Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes with coexisting nemaline myopathy: a case report.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes and nemaline myopathy are two rare genetic conditions. We report the first case reported in world literature with coexistence of both these rare disorders. CASE PRESENTATION: A 11-year-old previously healthy Sri Lankan male child, product of a nonconsanguineous marriage with normal development presented with acute onset short lasting recurring episodes of right-sided eye deviation with impaired consciousness. In between episodes he regained consciousness. Family history revealed a similar presentation in the mother at 36 years of age. Examination was significant for short stature and proximal upper and lower limb weakness. His plasma and cerebrospinal fluid lactate were elevated. Magnetic resonance imaging brain had evidence of an acute infarction in the right occipital territory. Sanger sequencing for common mitochondrial variants of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes confirmed this diagnosis. Whole exome sequencing revealed pathogenic compound heterozygous variants in NEB gene implicating in coexisting nemaline myopathy. Acute presentation was managed with supportive care, antiepileptics, and mitochondrial supplementation. Currently he is stable on daily supplementation of arginine and limb-strengthening physiotherapy. He is being monitored closely clinically and with serum lactate level. CONCLUSION: Genetic diseases are rare. Coexistence of two genetic conditions is even rarer. Genetic confirmation of diagnosis is imperative for prediction of complications, accurate management, and genetic counseling.

Clinical, biochemical, and molecular profiles of three Sri Lankan neonates with pyruvate carboxylase deficiency.

Objectives: Pyruvate carboxylase, a mitochondrial enzyme, catalyses the conversion of glycolytic end-product pyruvate to tricarboxylic acid cycle intermediate, oxaloacetate. Rare pyruvate carboxylase deficiency manifests in three clinical and biochemical phenotypes: neonatal onset type A, infantile onset type B and a benign C type. The objective of this case series is to expand the knowledge of overlapping clinical and biochemical phenotypes of pyruvate carboxylase deficiency. Case presentation: We report three Sri Lankan neonates including two siblings, of two unrelated families with pyruvate carboxylase deficiency. All three developed respiratory distress within the first few hours of birth. Two siblings displayed typical biochemical findings reported in type B. The other proband with normal citrulline, lysine, moderate lactate, paraventricular cystic lesions, bony deformities, and a novel missense, homozygous variant c.2746G>C [p.(Asp916His)] in the PC gene, biochemically favoured type A. Conclusions: Our findings indicate the necessity of prompt laboratory investigations in a tachypneic neonate with coexisting metabolic acidosis, as early recognition is essential for patient management and family counselling. Further case studies are required to identify overlapping symptoms and biochemical findings in different types of pyruvate carboxylase deficiency phenotypes.

Diagnostic outcome of pro bono neurogenetic diagnostic service in Sri Lanka: A wealth creation.

The inherited disease community in Sri Lanka has been widely neglected. This article aimed to present accumulated knowledge in establishing a pro bono cost-effective national, island-wide, free-of-charge molecular diagnostic service, suggesting a model for other developing countries. The project provided 637 molecular diagnostic tests and reports free of charge to a nation with limited resources. We pioneered the implementation of mobile clinics and home visits, where the research team acted as barefoot doctors with the concept of the doctor and the researcher at the patient's doorstep. Establishing pro bono, cost-effective molecular diagnostics is feasible in developing countries with limited resources and state funding through the effort of dedicated postgraduate students. This service could provide an accurate molecular diagnosis of Duchenne muscular dystrophy, Huntington's disease, Spinocerebellar ataxia, and Spinal muscular atrophy, a diagnostic yield of 54% (343/637), of which 43% (147/343) of the patients identified as amenable for available gene therapies. Initiated human resource development by double doctoral degree opportunities with international collaborations. Established a neurobiobank and a national registry in Sri Lanka, a rich and unique repository, wealth creation for translational collaborative research and sharing of information in neurological diseases, as well as a lodestar for aspiring initiatives from other developing countries.

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting.

BACKGROUND: The phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene. METHODOLOGY: Genetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data. RESULTS: In the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45-55 and 6-15. Exon 45-60 identified as a novel in-frame variation hotspot. Exon 45-59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations. CONCLUSION: DMD gene deletions and duplications are concentrated in exons 45-55 and 2-20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

Neurological disorders associated with COVID-19 in Sri Lanka.

BACKGROUND: Neurological manifestations of SARS-CoV-2 infection have been reported from many countries around the world, including the South Asian region. This surveillance study aimed to describe the spectrum of neurological disorders associated with COVID-19 in Sri Lanka. METHODS: COVID-19 patients manifesting neurological disorders one week prior and up to six weeks after infection were recruited from all the neurology centres of the government hospitals in Sri Lanka from May 2021 - May 2022. Data was collected using a structured data form that was electronically transmitted to a central repository. All patients were evaluated and managed by a neurologist. Data were analysed using simple descriptive analysis to characterise demographic and disease related variables, and simple comparisons and logistic regression were performed to analyse outcomes and their associations. RESULTS: One hundred and eighty-four patients with neurological manifestations associated with COVID-19 were recruited from all nine provinces in Sri Lanka. Ischaemic stroke (31%) was the commonest neurological manifestation followed by encephalopathy (13.6%), Guillain-Barre syndrome (GBS) (9.2%) and encephalitis (7.6%). Ischaemic stroke, encephalitis and encephalopathy presented within 6 days of onset of COVID-19 symptoms, whereas GBS and myelitis presented up to 10 days post onset while epilepsy and Bell palsy presented up to 20 - 40 days post onset. Haemorrhagic stroke presented either just prior to or at onset, or 10 - 25 days post onset of COVID-19 symptomatic infection. An increased frequency of children presenting with encephalitis and encephalopathy was observed during the Omicron variant predominant period. A poor outcome (no recovery or death) was associated with supplemental oxygen requirement during admission (Odds Ratio: 12.94; p = 0.046). CONCLUSIONS: The spectrum and frequencies of COVID-19 associated neurological disorders in Sri Lanka were similar to that reported from other countries, with strokes and encephalopathy being the commonest. Requiring supplemental oxygen during hospitalisation was associated with a poor outcome.

Duchenne Muscular Dystrophy from Brain to Muscle: The Role of Brain Dystrophin Isoforms in Motor Functions.

Brain function and its effect on motor performance in Duchenne muscular dystrophy (DMD) is an emerging concept. The present study explored how cumulative dystrophin isoform loss, age, and a corticosteroid treatment affect DMD motor outcomes. A total of 133 genetically confirmed DMD patients from Sri Lanka were divided into two groups based on whether their shorter dystrophin isoforms (Dp140, Dp116, and Dp71) were affected: Group 1, containing patients with Dp140, Dp116, and Dp71 affected (n = 98), and Group 2, containing unaffected patients (n = 35). A subset of 52 patients (Group 1, n = 38; Group 2, n = 14) was followed for up to three follow-ups performed in an average of 28-month intervals. The effect of the cumulative loss of shorter dystrophin isoforms on the natural history of DMD was analyzed. A total of 74/133 (56%) patients encountered developmental delays, with 66/74 (89%) being in Group 1 and 8/74 (11%) being in Group 2 (p < 0.001). Motor developmental delays were predominant. The hip and knee muscular strength, according to the Medical Research Council (MRC) scale and the North Star Ambulatory Assessment (NSAA) activities, "standing on one leg R", "standing on one leg L", and "walk", declined rapidly in Group 1 (p < 0.001 In the follow-up analysis, Group 1 patients became wheelchair-bound at a younger age than those of Group 2 (p = 0.004). DMD motor dysfunction is linked to DMD mutations that affect shorter dystrophin isoforms. When stratifying individuals for clinical trials, considering the DMD mutation site and its impact on a shorter dystrophin isoform is crucial.

Integrated genomic, proteomic and cognitive assessment in Duchenne Muscular Dystrophy suggest astrocyte centric pathology.

Introduction: Documented Duchenne Muscular Dystrophy (DMD) biomarkers are confined to Caucasians and are poor indicators of cognitive difficulties and neuropsychological alterations. Materials and methods: This study correlates serum protein signatures with cognitive performance in DMD patients of South Asian origin. Study included 25 DMD patients aged 6-16 years. Cognitive profiles were assessed by Wechsler Intelligence Scale for Children. Serum proteome profiling of 1317 proteins was performed in eight DMD patients and eight age-matched healthy volunteers. Results: Among the several novel observations we report, better cognitive performance in DMD was associated with increased serum levels of MMP9 and FN1 but decreased Siglec-3, C4b, and C3b. Worse cognitive performance was associated with increased serum levels of LDH-H1 and PDGF-BB but reduced GDF-11, MMP12, TPSB2, and G1B. Secondly, better cognitive performance in Processing Speed (PSI) and Perceptual Reasoning (PRI) domains was associated with intact Dp116, Dp140, and Dp71 dystrophin isoforms while better performance in Verbal Comprehension (VCI) and Working Memory (WMI) domains was associated with intact Dp116 and Dp140 isoforms. Finally, functional pathways shared with Alzheimer's Disease (AD) point towards an astrocyte-centric model for DMD. Conclusion: Astrocytic dysfunction leading to synaptic dysfunction reported previously in AD may be a common pathogenic mechanism underlying both AD and DMD, linking protein alterations to cognitive impairment. This new insight may pave the path towards novel therapeutic approaches targeting reactive astrocytes.

Gene therapy for selected neuromuscular and trinucleotide repeat disorders - An insight to subsume South Asia for multicenter clinical trials.

Background: In this article, the authors discuss how they utilized the genetic mutation data in Sri Lankan Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA) and Huntington's disease (HD) patients and compare the available literature from South Asian countries to identifying potential candidates for available gene therapy for DMD, SMA, SCA and HD patients. Methods: Rare disease patients (n = 623) with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy were genetically confirmed using Multiplex Ligation Dependent Probe Amplification (MLPA), and single plex PCR. A survey was conducted in the "Wiley database on Gene Therapy Trials Worldwide" to identify DMD, SMA, SCA, and HD gene therapy clinical trials performed worldwide up to April 2021. In order to identify candidates for gene therapy in other neighboring countries we compared our findings with available literature from India and Pakistan which has utilized the same molecular diagnostic protocol to our study. Results: From the overall cohort of 623 rare disease patients with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy, n = 343 (55%) [Muscular Dystrophy- 65%; (DMD-139, Becker Muscular Dystrophy -BMD-11), SCA type 1-3-53% (SCA1-61,SCA2- 23, SCA3- 39), HD- 52% (45) and SMA- 34% (22)] patients were positive for molecular diagnostics by MLPA and single plex PCR. A total of 147 patients in Sri Lanka amenable to available gene therapy; [DMD-83, SMA-15 and HD-49] were identified. A comparison of Sri Lankan finding with available literature from India and Pakistan identified a total of 1257 patients [DMD-1076, SMA- 57, and HD-124] from these three South Asian Countries as amenable for existing gene therapy trials. DMD, SMA, and HD gene therapy clinical trials (113 studies) performed worldwide up to April 2021 were concentrated mostly (99%) in High Income Countries (HIC) and Upper Middle-Income Countries (UMIC). However, studies on the potential use of anti-sense oligonucleotides (ASO) for treatment of SCAs have yet to reach clinical trials. Conclusion: Most genetic therapies for neurodegenerative and neuromuscular disorders have been evaluated for efficacy primarily in Western populations. No multicenter gene therapy clinical trial sites for DMD, SMA and HD in the South Asian region, leading to lack of knowledge on the safety and efficacy of such personalized therapies in other populations, including South Asians. By fostering collaboration between researchers, clinicians, patient advocacy groups, government and industry in gene therapy initiatives for the inherited-diseases community in the developing world would link the Global North and Global South and breathe life into the motto "Together we can make a difference".

Urine Organic Acid Analysis: Key Diagnostic Test for Fumaric Aciduria in a Sri Lankan Child.

Fumaric aciduria resulting from fumarate hydratase deficiency is a rare inherited disorder of the Krebs tricarboxylic acid cycle that is characterized by neurologic manifestations, a spectrum of brain abnormalities, and the excretion of fumaric acid in urine. We describe a 3 year old Sri Lankan boy who was referred at age 10 months with poor weight gain and hypotonia for further laboratory investigations. In addition to global developmental delay, there were noticeable dysmorphic features with a prominent forehead, low-set ears, micrognathia, and hypertelorism with persistent neutropenia. Urine organic acid assay revealed a massive elevation of fumaric acid on 2 occasions. Molecular analysis revealed a homozygous likely pathogenic missense variant, NM000143.3:c.1048C>T p. (Arg350Trp), in the FH gene, confirming the biochemical diagnosis. Our patient was the first patient in Sri Lanka molecularly diagnosed with fumaric aciduria. This case study highlights the importance of performing organic acid assays in children presenting with neurologic manifestations especially when these are suspected to have a metabolic basis.

Experience during COVID-19 lockdown and self-managing strategies among caregivers of children with epilepsy: A study from low middle income country.

PURPOSE: Abrupt halt of service provision due to pandemic state of COVID-19, significantly affected care of patients with chronic diseases like epilepsy; its impact being greater on caregivers of vulnerable groups such as children with epilepsy. We performed this study to describe difficulties posed by the lockdown to caregivers of children with epilepsy in a low-middle income country and describe their responses and self-management strategies to overcome difficulties and prepare for a recurrence. METHOD: A cross-sectional all-island survey was carried out at paediatric neurology centers in Sri Lanka. Data was gathered via a face-to-face interview after the lockdown period. Parental stress level was evaluated using a self-rating Stress Assessment Questionnaire. RESULTS: Caregivers of 140 children with epilepsy from seven centers served by paediatric neurologists were interviewed. Mean duration of epilepsy was 7.9 years(SD 4). Majority were on one (52.1 %) or two (20 %) anti-seizure medications regularly. The pandemic did not affect epilepsy control in majority (87.3 %), however, signficant proportion faced difficulties over regular reviews and presecription refills. Despite difficluties, 87.1 % of parents maintained dispensing anti-seizure medications to their child regularly. Caregivers demonstrated healthy self-management strategies such as awareness on medications and access methods to healthcare during lockdown and remained confident of accessability to services. Stress was experienced in < 5%. CONCLUSION: Lockdown status for COVID-19 did not significantly affect the control of epilepsy in children though it posed difficulties for regular reviews and obtaining medications. Self-management strategies will help caregivers to adopt to new-normal status and potential future outbreaks.

Protocol for the Sri Lankan Cerebral Palsy Register pilot study.

INTRODUCTION: Cerebral palsy (CP) describes a heterogeneous group of motor disorders resulting from disturbance in the developing brain. CP occurs in approximately 2.1 per 1000 live births in high-income countries, but in low- and middle-income countries (LMICs) the prevalence and severity of CP may be greater and aetiological risk factors different. In Sri Lanka, a LMIC, there have been no epidemiological studies of CP to date. Systematically collected data are required to identify opportunities for primary and secondary prevention, to plan and establish services to support children and adults with CP and their families and to act as a sampling frame for new research. Here we describe a pilot study protocol for a CP register in Sri Lanka. METHODS AND ANALYSIS: The aim of this study is to establish a CP register in Sri Lanka. We will use different surveillance methodologies in two provinces of Sri Lanka: hospital and community surveillance in the Western Province and community surveillance in the Eastern Province. A common record form will collect demographic, clinical and service data for children with CP <18 years living in these two provinces. Data will be transferred to a secure online data repository and used to describe the epidemiology of CP in these regions. We will describe the strengths and challenges of the surveillance mechanisms and estimate the resources required for ongoing hospital and community based surveillance in the Western and Eastern provinces and to include additional provinces across the country. ETHICS AND DISSEMINATION: This study has ethical clearance from The University of Kelaniya, National Health Research Council, the Institutional Ethics Review Committee of the Lady Ridgeway Hospital, Colombo South Teaching Hospital and the Director of the North Colombo Teaching Hospital. Results from this research will be disseminated through local and international conferences and through publications in peer-reviewed journals.

Uric acid, an important screening tool to detect inborn errors of metabolism: a case series.

BACKGROUND: Uric acid is the metabolic end product of purine metabolism in humans. Altered serum and urine uric acid level (both above and below the reference ranges) is an indispensable marker in detecting rare inborn errors of metabolism. We describe different case scenarios of 4 Sri Lankan patients related to abnormal uric acid levels in blood and urine. CASE 1: A one-and-half-year-old boy was investigated for haematuria and a calculus in the bladder. Xanthine crystals were seen in microscopic examination of urine sediment. Low uric acid concentrations in serum and low urinary fractional excretion of uric acid associated with high urinary excretion of xanthine and hypoxanthine were compatible with xanthine oxidase deficiency. CASE 2: An 8-month-old boy presented with intractable seizures, feeding difficulties, screaming episodes, microcephaly, facial dysmorphism and severe neuro developmental delay. Low uric acid level in serum, low fractional excretion of uric acid and radiological findings were consistent with possible molybdenum cofactor deficiency. Diagnosis was confirmed by elevated levels of xanthine, hypoxanthine and sulfocysteine levels in urine. CASE 3: A 3-year-10-month-old boy presented with global developmental delay, failure to thrive, dystonia and self-destructive behaviour. High uric acid levels in serum, increased fractional excretion of uric acid and absent hypoxanthine-guanine phosphoribosyltransferase enzyme level confirmed the diagnosis of Lesch-Nyhan syndrome. CASE 4: A 9-year-old boy was investigated for lower abdominal pain, gross haematuria and right renal calculus. Low uric acid level in serum and increased fractional excretion of uric acid pointed towards hereditary renal hypouricaemia which was confirmed by genetic studies. CONCLUSION: Abnormal uric acid level in blood and urine is a valuable tool in screening for clinical conditions related to derangement of the nucleic acid metabolic pathway.

Viral aetiologies of acute encephalitis in a hospital-based South Asian population.

BACKGROUND: The aetiological spectrum of acute encephalitis shows inter- and intra-geographical variations. We aimed to identify the viruses that cause infectious encephalitis in Sri Lanka, which represents a South Asian population. METHODS: A cross-sectional study was conducted among 99 patients with encephalitis/meningoencephalitis admitted to two tertiary-care hospitals in Colombo. Cerebrospinal fluid and serum were tested for conventional and emerging encephalitogenic viruses. Specific nucleic acid amplification and antibody assays were used to identify viruses. Plaque reduction neutralization test was done to confirm the diagnosis of West Nile virus (WNV). RESULTS: Patients' age ranged from 1 month to 73 years (mean = 24.91; SD = 21.33) with a male:female ratio of 1.75:1. A viral aetiology was identified in only 27.3%. These included dengue virus (40.7%), Japanese encephalitis virus (25.9%), varicella zoster virus, WNV and probable Epstein Barr virus (11.1% each). None were positive for herpes simplex viruses or cytomegalovirus. Screening for bacterial aetiologies was negative for all patients. There were no distinguishable clinical or laboratory findings between the different viral aetiologies. The case fatality rate was 7%, which was higher among patients with an identified viral aetiology. CONCLUSIONS: A viral aetiology was identified in only about a quarter of patients with encephalitis. Dengue virus accounted for the majority.