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.

Gaucher disease: single gene molecular characterization of one-hundred Indian patients reveals novel variants and the most prevalent mutation.

BACKGROUND: Gaucher disease is a rare pan-ethnic, lysosomal storage disorder resulting due to beta-Glucosidase (GBA1) gene defect. This leads to the glucocerebrosidase enzyme deficiency and an increased accumulation of undegraded glycolipid glucocerebroside inside the cells' lysosomes. To date, nearly 460 mutations have been described in the GBA1 gene. With the aim to determine mutations spectrum and molecular pathology of Gaucher disease in India, the present study investigated one hundred unrelated patients (age range: 1 day to 31 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality in some of the patients. METHODS: The biochemical investigation for the plasma chitotriosidase enzyme activity and ß-Glucosidase enzyme activity confirmed the Gaucher disease. The mutations were identified by screening the patients' whole GBA gene coding region using bidirectional Sanger sequencing. RESULTS: The biochemical analysis revealed a significant reduction in the ß-Glucosidase activity in all patients. Sanger sequencing established 71 patients with homozygous mutation and 22 patients with compound heterozygous mutation in GBA1 gene. Lack of identification of mutations in three patients suggests the possibility of either large deletion/duplication or deep intronic variations in the GBA1 gene. In four cases, where the proband died due to confirmed Gaucher disease, the parents were found to be a carrier. Overall, the study identified 33 mutations in 100 patients that also covers four missense mutations (p.Ser136Leu, p.Leu279Val, p.Gly383Asp, p.Gly399Arg) not previously reported in Gaucher disease patients. The mutation p.Leu483Pro was identified as the most commonly occurring Gaucher disease mutation in the study (62% patients). The second common mutations identified were p.Arg535Cys (7% patients) and RecNcil (7% patients). Another complex mutation Complex C was identified in a compound heterozygous status (3% patients). The homology modeling of the novel mutations suggested the destabilization of the GBA protein structure due to conformational changes. CONCLUSIONS: The study reports four novel and 29 known mutations identified in the GBA1 gene in one-hundred Gaucher patients. The given study establishes p.Leu483Pro as the most prevalent mutation in the Indian patients with type 1 Gaucher disease that provide new insight into the molecular basis of Gaucher Disease in India.

Rare cause of Hemophagocytic Lymphohistiocytosis due to mutation in PRF1 and SH2D1A genes in two children - a case report with a review.

BACKGROUND: Hemophagocytic Lymphohistiocytosis (HLH) is a rare, complex, life-threatening hyper-inflammatory condition due to over activation of lymphocytes mediated secretory cytokines in the body. It occurs as a primary HLH due to genetic defect that mostly occurs in the childhood and associated with early neonatal death. Secondary HLH is triggered by secondary to infection and can occur at any age. CASE PRESENTATION: The current report presents two cases of HLH. Case 1, three-months-old boy born to second degree consanguineous parents was clinically suspected with HLH. A pathogenic variant in exon 2 of PRF1 gene [c.386G > C (p.Trp129Ser); FLH-type2] was detected. The parents and the fetus under investigation were shown to be heterozygous carriers, while Case-1 was homozygous for the said variant. Case 2, a one and half-year old male child referred for work-up was born to non-consanguineous young parents. His HLH suspicion was in accordance with HLH-2004 Revised diagnostic guidelines (fulfilling 5/8 criteria). Molecular study revealed hemizygous likely pathogenic variant c.138-3C > G in intron 1 of SH2D1A gene. Both the mother and younger sister were confirmed to be the carrier of the same variant. CONCLUSION: This study has represented two rare cases of HLH carrying missense variant in PRF1 and splice site variant in SH2D1A gene. Detailed molecular analysis has helped the families with precise genetic counselling and prenatal diagnosis during subsequent pregnancy. It is advocated that male patients presenting with EBV-associated HLH may be screened for XLP that may lead to early diagnosis and therapeutic implication if any.

Batten disease: biochemical and molecular characterization revealing novel PPT1 and TPP1 gene mutations in Indian patients.

BACKGROUND: Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy. METHODS: The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants. RESULTS: Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal. CONCLUSION: The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

Worm infestation as a cause of severe anemia in a frail older adult: a case report.

Introduction: Worm infestations are a common occurrence in low-income countries. Anemia due to iron deficiency can be brought on by human intestinal worms. The authors report a case of an 86-year-old frail older adult with upper gastrointestinal (GI) bleeding caused by a worm infestation most likely to be hookworm. Case presentation: An 86-year-old male, presented to the Emergency Department with complaints of bilateral lower limb swelling and shortness of breath for 4 days associated with melena for 2 months. The authors made a provisional diagnosis of heart failure precipitated by anemia. Upper GI endoscopy revealed multiple whitish exudates, which are resistant to water jets. Multiple worms were noted in the second part of the duodenum. Based on clinical evaluation and endoscopy, the diagnosis of oesophagial candidiasis and iron deficiency anemia secondary to upper GI bleeding due to Hookworm infestation was made. Clinical discussion: In low-income countries, especially those involving the tropical area, worm infestation should be considered as an important cause of obscure acute GI bleeding and severe anemia. Usually, malignancy is suspected in an older adult with severe anemia but hookworm infestation is a treatable disease with a good prognosis and complete recovery. The most commonly used drugs for treatment are mebendazole and albendazole. In a low-income country with a high burden of worm infestations, empirical treatment of iron deficiency anemia with single dose albendazole has been recommended. Conclusion: Usually, severe anemia in an older adult is mostly attributed to an underlying malignancy. Our case serves as a good example of how a treatable condition can improve the quality of life in a frail older adult. Normally, there is a tendency to defer UGI endoscopy in frail elderly due to ageism. However, the diagnosis of a treatable cause of upper GI bleeding can be made by a simple upper GI endoscopy. Severe anemia due to hookworm infestation is treated effectively and quickly with albendazole and iron therapy.

Transformation of HHV-8-negative idiopathic multicentric Castleman disease into diffuse large B-cell lymphoma: A case report from Nepal.

Key Clinical Message: Idiopathic Castleman disease transforming into Diffuse Large B-cell Lymphoma has an aggressive course and can lead to mortality. Hence, early diagnosis and intervention are required. Abstract: Idiopathic Castleman disease transforming into non-Hodgkin lymphoma has an aggressive course, poor prognosis, and high mortality rate. Hence, early diagnosis and intervention are necessary. In a developing country like Nepal, where infectious diseases, particularly TB, are high, concomitant infection worsens the disease course. It also poses a diagnostic challenge as the clinical presentation may be similar.

A comparison of immunogenicity and safety of indigenously developed liquid (DTwPHB-Hib) pentavalent combination vaccine (Shan 5) with Easyfive (liq) and TritanrixHB + Hiberix (lyo) in Indian infants administered according to the EPI schedule.

The study was planned to assess and compare the immune response and safety of an indigenous DTPwHB-Hib pentavalent liquid combination vaccine (Shan 5) with Easyfive and TritanrixHB+ Hiberix, the two available pentavalent combination vaccines. Four hundred infants were randomized to receive three doses of either Shan 5 or one of the two comparators. Antibody analysis was performed prior to and four to six weeks post third vaccine dose. Solicited local and systemic events upto three days and unsolicited adverse events in the 30 days follow up period after each dose were recorded. A total of 365 subjects completed the study. Four to six weeks after third dose, 98.32% of the subjects in Shan 5 group had seroprotective Anti PRP-T IgG antibody concentrations (> or =0.15 microg/mL) as compared to 100% and 98.94% subjects in TritanrixHB + Hiberix and Easyfive groups respectively. Seroprotective levels for Anti-HBs (> or =10 mIU/mL) were observed in 97.77%, 97.83% and 98.94% subjects in Shan 5, TritanrixHB + Hiberix and Easyfive groups respectively. Comparable immune responses were observed for the three other components (D, T and P) in all the groups. Four Serious Adverse Events (SAEs) were reported (three with Shan 5 and one with Easyfive), all unrelated to the respective vaccines. Most commonly reported adverse events in all the groups were pain at injection site, mild fever (<103 degrees F) and minor swelling at injection site. The study proved that Shan 5 was safe and immunogenic compared to the two other licensed vaccines.

Immunogenicity and safety of an indigenously developed DTPw hepatitis B combination vaccine in Indian infants.

OBJECTIVE: To evaluate the safety and immunogenicity of an indigenous hepatitis B, diphtheria, tetanus and B. pertussis tetravalent vaccine (Shantetra) in comparison with Tritanrix HBTM in healthy Indian infants. DESIGN: Multicentric, randomized, single blind intention-to-treat study with 12-18 weeks of follow up period. SETTING: 5 out patient departments at tertiary care referral centers across India. PARTICIPANTS: 151 infants were randomized in a 2:1 ratio to recruit 101 in the Shantetra and 50 in the Tritanrix HBTM groups respectively. A total of 136 subjects completed the study. No patients were withdrawn from the study due to any adverse effects. INTERVENTIONS: Recruited subjects were randomized to receive three doses of either of the two DTPw-Hepatitis B combination vaccines as per the EPI schedule. MAIN OUTCOME MEASURES: Monitoring the humoral immune response (seroconversion rates) induced by each antigenic component three to six weeks after the last dose of vaccine in both the groups. RESULTS: Seroprotective immune response was observed in 98.9% subjects for diphtheria, tetanus and hepatitis B components in the Shantetra group as compared to 95.5% subjects in the Tritanrix HB group. Anti pertussis antibody response was seen in 89% and 91.1% in the Shantetra and Tritanrix HB groups, respectively. The commonly observed adverse events in both the groups were, pain at injection site, mild fever and transient crying. CONCLUSION: The safety and immunogenicity of indigenously developed DTPwHepatitis B combination vaccine was demonstrated in the present study.

Vaccinations in Primary Care.

Vaccination is amongst the best strategies to improve child survival and reduce morbidity. Vaccines represent the most cost effective and simple intervention to protect against distressing epidemics. There are mortality and morbidity related benefits derived from preventing infectious diseases through vaccination; these include financial benefits by avoiding hospitalization, preventing long-term disability and increased productivity. Ever since the invention of the first vaccine against smallpox by Edward Jenner in 1796, vaccination has become indispensable healthcare intervention and has saved millions of lives. Due to significant scientific progress, many vaccines are available and numerous are anticipated; however, vaccine preventable infectious diseases are still prevalent. Due to rapid pace of developments in the field of vaccination, providers must continue to update their knowledge. The present review is aimed at helping general practitioners understand routine vaccinations, their considerations, issues and side effects.

Molecular characterization and evaluation of complex rearrangements in a case of ring chromosome 15.

BACKGROUND: Ring chromosome 15 is a rare genetic entity. Only a few cases have been reported with characterization using molecular techniques. The clinical presentation is quite variable, as a result of differences in the breakpoints, haploinsufficiency of genes involved in deleted segment/s, level of mosaicism and ring instability resulting in a variability of rearrangement of genetic material. CASE PRESENTATION: The proband, a 2 months old boy, presented with small head size and facial dysmorphism. On examination microcephaly, triangular face, small anterior frontanelle, micrognathia, hypotonia, unilateral simian crease, hypertelorism, umbilical hernia, micropenis with mild phimosis were noted. Karyotype revealed 46,XY,r(15)(p11.2q26). Array-comparative genomic hybridization (aCGH) and targeted gene sequencing for microcephaly was carried out for genotype phenotype correlation. Array-CGH detected a 2.8 Mb terminal deletion at 15q26.3 along with a 496 kb interstitial micro-duplication, encompassing the IGF1R gene, in the affected genomic region, which was otherwise missed on conventional karyotype. CONCLUSION: The present study highlights the importance of aCGH in not only delineating specific phenotypes through accurate genotypic correlation but also in detection and evaluation of ring chromosome with unexpected complex rearrangements.

Isotopic correlation for 242Pu composition prediction: Multivariate regresssion approach.

Multivariate regression calibration using multiple linear regression (MLR), principle component regression (PCR) and partial least squares regression (PLSR) algorithm was performed on 238Pu, 239Pu, 240Pu and 241Pu atom% abundances to predict 242Pu isotopic abundance. The MLR algorithm was found to be the best among these three algorithms. The effect of 238Pu composition on the 242Pu abundance prediction was found to be small but significant especially for achieving high accuracy of <0.5%. PCR and PLSR generated nearly identical results and were inferior to the MLR results. A comparison of MLR results with those obtained by employing seven previously reported empirical methods revealed far superior prediction capability of MLR model. Among the seven empirical models, the best prediction capability was found for Bignan correlation containing 238Pu isotopic data. The study clearly demonstrates that the production of 238Pu and 242Pu has some small correlation and the use of 238Pu in isotopic correlation for 242Pu prediction is important to get accurate results.

Safety and immunogenicity of single dose live attenuated varicella vaccine (VR 795 Oka strain) in healthy Indian children: a randomized controlled study.

Varicella, an acute viral systemic infection that may cause lifelong latent infection with the potential for causing clinical reactivation, may be prevented by immunization. The present study was an open label, randomized, controlled, phase III, multicentre trial, conducted to evaluate and compare the safety, tolerability and immunogenicity of a freeze dried live attenuated Oka strain Varicella Vaccine (VR 795 Oka strain) with Varilrix (Oka-RIT strain) in children. A total of 268 healthy Indian children aged 12 months to 12 y with baseline VZV IgG antibody (<100 mIU/ mL) were enrolled, and 256 children completed the study. The extent of rise of VZV IgG antibody titer assessed as 3-fold and 4-fold rise from baseline was found to be significantly higher (89.1% and 85.2%) in the test group as compared to control group (73.4% and 61.7%). The post-vaccination GMT of the test group was significantly higher (112.5 mIU/mL) as compared with the control group (67.8 mIU/mL) (P < 0.001). The seroconversion rate considering the 5 gp ELISA units/ml equivalent to 10mIU/ml were similar in the control (96.5%) and the test (98.3%) groups. The adverse events were not different in the control and test groups (P > 0.05). The test live attenuated vaccine was found to be highly immunogenic, safe and comparable to Varilrix used in control arm.

Pertussis vaccine controversies and acellular pertussis vaccine.

Pertussis still continues to cause significant morbidity and mortality worldwide. Because of the high reactogenicity of whole cell pertussis vaccine, it had evoked public controversy in several countries. In 1970 Japan abandoned use of whole cell pertussis vaccine and mounted efforts to develop better vaccine. To date, nearly 24 acellular pertussis vaccines have been developed, using different number and quantity of components. No acellular vaccine is most or least immunogenic with respect to all included antigens. Vaccine efficacy and duration of immunity is comparable with whole cell pertussis vaccine. The adverse events are two thirds less compared to whole cell vaccine.

Lysosomal storage disorders.

This study was conducted to assess the variability of clinical expression of Lysosomal storage disorders (LSDs) and the selection of specific enzyme investigation to reach the differential diagnosis. Initially 150 children in the age range of 15 days to 13 years were screened for common metabolic disorder and based on screening results, clinical signs and symptoms, 30 children(4 mo-12 yr) of these were selected for the leukocyte enzyme study. Of these 21 were confirmed to have LSDs. The most common disorder was GM2-gangliosidosis (47.61%, 10/21) followed by mucopolysaccharidosis (33.33%; 7/21). All showed variable phenotypic expression. Metachromatic leukodystrophy (MLD) was observed in 9.5% (2/21) of children with arylsulphatase A enzyme deficiency, while two children had shown pseudodeficiency of arylsulphatase A. One case each of galactosialidosis and GMI-gangliosidosis were observed. We conclude that children with developmental delay, seizures, dysmorphic features and organomegaly, with or without positive urinary screening for common metabolic disorders, need to be investigated further for LSDs. Variability of clinical expression is commonly observed in LSDs which require further confirmation by specific leukocyte enzyme study.

Thoracoscopy in management of empyema thoracis in children.

The usual treatment for empyema in children varies from a simple thoracocentesis to thoracotomy and open decortication. We studied the role of thoracoscopy in the management of empyema thoracis in 10 immunocompetent children after failure of medical management. All children recovered well with an early removal of intercostal tube and reduced postoperative hospital stay and showed complete resolution of empyema on follow up. Thoracoscopy has come as a new ray of hope for the patients with empyema, with the advantages of complete evacuation, minimal pulmonary dysfunction, reduced pain and hospital stay.

Burden of lysosomal storage disorders in India: experience of 387 affected children from a single diagnostic facility.

Lysosomal storage disorders (LSDs) are considered to be a rare metabolic disease for the national health forum, clinicians, and scientists. This study aimed to know the prevalence of different LSDs, their geographical variation, and burden on the society. It included 1,110 children from January 2002 to December 2012, having coarse facial features, hepatomegaly or hepatosplenomegaly, skeletal dysplasia, neuroregression, leukodystrophy, developmental delay, cerebral-cerebellar atrophy, and abnormal ophthalmic findings. All subjects were screened for I-cell disease, glycolipid storage disorders (Niemann-Pick disease A/B, Gaucher), and mucopolysaccharide disorders followed by confirmatory lysosomal enzymes study from leucocytes and/or fibroblasts. Niemann-Pick disease-C (NPC) was confirmed by fibroblasts study using filipin stain. Various storage disorders were detected in 387 children (34.8 %) with highest prevalence of glycolipid storage disorders in 48 %, followed by mucopolysaccharide disorders in 22 % and defective sulfatide degradation in 14 % of the children. Less common defects were glycogen degradation defect and protein degradation defect in 5 % each, lysosomal trafficking protein defect in 4 %, and transport defect in 3 % of the patients. This study demonstrates higher incidence of Gaucher disease (16 %) followed by GM2 gangliosidosis that includes Tay-Sachs disease (10 %) and Sandhoff disease (7.8 %) and mucopolysaccharide disorders among all LSDs. Nearly 30 % of the affected children were born to consanguineous parents and this was higher (72 %) in children with Batten disease. Our study also demonstrates two common mutations c.1277_1278insTATC in 14.28 % (4/28) and c.964G>T (p.D322Y) in 10.7 % (3/28) for Tay-Sachs disease in addition to the earlier reported c.1385A>T (p.E462V) mutation in 21.42 % (6/28).