Background: Human angiotensin-converting enzyme 2 (ACE2), a type I transmembrane receptor physiologically acting as a carboxypeptidase enzyme within the renin-angiotensin system (RAS), is a critical mediator of infection by several severe acute respiratory syndrome (SARS) corona viruses. For instance, it has been demonstrated that ACE2 is the primary receptor for the SARS-CoV-2 entry to many human cells through binding to the viral spike S protein. Consequently, genetic variability in ACE2 gene has been suggested to contribute to the variable clinical manifestations in COVID-19. Many of those genetic variations result in missense variants within the amino acid sequence of ACE2. The potential effects of those variations on binding to the spike protein have been speculated and, in some cases, demonstrated experimentally. However, their effects on ACE2 protein folding, trafficking and subcellular targeting have not been established.Results: In this study we aimed to examine the potential effects of 28 missense variants (V801G, D785N, R768W, I753T, L731F, L731I, I727V, N720D, R710H, R708W, S692P, E668K, V658I, N638S, A627V, F592L, G575V, A501T, I468V, M383I, G173S, N159S, N149S, D38E, N33D, K26R, I21T, and S19P) distributed across the ACE2 receptor domains on its subcellular trafficking and targeting through combinatorial approach involving in silico analysis and experimental subcellular localization analysis. Our data show that none of the studied missense variants (including 3 variants predicted to be deleterious R768W, G575V, and G173S) has a significant effect on ACE2 intracellular trafficking and subcellular targeting to the plasma membrane.Conclusion: Although the selected missense variants display no significant change in ACE2 trafficking and subcellular localization, this does not rule out their effect on viral susceptibility and severity. Further studies are required to investigate the effect of ACE2 variants on its expression, binding, and internalization which might explain the variable clinical manifestations associated with the infection.

The variants of electron transfer flavoprotein (ETFA, ETFB) and ETF dehydrogenase (ETFDH) are the leading cause of glutaric aciduria type II (GA-II). In this study, we identified 13 patients harboring six variants of two genes associated with GA-II. Out of the six variants, four were missense, and two were frameshift mutations. A missense variant (ETFDH:p.Gln269His) was observed in a homozygous state in nine patients. Among nine patients, three had experienced metabolic crises with recurrent vomiting, abdominal pain, and nausea. In one patient with persistent metabolic acidosis, hypoglycemia, and a high anion gap, the ETFDH:p.Gly472Arg, and ETFB:p.Pro94Thrfs*8 variants were identified in a homozygous, and heterozygous state, respectively. A missense variant ETFDH:p.Ser442Leu was detected in a homozygous state in one patient with metabolic acidosis, hypoglycemia, hyperammonemia and liver dysfunction. The ETFDH:p.Arg41Leu, and ETFB:p.Ile346Phefs*19 variants were observed in a homozygous state in one patient each. Both these variants have not been reported so far. In silico approaches were used to evaluate the pathogenicity and structural changes linked with these six variants. Overall, the results indicate the importance of a newborn screening program and genetic investigations for patients with GA-II. Moreover, careful interpretation and correlation of variants of uncertain significance with clinical and biochemical findings are needed to confirm the pathogenicity of such variants.

Glutaric aciduria type II (GA-II) is a rare autosomal recessive disease caused by defects in electron transfer flavoprotein (ETF), ultimately causing insufficiencies in multiple acyl-CoA dehydrogenase (MAD). 3-phosphoglycerate dehydrogenase (3-PHGDH) deficiency, is another rare autosomal disorder that appears due to a defect in the synthesis of L-serine amino acid. Several mutations of ETFDH and PHGDH genes have been associated with different forms of GA-II and serine deficiency, respectively. In this study, we report a unique case of GA-II with serine deficiency using biochemical, genetic, and in silico approaches. The proband of Syrian descent had positive newborn screening (NBS) for GA-II. At two years of age, the patient presented with developmental regression, ataxia, and intractable seizures. Results of amino acid profiling demonstrated extremely low levels of serine. Confirmatory tests for GA-II and whole exome sequencing (WES) were performed to determine the etiology of intractable seizure. Sequencing results indicated a previously reported homozygous missense mutation, c.679 C>A (p.Pro227Thr) in the ETFDH gene and a novel missense homozygous mutation c.1219 T>C (p.Ser407Pro) in the PHGDH gene. In silico tools predicted these mutations as deleterious. Here, the clinical and biochemical investigations indicate that ETFDH:p.Pro227Thr and PHGDH:p.Ser407Pro variants likely underlie the pathogenesis of GA-II and serine deficiency, respectively. This study indicates that two rare autosomal recessive disorders should be considered in consanguineous families, more specifically in those with atypical presentation.