Early life involvement in C9orf72 repeat expansion carriers.

OBJECTIVES: The chromosome 9 open reading frame 72 gene (C9orf72) hexanucleotide repeat expansion (C9orf72RE) is the most common genetic cause of behavioural variant frontotemporal dementia (bvFTD). Since the onset of the C9orf72RE-associated disease is sometimes hard to define, we hypothesise that C9orf72RE may cause a lifelong neuropsychiatric vulnerability. The first aim of our study was to explore lifelong behavioural and personality characteristics in C9orf72RE. Second, we aimed to describe distinctive characteristics of C9orf72RE during disease course. METHODS: Out of 183 patients from the Amsterdam Dementia Cohort that underwent genetic testing between 2011 and 2018, 20 C9orf72RE bvFTD patients and 23 C9orf72RE negative bvFTD patients were included. Patients and their relatives were interviewed extensively to chart their biography. Data analysis was performed through a mixed-methods approach including qualitative and quantitative analyses. RESULTS: Education, type of professional career and number of intimate partners were not different between carriers and non-carriers. Carriers were more often described by their relatives as having 'fixed behavioural patterns in daily life' and with limited empathy already years before onset of bvFTD symptoms. In carriers, disease course was more often characterised by excessive buying and obsessive physical exercise than in non-carriers. CONCLUSION: This is the first study thoroughly exploring biographies of bvFTD patients with C9orf72RE, revealing that subtle personality traits may be present early in life. Our study suggests that C9orf72RE exerts a lifelong neuropsychiatric vulnerability. This may strengthen hypotheses of links between neurodevelopmental and neurodegenerative diseases. Moreover, the presence of a distinct C9orf72RE -associated syndrome within the FTD spectrum opens doors for investigation of vulnerable neuronal networks.

Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.

LRSAM1-mediated ubiquitylation is disrupted in axonal Charcot-Marie-Tooth disease 2P.

Charcot-Marie-Tooth (CMT) disease type 2 is a genetically heterogeneous group of inherited neuropathies characterized by motor and sensory deficits as a result of peripheral axonal degeneration. We recently reported a frameshift (FS) mutation in the Really Interesting New Gene finger (RING) domain of LRSAM1 (c.2121_2122dup, p.Leu708Argfs) that encodes an E3 ubiquitin ligase, as the cause of axonal-type CMT (CMT2P). However, the frequency of LRSAM1 mutations in CMT2 and the functional basis for their association with disease remains unknown. In this study, we evaluated LRSAM1 mutations in two large Dutch cohorts. In the first cohort (n = 107), we sequenced the full LRSAM1 coding exons in an unbiased fashion, and, in the second cohort (n = 468), we specifically sequenced the last, RING-encoding exon in individuals where other CMT-associated genes had been ruled out. We identified a novel LRSAM1 missense mutation (c.2120C > T, p.Pro707Leu) mapping to the RING domain. Based on our genetic analysis, the occurrence of pathogenic LRSAM1 mutations is estimated to be rare. Functional characterization of the FS, the identified missense mutation, as well as of another recently reported pathogenic missense mutation (c.2081G > A, p.Cys694Tyr), revealed that in vitro ubiquitylation activity was largely abrogated. We demonstrate that loss of the E2-E3 interaction that is an essential prerequisite for supporting ubiquitylation of target substrates, underlies this reduced ubiquitylation capacity. In contrast, LRSAM1 dimerization and interaction with the bona fide target TSG101 were not disrupted. In conclusion, our study provides further support for the role of LRSAM1 in CMT and identifies LRSAM1-mediated ubiquitylation as a common determinant of disease-associated LRSAM1 mutations.

Pseudodominant inheritance pattern in a family with CMT2 caused by GDAP1 mutations.

We report a family in which an autosomal dominantly inherited Charcot-Marie-Tooth (CMT) disease type 2 was suspected. The affected family members (proband, sister, father, and paternal aunt) showed intrafamilial clinical variability. The proband needed walking aids since adolescence because of generalized muscle weakness. The sister showed the same symptoms although to a lesser extent. The father and paternal aunt had foot deformity and atrophy of lower legs. A homozygous GDAP1 mutation was found in the proband and in the sister. Further testing showed compound heterozygous GDAP1 mutations in the father and paternal aunt. In this CMT2 family with a pseudodominant inheritance pattern DNA-diagnostics revealed the presence of both homozygous and compound heterozygous GDAP1 mutations. We recommend including multiple family members in genetic studies on CMT families.

tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia.

Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.

Myoclonus-dystonia: An under-recognized entity - Report of 5 cases.

Hereditary myoclonus-dystonia (DYT 11) is caused by the epsilon-sarcoglycan (SGCE) mutation. The clinical details and investigations of cases diagnosed with myoclonus-dystonia were reviewed. We describe 5 patients (3 families) with myoclonus-dystonia diagnosed at our center. Majority of the patients had the classical phenotype with few atypical features (adult-onset disease and onset in lower limbs). Four patients carried a mutant variant in the SGCE-gene. A diagnosis of myoclonus-dystonia should be considered in cognitively normal patients with early-onset myoclonus (that may occur both at rest and/or action) with or without dystonia and with or without psychiatric-disturbances.

Pathogenic variants in three families with distal muscle involvement.

Three families suspected of distal hereditary motor neuropathy underwent genetic screening with the aim to identify the molecular defect underlying the disease. The description of the identification reflects the shift in molecular diagnostics that was made during the last decades. Our candidate gene approach yielded a known pathogenic variant in BSCL2 (p.Asn88Ser) in one family, and via a CMT-capture, in HSPB1 (p.Arg127Trp), in addition to five other variations in Charcot-Marie-Tooth-related genes in the proband of the second family. In the third family, using whole exome sequencing, followed by linkage-by-location, a three base pair deletion in exon 33 of MYH7 (p.Glu1508del) was found, a reported pathogenic allele albeit for a myopathy. After identification of the causative molecular defect, cardiac examination was performed for patients of the third family and this demonstrated abnormalities in three out of five affected family members. Heterogeneity and expansion of clinical phenotypes beyond known characteristics requires a wider set of genes to be screened. Whole exome/genome analysis with limited prior clinical information may therefore be used to precede a detailed clinical evaluation in cases of large families, preventing screening of a too narrow set of genes, and enabling the identification of novel disease-associated genes. In our cases, the variants had been reported, and co-segregation analysis confirmed the molecular diagnosis.

Clinical, neuroradiological and genetic findings in pontocerebellar hypoplasia.

Pontocerebellar hypoplasia is a group of autosomal recessive neurodegenerative disorders with prenatal onset. The common characteristics are cerebellar hypoplasia with variable atrophy of the cerebellum and the ventral pons. Supratentorial involvement is reflected by variable neocortical atrophy, ventriculomegaly and microcephaly. Mutations in the transfer RNA splicing endonuclease subunit genes (TSEN54, TSEN2, TSEN34) were found to be associated with pontocerebellar hypoplasia types 2 and 4. Mutations in the mitochondrial transfer RNA arginyl synthetase gene (RARS2) were associated with pontocerebellar hypoplasia type 6. We studied a cohort of 169 patients from 141 families for mutations in these genes, of whom 106 patients tested positive for mutations in one of the TSEN genes or the RARS2 gene. In order to delineate the neuroradiological and clinical phenotype of patients with mutations in these genes, we compared this group with 63 patients suspected of pontocerebellar hypoplasia who were negative on mutation analysis. We found a strong correlation (P < 0.0005) between TSEN54 mutations and a dragonfly-like cerebellar pattern on magnetic resonance imaging, in which the cerebellar hemispheres are flat and severely reduced in size and the vermis is relatively spared. Mutations in TSEN54 are clinically associated with dyskinesia and/or dystonia and variable degrees of spasticity, in some cases with pure generalized spasticity. Nonsense or splice site mutations in TSEN54 are associated with a more severe phenotype of more perinatal symptoms, ventilator dependency and early death. In addition, we present ten new mutations in TSEN54, TSEN2 and RARS2. Furthermore, we show that pontocerebellar hypoplasia type 1 together with elevated cerebrospinal fluid lactate may be caused by RARS2 mutations.

Relationship Between Sporadic Behavioral Variant Frontotemporal Dementia and Primary Psychiatric Disorders: A Study in Families.

Background: Because the behavioral variant of frontotemporal dementia (bvFTD) shows major clinical overlap with primary psychiatric disorders (PPD) that affect similar neuroanatomical circuits, a common genetic vulnerability between FTD and PPD was hypothesized.Aims: We studied whether PPD are more prevalent in families of patients with sporadic frontotemporal dementia compared with healthy controls (HC), subjects with Alzheimer's disease (AD), and individuals with bipolar disorder (BD).Methods: In this case-control study performed between January 2013 and February 2019, we investigated the first-degree family history concerning depression, psychosis (including schizophrenia), BD, and autism spectrum disorder for 73 bvFTD patients, 153 patients with BD, 108 patients with AD, and 101 HC with a semistructured questionnaire (QFTD-NL 1.0) according to DSM-IV, DSM-5, or ICD-10 criteria.Results: Patients with bvFTD had a 2.58-fold higher odds of having a first-degree family member with depression compared to HC (P = .04). Furthermore, they showed 3.26-fold higher odds of having a first-degree relative with psychosis compared to HC (P = .09).Conclusions: Our results implicate a link between dementia, including sporadic bvFTD, and depression. Further study into the genetic overlap between bvFTD and PPD might provide clues to targeting common disease mechanisms.

The phenotype of the Gly94fsX222 PMP22 insertion.

Point mutations in PMP22 are relatively rare and the phenotype may vary from mild hereditary neuropathy with liability to pressure palsies (HNPP) to severe Charcot-Marie-Tooth type 1 (CMT1). We describe the phenotype of the Gly94fsX222 mutation in the PMP22 gene. Medical records of all patients were reviewed and 11 patients were re-examined. EMG was carried out in nine patients and nerve biopsy in one. Thirteen patients originating from seven families with a Gly94fsX222 mutation were included and consisted of 10 women and 3 men with a median age of 41 years (range 7-67). Five index patients were originally suspected of CMT1. Ten patients had abnormal motor skills during childhood. Nine patients had a history of pressure palsies. Involvement of the olfactory, trigeminal, facial, and pudendal nerves occurred in three patients. Twelve patients had pes cavus and one scoliosis. Distal anterior leg and distal arm weakness were found in 12 and 4 patients, respectively. Twelve patients had distal leg sensory abnormalities. Electrophysiological examination revealed a demyelinating sensorimotor neuropathy, both resembling CMT1 and HNPP. Sural nerve biopsy showed demyelinating neuropathy with presence of tomacula. More than three-fourths of the patients with Gly94fsX222 mutation demonstrated a CMT1 phenotype combined with transient deficits. Clinicians should test for this mutation in those patients exhibiting a generalised neuropathy combined with compressive like episodes.

Genome-wide association study of frontotemporal dementia identifies a C9ORF72 haplotype with a median of 12-G4C2 repeats that predisposes to pathological repeat expansions.

Genetic factors play a major role in frontotemporal dementia (FTD). The majority of FTD cannot be genetically explained yet and it is likely that there are still FTD risk loci to be discovered. Common variants have been identified with genome-wide association studies (GWAS), but these studies have not systematically searched for rare variants. To identify rare and new common variant FTD risk loci and provide more insight into the heritability of C9ORF72-related FTD, we performed a GWAS consisting of 354 FTD patients (including and excluding N = 28 pathological repeat carriers) and 4209 control subjects. The Haplotype Reference Consortium was used as reference panel, allowing for the imputation of rare genetic variants. Two rare genetic variants nearby C9ORF72 were strongly associated with FTD in the discovery (rs147211831: OR = 4.8, P = 9.2 × 10-9, rs117204439: OR = 4.9, P = 6.0 × 10-9) and replication analysis (P < 1.1 × 10-3). These variants also significantly associated with amyotrophic lateral sclerosis in a publicly available dataset. Using haplotype analyses in 1200 individuals, we showed that these variants tag a sub-haplotype of the founder haplotype of the repeat expansion that was previously found to be present in virtually all pathological C9ORF72 G4C2 repeat lengths. This new risk haplotype was 10 times more likely to contain a C9ORF72 pathological repeat length compared to founder haplotypes without one of the two risk variants (~22% versus ~2%; P = 7.70 × 10-58). In haplotypes without a pathologic expansion, the founder risk haplotype had a higher number of repeats (median = 12 repeats) compared to the founder haplotype without the risk variants (median = 8 repeats) (P = 2.05 × 10-260). In conclusion, the identified risk haplotype, which is carried by ~4% of all individuals, is a major risk factor for pathological repeat lengths of C9ORF72 G4C2. These findings strongly indicate that longer C9ORF72 repeats are unstable and more likely to convert to germline pathological C9ORF72 repeat expansions.

Congenital hypomyelinating neuropathy, a long term follow-up study in an affected family.

Congenital hypomyelinating neuropathy is a rare condition characterized by prenatal, neonatal or early infantile onset of hypotonia, paresis and areflexia. Most of the few patients described in literature die within the first years of life. Histopathologically there are no or thin myelin sheaths. Mutations have been described in the following genes, MPZ, EGR2, PMP22, and MTMR2. Here we describe a family with a heterozygous mutation in MPZ, confirmed in two generations.

Scaling of gene expression data allowing the comparison of different gene expression platforms.

Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce the need for duplicate experiments and facilitate a more extensive exchange of data within the research community. This requires a measure for the correspondence of the results from different gene expression platforms. To date, a number of cross-platform evaluations (including a few studies using SAGE and Affymetrix GeneChips) have been conducted showing a variable, but overall low, concordance using different overall correlation approaches, such as Up/Down classification, contingency tables, and correlation coefficients. Here, we demonstrate an approach to compare two platforms based on the calculation of the difference between expression ratios observed in each platform for each individual transcript. This approach results in a concordance measure per gene, as opposed to the commonly used overall concordance measures between platforms. This between-ratio difference is a filtering-independent measure for between-platform concordance. Moreover, the between-ratio difference per gene can be used to identify transcripts with similar regulation on both platforms.

Statistical comparison of two or more SAGE libraries: one tag at a time.

Several statistical tests have been introduced for the comparison of serial analysis of gene expression (SAGE) libraries to quantitatively analyze the differential expression of genes. As each SAGE library is only one measurement, the necessary information on biological variation or experimental precision is lacking. Therefore, each test includes its own approach to derive such a variance measure from the data set or a theoretical distribution. Because the confidence in tag counts depends on the library size, a test between two or more libraries should be based on original tag counts. When groups of libraries are compared, the test should determine that the proportion of a specific tag in all libraries is the same (null hypothesis), but also offer the possibility to detect specific differences between individual libraries and groups of libraries. The Z-test and the G-test encompass these characteristics and are described for the comparison of two libraries and (two or more) groups of libraries, respectively.

Serial analysis of gene expression (SAGE).

In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE procedure was described, which cannot only be used as a tool to generate gene expression profiles but also to identify new transcripts. SAGE data can easily be exchanged between laboratories and a large database with SAGE data is now available on the Internet.

RARS2 Mutations: Is Pontocerebellar Hypoplasia Type 6 a Mitochondrial Encephalopathy?

Mutations in the mitochondrial arginyl tRNA synthetase (RARS2) gene are associated with Pontocerebellar Hypoplasia type 6 (PCH6). Here we report two patients, compound heterozygous for RARS2 mutations, presenting with early onset epileptic encephalopathy and (progressive) atrophy of both supra- and infratentorial structures. Early pontocerebellar hypoplasia was virtually absent and respiratory chain (RC) defects could not be detected in muscle biopsies. Both patients carried a novel missense mutation c.1544A>G (p.(Asp515Gly)) in combination with either a splice site (c.297+2T>G) or a frameshift (c.452_454insC) mutation. The splice site mutation induced skipping of exon 4.These two patients expand the phenotypical spectrum associated with RARS2 mutations beyond the first report of PCH6 by Edvardson and colleagues. We propose to classify RARS2-associated phenotypes as an early onset mitochondrial encephalopathy, since this is more in agreement with both clinical presentation and underlying genetic cause.

Full transcriptome analysis of rhabdomyosarcoma, normal, and fetal skeletal muscle: statistical comparison of multiple SAGE libraries.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Improved treatment strategies have increased overall survival, but the response of approximately one-third of the patients is still poor. To increase the knowledge of RMS pathogenesis, we performed the first full transcriptome analysis of RMS using serial analysis of gene expression (SAGE). With a G-test for the simultaneous comparison of subsets of SAGE libraries of normal skeletal muscle, embryonal (ERMS) and alveolar (ARMS) RMS, we identified 251 differentially expressed genes. A literature-mining procedure demonstrated that 158 of these genes have not previously been associated with RMS or normal muscle. Gene Ontology (GO) analysis assigned 198 of the 251 genes to muscle-specific classes, including those involved in normal myogenic development, as well as tumor-related classes. Prominent GO classes were those associated with proliferation and actin reorganization, which are processes that play roles during early muscle development, muscle function, and tumor progression. Using custom microarrays, we confirmed the (up- or down-) regulation of 80% of 98 differentially expressed genes. Another SAGE library of 19- to 22-week-old fetal skeletal muscle was compared with the RMS and normal muscle transcriptomes. Cluster analysis showed that the RMS and fetal muscle SAGE libraries formed one cluster distinct from normal muscle samples. Moreover, the expression profile of 86% of the differentially expressed genes between normal muscle and RMS was highly similar in fetal muscle and RMS. In conclusion, the G-test is a robust tool for analyzing groups of SAGE libraries and correctly identifies genes marking the difference between fully differentiated skeletal muscle and RMS. This study not only substantiates the close association between embryonic myogenesis and RMS development but also provides a rich source of candidate genes to further elucidate the etiology of RMS or to identify diagnostic and/or prognostic markers.

Effective cauda equina decompression in two siblings with Charcot-Marie-Tooth disease type 1B.

Two siblings with Charcot-Marie-Tooth (CMT) 1B due to a c.517G>C (p.Gly173Arg) mutation in the MPZ gene both developed an acute cauda syndrome with unbearable back pain radiating to both legs, progressive muscle weakness of the legs, and saddle hypesthesia with fecal and urinary incontinence. MRI showed in both patients a lumbar spinal canal totally filled with hypertrophic caudal nerve roots. We performed acute decompression. Postoperatively, in both patients, the back pain resolved immediately, there was a significant improvement of both the paresis of the legs and the hypesthesia, and there was a full return of continence. There was no recurrence of acute symptoms during respectively 19 years and 1.5 years of follow-up. We conclude that in patients with CMT and a related cauda syndrome because of hypertrophic caudal nerve roots, acute decompression can be an effective and safe treatment with long-term efficacy.