Sandra Donkervoort

Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia

Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor-neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic but about 5–10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin 2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.

Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS/dementia

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of motor neurons. Mutations in the FUS gene were identified in patients with familial ALS (FALS) and patients with sporadic ALS (SALS) from a variety of genetic backgrounds. This work further explores the spectrum of FUS mutations in patients with FALS and patients with FALS with features of frontotemporal dementia (FALS/FTD) or parkinsonism and dementia (FALS/PD/DE). Methods: All exons of the FUS gene were sequenced in 476 FALS index cases negative for mutations in SOD1 and TARDBP. A total of 561–726 controls were analyzed for genetic variants observed. Clinical data from patients with FUS mutations were compared to those of patients with known SOD1 and TARDBP mutations. Results: We identified 17 FUS mutations in 22 FALS families, 2 FALS/FTD families, and 1 FALS/PD/DE family from diverse genetic backgrounds; 11 mutations were novel. There were 4 frameshift, 1 nonsense, and 1 possible alternate splicing mutation. Patients with FUS mutations appeared to have earlier symptom onset, a higher rate of bulbar onset, and shorter duration of symptoms than those with SOD1 mutations. Conclusions: FUS gene mutations are not an uncommon cause in patients with FALS from diverse genetic backgrounds, and have a prevalence of 5.6% in non-SOD1 and non-TARDBP FALS, and ∼4.79% in all FALS. The pathogenicity of some of these novel mutations awaits further studies. Patients with FUS mutations manifest earlier symptom onset, a higher rate of bulbar onset, and shorter duration of symptoms.

Genotype-Phenotype studies of VCP-associated Inclusion Body Myopathy with Paget Disease of Bone and/or Frontotemporal Dementia

Valosin containing protein (VCP) disease associated with inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype–phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring 10 missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and noncarriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter‐familial variation; and significant genotype–phenotype correlations were difficult to establish because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with an earlier onset of myopathy and Paget (p = 0.03). Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p = 0.03).We identified amyotrophic lateral sclerosis (ALS) was diagnosed in 13 individuals (8.9%) and Parkinsons disease in five individuals (3%); however, there was no genotypic correlation. This study represents the largest dataset of patients with VCP disease and expands our understanding of the natural history and provides genotype–phenotype correlations in this unique disease.

A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signalling pathway and causes Lenz microphthalmia syndrome

Introduction Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition. Methods and results Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells. Conclusions We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

Position of Glycine Substitutions in the Triple Helix of COL6A1, COL6A2, and COL6A3 is Correlated with Severity and Mode of Inheritance in Collagen VI Myopathies

Glycine substitutions in the conserved Gly‐X‐Y motif in the triple helical (TH) domain of collagen VI are the most commonly identified mutations in the collagen VI myopathies including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate (INT) phenotypes. We describe clinical and genetic characteristics of 97 individuals with glycine substitutions in the TH domain of COL6A1, COL6A2, or COL6A3 and add a review of 97 published cases, for a total of 194 cases. Clinical findings include severe, INT, and mild phenotypes even from patients with identical mutations. INT phenotypes were most common, accounting for almost half of patients, emphasizing the importance of INT phenotypes to the overall phenotypic spectrum. Glycine substitutions in the TH domain are heavily clustered in a short segment N‐terminal to the 17th Gly‐X‐Y triplet, where they are acting as dominants. The most severe cases are clustered in an even smaller region including Gly‐X‐Y triplets 10–15, accounting for only 5% of the TH domain. Our findings suggest that clustering of glycine substitutions in the N‐terminal region of collagen VI is not based on features of the primary sequence. We hypothesize that this region may represent a functional domain within the triple helix.

Clinical, Pathologic, and Mutational Spectrum of Dystroglycanopathy Caused by LARGE Mutations

Dystroglycanopathies are a subtype of congenital muscular dystrophy of varying severity that can affect the brain and eyes, ranging from Walker-Warburg syndrome with severe brain malformation to milder congenital muscular dystrophy presentations with affected or normal cognition and later onset. Mutations in dystroglycanopathy genes affect a specific glycoepitope on α-dystroglycan; of the 14 genes implicated to date, LARGE encodes the glycosyltransferase that adds the final xylose and glucuronic acid, allowing α-dystroglycan to bind ligands, including laminin 211 and neurexin. Only 11 patients with LARGE mutations have been reported. We report the clinical, neuroimaging, and genetic features of 4 additional patients. We confirm that gross deletions and rearrangements are important mutational mechanisms for LARGE. The brain abnormalities overshadowed the initially mild muscle phenotype in all 4 patients. We present the first comprehensive postnatal neuropathology of the brain, spinal cord, and eyes of a patient with a homozygous LARGE mutation at Cys443. In this patient, polymicrogyria was the predominant cortical malformation; densely festooned polymicrogyria were overlaid by a continuous agyric surface. In view of the severity of these abnormalities, Cys443 may be a functionally important residue in the LARGE protein, whereas the mutation p.Glu509Lys of Patient 1 in this study may confer a milder phenotype. Overall, these results expand the clinical and genetic spectrum of dystroglycanopathy.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Muscle contraction upon nerve stimulation relies on excitation–contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Mutation-Specific Effects on Thin Filament Length in Thin Filament Myopathy

Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation.

Next-generation sequencing still needs our generation's clinicians

The full arrival and broader availability of next-generation sequencing (NGS) is transforming the practice of medicine, including neurology. Compared with the traditional one-gene-at-a-time Sanger sequencing, NGS, or massively parallel sequencing, is a radically different approach to genetic sequencing. NGS allows for a large number of genes to be captured and sequenced in parallel, creating an enormous amount of data in a relatively short period of time at much lower cost “per gene.”

Mosaicism for dominant collagen 6 mutations as a cause for intrafamilial phenotypic variability.

Collagen 6‐related dystrophies and myopathies (COL6‐RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter‐ and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6‐RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild‐type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild‐type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6‐RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected.