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.

Charting the Course: Towards a Comprehensive Newborn Screening Program in India.

Integrating health interventions in a growing economy like India, with a birth cohort of 27 million/year, one-fifth of all childbirths, and approximately one-third of neonatal deaths globally, is a challenge. While mortality statistics are vital, intact survival and early preventive healthcare, such as newborn screening (NBS), are paramount. The appalling lack of information about the precise burden of metabolic errors at the state/national level or a mandated program encouraged a feasibility study of NBS in a prospective newborn cohort recruited in Delhi State (November 2014-April 2017) using a public-private partnership mode. The major determinants for effective implementation of universal NBS at the national level and limitations encountered are discussed in this report. Data to generate the 'core' panel for screening, sustained training of healthcare personnel, dissemination of the power of NBS to ensure neonatal/societal health to the public, and a 'national policy' emerge as priorities in a developing country.

Indian childhood cirrhosis: a retrospective study -redefining the older myths!

AIMS: This retrospective study emphasises the need of awareness for clinicopathological attributes of Indian childhood cirrhosis (ICC) in order to enable timely diagnosis and management. METHODS: This study was done on liver archival tissue of our department from the period of January 2016 to December 2022. Of these, cases of copper overload on paediatric biopsies were retrieved. The histopathological features were scrutinised independently by three pathologists, correlating with their clinico-radiological investigations. RESULTS: Five children in infancy to middle childhood presented with features of chronic liver disease in the form of jaundice and abdominal distention, were included in the study. Characteristic firm hepatomegaly with sharp margins and transaminitis was noted in all cases. Autoimmune, viral and metabolic workup were negative in all these patients except one which showed positive autoimmunity and another whose Coomb's test was positive. Normal ceruloplasmin levels and unremarkable slit lamp examination excluded the possibility of Wilson's disease. The histological features of marked ballooning degeneration with diffuse Mallory Denk, pericellular fibrosis, absence of steatosis and panlobular copper deposits clinched the diagnosis of ICC. CONCLUSIONS: ICC once believed to be extinct has still not vanished and remains underdiagnosed in routine practice. It is a rapidly fatal disease with a debatable pattern of inheritance and controversial role of copper as etiological agent. The clinical presentation is often deceptive and lack of awareness leads to misdiagnosis. Histopathological attributes are pathognomonic and possibility of ICC should be kept in all cases of cryptogenic cirrhosis.

Biodosimetric analysis of head and neck cancer patients undergoing radiotherapy by dicentric chromosome aberration assay.

BACKGROUND: Biodosimetry is the quantification of absorbed radiation dose using biological material obtained from an exposed individual. Radiation can cause different types of chromosomal aberrations, including stable aberrations like translocations and unstable ones like micronuclei, dicentric chromosomes (DC), acentric, and ring forms. Dicentric chromosome assay has become the "gold standard" for cytogenetic biodosimetry due to its reproducibility, specificity (low baseline rates), and sensitivity to low doses. Using existing calibration curves and models obtained from in vitro irradiation of blood, the yield of DCs can be used to estimate the average whole-body absorbed dose. PURPOSE: To evaluate and compare the in vivo dose-response relation of DC aberration formation in peripheral blood lymphocytes of head and neck cancer (HNC) patients undergoing radiotherapy (RT) alone, cisplatin-based chemoradiation (CCRT), accelerated fractionation RT (AFRT), and CCRT with gefitinib (GCRT). METHODOLOGY: This prospective observational and analytical study was conducted from 2018 to 2021 in the Department of Radiation Oncology and Genetic Lab of tertiary care, teaching hospital after approval from the Institutional Ethics Committee. Biodosimetric analysis was done weekly in patients undergoing RT (n = 20) versus CCRT (n = 20), CCRT (n = 12) versus AFRT (n = 12), and CCRT (n = 6) versus GCRT (n = 6). The yield of DCs was measured in blood samples taken before starting treatment, that is, day 0 and during RT on days 6, 11, and 16 in RT alone versus CCRT; on days 7 and 13 in CCRT versus AFRT; and days 6 and 11 in CCRT versus GCRT from a blood sample drawn 1-2 h after RT. Phytohemagglutinin-stimulated lymphocytes were cultured using heparinized blood in RPMI-1640 medium supplemented with fetal bovine serum. Cells were arrested at metaphase using demecolcine, harvested by centrifugation, mounted, and stained with Giemsa. Cytogenetic analysis was performed by analyzing at least 100 metaphases with well-spread chromosomes. DC aberrations and acentric fragments were identified and recorded. To standardize the findings as per the customized field for every patient, the mean DC yield per cm2 of the irradiated area was calculated and compared. RESULTS: The mean yield of DC/cm2 in the CCRT group was greater than the RT alone group by 16.33%, 28.57%, and 18.68% on days 6, 11, and 16 of treatment, respectively. This difference between the two groups at day 6 (P = 0.001), day 11 (P < 0.001), and day 16 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the AFRT group by 7.9% and 18.3% on days 7 and 13 of treatment, respectively. This difference at day 7 (P < 0.001) and day 13 (P < 0.001) was found to be statistically significant. The mean yield of DC/cm2 in the CCRT group was greater than the GCRT group by 22.7% and 21.8% on days 6 and 11 of treatment, respectively. The difference at day 6 (P = 0.01) was statistically significant but, on day 11 (P = 0.065) this difference was found insignificant. CONCLUSION: There is a dose-dependent increase in the yield of DCs in lymphocytes of HNC patients undergoing RT with subsequent fractions. Cisplatin-based chemoradiation is the superior method of treatment intensification radio-biologically proven by higher DC yield.

Inborn Errors of Ketogenesis: Novel Variants, Clinical Presentation, and Follow-Up in a Series of Four Patients.

Inborn errors of ketogenesis are rare disorders that result in acute and fulminant decompensation during lipolytic stress, particularly in infants and children. These include mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS) deficiency and HMG-CoA lyase (HMGCL) deficiency. In this series, we describe the clinical, biochemical, and molecular profiles of four patients along with dietary interventions and their outcomes on a long-term follow-up. Two patients each of HMGCS and HMGCL deficiency were evaluated with clinical history, biochemical investigations, including tandem mass spectrometry (TMS) and urine gas chromatography-mass spectrometry (GCMS). Molecular analysis was performed by whole-exome sequencing, as well as exon array validated by long-range polymerase chain reaction. All individuals were diagnosed with acute metabolic decompensation in the early infancy period except one with HMGCL deficiency who had the first presentation at 5 years of age. Central nervous system manifestations, severe metabolic acidosis, hyperammonemia, hypoglycemia with a normal lactate, and absence of urinary ketones were observed in all the affected individuals. The disorder was life-threatening in three individuals and one succumbed to the illness. TMS was nonspecific and urine GCMS revealed dicarboxylic aciduria in HMGCS deficiency. Both the patients with HMGCL deficiency demonstrated elevated 3 hydroxyisovaleryl carnitine levels in TMS and metabolites of leucine degradation in urine GCMS. We identified five novel variants that included a large deletion involving exon 2 in HMGCL gene. There was no evidence of long-term neurological sequelae in the living individuals. Diet with moderation of fat intake was followed in two individuals with HMGCS deficiency. Low leucine and protein diet with moderation of fat intake was followed in the individual with HMGCL deficiency. All affected individuals are thriving well with no further major metabolic decompensation.

Post-natal gestational age assessment using targeted metabolites of neonatal heel prick and umbilical cord blood: A GARBH-Ini cohort study from North India.

Background: Accurate assessment of gestational age (GA) and identification of preterm birth (PTB) at delivery is essential to guide appropriate post-natal clinical care. Undoubtedly, dating ultrasound sonography (USG) is the gold standard to ascertain GA, but is not accessible to the majority of pregnant women in low- and middle-income countries (LMICs), particularly in rural areas and small secondary care hospitals. Conventional methods of post-natal GA assessment are not reliable at delivery and are further compounded by a lack of trained personnel to conduct them. We aimed to develop a population-specific GA model using integrated clinical and biochemical variables measured at delivery. Methods: We acquired metabolic profiles on paired neonatal heel prick (nHP) and umbilical cord blood (uCB) dried blood spot (DBS) samples (n = 1278). The master data set consists of 31 predictors from nHP and 24 from uCB after feature selection. These selected predictors including biochemical analytes, birth weight, and placental weight were considered for the development of population-specific GA estimation and birth outcome classification models using eXtreme Gradient Boosting (XGBoost) algorithm. Results: The nHP and uCB full model revealed root mean square error (RMSE) of 1.14 (95% confidence interval (CI) = 0.82-1.18) and of 1.26 (95% CI = 0.88-1.32) to estimate the GA as compared to actual GA, respectively. In addition, these models correctly estimated 87.9 to 92.5% of the infants within ±2 weeks of the actual GA. The classification models also performed as the best fit to discriminate the PTB from term birth (TB) infants with an area under curve (AUC) of 0.89 (95% CI = 0.84-0.94) for nHP and an AUC of 0.89 (95% CI = 0.85-0.95) for uCB. Conclusion: The biochemical analytes along with clinical variables in the nHP and uCB data sets provide higher accuracy in predicting GA. These models also performed as the best fit to identify PTB infants at delivery.

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.