1. Genetic ancestry in MPS VII
The first case diagnosis of an MPS VII patient in Northern Brazil was in 2018. There are rare reports of the disease around other countries, and no more than 20 patients were found in Brazil [21]. MPS VII is a rare metabolic disorder. Therefore shared genotype referring to the most frequent variant (p.Leu176Phe) in the same country is an unusual case. Knowledge about the ancestry of populations has been considered an important tool to aid in the application of pharmacogenetics [22] and genomic medicine [23]. These themes contribute to the elucidation of existing differences in the clinic of certain diseases.
In this study, it is possible to observe the high European contribution followed by the Amerindian among patients with MPS VII from different regions. The results indicate that MPVII patients (n: 5) present a 52% contribution related to European ancestry, differing only statistically from African ancestry (p < 0.05). If the number of analyzed patients were more considerable, differences between ancestry contributions could be better estimated. The compound heterozygous patient had the highest European ancestry (60%), followed by Amerindian ancestry (22%) and African (18%).
The high contribution of Europeans to the Brazilian population has already been indicated in some studies [13, 24, 14]. However, there is divergence in its proportion between the different regions of Brazil, which is higher in the South and Southeast (77.7% and 73.7%, respectively), while in the North and Northeast regions, they indicated a contribution 69.7% and 60.06% for markers of European origin [25]. Although studies related to the Brazilian distribution agree on the genomic predominance of Amerindians in the North and Africans in the Northeast [26, 27].
There is some hypothesis considered in the initial design of this study. This hypothesis is related to the predominance of the p.Leu176Phe variant in the cohort of patients with MPS VII in Brazil. Possibly, the most prominent European ancestral contribution was more determinant for this group, and this variant originated from a founder effect caused by the migration of the Portuguese population or other European populations in one of the settlement events in the country.
Another hypothesis is related to the migration of populations from America. Mexico is a country that presented a high number of cases diagnosed with MPS VII as highlighted by Mendoza-Ruvalcaba et al. [28] (2020). Thus, there is a possibility that some populations have contributed to the spread of the variant in Brazil since there were migration events from North America to South America, and the Mexican population presents an ancestral contribution of Amerindians and Europeans [22].
In addition, it is necessary to consider some historical issues in the formation of the cities where patients with MPS VII were born. In Fig. 1, it is possible to notice two nearby cities in Bahia: Araci and Tucano. This proximity instigates the questioning of the potential social interactions of these places in recent centuries and, consequently, the perpetuation of the p.Leu176Phe variant. Giugliani et al. [9] (2021) emphasized the highest number of MPSVII cases in the northeast region, specifically in Bahia (5): 1 in Monte Santo, 2 in Araci, and 2 in Tucano. Except for Araci, where two brothers with MPS VII are confirmed in the same family, there are questions about the possible common origin connecting these locations to the incidence of the disease and whether the frequency of the pathogenic variant is higher than what is supposed in Bahia.
A similar situation has been reported with evidence of Iberian origin for one of the rarest variants (Ser341Arg) among patients with MPS IVA in the Northeast Region of Brazil [29]. However, the cohort of MPS VII patients in the Iberian Peninsula presents more diverse pathogenic variants than the one predominantly found in Brazil [30].
Brazil is an admixture country in terms of its ethnic composition, so there is the possibility that a founding mutation has occurred that has dispersed, causing the appearance of this same mutation in different regions of the country. Even if the origin of this mutation common among the patients comes from the emergence of independent mutations, a situation like this is unlikely given the need for this event to occur individually several times in different locations and due to the conservative state of the sequence region of amino acids where the p.Leu176Phe mutation is found [31]
2. GUSB expression and in silico prediction of the new variant
Considering the new variant (p.Leu292Pro), the differential expression for the MPSVII patient is more prominent than in the control group (Fig. 4). The fold change indicates a higher relative expression for GUSB. This result does not reflect the increase in activity of the β-glucuronidase enzyme since it showed lower in the patient: plasma was 0.7 nMol/h/mg (reference value 30–300 nmol/h/mL) and in leukocytes 0.9 nmol/h/mg protein (reference value 23–151 nmol/h/mg protein). These findings are new in the literature.
Twins with MPS VII, homozygous for p.Leu176Phe observed by Wu et al. [32] (1994), is a situation similar to this study. In this case, cell lines with this variant showed low β-glucuronidase activity and expressed the same amount of enzymatic protein as wild-type cell lines. For those strains that expressed four times the mutated protein, the β-glucuronidase enzymatic activity was higher than the wild-type strain in the early stages and decreased as time passed. According to this study, the occurrence of overexpression would be due cause by a non-pathogenic variant (p.Pro649Leu) that would partially activate a new site in the β-glucuronidase enzyme. Thus, correcting the deficient activity of the protein, at least caused by missense variants in MPS VII partly.
The modifications caused by the p.Leu176Phe variant in the β-glucuronidase enzyme have already been described in the study by Khan et al. [31] (2016) and other variants for MPS VII. This alteration causes changes in stability in a very conserved region of the protein due to the increase in the strength of hydrogen bonds. However, the actual cause of the increased expression has yet to be clarified. This discordance between GUSB gene mRNA levels and β-glucuronidase activity may be due to post-transcriptional regulation, similar to what was pointed out by the polymorphism in the study by Wu et al. [32] (1994).
Specifically regarding the individual analyses, the MPS VII patient has a higher amount of cDNA, indicating greater gene expression when compared to the control group. Other studies have suggested higher expression of the GUSB gene for certain diseases, such as inflammatory and liver diseases and some types of cancer, for example, [33, 34]. And as one of the biomarkers for changes in memory in Alzheimer's disease [35]. In the population, it is common to find expression variations for specific genes [36] due to a series of environmental or not factors. This fact occurred in 6 individuals in the control group who had high expression of the GUSB gene.
Regarding the similarity between the father/ MPS VII patient amplification cycles, it is possible to assume that the p.Leu176Phe variant, present in the father, interferes less in the expression of GUSB gene transcripts than the new variant (p.Leu292Pro) present in the mother of the patient. In this case, the mother showed a lower gene expression of the transcripts when compared to the patient and his father (Fig. 6B).
Interesting data is related to the assay of beta-glucuronidase protein in the patient's parents and the respective variants present in each one. The patient's mother (p. Leu292Pro) had beta-glucuronidase activity around 14.56 nmol/h/mL, below the reference value for this enzyme. On the other hand, the patient's father (p.Leu176Phe) had an enzyme activity of 43.5 nmol/h/mL. Possibly one of the variants interferes more with gene expression than the other and, consequently, with the protein phenotype.
The in silico study for the new mutation indicated the change to a shorter amino acid residue. And the variant is located in a region of the protein conserved among many species, such as Xenopus tropicalis (frog) and Danio rerio (zebrafish) (PolyPhen-2, access April 8, 2023) as well as other residues for other species. Thus, the substitution for proline in the protein sequence may be responsible for a more moderate MPS VII phenotype since it modifies the interaction with the protein domains.
The modifications caused by the replacement of the Leucine residue by one of Proline at position 292 allow new polar and non-polar alterations with nearby structures, such as with 293Met (Fig. 7). Furthermore, it can modify interactions with protein domains, such as the beta-galactosidase/glucuronidase superfamily. The regions referring to these amino acid residues are highly conserved among organisms (Fig. 8), and the amino acid proline has not been described among species.
Our study is the continuation of a previous work that identified a new variant in the GUSB gene in a patient with MPS VII [9]. This study is a pioneer in evaluating how ancestry can be an essential parameter to define the origin of variants in the GUSB gene in patients with MSVII in Brazil. There are several ways of interpreting the pathogenicity of a new variant of this, provided there are adequate resources for the analyses. Determining the frequency of the variant in a representative amount of the population would be an initial alternative, although it does not explain the damage to the protein structure. The in silico studies arise in a strategy to predict how much the substitution of Leucine for Proline can impair the three-dimensional model and the kinetic and structural properties of the GUSB protein, despite not informing the degree of pathogenicity of the variant. Thus, expression studies are necessary to understand how transcripts can confer greater or lesser stability to the enzyme in question. The next step would be to use in vitro models to evaluate how the new variant p.Leu292Pro interferes with the activity of the GUSB protein. The group of these tools would be proper for a better understanding of genotype and phenotype relationship in patients with MPS VII, mainly in Brazil.