Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort.

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340 nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6-3.9 and 7-20 nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9 months when stored at -80 °C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Analytical Method Validation for Estimation of Neurotransmitters (Biogenic Monoamines) from Cerebrospinal Fluid Using High Performance Liquid Chromatography.

Biogenic amine neurotransmitters such as serotonin and dopamine are essential for signaling in both central and peripheral nervous system. Their metabolism is a multistep pathway and any defect in this results in alteration in metabolites of serotonin 5-Hydroxyindole acetic acid (5HIAA) and dopamine homovanillic acid (HVA) and 3-O-Methyl Dopa (3-OMD). Estimation of these metabolites in cerebrospinal fluid (CSF) assists in diagnosis of neurotransmitter defects. Their estimation is technically demanding and is currently available only in referral centers. We aimed to optimize a method for analysis of 5HIAA, HVA and 3-OMD. A high performance liquid chromatography (HPLC) method with electro chemical detector (ECD) was standardized for estimation. Analysis for method validation, reference range verification and clinical correlation was performed. Linearity obtained for 5-HIAA, HVA and 3-OMD was 65.35-2615.0 nmoles/l, 68.62-2745.0 nmoles/l and 236.5-4730.0 nmoles/l respectively. The coefficient of variation for internal quality controls ranged from 5 to 14% and the external proficiency testing samples (n = 16) were within peer group range. CSF metabolite levels of samples for reference range analysis overlapped with age matched ranges reported in literature. Among the 40 suspected patients analyzed for clinical testing four were found to have a neurotransmitter defect. These patients were then confirmed with molecular testing and clinical correlation. The method is validated and can be adapted in a clinical laboratory with analytical competence in HPLC.

Cerebrospinal Fluid Biogenic Monoamine Analysis for Diagnosis of Primary Neurotransmitter Disorders.

Biogenic amine neurotransmitters metabolism is a multistep pathway with pterin and pyridoxal phosphate (vitamin B6) as cofactors. A defect in biogenic amine and cofactor metabolism and vesicular transporters result in a primary neurotransmitter disorders. These are a well-recognized groups of inherited disorders and often present with features overlapping with other neurological conditions. Their diagnosis is made by analysis of biogenic amine metabolites in cerebrospinal fluid (CSF) and other body fluids and respective enzyme assays. Many of these disorders are treatable and deficits can be reverted by timely intervention. CSF biogenic amine or cofactor metabolite analysis is one of the primary indicators of a neurotransmitter disorder. In this paper, 3 cases are reported-2 of cofactor deficiency and 1 with enzyme deficiency wherein biogenic amine estimation has assisted in diagnosis.

Paroxysmal Nonepileptic Events in a Pediatric Epilepsy Clinic.

AIMS: We aimed to study the frequency, age, and gender distribution of paroxysmal nonepileptic events (PNEs) in children referred to epilepsy clinic with the diagnosis of epilepsy. We also evaluated the therapeutic implications of correct diagnosis and co-existence of true epilepsy in this population. SETTINGS AND DESIGN: All new patients below 18 years attending the Pediatric epilepsy out-patient clinic of PD Hinduja hospital over 6 months were evaluated. MATERIALS AND METHODS: Patients with history of paroxysmal events characterized by abrupt changes in consciousness or behavior or movement were included. They were assessed on description of events aided by recorded videos. If the diagnosis was not confirmed by this preliminary evaluation, further investigations were advised. STATISTICAL ANALYSIS USED: Chi-square/Fisher's exact test was used to analyze differences between categorical variables and Kruskal-Wallis test between continuous variables. The data were analyzed by SAS University Edition. All significance tests were two-tailed with α <0.05. RESULTS: Two hundred new patients presenting with paroxysmal events were enrolled over 6 months. After diagnoses, 19% of these children had PNEs, 80% had epileptic events, and 1% remained undiagnosed. Common nonepileptic events seen were physiological in patients below 5 years and psychogenic in older children. Thirty-four percent of patients with PNEs were on anti-epileptic drugs (AEDs). After confirming nonepileptic attacks, only 2.6% patients needed AEDs for coexisting epilepsy which was statistically significant (P < 0.001) change in treatment. CONCLUSIONS: Epilepsy mimics are common in children and are often misdiagnosed causing undue stress. Correct diagnosis leads to a drastic change in management like withdrawal of drugs, commencing new treatment if needed, and appropriate referrals.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

BACKGROUND: Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. METHODS: The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. RESULTS: Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. CONCLUSION: The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.

Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting in progressive muscle weakness and atrophy. AIMS: The molecular analysis of two marker genes for spinal muscular atrophy (SMA) i.e, the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP) was conducted in 39 Indian patients with clinical symptoms of SMA. Out of these, 28 showed homozygous deletions and the phenotypic features of these SMA patients were compared with the corresponding genotypes. SETTINGS: A tertiary care teaching Hospital. DESIGN: This is a prospective hospital based study. MATERIALS AND METHODS: Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene. RESULTS: Exons 7 and 8 of SMN and NAIP (exon 5) were homozygously deleted in 73% of SMA I and 27% of SMA II patients. SMN exon 7 and 8 deletions without NAIP deletions were seen in 27% of type I SMA and 46% of SMA type II patients. Two patients of type III SMA showed single deletion of SMN exon 7 along with 27% of SMA type II patients. CONCLUSION: With the advent of molecular biology techniques, SMN gene deletion studies have become the first line of investigation for confirmation of a clinical diagnosis of SMA. The findings of homozygous deletions of exons 7 and/or 8 of SMN1 gene confirms the diagnosis of SMA, even in patients with atypical clinical features. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful for predicting the prognosis.

Recurrent acute transverse myelopathy: association with antiphospholipid antibody syndrome.

A seven-year-old boy presented with a second episode of acute transverse myelopathy. The first episode had responded dramatically to methylprednisolone. The manifestations of the second episode did not respond to methylprednisolone or IVIG. He showed persistently raised levels of antiphospholipid antibodies in the serum. Primary conditions like collagen vascular diseases, malignancy, exposure to drugs and HIV infection, which are known to be associated with the raised titers of these antibodies were ruled out clinically and by investigations. Recurrent transverse myelopathy is a rare event in childhood and reports of its association with Antiphospholipid Antibody Syndrome (APLAS) are scanty. The etiological role for these antibodies remains to be established. However, once the diagnosis is established, it may be prudent to treat the condition with agents and procedures to bring about a decrease in their titers. Long-term therapy to prevent thromboembolic complications of APLAS may also be instituted.