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    • The NGS of six Southern Brazil patients

    2022-10-02

    The NGS of six Southern Brazil patients revealed three different variants: c.472C > T (p.Arg158Trp); c.958G>A (p.Gly320Arg) and c.986T>C (p.Leu329Pro) (Table 2). The amino acids involved in these substitutions were evolutionarily conserved in different species (Fig. 1B). The amino PPT australia involved in the p.Arg158Trp substitution is a conserved amino acid located in an α-helix. It was suggested that this amino acid change could affect the secondary structure, altering the protein stability (Ijaz et al., 2017; Lebigot et al., 2015). However, our 3D modeling data do not indicate alterations in the secondary structure of the protein caused by this variant. Thus, other factors could affect the activity of the mutant enzyme. Small changes in the hydrophobicity of structure were evidenced, since the variant p.Arg158Trp changes a positively charged residue to a hydrophobic amino acid. This may be related to the pathogenicity of this variant, but additional data will be necessary to clarify this issue. The novel variants, c.958G>A PPT australia and c.986T>C, are located in exon 8 (Fig. 2). According to the guidelines issued by the American College of Medical Genetics and Genomics (Richards et al., 2015), the c.986T>C variant is likely pathogenic, and the c.958G>A is pathogenic. In addition to that, the two novel variants were absent in two different databases: gnomAD and ABraOM (Naslavsky et al., 2017). The c.958G>A and c.986T>C variants cause alterations of a Glycine to an Arginine and a Leucine to a Proline at positions 320 and 329, respectively. Both occur in the C-terminal region, which is a nonconserved region, although some sites are evolutionarily conserved at these two positions. Expression analyses of c.966delC and c.960_961insG pathogenic variants, located in the same region of the novel alleles, demonstrated that the changes result in an inactive protein (Herzog et al., 1999). This reinforces the importance of this region for FBPase activity. The 3D model demonstrated that these variants do not cause alterations in the protein structure but can be drastic for their physical and chemical properties. The p.Gly320Arg and p.Leu329Pro variants involve the particular cases of the amino acids Glycine and Proline. Thus, the hydrogen of Glycine is replaced by a large, positively charged sidechain, while in the p.Leu329Pro variant, a hydrophobic sidechain is replaced by the structurally restricted Proline. These changes may have indirect effects on protein conformation by transmission of local changes along the protein backbone. However, the hydrophobicity analyses showed no significant alterations in this property in the mutant proteins. Therefore, more in-depth investigations of mutant protein conformational behavior are necessary. The mechanism involved in the novel alterations is not clear, but FBPase activity in in vivo and in silico analyses indicate their pathogenicity. Beyond the level of the enzyme activity, it was not possible to associate the alterations with other symptoms of patients. Patients with the same genotype present different forms of the disease, such as age onset, mental and muscular symptoms, or hepatomegaly. As in other studies, the genotype cannot predict the symptom sets of patients with FBPase deficiency (Li et al., 2017). The genotype-phenotype correlation is complicated by FBPase deficiency because most of the variants are private, and the symptoms are very heterogeneous. In this study, the majority of patients were homozygous, and only one of them had consanguineous parents. Although it was not possible to obtain DNA samples from every parent, and, so, to perform segregation analyses, manual review of the raw data indicates the absence of large deletions in the patients. Other studies described homozygous patients who apparently had non-consanguineous parents who were born in small villages (Herzog et al., 1999; Lu et al., 2017; Moey et al., 2018). The RS was populated from the eighteenth century predominantly by Portuguese and Azorean immigrations and with non-Iberian Europeans influx only in the nineteenth century. These founding groups remained isolated mainly because of their religious and cultural characteristics. Studies show that even geographically close population groups have maintained preferential marriages according to the country of origin and religion (De Oliveira et al., 2013). In this sense, it is not unexpected the presence of rare and private mutations with common ancestry in couples who do not declare themselves consanguineous. Add to this the fact that the three cities where these cases occurred have Portuguese ancestry.