Annu Aggarwal

Indian-Subcontinent NBIA: Unusual Phenotypes, Novel PANK2 Mutations, and Undetermined Genetic Forms

Neurodegeneration with brain iron accumulation (NBIA) is etiologically, clinically, and by imaging a heterogeneous group including NBIA types 1 [pantothenate kinase‐associated neurodegeneration (PKAN)] and 2 (PLA2G6‐associated neurodegeneration), neuroferritinopathy, and aceruloplasminaemia. Data on genetically defined Indian‐subcontinent NBIA cases are limited. We report 6 patients from the Indian‐subcontinent with a movement disorder and MRI basal ganglia iron deposition, compatible with diagnosis of an NBIA syndrome. All patients were screened for abnormalities in serum ceruloplasmin and ferritin levels and mutations in NBIA‐associated genes [pantothenate kinase 2 (PANK2), PLA2G6 and ferritin light chain (exon 4)]. We present clinical, imaging and genetic data correlating phenotype–genotype relations. Four patients carried PANK2 mutations, two of these were novel. The clinical phenotype was mainly dystonic with generalized dystonia and marked orobulbar features in the 4 adolescent‐onset cases. One of the four had a late‐onset (age 37) unilateral jerky postural tremor. His mutation, c.1379C>T, appears associated with a milder phenotype. Interestingly, he developed the eye‐of‐the‐tiger sign only 10 years after onset. Two of the six presented with adult‐onset levodopa (L‐dopa)‐responsive asymmetric re‐emergent rest tremor, developing L‐dopa‐induced dyskinesias, and good benefit to deep brain stimulation (in one), thus resembling Parkinsons disease (PD). Both had an eye‐of‐the‐tiger sign on MRI but were negative for known NBIA‐associated genes, suggesting the existence of further genetic or sporadic forms of NBIA syndromes. In conclusion, genetically determined NBIA cases from the Indian subcontinent suggest presence of unusual phenotypes of PANK2 and novel mutations. The phenotype of NBIA of unknown cause includes a PD‐like presentation.

Dystonic opisthotonus: a "red flag" for neurodegeneration with brain iron accumulation syndromes?

Back arching was reported in one of the very first patients with neurodegeneration with brain iron accumulation syndrome (NBIAs) published in 1936. However, recent reports have mainly focused on the genetic and imaging aspects of these disorders, and the phenotypic characterization of the dystonia has been lost. In evaluating patients with NBIAs in our centers, we have observed that action‐induced dystonic opisthotonus is a common and characteristic feature of NBIAs. Here, we present a case series of patients with NBIAs presenting this feature demonstrated by videos. We suggest that dystonic opisthotonus could be a useful “red flag” for clinicians to suspect NBIAs, and we discuss the differential diagnosis of this feature. This would be particularly useful in identifying patients with NBIAs and no iron accumulation as yet on brain imaging (for example, as in phospholipase A2, group IV (cytosolic, calcium‐independent) [PLA2G6]‐related disorders), and it has management implications.

Update on Wilson Disease

Wilson disease (WD) is an inherited disorder of chronic copper toxicosis characterized by excessive copper deposition in the body, primarily in the liver and the brain. It is a progressive disease and fatal if untreated. Excessive copper accumulation results from the inability of liver to excrete copper in bile. Copper is an essential trace metal and has a crucial role in many metabolic processes. Almost all of the body copper is protein bound. In WD, the slow but relentless copper accumulation overwhelms the copper chaperones (copper-binding proteins), resulting in high levels of free copper and copper-induced tissue injury. Liver is the central organ for copper metabolism, and copper is initially accumulated in the liver but over time spills to other tissues. WD has protean clinical manifestations mainly attributable to liver, brain, and osseomuscular impairment. Diagnosis of WD is challenging and based on combination of clinical features and laboratory tests. Identification of various high-frequency mutations identified in different population studies across the world has revived interest in developing DNA chips for rapid genetic diagnosis of WD. All symptomatic and all presymptomatic patients require lifelong decoppering with careful clinical tracking. Decoppering ensures that presymptomatic individuals remain symptom free. With judicious decoppering, given time, even patients with severe neurological disability improve and can return to normal life and resume school or work at par with their peers. Treatment regimens and tracking patients using the WD-specific Global Assessment Scale for WD (GAS for WD) are discussed.

Wilson Disease Mutation Pattern with Genotype-Phenotype Correlations from Western India: Confirmation of p.C271* as a Common Indian Mutation and Identification of 14 Novel Mutations

Wilson disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene, with over 600 mutations described. Identification of mutations has made genetic diagnosis of WD feasible in many countries. The heterogeneity of ATP7B mutants is, however, yet to be identified in the Indian population. We analyzed the mutational pattern of WD in a large region of Western India. We studied patients (n = 52) for ATP7B gene mutations in a cohort of families with WD and also in first‐degree relatives (n = 126). All 21 exon–intron boundaries of the WD gene were amplified and directly sequenced. We identified 36 different disease‐causing mutations (31 exonic and five intronic splice site variants). Fourteen novel mutations were identified. Exons 2, 8, 13, 14, and 18 accounted for the majority of mutations (86.4%). A previously recognized mutation, p.C271*, and the novel mutation p.E122fs, were the most common mutations with allelic frequencies of 20.2% and 10.6%, respectively. Frequent homozygous mutations (58.9%) and disease severity assessments allowed analysis of genotype–phenotype correlations. Our study significantly adds to the emerging data from other parts of India suggesting that p.C271* may be the most frequent mutation across India, and may harbor a moderate to severely disabling phenotype with limited variability.

The Pragmatic Treatment of Wilson's Disease

Wilsons disease (WD) is a potentially fatal disorder of chronic copper toxicity, primarily affecting the liver and the brain. Judicious treatment can restore health and longevity, even in patients with severe neurological impairment. However, the disease is associated with considerable morbidity and mortality resulting from delay in diagnosis, and difficulty in pacing the medical treatment. In this article, we briefly review the diagnosis and treatment options for WD and share our experience in managing patients with WD. We focus on decoppering (copper chelation) treatment of WD and outline pragmatic strategies for patient management designed to recognize and minimize adverse effects while ensuring treatment compliance and effectiveness.

The Effect of Zinc and D-Penicillamine in a Stable Human Hepatoma ATP7B Knockout Cell Line

Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

AIM To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.

Commonly used gastrointestinal drugs.

This chapter reviews the spectrum and mechanisms of neurologic adverse effects of commonly used gastrointestinal drugs including antiemetics, promotility drugs, laxatives, antimotility drugs, and drugs for acid-related disorders. The commonly used gastrointestinal drugs as a group are considered safe and are widely used. A range of neurologic complications are reported following use of various gastrointestinal drugs. Acute neurotoxicities, including transient akathisias, oculogyric crisis, delirium, seizures, and strokes, can develop after use of certain gastrointestinal medications, while disabling and pervasive tardive syndromes are described following long-term and often unsupervised use of phenothiazines, metoclopramide, and other drugs. In rare instances, some of the antiemetics can precipitate life-threatening extrapyramidal reactions, neuroleptic malignant syndrome, or serotonin syndrome. In contrast, concerns about the cardiovascular toxicity of drugs such as cisapride and tegaserod have been grave enough to lead to their withdrawal from many world markets. Awareness and recognition of the neurotoxicity of gastrointestinal drugs is essential to help weigh the benefit of their use against possible adverse effects, even if uncommon. Furthermore, as far as possible, drugs such as metoclopramide and others that can lead to tardive dyskinesias should be used for as short time as possible, with close clinical monitoring and patient education.

Facial Reflex Hyperexcitability in Geniospasm Suggests a Brainstem Origin

Geniospasm (OMIM 190100) is characterized by spontaneous repetitive involuntary contraction of mentalis muscles resulting in quivering or trembling of the chin that is intensified by stress or anxiety. The movements are first noticed in infancy or childhood and usually abate by late adulthood. Although geniospasm is a benign disorder the movements cause embarrassment and significant social anxiety. The nosology of the movements of geniospasm has been variously described as myokymia, tremor, and myoclonus but their origin remains unclear. 3 This report examines mentalis activity and facial nerve excitability in a patient with geniospasm. A 42-year-old man from a four generation Australian family with six affected members with geniospasm was studied. He had been aware of semicontinuous chin quivering since childhood. Neurological examination revealed bilateral continuous, semirhythmic chin quivering. There was no facial weakness. Drugs and left lower peripheral facial nerve surgery had no effect on the movements. Botulinum toxin (Botox, Allergan) injections (30 units in each mentalis muscle) every 8 to 10 months abolished the movements. Muscle activity was recorded from bilateral mentalis and orbicularis oculi (OO) muscles using needle and surface electrodes on a multichannel electromyography (EMG) system (Nicolet Viking III, Madison, WI). The supraorbital nerves (SON) were stimulated in the supraorbital groove at three to five times the sensory threshold and responses recorded from surface electrodes over both OO and mentalis muscles. Response latency was measured by visual inspection. Responses to facial nerve stimulation at the tragus were recorded in ipsilateral mentalis muscles. The mentalis muscle activity responsible for geniospasm comprised brief motor unit action potentials of normal morphology, 10 to 12 milliseconds duration and amplitude 50 to 100 lV, discharging arrhythmically at five to eight per second. Both sides were involved. There were no high frequency discharges or grouped discharges of myokymia. Voluntary contraction produced normal recruitment pattern that interrupted geniospasm. Stimulation of the facial nerves at the tragus evoked mentalis responses of normal morphology and latency (without late responses). Stimulation of the SON evoked a series of reflex responses in the facial muscles. Normal appearing blink reflexes (R1 and R2) were recorded from bilateral OO (not shown). Responses corresponding to R1, R2, and R3 were recorded from both mentalis (Fig. 1). Needle EMG confirmed the responses corresponding to the R2 and R3 were bilateral but R1 responses were more variable and largely unilateral (the anatomy of mentalis allowed surface EMG recording to pick up activity from both sides of the muscle). Spread of R2 responses beyond OO muscles is described in normal subjects with high stimulus intensities and in patients with synkinesis because of aberrant facial nerve regeneration. It is suggested in geniospasm spread of R2 and R3 responses to mentalis indicates facial nuclear hyperexcitability. The characteristics of geniospasm in this patient were similar to those described previously, 3 including spread of FIG. 1. Rectified surface EMG recordings from the mentalis muscles following stimulation of the supraorbital nerves (SON) (each trace represents the average of 10 trials). Unilateral SON stimulation evoked mentalis responses at latency 14 milliseconds corresponding to the R1 response of the blink reflex and bilateral responses at 42 milliseconds (corresponding to the R2 response of the blink reflex) and 90 milliseconds (corresponding to the R3 response of the blink reflex). The extent of facial muscle activity suggests facial nuclear hyperexcitability.

Stand alone mechanical thrombectomy (with penumbra system) for acute ischemic stroke based on MR imaging: Single center experience

BACKGROUND There is dismal rate of recanalization following intravenous thrombolysis of large vessel occlusive ischemic stroke. Trials on use of mechanical clot retrievers in acute ischemic stroke have used time from onset and clinical deficit at presentation as the main indications for intervention. MATERIALS AND METHODS Retrospective analysis of case records of acute stroke seen between May 2009 and October 2011 was done. It revealed 23 patients with acute ischemic stroke treated by mechanical thrombectomy using Penumbra system (PS). We used magnetic resonance (MR) imaging in correlation with clinical presentation to determine patients likely to benefit from recanalization and accordingly offered or at times deferred revascularization. A comparison of approach and outcomes was done with other relevant trials/reports. RESULTS Recanalization was achieved in all but one patient. Median modified Rankin Scale (mRS) score at 90 days was 2. Good clinical outcome (mRS ≤ 2) was achieved in 56.5% compared with 25% in Penumbra pivotal trial and 36% in multi Mechanical Embolus Removal in Cerebral Ischemia (multi MERCI) trial. All cause mortality was 13.04%. Symptomatic intracerebral hemorrhage (ICH) occurred in two patients (8.6%). CONCLUSION Analysis of our results suggests that PS is safe and effective (91.3%) in recanalizing cerebral vessels without concomitant thrombolytics.