To date, this is the first study in Brazil to identify variants in the BCKDHA, BCKDHB, and DBT genes in clinically and biochemically diagnosed MSUD patients enrolled in the MSUD Network. We found that out of the 42 alleles, the disease-causing variant was located in the BCKDHA gene in 23.8% (5 patients), in the BCKDHB gene in 57.1% (12 patients), and in the DBT gene in 19.1% (4 patients).
Some of the novel variants have already been detected in database projects involving the search for variants in a large number of individuals but never related to patients. We investigate the variants in “The Exome Aggregation Consortium” – ExAC – composed of 60,706 unrelated individuals, and the Online Archive of Brazilian Mutations – AbraOM – composed of 609 elderly individuals (16,17). Three of 11 novel variants were in ExAC, all in very low frequencies (Table 2).
Nellis and Danner (6) studied 63 patients diagnosed clinically with MSUD and found the following frequency of mutations: 33% in the BCKDHA gene, 38% in the BCKDHB gene, and 19% in the DBT gene. In agreement with the results of the present study, most patients (of varied ethnicity) evaluated by Henneke et al. (22) presented mutations of the subtype Ib, followed by Ia, with the least frequent mutation occurring in the DBT gene (subtype II). In a cohort of 32 unrelated Turkish patients, out of 64 alleles, the disease-causing mutation was located in the BCKDHA gene in 37% (12 patients), in the BCKDHB gene in 44% (14 patients), and in the DBT gene in 19% (6 patients) (23). However, Quental et al. (5), in a study with 30 Portuguese patients, found 17 mutations with no difference in the frequency of these genes, with six in the BCKDHA gene, five in the BCKDHB gene, and six in the DBT gene. A study published by Abiri et al. (24) investigating 20 Iranian families of MSUD patients, six patients demonstrated homozygous haplotype for the BCKDHA gene, nine for the BCKDHB gene, and two for the DBT gene. Gupta et al. (26), studying 24 unrelated Indian patients, found 20 mutations in 22 of the patients, with 11 novel mutations - four in the BCKDHA gene, six in the BCKDHB gene, with a novel mutation identified in the DBT gene.
Li et al. (27) described eight cases of MSUD (in four females and four males) from unrelated Chinese families who were diagnosed through serum BCAA and genetic analysis between nine days to one year and eight months of life (six patients presented the neonatal form of the disease); 12 different mutations were found with six in the BCKDHA gene, five in the BCKDHB gene, and one in the DBT gene, of which only one mutation located in the BCKDHA gene had been reported in the literature.
In agreement with the present study, all the Spanish patients studied (belonging to a cohort of 33 MSUD patients) by Rodríguez-Pombo et al. (8) who had a mutation in the BCKDHB gene (subtype Ib) also had the classical phenotype; however, inconsistencies were found between the biochemical parameters of the patients and their clinical phenotype, as in the case of three patients who were homozygotic for the same mutation and exhibited a residual enzymatic activity variable between < 1 to 13%.
Abiri et al. (28) reported six Iranian patients who were homozygous for mutations in the BCKDHA gene, demonstrating that most of the mutations found were in exons 2, 4, and 6. Recently, 20 new mutations were identified in the BCKDHA, BCKDHB, and DBT genes of 52 MSUD patients from Saudi Arabia, with no mutation in the DLD gene identified (29).
The leucine levels at diagnosis were high and are, in the majority of the studied patients, considered critical, and can produce irreversible damage or even death of the patient (30). However, the results of a Brazilian study (31) on the MSUD panorama over the last two decades in the country did not indicate a significant association between the severity of developmental delay and leucine levels at diagnosis; this can be attributed to the fact that early diagnosis and long-term metabolic control are considered more decisive factors for psychomotor and cognitive development than leucine levels at the time of diagnosis. Therefore, regarding the characteristics and clinical situation of the patients, it is thought that the association of phenotype and genotype is not ideal in that neurological deterioration is directly associated with delayed diagnosis and thus the absence of appropriate nutritional support, which is essential for the control of BCAA serum levels as well as those of their metabolites (metabolic control).
Although some patients were diagnosed in the first month of life, as can be seen in Table 1, diagnosis was not made at the beginning of the presentation of symptoms in the majority of patients (only patients 6, 11, and 15 were diagnosed at the time at which symptoms appeared and patient 18 was diagnosed when still asymptomatic). In Brazil, the time between diagnosis and receipt of the metabolic formula is long and variable (31), which can also be observed in the registry of the clinical files of patients in our study. Only four patients (patients 4, 6, 11, and 18) received the metabolic formula for treatment of MSUD immediately after diagnosis; receipt varied for the other patients between ten days and two months after diagnosis, delaying appropriate treatment of the disease. When patients are diagnosed in the acute phase of the disease during a hospital stay due to metabolic decompensation, treatment with a metabolic formula is started at the time of diagnosis only if the formula is available at the admitting hospital. In contrast, patients diagnosed in the non-acute phase of the disease and treated on an outpatient basis are prone to receive the formula late, with the guarantee of access to the metabolic formula occurring only through judicial measures (31). These facts reinforce the need to include neonatal screening for MSUD in the Brazilian Newborn Screening Program.
Molecular genetics analysis is critical for the accurate diagnosis of MSUD patients, can aid in identifying variant phenotypes and offer guidance on prognosis and treatment. In addition, the molecular screening of affected patients provides epidemiological data for implementation of the test in populations, facilitating prenatal diagnosis in families at risk (28,26).
Flaschker et al. (32), who described mutations in 15 individuals from Germany, Austria, and Switzerland with different phenotypic variants of MSUD, concluded that in the cohort of patients analyzed, the most severe enzymatic and clinical phenotypes of the disease forms (MSUD variants) were associated mainly with specific genotypes in the BCKDHA gene, whereas milder enzymatic and clinical phenotypes were associated with specific genotypes in the BCKDHB and DBT genes; the results of this study led to the conclusion that genotyping may be predictive of metabolic and clinical phenotype and may have a prognostic value, particularly in individuals with a variant of MSUD identified in neonatal screening such that early treatment will effectively delay the natural course of the disease.
However, in a study by Gupta et al. (26), it was not possible to establish any genotype and phenotype correlation in Indian MSUD patients; most of the cases (66.6%) had the classic neonatal type of MSUD, most of the classic neonatal patients (10 of 14 cases) having mutations in the BCKDHB gene.
Patients with the intermittent form of the disease, evaluated by Tsuruta et al. (33), presented mutations exclusively in the DBT gene, giving rise to low but significant residual activity of the BCKD complex. However, in the present study, no patient with the genetic subtype EII presented this phenotypic form of the disease.
Wang et al. (34) suggested that DNA sequencing associated with in silico analysis is a simple and rapid method to predict the severity of MSUD and that neonatal screening has been demonstrated to be an effective approach for screening MSUD patients in a diverse ethnic population (32), allowing early intervention with dietary therapy (34). The identification of different missense-type mutations provides valuable evidence for the accuracy of the analysis, as well as the determination of its effect on the catalytic subunit. DNA sequencing analysis, together with in silico analysis, has been suggested to predict the clinical manifestations of MSUD, since it is a simple and rapid method (34).
According to Feier et al. (12), liver transplantation provides metabolic control, allowing the patient to resume a normal diet and avoid further neurological damage, helping the individual's neurodevelopment as well as eliminating the chance of cerebral edema and death. In this case report, Feier et al. (12) described the situation of a 2-year-old child with MSUD who underwent liver transplantation (domino transplantation); the donor was the patient's mother. The use of living relative donors has been controversial because parents are mandatory heterozygotes, but in this case, the data suggest that the use of a donor associated with MSUD was effective. Although the traditional treatment during the maintenance phase is basically the dietary restriction of BCAA and supplementation with thiamine and a food formula without BCAA, liver transplantation is indicated in the literature as a good treatment option (12,13,18).
No somatic cell complementation studies were performed to assign enzymatic subtypes Ia, Ib or II, which can be a limitation. Another limitation of this study is about the ability or lack thereof of performing deletion/duplication analysis at the exon or whole gene level, and this can be related to not being able to make a molecular diagnosis in some of the patients studied. In addition, other molecular characteristics (type and location of mutation) were not considered in this analysis of this study.