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Resumen Introduccion La ARCA1/SCAR8 es una heredoataxia recesiva causada por mutaciones en el gen SYNE1, que fue descrita inicialmente en familias francocanadienses (Quebec) con un sindrome cerebeloso puro. En la actualidad se reporta cada vez mas este tipo de ataxia en otras partes del mundo y con un fenotipo muy variable. Recientemente se han notificado casos de distrofia muscular, artrogriposis y miocardiopatia por mutaciones de este gen. Objetivos Describir los hallazgos clinicos y moleculares en 3 familias espanolas de diferente origen geografico, las primeras en las que se confirmo el diagnostico de ARCA1/SCAR8 con analisis molecular. Material y metodos Evaluacion clinica, pruebas paraclinicas y estudio genetico en 4 pacientes (3 varones y una mujer), diagnosticados en distintos servicios de neurologia espanoles. Resultados Los sintomas cerebelosos comenzaron en todos los casos en la tercera-cuarta decadas. Tras 15 anos de evolucion, 3 pacientes presentaban un sindrome cerebeloso puro similar a la descripcion original, mientras que un paciente con mas de 30 anos de evolucion presentaba tambien paralisis de mirada vertical, afectacion piramidal y moderado deterioro cognitivo. El estudio de resonancia magnetica mostro en todos los casos atrofia restringida al cerebelo. La secuenciacion de SYNE1 permitio identificar distintas variantes patogenicas en cada familia. Conclusiones La ARCA1/SCAR8 tiene una distribucion mundial con una gran diversidad de mutaciones en SYNE1. Ademas de ataxia puede dar lugar a un complejo fenotipo de inicio y gravedad muy variable.

Introduction In 2009, we described a possible founder effect of autosomal dominant Segawa disease in Cordoba (Spain) due to mutation c.265C>T (p. Q89*) in the GCH1 gene. We present a retrospective multicentre study aimed at improving our knowledge of Segawa disease in Spain and providing a detailed phenotypic-genotypic description of patients. Methods Clinical-genetic information were obtained from standardized questionnaires that were completed by the neurologists attending children and/or adults from 16 Spanish hospitals. Results Eighty subjects belonging to 24 pedigrees had heterozygous mutations in GCH1. Seven genetic variants have been described only in our cohort of patients, 5 of which are novel mutations. Five families not previously described with p. Q89* were detected in Andalusia due to a possible founder effect. The median latency to diagnosis was 5 years (IQR 0–16). The most frequent signs and/or symptoms were lower limb dystonia (38/56, 67.8%, p = 0.008) and diurnal fluctuations (38/56, 67.8%, p = 0.008). Diurnal fluctuations were not present in the phenotypes other than dystonia. Fifty-three of 56 symptomatic patients were treated with a levodopa/decarboxylase inhibitor for (mean ± SD) 12.4 ± 8.12 years, with 81% at doses lower than 350 mg/day (≤5 mg/kg/d in children). Eleven of 53 (20%) patients had non-responsive symptoms that affected daily life activities. Dyskinesias (4 subjects) were the most prominent adverse effects. Conclusion This study identifies 5 novel mutations and supports the hypothesis of a founder effect of p. Q89* in Andalusia. New insights are provided for the phenotypes and long-term treatment responses, which may improve early recognition and therapeutic management.

Objective: SPG4 is an autosomal dominant pure form of hereditary spastic paraplegia (HSP) caused by mutations in the SPAST gene. HSP is considered an upper motor neuron disorder characterized by progressive retrograde degeneration, or "dying-back" phenomenon, of the corticospinal tract's longest axons. Neuroimaging studies mainly focus on white matter changes and, although previous studies reported cortical thinning in complicated HSP forms, cortical changes remain unclear in SPG4 patients. This work aimed to compare changes in white matter microstructure and cortical thickness between 12 SPG4 patients and 22 healthy age-matched controls. We also explore whether white matter alterations are related to cortical thickness and their correlation with clinical symptoms. Methods: we used fixel-based analysis, an advanced diffusion-weighted imaging technique, and probabilistic tractography of the corticospinal tracts. We also analyzed cortical morphometry using whole-brain surface-based and atlas-based methods in sensorimotor areas. Results: SPG4 patients showed bilateral involvement in the corticospinal tracts; this was more intense in the distal portion than in the upper segments and was associated with the degree of clinical impairment. We found a significant correlation between disease severity and fiber density and cross-section of the corticospinal tracts. Furthermore, corticospinal tract changes were significantly correlated with bilateral cortical thinning in the precentral gyrus in SPG4 patients. Conclusions: Our data point to axonal damage of the corticospinal motor neurons in SPG4 patients might be related to cortical thinning in motor regions.

SPG4 is an autosomal dominant pure form of hereditary spastic paraplegia (HSP) caused by mutations in the SPAST gene. HSP is considered an upper motor neuron disorder characterized by progressive spasticity and weakness of the lower limbs caused by degeneration of the corticospinal tract. In other neurodegenerative motor disorders, the thalamus and basal ganglia are affected, with a considerable impact on disease progression. However, only a few works have studied these brain structures in HSP, mainly in complex forms of this disease. Our research aims to detect potential alterations in the volume and shape of the thalamus and various basal ganglia structures by comparing 12 patients with pure HSP and 18 healthy controls. We used two neuroimaging procedures: automated segmentation of the subcortical structures (thalamus, hippocampus, caudate nucleus, globus pallidus, and putamen) in native space and shape analysis of the structures. We found a significant reduction in thalamic volume bilaterally, as well as an inward deformation, mainly in the sensory-motor thalamic regions in patients with pure HSP and a mutation in SPG4. We also observed a significant negative correlation between the shape of the thalamus and clinical scores (the Spastic Paraplegia Rating Scale score and disease duration). Moreover, we found a ‘Group × Age’ interaction that was closely related to the severity of the disease. No differences in volume or in shape were found in the remaining subcortical structures studied. Our results suggest that changes in structure of the thalamus could be an imaging biomarker of disease progression in pHSP.

SPG4 is an autosomal dominant pure form of hereditary spastic paraplegia (HSP) caused by mutations in the SPAST gene. HSP is considered an upper motor neuron disorder characterized by progressive spasticity and weakness of the lower limbs caused by degeneration of the corticospinal tract. In other neurodegenerative motor disorders, the thalamus and basal ganglia are affected, with a considerable impact on disease progression. However, only a few works have studied these brain structures in HSP, mainly in complex forms of this disease. Our research aims to detect potential alterations in the volume and shape of the thalamus and various basal ganglia structures by comparing 12 patients with pure HSP and 18 healthy controls. We used two neuroimaging procedures: automated segmentation of the subcortical structures (thalamus, hippocampus, caudate nucleus, globus pallidus, and putamen) in native space and shape analysis of the structures. We found a significant reduction in thalamic volume bilaterally, as well as an inward deformation, mainly in the sensory-motor thalamic regions in patients with pure HSP and a mutation in SPG4. We also observed a significant negative correlation between the shape of the thalamus and clinical scores (the Spastic Paraplegia Rating Scale score and disease duration). Moreover, we found a 'Group × Age' interaction that was closely related to the severity of the disease. No differences in volume or in shape were found in the remaining subcortical structures studied. Our results suggest that changes in structure of the thalamus could be an imaging biomarker of disease progression in pHSP.

SCA36 is an autosomal dominant spinocerebellar ataxia (SCA) affecting many families from Costa da Morte, a northwestern region of Spain. It is caused by an intronic GGCCTG repeat expansion in NOP56. In order to characterize the cognitive and affective manifestations of this cerebellar disease, a group of 30 SCA36 mutation carriers (11 preataxic and 19 ataxic patients) were assessed with a comprehensive battery of standardized tests. Phonological verbal fluency – but not semantic fluency – was already mildly impaired in preataxic subjects. In ataxic patients, both phonological and semantic fluencies were significantly below normal. Depression, while more frequent and prominent in ataxic patients, was also often present in the preataxic stage. This is the first systematic study supporting the presence of a mild cerebellar cognitive and affective syndrome in SCA36. Routine evaluation of cognitive and emotional spheres in SCA36 patients as well as asymptomatic mutation carriers should allow early detection and timely therapeutic intervention.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.

Background The spinocerebellar ataxias (SCAs) form a clinically, genetically, and pathological heterogeneous group of autosomal-dominant degenerative diseases. In particular, SCA36 is characterized by a late-onset, slowly progressive cerebellar syndrome typically associated with sensorineural hearing loss. This study was aimed at analyzing the neurodegenerative process underlying SCA36 through fluorodeoxyglucose positron emission tomography (FDG-PET) and MRI scans. Methods Twenty SCA36 patients underwent a study consisting of FDG-PET and MRI scans. Clinical motor evaluation was performed through the Scale for the Assessment and Rating of Ataxia (SARA). FDG-PET was carried out using a voxel-by-voxel and region-of-interest analysis. MRI evaluation was based on visual inspection and volumetric analysis. Results SARA ranged from 0 to 24.5 (4 patients asymptomatic, 3 with unspecific symptoms, and 13 with cerebellar signs). FDG-PET revealed hypometabolism in the asymptomatic stage in the vermis and right cerebellar hemisphere. In the ataxic stage, hypometabolism spread to both cerebellar hemispheres and the brain stem. MRI was normal in asymptomatic and preataxic individuals and showed superior cerebellar vermis atrophy early in the ataxic stage, diffuse cerebellar atrophy some years into the disease course, and a pattern of olivopontocerebellar atrophy in the oldest patients. There was no significant cerebellar atrophy in patients younger than 50 years. Conclusions We present the first FDG-PET study of SCA36 and one of the largest neuroimaging study of SCAs. Our results revealed neuronal dysfunctions in the vermis and right cerebellar hemisphere as soon as a decade before the onset of motor symptoms. In the ataxic stage, dysfunctions spread to both hemispheres and the brain stem. © 2016 International Parkinson and Movement Disorder Society.

Introduction The pathophysiology of PD (Parkinson's disease) has been related to the ubiquitin proteasome system and oxidative stress. Parkin acts as ubiquitin ligase on several substrates. Because genetic variants often have different frequencies across populations, population specific analyses are necessary to complement and validate results from genome-wide association studies. Methods We carried out an association study with genes coding for parkin substrates and cellular stress components in the Galician population (Northern Spain). SNCA and MAPT SNPs were also analyzed. We studied 75 SNPs in a discovery sample of 268 PD patients and 265 controls from Galicia. A replication sample of 271 patients and 260 controls was recruited from Mexico City. Results We observed significant association between PD and SNPs in MAPT. Nominal p-values < 0.05 were obtained in the Galician cohort for SNPs in SYT11, coding for synaptotagmin XI. These results were replicated in the Mexican sample. Discussion The associated markers lie within a ~ 140 kb strong linkage disequilibrium segment that harbors several candidate genes, including SYT11. SNPs from the GBA-SYT11-RAB25 region have been previously associated with PD, however the functionally relevant variants remain unknown. Our data support a likely role of genetic factors within 1q22 in PD susceptibility.

Primary familial brain calcification (PFBC) is a neurodegenerative disease with characteristic calcium deposits in the basal ganglia and other brain regions. The disease usually presents as a combination of abnormal movements, cognitive and psychiatric manifestations, clinically indistinguishable from other adult-onset neurodegenerative disorders. The differential diagnosis must be established with genetic and nongenetic disorders that can also lead to calcium deposits in encephalic structures. In the past years PFBC causal mutations have been discovered in genes related to calcium phosphate homeostasis (SLC20A2, XPR1) and in genes involved with endothelial function and integrity (PDGFB, PDGFRB). The most frequently mutated gene is SLC20A2, where mutations can affect any domain of the protein. There is no clearcut relationship between the specific mutation/gene, onset age, neuroimaging pattern, and severity of clinical manifestations. The discovery of the genetic basis of PFBC provides not only a diagnostic tool, but also an insight into the pathomechanisms and potential therapeutic trials for this rare disease.

Primary familial brain calcification (PFBC) is a neurodegenerative disease with characteristic calcium deposits in the basal ganglia and other brain regions. The disease usually presents as a combination of abnormal movements, cognitive and psychiatric manifestations, clinically indistinguishable from other adult-onset neurodegenerative disorders. The differential diagnosis must be established with genetic and nongenetic disorders that can also lead to calcium deposits in encephalic structures. In the past years PFBC causal mutations have been discovered in genes related to calcium phosphate homeostasis (SLC20A2, XPR1) and in genes involved with endothelial function and integrity (PDGFB, PDGFRB). The most frequently mutated gene is SLC20A2, where mutations can affect any domain of the protein. There is no clearcut relationship between the specific mutation/gene, onset age, neuroimaging pattern, and severity of clinical manifestations. The discovery of the genetic basis of PFBC provides not only a diagnostic tool, but also an insight into the pathomechanisms and potential therapeutic trials for this rare disease.

Objective:To assess the prevalence and clinical features of individuals affected by spinocerebellar ataxia 36 (SCA36) at a large tertiary referral center in the United States.Methods:A total of 577 patients with undiagnosed sporadic or familial cerebellar ataxia comprehensively evaluated at a tertiary referral ataxia center were molecularly evaluated for SCA36. Repeat primed PCR and fragment analysis were used to screen for the presence of a repeat expansion in the NOP56 gene.Results:Fragment analysis of triplet repeat primed PCR products identified a GGCCTG hexanucleotide repeat expansion in intron 1 of NOP56 in 4 index cases. These 4 SCA36-positive families comprised 2 distinct ethnic groups: white (European) (2) and Asian (Japanese [1] and Vietnamese [1]). Individuals affected by SCA36 exhibited typical clinical features with gait ataxia and age at onset ranging between 35 and 50 years. Patients also suffered from ataxic or spastic limbs, altered reflexes, abnormal ocular movement, and cognitive impairment.Conclusions:In a US population, SCA36 was observed to be a rare disorder, accounting for 0.7% (4/577 index cases) of disease in a large undiagnosed ataxia cohort.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37. We thank the families who participated in this study. We are grateful to Goncalo Abecasis, Miguel Costa, Tito Vieira, and Andre Torres for help with MERLIN analysis; Beatriz Sobrino, Jorge Amigo, and Pilar Cacheiro for next-generation sequencing analysis, performed at the Santiago de Compostela node of the Spanish National Genotyping Center; Nuno Santarem and Anabela Cordeiro-da-Silva for assistance with cloning; Antonio Amorim, Laura Vilarinho, and Paula Jorge for samples from the Portuguese population; and Paula Magalhaes from the Institute for Molecular and Cell Biology Cell Culture and Genotyping Core for DNA extraction. This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI) of Portugal 2020 and by Portuguese funds through the Fundacao para a Ciencia e a Tecnologia (FCT) and Ministerio da Ciencia, Tecnologia, e Inovacao in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274); and by FCT grant PTDC/SAU-GMG/098305/2008 to I.S. A. I.S. was the recipient of an FCT scholarship (SFRH/BD/30702/2006). J.R.L. was supported by scholarships from PEst-C/SAU/LA0002/2013 and the European Molecular Biology Organization (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also financed by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigacao e Inovacao em Saude (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through FEDER, and by the Fondo de Investigacion Sanitaria of the Instituto de Salud Carlos III (grant PI12/00742).

Introduction: Variant detection protocols for clinical next-generation sequencing (NGS) need application-specific optimization. Our aim was to analyze the performance of single nucleotide variant (SNV) and copy number (CNV) detection programs on an NGS panel for a rare disease. Methods: Thirty genes were sequenced in 83 patients with hereditary spastic paraplegia. The variant calls obtained with LifeScope, GATK UnifiedGenotyper and GATK HaplotypeCaller were compared with Sanger sequencing. The calling efficiency was evaluated for 187 (56 unique) SNVs and indels. Five multiexon deletions detected by multiple ligation probe assay were assessed from the NGS panel data with ExomeDepth, panelcn.MOPS and CNVPanelizer software. Results: There were 48/51 (94%) SNVs and 1/5 (20%) indels consistently detected by all the calling algorithms. Two SNVs were not detected by any of the callers because of a rare reference allele, and one SNV in a low coverage region was only detected by two algorithms. Regarding CNVs, ExomeDepth detected 5/5 multi-exon deletions, panelcn.MOPs 4/5 and only 3/5 deletions were accurately detected by CNVPanelizer. Conclusions: The calling efficiency of NGS algorithms for SNVs is influenced by variant type and coverage. NGS protocols need to account for the presence of rare variants in the reference sequence as well as for ambiguities in indel calling. CNV detection algorithms can be used to identify large deletions from NGS panel data for diagnostic applications; however, sensitivity depends on coverage, selection of the reference set and deletion size. We recommend the incorporation of several variant callers in the NGS pipeline to maximize variant detection efficiency.

Certain mitochondrial DNA (mtDNA) variants and haplogroups have been found to be associated with neurological disorders. Several studies have suggested that mtDNA variation could have an etiologic role in these disorders by affecting the ATP production on high-energy demanding organs, such as the brain. We have analyzed 15 mtDNA SNPs (mtSNPs) in five cohorts of cases presenting Alzheimer disease (AD), Parkinson disease (PD), and migraine, and in controls, to evaluate the role mtDNA variation in disease risk. Association tests were undertaken both for mtSNPs and mitochondrial haplogroups. No significant association was detected for any mtSNP or haplogroup in AD and PD cohorts. Two mtSNPs were associated with one migraine cohort after correcting for multiple tests, namely, T4216C and G13708A and haplogroup J (FDR q-value = 0.02; Santiago's cohort). However, this association was not confirmed in a second replication migraine series. A review of the literature reveals the existence of inconsistent findings and methodological shortcomings affecting a large proportion of mtDNA association studies on AD, PD, and migraine. A detailed inspection of the literature highlights the need for performing more rigorous methodological and statistical standards in mtDNA genetic association studies aimed to avoid false positive results of association between mtDNA variants and neurological diseases.

The field of medical genomics involves translating high throughput genetic methods to the clinic, in order to improve diagnostic efficiency and treatment decision making. Technical questions related to sample enrichment, sequencing methodologies and variant identification and calling algorithms, still need careful investigation in order to validate the analytical step of next generation sequencing techniques for clinical applications. However, the main foreseeable challenge will be interpreting the clinical significance of the variants observed in a given patient, as well as their significance for family members and for other patients.Every step in the variant interpretation process has limitations and difficulties, and its quote of contribution to false positive and false negative results. There is no single piece of evidence enough on its own to make firm conclusions on the pathogenicity and disease causality of a given variant.A plethora of automated analysis software tools is being developed that will enhance efficiency and accuracy. However a risk of misinterpretation could derive from biased biorepository content, facilitated by annotation of variant functional consequences using previous datasets stored in the same or linked repositories. In order to improve variant interpretation and avoid an exponential accumulation of confounding noise in the medical literature, the use of terms in a standard way should be sought and requested when reporting genetic variants and their consequences. Generally, stepwise and linear interpretation processes are likely to overrate some pieces of evidence while underscoring others. Algorithms are needed that allow a multidimensional, parallel analysis of diverse lines of evidence to be carried out by expert teams for specific genes, cellular pathways or disorders.

The diagnostic gap for rare neurodegenerative diseases is still considerable, despite continuous advances in gene identification. Many novel Mendelian genes have only been identified in a few families worldwide. Here we report the identification of an autosomal-dominant gene for hereditary spastic paraplegia (HSP) in 10 families that are of diverse geographic origin and whose affected members all carry unique truncating changes in a circumscript region of UBAP1 (ubiquitin-associated protein 1). HSP is a neurodegenerative disease characterized by progressive lower-limb spasticity and weakness, as well as frequent bladder dysfunction. At least 40% of affected persons are currently undiagnosed after exome sequencing. We identified pathological truncating variants in UBAP1 in affected persons from Iran, USA, Germany, Canada, Spain, and Bulgarian Roma. The genetic support ranges from linkage in the largest family (LOD = 8.3) to three confirmed de novo mutations. We show that mRNA in the fibroblasts of affected individuals escapes nonsense-mediated decay and thus leads to the expression of truncated proteins; in addition, concentrations of the full-length protein are reduced in comparison to those in controls. This suggests either a dominant-negative effect or haploinsufficiency. UBAP1 links endosomal trafficking to the ubiquitination machinery pathways that have been previously implicated in HSPs, and UBAP1 provides a bridge toward a more unified pathophysiology.

Hereditary spastic paraplegias constitute a heterogeneous group of neurodegenerative diseases encompassing pure and complicated forms, for which at least 52 loci and 31 causative genes have been identified. Although mutations in the SPAST gene explain approximately 40% of the pure autosomal dominant forms, molecular diagnosis can be challenging for the sporadic and recessive forms, which are often complicated and clinically overlap with a broad number of movement disorders. The validity of exome sequencing as a routine diagnostic approach in the movement disorder clinic needs to be assessed. The main goal of this study was to explore the usefulness of an exome analysis for the diagnosis of a complicated form of spastic paraplegia. Whole-exome sequencing was performed in two Spanish siblings with a neurodegenerative syndrome including upper and lower motor neuron, ocular and cerebellar signs. Exome sequencing revealed that both patients carry a novel homozygous nonsense mutation in exon 15 of the SPG11 gene (c.2678G>A; p.W893X), which was not found in 584 Spanish control chromosomes. After many years of follow-up and multiple time-consuming genetic testing, we were able to diagnose these patients by making use of whole-exome sequencing, showing that this is a cost-efficient diagnostic tool for the movement disorder specialist.

PLA2G6-associated neurodegeneration (PLAN) and hereditary spastic paraplegia (HSP) are 2 groups of heterogeneous neurodegenerative diseases. In this study, we report PLA2G6 gene mutations in 3 families from Turkey, Morocco, and Romania. Two affected Turkish siblings presenting HSP adds the disease to PLAN phenotypes. They were homozygous for the PLA2G6 missense c.2239C>T, p.Arg747Trp variant and the ages of onset were 9 and 21. Parkinsonism, dystonia or cognitive decline were not the clinical elements in these patients contrary to the cases that has been previously reported with the same variant, however, iron accumulation was evident in their cranial magnetic resonance imaging. The Moroccan patient was homozygous for a novel missense c.1786C>T, p.Leu596Phe variant and the Romanian patient had 2 novel mutations; c.1898C>T, p.Ala633Val and c.1765_1768del, p.Ser589ThrfsTer76. Both of these patients conformed better to childhood onset PLAN with the age of onset at 4 and 7 years, respectively. Interestingly, all identified mutations were affecting the highly conserved patatin-like phospholipase domain of the PLA2G6 protein.

The spinocerebellar ataxias (SCAs) form a clinically, genetically, and pathological heterogeneous group of autosomal-dominant degenerative diseases. In particular, SCA36 is characterized by a late-onset, slowly progressive cerebellar syndrome typically associated with sensorineural hearing loss. This study was aimed at analyzing the neurodegenerative process underlying SCA36 through fluorodeoxyglucose positron emission tomography (FDG-PET) and MRI scans.Twenty SCA36 patients underwent a study consisting of FDG-PET and MRI scans. Clinical motor evaluation was performed through the Scale for the Assessment and Rating of Ataxia (SARA). FDG-PET was carried out using a voxel-by-voxel and region-of-interest analysis. MRI evaluation was based on visual inspection and volumetric analysis.SARA ranged from 0 to 24.5 (4 patients asymptomatic, 3 with unspecific symptoms, and 13 with cerebellar signs). FDG-PET revealed hypometabolism in the asymptomatic stage in the vermis and right cerebellar hemisphere. In the ataxic stage, hypometabolism spread to both cerebellar hemispheres and the brain stem. MRI was normal in asymptomatic and preataxic individuals and showed superior cerebellar vermis atrophy early in the ataxic stage, diffuse cerebellar atrophy some years into the disease course, and a pattern of olivopontocerebellar atrophy in the oldest patients. There was no significant cerebellar atrophy in patients younger than 50 years.We present the first FDG-PET study of SCA36 and one of the largest neuroimaging study of SCAs. Our results revealed neuronal dysfunctions in the vermis and right cerebellar hemisphere as soon as a decade before the onset of motor symptoms. In the ataxic stage, dysfunctions spread to both hemispheres and the brain stem. © 2016 International Parkinson and Movement Disorder Society.

Spinocerebellar ataxia 36 has been recently described in Japanese families as a new type of spinocerebellar ataxia with motor neuron signs. It is caused by a GGCCTG repeat expansion in intron 1 of NOP56 . Family interview and document research allowed us to reconstruct two extensive, multigenerational kindreds stemming from the same village (Costa da Morte in Galicia, Spain), in the 17th century. We found the presence of the spinocerebellar ataxia 36 mutation co-segregating with disease in these families in whom we had previously identified an ∼0.8 Mb linkage region to chromosome 20 p. Subsequent screening revealed the NOP56 expansion in eight additional Galician ataxia kindreds. While normal alleles contain 5–14 hexanucleotide repeats, expanded alleles range from ∼650 to 2500 repeats, within a shared haplotype. Further expansion of repeat size was frequent, especially upon paternal transmission, while instances of allele contraction were observed in maternal transmissions. We found a total of 63 individuals carrying the mutation, 44 of whom were confirmed to be clinically affected; over 400 people are at risk. We describe here the detailed clinical picture, consisting of a late-onset, slowly progressive cerebellar syndrome with variable eye movement abnormalities and sensorineural hearing loss. There were signs of denervation in the tongue, as well as mild pyramidal signs, but otherwise no signs of classical amyotrophic lateral sclerosis. Magnetic resonance imaging findings were consistent with the clinical course, showing atrophy of the cerebellar vermis in initial stages, later evolving to a pattern of olivo-ponto-cerebellar atrophy. We estimated the origin of the founder mutation in Galicia to have occurred ∼1275 years ago. Out of 160 Galician families with spinocerebellar ataxia, 10 (6.3%) were found to have spinocerebellar ataxia 36, while 15 (9.4%) showed other of the routinely tested dominant spinocerebellar ataxia types. Spinocerebellar ataxia 36 is thus, so far, the most frequent dominant spinocerebellar ataxia in this region, which may have implications for American countries associated with traditional Spanish emigration. * Abbreviations : SCA : spinocerebellar ataxia SNP : single nucleotide polymorphism TP-PCR : triplet repeat primed polymerase chain reaction

Familial idiopathic basal ganglia calcification (IBGC) is a genetic condition with a wide spectrum of neuropsychiatric symptoms, including parkinsonism and dementia. Here, we identified mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), in IBGC-affected families of varied ancestry, and we observed significantly impaired phosphate transport activity for all assayed PiT2 mutants in Xenopus laevis oocytes. Our results implicate altered phosphate homeostasis in the etiology of IBGC.

Neurofibromatosis type 1 (NF1) is a multisystem disease with autosomal dominant inheritance and complete penetrance diagnosed by clinical findings. Cutaneous neurofibromas are present in almost all adult patients in the dermis, epidermis or along the peripheral nerves. Plexiform neurofibromas are subcutaneous or deep lesions involving nerve plexuses or roots. Neurofibromas can degenerate into malignant tumors, with important prognostic implications. NF1 shows a broad clinic variability even within a single family. Exceptions are cases reporting the in-frame microdeletion c.2970_2972delAAT, presenting with the typical pigmentary features of NF1, but no cutaneous or plexiform neurofibromas. We report a patient with a de novo c.2970_2972delAAT mutation who had few café-au-lait spots, only 2 of which measured >15 mm, axillary and submammary freckling, a flat angioma extending over the neck, arm and trunk, a high arched palate, micrognathia, macrocephaly, pes cavus and scoliosis. There was complete absence of observable cutaneous neurofibromas as well as external plexiform neurofibromas. She had had epileptic seizures since childhood; however, a diagnosis of NF1 had not been confirmed until she was 38, partly due to the paucity of characteristic cutaneous stigmata. We confirm the association of the c.2970_2972delAAT mutation in NF1 with a particular clinical phenotype, especially with lack of detectable neurofibromas. For an appropriate management of patients and family counseling, molecular study of the NF1 gene should be considered in patients not fulfilling NIH criteria when other features suggestive of NF1 are present. In the absence of neurofibromas, starting NF1 testing with the screening of exon 17 may be worthwhile.

Resumen: Introducción: La expansión de los estudios genéticos está transformando la práctica de la Neurología y enfrenta a los servicios clínicos con nuevos retos, como la articulación del asesoramiento genético. La amplitud de los conocimientos tanto clínicos como moleculares precisos, así como la necesidad de una evaluación psicológica y apoyo familiar, especialmente en los análisis predictivos y planificación reproductiva, hacen necesario un enfoque multidisciplinar. Desarrollo: Las características principales de las enfermedades neurodegenerativas de base genética son el elevado nivel de especialización requerido —por tratarse de enfermedades poco comunes y de difícil diagnóstico— junto con su carácter generalmente progresivo, la ausencia de tratamientos eficaces, la problemática generada por la posibilidad de estudios predictivos y la interpretación de los resultados genéticos. El objetivo del asesoramiento genético es proporcionar la información suficiente y objetiva para que cada individuo pueda tomar sus propias decisiones sobre el estudio genético. Debe incluir la evaluación de aspectos psicológicos y de comunicación familiar. El programa PICOGEN del Hospital Clinic de Barcelona para el análisis y asesoramiento genético en demencias es un buen ejemplo de una estrategia integrada capaz de abordar esta nueva situación asistencial en Neurología. Lamentablemente, este programa es una excepción en España y los pacientes con enfermedades neurogenéticas y sus familias no tienen garantizada habitualmente una asistencia adecuada. Conclusiones: El asesoramiento genético es un acto clínico per se, que precisa de un espacio, tiempo y recursos suficientes. Implica una participación multidisciplinar, atención a los aspectos psicológicos y familiares y no se puede llevar a cabo correctamente en el seno de una consulta rutinaria estándar de Neurología. Es necesaria una legislación que garantice la adecuada articulación del asesoramiento genético en nuestro país con criterios de calidad y equidad. Esto incluye la regulación de la formación de los profesionales necesarios, la clarificación de competencias y la dotación de recursos a las instituciones para el desarrollo de estos programas. Abstract: Introduction: The generalization of genetic studies is transforming the practice of Neurology and confronts the clinical departments with new challenges, such as the organization of genetic counseling. The requirement of specialized knowledge, both clinical and molecular, as well as the need for psychological evaluation and family support, especially for predictive testing and reproductive planning, makes a multidisciplinary approach mandatory. Development: The main characteristics of genetic neurodegenerative diseases are the high level of required specialization – since these disorders are often rare and of difficult diagnosis – together with a generally progressive course, unavailability of effective treatment, the issues generated by predictive testing and the interpretation of genetic testing. The aim of genetic counseling is to provide sufficient and objective information for each individual to make their own decision on genetic testing. It must touch upon psychological aspects and family communication. The PICOGEN program from the Clinic Hospital in Barcelona for genetic testing and counseling of dementias is a good example of integrated strategy capable of managing this new clinical scenario in neurology. Unfortunately, this program is an exception in Spain and the patients with neurogenetic disorders and their families usually do not have guaranteed access to an appropriate care. Conclusions: Genetic counseling is a unique clinical activity that requires provision of enough time, space and resources to be developed. It implies multidisciplinary participation, due attention to psychological and family issues, and cannot be carried out adequately in a routine Neurology clinic. Legislation is needed to promote a correct articulation of genetic counseling in our country with guarantee of quality and equity. This includes training of the necessary health professionals, clarification of competences and provision of resources to the institutions for the development of such programs. Palabras clave: Análisis genéticos, Asesoramiento genético, Neurogenética, Legislación, Enfermedades raras, Test predictivos, Keywords: Genetic tests, Genetic counseling, Neurogenetics, Legislation, Rare disorders, Predictive testing

Introduction: The generalization of genetic studies is transforming the practice of Neurology and confronts the clinical departments with new challenges, such as the organization of genetic counselling. The requirement of specialized knowledge, both clinical and molecular, as well as the need for psychological evaluation and family support, especially for predictive testing and reproductive planning, makes a multidisciplinary approach mandatory. Development: The main characteristics of genetic neurodegenerative diseases are the high level of required specialization - since these disorders are often rare and of difficult diagnosis - together with a generally progressive course, unavailability of effective treatment, the issues generated by predictive testing and the interpretation of genetic testing. The aim of genetic counselling is to provide sufficient and objective information for each individual to make their own decision on genetic testing. It must touch upon psychological aspects and family communication. The PICOGEN program from the Clinic Hospital in Barcelona for genetic testing and counselling of dementias is a good example of integrated strategy capable of managing this new clinical scenario in neurology. Unfortunately, this program is an exception in Spain and the patients with neurogenetic disorders and their families usually do not have guaranteed access to an appropriate care. Conclusions: Genetic counselling is a unique clinical activity that requires provision of enough time, space and resources to be developed. It implies multidisciplinary participation, due attention to psychological and family issues, and cannot be carried out adequately in a routine Neurology clinic. Legislation is needed to promote a correct articulation of genetic counselling in our country with guarantee of quality and equity. This includes training of the necessary health professionals, clarification of competences and provision of resources to the institutions for the development of such programs. Resumen: Introducción: La expansión de los estudios genéticos está transformando la práctica de la Neurología y enfrenta a los servicios clínicos con nuevos retos, como la articulación del asesoramiento genético. La amplitud de los conocimientos tanto clínicos como moleculares precisos, así como la necesidad de una evaluación psicológica y apoyo familiar, especialmente en los análisis predictivos y planificación reproductiva, hacen necesario un enfoque multidisciplinar. Desarrollo: Las características principales de las enfermedades neurodegenerativas de base genética son el elevado nivel de especialización requerido —por tratarse de enfermedades poco comunes y de difícil diagnóstico— junto con su carácter generalmente progresivo, la ausencia de tratamientos eficaces, la problemática generada por la posibilidad de estudios predictivos y la interpretación de los resultados genéticos. El objetivo del asesoramiento genético es proporcionar la información suficiente y objetiva para que cada individuo pueda tomar sus propias decisiones sobre el estudio genético. Debe incluir la evaluación de aspectos psicológicos y de comunicación familiar. El programa PICOGEN del Hospital Clínic de Barcelona para el análisis y asesoramiento genético en demencias es un buen ejemplo de una estrategia integrada capaz de abordar esta nueva situación asistencial en Neurología. Lamentablemente, este programa es una excepción en España y los pacientes con enfermedades neurogenéticas y sus familias no tienen garantizada habitualmente una asistencia adecuada. Conclusiones: El asesoramiento genético es un acto clínico per se, que precisa de un espacio, tiempo y recursos suficientes. Implica una participación multidisciplinar, atención a los aspectos psicológicos y familiares y no se puede llevar a cabo correctamente en el seno de una consulta rutinaria estándar de Neurología. Es necesaria una legislación que garantice la adecuada articulación del asesoramiento genético en nuestro país con criterios de calidad y equidad. Esto incluye la regulación de la formación de los profesionales necesarios, la clarificación de competencias y la dotación de recursos a las instituciones para el desarrollo de estos programas. Keywords: Genetic tests, Genetic counselling, Neurogenetics, Legislation, Rare disorders, Predictive testing, Palabras clave: Análisis genéticos, Asesoramiento genético, Neurogenética, Legislación, Enfermedades raras, Test predictivos

Other participants at the EMQN Best Practice Meeting and/or at the electronic discussions thereafter were Isabel Alonso, Anna Andersson, David Barton, Nazli Bazac, Kyproula Christodoulou, Luís Correia, Mark Davis, Mary Davis, Rob Elles, Marina Frontali, Javier Garcia-Planells, Paola Giunti, Petra Hämäläinen, Jenni Jonasson, Outi Kamarainen, Nina Larsson, Eric Leguern, Monique Losekoot, Carla Martins, Michael Morris, Clemens Müller-Reible, Simon Patton, M Luiza Saraiva-Pereira, Jorge Pinto-Basto, Beatriz Quintáns, Simon C Ramsden, Anna Ravani, Laura Rooke, Isabel Silveira, Richard Sinke, Su Stenhouse, Laura Stewart, Katrien Storm, Anna Sulek-Piatkowska, Francine Thonney, Victor Volpini, Jon Warner, Helga Weirich, Stefan Wieczorek and Christine Zühlke.

Hereditary ataxias are clinically and genetically heterogeneous neurodegenerative disorders. Although many ataxia genes have been identified, about 50 % of cases await the identification of the genetic cause [1]. High-throughput sequencing, namely whole-exome sequencing (WES), has changed the paradigm of genetic analysis of rare heterogeneous Mendelian disorders [2], and allowed the identification of STUB1 mutations as the cause of autosomal recessive ataxia syndromes [3–7]. We report on clinical and pathological features of a family with spastic ataxia due to novel STUB1

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC. Lung GO Sequencing Project Lung GO Sequencing Project Women's Health Initiative (WHI) Sequencing Project Broad GO Sequencing Project Seattle GO Sequencing Project Heart GO Sequencing Project United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Neurological Disorders & Stroke (NINDS) Association Francaise contre les Myopathies Ligue Nationale contre le Cancer (Comite de l'Herault) Fondation pour la Recherche Medicale FEDER European Union Languedoc-Roussillon grant National Institute of Neurological Disorders and Stroke Informatics Center for Neurogenetics and Neurogenomics Fondation pour la Recherche Medicale Institut National du Cancer (INCA) France Labex GR-Ex Labex EpiGenMed French National Research Agency (ANR) Institut National de la Sante et de la Recherche Medicale (Inserm) European Commission Instituto de Salud Carlos III (ISCIII) INNOPHARMA project MINECO-USC Servizo Galego de Saúde (SERGAS) National Council for Scientific and Technological Development (CNPq) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Mental Health (NIMH) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Neurological Disorders & Stroke (NINDS) University Hospital of Rouen French CNR-MAJ United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Heart Lung & Blood Institute (NHLBI) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Mental Health (NIMH) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Neurological Disorders & Stroke (NINDS)

The Spanish and Portuguese ISFG Working Group (GEP-ISFG) carried out a collaborative exercise in order to asses the performance of two Y chromosome STR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The groups that reported correct results in all the systems were also asked to analyse a population sample in order to evaluate the informative content of these STRs in different populations. A total of 1020 males out of 13 population samples from Argentina, Brazil, Costa Rica, Macao, Mozambique, Portugal and Spain were analysed for all the loci included in the present study. Haplotype and allele frequencies of these eight Y-STRs were estimated in all samples. The lowest haplotype diversity was found in the Lara (Argentina) population (95.44%) and the highest (99.90%) in Macao (China). Pairwise haplotype analysis showed the relative homogeneity of the Iberian origin samples, in accordance with what was previously found in the European populations for other Y-STR haplotypes (http://www.ystr.org). As expected, the four non-Caucasian samples, Macao (Chinese), Mozambique (Africans), Costa Rica (Africans) and Argentina (Lara, Amerindians), show highly significant Φst values in the pairwise comparisons with all the Caucasian samples. © 2003 Elsevier Ireland Ltd. All rights reserved.

A set of seven common Y-chromosome specific microsatellites has been used in the present report to type 308 individuals of 16 African population groups from Mozambique. These microsatellites include the DYS19, DYS390, DYS391, DYS392, DYS393, DYS389I and DYS389II systems. The population structure was analysed and therefore genetic distances and several diversity indices were computed and compared with other populations around the world. The results obtained in the present study show that these populations share common characteristics with other available Bantu-speaking African population groups and clear differences with non-African ones. The combination of the analysis of Y-chromosome STRs with other more slowly mutating Y-chromosome polymorphisms (biallelic markers), mtDNA sequences and autosomal data for the same populations, will allow the analysis of human evolution in paternal lineages in different time scales. The forensic implications of these results are discussed. D 2003 Elsevier Science B.V. All rights reserved.

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs. Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

In last years, interest in Single Nucleotide Polymorphism (SNP) has increased due to their numerous applications: in medical genetics, in human and evolutionary genetics but also in forensic field. Polymorphisms placed in the mitochondrial genome are particularly important in a forensic context in part due to the fact that it is in high copy number per cell. Consequently, mtDNA is especially useful to the study of degraded samples with little or no nuclear DNA as being old skeletal remains and hair shafts. On the other hand, mtDNA SNPs have demonstrate its usefulness for human population studies. The development of new methodologies for high-throughput SNPs analysis is one of the most promising areas in genetics. Here, we describe a rapid and robust assay to simultaneously genotype 17 SNPs of mitochondrial DNA by minisequencing reaction using SNaPshot (Applied Biosystems). The methodology here described shows high accuracy and sensitivity, and avoid the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the requirement of high quantities of DNA template. D 2003 Elsevier B.V. All rights reserved.

In order to assess the microgeographic heterogeneity of Y chromosome lineage's distribution in the north-western part of Iberian Peninsula, 409 male samples from the five districts of Northern Portugal and two districts of Southern Galicia were typed for 15 Y chromosome microsatellite markers. The results suggest that, in NW Iberia, barriers other than political were more important in shaping present Y chromosome lineage substructuring.

Familial idiopathic basal ganglia calcification (IBGC) or Fahr's disease is a rare neurodegenerative disorder characterized by calcium deposits in the basal ganglia and other brain regions, which is associated with neuropsychiatric and motor symptoms. Familial IBGC is genetically heterogeneous and typically transmitted in an autosomal dominant fashion. We performed a mutational analysis of SLC20A2, the first gene found to cause IBGC, to assess its genetic contribution to familial IBGC. We recruited 218 subjects from 29 IBGC-affected families of varied ancestry and collected medical history, neurological exam, and head CT scans to characterize each patient's disease status. We screened our patient cohort for mutations in SLC20A2. Twelve novel (nonsense, deletions, missense, and splice site) potentially pathogenic variants, one synonymous variant, and one previously reported mutation were identified in 13 families. Variants predicted to be deleterious cosegregated with disease in five families. Three families showed nonsegregation with clinical disease of such variants, but retrospective review of clinical and neuroimaging data strongly suggested previous misclassification. Overall, mutations in SLC20A2 account for as many as 41 % of our familial IBGC cases. Our screen in a large series expands the catalog of SLC20A2 mutations identified to date and demonstrates that mutations in SLC20A2 are a major cause of familial IBGC. Non-perfect segregation patterns of predicted deleterious variants highlight the challenges of phenotypic assessment in this condition with highly variable clinical presentation.

The C9ORF72 Spanish Study Group, et al., A hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) can cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). We assessed its frequency in 781 sporadic ALS (sALS) and 155 familial ALS (fALS) cases, and in 248 Spanish controls. We tested the presence of the reported founder haplotype among mutation carriers and in 171 Ceph Europeans from Utah (CEU), 170 Yoruba Africans, 81 Han Chinese, and 85 Japanese subjects. The C9orf72 expansion was present in 27.1% of fALS and 3.2% of sALS. Mutation carriers showed lower age at onset (P = 0.04), shorter survival (P = 0.02), greater co-occurrence of FTD (P = 8.2 × 10-5), and more family history of ALS (P = 1.4 × 10-20), than noncarriers. No association between alleles within the normal range and the risk of ALS was found (P = 0.12). All 61 of the mutation carriers were tested and a patient carrying 28 hexanucleotide repeats presented with the founder haplotype. This haplotype was found in 5.6% Yoruba Africans, 8.9% CEU, 3.9% Japanese, and 1.6% Han Chinese chromosomes. © 2012 Wiley Periodicals, Inc., We acknowledge the ALS Research Spanish Foundation (FUNDELA) and the UTE project FIMA (Spain) for their help to P.P. Contract grant sponsors: Neuromuscular Database Project, CIBERNED (PI 2010/11); MICINN (SAF2010-10434); ISCIII (PI10/00092 and EC08/00049).

Hereditary spastic paraplegias (HSPs) constitute a heterogeneous group of neurological disorders, characterized primarily by progressive spasticity and weakness of the lower limbs. HSPs are caused by mutations in multiple genes (at least 48 loci and 28 causative genes). The clinical spectrum of HSPs is wide and important differences have been reported between patients with distinct mutations in the same gene, or even between different family members bearing the same mutation. Many patients with HSP present clinical deficits related to the involvement of neuronal systems other than corticospinal tracts, namely, peripheral nerves, sensory, or cerebellar pathways. These cases may be difficult to differentiate from other neurological diseases (e.g., hereditary ataxias), also genetically and clinically heterogeneous. As an illustration of how overlapping this genotype–phenotype relationship is, and the difficulties that it brings upon the development of neurogenetic algorithms and databases, we review the main clinical and genetic features of HSPs, and summarize reports on cases of triplet-repeat spinocerebellar ataxias that can mimic HSP phenotypes. This complex scenario makes the necessity of high-quality, curated mutation databases even more urgent, in order to develop adequate diagnostic guidelines, correct interpretation of genetic testing, and appropriate genetic counseling. Hum Mutat 33:1315–1323, 2012. © 2012 Wiley Periodicals, Inc.

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.

Background: R0 embraces the most common mitochondrial DNA (mtDNA) lineage in West Eurasia, namely, haplogroup H (,40%). R0 sub-lineages are badly defined in the control region and therefore, the analysis of diagnostic coding region polymorphisms is needed in order to gain resolution in population and medical studies. Methodology/Principal Findings: We sequenced the first hypervariable segment (HVS-I) of 518 individuals from different North Iberian regions. The mtDNAs belonging to R0 (,57%) were further genotyped for a set of 71 coding region SNPs characterizing major and minor branches of R0. We found that the North Iberian Peninsula shows moderate levels of population stratification; for instance, haplogroup V reaches the highest frequency in Cantabria (north-central Iberia), but lower in Galicia (northwest Iberia) and Catalonia (northeast Iberia). When compared to other European and Middle East populations, haplogroups H1, H3 and H5a show frequency peaks in the Franco-Cantabrian region, declining from West towards the East and South Europe. In addition, we have characterized, by way of complete genome sequencing, a new autochthonous clade of haplogroup H in the Basque country, named H2a5. Its coalescence age, 15.668 thousand years ago (kya), dates to the period immediately after the Last Glacial Maximum (LGM). Conclusions/Significance: In contrast to other H lineages that experienced re-expansion outside the Franco-Cantabrian refuge after the LGM (e.g. H1 and H3), H2a5 most likely remained confined to this area till present days. This research received support from Xunta de Galicia (Grupos Emerxentes; 2008/XA122), two grants from the Fundación de Investigación Médica Mutua Madrileña, and a grant from the Ministerio de Ciencia e Innovación (SAF2008-02971) given to AS SI

We describe a rapid and robust assay to genotype mitochondrial deoxyribonucleic acid (mtDNA) coding region single nucleotide polymorphism (SNPs) using the SNaPshot (Applied Biosystems, Foster City, CA) minisequencing reaction kit. A protocol for mtDNA SNaPshot typing is described in detail, although we emphasize that this method allows great flexibility in the implementation of whatever set of mtDNA SNPs. We discuss the utility of our selection of mtDNA SNPs for molecular anthropologists and forensic geneticists. Firstly, these SNPs allow allocating common mitochondrial West Eurasian haplotypes into their corresponding branches of the mtDNA skeleton, with special attention to the subdivision of sequences belonging to haplogroup H, the most frequent European haplogroup (40-50%) and the worst phylogenetically characterized in the first and second hypervariable segments (HVS-I/II; by far, the most common segments analyzed by sequencing). Second, the polymorphic positions selected for this multiplex reaction considerably increase the discrimination power of current mitochondrial analysis in the forensic field. The method shows high accuracy and robustness, avoiding both the use of alternative time-consuming classical strategies (i.e., restriction fragment length polymorphism typing) and the requirement of high quantities of DNA template.

We describe a rapid and robust assay to genotype mitochondrial deoxyribonucleic acid (mtDNA) coding region single nucleotide polymorphism (SNPs) using the SNaPshot (Applied Biosystems, Foster City, CA) minisequencing reaction kit. A protocol for mtDNA SNaPshot typing is described in detail, although we emphasize that this method allows great flexibility in the implementation of whatever set of mtDNA SNPs. We discuss the utility of our selection of mtDNA SNPs for molecular anthropologists and forensic geneticists. Firstly, these SNPs allow allocating common mitochondrial West Eurasian haplotypes into their corresponding branches of the mtDNA skeleton, with special attention to the subdivision of sequences belonging to haplogroup H, the most frequent European haplogroup (40-50%) and the worst phylogenetically characterized in the first and second hypervariable segments (HVS-I/II; by far, the most common segments analyzed by sequencing). Second, the polymorphic positions selected for this multiplex reaction considerably increase the discrimination power of current mitochondrial analysis in the forensic field. The method shows high accuracy and robustness, avoiding both the use of alternative time-consuming classical strategies (i.e., restriction fragment length polymorphism typing) and the requirement of high quantities of DNA template.

Objective To investigate the genetic effect of a new mutation found in exon 17 of the myophosphorylase ( PYGM ) gene as a cause of McArdle disease (also known as type 5 glycogenosis). Patients A Spanish patient with McArdle disease was screened for 3 common mutations in the PYGM gene (R49X, W797R, and G204S), as previously described. The patient was heterozygous for R49X. To find other mutations, the coding sequence of the entire PYGM gene was sequenced. The carrier status of his relatives was also studied. Results A novel rare mutation was found in codon 691 of exon 17. This is an insertion/deletion (indel) and consists simultaneously of a deletion of 2 bases and an insertion of 3 bases (691delCC/insAAA). A restriction analysis was designed to simplify the detection method. Conclusions The 691delCC/insAAA is the third indel described in the PYGM gene. Indels represent 0.95% of the total reported mutations in the Human Gene Mutation Database. The molecular origin of this mutation is not fully understood. These findings point again to the allelic heterogeneity of McArdle disease.

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.

Haplotype, allele frequencies and population data of eight Y-chromosome STR loci, DYS437, DYS438, DYS439, GATA A10, GATA A7.1, GATA A7.2, GATA C4 and GATA H4, were determined from a sample of 212 unrelated male individuals from Galicia (NW of Spain).