His complete blood count, at time of study was WBC 9.1 x 103/ul, RBC-2.14x106/ul, HB-6.2g/dl, HCT-18.5%, MCV-86.4fl, MCH-28.5pg, MCHC-33 g/dl, PLT-111x103/ul, RDW-23.9%. He showed elevated indirect bilirubin, LDH, and reticulocyte count (8.8%), total bilirubin- 2.3mg/dl, indirect bilirubin-1.5mg/dl, LDH-3400 IU/L. All investigations were suggestive of haemolytic crisis. RBC membranopathies were found to be normal reveals by the EMA test showed − 980 MCF (Mean channel fluorescence). This implies that he would be a case of RBC enzymopathy. Although the common red metabolic enzymes study were found to be normal. G6PD-5.6 IU/gHb, PK-9.1 IU/gHb, GPI-59.6 IU/gHb. The haematological biochemical and molecular data of the proband and parents are summarized in Table-2.
The proband sample was subjected to targeted NGS. The human reference genome (GRCh37/hg19) and obtained sequence are aligning by the BWA software and Picard and GATK version 3.6 used to analyze to identify relevant variants associated with the clinical presentations. The clinically relevant variant was annotated with the published literature and aggregates the information using databases likes ClinVar, OMIM, GWAS, HGMD, and SwissVar. NGS result revealed a novel homozygous substitution in exon 4 of AK1 gene - c.301C > A, p. Gln101Lys. DNA Sanger sequencing confirmed the homozygosity status of the proband and heterozygous in the parents (Figure-1). This was later confirmed with both the parent’s DNA sample and validated by DNA Sanger sequencing, also further confirmed by measuring enzyme AK enzyme activity in a freshly collected blood sample. AK activity was found to be (38.0 IU/g Hb) deficient, mother showed 192.0 IU/gHb and the father showed 208.0 IU/gHb activity, whereas 50 normal healthy controls showed in the range of 297–360 IU/gHb. Further, the family decided to undergo prenatal diagnosis at the time of the second pregnancy based on the results of the proband. Subsequent biochemical analysis of the proband and the parents confirmed the molecular study result.
The fetus, as well as parent genomic DNA, was subjected to DNA Sanger sequencing based on the identified variant in the AK1 gene in Exon-4 c.301C > A, p. Gln101Lys in the index case by NGS. The fetus and both the parents were heterozygous, whereas only the proband was homozygous for the aforementioned variant. After reconfirmation from KB staining that there is no indication of the remnant of the mother's sample in the fetus, the results were discussed with the couples. The couples were thoroughly counselled and given necessary genetic advice. The pregnancy was continued and the normal healthy child was born post gestation period of 9 months.
This novel variant changes the structure of AK protein as it has a deleterious effect which was confirmed by bioinformatics tools. According to the available list of mutations in HGMD, this substitution has not been mentioned in the database. All the prediction tools -Polyphen-2, SIFT, PROVEAN, Mutation taster, Mutation Assessor, PMUT, MutPred2, have been explored to evaluate the effect of the variants on the structure of the protein. Table-3 summarizes the multiple bioinformatics software’s prediction results of p. Gln101Lys. The entire prediction tool suggested the damaging effect of the amino acid change from glutamine to lysine. AK1 protein (PDB ID-1Z83) consists of three chains A, B, C, spanning to the length of 194 residues. Each chain consists of one large central "CORE" domain and two small peripheral domains, NMP binding domain, and the LID domain. Upon ATP binding, the LID domain closes over the phosphoryl transfer site. The amino acid residue position Q101 is an important AMP binding site, along with 39,44,138, 149 residues. Any changes at these AMP binding sites possibly hamper the catalytic cycle of the enzyme. Figure 2A showed complete ribbon representation of protein (PDB ID-1Z83) with chain A, B, C which highlights the three chains of the AK1 enzyme, along with insight at the Q101 position’s helical structure and amino acid change from wild type (glutamine) to mutant type (lysine), (B) Secondary structure of the protein (PDB ID-1Z83) showing amino acid residue at position 101(Q101), (C) Wild type amino acid residue Glutamine 101 (Q101), (D) Mutant type amino acid residue Lysine101 (K101) and (E) showed the residue (Q) at positions 101 of AK-1 is highly conserved across species. Bioinformatics prediction tools have confirmed the damaging effect of the variant on the structure of the enzyme and demonstrated its conversation throughout the species [10–11].