Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India.

PURPOSE: Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. METHODS: Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband's and parent's(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). RESULTS: Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. CONCLUSIONS: We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Current Indicators of Nutritional Care in Children with Type 1 Diabetes in India: Do we Need a National Nutritional Guideline?

BACKGROUND: Nutrition is an important pillar of management in children with type 1 diabetes. Indian food is heavily influenced by region, religion, traditions, seasons, and cultural choices. This survey was done to assess current practices and the need for India specific nutritional guidelines for children with type 1 diabetes. MATERIALS AND METHODS: Two 12-item questionnaires were administered to forty health-care professionals across India. The first questionnaire evaluated current clinical practice indicators for nutrition in these children and second assessed practices for counseling a child on dietary habits. RESULTS: There is great heterogeneity across the country with regard to dietary advice offered to children with type 1 diabetes. 97.5% of the respondents feel there is a need for an Indian dietary guideline for children with type 1 diabetes. CONCLUSION: There is need of India specific nutritional guidelines that should be made considering key variants such as age, region, cultural preference, economic burden and psychosocial beliefs, to offer guidance to diabetes care professionals.

Clinical and Molecular Characterization of Prader-Willi Syndrome.

OBJECTIVES: To describe the clinical presentations and molecular diagnosis to aid the clinicians in early diagnosis and appropriate management of Prader-Willi syndrome (PWS). METHODS: Thirty-four clinically diagnosed PWS cases were enrolled after obtaining informed consent/assent. Demographic details, clinical data and anthropometry were recorded using structured proforma. The facial dysmorphology was evaluated. Appropriate genetic testing was performed to confirm the diagnosis. RESULTS: At diagnosis, the most common clinical features included obesity (59%) and short stature (53%). Distinct dysmorphic features were observed in 67%. Neonatal hypotonia with feeding difficulty, delayed development in infancy and childhood behavioral problems were reported in 94%, 94% and 74% respectively. Food seeking behavior and hyperphagia was reported in 67%. Seizures were reported in 47%. All children had underdeveloped external genitalia. Growth hormone (GH) deficiency and impaired glucose tolerance were found in 56% and 50% respectively. Sleep related problems were seen in 67%. Skin and rectal picking were reported in 67%. FISH confirmed micro-deletion was found in 64.7% and abnormal methylation in 35%, of which uniparental disomy was confirmed in 14.7%. CONCLUSIONS: Clinical suspicion is vital for early detection of PWS. Confirmation of the diagnosis requires complex multi-tier molecular genetic testing.

Weight Gain and Height Growth during Infancy, Childhood, and Adolescence as Predictors of Adult Cardiovascular Risk.

OBJECTIVES: To investigate independent relationships of childhood linear growth (height gain) and relative weight gain to adult cardiovascular disease (CVD) risk traits in Asian Indians. STUDY DESIGN: Data from 2218 adults from the Vellore Birth Cohort were examined for associations of cross-sectional height and body mass index (BMI) and longitudinal growth (independent conditional measures of height and weight gain) in infancy, childhood, adolescence, and adulthood with adult waist circumference (WC), blood pressure (BP), insulin resistance (homeostatic model assessment-insulin resistance [HOMA-IR]), and plasma glucose and lipid concentrations. RESULTS: Higher BMI/greater conditional relative weight gain at all ages was associated with higher adult WC, after 3 months with higher adult BP, HOMA-IR, and lipids, and after 15 years with higher glucose concentrations. Taller adult height was associated with higher WC (men ß = 2.32 cm per SD, women ß = 1.63, both P < .001), BP (men ß = 2.10 mm Hg per SD, women ß = 1.21, both P ≤ .001), and HOMA-IR (men ß = 0.08 log units per SD, women ß = 0.12, both P ≤ .05) but lower glucose concentrations (women ß = -0.03 log mmol/L per SD P = .003). Greater height or height gain at all earlier ages were associated with higher adult CVD risk traits. These positive associations were attenuated when adjusted for adult BMI and height. Shorter length and lower BMI at birth were associated with higher glucose concentration in women. CONCLUSIONS: Greater height or weight gain relative to height during childhood or adolescence was associated with a more adverse adult CVD risk marker profile, and this was mostly attributable to larger adult size.

Diabetic ketoacidosis in children and adolescents.

Diabetic ketoacidosis (DKA) is considered to be a common presentation of both type 1 diabetes mellitus and type 2 diabetes mellitus in children and adolescents. DKA arises due to lack of adequate insulin in the body. Insulin stops the use of fat as an energy source by inhibiting the peptide hormone glucagon. Without insulin, glucagon levels rise resulting in the release of free fatty acids from adipose tissue, as well as amino acids from muscle cells. Neurological observations should be made for warning signs and symptoms of cerebral edema, and capillary blood glucose concentration should be measured on an hourly basis. Every 2-4 h electrolytes, blood gases, and beta-hydroxybutyrate should be measured. Cerebral edema occurs in 0.5-0.9% of all episodes of DKA. It is considered to be a major cause of death in childhood DKA. Treatment of cerebral edema should be prompt and immediate. Successful DKA management in children depends upon swift diagnosis, meticulous monitoring of clinical and biochemical parameters with prompt intervention.

Type 1 diabetes: Awareness, management and challenges: Current scenario in India.

Type 1 diabetes mellitus (T1DM) has a wide presence in children and has a high mortality rates. The disease, if left unmanaged, poses various challenges to the patient and healthcare providers, including development of diabetic complications and thus decreasing the life expectancy of the affected child. The challenges of T1DM include awareness of the disease that is very poor among the general public and also in parents of T1DM children along with the health care professionals. The challenge of lack of awareness of T1DM can be met by increasing public awareness programs, conducting workshops for diabetes educators regarding T1DM in children, newsletters, CMEs, online courses, and by structured teaching modules for diabetes educators. Diagnosis of T1DM was a challenge a few decades ago but the situation has improved today with diagnostic tests and facilities, made available even in villages. Investigation facilities and infrastructure, however, are very poor at the primary care level, especially in rural areas. Insulin availability, acceptability, and affordability are also major problems, compounded by the various types of insulin that are available in the market with a varied price range. But effective use of insulin remains a matter of utmost importance.

Clinical and molecular characterization of neonatal diabetes and monogenic syndromic diabetes in Asian Indian children.

Mutations in the pancreatic ATP sensitive K(+) channel proteins [sulfonyluea receptor 1 (SUR1) and inward rectifier K(+) channel Kir6.2 (Kir6.2), encoded by ATP-binding cassette transporter subfamily C member 8 (ABCC8) and potassium channel J11 (KCNJ11), respectively], are the most common cause of neonatal diabetes. We describe the clinical presentation and molecular characterization of Asian Indian children with neonatal diabetes mellitus and monogenic syndromes of diabetes. We sequenced KCNJ11, ABCC8 and insulin (INS) genes in 33 unrelated Indian probands with onset of diabetes below one year of age. A total of 12 mutations were identified which included ABCC8 mutations in seven, KCNJ11 mutations in three and INS mutations in two children. The Asp212Tyr mutation in ABCC8 was novel. We also detected two novel mutations (Val67Met and Leu19Arg) in children with syndromic forms of diabetes like Berardinelli Seip syndrome [1-acyl-sn-glycerol-3-phosphate acyltransferase beta (AGPAT2)] and Fanconi Bickel syndrome [solute carrier family 2A2 (SLC2A2)]. Children carrying the KCNJ11 (Cys42Arg, Arg201Cys) and ABCC8 (Val86Ala, Asp212Tyr) mutations have been successfully switched over from insulin therapy to oral sulfonylurea. Our study is the first large genetic screening study of neonatal diabetes in India.

Does 3-day course of oral amoxycillin benefit children of non-severe pneumonia with wheeze: a multicentric randomised controlled trial.

BACKGROUND: WHO-defined pneumonias, treated with antibiotics, are responsible for a significant proportion of childhood morbidity and mortality in the developing countries. Since substantial proportion pneumonias have a viral etiology, where children are more likely to present with wheeze, there is a concern that currently antibiotics are being over-prescribed for it. Hence the current trial was conducted with the objective to show the therapeutic equivalence of two treatments (placebo and amoxycillin) for children presenting with non-severe pneumonia with wheeze, who have persistent fast breathing after nebulisation with salbutamol, and have normal chest radiograph. METHODOLOGY: This multi-centric, randomised placebo controlled double blind clinical trial intended to investigate equivalent efficacy of placebo and amoxicillin and was conducted in ambulatory care settings in eight government hospitals in India. Participants were children aged 2-59 months of age, who received either oral amoxycillin (31-54 mg/Kg/day, in three divided doses for three days) or placebo, and standard bronchodilator therapy. Primary outcome was clinical failure on or before day- 4. PRINCIPAL FINDINGS: We randomized 836 cases in placebo and 835 in amoxycillin group. Clinical failures occurred in 201 (24.0%) on placebo and 166 (19.9%) on amoxycillin (risk difference 4.2% in favour of antibiotic, 95% CI: 0.2 to 8.1). Adherence for both placebo and amoxycillin was >96% and 98.9% subjects were followed up on day- 4. Clinical failure was associated with (i) placebo treatment (adjusted OR = 1.28, 95% CI: 1.01 to1.62), (ii) excess respiratory rate of >10 breaths per minute (adjusted OR = 1.51, 95% CI: 1.19, 1.92), (iii) vomiting at enrolment (adjusted OR = 1.49, 95% CI: 1.13, 1.96), (iv) history of use of broncho-dilators (adjusted OR = 1.71, 95% CI: 1.30, 2.24) and (v) non-adherence (adjusted OR = 8.06, 95% CI: 4.36, 14.92). CONCLUSIONS: Treating children with non-severe pneumonia and wheeze with a placebo is not equivalent to treatment with oral amoxycillin. TRIAL REGISTRATION: ClinicalTrials.gov NCT00407394.

Trends in human birth weight across two successive generations.

OBJECTIVE: To determine the correlation between parental and offspring birthweight (BW) in India. METHODS: The study involved two birth cohorts of successive generations. The parental cohort comprised of 472 fathers and 422 mothers from an earlier study. Details of their anthropometry at birth and in adulthood were available. 1525 children born to them comprised the offspring cohort. BW was obtained from hospital records for the offspring cohort. Odds ratios and regression coefficients were calculated to estimate the risks of a low birth weight (LBW) parent producing a LBW baby and quantitate the effects after adjusting for confounders. RESULTS: A LBW mother had a 2.8 times risk (95%CI 1.2-6.4) of delivering a LBW baby (p=0.02) and a LBW father was twice as likely to produce a LBW baby (OR 2.2; 95%CI 1.0 - 4.8; p=0.05). Every 100g increase in maternal BW was associated with an increase in offspring BW of 14 g; the equivalent figure for paternal BW was 18.1g (p< 0.001 for both). Between the generations, the incidence of LBW decreased from 19.7% to 17.2% (p=0.1). Mean BW increased in males (2846 g vs 2861 g; p=0.59) but not in females (2790 g vs 2743 g; p=0.08). CONCLUSION: Both maternal and paternal BW are strong determinants of offspring BW. The effect of mothers' BW on offspring BW is weaker than that seen in developed nations. Stronger intrauterine constraint exhibited by Indian women secondary to a higher prevalence of growth restriction in utero may be responsible. Paternal effects may be governed by paternal genes inherited by the offspring.

Safety and reactogenicity of a low dose diphtheria tetanus acellular pertussis vaccine (Boostrix) in pre-school Indian children.

OBJECTIVE: To evaluate the safety and reactogenicity of a reduced-antigen-content combined Diphtheria Tetanus Acellular Pertussis (dTpa) vaccine in Indian preschool children. METHODS: GlaxoSmithKline Biologicals combination dTpa vaccine was administered as a single booster dose to 347 children aged 46 years in seven centers across India. All children were subsequently followed up for two weeks for safety and reactogenicity assessment. RESULTS: A total of 345 subjects completed the study and two subjects were lost to follow-up. One serious adverse event (head injury) unrelated to vaccination was reported. Otherwise, all subjects were in good health throughout the study period. Three subjects (0.9%) reported transient general symptoms (such as irritability and drowsiness), which prevented normal activity. Pain at injection site, swelling and redness was reported in 31.1%, 18.2% and 8.9% subjects respectively. Five subjects (1.4%) reported severe pain preventing normal movement. This resolved within 48 hours in all cases. There were no other severe local reactions including large injection site reactions. CONCLUSION: The reduced antigen content combined dTpa vaccine is safe and well tolerated in Indian pre-school children.

Efficacy of zinc in the treatment of severe pneumonia in hospitalized children <2 y old.

BACKGROUND: Severe pneumonia remains a leading cause of morbidity and mortality in undernourished young children in developing countries. OBJECTIVE: This study evaluated the effect of adjuvant zinc therapy on recovery from severe pneumonia by hospitalized children in southern India who were receiving standard antibiotic therapy. DESIGN: This randomized, double-blind, placebo-controlled clinical trial was conducted at the Christian Medical College Hospital, an 1800-bed teaching hospital in Tamilnadu, India. Enrollment and follow-up occurred between September 2003 and August 2004. Children aged 2-23 mo (n = 299) who were hospitalized with severe pneumonia were randomly assigned to receive 10-mg tablets of zinc sulfate or placebo twice a day during hospitalization, along with standard therapy for severe pneumonia. All clinical signs and symptoms of pneumonia were assessed and recorded at 8-h intervals. RESULTS: There were no clinical or statistically significant differences in the duration of tachypnea, hypoxia, chest indrawing, inability to feed, lethargy, severe illness, or hospitalization. Zinc supplementation was associated with a significantly longer duration of pneumonia in the hot season (P = 0.015). CONCLUSIONS: Zinc supplementation had no overall effect on the duration of hospitalization or of clinical signs associated with severe infection in young children hospitalized for severe pneumonia in southern India. This finding differs from the results of 2 previously reported trials wherein zinc supplementation was associated with a shorter period of recovery from severe pneumonia. Given the conflicting results, further research in representative settings is required to help clarify the role of zinc in the treatment of severe pneumonia.

Amylase-resistant starch as adjunct to oral rehydration therapy in children with diarrhea.

BACKGROUND: Oral rehydration solution (ORS) for treatment of diarrhea relies on enhancement of small intestinal sodium and fluid absorption to correct dehydration. Amylase-resistant starch added to ORS significantly reduced the duration and severity of diarrhea in adults with cholera, presumably by generation of short-chain fatty acids in the colon and enhancement of colonic sodium and fluid absorption. The present study was initiated to determine whether addition of amylase-resistant starch to standard World Health Organization glucose-ORS (G-ORS) would reduce the duration of diarrhea and fecal fluid losses in children with acute diarrhea. METHODS: One hundred eighty-three children (6 months to 3 years) with acute watery diarrhea were randomized to receive either standard treatment with G-ORS or G-ORS with additional amylase-resistant starch, HAMS (HAMS-ORS, 50g/L). Stool weight and consistency were monitored serially until development of formed stool or development of treatment failure defined as either the need for unscheduled intravenous fluid therapy or diarrhea longer than 72 hours. RESULTS: Five of the subjects were lost to follow up. In 178 remaining children (87 HAMS-ORS and 91 G-ORS) with evaluable data, time from enrolment to last unformed stool was significantly less in children receiving HAMS-ORS (median, 6.75 hours; 95% confidence interval, 4.27-9.22) than in children treated with G-ORS (12.80 hours, 8.69-16.91) (P = 0.0292). Time to first formed stool was also significantly shorter in children receiving HAMS-ORS (median, 18.25 hours; 95% confidence interval, 13.09-23.41) compared with children receiving G-ORS (median, 21.50 hours; 95% confidence interval, 17.26-25.74) (P = 0.0440). The total amount of ORS consumed was similar in both groups. There was a trend toward lower mean stool weight in first 24 hours (P = 0.0752) as well as total diarrheal stool weight (P = 0.0926) in patients in the HAMS group compared with the G-ORS group. CONCLUSION: In children with acute diarrhea, the addition of amylase-resistant starch to glucose ORS significantly shortened duration of diarrhea compared with standard treatment.

Massive powder aspiration in a toddler.

We report a case of aspiration of calcium carbonate powder by a toddler. Bronchoscopic removal of aspirated contents resulted in favourable outcome.

Neurologic deterioration in a child with Wilson's disease on penicillamine therapy.

Penicillamine is the standard therapy for Wilson's disease in children. We report an 8-year-old-girl with liver disease due to Wilson's disease who developed extrapyramidal symptoms following administration of penicillamine. Symptoms resolved within 20 hours of stopping the drug but recurred within 24 hours when gradually increasing small doses were recommenced.

Effect of Vitamin A supplementation on the immune response to measles vaccination.

A randomized controlled trial was conducted in 395 infants aged 9-12 months to determine the effect of Vitamin A supplementation on concurrently administered measles vaccine. Antibody response was measured using the plaque reduction neutralization assay. No statistically significant differences were demonstrated between the immune response in Vitamin A supplemented and unsupplemented children. Unlike some recent studies, we were unable to demonstrate an immune enhancing effect of Vitamin A supplementation. On the contrary, among children who were given Vitamin A, a lower, but statistically non-significant, proportion had protective antibody levels 6 months after vaccination.