Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

KCTD7-related progressive myoclonic epilepsy: Report of 42 cases and review of literature.

OBJECTIVE: KCTD7-related progressive myoclonic epilepsy (PME) is a rare autosomal-recessive disorder. This study aimed to describe the clinical details and genetic variants in a large international cohort. METHODS: Families with molecularly confirmed diagnoses of KCTD7-related PME were identified through international collaboration. Furthermore, a systematic review was done to identify previously reported cases. Salient demographic, epilepsy, treatment, genetic testing, electroencephalographic (EEG), and imaging-related variables were collected and summarized. RESULTS: Forty-two patients (36 families) were included. The median age at first seizure was 14 months (interquartile range = 11.75-22.5). Myoclonic seizures were frequently the first seizure type noted (n = 18, 43.9%). EEG and brain magnetic resonance imaging findings were variable. Many patients exhibited delayed development with subsequent progressive regression (n = 16, 38.1%). Twenty-one cases with genetic testing available (55%) had previously reported variants in KCTD7, and 17 cases (45%) had novel variants in KCTD7 gene. Six patients died in the cohort (age range = 1.5-21 years). The systematic review identified 23 eligible studies and further identified 59 previously reported cases of KCTD7-related disorders from the literature. The phenotype for the majority of the reported cases was consistent with a PME (n = 52, 88%). Other reported phenotypes in the literature included opsoclonus myoclonus ataxia syndrome (n = 2), myoclonus dystonia (n = 2), and neuronal ceroid lipofuscinosis (n = 3). Eight published cases died over time (14%, age range = 3-18 years). SIGNIFICANCE: This study cohort and systematic review consolidated the phenotypic spectrum and natural history of KCTD7-related disorders. Early onset drug-resistant epilepsy, relentless neuroregression, and severe neurological sequalae were common. Better understanding of the natural history may help future clinical trials.

Cerebral visual impairment and effect of phase-wise stimulation strategies-An interim analysis.

Context: Cerebral visual impairment (CVI) is an overarching term, defined as a brain-based visual impairment with onset in childhood, unexplained by an ocular disorder and associated with unique visual and behavioral characteristics. Good vision and awareness of visual function in a child are highly essential as neuroplasticity is maximum in the first three years of life and response to intervention is utmost in this period. Awareness is lacking regarding CVI, and the diagnosis is largely missed. This can be easily addressed if a structured approach is employed. Purpose: This study aims to evaluate the etiology and radiological correlation with the severity of CVI and outcome after structured intervention in children with CVI. Settings and Design: Prospective-interventional study. Methods and Material: Children attending the Child Development Centre (CDC) of a tertiary care hospital in North Karnataka and diagnosed with CVI in the age group of six months to 12 years and meeting the sampling criteria were screened and enrolled consecutively after obtaining parental consent/assent. Statistical analysis used is nonparametric test with SPSS software. Results: Age showed a significant association with the phase of CVI. Perinatal insult was associated significantly with the severity of CVI. Magnetic resonance imaging (MRI) findings did not hamper the recovery of CVI. Conclusions: Enrolment in early intervention programs tailored according to child's specific needs should be encouraged, with stress on ophthalmic screening of preterm and high-risk babies with perinatal hypoxia and history of convulsions, as early as six months.

ARV1 Gene: A Novel Cause of Autosomal Recessive Cerebellar Ataxia with Elevated Alpha Fetoprotein.

ARV1 mutation is known to present as developmental and epileptic encephalopathy (DEE)-38. However, the phenotypic spectrum has been expanding ever since it was reported in 2016. Along with seizures and developmental delay, other unique clinical features include ophthalmological abnormalities and movement disorders in the form of ataxia and dystonia, especially in those with missense mutation. These manifestations closely mimic ataxia telangiectasia. Elevation of alpha-fetoprotein levels is an important investigative marker in the diagnosis of ataxia telangiectasia and ataxia with oculomotor apraxia syndromes. ARV1 can also be associated with increased alpha-fetoprotein. There are no reports evaluating alpha-fetoprotein levels in cases with ARV1 mutation, which is significant in the context of ocular abnormalities with ataxia. We report a case of ARV1 mutation presenting with ataxia, ocular abnormalities, and elevated alpha-fetoprotein levels, thus mimicking autosomal recessive cerebellar ataxias. This study provides a comprehensive literature review of the cases reported so far, thus expanding the understanding of the spectrum of presentation, and helps in correlating the clinical picture with the underlying causative genetic mutation. ARV1 gene is another example of one gene with phenotypic pleiotropy. Though presentation with DEE is common, a few, especially those with missense mutations, can present with ataxia and ocular abnormalities. All cases presenting with ataxia who have increased alpha-fetoprotein levels and seizures should be tested for the ARV1 gene, when testing for ataxia genes is negative. The underlying genetic mechanism can explain the varying clinical manifestations of the ARV1 gene.

Clinical Profile and Treatment Outcomes of Hypermanganesemia with Dystonia 1 and 2 among 27 Indian Children.

Background: Hypermanganesemia with dystonia 1 and 2 (HMNDYT1 and 2) are rare, inherited disorders of manganese transport. Objectives: We aimed to describe clinical, laboratory features, and outcomes among children with HMNDYT. Methods: We conducted a retrospective multicenter study involving tertiary centers across India. We enrolled children between 1 month to 18 years of age with genetically confirmed/clinically probable HMNDYT. Clinical, laboratory profile, genetic testing, treatment details, and outcomes scored by treating physicians on a Likert scale were recorded. Results: We enrolled 27 children (19 girls). Fourteen harbored SLC30A10 mutations; nine had SLC39A14 mutations. The SLC39A14 cohort had lower median age at onset (1.3 [interquartile range (IQR), 0.7-5.5] years) versus SLC30A10 cohort (2.0 [IQR, 1.5-5.1] years). The most frequent neurological features were dystonia (100%; n = 27), gait abnormality (77.7%; n = 21), falls (66.7%; n = 18), and parkinsonism (59.3%; n = 16). Median serum manganese (Mn) levels among SLC39A14 (44.9 [IQR, 27.3-147.7] mcg/L) cohort were higher than SLC30A10 (29.4 [17.1-42.0] mcg/L); median hemoglobin was higher in SLC30A10 (16.3 [IQR, 15.2-17.5] g/dL) versus SLC39A14 cohort (12.5 [8.8-13.2] g/dL). Hepatic involvement and polycythaemia were observed exclusively in SLC30A10 variants. A total of 26/27 children underwent chelation with disodium calcium edetate. Nine demonstrated some improvement, three stabilized, two had marked improvement, and one had normalization. Children with SLC39A14 mutations had poorer response. Two children died and nine were lost to follow-up. Conclusions: We found female predominance. Children with SLC39A14 mutations presented at younger age and responded less favorably to chelation compared to SLC30A10 mutations. There is emerging need to better define management strategies, especially in low resource settings.

Which is the Most Common Physiologic Type of Cerebral Palsy?

Introduction: Spastic cerebral palsy (CP) is the commonest physiological type according to literature which comes mainly from the developed countries where prematurity is a common cause for cerebral palsy. In developing countries like India, the leading causes of cerebral palsy are birth asphyxia, infections, and hyperbilirubinemia and, hence, the physiological type of CP is likely to be different. However, the data from our country is scant. Methods: 103 consecutive treatment-naive CP patients attending pediatric neurology clinic were evaluated in detail using an objective tool, hypertonia assessment tool (HAT) over a period of 6months. Based on the predominant tone, the cases were classified as spastic, dyskinetic, ataxic/hypotonic, and mixed. The type of cerebral palsy was correlated with perinatal details and neuroimaging findings. Results: Out of 103 children, the most common physiological type of CP seen was of dyskinetic type [54 (52.4%)], followed by spastic CP in 30 (29.1%) and mixed (dyskinetic+spastic) CP in 19 (18.4%) children. The most common cause for dyskinetic CP was perinatal asphyxia 33 (61%); for spastic CP was prematurity 17 (56.7%) and; for mixed CP, the main cause was perinatal asphyxia 12 (63.2%). The main neuroimaging finding in predominant dyskinetic CP was basal ganglia/thalamus involvement followed by pericentral and perirolandic gliosis, whereas in spastic CP, it was periventricular leucomalacia. In mixed CP, there was multicystic encephalomalacia. Conclusions: Dyskinetic CP either as predominant type or along with spasticity is the most common physiological type of CP in India and is due to birth-asphyxia, hyperbilirubinemia, hypoglycemia, and infections.

Disorders of Tetrahydrobiopterin Metabolism: Experience from South India.

BACKGROUND: Disorders of tetrahydrobiopterin metabolism represent a rare group of inherited neurotransmitter disorders that manifests mainly in infancy or childhood with developmental delay, neuroregression, epilepsy, movement disorders, and autonomic symptoms. METHODOLOGY: A retrospective review of genetically confirmed cases of disorders of tetrahydrobiopterin metabolism over a period of three years (Jan 2018 to Jan 2021) was performed across two paediatric neurology centres from South India. RESULTS: A total of nine patients(M:F=4:5) fulfilled the eligibility criteria. The genetic variants detected include homozygous mutations in the QDPR(n=6), GCH1(n=2), and PTS(n=1) genes. The median age at onset of symptoms was 6-months(range 3-78 months), while that at diagnosis was 15-months (8-120 months), resulting in a median delay in diagnosis of 9-months. The main clinical manifestations included neuroregression (89%), developmental delay(78%), dystonia(78%) and seizures(55%). Management strategies included a phenylalanine restricted diet, levodopa/carbidopa, 5-Hydroxytryphtophan, and folinic acid. Only, Patient-2 afforded and received BH4 supplementation at a sub-optimal dose later in the disease course. We had a median duration of follow up of 15 months (range 2-48 months). Though the biochemical response has been marked; except for patients with GTPCH deficiency, only mild clinical improvement was noted with regards to developmental milestones, seizures, or dystonia in others. CONCLUSION: Tetrahydrobiopterin deficiencies represent a rare yet potentially treatable cause for non-phenylketonuria hyperphenylalaninemia with better outcomes when treated early in life. Screening for disorders of biopterin metabolism in patients with hyperphenylalaninemia prevents delayed diagnosis. This study expands the genotype-phenotype spectrum of patients with disorders of tetrahydrobiopterin metabolism from South India.

Antenatal presentation and supratentorial brain abnormalities in a child with Poretti-Boltshauser syndrome.

Autosomal recessively inherited Poretti-Boltshauser syndrome (PBS) with loss-of-function variants in the LAMA1 gene are characterized by motor and speech developmental delay, high myopia, and cerebellar dysplasia with cysts without any supratentorial abnormalities on neuroimaging. There is no muscular involvement. We report an eight months child with genetically confirmed PBS who presented with antenatally detected ventriculomegaly and had global developmental delay, focal seizures, myopic degeneration of fundi. Neuroimaging showed asymmetric ventriculomegaly and lissencephaly in bilateral temporal horns along with cerebellar dysplasia and cysts. These supratentorial abnormalities and antenatal presentation as ventriculomegaly have not been reported earlier. Child also had a small subaortic ventricular septal defect.

Clinical Characteristics, Molecular Profile, and Outcomes in Indian Patients with Glutaric Aciduria Type 1.

Glutaric acidemia type 1 (GA-1, OMIM 231670) is an autosomal recessive inborn error of metabolism caused by the deficiency of glutaryl-coenzyme A (CoA) dehydrogenase with most children presenting in infancy with encephalopathy, dystonia, and macrocephaly. In this article, we presented the clinical characteristics, molecular profile, and outcomes in 29 unrelated families with affected children (30 cases total). The mean age at onset of illness was 10 months (±14.58), whereas the mean age at referral for molecular diagnosis was 29.44 months (±28.11). Patients were residents of nine different states of India. Clinical presentation varied from acute encephalitis followed by neuroregression and chronic/insidious developmental delay. Neurological sequelae varied from asymptomatic (no sequelae, 2 patients) to moderate (5 patients) and severe (23 patients) sequelae. All patients underwent blood tandem mass spectrometry (TMS on dried blood spots) and/or urine gas chromatography mass spectrometry (GCMS). Neuroimaging demonstrated batwing appearance in 95% cases. Sanger's sequencing of GCDH , covering all exons and exon-intron boundaries, was performed for all patients. Variants identified include 15 novel coding variants: p.Met100Thr, p.Gly107Ser, p.Leu179Val, p.Pro217Ser, p. Phe236Leufs*107, p.Ser255Pro, p.Met266Leufs*2, p.Gln330Ter, p.Thr344Ile, p.Leu345Pro, p.Lys377Arg, p.Leu424Pro, p.Asn373Lys, p.Lys377Arg, p.Asn392Metfs*9, and nine known genetic variants such as p.Arg128Gln, p.Leu179Arg, p.Trp225Ter, p.Met339Val, p.Gly354Ser, p.Arg402Gln, p.Arg402Trp, p.His403Tyr, and p.Ala433Val (Ensembl transcript ID: ENST00000222214). Using in silico analysis, genetic variants were shown to be affecting the residues responsible for homotetramer formation of the glutaryl-CoA dehydrogenase protein. Treatment included oral carnitine, riboflavin, protein-restricted diet, lysine-deficient special formulae, and management of acute crises with intravenous glucose and hydration. However, the mortality (9/30, 27.58%) and morbidity was high in our cohort with only two patients affording the diet. Our study is the largest multicentric, genetic variant-proven series of glutaric aciduria type 1 from India till date.

Management of Neurocysticercosis in Children: Association of Child Neurology Consensus Guidelines.

JUSTIFICATION: Neurocysticercosis (NCC) is a significant problem in India and other developing countries; however, several aspects of this disease have no clear, practical guidelines. There is a need for pragmatic guidelines, summarizing the available evidence, and filling in the gaps in evidence with expert advice to manage children with neurocysticercosis. PROCESS: An expert group (16 members) and a writing group (8 members) was constituted, consisting of members with varied expertise. It included pediatric neurologists (18), neurologist (1), Neuroradiologists (4), and a parasitologist (1). The writing group divided the six topics and reviewed the literature on the topics individually to determine the clinical questions for which no clear guidance was available from the literature. The experts were then contacted and opinions were obtained online. The Delphi consensus method was adopted to arrive at a general consensus regarding various questions, with both the experts and the writing group members contributing. The final guidelines were then drafted by the writing group. RECOMMENDATIONS: Diagnosis of NCC should be based on clinical history and neuroimaging. Contrast-enhanced magnetic resonance imaging of the brain is the modality of choice. For single enhancing lesion, albendazole therapy for 10-14 days is recommended, and it should be combined with praziquantel for 10-14 days for more than one ring-enhancing lesions. For persistent lesion, the same dose and duration of albendazole or concurrent administration of albendazole and praziquantel should be given. Pulse intravenous steroids should be used to reduce the acute symptomatic edema in children with cysticercal encephalitis. Carbamazepine or oxcarbazepine are best suited for seizure prophylaxis for those who present with seizures; phenytoin and levetiracetam are the other alternatives. In the case of NCC presenting with symptoms other than seizures, there appears to be no role for routine anti-seizure medication prophylaxis. For a single ring-enhancing lesion, six months of anti-seizure medication is sufficient if the lesion resolves on follow-up. Those with persistent lesions, calcification, or multiple lesions, require a longer treatment duration of at least 24 months.

Neuronal Ceroid Lipofuscinoses in Children.

BACKGROUND: The neuronal ceroid lipofuscinoses (NCL) constitute a group of gray matter neurodegenerative disorders characterized by the accumulation of ceroid lipopigment in lysosomes in neurons and other cell types. There are very few published studies on NCL from India, especially in children. METHODS: A retrospective study of confirmed patients of NCL diagnosed over a period of 10 years from January 2019 to December 2019. RESULTS: Fifty children had a definitive diagnosis of NCL based on enzymatic studies or genetic testing using next-generation sequencing. Around 15 children were diagnosed to have CLN-1 (ceroid lipofuscinoses, neuronal-1) based on palmitoyl protein thioesterase-1 deficiency; 24 children were diagnosed with CLN2 (ceroid lipofuscinoses, neuronal-2) based on deficient tripeptidyl-peptidase-1 activity; three patients were diagnosed as CLN6, five patients as CLN7, one case each of CLN8, CLN11, and CLN14 based on genetic testing. Clinical presentation was quite varied and included refractory seizures, developmental delay/regression, and abnormal movements. Visual failure was not common in the present case series. Neuroimaging patterns in different types of NCL were different. All children had a progressive downhill course resulting in death in many over a period of 5-10 years of disease onset. CONCLUSION: NCL is not uncommon and diagnosis can be suspected based on clinical investigations and neuroimaging findings. Diagnosis can be confirmed by enzymatic assays or genetic testing.