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.

Burden of rare genetic disorders in India: twenty-two years' experience of a tertiary centre.

BACKGROUND: Rare disorders comprise of ~ 7500 different conditions affecting multiple systems. Diagnosis of rare diseases is complex due to dearth of specialized medical professionals, testing labs and limited therapeutic options. There is scarcity of data on the prevalence of rare diseases in different populations. India being home to a large population comprising of 4600 population groups, of which several thousand are endogamous, is likely to have a high burden of rare diseases. The present study provides a retrospective overview of a cohort of patients with rare genetic diseases identified at a tertiary genetic test centre in India. RESULTS: Overall, 3294 patients with 305 rare diseases were identified in the present study cohort. These were categorized into 14 disease groups based on the major organ/ organ system affected. Highest number of rare diseases (D = 149/305, 48.9%) were identified in the neuromuscular and neurodevelopmental (NMND) group followed by inborn errors of metabolism (IEM) (D = 47/305; 15.4%). Majority patients in the present cohort (N = 1992, 61%) were diagnosed under IEM group, of which Gaucher disease constituted maximum cases (N = 224, 11.2%). Under the NMND group, Duchenne muscular dystrophy (N = 291/885, 32.9%), trinucleotide repeat expansion disorders (N = 242/885; 27.3%) and spinal muscular atrophy (N = 141/885, 15.9%) were the most common. Majority cases of ß-thalassemia (N = 120/149, 80.5%) and cystic fibrosis (N = 74/75, 98.7%) under the haematological and pulmonary groups were observed, respectively. Founder variants were identified for Tay-Sachs disease and mucopolysaccharidosis IVA diseases. Recurrent variants for Gaucher disease (GBA:c.1448T > C), ß-thalassemia (HBB:c.92.+5G > C), non-syndromic hearing loss (GJB2:c.71G > A), albinism (TYR:c.832 C > T), congenital adrenal hyperplasia (CYP21A2:c.29-13 C > G) and progressive pseudo rheumatoid dysplasia (CCN6:c.298T > A) were observed in the present study. CONCLUSION: The present retrospective study of rare disease patients diagnosed at a tertiary genetic test centre provides first insight into the distribution of rare genetic diseases across the country. This information will likely aid in drafting future health policies, including newborn screening programs, development of target specific panel for affordable diagnosis of rare diseases and eventually build a platform for devising novel treatment strategies for rare diseases.

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.

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.

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.

Plasma chitotriosidase activity in children with lysosomal storage disorders.

Chitotriosidase (ChT) is an enzyme that is selectively activated in tissue macrophage. This property of ChT makes it a potential marker for many disease process and prognostication. Present study has been carried out to know the significance of ChT as a screening marker in lysosomal storage disorders (LSDs) where tissue macrophage activation is commonly observed due to accumulation of substrate in various organs of the body. Study comprises of 20 healthy children in the age range of 10 days to 5 yrs and 56 children in the age range of 2.5 months to 13 yrs with regression of milestones, skeletal dysplasia, neuroregression and hepatosplenomegaly were selected for plasma ChT who had confirmed LSDs as carried out by specific lysosomal enzyme study from the leukocytes or fibroblasts. Plasma ChT was 55.21 +/- 20.81 nmol/ml/hr in twenty healthy age matched controls. Plasma ChT level was 42.88 to 79.78 nmol/ml/hr in thirteen of 56 (23.21%) children with LSDs like Morquio-B, Pompe, Metachromatic leucodystrophy (MLD), Sandhoff and Niemann-Pick disease type C (NPD-C). While in 43 (76.78%) children it was in the range of 213.74 to 23,511.40 nmol/ml/hr. who had LSDs like Morquio-B, Sly syndrome, MLD, GM2 Gangliosidosis, NPD-A/B and Gaucher disease (GD). Marked elevated ChT (4,000 to 23,511 nmol/ml/hr) was observed in all cases of GD (n=7) and NDP-A/B. It can be concluded from the present study that moderately raised activity of ChT can be utilized as a positive predictive test for certain LSD's. Those with marked elevated ChT have confirmed GD or NPD-A/B making it a strong screening marker for this group of diseases.

Antimicrobial stewardship in institutions and office practices.

Medically inappropriate, ineffective and economically inefficient use of antimicrobials is commonly observed in the health care units throughout the world especially in the developing countries. Antimicrobial stewardship programs attempt to balance the demand for these life-saving drugs with the need to preserve their future efficacy. A comprehensive evidence-based stewardship program should include elements chosen from the recommendations based on local antimicrobials use and resistance problems and on available resources that may differ, depending on the size of the institution or clinical setting. For success of antibiotic stewardship it is essential to increase awareness amongst medical professionals. Discipline in antimicrobial prescribing is most vital in clinical settings. A careful assessment of the benefits of prescribing against the risk of non-prescribing of antibiotics should be considered. It should be an endeavor of every physician to justify antibiotic prescription in case of empirical use. Integration of advanced information technology into antimicrobial stewardship programs holds the potential to both reduce antimicrobial overuse and improve outcomes.