Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Genetic variability in sporadic amyotrophic lateral sclerosis.

With the advent of gene therapies for amyotrophic lateral sclerosis (ALS), there is a surge in gene testing for this disease. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging, given the complex genetic architecture of sALS, for which there are genetic variants with large and small effect sizes. Guidelines for the interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the American College of Medical Genetics and Genomics (ACMG) criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (odds ratio 1.75; P-value 1.64 × 10-5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS patients. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS.

The Effect of SMN Gene Dosage on ALS Risk and Disease Severity.

OBJECTIVE: The role of the survival of motor neuron (SMN) gene in amyotrophic lateral sclerosis (ALS) is unclear, with several conflicting reports. A decisive result on this topic is needed, given that treatment options are available now for SMN deficiency. METHODS: In this largest multicenter case control study to evaluate the effect of SMN1 and SMN2 copy numbers in ALS, we used whole genome sequencing data from Project MinE data freeze 2. SMN copy numbers of 6,375 patients with ALS and 2,412 controls were called from whole genome sequencing data, and the reliability of the calls was tested with multiplex ligation-dependent probe amplification data. RESULTS: The copy number distribution of SMN1 and SMN2 between cases and controls did not show any statistical differences (binomial multivariate logistic regression SMN1 p = 0.54 and SMN2 p = 0.49). In addition, the copy number of SMN did not associate with patient survival (Royston-Parmar; SMN1 p = 0.78 and SMN2 p = 0.23) or age at onset (Royston-Parmar; SMN1 p = 0.75 and SMN2 p = 0.63). INTERPRETATION: In our well-powered study, there was no association of SMN1 or SMN2 copy numbers with the risk of ALS or ALS disease severity. This suggests that changing SMN protein levels in the physiological range may not modify ALS disease course. This is an important finding in the light of emerging therapies targeted at SMN deficiencies. ANN NEUROL 2021;89:686-697.

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). RESULTS: Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific disease-associated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. CONCLUSIONS: To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance.

Mutations in the tail and rod domains of the neurofilament heavy-chain gene increase the risk of ALS.

OBJECTIVE: Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly explored. Still, NEFH is commonly included in genetic screening panels worldwide. We therefore aimed to determine if NEFH variants modify ALS risk. METHODS: Genetic data of 11,130 people with ALS and 7,416 controls from the literature and Project MinE were analysed. We performed meta-analyses of published case-control studies reporting NEFH variants, and variant analysis of NEFH in Project MinE whole-genome sequencing data. RESULTS: Fixed-effects meta-analysis found that rare (MAF <1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.55, 95% CI 2.13-9.71, p < 0.0001). In Project MinE, ultrarare NEFH variants increased ALS risk (OR 1.37 95% CI 1.14-1.63, p = 0.0007), with rod domain variants (mostly intronic) appearing to drive the association (OR 1.45 95% CI 1.18-1.77, pMadsen-Browning = 0.0007, pSKAT-O = 0.003). While in the tail domain, ultrarare (MAF <0.1%) pathogenic missense variants were also associated with higher risk of ALS (OR 1.94, 95% CI 0.86-4.37, pMadsen-Browning = 0.039), supporting the meta-analysis results. Finally, several tail in-frame deletions were also found to affect disease risk, however, both protective and pathogenic deletions were found in this domain, highlighting an intricate architecture that requires further investigation. INTERPRETATION: We showed that NEFH tail missense and in-frame deletion variants, and intronic rod variants are risk factors for ALS. However, they are not variants of large effect, and their functional impact needs to be clarified in further studies. Therefore, their inclusion in routine genetic screening panels should be reconsidered.

The GENESIS database and tools: A decade of discovery in Mendelian genomics.

In the past decade, human genetics research saw an acceleration of disease gene discovery and further dissection of the genetic architectures of many disorders. Much of this progress was enabled via data aggregation projects, collaborative data sharing among researchers, and the adoption of sophisticated and standardized bioinformatics analyses pipelines. In 2012, we launched the GENESIS platform, formerly known as GEM.app, with the aims to 1) empower clinical and basic researchers without bioinformatics expertise to analyze and explore genome level data and 2) facilitate the detection of novel pathogenic variation and novel disease genes by leveraging data aggregation and genetic matchmaking. The GENESIS database has grown to over 20,000 datasets from rare disease patients, which were provided by multiple academic research consortia and many individual investigators. Some of the largest global collections of genome-level data are available for Charcot-Marie-Tooth disease, hereditary spastic paraplegia, and cerebellar ataxia. A number of rare disease consortia and networks are archiving their data in this database. Over the past decade, more than 1500 scientists have registered and used this resource and published over 200 papers on gene and variant identifications, which garnered >6000 citations. GENESIS has supported >100 gene discoveries and contributed to approximately half of all gene identifications in the fields of inherited peripheral neuropathies and spastic paraplegia in this time frame. Many diagnostic odysseys of rare disease patients have been resolved. The concept of genomes-to-therapy has borne out for a number of such discoveries that let to rapid clinical trials and expedited natural history studies. This marks GENESIS as one of the most impactful data aggregation initiatives in rare monogenic diseases.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS. Methods: Samples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression. Results: There were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 × 10-12), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0×10-7). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0×10-4). Discussion: Although telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS.

The SOD1-mediated ALS phenotype shows a decoupling between age of symptom onset and disease duration.

Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability.

Structural variation analysis of 6,500 whole genome sequences in amyotrophic lateral sclerosis.

There is a strong genetic contribution to Amyotrophic lateral sclerosis (ALS) risk, with heritability estimates of up to 60%. Both Mendelian and small effect variants have been identified, but in common with other conditions, such variants only explain a little of the heritability. Genomic structural variation might account for some of this otherwise unexplained heritability. We therefore investigated association between structural variation in a set of 25 ALS genes, and ALS risk and phenotype. As expected, the repeat expansion in the C9orf72 gene was identified as associated with ALS. Two other ALS-associated structural variants were identified: inversion in the VCP gene and insertion in the ERBB4 gene. All three variants were associated both with increased risk of ALS and specific phenotypic patterns of disease expression. More than 70% of people with respiratory onset ALS harboured ERBB4 insertion compared with 25% of the general population, suggesting respiratory onset ALS may be a distinct genetic subtype.

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.

Phenotypic differences in a large family with Kennedy's disease from the Middle Black Sea region of Turkey.

We report the clinical and electrophysiological features of a large Turkish family with genetically confirmed X-linked spinal and bulbar muscular atrophy (SBMA). Family members were identified by field work. A detailed history was obtained from each subject, and each subject received a detailed neurological examination. To confirm the CAG repeat expansion in the AR gene, genomic DNA was extracted from the peripheral blood of patients. The family consisted of 128 individuals over five generations, with two consanguineous parents, one slightly affected female, and 12 affected males with SBMA. We studied the five surviving male patients and one surviving female carrier. The age at disease onset, phenotypic features, and disease severity varied among the family members. DNA analysis was performed on five individuals, belonging to five generations of the family. Four affected males and a slightly affected female carrier were shown to carry an expanded CAG repeat in the androgen receptor gene. This family report is consistent with previous studies suggesting that SBMA may be present with a wide clinical spectrum in affected family members. Further descriptions of SBMA affected families with different ethnic backgrounds may assist in identifying possible phenotypic and genetic features of the disease.

Two new beta-thalassemia deletions compromising prenatal diagnosis in an Italian and a Turkish couple seeking prevention.

When the molecular background of couples requesting prevention is unclear, family analysis and tools to define rare mutations are essential. We report two novel deletion defects observed in an Italian and in a Turkish couple. The first proband presented with microcytic hypochromic parameters without iron deficiency, a normal HbA(2) and an elevated HbF (10.6%). His father presented with a similar phenotype and his wife was heterozygous for the common Mediterranean codon 39 (HBB:c.118C>T) mutation. Having excluded point mutations and common deletions, Multiplex Ligation-dependent Probe Amplification was performed revealing an unknown Ggamma(Agammadeltabeta)(0)-thalassemia defect spanning from the Agamma gene to downstream of the beta-globin gene provisionally named Leiden 69.5 kb deletion. In the second case, the wife presented with a mild thalassemic picture, normal HbA(2), elevated HbF (18.5%) and a beta/alpha globin chain synthesis ratio of 0.62, without iron deficiency or any known beta-thalassemia defect, while the husband was a simple carrier of the common Mediterranean IVS-I-110 (HBB:c.93-21 G>A) mutation. A new large deletion involving the beta-gene and part of the delta-gene was identified by Multiplex Ligation-dependent Probe Amplification provisionally named "Leiden 7.4 kb".

Mitochondrial pathology in muscle of a patient with a novel parkin mutation.

Mutations of the parkin gene on chromosome 6 cause early-onset parkinsonism. Myopathy has not been reported to be a feature of this condition. Here we report the muscle biopsy findings of a 53-year-old man with a novel parkin gene mutation (IVS-9-1 deletion). His symptoms were characterized by typical early-onset, dopa-responsive, and slowly progressive parkinsonism. Parkin gene analysis revealed a homozygous IVS-9-1 deletion in the proband and his sibling. The unusual feature was hypertrophy of bilateral thigh muscles in the proband. Muscle biopsy from the biceps brachii muscle showed abundant cytochrome oxidase (COX) (-) fibers. This is the first report on the coexistence of a myopathy with COX deficiency with parkin disease and may shed light on the function of parkin in muscle.

Shunt Tapping Versus Lumbar Puncture for Evaluating Cerebrospinal Fluid Infections in a Pediatric Population.

AIM: To compare the results of lumbar puncture (LP) and shunt tapping in pediatric patients with suspected ventriculoperitoneal shunt infection. MATERIAL AND METHODS: Medical records of pediatric patients with suspected shunt infections were retrospectively analyzed. All patients had cerebrospinal fluid samples obtained either via shunt tapping, LP or both. The diagnosis of infection was made when at least one cerebrospinal fluid had positive culture results. The patients with negative cerebrospinal fluid culture results were followed up for at least 6 months to monitor the occurrence of central nervous system infection. RESULTS: There were 20 patients in the study (12 males, 8 females). Cerebrospinal fluid was obtained by shunt tapping in 11, by lumbar puncture in 9 and by both methods in one patient. Thirteen patients [ Shunt tapping: 5/11 (45%), LP: 7/9 (78%), Both: 1) ] were diagnosed with shunt infection on the basis of cerebrospinal fluid culture. Seven patients with negative cerebrospinal fluid culture were found to have infections unrelated to shunts and did not show evidence of cerebrospinal fluid infection during the follow-up period. Although the percentage of detecting the infection was higher in LP group, both groups showed negative predictive value of 100%. CONCLUSION: Both shunt tapping and LP are effective in establishing the diagnosis of shunt infection in suspected patients.

Spinocerebellar ataxia type 2 in a Turkish family.

Autosomal dominant spinocerebellar ataxias are neurodegenerative disorders that generally present in adulthood. Due to extreme expansion of the repeat size during spermatogenesis, they can also be observed in childhood. The diagnosis in childhood is very difficult in the absence of family history. Here we describe an 8-year-old girl with spinocerebellar ataxia type 2 who presented with progressive ataxia, cognitive deficits, and dysarthria. A detailed family history exhibited similarly affected cases on the paternal side. Molecular testing for spinocerebellar ataxia type 2 revealed abnormal "cytosineadenine-guanosine" expansion in all affected family members. The number of cytosine-adenine-guanosine repeats in the index case was 70. The mean size of expansion in the relatives of the patient was 42 (39-46). This finding explains the early onset of symptoms in the index case.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].