Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Background & objectives: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics. Methods: Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing. Results: The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (Tm) values. Variations in Ct and Tm values among the two alleles were observed in both rs3014866 (Ct: C allele - 24±1, T allele - 27±1; Tm: C allele - 82.5±0.3, T allele - 86.3±0.2) and rs2149356 (Ct: C allele - 24±1, A allele - 26±1; Tm: C allele - 79.4±0.2, A allele - 80.4±0.3). Based on the variations, homozygous and heterozygous alleles were detected. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR. Interpretation & conclusions: In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping

Oxidative stress in perinatal asphyxia in relation to outcome.

Objective. To study the serum levels of oxidative stress markers - malondialdehyde (MDA) and protein carbonyl in babies with perinatal asphyxia and to correlate their levels with the outcome in terms of mortality and neurodevelopmental sequelae. Methods. A group of 40 term AGA (appropriate for gestational age) infants with perinatal asphyxia were selected as cases and same number of healthy babies as controls. Serum levels of oxidative stress markers - malondialdehyde and protein carbonyl were determined in cord blood and at 48 hours of life. Their levels were correlated with the outcome of perinatal asphyxia in terms of mortality and the long term neurological outcome. Results. MDA and protein carbonyl, in cord blood were significantly higher among cases (5.88±1.40 μmol/L and 1.50±0.48 nmol/mg of protein respectively) than controls (3.11±0.82 μmol/L and 0.83±0.19 nmol/mg of protein respectively). Among the cases, MDA and protein carbonyl values at 48 hours of life (7.52 ± 1.06 μmol/L and 2.91 ± 0.62 nmol/mg of protein respectively) were significantly higher than those at birth. MDA at birth and 48 hours was significantly higher among babies who had seizures than those who remained seizure free. These values were also significantly higher in babies who expired as compared to those who survived. Protein carbonyl values though higher in those who had seizures and in those who expired, were not statistically significant from controls. MDA and protein carbonyl at birth and 48 hours were higher in babies with developmental delay but the association was not statistically significant. Conclusions. In hypoxic ischemic encephalopathy (HIE), oxidative stress markers MDA and protein carbonyl are high at birth and rise further at 48 hours and the values correlate with the morbidity and mortality. Therefore, determining the serum levels of oxidative stress markers MDA and protein carbonyl will be of benefit in predicting the outcome in perinatal asphyxia.

Fulminant hepatic failure in an infant with severe dengue infection.

Fulminant hepatic failure due to dengue infection is rare, although mild liver dysfunction is common. Here we report a fatal case of fulminant hepatitis in an infant infected with dengue 3 serotype. Attention must be given to the use of hepatotoxic drugs in some cases of dengue especially in infants.

Cardiac enzyme levels in myocardial dysfunction in newborns with perinatal asphyxia.

OBJECTIVE: To study the usefulness of cardiac enzymes in evaluating myocardial damage in perinatal asphyxia. METHODS: Thirty term babies with perinatal asphyxia and without any congenital malformations were selected as cases. They were compared with thirty healthy term babies without asphyxia. Myocardial dysfunction was evaluated using clinical, electrocardiography, echocardiography and cardiac enzymes i.e, troponin-T and CK-MB levels. RESULTS: Among the 30 cases 23 had evidence of myocardial involvement while one baby in the control group had ECG evidence compatible with cardiac involvement. Cardiac enzymes were significantly increased in babies with perinatal asphyxia. The mean level of C-troponin-T among cases and controls were 0.22+/-0.28 and 0.003+/-0.018 while CK-MB levels were 121+/-77.4 IU/L and 28.8 +/- 20.2 IU/L respectively. C-troponin-T had higher sensitivity and specificity compared to CK-MB levels. Moreover, C troponin-T levels correlated well with severity and outcome in babies with perinatal asphyxia. CONCLUSION: C-Tropopnin assay is useful in evaluating the severity of myocardial damage and outcome in perinatal asphyxia.

Short rib polydactyly syndrome - type 2 (Majewski).

Short rib polydactyly syndromes (SRPS) are a group of inherited autosomal recessive skeletal dysplasias. It is characterized by the triad of micromelia, polydactyly and short horizontal ribs with or without visceral involvement. We report a case of SRPS-2 in a fresh stillborn and discuss the differential diagnosis.

Effect of pregnancy induced hypertension on mothers and their babies.

OBJECTIVE: To study the hematological profile of mothers with pregnancy induced hypertension and their infants. METHODS: The effects of maternal hypertension on the hematological profile of neonates were studied in 50 cases comparing the values with that of infants born to normotensive mothers. RESULTS: There was higher number of preterm, Intra-Uterine Growth Restriction (IUGR) and Small for Gestational Age (SGA) babies among the infants of hypertensive mothers. There was a significantly higher incidence of thrombocytopenia and nucleated RBCs seen in these babies. Significant neutropenia was not documented and there was no increased incidence of bleeding when compared to controls. CONCLUSION: Although there were significant changes in the hematological profile of infants born to hypertensive mothers, there was no significant increase in neonatal morbidity as a result of these changes.

Neural tube defects in Pondicherry.

OBJECTIVE: This study was carried out to identify the trend and the frequency of neural tube defects from July 1998 to June 2004. METHODS: A total of 310 babies were born with neural tube defects with the overall frequency of 5.7/1000 births compared to 2.3/1000 births observed earlier in our hospital. RESULTS: The most common defect was spina bifida (54.8%) followed by anencephaly (31.6%), and encephalocele (11.6%). More neural tube defects were observed in female and low birth weight babies, still births and unbooked mothers. Neural tube defect was significantly higher among babies born to parents of consanguineous marriage (p< 0.01). Associated congenital defects were observed in thirty nine (12.6%) cases. CONCLUSION: The rise in the frequency of NTDS may indicate the current trend of NTDs in Southern India. A further prospective study is desired to measure the effectiveness of regular folic acid supplementation in bringing down this frequency.

Value of nucleated red blood cells in predicting severity and outcome of perinatal asphyxia.

The objective of the study was to assess nucleated red blood cell counts in cord blood in a group of asphyxiated infants, and to determine its predictive value for short-term outcome. A prospective case control study was undertaken on cord blood samples collected from fifty six term neonates with perinatal asphyxia and an equal number of normal appropriately matched controls for cord blood pH and nucleated RBC counts. Babies were followed up in nursery till discharge. Statistical analysis employed were ANOVA test, logistic and linear regression analysis. There was a significant increase in the number of nucleated red blood cells in cases as compared to controls. Low Apgar, cord blood pH and neonatal outcome correlated well with nucleated RBC counts. Nucleated red blood cell count at birth is a useful predictor of severity and short-term outcome of perinatal asphyxia.

Hodgkins disease in relapse presenting as bone tumor.

A 10-year-old male child presented with multiple lymph node swellings. A diagnosis of Hodgkins disease was made on histopathological examination. The patient developed relapse six months after his last chemotherapy as a solitary bone tumor, which is rare. Immunohistochemical evaluation helped for the correct typing of Hodgkins disease.