Repeat expansions in AR, ATXN1, ATXN2 and HTT in Norwegian patients diagnosed with amyotrophic lateral sclerosis.

Genetic repeat expansions cause neuronal degeneration in amyotrophic lateral sclerosis as well as other neurodegenerative disorders such as spinocerebellar ataxia, Huntington's disease and Kennedy's disease. Repeat expansions in the same gene can cause multiple clinical phenotypes. We aimed to characterize repeat expansions in a Norwegian amyotrophic lateral sclerosis cohort. Norwegian amyotrophic lateral sclerosis patients (n = 414) and neurologically healthy controls adjusted for age and gender (n = 713) were investigated for repeat expansions in AR, ATXN1, ATXN2 and HTT using short read exome sequencing and the ExpansionHunter software. Five amyotrophic lateral sclerosis patients (1.2%) and two controls (0.3%) carried ≥36 repeats in HTT (P = 0.032), and seven amyotrophic lateral sclerosis patients (1.7%) and three controls (0.4%) carried ≥29 repeats in ATXN2 (P = 0.038). One male diagnosed with amyotrophic lateral sclerosis carried a pathogenic repeat expansion in AR, and his diagnosis was revised to Kennedy's disease. In ATXN1, 50 amyotrophic lateral sclerosis patients (12.1%) and 96 controls (13.5%) carried ≥33 repeats (P = 0.753). None of the patients with repeat expansions in ATXN2 or HTT had signs of Huntington's disease or spinocerebellar ataxia type 2, based on a re-evaluation of medical records. The diagnosis of amyotrophic lateral sclerosis was confirmed in all patients, with the exception of one patient who had primary lateral sclerosis. Our findings indicate that repeat expansions in HTT and ATXN2 are associated with increased likelihood of developing amyotrophic lateral sclerosis. Further studies are required to investigate the potential relationship between HTT repeat expansions and amyotrophic lateral sclerosis.

Clinical characteristics and proteome modifications in two Charcot-Marie-Tooth families with the AARS1 Arg326Trp mutation.

BACKGROUND: Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1. The pathogenic mechanism underlying AARS1-related neuropathy is not known. METHODS: From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an "in-house" next-generation sequencing panel. DNA from patient's family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls. RESULTS: Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74-98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842. CONCLUSION: This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy.

Heterozygous variants in ZBTB7A cause a neurodevelopmental disorder associated with symptomatic overgrowth of pharyngeal lymphoid tissue, macrocephaly, and elevated fetal hemoglobin.

By clinical whole exome sequencing, we identified 12 individuals with ages 3 to 37 years, including three individuals from the same family, with a consistent phenotype of intellectual disability (ID), macrocephaly, and overgrowth of adenoid tissue. All 12 individuals harbored a rare heterozygous variant in ZBTB7A which encodes the transcription factor Zinc finger and BTB-domain containing protein 7A, known to play a role in lympho- and hematopoiesis. ID was generally mild. Fetal hemoglobin (HbF) fraction was elevated 2.2%-11.2% (reference value <2% in individuals > 6 months) in four of the five individuals for whom results were available. Ten of twelve individuals had undergone surgery at least once for lymphoid hypertrophy limited to the pharynx. In the most severely affected individual (individual 1), airway obstruction resulted in 17 surgical procedures before the age of 13 years. Sleep apnea was present in 8 of 10 individuals. In the nine unrelated individuals, ZBTB7A variants were novel and de novo. The six frameshift/nonsense and four missense variants were spread throughout the gene. This is the first report of a cohort of individuals with this novel syndromic neurodevelopmental disorder.

Genotype-phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders.

BACKGROUND: We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. METHODS: Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. RESULTS: We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. CONCLUSIONS: Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

NEXMIF encephalopathy: an X-linked disorder with male and female phenotypic patterns.

PURPOSE: Pathogenic variants in the X-linked gene NEXMIF (previously KIAA2022) are associated with intellectual disability (ID), autism spectrum disorder, and epilepsy. We aimed to delineate the female and male phenotypic spectrum of NEXMIF encephalopathy. METHODS: Through an international collaboration, we analyzed the phenotypes and genotypes of 87 patients with NEXMIF encephalopathy. RESULTS: Sixty-three females and 24 males (46 new patients) with NEXMIF encephalopathy were studied, with 30 novel variants. Phenotypic features included developmental delay/ID in 86/87 (99%), seizures in 71/86 (83%) and multiple comorbidities. Generalized seizures predominated including myoclonic seizures and absence seizures (both 46/70, 66%), absence with eyelid myoclonia (17/70, 24%), and atonic seizures (30/70, 43%). Males had more severe developmental impairment; females had epilepsy more frequently, and varied from unaffected to severely affected. All NEXMIF pathogenic variants led to a premature stop codon or were deleterious structural variants. Most arose de novo, although X-linked segregation occurred for both sexes. Somatic mosaicism occurred in two males and a family with suspected parental mosaicism. CONCLUSION: NEXMIF encephalopathy is an X-linked, generalized developmental and epileptic encephalopathy characterized by myoclonic-atonic epilepsy overlapping with eyelid myoclonia with absence. Some patients have developmental encephalopathy without epilepsy. Males have more severe developmental impairment. NEXMIF encephalopathy arises due to loss-of-function variants.

De Novo Variants in TAOK1 Cause Neurodevelopmental Disorders.

De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs.

Genetic and Phenotypic Characterization of Community Hospital Patients With QT Prolongation.

Background Congenital long- QT syndrome ( LQTS ) is a genetic disorder characterized by prolongation of the corrected QT interval ( QT c) on an ECG . The aim of the present study was to estimate the prevalence of pathogenic and likely pathogenic sequence variants in patients who had at least 1 ECG with a QT c ≥500 ms. Methods and Results Telemark Hospital Trust is a community hospital within the Norwegian national health system, serving ≈173 000 inhabitants. We searched the ECG database at Telemark Hospital Trust, Norway, from January 2004 to December 2014, and identified 1531 patients with at least 1 ECG with a QT c ≥500 ms. At the time of inclusion in this study (2015), 766 patients were alive. A total of 733 patients were invited to participate, and 475 accepted. The 17 genes that have been reported to cause monogenic LQTS were sequenced among the patients. Pro- QT c score was calculated for each patient. A molecular genetic cause of LQTS was detected in 31 (6.5%) of 475 patients. These patients had a lower pro- QT c score than those without pathogenic or likely pathogenic variants (1.7±1.0 versus 2.8±1.6; P<0.001). Conclusions Compared with the general population, hospitalized patients with a QT c ≥500 ms in at least 1 ECG recording had an increased likelihood for pathogenic and likely pathogenic variants in LQTS genes. We recommend increased awareness of the possibility of LQTS in patients with at least 1 ECG with a QT c ≥500 ms.

Next-generation sequencing of the monogenic obesity genes LEP, LEPR, MC4R, PCSK1 and POMC in a Norwegian cohort of patients with morbid obesity and normal weight controls.

BACKGROUND: Rare sequence variants in at least five genes are known to cause monogenic obesity. In this study we aimed to investigate the prevalence of, and characterize, rare coding and splice site variants in LEP, LEPR, MC4R, PCSK1 and POMC in patients with morbid obesity and normal weight controls. METHOD: Targeted next-generation sequencing of all exons in LEP, LEPR, MC4R, PCSK1 and POMC was performed in 485 patients with morbid obesity and 327 normal weight population-based controls from Norway. RESULTS: In total 151 variants were detected. Twenty-eight (18.5%) of these were rare, coding or splice variants and five (3.3%) were novel. All individuals, except one control, were heterozygous for the 28 variants, and the distribution of the rare variants showed a significantly higher carrier frequency among cases than controls (9.9% vs. 4.9%, p=0.011). Four variants in MC4R were classified as pathogenic or likely pathogenic. CONCLUSION: Four cases (0.8%) of monogenic obesity were detected, all due to MC4R variants previously linked to monogenic obesity. Significant differences in carrier frequencies among patients with morbid obesity and normal weight controls suggest an association between heterozygous rare coding variants in these five genes and morbid obesity. However, additional studies in larger cohorts and functional testing of the novel variants identified are required to confirm the findings.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

Clinical exome sequencing ­ Norwegian findings.

BACKGROUND: New DNA-sequencing technology is revolutionising medical diagnostics. Through the use of exome sequencing, it is now possible to sequence all human genes in parallel. This technology has been widely used in research over the last few years and is now also being applied to diagnostics. The aim of this study was to systematically examine initial experiences with diagnostic exome sequencing in Norway. MATERIAL AND METHOD: This is a retrospective observational study of the results of all exome sequencing performed by the Section of Medical Genetics at Telemark Hospital between December 2012 and October 2014, and includes 125 persons in 46 families. The majority of these families were being investigated for a syndrome (n = 35, 76%) or neurological disease (n = 9, 20%). RESULTS: Exome sequencing detected pathogenic sequence variants in 15 of 46 probands, and variants of unknown significance in 12 probands. Of the 100 patients who stated their wishes regarding feedback of any incidental findings, six indicated that they did not wish to receive such information. There were no incidental findings in this study, but neither were such sequence variants actively looked for. INTERPRETATION: Exome sequencing can enable more patients with syndromes or neurological diseases to receive a causal diagnosis, and to receive this diagnosis at an earlier stage. However, the patients in this study were quite highly selected, and the results must therefore be interpreted with caution.

Hereditary peripheral neuropathies diagnosed by next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) is a genetic technique used to determine the order of nucleotides in DNA. The technique has proved to be more efficient than the traditional method, Sanger sequencing, for sequencing multiple genes. NGS is now being used to diagnose disorders in which multiple genes are involved. This study has examined whether next-generation sequencing produces a greater number of positive diagnoses than its traditional counterpart in patients with suspected hereditary peripheral neuropathy. MATERIAL AND METHOD: This study is a retrospective review of samples from 103 patients investigated for hereditary peripheral neuropathy, received by Telemark Hospital in the period 2012-14. After exclusion of duplication/deletion of PMP22, 96 samples were analysed by NGS with physical enrichment of 52 hereditary peripheral neuropathy genes. RESULTS: A genetic cause was identified in 35 patients (34%) with peripheral neuropathy, of which 28 (27%) were point mutations identified by NGS. INTERPRETATION: Of the pathogenic point mutations identified in this study, 12 were in genes that would previously have been analysed by Sanger sequencing in our department, whereas 16 were in genes that would not previously have been tested.

Genetic diagnosis of Charcot-Marie-Tooth disease in a population by next-generation sequencing.

Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited neuropathy. Today more than 40 CMT genes have been identified. Diagnosing heterogeneous diseases by conventional Sanger sequencing is time consuming and expensive. Thus, more efficient and less costly methods are needed in clinical diagnostics. We included a population based sample of 81 CMT families. Gene mutations had previously been identified in 22 families; the remaining 59 families were analysed by next-generation sequencing. Thirty-two CMT genes and 19 genes causing other inherited neuropathies were included in a custom panel. Variants were classified into five pathogenicity classes by genotype-phenotype correlations and bioinformatics tools. Gene mutations, classified certainly or likely pathogenic, were identified in 37 (46%) of the 81 families. Point mutations in known CMT genes were identified in 21 families (26%), whereas four families (5%) had point mutations in other neuropathy genes, ARHGEF10, POLG, SETX, and SOD1. Eleven families (14%) carried the PMP22 duplication and one family carried a MPZ duplication (1%). Most mutations were identified not only in known CMT genes but also in other neuropathy genes, emphasising that genetic analysis should not be restricted to CMT genes only. Next-generation sequencing is a cost-effective tool in diagnosis of CMT improving diagnostic precision and time efficiency.

Familial visceral myopathy diagnosed by exome sequencing of a patient with chronic intestinal pseudo-obstruction.

A 55-year-old woman with a history of bowel dysmotility presented with abdominal distension and peritonitis. Family history included premature deaths with intestinal symptomatology, suggesting autosomal dominant inheritance. Computed tomography showed a distended small bowel. Symptoms were alleviated by enterocutaneous stomas. Initial ileal biopsy suggested neuropathy; however, exome sequencing revealed an Arg148Ser mutation in the enteric smooth muscle actin gamma 2 (ACTG2) gene. Histological reassessment showed abnormal muscularis propria and smooth muscle actin, with the same findings in sibling, confirming familial visceral myopathy. Thus, noninvasive genomic analysis can provide early and specific diagnosis of familial visceral myopathy, which may help to avoid inappropriate surgery.

Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and ß-zearalenol.

α- and ß-zearalenol (α-ZOL and ß-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of α-ZOL and ß-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by α-ZOL and ß-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.

Relative quantification of the proteomic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model.

Zearalenone (ZEN) is a mycotoxin with endocrine disrupting effects having vast economic implications in e.g. pig farming. Structurally, ZEN resembles 17ß-estradiol, and thus is able to bind to estrogen receptors (ER) in target cells. Because of this, it is also classified as a non-steroidal estrogen, a phytoestrogen, a mycoestrogen, and a growth promoter. Quantitative proteomic analysis was undertaken using stable-isotope labeling by amino acids in cell culture (SILAC) upon exposure of the steroidogenesis cell model H295R with ZEN to elucidate its effect on protein regulation. ZEN significantly regulated 21 proteins, including proteins with known endocrine disrupting effects and several oncogenes. In addition, network analysis using Ingenuity Pathway Analysis showed that ZEN affected the oxidative phosphorylation pathway and the mitochondrial dysfunction pathway, both previously reported to be involved in endocrine dysfunction.

Identification of surface proteins in Enterococcus faecalis V583.

BACKGROUND: Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. RESULTS: Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31) or to be secreted (5). Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. CONCLUSIONS: The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5) and surface (31) proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.