Parkin truncating variants result in a loss-of-function phenotype

Parkinson disease (PD) is the second most common neurodegenerative disorder. Most cases of PD are sporadic, while 5–10% have a known genetic basis. Variants in the PARK2 gene are the most frequent cause of autosomal recessive juvenile-onset PD. PARK2 encodes parkin, a multi-domain protein that functions as an ubiquitin E3 ligase. Numerous variants spanning all parkin domains have been identified, although the pathogenic relevance for several of those remains unclear. In this study, we aimed to functionally characterize two truncating parkin variants: N52Mfs*29, which is highly prevalent in the Portuguese and Spanish populations, and L358Rfs*77, recently identified in the Portuguese population. Our results indicate that both variants are prematurely degraded by the proteasome, even though proteins levels are still moderate. We also showed that they are aggregation-prone and lead to mislocalized parkin. Interestingly, the L358Rfs*77 variant is mislocalized to the nucleus, which was never reported for parkin variants. While N52Mfs*29 impaired self-ubiquitination activity, the L358Rfs*77 variant seemed to retain it. Both variants, however, fail to ubiquitinate p62 substrate and did not relocalize to depolarized mitochondria. Therefore, we conclude that parkin truncating variants cause loss of parkin function, thus showing their causative role in PD pathogenesis.This work was funded by FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE 2020 and by Nacional funds through the Fundação para a Ciência e Tecnologia - FCT [COMPETE: POCI-01-0145-FEDER-007440]. This work was also funded in part by the FCT grant FCT-ANR/BEX-GMG/0008/2013 and the Porto Neurosciences and Neurologic Disease Research Initiative at the i3S (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, also through FEDER. The authors also acknowledge the support of the i3S Scientific Platform Advanced Light Microscopy, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122). MS, SM and CP were the recipients of fellowships (SFRH/BPD/116046/2016, SFRH/ BD/87189/2012 and SFRH/BD/90048/2012) from the FCT supported by POPH/MCTES funding

Novel mag variant causes cerebellar ataxia with oculomotor apraxia: Molecular basis and expanded clinical phenotype

Homozygous variants in MAG, encoding myelin-associated glycoprotein (MAG), have been associated with complicated forms of hereditary spastic paraplegia (HSP). MAG is a glycoprotein member of the immunoglobulin superfamily, expressed by myelination cells. In this study, we identified a novel homozygous missense variant in MAG (c.124T>C; p.Cys42Arg) in a Portuguese family with early-onset autosomal recessive cerebellar ataxia with neuropathy and oculomotor apraxia. We used homozygosity mapping and exome sequencing to identify the MAG variant, and cellular studies to confirm its detrimental effect. Our results showed that this variant reduces protein stability and impairs the post-translational processing (N-linked glycosylation) and subcellular localization of MAG, thereby associating a loss of protein function with the phenotype. Therefore, MAG variants should be considered in the diagnosis of hereditary cerebellar ataxia with oculomotor apraxia, in addition to spastic paraplegia.This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020. It was also funded by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE 2020 and by Nacional funds through the FCT [COMPETE: POCI-01-0145-FEDER-007440]. This work was also funded in part by the FCT grant FCT-ANR/BEX-GMG/0008/2013 and the Porto Neurosciences and Neurologic Disease Research Initiative at the i3S (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, also through FEDER. The authors also acknowledge the support of the i3S Scientific Platform Advanced Light Microscopy, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122) and GenomePT (POCI-01-0145-FEDER-022184). MS was the recipient of a fellowship (SFRH/BPD/116046/2016) from the FCT supported by POPH/MCTES funding

BDNF and CGRP interaction: implications in migraine susceptibility

Abstract OBJECTIVES: Migraine pathophysiology involves several pathways. Our aims were to explore a possible role of the brain-derived neurotrophic factor gene (BDNF) in migraine susceptibility; to study, for the first time, the calcitonin gene-related peptide gene (CGRP); and a possible interaction between the two. METHODS: Using a case-control approach, four tagging single nucleotide polymorphisms (SNPs) (rs7124442, rs6265, rs11030107, and rs2049046) of BDNF and one tagging SNP-rs1553005-of CGRP were analyzed in 188 cases and 287 controls. A multivariable logistic regression was performed, adjusting for gender. Allelic and haplotypic frequencies were estimated. Interaction was assessed by a stepwise multivariable-logistic regression and confirmed by a multifactor dimensionality reduction analysis. RESULTS: No significant main effects were found; however, a significant interaction was found between BDNF and CGRP, showing an increased risk for the AT-genotype of rs2049046 and the GC-genotype of rs1553005 (odds ratio=1.88, 95% confidence interval: 1.20-2.93) for migraineurs. CONCLUSION: Our data support the hypothesis of an interaction between BDNF and CGRP in migraine susceptibility that should be further explored

Fabrication of low electrical percolation threshold multi-walled carbon nanotube sensors using magnetic patterning

Soft robotics is an expanding area with multiple applications; however, building low-cost, soft, and flexible robots requires the development of sensors that can be directly integrated into the soft robotics fabrication process. Thus, the motivation for this work was the design of a low-cost fabrication process of flexible sensors that can detect touch and deformation. The fabrication process proposed uses a flexible polymer nanocomposite with permanent magnets strategically placed where the conductive electrodes should be. The nanocomposite is based on poly(dimethylsiloxane) (PDMS) and multi-walled carbon nanotubes (MWCNTs). The MWCNT contains ferromagnetic impurities remaining from the synthesis process, which can be used for magnetic manipulation. Several electrode geometries were successfully simulated and tested. The magnetic patterning was simulated, allowing the fabrication of conductive patterns within the composite. This fabrication process allowed the reduction of the electrical resistivity of the nanocomposites as compared to the composites with homogeneous MWCNT dispersion. It also allowed the fabrication of piezoresistive and triboelectric sensors at MWCNT concentration as low as 0.5 wt.%. The fabrication process proposed is flexible, allows the development of sensors for soft robotics, as well as monitoring large and unconventional areas, and may be adapted to different mould shapes and polymers at low cost.This research is part of the PhD project at the Doctoral Program in Advanced Materials and Processing—FEUP. We would like to thank CeNTI for providing resources (labs, equipment and consumables) to perform the fabrication and characterisation of the samples. The authors thank CEMUP for expert assistance (Rui Rocha) with SEM-EDS. IPC acknowledges the support of FCT through National Funds References UIDB/05256/2020 and UIDP/05256/2020

A Trans-acting Factor May Modify Age at Onset in Familial Amyloid Polyneuropathy ATTRV30M in Portugal

Although all familial amyloid polyneuropathy (FAP) ATTRV30M patients carry the same causative mutation, early (<40) and late-onset forms (≥50 years) of FAP may coexist in the same family. However, this variability in age at onset is still unexplained. To identify modifiers closely linked to the TTR locus that may in part be associated with age at onset of FAP ATTRV30M, in particular in a group of very early-onset patients (≤30 years) when compared with late-onset individuals. A clinical genetic study at a referral center comprising a sample of 910 Portuguese individuals includes 589 Val30Met carriers, 102 spouses, and 189 controls from the general population. Haplotype analysis was performed, using eight intragenic single nucleotide polymorphisms (SNPs) at the TTR locus. We compared haplotypes frequency in FAP samples and controls and in parent-offspring pairs using appropriated statistical analysis. Haplotype A was the most common in the general population. Noteworthy, haplotype C was more frequent in early-onset (<40) than in late-onset patients (≥50 years) (p = 0.012). When comparing allelic frequencies of each SNP within haplotype C between "very early" (≤30 years) and late-onset (≥50 years) cases, the A allele of rs72922947 was associated with an earlier onset (p = 0.009); this remained significant after a permutation-based correction. Also, the heterozygous genotype (GA) for this SNP was associated with a decrease in mean age at onset of 8.6 years (p = 0.014). We found a more common haplotype (A) linked to the Val30Met variant and a possible modulatory trans effect on age at onset. These findings may lead to potential therapeutical targets.This work was supported by grants of Fundação para a Ciência eTecnologia,FCT[PTDC/SAU-GMG/100240/2008andPEsT], cofunded byERDF andCOMPETE; and byFinanciamentoPlurianual de Unidades de Investigação (FCT). Conflict of Interest M.A.F. and D. S. have received research support from a FCT fellowship (SFRH/BD/101352/2014 and SFRH/BD/91160/2012, respectively). T.C.’sinstitution hasreceived support from FoldRx Pharmaceuticals, which was acquired by Pfizer Inc. in October 2010; T. C. hasserved on the scientific advisory board of PfizerInc. and received funding from Pfizer Inc. for scientific meeting expenses (travel, accommodations,and registration).ShecurrentlyservesontheTHAOS(natural historydiseaseregistry)scientific advisory board.J.S.,I.A.,A.S.,and C. L. report no disclosures

Improvement in the molecular diagnosis of Machado-Joseph disease

Abstract BACKGROUND: Direct detection of the gene mutation allows for the confirmation of the clinical diagnosis of Machado-Joseph disease (MJD), the most frequent cause of autosomal dominant spinocerebellar ataxia worldwide. OBJECTIVE: To address the main difficulties in our national MJD predictive testing program. The first was the emergence of intermediate alleles, for which it is not yet possible to determine whether they will cause disease. The second was the issue of homoallelism, ie, homozygosity for 2 normal alleles with exactly the same (CAG)(n) length, which occurs in about 10% of all test results. METHODS: A large pedigree with 1 affected patient carrying a 71 and a 51 CAG repeat and 2 asymptomatic relatives carrying the 51 CAG repeat and normal-size alleles underwent clinical and molecular studies. Intragenic haplotypes for these alleles were determined. A representative sample of the healthy population in the region was obtained to assess the distribution of the normal (CAG)(n) length. We established the genotype for 4 intragenic polymorphisms in the gene for MJD (MJD1) in 21 homoallelic individuals, to distinguish their 2 normal chromosomes. In addition, we developed a new Southern blot method to completely exclude cases of nonamplification of expanded alleles in the homoallelic individuals. RESULTS: The study of the family in which the 51 CAG repeat was found suggests that the allele is apparently not associated with disease. These intermediate alleles were not present in a large sample of the healthy population from the same region. Intragenic polymorphisms allowed distinction of the 2 different normal alleles in all cases of homoallelism. The absence of an expanded allele was also confirmed by Southern blot. CONCLUSIONS: We propose an improved protocol for molecular testing for MJD. These strategies, developed to overcome the practical difficulties mostly in the presymptomatic and prenatal diagnosis of MJD, should prove useful for other polyglutamine-related disorders

Life paths of patients with transthyretin-related familial amyloid polyneuropathy Val30Met: a descriptive study

Transthyretin-related familial amyloid polyneuropathy Val30Met is a fatal progressive disease. It is a rare hereditary amyloidosis, manifesting as a sensorimotor neuropathy and autonomic dysfunction. It begins during adulthood and is a disabling disease, posing a great psychological burden to patients and their families. Our aim was to describe and characterize life events related to the disease and discuss its psychosocial implications. Social and demographic data and a questionnaire on history of family and personal disease, and biographic events, were applied to 209 subjects attending an outpatient specialized clinic. Descriptive and statistical analyses were performed. They were 84 men and 127 women belonging to three groups: pre-symptomatic carriers, patients, and subjects with no established diagnosis. Most subjects were married/lived with a partner and had children (mean of 4). Most (96.3%) had contact with the disease before having a diagnosis; the affected or at-risk parent was the mother in 53.8% and the father in 43.3%; 71.8% of these had deceased. At their parent’s death, many subjects were aged under 10 (9.9%), 10–14 (15.5%), or 15–24 years (31.7%). Most were under age 14 (44.9%) at their parent’s disease onset; 37.2% referred this brought life changes with psychological and familial impact; most had been parent’s caregivers; 7.5% had not been raised by the parents. Some (8.4%) declined to know their genetic tests results for over 1 year. Parent’s disease and death are very common early in these patient’s lives. During childhood or youth, many subjects became caregivers, implying changes in family roles. This disease and its life implications pose a significant psychosocial burden since childhood. TTR-FAP patients and their relatives are highly vulnerable to emotional stress and psychopathology during their lifetime. Psychological and psychiatric support, implying a multidisciplinary group, must thus be available for all of them

Evidence of syntaxin 1A involvement in migraine susceptibility: a Portuguese study.

Abstract OBJECTIVE: To confirm syntaxin 1A as a risk factor for migraine, given that syntaxin 1A interacts with several factors in migraine pathophysiology. DESIGN: Case-control approach. SETTING: An outpatient clinic. PARTICIPANTS: In a sample of 188 migraineurs (111 without aura and 77 with aura) and 287 migraine-free controls, 3 tagging SNPs of STX1A (rs3793243, rs941298, and rs6951030) were analyzed. A backward stepwise multiple logistic regression was performed. Allelic and haplotypic frequencies were compared between cases and controls. RESULTS: We found that rs941298 and rs6951030 were risk factors for migraines. In particular, the TT genotype of rs941298 is associated with an increased risk of both migraine in general and migraine without aura; the GG and GT genotypes for rs6951030 are also associated with migraine, while the GT genotype of rs6951030 was found to be significant in the migraine without aura group. The single-nucleotide polymorphism rs3793243 did not show any significant association. In the haplotype-based analysis, we found an underrepresentation of the T-C-T haplotype (rs3793243-rs941298-rs6951030) in the global sample and in migraine without aura group. We found an enrichment of the G allele of rs6951030 for female migraineurs only. CONCLUSIONS: We confirmed the involvement of syntaxin 1A in migraine susceptibility regarding rs941298. In addition, we found rs6951030 to also be associated in Portuguese migraine patients. Sex differences should be further explored to disentangle a possible sex susceptibility in syntaxin 1

Monozygotic twin sisters discordant for familial hemiplegic migraine

Background: The high concordance rate of migraine in monozygotic twin pairs has long been recognised. In the current study, we present a monozygotic twin pair discordant for familial hemiplegic migraine (FHM). Case presentations: We evaluated 12 adult family members in 2012. The twin pair was separately examined by neurologists at different time points. Mutation screening was performed for known FHM-related genes. The monozygosity of the twins was verified. Eleven individuals had a history of migraine or paroxysmal neurological symptoms, including four patients with motor aura. No mutations were detected in the CACNA1A, ATP1A2, SCN1A, PRRT2 or NOTCH3 genes. The monozygotic twin sisters, aged 52, were discordant for age of onset, motor aura and neuropsychological aura (forced thinking). Overall, the family members presented a wide range of phenotypical features. Conclusions: Familial hemiplegic migraine is a monogenic disorder that is distinct from migraine with typical aura. However, in certain families with motor aura, such as this one, it is possible that the most severe phenotype is caused by an unlikely combination of polygenic traits and non-genetic factors. In these kindreds, we propose that hemiplegic aura is only a severe and complex form of typical aura

Mutational mechanism for DAB1 (ATTTC) n insertion in SCA37: ATTTT repeat lengthening and nucleotide substitution

Dynamic mutations by microsatellite instability are the molecular basis of a growing number of neuromuscular and neurodegenerative diseases. Repetitive stretches in the human genome may drive pathogenicity, either by expansion above a given threshold, or by insertion of abnormal tracts in nonpathogenic polymorphic repetitive regions, as is the case in spinocerebellar ataxia type 37 (SCA37). We have recently established that this neurodegenerative disease is caused by an (ATTTC)n insertion within an (ATTTT)n in a noncoding region of DAB1. We now investigated the mutational mechanism that originated the (ATTTC)n insertion within an ancestral (ATTTT)n . Approximately 3% of nonpathogenic (ATTTT)n alleles are interspersed by AT-rich motifs, contrarily to mutant alleles that are composed of pure (ATTTT)n and (ATTTC)n stretches. Haplotype studies in unaffected chromosomes suggested that the primary mutational mechanism, leading to the (ATTTC)n insertion, was likely one or more T>C substitutions in an (ATTTT)n pure allele of approximately 200 repeats. Then, the (ATTTC)n expanded in size, originating a deleterious allele in DAB1 that leads to SCA37. This is likely the mutational mechanism in three similar (TTTCA)n insertions responsible for familial myoclonic epilepsy. Because (ATTTT)n tracts are frequent in the human genome, many loci could be at risk for this mutational process.We are grateful to the families and individuals who participated in this work. We thank Patricia Ribeiro for technical assistance. This study was financed by Fundo Europeu de Desenvolvimento Regional (FEDER), through the COMPETE 2020 Operational Pro- gram for Competitiveness and Internationalization (POCI) of Portugal 2020, and by the Fundacão para a Ciência e a Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (Portugal), in the framework of the project POCI-01-0145-FEDER-029255; (PTDC/MED-GEN/29255/2017) to I.S. J.R.L. and C.L.O. were sup- ported by scholarships from PEst-C/SAU/LA0002/2013. S.M. is funded by the project IF/00930/2013/ CP1184/CT0002 from FCT. This work was also funded by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigação e Inovação em Saúde (Norte-01-0145-FEDER- 000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTU- GAL 2020 Partnership Agreement with FEDER