Path to Facilitate the Prediction of Functional Amino Acid Substitutions in Red Blood Cell Disorders – A Computational Approach

A major area of effort in current genomics is to distinguish mutations that are functionally neutral from those that contribute to disease. Single Nucleotide Polymorphisms (SNPs) are amino acid substitutions that currently account for approximately half of the known gene lesions responsible for human inherited diseases. As a result, the prediction of non-synonymous SNPs (nsSNPs) that affect protein functions and relate to disease is an important task.In this study, we performed a comprehensive analysis of deleterious SNPs at both functional and structural level in the respective genes associated with red blood cell metabolism disorders using bioinformatics tools. We analyzed the variants in Glucose-6-phosphate dehydrogenase (G6PD) and isoforms of Pyruvate Kinase (PKLR & PKM2) genes responsible for major red blood cell disorders. Deleterious nsSNPs were categorized based on empirical rule and support vector machine based methods to predict the impact on protein functions. Furthermore, we modeled mutant proteins and compared them with the native protein for evaluation of protein structure stability.We argue here that bioinformatics tools can play an important role in addressing the complexity of the underlying genetic basis of Red Blood Cell disorders. Based on our investigation, we report here the potential candidate SNPs, for future studies in human Red Blood Cell disorders. Current study also demonstrates the presence of other deleterious mutations and also endorses with in vivo experimental studies. Our approach will present the application of computational tools in understanding functional variation from the perspective of structure, expression, evolution and phenotype

Neonatal Hyperbilirubinemia in infants with <it>G6PD c.563C > T</it><it>Variant</it>

<p>Abstract</p> <p>Background</p> <p>There is a strong correlation between glucose-6-phosphate dehydrogenase (G6PD) deficiency and neonatal hyperbilirubinemia with a rare but potential threat of devastating acute bilirubin encephalopathy. G6PD deficiency was observed in 4–14% of hospitalized icteric neonates in Pakistan. <it>G6PD c.563C > T</it> is the most frequently reported variant in this population. The present study was aimed at evaluating the time to onset of hyperbilirubinemia and the postnatal bilirubin trajectory in infants having <it>G6PD c.563C > T.</it></p> <p>Methods</p> <p>This was a case–control study conducted at The Aga Khan University, Pakistan during the year 2008. We studied 216 icteric male neonates who were re-admitted for phototherapy during the study period. No selection was exercised. Medical records showed that 32 were G6PD deficient while 184 were G6PD normal. Each infant was studied for birth weight, gestational age, age at the time of presentation, presence of cephalhematoma, sepsis and neurological signs, peak bilirubin level, age at peak bilirubin level, days of hospitalization, whether phototherapy or exchange blood transfusion was initiated, and the outcome. During hospital stay, each baby was tested for complete blood count, reticulocyte count, ABO and Rh blood type, direct antiglobulin test and quantitative G6PD estimation [by kinetic determination of G6PDH]. <it>G6PDgenotype</it> was analyzed in 32 deficient infants through PCR-RFLP analysis and gene sequencing.</p> <p>Results</p> <p><it>G6PD variants c.563C > T</it> and <it>c.131 C > G</it> were observed in 21 (65%) and three (9%) of the 32 G6PD deficient infants, respectively. DNA of eight (25%) newborns remained uncharacterized. In contrast to G6PD normal neonates, infants with <it>c.563C > T</it> variant had significantly lower enzyme activity (mean ± 1SD; 0.3 ± 0.2 U/gHb vs. 14.0 ± 4.5 U/gHb, <it>p</it> < 0.001) experienced higher peak levels of total serum bilirubin (mean ± 1SD; 16.8 ± 5.4 mg/dl vs. 13.8 ± 4.6 mg/dl, <it>p =</it> 0.008) which peaked earlier after birth (mean ± 1SD 2.9 ± 1.6 vs. 4.3 ± 2.3 days, <it>p =</it> 0.007). No statistically significant difference was observed in mean weight, age at presentation, hemoglobin, reticulocyte count, TSH level, hospital stay or in the frequency of initiation of phototherapy or blood exchange between the two groups.</p> <p>Conclusions</p> <p>We concluded that infants with <it>G6PD c.563C > T</it> variant developed jaundice earlier than infants with normal G6PD enzyme levels. Compared to G6PD normal infants, <it>G6PD c.563C > T</it> carrying infants had significantly low G6PD activity.</p