Sandeep Jayawant

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic diet.

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US

Ephedrine treatment in congenital myasthenic syndrome due to mutations in DOK7

620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

Nemaline myopathy caused by absence of α‐skeletal muscle actin

Background: Mutations in the postsynaptic adaptor protein Dok-7 underlie congenital myasthenic syndrome (CMS) with a characteristic limb girdle pattern of muscle weakness. Patients usually do not respond to or worsen with the standard CMS treatments: cholinesterase inhibitors and 3,4-diaminopyridine. However, anecdotal reports suggest they may improve with ephedrine. Methods: This was an open prospective follow-up study to determine muscle strength in response to ephedrine in Dok-7 CMS. Patients were first evaluated as inpatients for suitability for a trial of treatment with ephedrine. The response was assessed at 2 and 6 to 8 months follow-up clinic visits using a quantitative myasthenia gravis (severity) score (QMG) and mobility measures. Results: Ten out of 12 of the cohort with DOK7 mutations tolerated ephedrine. We noted a progressive response to treatment over the 6 to 8 months assessment period with a significant improvement at the final QMG score (p = 0.009). Mobility scores also improved (p = 0.0006). Improvements in the subcomponents of the QMG score that measured proximal muscle function (those muscle groups most severely affected) were most marked, and in some cases were dramatic. All patients reported enhanced activities of daily living at 6–8 months. Conclusion: Ephedrine appears to be an effective treatment for Dok-7 CMS. It is well-tolerated by most patients and improvement in strength can be profound. Determining the long-term response and the most effective dosing regimen will require further research. Classification of evidence: This study provides Class IV evidence that ephedrine given at doses between 15 and 90 mg/day improves muscle strength in patients with documented mutations in DOK7.

Neuroradiological aspects of subdural haemorrhages

To investigate seven congenital myopathy patients from six families: one French Gypsy, one Spanish Gypsy, four British Pakistanis, and one British Indian. Three patients required mechanical ventilation from birth, five died before 22 months, one is ventilator‐dependent, but one, at 30 months, is sitting with minimal support. All parents were unaffected.

De novo point mutations in patients diagnosed with ataxic cerebral palsy.

Aims: To review the neuroimaging of a series of infants and young children admitted to hospital with subdural haemorrhage (SDH). Methods: Neuroradiological investigations of 74 children under 2 years of age, from South Wales and southwest England, in whom an SDH or subdural effusion had been diagnosed between 1992 and 2001, were reviewed. Two paediatric neuroradiologists blinded to the original radiological report reviewed all the relevant images. Results: Neuroradiological review of images identified radiological features which were highly suggestive of non-accidental head injury (NAHI). Interhemispheric haemorrhages and SDHs in multiple sites or of different densities were almost exclusively seen in NAHI. MRI was more sensitive in identifying SDHs of different signal characteristics, posterior and middle cranial fossa bleeds, and parenchymal changes in the brain. CT scans, if performed with suboptimal protocols, were likely to miss small subdural bleeds. Conclusions: Guidelines for neuroimaging in suspected NAHI are recommended. A radiologist with experience in NAHI should report or review these scans. The initial investigation should be CT, but MRI will also be necessary in most cases. Head CT should be an integral part of the skeletal survey in all infants less than 6 months of age referred for child protection investigation, and in children less than 2 years where child abuse is suspected and there are neurological signs, retinal haemorrhages, or fractures.

Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies

Cerebral palsy is commonly attributed to perinatal asphyxia. However, Schnekenberg et al. describe here four individuals with ataxic cerebral palsy likely due to de novo dominant mutations associated with increased paternal age. Therefore, patients with cerebral palsy should be investigated for genetic causes before the disorder is ascribed to asphyxia.

Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development

The development of next generation sequencing (NGS) has radically transformed the scientific landscape, making it possible to sequence the exome of any given individual in a cost-effective way. The power of this approach has been demonstrated by a number of groups who have identified pathogenic mutations in small pedigrees that have been resistant to traditional genetic mapping. Recently it has become clear that exome sequencing has great potential with respect to sporadic disease and the identification of de novo mutations. This is highlighted by studies reporting whole-exome sequencing of patient–parental trios affected by learning disability, autism and schizophrenia. It is widely anticipated that the introduction of this technique into a clinical setting will revolutionise genetic diagnosis. However, the sensitivity of NGS exome sequencing is currently unclear. Here, we describe the exome sequencing of DNA samples from a patient with double cortex syndrome and her parents, resulting in the detection of a mosaic splicing mutation in LIS1. This variant was found at an allele frequency of just 18%, demonstrating that NGS methods have the capacity to identify pathogenic mosaic mutations present at a low level.

Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as “Lincoln ataxia,” because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

Early use of high‐dose riboflavin in a case of Brown–Vialetto–Van Laere syndrome

Congenital myopathy with fibre type disproportion (CFTD) has been associated with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. We report the clinico-pathological and electrophysiological features of 2 unrelated cases with heterozygous TPM3 mutation. Case 1 is a 19-year-old lady who presented with motor delay in infancy, respiratory failure in early teens requiring non-invasive ventilation despite being ambulant, ptosis, axial more than proximal weakness and scoliosis. Case 2 is a 7-year-old boy with hypotonia, feeding difficulties, motor delay and scoliosis, also requiring non-invasive ventilation while ambulant. Muscle biopsies in both cases showed fibre type disproportion. Muscle MRI (Case 1) showed mild uniformly increased interstitial tissue in and around the muscles. Sequencing of TPM3 in case 1 revealed a previously described heterozygous c.503G > A(pArg168His) missense variant in exon 5 and a novel heterozygous missense mutation c.521A > C(pGlu174Ala), also in exon 5, in case 2. A mild abnormality in the single fibre EMG was documented on electrophysiology in both cases. These cases highlight the neuromuscular transmission defect in CFTD secondary to TPM3 mutations.