Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to ß-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into ß-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Efficacy and safety of onasemnogene abeparvovec in children with spinal muscular atrophy type 1: real-world evidence from 6 infusion centres in the United Kingdom.

Background: Real-world data on the efficacy and safety of onasemnogene abeparvovec (OA) in spinal muscular atrophy (SMA) are needed, especially to overcome uncertainties around its use in older and heavier children. This study evaluated the efficacy and safety of OA in patients with SMA type 1 in the UK, including patients ≥2 years old and weighing ≥13.5 kg. Methods: This observational cohort study used data from patients with genetically confirmed SMA type 1 treated with OA between May 2021 and January 2023, at 6 infusion centres in the United Kingdom. Functional outcomes were assessed using age-appropriate functional scales. Safety analyses included review of liver function, platelet count, cardiac assessments, and steroid requirements. Findings: Ninety-nine patients (45 SMA therapy-naïve) were treated with OA (median age at infusion: 10 [range, 0.6-89] months; median weight: 7.86 [range, 3.2-20.2] kg; duration of follow-up: 3-22 months). After OA infusion, mean ± SD change in CHOP-INTEND score was 11.0 ± 10.3 with increased score in 66/78 patients (84.6%); patients aged <6 months had a 13.9 points higher gain in CHOP-INTEND score than patients ≥2 years (95% CI, 6.8-21.0; P < 0.001). Asymptomatic thrombocytopenia (71/99 patients; 71.7%), asymptomatic troponin-I elevation (30/89 patients; 33.7%) and transaminitis (87/99 patients; 87.9%) were reported. No thrombotic microangiopathy was observed. Median steroid treatment duration was 97 (range, 28-548) days with dose doubled in 35/99 patients (35.4%). There were 22.5-fold increased odds of having a transaminase peak >100 U/L (95% CI, 2.3-223.7; P = 0.008) and 21.2-fold increased odds of steroid doubling, as per treatment protocol (95% CI, 2.2-209.2; P = 0.009) in patients weighing ≥13.5 kg versus <8.5 kg. Weight at infusion was positively correlated with steroid treatment duration (r = 0.43; P < 0.001). Worsening transaminitis, despite doubling of oral prednisolone, led to treatment with intravenous methylprednisolone in 5 children. Steroid-sparing immunosuppressants were used in 5 children to enable steroid weaning. Two deaths apparently unrelated to OA were reported. Interpretation: OA led to functional improvements and was well tolerated with no persistent clinical complications, including in older and heavier patients. Funding: Novartis Innovative Therapies AG provided a grant for independent medical writing services.

Long-term Natural History of Pediatric Dominant and Recessive RYR1-Related Myopathy.

BACKGROUND AND OBJECTIVES: RYR1-related myopathies are the most common congenital myopathies, but long-term natural history data are still scarce. We aim to describe the natural history of dominant and recessive RYR1-related myopathies. METHODS: A cross-sectional and longitudinal retrospective data analysis of pediatric cases with RYR1-related myopathies seen between 1992-2019 in 2 large UK centers. Patients were identified, and data were collected from individual medical records. RESULTS: Sixty-nine patients were included in the study, 63 in both cross-sectional and longitudinal studies and 6 in the cross-sectional analysis only. Onset ranged from birth to 7 years. Twenty-nine patients had an autosomal dominant RYR1-related myopathy, 31 recessive, 6 de novo dominant, and 3 uncertain inheritance. Median age at the first and last appointment was 4.0 and 10.8 years, respectively. Fifteen% of patients older than 2 years never walked (5 recessive, 4 de novo dominant, and 1 dominant patient) and 7% lost ambulation during follow-up. Scoliosis and spinal rigidity were present in 30% and 17% of patients, respectively. Respiratory involvement was observed in 22% of patients, and 12% needed ventilatory support from a median age of 7 years. Feeding difficulties were present in 30% of patients, and 57% of those needed gastrostomy or tube feeding. There were no anesthetic-induced malignant hyperthermia episodes reported in this cohort. We observed a higher prevalence of prenatal/neonatal features in recessive patients, in particular hypotonia and respiratory difficulties. Clinical presentation, respiratory outcomes, and feeding outcomes were consistently more severe at presentation and in the recessive group. Conversely, longitudinal analysis suggested a less progressive course for motor and respiratory function in recessive patients. Annual change in forced vital capacity was -0.2%/year in recessive vs -1.4%/year in dominant patients. DISCUSSION: This clinical study provides long-term data on disease progression in RYR1-related myopathies that may inform management and provide essential milestones for future therapeutic interventions.

Food protein-derived amyloids do not accelerate amyloid ß aggregation.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-ß (Aß), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aß. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aß in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aß pathology and Alzheimer's disease.

New treatments in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe neurodegenerative condition due to recessive mutations in the SMN1 gene resulting in insufficiency of survival motor neuron (SMN) protein. Lack of SMN protein results in irreversible degeneration of lower motor neurons and consequential muscle atrophy and weakness. SMN2, a SMN1 homologue, produces low levels of functional SMN protein with the potential to partially compensate SMN1 loss. Several compounds have been shown to successfully restore SMN protein production in motor neurons, either by enhancing SMN2 gene function or by direct replacement of the SMN1 gene. Clinical trials of these compounds have demonstrated the potential to substantially alter the natural history of SMA and have led to their implementation into clinical practice. To date, 3 novel drugs, nusinersen, onasemnogene aberparvovec and risdiplam, have received marketing authorisation for SMA treatment by several authorities including Food and Drug Administration and European Medicines Agency. While implementing these drugs into daily clinical practice, clinicians face a number of new challenges, including identifying the most advantageous treatment for any individual, optimisation of outcomes and management of a modified SMA phenotype. Considering that treatment initiation at the pre-symptomatic or paucisymptomatic stage appears to be associated with better outcomes, health services need to support early diagnosis for this now treatable condition. This review aims to give an overview of the current therapeutic landscape of SMA, to provide an understanding of current practice of SMA management and to help increase awareness of the imminent need for urgent early diagnosis at the pre-symptomatic stage.

Prediagnosis pathway benchmarking audit in patients with Duchenne muscular dystrophy.

OBJECTIVE: To describe age and time at key stages in the Duchenne muscular dystrophy (DMD) prediagnosis pathway at selected centres to identify opportunities for service improvement. DESIGN: A multicentre retrospective national audit. SETTING: Nine tertiary neuromuscular centres across the UK and Ireland. A prior single-centre UK audit of 20 patients with no DMD family history provided benchmark criteria. PATIENTS: Patients with a definitive diagnosis of DMD documented within 3 years prior to December 2018 (n=122). MAIN OUTCOME MEASURES: Mean age (months) at four key stages in the DMD diagnostic pathway and mean time (months) of presentational and diagnostic delay, and time from first reported symptoms to definitive diagnosis. Type of symptoms was also recorded. RESULTS: Overall, mean age at definitive diagnosis, age at first engagement with healthcare professional (HCP) and age at first reported symptoms were 53.9±29.7, 49.9±28.9 and 36.4±26.8 months, respectively. The presentational delay and time to diagnosis were 21.1 (±21.1) and 4.6 (±7.9) months, respectively. The mean time from first reported symptoms to definitive diagnosis was 24.2±20.9. The percentages of patients with motor and/or non-motor symptoms recorded were 88% (n=106/121) and 47% (n=57/121), respectively. CONCLUSIONS: Majority of data mirrored the benchmark audit. However, while the time to diagnosis was shorter, a presentational delay was observed. Failure to recognise early symptoms of DMD could be a contributing factor and represents an unmet need in the diagnosis pathway. Methods determining how to improve this need to be explored.

KIAA0753-related skeletal ciliopathy: a ninth case, extending the phenotype and reporting a novel variant.

KIAA0753-related skeletal ciliopathy is a recently described recessive disorder causing skeletal dysplasia and overlapping features of certain ciliopathies; Joubert, Jeune and Oro-facial-digital syndromes. We describe a ninth case that expands the phenotype; a 10-year-old girl with rhizomelic short stature (-5.6 SD), macrocephaly, developmental delay, CNS anomalies (thin corpus callosum, bilateral ventriculomegaly), cone-rod dystrophy, nystagmus, mild conductive hearing loss and recurrent chest infections secondary to confirmed ciliary dyskinesia. Testing for FGFR3 achondroplasia-related hotspots and mucopolysaccharidosis were negative. Whole-exome sequencing, aged eight, via skeletal dysplasia panel analysis and subsequent whole-genome sequencing (via the 100,000 genomes project) found no cause. WGS data reanalysis using exomiser uncovered compound heterozygous pathogenic KIAA0753 variants (frameshift and splice site). Further clinical and radiological surveys were consistent with the expected phenotype. We discuss the emerging phenotype of this uncommon disorder. This report details the sixth published case of skeletal dysplasia in all cases of KIAA0753-related disease and the first case to describe a novel c.1830-2A>G splice variant. Our case is the eldest woman reported to date (aged ten years) and the only known case to report associated hearing loss, leg-length discrepancy, pectus carinatum, respiratory ciliary dyskinesia and late-onset (9 years old) neuro-degenerative regression.

Systemic Inflammation Is Associated With Neurologic Involvement in Pediatric Inflammatory Multisystem Syndrome Associated With SARS-CoV-2.

OBJECTIVE: Pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) is a severe immune-mediated disorder. We aim to report the neurologic features of children with PIMS-TS. METHODS: We identified children presenting to a large children's hospital with PIMS-TS from March to June 2020 and performed a retrospective medical note review, identifying clinical and investigative features alongside short-term outcome of children presenting with neurologic symptoms. RESULTS: Seventy-five patients with PIMS-TS were identified, 9 (12%) had neurologic involvement: altered conciseness (3), behavioral changes (3), focal neurology deficits (2), persistent headaches (2), hallucinations (2), excessive sleepiness (1), and new-onset focal seizures (1). Four patients had cranial images abnormalities. At 3-month follow-up, 1 child had died, 1 had hemiparesis, 3 had behavioral changes, and 4 completely recovered. Systemic inflammatory and prothrombotic markers were higher in patients with neurologic involvement (mean highest CRP 267 vs 202 mg/L, p = 0.05; procalcitonin 30.65 vs 13.11 µg/L, p = 0.04; fibrinogen 7.04 vs 6.17 g/L, p = 0.07; d-dimers 19.68 vs 7.35 mg/L, p = 0.005). Among patients with neurologic involvement, these markers were higher in those without full recovery at 3 months (ferritin 2284 vs 283 µg/L, p = 0.05; d-dimers 30.34 vs 6.37 mg/L, p = 0.04). Patients with and without neurologic involvement shared similar risk factors for PIMS-TS (Black, Asian and Minority Ethnic ethnicity 78% vs 70%, obese/overweight 56% vs 42%). CONCLUSIONS: Broad neurologic features were found in 12% patients with PIMS-TS. By 3-month follow-up, half of these surviving children had recovered fully without neurologic impairment. Significantly higher systemic inflammatory markers were identified in children with neurologic involvement and in those who had not recovered fully.

Structural characterisation of amyloid-like fibrils formed by an amyloidogenic peptide segment of ß-lactoglobulin.

Protein nanofibrils (PNFs) represent a promising class of biobased nanomaterials for biomedical and materials science applications. In the design of such materials, a fundamental understanding of the structure-function relationship at both molecular and nanoscale levels is essential. Here we report investigations of the nanoscale morphology and molecular arrangement of amyloid-like PNFs of a synthetic peptide fragment consisting of residues 11-20 of the protein ß-lactoglobulin (ß-LG11-20), an important model system for PNF materials. Nanoscale fibril morphology was analysed by atomic force microscopy (AFM) that indicates the presence of polymorphic self-assembly of protofilaments. However, observation of a single set of 13C and 15N resonances in the solid-state NMR spectra for the ß-LG11-20 fibrils suggests that the observed polymorphism originates from the assembly of protofilaments at the nanoscale but not from the molecular structure. The secondary structure and inter-residue proximities in the ß-LG11-20 fibrils were probed using NMR experiments of the peptide with 13C- and 15N-labelled amino acid residues at selected positions. We can conclude that the peptides form parallel ß-sheets, but the NMR data was inconclusive regarding inter-sheet packing. Molecular dynamics simulations confirm the stability of parallel ß-sheets and suggest two preferred modes of packing. Comparison of molecular dynamics models with NMR data and calculated chemical shifts indicates that both packing models are possible.

Respiratory Trajectories in Type 2 and 3 Spinal Muscular Atrophy in the iSMAC Cohort Study.

OBJECTIVE: To describe the respiratory trajectories and their correlation with motor function in an international pediatric cohort of patients with type 2 and nonambulant type 3 spinal muscular atrophy (SMA). METHODS: This was an 8-year retrospective observational study of patients in the International SMA Consortium (iSMAc) natural history study. We retrieved anthropometrics, forced vital capacity (FVC) absolute, FVC percent predicted (FVC%P), and noninvasive ventilation (NIV) requirement. Hammersmith Functional Motor Scale (HFMS) and revised Performance of Upper Limb (RULM) scores were correlated with respiratory function. We excluded patients in interventional clinical trials and on nusinersen commercial therapy. RESULTS: There were 437 patients with SMA: 348 with type 2 and 89 with nonambulant type 3. Mean age at first visit was 6.9 (±4.4) and 11.1 (±4) years. In SMA type 2, FVC%P declined by 4.2%/y from 5 to 13 years, followed by a slower decline (1.0%/y). In type 3, FVC%P declined by 6.3%/y between 8 and 13 years, followed by a slower decline (0.9%/y). Thirty-nine percent with SMA type 2% and 9% with type 3 required NIV at a median age 5.0 (1.8-16.6) and 15.1 (13.8-16.3) years. Eighty-four percent with SMA type 2% and 80% with type 3 had scoliosis; 54% and 46% required surgery, which did not significantly affect respiratory decline. FVC%P positively correlated with HFMS and RULM scores in both subtypes. CONCLUSIONS: In SMA type 2 and nonambulant type 3, lung function declines differently, with a common leveling after age 13 years. Lung and motor function correlated in both subtypes. Our data further define the milder SMA phenotypes and provide information to benchmark the long-term efficacy of new treatments for SMA.

Intrinsically disordered protein as carbon nanotube dispersant: How dynamic interactions lead to excellent colloidal stability.

The rich pool of protein conformations combined with the dimensions and properties of carbon nanotubes create new possibilities in functional materials and nanomedicine. Here, the intrinsically disordered protein α-synuclein is explored as a dispersant of single-walled carbon nanotubes (SWNTs) in water. We use a range of spectroscopic methods to quantify the amount of dispersed SWNT and to elucidate the binding mode of α-synuclein to SWNT. The dispersion ability of α-synuclein is good even with mild sonication and the obtained dispersion is very stable over time. The whole polypeptide chain is involved in the interaction accompanied by a fraction of the chain changing into a helical structure upon binding. Similar to other dispersants, we observe that only a small fraction (15-20%) of α-synuclein is adsorbed on the SWNT surface with an average residence time below 10 ms.

Unusual Presentations of Dystrophinopathies in Childhood.

X-linked recessive mutations in the dystrophin gene are one of the most common causes of inherited neuromuscular disorders in humans. Duchenne muscular dystrophy, the most common phenotype, and Becker muscular dystrophy are often recognizable by certain clinical features; however, less frequent presentations require a higher degree of suspicion. In this article, we describe a series of 6 children (4 boys, 2 girls) referred to a tertiary pediatric neuromuscular clinic for isolated elevated creatine kinase levels (range: 720-7000 IU/L) identified on initial assessment for otherwise unexplained transaminase elevations (n = 2), a social communication disorder (n = 3), and exertional myalgia and/or rhabdomyolysis (n = 1). There was no preceding family history of neuromuscular disease. One boy had an additional history of severe cerebral palsy and cyclical vomiting, and 1 girl had a history of maternal hepatitis C. There was no significant weakness at presentation, and the majority remained stable over a prolonged period of follow-up (age range at last follow-up: 9-16 years). All 6 children were found to carry dystrophin gene mutations resulting in milder phenotypes. This series highlights that dystrophinopathies may not uncommonly present with features distinct from the classic Duchenne muscular dystrophy and Becker muscular dystrophy phenotypes in both boys and girls. Pediatricians should be aware of such atypical presentations to initiate a timely and adequate diagnostic process. Establishing the correct genetic diagnosis of a dystrophinopathy is important to allow appropriate genetic counseling, to implement relevant surveillance and management strategies, and to avoid unnecessary investigations in search of an incorrect alternative diagnosis.

DFT calculations in the assignment of solid-state NMR and crystal structure elucidation of a lanthanum(iii) complex with dithiocarbamate and phenanthroline.

The molecular, crystal, and electronic structures as well as spectroscopic properties of a mononuclear heteroleptic lanthanum(iii) complex with diethyldithiocarbamate and 1,10-phenanthroline ligands (3 : 1) were studied by solid-state 13C and 15N cross-polarisation (CP) magic-angle-spinning (MAS) NMR, X-ray diffraction (XRD), and first principles density functional theory (DFT) calculations. A substantially different powder XRD pattern and 13C and 15N CP-MAS NMR spectra indicated that the title compound is not isostructural to the previously reported analogous rare earth complexes with the space group P21/n. Both 13C and 15N CP-MAS NMR revealed the presence of six structurally different dithiocarbamate groups in the asymmetric unit cell, implying a non-centrosymmetric packing arrangement of molecules. This was supported by single-crystal X-ray crystallography showing that the title compound crystallised in the triclinic space group P1[combining macron]. In addition, the crystal structure also revealed that one of the dithiocarbamate ligands has a conformational disorder. NMR chemical shift calculations employing the periodic gauge including projector augmented wave (GIPAW) approach supported the assignment of the experimental 13C and 15N NMR spectra. However, the best correspondences were obtained with the structure where the atomic positions in the X-ray unit cell were optimised at the DFT level. The roles of the scalar and spin-orbit relativistic effects on NMR shielding were investigated using the zeroth-order regular approximation (ZORA) method with the outcome that already the scalar relativistic level qualitatively reproduces the experimental chemical shifts. The electronic properties of the complex were evaluated based on the results of the natural bond orbital (NBO) and topology of the electron density analyses. Overall, we apply a multidisciplinary approach acquiring comprehensive information about the solid-state structure and the metal-ligand bonding of the heteroleptic lanthanum complex.

Acute flaccid weakness with myelopathy and peripheral nerve involvement in 2 children: Recent characterization of a previously observed phenomenon.

BACKGROUND: Acute flaccid weakness may be the first presentation of acute transverse myelitis (ATM), an immune-mediated central nervous system disorder or may be the first presentation of anterior horn cell syndrome or peripheral nervous system disease. CASE REPORTS: We describe two previously healthy female infants who presented with acute flaccid paralysis and encephalopathy. Neuroimaging revealed central cord signal changes in both cases and surprisingly electrophysiological studies performed revealed a generalized axonal motor neuropathy as well. CONCLUSION: Clinical, radiological and neurophysiological assessment are important to aid in the diagnosis and subsequent management of children with overlapping inflammatory peripheral and central nervous system syndromes.

Effect of methylene group insertions on the structural rigidity of Aib containing helices.

Nonprotein amino acids are being extensively used in the design of synthetic peptides to create new structure mimics. In this study we report the effect of methylene group insertions in a heptapeptide Boc-Ala1-Leu2-Aib3-Xxx4-Ala5-Leu6-Aib7-OMe which nicely folds into a mixed 310 -/α-helical structure when Xxx= Ala. Analogs of this peptide have been made and studied by replacing central Xxx4 residue with Glycine (α-residue), ß-Alanine (ß-Αla), γ-aminobutyric acid (Gaba), and ε-aminocaproic acid (ε-Aca). NMR and circular dichroism were used to study the solution structure of these peptides. Crystals of the peptides containing alanine, ß-Αla, and Gaba reveal that increasing the number of central methylene (-CH2 -) groups introduces local perturbations even as the helical structure is retained.

Congenital myopathies: Natural history of a large pediatric cohort.

OBJECTIVE: To assess the natural history of congenital myopathies (CMs) due to different genotypes. METHODS: Retrospective cross-sectional study based on case-note review of 125 patients affected by CM, followed at a single pediatric neuromuscular center, between 1984 and 2012. RESULTS: Genetic characterization was achieved in 99 of 125 cases (79.2%), with RYR1 most frequently implicated (44/125). Neonatal/infantile onset was observed in 76%. At birth, 30.4% required respiratory support, and 25.2% nasogastric feeding. Twelve percent died, mainly within the first year, associated with mutations in ACTA1, MTM1, or KLHL40. All RYR1-mutated cases survived and did not require long-term ventilator support including those with severe neonatal onset; however, recessive cases were more likely to require gastrostomy insertion (p = 0.0028) compared with dominant cases. Independent ambulation was achieved in 74.1% of all patients; 62.9% were late walkers. Among ambulant patients, 9% eventually became wheelchair-dependent. Scoliosis of variable severity was reported in 40%, with 1/3 of (both ambulant and nonambulant) patients requiring surgery. Bulbar involvement was present in 46.4% and required gastrostomy placement in 28.8% (at a mean age of 2.7 years). Respiratory impairment of variable severity was a feature in 64.1%; approximately half of these patients required nocturnal noninvasive ventilation due to respiratory failure (at a mean age of 8.5 years). CONCLUSIONS: We describe the long-term outcome of a large cohort of patients with CMs. While overall course is stable, we demonstrate a wide clinical spectrum with motor deterioration in a subset of cases. Severity in the neonatal/infantile period is critical for survival, with clear genotype-phenotype correlations that may inform future counseling.

Prospective study of POLG mutations presenting in children with intractable epilepsy: prevalence and clinical features.

PURPOSE: To assess the frequency and clinical features of childhood-onset intractable epilepsy caused by the most common mutations in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma. METHODS: Children presenting with nonsyndromic intractable epilepsy of unknown etiology but without documented liver dysfunction at presentation were eligible for this prospective, population-based study. Blood samples were analyzed for the three most common POLG mutations. If any of the three tested mutations were found, all the exons and the exon-intron boundaries of the POLG gene were sequenced. In addition, we retrospectively reviewed the notes of patients presenting with intractable epilepsy in which we had found POLG mutations. All available clinical data were collected by questionnaire and by reviewing the medical records. KEY FINDINGS: We analyzed 213 blood DNA samples from patients fulfilling the inclusion criteria of the prospective study. Among these, five patients (2.3%) were found with one of the three common POLG mutations as homozygous or compound heterozygous states. In addition, three patients were retrospectively identified. Seven of the eight patients had either raised cerebrospinal fluid (CSF) lactate (n = 3) or brain magnetic resonance imaging (MRI) changes (n = 4) at presentation with intractable epilepsy. Three patients later developed liver dysfunction, progressing to fatal liver failure in two without previous treatment with sodium valproate (VPA). Furthermore, it is worth mentioning that one patient presented first with an autism spectrum disorder before seizures emerged. SIGNIFICANCE: Mutations in POLG are an important cause of early and juvenile onset nonsyndromic intractable epilepsy with highly variable associated manifestations including autistic features. This study emphasizes that genetic testing for POLG mutations in patients with nonsyndromic intractable epilepsies is very important for clinical diagnostics, genetic counseling, and treatment decisions because of the increased risk for VPA-induced liver failure in patients with POLG mutations. We recommend POLG gene testing for patients with intractable seizures and at least one elevated CSF lactate or suggestive brain MRI changes (predominantly abnormal T2 -weighted thalamic signal) with or without status epilepticus, epilepsia partialis continua, or liver manifestations typical for Alpers disease, especially when the disease course is progressive.

Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations.

We investigated clinical and cellular phenotypes of 24 children with mutations in the catalytic (alpha) subunit of the mitochondrial DNA (mtDNA) gamma polymerase (POLG1). Twenty-one had Alpers syndrome, the commonest severe POLG1 autosomal recessive phenotype, comprising hepatoencephalopathy and often mtDNA depletion. The cellular mtDNA content reflected the genotype more closely than did clinical features. Patients with tissue depletion of mtDNA all had at least one allele with either a missense mutation in a catalytic domain or a nonsense mutation. Four out of 12 patients exhibited a progressive, mosaic pattern of mtDNA depletion in cultured fibroblasts. All these patients had mutations in a catalytic domain in both POLG1 alleles, in either the polymerase or exonuclease domain or both. The tissue mtDNA content of patients who had two linker mutations was normal, and their phenotypes the mildest. Epilepsy and/or movement disorder were major features in all 21. Previous studies have implicated replication stalling as a mechanism for mtDNA depletion. The mosaic cellular depletion that we have demonstrated in cell cultures may be a manifestation of severe replication stalling. One patient with a severe cellular and clinical phenotype was a compound heterozygote with POLG1 mutations in the polymerase and exonuclease domain intrans. This suggests that POLG1 requires both polymerase and 3'-5' exonuclease activity in the same molecule. This is consistent with current functional models for eukaryotic DNA polymerases, which alternate between polymerizing and editing modes, as determined by competition between these two active sites for the 3' end of the DNA.