Hypomorphic mutations in POLR3A are a frequent cause of sporadic and recessive spastic ataxia.

Despite extensive efforts, half of patients with rare movement disorders such as hereditary spastic paraplegias and cerebellar ataxias remain genetically unexplained, implicating novel genes and unrecognized mutations in known genes. Non-coding DNA variants are suspected to account for a substantial part of undiscovered causes of rare diseases. Here we identified mutations located deep in introns of POLR3A to be a frequent cause of hereditary spastic paraplegia and cerebellar ataxia. First, whole-exome sequencing findings in a recessive spastic ataxia family turned our attention to intronic variants in POLR3A, a gene previously associated with hypomyelinating leukodystrophy type 7. Next, we screened a cohort of hereditary spastic paraplegia and cerebellar ataxia cases (n = 618) for mutations in POLR3A and identified compound heterozygous POLR3A mutations in ∼3.1% of index cases. Interestingly, >80% of POLR3A mutation carriers presented the same deep-intronic mutation (c.1909+22G>A), which activates a cryptic splice site in a tissue and stage of development-specific manner and leads to a novel distinct and uniform phenotype. The phenotype is characterized by adolescent-onset progressive spastic ataxia with frequent occurrence of tremor, involvement of the central sensory tracts and dental problems (hypodontia, early onset of severe and aggressive periodontal disease). Instead of the typical hypomyelination magnetic resonance imaging pattern associated with classical POLR3A mutations, cases carrying c.1909+22G>A demonstrated hyperintensities along the superior cerebellar peduncles. These hyperintensities may represent the structural correlate to the cerebellar symptoms observed in these patients. The associated c.1909+22G>A variant was significantly enriched in 1139 cases with spastic ataxia-related phenotypes as compared to unrelated neurological and non-neurological phenotypes and healthy controls (P = 1.3 × 10-4). In this study we demonstrate that (i) autosomal-recessive mutations in POLR3A are a frequent cause of hereditary spastic ataxias, accounting for about 3% of hitherto genetically unclassified autosomal recessive and sporadic cases; and (ii) hypomyelination is frequently absent in POLR3A-related syndromes, especially when intronic mutations are present, and thus can no longer be considered as the unifying feature of POLR3A disease. Furthermore, our results demonstrate that substantial progress in revealing the causes of Mendelian diseases can be made by exploring the non-coding sequences of the human genome.

High Frequency of Pathogenic Rearrangements in SPG11 and Extensive Contribution of Mutational Hotspots and Founder Alleles.

Biallelic loss-of-function mutations in SPG11 cause a wide spectrum of recessively inherited, neurodegenerative disorders including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis, and Charcot-Marie-Tooth disease. By comprehensive screening of three large cohorts of HSP index patients, we identified 83 alleles with "small" mutations and 13 alleles that carry large genomic rearrangements. Including relevant data from previous studies, we estimate that copy number variants (CNVs) account for ∼19% of pathogenic SPG11 alleles. The breakpoints for all novel and some previously reported CNVs were determined by long-range PCR and sequencing. This revealed several Alu-associated recombination hotspots. We also found evidence for additional mutational mechanisms, including for a two-step event in which an Alu retrotransposition preceded the actual rearrangement. Apparently independent samples with identical breakpoints were analyzed by microsatellite PCRs. The resulting haplotypes suggested the existence of two rearrangement founder alleles. Our findings widen the spectra of mutations and mutational mechanisms in SPG11, underscore the pivotal role played by Alus, and are of high diagnostic relevance for a wide spectrum of clinical phenotypes including the most frequent form of recessive HSP.

Genetic analysis of polymorphisms in the kalirin gene for association with age-at-onset in European Huntington disease patients.

BACKGROUND: Huntington disease (HD) is caused by an expanded CAG repeat in the HD gene. Although the length of the CAG repeat strongly correlates with the age-at-onset (AAO), AAO in HD individuals may differ dramatically in spite of similar expanded CAG repeat lengths. Additional genetic or environmental factors are thought to influence the disease onset. Several modifier genes have been discovered so far but they do not fully explain the variability of AAO in HD. To potentially identify a novel genetic modifier, we analyzed single nucleotide polymorphisms (SNPs) in the kalirin (KALRN) gene. Kalirin is a protein crucially involved in spine plasticity and its interaction with huntingtin-associated protein-1 (HAP-1) and a potential protein dysfunction might contribute to spine pathogenesis in HD. METHODS: The selected SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and association of SNPs with AAO was investigated with the framework of linear models in an analysis of variance and covariance. RESULTS: Eleven SNPs in the kalirin gene were examined in an association study in European HD patients. The ten coding SNPs under investigation were monomorphic, whereas SNP rs10934657 in the promoter region showed a minor allele frequency >1%. An analysis of covariance together with the influence of the expanded HD allele was applied in 680 HD patients. SNP rs10934657 did not affect the AAO of the examined HD population. CONCLUSIONS: The results did not reveal an association between the analyzed kalirin polymorphisms and the AAO in HD. However, it does not exclude other SNPs of the kalirin gene as susceptible genetic modifiers.

Periphilin is a novel interactor of synphilin-1, a protein implicated in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein and its interactor synphilin-1 are major components of these inclusions. Rare mutations in the alpha-synuclein and synphilin-1 genes have been implicated in the pathogenesis of PD; however, the normal function of these proteins is far from being completely elucidated. We, thus, searched for novel synphilin-1-interacting proteins and deciphered periphilin as new interactor. Periphilin isoforms are involved in multiple cellular functions in vivo, and the protein is broadly expressed during embryogenesis and in the adult brain. We show that periphilin displays an overlapping expression pattern with synphilin-1 in cellular and animal models and in Lewy bodies of PD patients. Functional studies demonstrate that periphilin, as previously shown for synphilin-1, displays an antiapoptotic function by reducing caspase-3 activity. Searching for mutations in the periphilin gene, we detected a K69E substitution in two patients of a PD family. Taken together, these findings support for the first time an involvement of periphilin in PD.

Huntingtin-associated protein-1 is a modifier of the age-at-onset of Huntington's disease.

A polyglutamine repeat expansion of more than 36 units in a protein called huntingtin (htt) is the only known cause of Huntington's disease (HD). The expanded repeat length is inversely correlated with the age-at-onset (AAO), however, the onset age among HD patients with CAG repeats below 60 units varies considerably. In addition to environmental factors, genetic factors different from the expanded CAG repeat length can modify the AAO of HD. We hypothezised that htt interacting proteins might contribute to this variation in the AAO and investigated human htt-associated protein-1 (HAP1) using genetic and functional assays. We identified six polymorphisms in the HAP1 gene including one that substitutes methionine (M441) for threonine (T441) at amino acid 441. Analyzing 980 European HD patients, we found that patients homozygous for the M441 genotype show an 8-year delay in the AAO. Functional assays demonstrated that human M441-HAP1 interacts with mutant htt more tightly than does human T441-HAP1, reduces soluble htt degraded products and protects against htt-mediated toxicity. We thus provide genetic and functional evidence that the M441-HAP1 polymorphism modifies the AAO of HD.

Periphilin is strongly expressed in the murine nervous system and is indispensable for murine development.

Periphilin is involved in multiple processes in vivo. To explore its physiological role from an organismic perspective, we generated mice with a gene trap insertion in the periphilin-1 gene. Based on beta-gal reporter activity, a widespread periphilin expression was evident, especially in the developing somites and limbs, the embryonic nervous system, and the adult brain. In accordance with this broad expression, homozygous deficiency of periphilin was lethal in early embryogenesis. Mice with a heterozygous deficiency did not show any abnormalities of brain morphology and function, neither histologically nor regarding the transcriptome. Interestingly, the reduction of the periphilin-1 gene dosage was compensated by an increased expression of the remaining wild-type allele in the brain. These results point to an indispensable function of periphilin during murine development and an important role in the nervous system, reflected by a strong and tightly regulated expression in the murine brain.

Genetic analysis of coding SNPs in blood-brain barrier transporter MDR1 in European Parkinson's disease patients.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic inclusions (Lewy bodies). Iron, which is elevated in the substantia nigra of PD patients, seems to be of pivotal importance, because of its capacity to enhance the amplification of reactive oxygen species. As iron enters and exits the brain via transport proteins in the blood-brain barrier (BBB), these proteins may represent candidates for a genetic susceptibility to PD. P-glycoprotein (P-gp) is one important efflux pump in the BBB. There is evidence that the function of P-gp is impaired in PD patients. In the current study we examined ten coding single nucleotide polymorphisms in the multidrug resistance gene 1 (MDR1) encoding P-gp to assess whether certain genotypes are associated with PD. However, genotyping of 300 PD patients and 302 healthy controls did not reveal a significant association between coding MDR1 gene polymorphisms and PD.

Genetic analysis of heme oxygenase-1 (HO-1) in German Parkinson's disease patients.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic inclusions (Lewy bodies). Iron, which is elevated in the substantia nigra (SN) of PD patients, seems to be of pivotal importance, because of its capacity to enhance the amplification of reactive-oxygen species. Therefore, it is tempting that the iron-releasing key enzyme in heme catabolism, heme oxygenase-1 (HO-1), may represent a candidate for a genetic susceptibility to PD. In the current study, we examined a (GT)n fragment length polymorphism in the promoter region, as well as three coding SNPs in the HO-1 gene in order to assess if certain genotypes are associated with PD. Furthermore, peripheral blood expression levels of HO-1 in PD patients and healthy probands were compared. However, our analyses did not reveal a significant association of these genetic markers in the HO-1 gene with an increased susceptibility to PD.

The proteasomal subunit S6 ATPase is a novel synphilin-1 interacting protein--implications for Parkinson's disease.

Synphilin-1 is linked to Parkinson's disease (PD), based on its role as an alpha-synuclein (PARK1)-interacting protein and substrate of the ubiquitin E3 ligase Parkin (PARK2) and because of its presence in Lewy bodies (LB) in brains of PD patients. We found that overexpression of synphilin-1 in cells leads to the formation of ubiquitinated cytoplasmic inclusions supporting a derangement of the ubiquitin-proteasome system in PD. We report here a novel specific interaction of synphilin-1 with the regulatory proteasomal protein S6 ATPase (tbp7). Functional characterization of this interaction on a cellular level revealed colocalization of S6 and synphilin-1 in aggresome-like intracytoplasmic inclusions. Overexpression of synphilin-1 and S6 in cells caused reduced proteasomal activity associated with a significant increase in inclusion formation compared to cells expressing synphilin-1 alone. Steady-state levels of synphilin-1 in cells were not altered after cotransfection of S6 and colocalization of synphilin-1-positive inclusions with lysosomal markers suggests the presence of an alternative lysosomal degradation pathway. Subsequent immunohistochemical studies in brains of PD patients identified S6 ATPase as a component of LB. This is the first study investigating the physiological role of synphilin-1 in the ubiquitin proteasome system. Our data suggest a direct interaction of synphilin-1 with the regulatory complex of the proteasome modulating proteasomal function.

Uniparental disomy determined by whole-exome sequencing in a spectrum of rare motoneuron diseases and ataxias.

BACKGROUND: The genetic causes of many rare inherited motoneuron diseases and ataxias (MND and ATX) remain largely unresolved, especially for sporadic patients, despite tremendous advances in gene discovery. Whole exome data is often available for patients, but it is rarely evaluated for unusual inheritance patterns, such as uniparental disomy (UPD). UPD is the inheritance of two copies of a chromosomal region from one parent, which may generate homozygosity for a deleterious recessive variant from only one carrier-parent. Detection of UPD-caused homozygous disease-causing variants is detrimental to accurate genetic counseling. Whole-exome sequencing can allow for the detection of such events. METHODS: We systematically studied the exomes of a phenotypically heterogeneous cohort of unresolved cases (n = 96 families) to reveal UPD events hindering a diagnosis and to evaluate the prevalence of UPD in recessive MND and ATX. RESULTS: One hereditary spastic paraplegia case harbored homozygous regions spanning 80% of chromosome 16. A homozygous disease-causing mutation in the SPG35 disease gene was then identified within this region. CONCLUSION: This study demonstrates the ability to detect UPD in exome data of index patients. Our results suggest that UPD is a rare mechanism for recessive MND and ATX.

Uniparental disomy of chromosome 16 unmasks recessive mutations of FA2H/SPG35 in 4 families.

OBJECTIVE: Identifying an intriguing mechanism for unmasking recessive hereditary spastic paraplegias. METHOD: Herein, we describe 4 novel homozygous FA2H mutations in 4 nonconsanguineous families detected by whole-exome sequencing or a targeted gene panel analysis providing high coverage of all known hereditary spastic paraplegia genes. RESULTS: Segregation analysis revealed in all cases only one parent as a heterozygous mutation carrier whereas the other parent did not carry FA2H mutations. A macro deletion within FA2H, which could have caused a hemizygous genotype, was excluded by multiplex ligation-dependent probe amplification in all cases. Finally, a microsatellite array revealed uniparental disomy (UPD) in all 4 families leading to homozygous FA2H mutations. UPD was confirmed by microarray analyses and methylation profiling. CONCLUSION: UPD has rarely been described as causative mechanism in neurodegenerative diseases. Of note, we identified this mode of inheritance in 4 families with the rare diagnosis of spastic paraplegia type 35 (SPG35). Since UPD seems to be a relevant factor in SPG35 and probably additional autosomal recessive diseases, we recommend segregation analysis especially in nonconsanguineous homozygous index cases to unravel UPD as mutational mechanism. This finding may bear major repercussion for genetic counseling, given the markedly reduced risk of recurrence for affected families.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS): expanding the genetic, clinical and imaging spectrum.

BACKGROUND: Mutations in SACS, leading to autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), have been identified as a frequent cause of recessive early-onset ataxia around the world. Here we aimed to enlarge the spectrum of SACS mutations outside Quebec, to establish the pathogenicity of novel variants, and to expand the clinical and imaging phenotype. METHODS: Sequencing of SACS in 22 patients with unexplained early-onset ataxia, assessment of novel SACS variants in 3.500 European control chromosomes and extensive phenotypic investigations of all SACS carriers. RESULTS: We identified 11 index patients harbouring 17 novel SACS variants. 9/11 patients harboured two variants of at least probable pathogenicity which were not observed in controls and, in case of missense mutations, were located in highly conserved domains. These 9 patients accounted for at least 11% (9/83) in our series of unexplained early onset ataxia subjects. While most patients (7/9) showed the classical ARSACS triad, the presenting phenotype reached from pure neuropathy (leading to the initial diagnosis of Charcot-Marie-Tooth disease) in one subject to the absence of any signs of neuropathy in another. In contrast to its name "spastic ataxia", neither spasticity (absent in 2/9=22%) nor extensor plantar response (absent in 3/9=33%) nor cerebellar ataxia (absent in 1/9=11%) were obligate features. Autonomic features included urine urge incontinence and erectile dysfunction. Apart from the well-established MRI finding of pontine hypointensities, all patients (100%) showed hyperintensities of the lateral pons merging into the (thickened) middle cerebellar peduncles. In addition, 63% exhibited bilateral parietal cerebral atrophy, and 63% a short circumscribed thinning of the posterior midbody of the corpus callosum. In 2 further patients with differences in important clinical features, VUS class 3 variants (c.1373C>T [p.Thr458Ile] and c.2983 G>T [p.Val995Phe]) were identified. These variants were, however, also observed in controls, thus questioning their pathogenic relevance. CONCLUSIONS: We here demonstrate that each feature of the classical ARSACS triad (cerebellar ataxia, spasticity and peripheral neuropathy) might be missing in ARSACS. Nevertheless, characteristic MRI features - which also extend to supratentorial regions and involve the cerebral cortex - will help to establish the diagnosis in most cases.

The Guanine nucleotide exchange factor kalirin-7 is a novel synphilin-1 interacting protein and modifies synphilin-1 aggregate transport and formation.

Synphilin-1 has been identified as an interaction partner of α-synuclein, a key protein in the pathogenesis of Parkinson disease (PD). To further explore novel binding partners of synphilin-1, a yeast two hybrid screening was performed and kalirin-7 was identified as a novel interactor. We then investigated the effect of kalirin-7 on synphilin-1 aggregate formation. Coexpression of kalirin-7 and synphilin-1 caused a dramatic relocation of synphilin-1 cytoplasmic small inclusions to a single prominent, perinuclear inclusion. These perinuclear inclusions were characterized as being aggresomes according to their colocalization with microtubule organization center markers, and their formation was microtubule-dependent. Furthermore, kalirin-7 increased the susceptibility of synphilin-1 inclusions to be degraded as demonstrated by live cell imaging and quantification of aggregates. However, the kalirin-7-mediated synphilin-1 aggresome response was not dependent on the GEF activity of kalirin-7 since various dominant negative small GTPases could not inhibit the formation of aggresomes. Interestingly, the aggresome response was blocked by HDAC6 catalytic mutants and the HDAC inhibitor trichostatin A (TSA). Moreover, kalirin-7 decreased the level of acetylated α-tubulin in response to TSA, which suggests an effect of kalirin-7 on HDAC6-mediated protein transportation and aggresome formation. In summary, this is the first report demonstrating that kalirin-7 leads to the recruitment of synphilin-1 into aggresomes in a HDAC6-dependent manner and also links kalirin-7 to microtubule dynamics.

Development and adult morphology of the eye lens in the zebrafish.

The zebrafish has become an important vertebrate model organism to study the development of the visual system. Mutagenesis projects have resulted in the identification of hundreds of eye mutants. Analysis of the phenotypes of these mutants relies on in depth knowledge of the embryogenesis in wild-type animals. While the morphological events leading to the formation of the retina and its connections to the central nervous system have been described in great detail, the characterization of the development of the eye lens is still incomplete. In the present study, we provide a morphological description of embryonic and larval lens development as well as adult lens morphology in the zebrafish. Our analyses show that, in contrast to other vertebrate species, the zebrafish lens delaminates from the surface ectoderm as a solid cluster of cells. Detachment of the prospective lens from the surface ectoderm is facilitated by apoptosis. Primary fibre cell elongation occurs in a circular fashion resulting in an embryonic lens nucleus with concentric shells of fibres. After formation of a monolayer of lens epithelial cells, differentiation and elongation of secondary lens fibres result in a final lens morphology similar to that of other vertebrate species. As in other vertebrates, secondary fibre cell differentiation includes the programmed degradation of nuclei, the interconnection of adjacent fibres via protrusions at the fibre cells' edges and the establishment of gap junctions between lens fibre cells. The very close spacing of the nuclei of the differentiating secondary fibres in a narrow zone close to the equatorial epithelium, however, suggests that secondary fibre cell differentiation deviates from that described for mammalian or avian lenses. In summary, while there are similarities in the development and final morphology of the zebrafish lens with mammalian and avian lenses, there are also significant differences, suggesting caution when extrapolating findings on the zebrafish to, for example, human lens development or function.