FBPase deficiency is a very
FBPase deficiency is a very rare inborn error of fructose salidroside of autosomal recessive inheritance. This disorder usually occurs in the first year of life through ketotic hypoglycemia and lactic acidosis triggered by catabolic episodes such as prolonged fasting or febrile infections (Steinmann et al., 2012). The biochemical manifestations of FBPase deficiency include lactate accumulation accompanied by a decreased pH and an increased lactate/pyruvate ratio, hyperalaninemia, hyperketonemia, glyceroluria, increased levels of acids uric and free fatty acids and pseudo-hypertriglyceridemia (Afroze et al., 2013; Morris et al., 1995; Steinmann et al., 2012). The diagnosis of FBPase deficiency is based on clinical manifestations and laboratory findings. However, this can be complicated by nonspecific presentation. In addition, the more reliable enzymatic test is the measure of FBPase activity in hepatic tissue, an invasive test. Thus, pathogenic variants analysis of the FBP1 gene has been employed for the definitive diagnosis with clinical presentation (Asberg et al., 2010). To date, 48 pathogenic variants have been described in FBP1 in patients with FBPase deficiency, the majority being private variants (Human Gene Mutation Database - HGMD®).
Results The main clinical information of the included patients is summarized in Table 1.
Discussion FBPase deficiency is a rare inborn error of metabolism involving the gluconeogenesis pathway, which is reported in patients with different ethnic backgrounds. The estimated incidence of FBPase deficiency is variable among 1:350,000–1:900,000 in European countries (Visser et al., 2004). However, studies evaluating patients of the American continent are scarce; only one study included patients of North American origin (Herzog et al., 2001). Therefore, the present study is the first one to describe FBPase-deficient patients from Latin America. The patients analyzed come from the Rio Grande do Sul (RS), Southern Brazil, a region that presents a predominantly European genetic background, as well as the other geographic regions of the country but to a higher proportion (Rodrigues de Moura et al., 2015). The state population is 10,693,929 (Census 2010 IBGE – Brazilian Institute of Geography and Statistics), considering the number of samples it can be inferred a prevalence of FBPase deficiency of 1:1,782,321, approximately. Probably not all patients in the state were sampled, but the reduced sample size may indicate the rarity of the disorder in the Brazilian population. The genetic analysis of the FBP1 has been performed as a noninvasive method to diagnosis FBPase deficiency. Since 1995, when the first pathogenic variant in FBP1 was detected in a Japanese patient (Kikawa et al., 1995), 48 pathogenic variants (Fig. 2) were described in FBPase deficiency patients around the world (Human Gene Mutation Database - HGMD®). In most published cases, FBP1 has been evaluated by automated Sanger sequencing (Asberg et al., 2010; Ijaz et al., 2017; Moey et al., 2018). This study applied NGS to perform the genetic diagnosis of patients with FBPase deficiency. The analyses failed to detect one allele in patient C (Table 2). Failures in the amplification process of exons 2 and 5 resulted in a low sequencing depth of these regions (Supplementary Table 1), which led to the nondetection of the variant c.472C>T in exon 5 in this patient. Analyses carried out excluded the presence of a polymorphism in the NGS primer annealing site and secondary structures of PCR product interfering in the amplification of exon 5. The analysis of the NGS reverse primer sequence revealed that it has three tandem repeats of the “GAT” sequence. On exon 2, the NGS forward primer was designed with three tandem repeats of the dinucleotide “CT”. These repeats in the primers may cause slipping along the primed segment of DNA or secondary structures, such as hairpin loops (Lorenz, 2012), that affect its appropriate annealing. Therefore, we performed automated Sanger sequencing of exons 2 and 5 in all patients, confirming the NGS findings and the genotype of patient C. Cases such as this one reinforce the American College of Medical Genetics and Genomics recommendation for the use of a second test to confirm the variants reported by NGS when the sequencing depth of the region is not appropriate (Baudhuin et al., 2015). Despite this, NGS has been shown to be a suitable tool for the molecular diagnosis of metabolic disorders, including FBPase deficiency (Li et al., 2017). In the meantime, particular attention to the quality of sequencing is necessary.