CARDIOVASCULAR JOURNAL OF AFRICA • Volume 33, No 5, September/October 2022 AFRICA 283 any medical care being administered. No written clinical history/ records were available, however his mother reported him having a recent cold for which she administered cough medication. The mother also reported an increase in crying and that the infant struggled to feed. Due to the sudden and unexpected nature surrounding the death, the body was admitted to the Pretoria Medico-Legal Laboratory for further medico-legal investigation, in accordance with the Inquests Act 58 of 1959. A complete macroscopic autopsy examination was conducted, which externally revealed the deceased to be of average physique and nutritional state. No injuries were noted on the body. Upon internal examination, the intracranial examination showed no gross pathological changes. Examination of the heart revealed no abnormalities involving the epicardium. The myocardium and heart valves appeared normal. The lungs appeared congested and oedematous. On cut surfaces, the lungs showed sharply defined edges, had a friable texture and contained muco-purulent fluid. Examination of the stomach revealed contents of a milk-like residue and it was noted that the gastric mucosa appeared normal. As a result, no macroscopic cause of death could be identified at autopsy. Toxicology results revealed only trace amounts of theophylline, a bronchodilator, which is in keeping with the history of cough medication administered to the infant. No sedatives could be detected in the blood specimen. Histological examination of the thymus, brain and heart showed no obvious pathological changes. Sections of the heart showed no evidence of myocyte hypertrophy, nucleomegally or interstitial fibrosis. Sections of the lungs showed a mild mononuclear interstitial infiltrate with thickening, congestion and focal haemorrhage (Fig. 1). Focal intra-alveolar neutrophilic exudate was also noted in the lungs, as seen in Fig. 2. The features noted in the lungs were found to be in keeping with an acute bacterial pneumonia. Henceforth, the primary medical cause of death was acute bacterial pneumonia. Genetic testing Due to the diagnosis of an acute bacterial pneumonia as the cause of death, this infant case study was included in our larger study. For this case, an archived, formalin-fixed, paraffin-embedded (FFPE) myocardial tissue sample, obtained from the original autopsy 10 years prior to this study, for histology purposes, was subsequently subjected to retrospective post mortem genetic testing of the SCN5A gene. DNA was extracted from the FFPE myocardial tissue sample using the QIAamp DNA FFPE tissue kit (Qiagen). After extraction, the concentration and purity of the DNA sample was determined spectrophotometrically (NanoDrop spectrophotometer, Thermo Scientific). Thirty-nine primer pairs were used for amplification of 28 exons of the SCN5A gene.13 High-resolution melt real-time polymerase chain reaction (PCR) amplifications were performed using SensiFast HRM mastermix (Bioline) on the RotorGene Q (Qiagen). Following optimisation, DNA concentrations used per reaction averaged 50 to 80 ng. Final primer concentrations were 10 pmol. Thermal cycling conditions followed SensiFast guidelines, and annealing temperatures were dictated by the primers. Highresolution melting (HRM) analysis was performed, and control and case study samples were compared. All amplicons that showed variation on HRMwere subjected to sequencing (Inqaba Biotec). Sequencing results were analysed using CLC Main Workbench 5 software (CLC Bio®) and were aligned with the SCN5A gene sequences from GenBank (SCN5A NG_008934.1; NM_001160161.1 and NP_001092874.1) [National Center of Biotechnology and Information (NCBI)]. Polymorphism phenotyping v2 (PolyPhen2) was used to determine the probability of pathogenicity for novel identified variations. Results Genetic analysis revealed two different variations in exon 28 of the SCN5A gene. The first was a novel heterozygous variation (c.5566G>A) in the coding DNA sequence. This missense variation leads to a G>A nucleotide change in codon 1856, with an amino acid change of alanine (Ala) to threonine (Thr) (p.A1856T) (Fig. 3). Due to the functional difference between these two amino acids, the possibility of this variation affecting the protein structure is high. The PolyPhen-2 online algorithm predicted this variant to be probably damaging with a score of 1.000. Fig. 1. Haematoxylin and eosin stain of the lungs tissue shows a mild mononuclear interstitial infiltrate with thickening, congestion and fresh focal haemorrhage. Fig. 2. Haematoxylin and eosin-stained slide of the lungs showing a mixed intra-alveolar infiltrate chiefly composed of macrophages, neutrophils, fresh haemorrhage and oedema.
RkJQdWJsaXNoZXIy NDIzNzc=