also highlights the need for further studies using large numbers of patients and controls in order to elucidate the effect(s) of GBA mutations on PD clinical manifestations and their role in the molecular mechanisms of PD pathogenesis.

Parkinson's disease (PD), a common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and their terminations in the basal ganglia, is thought to be related to genetic and environmental factors. Although the pathophysiology of PD neurodegeneration remains unclear, protein misfolding, mitochondrial abnormalities, glutamate dysfunction and/or oxidative stress have been implicated. In this study, we report that a rare T1492G variant in GLUD2, an X-linked gene encoding a glutamate dehydrogenase (a mitochondrial enzyme central to glutamate metabolism) that is expressed in brain (hGDH2), interacted significantly with age at PD onset in Caucasian populations. Individuals hemizygous for this GLUD2 coding change that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 developed PD 6–13 years earlier than did subjects with other genotypes in two independent Greek PD groups and one North American PD cohort. However, this effect was not present in female PD patients who were heterozygous for the DNA change. The variant enzyme, obtained by substitution of Ala for Ser445, showed an enhanced basal activity that was resistant to GTP inhibition but markedly sensitive to modification by estrogens. Thus, a gain-of-function rare polymorphism in hGDH2 hastens the onset of PD in hemizygous subjects, probably by damaging nigral cells through enhanced glutamate oxidative dehydrogenation. The lack of effect in female heterozygous PD patients could be related to a modification of the overactive variant enzyme by estrogens.

Mammalian glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is thought to localize to the mitochondrial matrix, although there are also suggestions for the extramitochondrial presence of this protein. Whereas GDH in mammals is encoded by the GLUD1 gene, humans and the great apes have, in addition, a GLUD2 gene showing a distinct expression pattern. The encoded hGDH1 and hGDH2 isoenzymes are highly homologous, but their leader sequences are more divergent. To explore their subcellular targeting, we constructed expression vectors in which hGDH1 or hGDH2 was fused with the enhanced green fluorescent protein (EGFP) and used these to transfect COS 7, HeLa, CHO, HEK293, or neuroblastoma SHSY-5Y cells. Confocal microscopy revealed GDH-EGFP fluorescence in the cytoplasm within coarse structures. Cotransfection experiments using organelle-specific markers revealed that hGDH1 or hGDH2 colocalized with the mitochondrial marker DsRed2-Mito and to a lesser extent with the endoplasmic reticulum marker DsRed2-ER. Western blots detected two GDH-EGFP specific bands: a ~90 kDa band and a ~95 kDa band associated with the mitochondria and the endoplasmic reticulum containing cytosol, respectively. Deletion of the signal sequence, while altering drastically the fluoresce distribution within the cell, prevented GDH from entering the mitochondria, with the ~90 kDa band being retained in the cytosol. In addition, the deletion eliminated the ~95 kDa band from cell lysates, thus confirming that it represents the full-length GDH. Hence, while most of the hGDHs translocate into the mitochondria (a process associated with cleavage of the signal sequence), part of the protein localizes to the endoplasmic reticulum, probably serving additional functions.

In all mammals, glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is encoded by a single gene (GLUD1 in humans) which is expressed widely (housekeeping). Humans and other primates also possess a second gene, GLUD2, which encodes a highly homologous GDH isoenzyme (hGDH2) expressed predominantly in retina, brain and testis. There is evidence that GLUD1 was retro-posed

Aim: Studying exceptional responders to standard treatments may provide broader insights into the genetic mechanisms underlying such responses and suggest therapeutic options that weren’t previously apparent. In this study we unravelled the genetic signature of undifferentiated sarcoma in order to disclose the molecular mechanisms that may underlie the patient’s exquisite sensitivity to Pazopanib. Methods: A female patient with massive undifferentiated gynecological sarcoma experienced a dramatic response to Pazopanib that made debulking surgery possible. We performed whole exome sequencing on DNA isolated from both blood and formalin fixed paraffin-embedded (FFPE) tumour employing Ion Torrent technology. Variant annotation was performed using the Ion ReporterTM v5.0 (MA, USA) tool after application of specific filters. Subsequent curation of annotated data was completed using a combined semi-automated data analyses workflow developed by Minotech Genomics Facility with the implementation of two in house-developed lists regarding 24 pazopanib-related genes, and 96 angiogenesis-associated genes. We also performed targeted sequencing of Ion Ampliseq Comprehensive Cancer panel (CCP) that is strategically designed to interrogate coding DNA sequences and splice variants across 409 tumour suppressor genes and oncogenes. Results: Whole exome sequencing was conducted at 20x coverage for 91.9% and 60.5% of >19,000 genes for germline and FFPE DNA samples, respectively. Targeted sequencing was conducted at 20x for >97% of 409 genes of the comprehensive cancer panel. Comparative analyses revealed two variants common in germline and FFPE samples, localized in LTK(p.Val541Ile) and FGFR4 (p.Gly388Arg). Interestingly, examination of the annotated data for biologically plausible mechanisms revealed 2 yet uncharacterized heterozygous variants in two other members of the FGFR family, FGFR2 (c.2183A>G, p.Asn728Ser) and FGFR3 (c.2050G>A, p.Val684Ile) that were present only in the FFPE samples. Even though both variants reside within the catalytic tyrosine kinase domains, they are predicted to exert tolerated effects on the encoded FGFR2 and FGFR3 proteins. Since, Asn728Ser is highly conserved among different kinases of the FGFR family we chose this variant to predict a possible effect on Pazopanib binding. Specifically, protein structure simulation of FGFR2 revealed that asparagine residue at position 728 is located in the external side of the drug binding cavity and is thus predicted to not affect Pazopanib binding. Conclusion: We pursue the possible genetic or somatic modifiers underling the excessive response to Pazopanib. The detection of two, yet uncharacterised, variants in known Pazopanib targets highlights the potential of next generation sequencing in disclosing the genetic signature of undifferentiated sarcomas in relation to the activity and effectiveness of certain therapeutic agents. Note: This abstract was not presented at the meeting. Citation Format: Emmanouil Saloustros, Helen Latsoudis, Despoina Vassou, Galateia Kallergi, Irene Stratidaki, Niki-Antonia Gounalaki, Vasiliki Fadouloglou, Ioannis Drositis, Emmanouil Kontopodis, Kitty Pavlakis, Dimitrios Mavroudis, Nikolaos Androulakis, Dimitrios Kafetzopoulos. The pursue of genetic mechanisms underlying supreme response to pazopanib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2017-3740

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6–13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme.

Glutamate dehydrogenase (GDH) in human exists in GLUD1 and GLUD2 gene-encoded isoforms (hGDH1 and hGDH2, respectively), differing in their regulation and tissue expression pattern. Whereas hGDH1 is subject to GTP control, hGDH2 uses for its regulation, a novel molecular mechanism not requiring GTP. This is based on the ability of hGDH2 to maintain a baseline activity of

Mitochondrial complex I deficiency has been implicated in the pathogenesis of Parkinson's disease (PD), but as yet no mitochondrial DNA (mtDNA) variations have been identified that could account for the impaired complex I activity. On the other hand, it has been suggested that mtDNA polymorphisms (mtSNPs) or haplogroups may modify the risk of developing PD. Here, we determined the distributions of ten mtSNPs that define the nine major European haplogroups among 224 PD patients and 383 controls from Crete, an island of 0.6 million inhabitants who share a similar genetic background and a common environment. The recruitment of patients and controls was restricted to individuals of Cretan origin for at least three generations from both parental sides in order to avoid population admixture and subsequent genetic heterogeneity. We found no mtSNP or mtDNA haplogroup that predisposes to PD, although there was a trend for haplogroups J, T, U and I and the supercluster of haplogroups UKJT to be slightly underrepresented in our PD patients as compared to controls. While a combination of common mtSNPs (present inor=5% of the general population) may decrease the chance of developing PD, this effect was minor in the Cretan population.

MicroRNA signature of THP1 cells revealed a 5.9-fold decreased expression of miR-4520a following siRNA-mediated knockdown of MEFV gene that encodes pyrin [1].

Background MicroRNA signature of THP1 cells revealed a 5.9 fold decreased expression of miR-4520a following siRNA-mediated knockdown of MEFV gene that encodes pyrin (1). Objectives We herein sought to validate the expression levels of miR-4520a in monocytes isolated from peripheral blood mononuclear cells (PBMCs) of FMF patients. Methods Dual luciferase assay and t-test analyses were used to determine the predicted target of miR-4520a, Rheb, after cloning its 3'UTR region containing miR-4520a recognition element into PGL3-prom vector. The expression levels of pyrin, miR-4520a and its putative target Rheb were validated in monocytes from FMF patients (n=10) and compared with healthy controls (n=8). Patients were off colchicine for two days (attack-free period) and monocytes were isolated from PBMCs. Total RNA together with the respective miRNA enriched fractions were isolated from monocytes and used for mRNA and miR-4520a quantitation by real-time quantitative PCR. Expression levels of mRNAs and miRNA were determined with the 2-ΔΔCt method after normalizing to 18S RNA and RNU6B housekeeping genes, respectively. Protein levels of pyrin and Rheb were detected by western blotting. Results The relative expression levels of miR-4520a were variable among FMF patients and not significantly different between patients and controls. Stratification of patients group by genotype revealed an intriguing difference in miR-4520a relative expression. Specifically, miR-4520a expression was lower in carriers of M694V variant (combined group of homo- and heterozygotes), whereas the rest of FMF genotypes had higher levels (p

Mutations of the LRRK2 gene that encodes the protein dardarin occur in Parkinson disease (PD)1 with the G2019S change being the most common cause of hereditary parkinsonism. While the G2019S mutation phenotype is that of typical PD,2 three different amino acid substitutions at residue 1441 (4322G>A: [R1441H], 4321C>T: [R1441C], 4321C>G: [R1441G]) have been associated with pleomorphic phenotypic expressions.3 There is evidence, however, that not all LRRK2 mutations are sufficient to cause PD and that additional genetic or environmental factors may be involved. An oligogenic inheritance for PD has also been suggested in a study of 247 index patients with PD from Crete.4 Here we searched for susceptibility genes to PD in our cohort of 266 cases with PD and 13 patients with progressive supranuclear palsy (PSP) by testing them for LRRK2 mutations. PD was diagnosed by established criteria.4 PSP was diagnosed by the National Institute of Neurologic Disorders and Stroke–SPSP criteria, requiring early postural instability and falls, vertical supranuclear palsy, pseudobulbar palsy, and akinetic-rigid parkinsonism (with prominent axial rigidity) unrelieved by levodopa. Controls (n = 300) were healthy subjects natives of Crete. All patients and controls were analyzed for five LRRK2 …

Background: Horizontal gaze palsy and progressive scoliosis (HGPPS) is caused by mutations of the ROBO3 gene, which encodes a receptor associated with axonal guidance during development. Although there is evidence for uncrossed cuneatal and corticospinal tracts in HGPPS, it is unclear whether other central nervous system pathways are involved. Objective: To study two patients with HGPPS homozygotic for the ROBO3 E319K mutation using a variety of neurophysiological and neuropsychological tests. Methods: A battery of neuropsychological tests was applied to assess various cognitive and perceptual functions. The corticospinal, somatosensory and auditory pathways were evaluated using appropriate neurophysiological tests. To access motor pathways to the neck muscles, electromyographic recordings were obtained from the sternocleidomastoideus and splenius capitis muscle during active head rotation. Results: Both patients performed normally on manual dexterity, complex sensory and visuospatial functions, reading and general intelligence tests. Motor evoked potentials in both patients showed uncrossed corticospinal tracts for the extremities, although in one patient, electromyography indicated pyramidal tract crossing for the neck muscles. Although somatosensory evoked potentials showed uncrossed somatosensory fibres subserving proprioception and light touch, right median nerve somatosensory evoked potential in one patient indicated a partial lemniscal crossing. Sympathetic skin response and blink reflex showed a midline crossing of the spinothalamic and quintothalamic tracts. Brain stem auditory evoked potentials indicated a lack of crossing in the level of the trapezoid body. Conclusions: Our patients with the ROBO3 E319Κ mutation show normal perceptual and cognitive functions and have both crossed and uncrossed motor, sensory and auditory pathways.

Various deletions and point mutations in the Parkin gene have been strongly associated with Parkinson’s disease (PD) and parkinsonism, especially when the onset occurs at a young age. In this study, we screened 25 ‘early-onset’ (2 = 6.54, p = 0.011, odds ratio = 1.61, 95% confidence interval, CI, 1.10–2.37), as was the 167Asn genotype (17.3 vs. 11.3%; p = 0.04). The frequency of the 167Ser genotype was significantly lower in PD patients than in controls when compared with that of the other two genotypes combined (χ2 = 7.84, p = 0.005, odds ratio = 0.46, 95% CI 0.25 – 0.82). No significant differences in the frequencies of the allele and genotypes were found between patients classified into two groups according to symptoms at onset (χ2 = 0.191, p = 0.66, odds ratio = 1.12, 95% CI 0.65–1.95; χ2 = 0.24, p = 0.887) or age of onset (p = 0.787). In summary, homozygous deletion mutations and point mutations in exons 1–12 of the Parkin gene were not detected in this Han Chinese population, although we cannot exclude compound heterozygous deletions. In addition, our study suggests that the variant 167Asn increases the risk of developing PD.

Background Mutations in MEFV gene encoding pyrin [1] account for Familial Mediterranean fever FMF, a recessively inherited disease but the high number of heterozygote patients is puzzling and requires additional investigation [2,3]. Objectives Elucidation of the pathophysiology of the disease, through the identification of novel genes and miRNAs that interact with or regulate the MEFV gene. Methods Silencing of MEFV gene by AccellsiRNAs was performed in THP-1 cell line that expresses endogenous pyrin. Global changes in mRNA and microRNA expression were assessed by GeneChip HuGene-1.0-ST-v1 and GeneChipmiRNA 3.0 Array, respectively. Putative target genes of the differentially regulated miRNAs were identified by using appropriate algorithms (TargetScan, PicTar). Functional classifications and biological processes of differentially expressed mRNAs and miRNA target genes were assigned according to Gene Ontology (GO) and the updated Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. mRNA and miRNA target genetic or protein interactions were constructed using Cytoscape 3.0.2 (Plugins GeneMania and BioGrid). Results We achieved 62% knockdown of the MEFV mRNA as measured by Taqman PCR, following siRNA-mediated knockdown of the respective gene.Global transcriptome analysis revealed 89 differentially expressed genes and 25 deregulated miRNAs involved in inflammatory-mediated responses, cell cycle, cytoskeleton/microtubule dynamics, intracellular trafficking and survival. Specifically, miR-4520a and miR-19b demonstrate altered expression levels during TLR-mediated signaling, which corroborate the role of pyrin in the regulation of inflammatory responses. Interestingly, the aforementioned deregulated miRNAs are also shown to play a significant role in intracellular membrane trafficking and subsequently autophagy through regulation of small RabGTPases. Bioinformatic analyses predicted high cooperativity within the significantly deregulated miRNAs, with the clustering of 8 (out of 11) members of the miR-378/422a superfamily being the most pertinent example. Conclusions These findings reiterate the involvement of pyrin in innate immune responses and suggest a potential role of this protein in autophagy, probably through regulation of membrane trafficking RabGTPases. Validation of this data in monocytes from FMF patients is in progress. References The International FMF Consortium (1997). Cell 90:797–807. Booty M.G., Chae J.J., et al (2009). Ann. Rheum. Dis. 60:1851-1861. Touitou I, Picot MC, Domingo C, et al. (2001). Arthritis Rheum. 44:163–169. Acknowledgements Mir-Farzin Mashreghi, Helen Latsoudis and Joachim Gruen contributed equally. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2470

The Roc domain of the Lrrk2 protein harbors two pathogenic mutations which cause autosomal dominant parkinsonism (R1441C and R1441G). A third putatively pathogenic variant (R1441H) has been identified in four probands of diverse ethnicity with parkinsonism. Herein we show that the R1441H substitutions lie on different haplotypes within our patients, confirming this codon as a mutational hotspot. The absence of this variant in control subjects and the presence of two other pathogenic variants at this amino acid position collectively support the contention that 81441 H is a pathogenic substitution. (C) 2008 Elsevier Ltd. All rights reserved.

Background Transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases, are thought to be caused by an abnormal isoform of a naturally occurring protein known as cellular prion protein, PrPC. The abnormal form of prion protein, PrPSc accumulates in the brain of affected individuals. Both isoforms are encoded by the same prion protein gene (PRNP), and the structural changes occur post-translationally. Certain mutations in the PRNP gene result in genetic TSEs or increased susceptibility to TSEs. Case presentation A 70 year old woman was admitted to the hospital with severe confusion and inability to walk. Relatives recognized memory loss, gait and behavioral disturbances over a six month period prior to hospitalization. Neurological examination revealed Creutzfeldt-Jakob disease (CJD) related symptoms such as incontinence, Babinski sign and myoclonus. EEG showed periodic sharp waves typical of sporadic CJD and cerebrospinal fluid analysis (CSF) was positive for the presence of the 14-3-3-protein. As the disease progressed the patient developed akinetic mutism and died in the tenth month after onset of the disease symptoms. Unfortunately, no autopsy material was available. PRNP sequencing showed the occurrence of a point mutation on one allele at codon 193, which is altered from ACC, coding for a threonine, to ATC, encoding an isoleucine (T193I). Conclusion Here we report a novel mutation of the PRNP gene found in an elderly female patient resulting in heterozygosity for isoleucine and threonine at codon 193, in which normally homozygosity for threonine is expected (T193). The patient presented typical clinical symptoms of CJD. EEG findings and the presence of the 14-3-3 protein in the CSF, contributed to CJD diagnosis, allowing the classification of this case as a probable CJD according to the World Health Organization (WHO) accepted criteria.