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Novel Mutations in TSEN54 in Pontocerebellar Hypoplasia Type 2
Abstract
Pontocerebellar hypoplasias represent a group of neurodegenerative autosomal recessive disorders characterized by hypoplasia/atrophy of the cerebellum, hypoplastic ventral pons, and microcephaly and associated with various clinical features. Pontocerebellar hypolasia type 2 is the most common form, and different mutations in genes encoding subunits of the transfer ribonucleic acid (RNA)-splicing endonuclease (TSEN) complex were identified in patients. The authors report clinical, imaging, and molecular studies in 2 unrelated patients with different clinical pictures of the pontocerebellar hypoplasia type 2 spectrum and novel mutations in TSEN54, aiming to further define the clinical spectrum of the disease and possible indicators of more favorable progression. They identified a novel missense mutation c.355T>G/p.Y119D in compound heterozygosity with the "common" c.919G>T/p.A307S (patient 1) and a novel homozygous c.7ins6(CCGGAG)/p.E2-P3insPE variant (patient 2). An expanded array of mutations might contribute in defining possible differences in severity and phenotype-genotype correlations.
... Among the many human neurological diseases, there is a class of disorders called Pontocerebellar Hypoplasia (PCH). Interestingly, several subytpes of PCH are associated with mutations in genes that encode the TSEN complex (Battini et al., 2014;Breuss et al., 2016;Budde et al., 2008;Cassandrini et al., 2010;Namavar et al., 2011a;2011b;Valayannopoulos et al., 2012). Although the precise disease mechanisms are not well understood, each PCH subtype exhibits structural abnormalities in the brain, including microcephaly. ...
... However, despite the lack of such specific mutations in our models, we observed PCH-related phenotypes in nearly all of the mutants tested. Strikingly, these animals all exhibited microcephaly (Fig. 4), similar to established PCH phenotypes (Battini et al., 2014;Breuss et al., 2016;Budde et al., 2008;Namavar et al., 2011a). Although tRNA splicing is implicated in PCH (Breuss et al., 2016;Karaca et al., 2014;Schaffer et al., 2014), it is possible that these diseases are caused by a non-tRNA splicing function of the TSEN complex or its presumptive regulatory factor, CLP1. ...
Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.
... Hypoplasia (Battini et al., 2014;Karaca et al., 2014;Schaffer et al., 2014). The efficiency of tRNA splicing due to the CLP1/TSEN complex was shown to be clearly decreased in these patients. ...
Mutations in genes encoding these tRNA modifying enzymes have recently been linked to neurodevelopmental diseases, such as intellectual disability or epilepsy. However, the functional mechanisms by which they lead to such diseases are still poorly understood. During my thesis, I studied two tRNA modifying heterodimers with variants associated with these types of diseases: ADAT2/3, which modifies adenosine to inosine at position 34 (I34), and METTL1/WDR4 which catalyzes the formation of N (7) -methylguanosine at position 46 (m7G46). My thesis work identifies these two complexes as regulators of key processes for the development of the cerebral cortex, such as radial migration and neurogenesis, in mice and humans. In addition, I have identified new variants in ADAT3, WDR4 and METTL1 and I have shown how these mutations can lead to neurodevelopmental disorders.
... Введение Понтоцеребеллярные гипоплазии (ПЦГ) -группа редких моногенных нейродегенеративных заболеваний, характеризующихся гипоплазией мозжечка и варолиева моста головного мозга, формирующихся внутриутробно. Клинические проявления большинства генетических вариантов ПЦГ возникают с рождения и характеризуются прогрессирующей микроцефалией, расстройствами дыхания, экстрапирамидными и пирамидными симптомами, изменениями мышечного тонуса, судорогами и задержкой психомоторного развития [1][2][3][4]. Распространенность заболеваний этой группы неизвестна, однако, по данным литературы, частота встречаемости ПЦГ 2А типа составляет 1 : 200 тыс. новорожденных [5][6][7]. ...
Introduction . The description of the clinical and genetic characteristics of eight patients with autosomal-recessive variant pontocerebellar hypoplasia due to mutations in the TSEN54 gene.
Purpose . Description of clinical and genetic characteristics of Russian patients with type 2A and type 4 of pontocerebellar hypoplasia.
Materials and methods . The diagnosis of pontocerebellar hypoplasia was established on the basis of the specific features of clinical manifestations and detection of mutations in the gene TSEN54 based on the analysis of the results of exome sequencing.
Results . 8 patients with pontocerebellar hypoplasia caused by mutations in the TSEN54 gene were identified.
Discussion . Based on the features of clinical manifestations and severity of the disease in 5 patients diagnosed pontocerebellar hypoplasia type 2A, and in 3 patients – type 4. In patients with type 2A of pontocerebellar hypoplasia discovered mutation c. 919G>T (p.Ala307Ser) in a homozygous state. Patients with type 4 of pontocerebellar hypoplasia this mutation is detected in the compound heterozygous state with c.670_671delAA (p.Lys224fs) and c.1264C>T (p.Gln422fs).
Conclusion . The obtained results allow us to conclude that, as well as in European populations, the mutation c.919G>T (p. Ala307Ser) is a major in Russian patients with pontocerebellar hypoplasia 2A and 4 types, which account for about half of all cases of this disease group. The search for this mutation should be the first stage of molecular genetic diagnosis in patients with clinical and magnetic resonance signs of pontocerebellar hypoplasia.
... An interesting aspect of the tRNA splicing pathway is that mutations in human tRNA processing factors have been shown to cause neurological disease. For example, there are several clinical reports of mutations in nearly any member of the human TSEN complex that lead to pontocerebellar hypoplasia (54)(55)(56)(57)(58)(59)(60). Furthermore, homozygous mutation of the CLP1 kinase gene in consanguineous Turkish families causes similar phenotypes (61, 62). ...
Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis -acting elements required for tricRNA formation in both human and fly cells. We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient. Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identify trans -acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players in Drosophila tricRNA biogenesis.
tRNA splicing endonuclease (TSEN) has a well-characterized role in tRNA splicing, but also other functions. For yeast TSEN, these other functions include degradation of a subset of mRNAs that encode mitochondrial proteins and an unknown essential function. In this study, we use yeast genetics to characterize the unknown tRNA-independent function(s) of TSEN. Using a high-copy suppressor screen we found that sen2 mutants can be suppressed by overexpression of SEN54. This effect was seen both for tRNA-dependent and tRNA-independent functions indicating SEN54 is a general suppressor of sen2, likely through structural stabilization. A spontaneous suppressor screen identified mutations in the intron-debranching enzyme, Dbr1, as tRNA-splicing independent suppressors. Transcriptome analysis showed sen2 mutation activates the Gcn4 stress response. These Gcn4 target transcripts decreased considerably in the sen2 dbr1 double mutant. We propose that Dbr1 and TSEN may compete for a shared substrate, which TSEN normally processes into an essential RNA, while Dbr1 initiates its degradation. These data provide further insight into the essential function(s) of TSEN. Importantly, single amino acid mutations in TSEN cause the generally fatal neuronal disease pontocerebellar hypoplasia (PCH). The mechanism by which defects in TSEN cause this disease is unknown and our results reveal new possible mechanisms.
Transfer RNAs (tRNAs) represent the most abundant class of RNA molecules in the cell and are key players during protein synthesis and cellular homeostasis. Aberrations in the extensive tRNA biogenesis pathways lead to severe neurological disorders in humans. Mutations in the tRNA splicing endonuclease (TSEN) and its associated RNA kinase cleavage factor polyribonucleotide kinase subunit 1 (CLP1) cause pontocerebellar hypoplasia (PCH), a heterogeneous group of neurodegenerative disorders, that manifest as underdevelopment of specific brain regions typically accompanied by microcephaly, profound motor impairments, and child mortality. Recently, we demonstrated that mutations leading to specific PCH subtypes destabilize TSEN in vitro and cause imbalances of immature to mature tRNA ratios in patient‐derived cells. However, how tRNA processing defects translate to disease on a systems level has not been understood. Recent findings suggested that other cellular processes may be affected by mutations in TSEN/CLP1 and obscure the molecular mechanisms of PCH emergence. Here, we review PCH disease models linked to the TSEN/CLP1 machinery and discuss future directions to study neuropathogenesis. Impaired transfer RNA (tRNA) splicing has been postulated as main driver for the orphan neurodegenerative disorder pontocerebellar hypoplasia (PCH). Recent evidence from animal model systems suggested that messenger RNA (mRNA) processing defects are also associated with PCH. To comprehensively understand the molecular mechanisms that lead to PCH, a multidisciplinary approach is needed.
Study Objective: to investigate the association of dyspepsia and heartburn in school-age children. Study Design: A continuous epidemiological screening of heartburn and dyspepsia in school-age children was carried out using a single- point (transverse) method. Material and Methods. A total of 328 children were examined (163 boys and 165 girls, mean age 12.4 years). Heartburn was determined according to the recommendations of the international pediatric consensus on gastroesophageal reflux disease. Dyspepsia was diagnosed in accordance with the pediatric section of the Rome IV criteria. Study Results. The prevalence of dyspepsia syndrome was 14.0%, heartburn — 10.4%. Increasing age in children was a risk factor for dyspepsia. The frequency of heartburn was registered in 39.1% of patients with dyspepsia and only in 5.7% of those without dyspepsia (p < 0.001). The prevalence of overlap syndrome of dyspepsia and heartburn in school-age children was 5.5%. Conclusion. The association of heartburn and dyspepsia in school-age children is a real problem that requires the attention of practitioners. Keywords: gastroesophageal reflux disease, dyspepsia, overlap syndrome, children.
Background
Pontocerebellar hypoplasias (PCH) comprise a group of genetically heterogeneous disorders characterised by concurrent hypoplasia of the pons and the cerebellum and variable clinical and imaging features. The current classification includes 13 subtypes, with ~20 known causative genes. Attempts have been made to delineate the phenotypic spectrum associated to specific PCH genes, yet clinical and neuroradiological features are not consistent across studies, making it difficult to define gene-specific outcomes.
Methods
We performed deep clinical and imaging phenotyping in 56 probands with a neuroradiological diagnosis of PCH, who underwent NGS-based panel sequencing of PCH genes and MLPA for CASK rearrangements. Next, we conducted a phenotype-based unsupervised hierarchical cluster analysis to investigate associations between genes and specific phenotypic clusters.
Results
A genetic diagnosis was obtained in 43 probands (77%). The most common causative gene was CASK , which accounted for nearly half cases (45%) and was mutated in females and occasionally in males. The European founder mutation p.Ala307Ser in TSEN54 and pathogenic variants in EXOSC3 accounted for 18% and 9% of cases, respectively. VLDLR , TOE1 and RARS2 were mutated in single patients. We were able to confirm only few previously reported associations, including jitteriness and clonus with TSEN54 and lower motor neuron signs with EXOSC3 . When considering multiple features simultaneously, a clear association with a phenotypic cluster only emerged for EXOSC3 .
Conclusion
CASK represents the major PCH causative gene in Italy. Phenotypic variability associated with the most common genetic causes of PCH is wider than previously thought, with marked overlap between CASK and TSEN54 -associated disorders.
Pontocerebeliarinė hipoplazija (PCH) – tai grupė retų autosominiu recesyviniu būdu paveldimų neurodegeracinių sutrikimų, prasidedančių intrauteriniu periodu ir pasireiškiančių smegenėlių hipoplazija ar atrofija, mikrocefalija, ventralinės tilto dalies hipoplazija bei įvairiais klinikiniais simptomais. Šiai patologijai būdingi pirmieji klinikiniai simptomai pasireiškia jau neonataliniu arba kūdikystės periodu. Stebima progresuojanti mikrocefalija, rijimo ir čiulpimo refleksų sutrikimai, naujagimio neramumas, generalizuotas klonusas, nepakankamas valingų judesių ir kognityvinių funkcijų vystymasis, distonija. Magnetinio rezonanso tomografijos tyrimas (MRT), kartu su klinikiniais požymiais, yra labai reikšmingas įtariant ir diagnozuojant PCH. Būdingiausi PCH2 tipo MRT požymiai: koronariniuose pjūviuose stebima smegenėlių „laumžirgio“ konfigūracija, nestebimas tilto iškyšulys arba matomas ryškus jo sumažėjimas, įvairaus laipsnio smegenų žievės atrofija, vėluojanti mielinizacija. Pontocerebeliarinės hipoplazijos 2A subtipo diagnozei patvirtinti reikalingi genetiniai tyrimai – nustatytas homozigotinis aminorūgštį keičiantis pokytis TSEN54 gene. Šiame straipsnyje pristatomas paciento, sergančio PCH2A subtipu, klinikinis atvejis, kai diagnozė buvo įtarta, remiantis klinikiniais bei MRT požymiais, ir patvirtinta nustačius homozigotinį aminorūgštį keičiantį pokytį TSEN54 gene.
We report a hereditary leukodystrophy in Standard Schnauzer puppies. Clinical signs occurred shortly after birth or started at an age of under 4 weeks and included apathy, dysphoric vocalization, hypermetric ataxia, intension tremor, head tilt, circling, proprioceptive deficits, seizures and ventral strabismus consistent with a diffuse intracranial lesion. Magnetic resonance imaging revealed a diffuse white matter disease without mass effect. Macroscopically, the cerebral white matter showed a gelatinous texture in the centrum semiovale. A mild hydrocephalus internus was noted. Histopathologically, a severe multifocal reduction of myelin formation and moderate diffuse edema without inflammation was detected leading to the diagnosis of leukodystrophy. Combined linkage analysis and homozygosity mapping in two related families delineated critical intervals of approximately 29 Mb. The comparison of whole genome sequence data of one affected Standard Schnauzer to 221 control genomes revealed a single private homozygous protein changing variant in the critical intervals, TSEN54:c.371G>A or p.(Gly124Asp). TSEN54 encodes the tRNA splicing endonuclease subunit 54. In humans, several variants in TSEN54 were reported to cause different types of pontocerebellar hypoplasia. The genotypes at the c.371G>A variant were perfectly associated with the leukodystrophy phenotype in 12 affected Standard Schnauzers and almost 1000 control dogs from different breeds. These results suggest that TSEN54:c.371G>A causes the leukodystrophy. The identification of a candidate causative variant enables genetic testing so that the unintentional breeding of affected Standard Schnauzers can be avoided in the future. Our findings extend the known genotype-phenotype correlation for TSEN54 variants.
Pontocerebellar hypoplasia (PCH) type 2 is a very rare autosomal recessive neurodegenerative disorder with prenatal onset that disrupts brain development. We present three patients (two siblings and one unrelated child) with PCH 2 linked to the most common mutation c.919G > T (p.Ala307Ser) in TSEN54 gene. The disease started soon after birth with feeding difficulties, extrapyramidal symptoms, psychomotor retardation, progressive microcephaly. Two of the patients were diagnosed with dyskinetic cerebral palsy (CP) at first. Despite the neurodegenerative character of PCH 2, the absence of regression and even some developmental progress in few patients, might erroneously lead to the incorrect diagnosis of dyskinetic CP. Megacisterna magna on brain ultrasound makes the diagnosis of PCH 2 highly probable and should prompt further imaging with MRI. MRI findings of PCH are pivotal for the diagnosis. Genetic testing for the most common mutation in TSEN54 gene should also be performed. Correct diagnosis of PCH 2 is essential not only for the prognosis of the patient, but also for prenatal diagnosis in future pregnancies. Knowledge of the clinical picture of PCH 2 will lead to correct and timely diagnosis. Advanced neuroimaging procedures and molecular genetic techniques provide valuable tools for prompt diagnosis of rare, but clinically important, neurogenetic imitators of CP.
Degenerative disorders of the newborn are relatively rare. Nonetheless, they are important to recognize because a few are treatable when diagnosed early. Dividing these disorders into those that primarily affect gray matter, white matter, or both is useful. Disorders of gray matter are characterized by seizures, myoclonus, spikes, or sharp activity on electroencephalogram, failure of cognitive development, and retinal disease. Some are accompanied by visceral storage including GM1 gangliosidosis, GM2 gangliosidosis, Niemann-Pick disease, Gaucher disease, Farber disease, and infantile sialic acid storage disease. Other gray matter disorders such as Tay-Sachs disease, congenital neuronal ceroid-lipofuscinosis, Alpers disease, and Menkes disease lack visceral storage. White matter disorders are characterized by marked motor deficits and slow activity on electroencephalogram and include Canavan disease, Alexander disease, Krabbe disease, Pelizaeus-Merzbacher disease, and Aicardi-Goutieres disease. Disorders that affect both white and gray matter often target subcellular structures (mitochondria, peroxisomes), target amino acid or neurotransmitter synthesis pathways, and can have regional selectivity for the cerebellum, pons, or basal ganglia. Examples include neonatal adrenoleukodystrophy, Zellweger syndrome, Leigh syndrome, mitochondrial encephalopathies, congenital disorders of glycosylation, pontocerebellar hypoplasias, neurotransmitter defects including serine synthesis deficiency, and Rett syndrome in males. We discuss each of these disorders briefly, focusing on clinical features, diagnosis, genetics, neuropathology, neuroimaging findings, current treatments, and emerging therapies.
Human La antigen (Sjögren's syndrome antigen B, SSB) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3' exonucleases, the ends of precursor-tRNAs and other transcripts synthesized by RNA polymerase III, and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed earlier that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing although pathway(s) involved in neuronal loss thereafter was unknown. Here we demonstrate La is stably associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear accumulation of 5.8S rRNA in La cKO is supported by ten-fold increase in a pre-5.8S rRNA intermediate. To identify pathways involved in subsequent neurodegeneration and loss of brain mass in the cKO cortex we employed mRNA-Seq, immunohistochemistry and other approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO cortex. Immunohistochemistry including temporal analyses demonstrated neurodegeneration followed by astrocyte invasion associated with immune response and decreasing cKO cortex size over time. Thus, deletion of La from post-mitotic neurons results in defective pre-tRNA and pre-rRNA processing, and progressive neurodegeneration with loss of cortical brain mass.
Background: The term Pontocerebellar Hypoplasias collectively refers to a group of rare, heterogeneous and progressive disorders, which are frequently inherited in an autosomal recessive manner and usually have a prenatal onset. Mutations in the SEPSECS gene, leading to deficiency in selenoprotein biosynthesis, have been identified in recent times as the molecular etiology of different pre/perinatal onset neurological phenotypes, including cerebello-cerebral atrophy, Pontocerebellar hypoplasia type 2D and progressive encephalopathy with elevated lactate. These disorders share a similar spectrum of central (e.g., brain neurodegeneration with grey and white matter both involved) and peripheral (e.g., spasticity due to axonal neuropathy) nervous system impairment. Case presentation: We hereby describe a 9-year-old boy with (i) a typical Pontocerebellar hypoplasia type 2D phenotype (e.g. profound mental retardation, spastic quadriplegia, ponto-cerebellar hypoplasia and progressive cerebral atrophy); (ii) optic nerve atrophy and (iii) mild secondary mitochondrial myopathy detected by muscle biopsy and respiratory chain enzyme analysis. We performed whole exome sequencing which identified a homozygous mutation of the
SEPSECS gene (c.1001T>C), confirming the clinical suspect of Pontocerebellar hypoplasia type 2D. Conclusion: This report further corroborates the notion of a potential secondary mitochondrial dysfunction in the context of selenoprotein biosynthesis deficiency and also adds optic nerve atrophy as a new potential clinical feature within the SEPSECS-associated clinical spectrum. These findings suggest the presence of a possible shared genetic etiology among similar clinical entities characterized by the combination of progressive cerebello-cerebral and optic nerve atrophy and also stress the biological importance of selenoproteins in the regulation of neuronal and metabolic homeostasis.
The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.
Pontocerebellar hypoplasia (PCH) is a clinically and genetically heterogeneous group of autosomal recessively inherited neurodevelopmental disorders. Following the rapidly increasing number of genes identified in different subtypes, the clinical spectrum has been broadened to completely different neurological phenotypes. In this review we will address the clinical picture, neuroradiological, pathoanatomic, and genetic findings in the currently known PCH subtypes. (c) 2014 Wiley Periodicals, Inc.
RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.
Pontocerebellar Hypoplasia (PCH) is group of very rare, inherited progressive neurodegenerative disorders with prenatal onset. Up to now seven different subtypes have been reported (PCH1-7). The incidence of each subtype is unknown. All subtypes share common characteristics, including hypoplasia/atrophy of cerebellum and pons, progressive microcephaly, and variable cerebral involvement. Patients have severe cognitive and motor handicaps and seizures are often reported. Treatment is only symptomatic and prognosis is poor, as most patients die during infancy or childhood. The genetic basis of different subtypes has been elucidated, which makes prenatal testing possible in families with mutations. Mutations in three tRNA splicing endonuclease subunit genes were found to be responsible for PCH2, PCH4 and PCH5. Mutations in the nuclear encoded mitochondrial arginyl- tRNA synthetase gene underlie PCH6. The tRNA splicing endonuclease, the mitochondrial arginyl- tRNA synthetase and the vaccinia related kinase1 are mutated in the minority of PCH1 cases. These genes are involved in essential processes in protein synthesis in general and tRNA processing in particular. In this review we describe the neuroradiological, neuropathological, clinical and genetic features of the different PCH subtypes and we report on in vitro and in vivo studies on the tRNA splicing endonuclease and mitochondrial arginyl-tRNA synthetase and discuss their relation to pontocerebellar hypoplasia.
Pontocerebellar hypoplasia (PCH) is a group of autosomal recessive neurodegenerative disorders characterized by prenatal onset of stunted brain growth and progressive atrophy predominantly affecting cerebellum, pons and olivary nuclei, and to a lesser extent also the cerebral cortex. Six subtypes (PCH1-6) were described and genes for four types (PCH1, 2, 4 and 6) were identified. Mutations in the tRNA splicing endonuclease subunit (TSEN) genes 54, 2 and 34 are found in PCH2 and PCH4. One family with severe prenatal onset of PCH has been the only representative of PCH5 published so far, and the molecular genetic status of PCH5 has not been ascertained until now. We screened the previously reported PCH5 family for mutations in the TSEN54 gene. The PCH5 patient was found to be the result of compound heterozygosity for the common TSEN54 mutation (p.A307S) plus a novel splice site mutation. The mutations associated with PCH5 are similar to what has been reported in PCH4. Thus, PCH5, PCH4 and PCH2 represent a spectrum of clinical manifestations caused by different mutations in the TSEN genes. We, therefore, propose to classify PCH2, PCH4 and PCH5 as TSEN mutation spectrum disorders.
Pontocerebellar hypoplasia is a group of autosomal recessive neurodegenerative disorders with prenatal onset. The common characteristics are cerebellar hypoplasia with variable atrophy of the cerebellum and the ventral pons. Supratentorial involvement is reflected by variable neocortical atrophy, ventriculomegaly and microcephaly. Mutations in the transfer RNA splicing endonuclease subunit genes (TSEN54, TSEN2, TSEN34) were found to be associated with pontocerebellar hypoplasia types 2 and 4. Mutations in the mitochondrial transfer RNA arginyl synthetase gene (RARS2) were associated with pontocerebellar hypoplasia type 6. We studied a cohort of 169 patients from 141 families for mutations in these genes, of whom 106 patients tested positive for mutations in one of the TSEN genes or the RARS2 gene. In order to delineate the neuroradiological and clinical phenotype of patients with mutations in these genes, we compared this group with 63 patients suspected of pontocerebellar hypoplasia who were negative on mutation analysis. We found a strong correlation (P < 0.0005) between TSEN54 mutations and a dragonfly-like cerebellar pattern on magnetic resonance imaging, in which the cerebellar hemispheres are flat and severely reduced in size and the vermis is relatively spared. Mutations in TSEN54 are clinically associated with dyskinesia and/or dystonia and variable degrees of spasticity, in some cases with pure generalized spasticity. Nonsense or splice site mutations in TSEN54 are associated with a more severe phenotype of more perinatal symptoms, ventilator dependency and early death. In addition, we present ten new mutations in TSEN54, TSEN2 and RARS2. Furthermore, we show that pontocerebellar hypoplasia type 1 together with elevated cerebrospinal fluid lactate may be caused by RARS2 mutations.
The RosettaBackrub server (http://kortemmelab.ucsf.edu/backrub) implements the Backrub method, derived from observations of alternative conformations in high-resolution protein crystal
structures, for flexible backbone protein modeling. Backrub modeling is applied to three related applications using the Rosetta
program for structure prediction and design: (I) modeling of structures of point mutations, (II) generating protein conformational
ensembles and designing sequences consistent with these conformations and (III) predicting tolerated sequences at protein–protein
interfaces. The three protocols have been validated on experimental data. Starting from a user-provided single input protein
structure in PDB format, the server generates near-native conformational ensembles. The predicted conformations and sequences
can be used for different applications, such as to guide mutagenesis experiments, for ensemble-docking approaches or to generate
sequence libraries for protein design.
Intrinsically unstructured/disordered proteins and domains (IUPs) lack a well-defined three-dimensional structure under native
conditions. The IUPred server presents a novel algorithm for predicting such regions from amino acid sequences by estimating
their total pairwise interresidue interaction energy, based on the assumption that IUP sequences do not fold due to their
inability to form sufficient stabilizing interresidue interactions. Optional to the prediction are built-in parameter sets
optimized for predicting short or long disordered regions and structured domains.
Availability: The IUPred server is available for academic users at http://iupred.enzim.hu
Contact: zsuzsa{at}enzim.hu
PrDOS is a server that predicts the disordered regions of a protein from its amino acid sequence (http://prdos.hgc.jp). The server accepts a single protein amino acid sequence, in either plain text or FASTA format. The prediction system is
composed of two predictors: a predictor based on local amino acid sequence information and one based on template proteins.
The server combines the results of the two predictors and returns a two-state prediction (order/disorder) and a disorder probability
for each residue. The prediction results are sent by e-mail, and the server also provides a web-interface to check the results.
Many protein regions and some entire proteins have no definite tertiary structure, existing instead as dynamic, disorder ensembles under different physiochemical circumstances. Identification of these protein disorder regions is important for protein production, protein structure prediction and determination, and protein function annotation. A number of different disorder prediction software and web services have been developed since the first predictor was designed by Dunker's lab in 1997. However, most of the software packages use a pre-defined threshold to select ordered or disordered residues. In many situations, users need to choose ordered or disordered residues at different sensitivity and specificity levels.
Here we benchmark a state of the art disorder predictor, DISpro, on a large protein disorder dataset created from Protein Data Bank and systematically evaluate the relationship of sensitivity and specificity. Also, we extend its functionality to allow users to trade off specificity and sensitivity by setting different decision thresholds. Moreover, we compare DISpro with seven other automated disorder predictors on the 95 protein targets used in the seventh edition of Critical Assessment of Techniques for Protein Structure Prediction (CASP7). DISpro is ranked as one of the best predictors.
The evaluation and extension of DISpro make it a more valuable and useful tool for structural and functional genomics.
Orthologous genes with deep phylogenetic histories are likely to retain similar regulatory features. In this report we utilize orthology assignments for pairs of genes co-regulated by bidirectional promoters to map the ancestral history of the promoter regions.
Our mapping of bidirectional promoters from humans to fish shows that many such promoters emerged after the divergence of chickens and fish. Furthermore, annotations of promoters in deep phylogenies enable detection of missing data or assembly problems present in higher vertebrates. The functional importance of bidirectional promoters is indicated by selective pressure to maintain the arrangement of genes regulated by the promoter over long evolutionary time spans. Characteristics unique to bidirectional promoters are further elucidated using a technique for unsupervised classification, known as ESPERR.
Results of these analyses will aid in our understanding of the evolution of bidirectional promoters, including whether the regulation of two genes evolved as a consequence of their proximity or if function dictated their co-regulation.
The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the
splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal
structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of
ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results
suggest an evolutionary pathway for splice site recognition.
Mutations in genes encoding subunits of the tRNA-splicing endonuclease (TSEN) complex were identified in patients with pontocerebellar hypoplasia 2 (PCH2) and pontocerebellar hypoplasia 4 (PCH4).
We report molecular genetic findings in 12 Italian patients with clinical and MRI findings compatible with PCH2 and PCH4.
We retrospectively selected a cohort of 12 children from 9 Italian families with MRI of hypoplastic pontocerebellar structures and clinical manifestations suggesting either PCH2 or PCH4 and submitted them to direct sequencing of the genes encoding the 4 subunits of the TSEN complex, namely TSEN54, TSEN34, TSEN15, and TSEN2.
In a cohort of 12 children, we detected the common p.A307S mutation in TSEN54 in 9/12 available patients from nine unrelated families. We also detected a novel c.1170_1183del (p. V390fs39X) in compound heterozygosity with the common p.A307S in a child with a severe PCH4 phenotype. In another severely affected patient, the second mutant allele was not identified. Two sibs without mutations in the TSEN complex were unlinked to the PCH3 locus. In addition to typical clinical and neuroradiologic features of PCH2, both children were affected by a tubulopathy resembling Bartter syndrome.
We confirm that the common p.A307S mutation in TSEN54 is responsible for most of the patients with a PCH2 phenotype. The presence of a heterozygous in/del variant correlates with a more severe phenotype as PCH4. In addition, we describe a new clinical form of PCH in 2 sibs with clinical and MRI features of PCH2.
Protein intrinsic disorder is becoming increasingly recognized in proteomics research. While lacking structure, many regions of disorder have been associated with biological function. There are many different experimental methods for characterizing intrinsically disordered proteins and regions; nevertheless, the prediction of intrinsic disorder from amino acid sequence remains a useful strategy especially for many large-scale proteomic investigations. Here we introduced a consensus artificial neural network (ANN) prediction method, which was developed by combining the outputs of several individual disorder predictors. By eight-fold cross-validation, this meta-predictor, called PONDR-FIT, was found to improve the prediction accuracy over a range of 3 to 20% with an average of 11% compared to the single predictors, depending on the datasets being used. Analysis of the errors shows that the worst accuracy still occurs for short disordered regions with less than ten residues, as well as for the residues close to order/disorder boundaries. Increased understanding of the underlying mechanism by which such meta-predictors give improved predictions will likely promote the further development of protein disorder predictors. Access to PONDR-FIT is available at www.disprot.org.
The spinal muscular atrophies (SMAs) are a genetically and clinically heterogeneous group of disorders characterized by degeneration and loss of anterior horn cells in the spinal cord, leading to muscle weakness and atrophy. Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH, also known as pontocerebellar hypoplasia type 1 [PCH1]) is one of the rare infantile SMA variants that include additional clinical manifestations, and its genetic basis is unknown. We used a homozygosity mapping and positional cloning approach in a consanguineous family of Ashkenazi Jewish origin and identified a nonsense mutation in the vaccinia-related kinase 1 gene (VRK1) as a cause of SMA-PCH. VRK1, one of three members of the mammalian VRK family, is a serine/threonine kinase that phosphorylates p53 and CREB and is essential for nuclear envelope formation. Its identification as a gene involved in SMA-PCH implies new roles for the VRK proteins in neuronal development and maintenance and suggests the VRK genes as candidates for related phenotypes.
Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.
The syndrome of autosomal recessive pontocerebellar hypoplasia, microcephaly, severely impaired mental and motor development, and extrapyramidal dyskinesia is a distinct system degeneration, previously designated pontocerebellar hypoplasia type 2 (PCH-2). To further characterize its clinical and neuroimaging features, we compiled data from 10 nonrelated pedigrees. Six pedigrees were Dutch, two Swedish, and two German. All 16 patients showed an identical profile of virtually absent developmental milestones, early-onset severe chorea, and microcephaly together with pontocerebellar hypoplasia. Family distribution supports autosomal recessive transmission. The present data support the PCH-2 phenotype as a distinct neurogenetic entity.
We describe a comparative protein modelling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability density functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equivalent residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain C alpha-C alpha distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the molecular pdf such that the model violates the input restraints as little as possible. The molecular pdf is derived as a combination of pdfs restraining individual spatial features of the whole molecule. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modelling of trypsin from two other serine proteinases.
The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla. In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA. We have cloned the genes encoding all four protein subunits of endonuclease. Each gene is essential. Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids. This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other Archaea. Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively. The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.
An original article describes a sibship with early fatal pontocerebellar hypoplasia of a yet unclassified type, with the accompanying features of polyhydramnios and neonatal myoclonus. Autopsy in one patient excluded spinal anterior horn involvement, which argues against pontocerebellar hypoplasia type I (PCH-1). The present PCH classification and literature are briefly reviewed. Four previous publications bear similarity to the present report. Definite classification as a genetically separate entity, however, remains elusive pending localization and identification of the gene(s) involved.
The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.
Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. They are involved in development of neural tissues and the neural crest, in left-right axis patterning, in somite development, and in formation of the cerebellum. In addition to their roles in cell-fate specification, zic genes also promote cell proliferation. Further, they are expressed in postmitotic cells of the cerebellum and in retinal ganglion cells. Efforts to determine the role of individual zic genes within an array of developmental and cellular processes are complicated by overlapping patterns of zic gene expression and strong sequence conservation within this gene family. Nevertheless, substantial progress has been made. This review summarizes our knowledge of the molecular events that govern the activities of zic family members, including emerging relationships between upstream signaling pathways and zic genes. In addition, advancements in our understanding of the molecular events downstream of Zic transcription factors are reviewed. Despite significant progress, however, much remains to be learned regarding the mechanisms through which zic genes exert their function in a variety of different contexts.
Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.
Poll-The BT, Baas F. Classification, diagnosis and potential mechanisms in pontocerebellar hypopla-sia
- Y Namavar
- Barth
- Pg
Namavar Y, Barth PG, Poll-The BT, Baas F. Classification, diagnosis and potential mechanisms in pontocerebellar hypopla-sia. Orphanet J Rare Dis. 2011;6:50.