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Spastic ataxias are rare neurogenetic disorders involving spinocerebellar and pyramidal tracts. Many genes are involved. Among them, CAPN1, when mutated, is responsible for a complex inherited form of spastic paraplegia (SPG76). We report the largest published series of 21 novel patients with nine new CAPN1 disease-causing variants and their clinical characteristics from two European university hospitals (Paris and Stockholm). After a formal clinical examination, causative variants were identified by next-generation sequencing and confirmed by Sanger sequencing. CAPN1 variants are a rare cause (~ 1.4%) of young-adult-onset spastic ataxia; however, together with all published cases, they allowed us to better describe the clinical and genetic spectra of this form. Truncating variants are the most frequent, and missense variants lead to earlier age at onset in favor of an additional deleterious effect. Cerebellar ataxia with cerebellar atrophy, dysarthria and lower limb weakness are often associated with spasticity. We also suggest that cognitive impairment and depression should be assessed specifically in the follow-up of SPG76 cases.

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) α-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of "extra-target" RAS suggests the need for RAS screening in all three DAA target regions., Competing Interests: Declaration of competing interest We declare that no public or private company has had any role in the study design, data collection, experimental work, data analysis, decision to publish, or preparation of the manuscript. Roche Diagnostics S.L. provided support in the form of a salary for one of the authors [Josep Gregori], but the company did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. No other Competing Interests to declare. Thus, our adherence to Antiviral Research policies on sharing data and materials is not altered., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background Permanent primary congenital hypothyroidism (CH) can be caused by thyroid dysgenesis or dyshormonogenesis. A molecular genetic study is recommended in dyshormonogenesis, in syndromic hypothyroidism and when there is a family history of CH. The aim of this study was to identify a monogenic etiology for CH in selected individuals from a cohort of primary permanent CH. Methods From an initial cohort of 79 patients with permanent CH (3-19 years), 11 patients were selected for molecular analyses. Nine patients with dyshormonogenesis (normal in-situ gland or goiter) were screened for causative variants, by next-generation sequencing (NGS), in 28 genes known to be responsible for CH. One patient with a family history of CH was screened for the paired-box gene 8 (PAX8) gene and another patient with a syndromic CH was screened for the NKX2-1 gene. Results We found a monogenic basis of disease in eight patients, involving the thyroid peroxidase (TPO) gene (four patients), the thyroglobulin (TG) gene (two patients), and the PAX8 and NKX2-1 genes (one patient each). Two patients were heterozygotes, one harboring a variant in the TG gene and the other in the SLC5A5 gene. In one patient, we found no potential causative variants in any of the 28 genes screened. We described five novel variants: three in the TG gene, one in the NKX2-1 and one in the SLC5A5 gene, all of them classified as pathogenic. Conclusions In eight of the 11 screened patients, a monogenic disease was found. These results highlight the advantage of using an NGS panel and provide further data regarding the molecular basis of CH.

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery., (Copyright © 2019. Published by Elsevier B.V.)

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Hereditary spastic paraplegias (HSP) are neurodegenerative disorders characterized by lower limb spasticity and weakness that can be complicated by other neurological or non-neurological signs. Despite a high genetic heterogeneity (>60 causative genes), 40-70% of the families remain without a molecular diagnosis. Analysis of one of the pioneer cohorts of 193 HSP families generated in the early 1990s in Portugal highlighted that SPAST and SPG11 are the most frequent diagnoses. We have now explored 98 unsolved families from this series using custom next generation sequencing panels analyzing up to 70 candidate HSP genes. We identified the likely disease-causing variant in 20 of the 98 families with KIF5A being the most frequently mutated gene. We also found 52 variants of unknown significance (VUS) in 38% of the cases. These new diagnoses resulted in 42% of solved cases in the full Portuguese cohort (81/193). Segregation of the variants was not always compatible with the presumed inheritance, indicating that the analysis of all HSP genes regardless of the inheritance mode can help to explain some cases. Our results show that there is still a large set of unknown genes responsible for HSP and most likely novel mechanisms or inheritance modes leading to the disease to be uncovered, but this will require international collaborative efforts, particularly for the analysis of VUS.

More than 600 human disorders afflict the nervous system. Of these, neurodegenerative diseases are usually characterised by onset in late adulthood, progressive clinical course, and neuronal loss with regional specificity in the central nervous system. They include Alzheimer's disease and other less frequent dementias, brain cancer, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), Huntington's disease, and Prion diseases, among others. Neurodegeneration usually affects, but is not limited to, the cerebral cortex, intracranial white matter, basal ganglia, thalamus, hypothalamus, brain stem, and cerebellum. Although the majority of neurodegenerative diseases are sporadic, Mendelian inheritance is well documented. Intriguingly, the clinical presentations and neuropathological findings in inherited neurodegenerative forms are often indistinguishable from those of sporadic cases, suggesting that converging genomic signatures and pathophysiologic mechanisms underlie both hereditary and sporadic neurodegenerative diseases. Unfortunately, effective therapies for these diseases are scarce to non-existent. In this chapter, we highlight the clinical and genetic features associated with the rare inherited forms of neurodegenerative diseases, including ataxias, multiple system atrophy, spastic paraplegias, Parkinson's disease, dementias, motor neuron diseases, and rare metabolic disorders.

Hereditary spastic paraplegias are heterogeneous neurological disorders characterized by a pyramidal syndrome with symptoms predominantly affecting the lower limbs. Some limited pyramidal involvement also occurs in patients with an autosomal recessive neurocutaneous syndrome due to ALDH18A1 mutations. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthase (P5CS), an enzyme that catalyses the first and common step of proline and ornithine biosynthesis from glutamate. Through exome sequencing and candidate gene screening, we report two families with autosomal recessive transmission of ALDH18A1 mutations, and predominant complex hereditary spastic paraplegia with marked cognitive impairment, without any cutaneous abnormality. More interestingly, we also identified monoallelic ALDH18A1 mutations segregating in three independent families with autosomal dominant pure or complex hereditary spastic paraplegia, as well as in two sporadic patients. Low levels of plasma ornithine, citrulline, arginine and proline in four individuals from two families suggested P5CS deficiency. Glutamine loading tests in two fibroblast cultures from two related affected subjects confirmed a metabolic block at the level of P5CS in vivo. Besides expanding the clinical spectrum of ALDH18A1-related pathology, we describe mutations segregating in an autosomal dominant pattern. The latter are associated with a potential trait biomarker; we therefore suggest including amino acid chromatography in the clinico-genetic work-up of hereditary spastic paraplegia, particularly in dominant cases, as the associated phenotype is not distinct from other causative genes., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Importance: Hereditary spastic paraplegias (HSPs) are a group of diseases caused by corticospinal tract degeneration. Mutations in 3 genes (SPG4, SPG3, and SPG31) are said to be the cause in half of the autosomal dominant HSPs (AD-HSPs). This study is a systematic review of families with HSP resulting from a population-based survey. Novel genotype-phenotype correlations were established., Objective: To describe the clinical, genetic, and epidemiological features of Portuguese AD-HSP families., Design: Retrospective medical record review., Setting: A population-based systematic survey of hereditary ataxias and spastic paraplegias conducted in Portugal from 1993 to 2004., Participants: Families with AD-HSP., Main Outcome Measure: Mutation detection in the most prevalent genes., Results: We identified 239 patients belonging to 89 AD-HSP families. The prevalence was 2.4 in 100 000. Thirty-one distinct mutations (26 in SPG4, 4 in SPG3, and 1 in SPG31) segregated in 41% of the families (33.7%, 6.2%, and 1.2% had SPG4, SPG3 and SPG31 mutations, respectively). Seven of the SPG4 mutations were novel, and 7% of all SPG4 mutations were deletions. When disease onset was before the first decade, 31% had SPG4 mutations and 27% had SPG3 mutations. In patients with SPG4 mutations, those with large deletions had the earliest disease onset, followed by those with missense, frameshift, nonsense, and alternative-splicing mutations. Rate of disease progression was not significantly different among patients with SPG3 and SPG4 mutations in a multivariate analysis. For patients with SPG4 mutations, disease progression was worst in patients with later-onset disease., Conclusions and Relevance: The prevalence of AD-HSP and frequency of SPG3 and SPG4 mutations in the current study were similar to what has been described in other studies except that the frequency of SPG4 deletions was lower. In contrast, the frequency of SPG31 mutations in the current study was rare compared with other studies. The most interesting aspects of this study are that even in patients with early-onset disease the probability of finding a SPG4 mutation was higher than for patients with SPG3 mutations; there was no difference in disease progression with genotype but an association with the age at onset; 7 new SPG4 mutations were identified; and for the first time, to our knowledge, the nature of the SPG4 mutations was found to predict the age at onset.

Purpose: Hereditary spastic paraplegias compose a group of neurodegenerative disorders with a large clinical and genetic heterogeneity. Among the autosomal recessive forms, spastic paraplegia type 11 is the most common., Methods: To better understand the spastic paraplegia type 11 mutation spectrum, we studied a group of 54 patients with hereditary spastic paraplegia. Mutation screening was performed by PCR amplification of SPG11 coding regions and intron boundaries, followed by sequencing. For the detection of large gene rearrangements, we performed multiplex ligation-dependent probe amplification., Results: We report 13 families with spastic paraplegia type 11 carrying either novel or previously identified mutations. We describe a complex entire SPG11 rearrangement and show that large gene rearrangements are frequent among patients with spastic paraplegia type 11. Moreover, we mapped the deletion breakpoints of three different large SPG11 deletions and provide evidence for Alu microhomology-mediated exon deletion., Conclusion: Our analysis shows that the high number of repeated elements in SPG11 together with the presence of recombination hotspots and the high intrinsic instability of the 15q locus all contribute toward making this genomic region more prone to large gene rearrangements. These findings enlarge the amount of data relating repeated elements with neurodegenerative disorders and highlight their importance in human disease and genome evolution.

The fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expansions of 55-200 CGG repeats in the 5'UTR of the FMR1 gene. These FMR1 premutation expansions have relatively high frequency in the general population. To estimate the frequency of FMR1 premutations among Portuguese males with non-familial, late-onset movement disorders of unknown etiology, we assessed CGG repeat size in males with disease onset after the age of 50 and negative or unknown family history for late-onset movement disorders, who were sent for SCA, HD, or PD genetic testing at a reference laboratory. The selected patients had a primary clinical diagnosis based on one of the following cardinal features of FXTAS: ataxia, tremor, or cognitive decline. A total of 86 subjects were genotyped for the CGG repeat in the FMR1 gene. We detected one patient with an expansion in the premutation range. The frequency of FMR1 premutations was 1.9% (1/54) in our group of patients with ataxia as the primary clinical feature, and 1.2% (1/86) in the larger movement disorders group. In the family of the FXTAS case, premutation-transmitting females presented a history of psychiatric symptoms, suggesting that, given the wide phenotypical expression of the premutation in females, neuropsychiatric surveillance is necessary. In conclusion, genetic testing for FXTAS should be made available to patients with adult-onset movement disorders to enable adequate genetic counseling to family members.

Objectives/hypothesis: Proteins p53 and cyclin D1 play a crucial role in cell cycle control. Protein p53 mutations are one of the most common genetic alterations in human cancer, and cyclin D1 gene amplification has been found to be associated with poor prognosis in different types of tumors. Functional alterations of these proteins may play an important role both in the carcinogenesis of squamous carcinomas of the head and neck and in the clinical evolution of these tumors. The objective of the present study was to evaluate whether the presence of p53 and/or cyclin D1 proteins (detected by immunohistochemical analysis) could serve as relevant variables for the assessment of the prognosis of laryngeal squamous cell carcinoma., Study Design: Retrospective multicentric study., Methods: Paraffin-embedded biopsy specimens from 62 human epidermoid laryngeal carcinomas were randomly selected. The expression of p53 and cyclin D1 was examined by means of immunohistochemical analysis with a view to evaluating whether there is a correlation between the aberrant expression of these proteins and disease prognosis., Results: In the sample, the presence of immunohistochemically detectable p53 is associated with shorter survival and disease-free intervals, as shown in Kaplan-Meier survival curve analysis. Indeed, multivariate statistical analysis revealed that the accumulation of p53 is an independent prognostic factor, which is associated with shorter survival. This association was not evident in the case of cyclin D1., Conclusion: A more precise prognosis of patients with laryngeal epidermoid carcinomas could be achieved by taking into account the presence of p53 (as assayed by immunohistochemical analysis) in biopsy tissue