Only 5% of the patients in this study presented with an intermediate form of MSUD, while the majority, 95%, had the classic form of the disease. These classifications were made primarily as a result of the early onset of symptoms, with a high prevalence of homozygous mutations, 61%. It is interesting that none of the patients had consanguinity involving the parents, although in two cases there were historical reports of illness in the family. This fact might be linked to the greater genetic homogeneity in the Chilean population originating from their indigenous origin. The overwhelming majority of Chileans are the product of varying degrees of admixture between European ethnic groups (predominantly Spaniards) with Amerindian, peoplesindigenous to Chile's territory.
Although the historical mixing of Europeans and Amerindians is evident across all social strata in the Chilean population, there is a strong correlation between the ratio of a Chilean's European and Amerindian genetic components and his or her socioeconomic situation (16).
There is a marked increase in Amerindian ancestry in the lower social classes in Chile, with an increasing European component in the upper classes. Indigenous inheritance, whether cultural or genetic, is most pronounced in rural areas and in aspects of the culture including Chilean cuisine and the Chilean Spanish language (17).
The largest portion of the participants in this study were from Santiago (44%), Chile`s capital. With 73% considered lower class and 27% middle class. Their ancestry was predominantly Spanish (88% of cases). Although none of them had a surname of indigenous origin, it is known that the vast majority of this population is a genetic admix.
An autosomal DNA study from 2014 found that Chile possesses a gene pool with an average admix of 51.85% (± 5.44%) European, 44.34% (± 3.9%) Amerindian, and 3.81% (± 0.45%) African. This study was conducted across all regions of Chile. When this data was stratified by social class and region they were able to show that the average Santiago residents admix was strongly influenced by their social class. With the lower class exhibiting an admix of 51% European and 49% Amerindian, the middle class 70% European and 30% Amerindian and the upper class 91% European and 9% Amerindian (18).
MSUD is initially identified by clinical examinations and is often first suspected following detection of the peculiar maple syrup odor in patient’s urine. BCAA serum analysis is the most convenient method for diagnosis and is primarily done during neonatal screening. If a patient has high levels of leucine, valine, isoleucine, or alloisoleucine (>5 mm/L), MSUD must be part of a clinicians differential diagnosis (19). In addition, levels of leucine greater than 1,000 µmol/L are considered critical, as they can result in long term organ damage, or even lead to death (20). Leucine levels within our study ranged from 440 to 3962 µmol/L at the time of diagnosis, and most of the participants (72%) presented with leucine values of more than 1,000 µmol/L.
The BCKDHB gene harbored the majority of the mutations, with six of the eight mutations from this cohort located in the E1b subunit. The Spanish mutation Ile214Lys was the known mutation with the highest incidence and was detected in 61% of the patients. The novel mutation with the highest incidence was Pro240Thr which was identified in 16% of the samples. Notably both mutations occur in exon 6 of BCKDHB.
According to Rodríguez-Pombo and collaborators (2006), the p.Ile214Lys mutation is responsible for reducing the stability of the coding protein causing a classic form of the disease. In our findings, we observed symptom variability in patients with this mutation: patients 5, 9, 11, 12, and 18 had a homozygous p.Ile214Lys mutation, while children 9 and 11 presented with serious NPMDD, child five exhibited only mild NPMDD.
Other factors that need to be considered include the level of leucine and the evolution of the disease. It was observed that patient five, with a leucine value of 2600 μmol/L at diagnosis, showed only light NPMDD, while patient 11, with a leucine level of 440 μmol/L had serious NPMDD. Therefore, we found no association between the initial leucine levels and disease severity. Patients 11 and 12 are siblings, have homozygous p.Ile214Lys mutations and their initial values of leucine were very different. Patient 12 is the eldest, born in 1997; and it took 90 days to establish his MSUD diagnosis, while his younger brother, born in 2006, obtained a diagnosis in only 9 days. This rapid diagnosis was probably aided by the history of illness in the family, improvements in diagnostic testing for MSUD, as well as the previous genetic counseling that this family had already received. The earlier diagnosis almost certainly helped reduce the severity of MSUD.
This kind of symptom variability was also observed in the p.Pro240Thr variant. When this appeared as a homozygous mutation it manifested as serious NPMDD in patient 8 but light NPMDD in patient 15.
High levels of leucine are seen at diagnosis in most critical patients, and this can result in permanent organ damage or even death (21). The results of a Brazilian study (20) on the spectrum of MSUD over the last two decades found no significant association between the severity of mental developmental delays and the levels of leucine at the time of diagnosis, which is consistent with our observations. This can be attributed to the fact that long-term metabolic control is a more important factor in cognitive and psychomotor development than initial levels of leucine (21).
When evaluating the novel mutations p.Thr338Ile, p.Gly336Ser, and p.Gly406Asp and the age of diagnosis in the participants, most of these mutations seem to result in classic MSUD, with early onset of symptoms. Only p.Pro240Thr was identified in patients (1) with later disease onset, favoring an intermediate phenotype classification. However, one of the major problems observed here is the time between onset of symptoms and the diagnosis in locations without neonatal screening for MSUD, like in Chile. One of the patients with a homozygous p.Pro240Thr mutation was classified as an intermediate phenotype because of the diagnosis age but this patient remained in hospital for 90 days. His diagnosis was at 210 days, but how much time passed between onset of symptoms and diagnosis is unknown. It is worth noting, as discussed before, that the BCAA values and clinical evolution of this patient was not consistent with that described in the literature for intermediate phenotypes. Thus, a patient’s clinical situation is not necessarily the best way to correlate phenotype and genotype, because neurological deterioration is directly associated with the absence of early diagnosis and, therefore, with the absence of adequate nutritional treatment, which is fundamental to the control of BCAA concentration. In a study by Morton et al. (2002), in which patients had prompt access to a metabolic formula, and where the clinical protocol was followed in the acute early stages of the disease, patient outcomes were better and patients were able to reach more age appropriate developmental milestones (22).
Although some patients were diagnosed in the first month of life, it is known that the time between diagnosis and receipt of metabolic formula can be long and variable.
Therefore, if MSUD was diagnosed more rapidly, by neonatal screening, perhaps it would be possible to establish genotype-phenotype associations more efficiently. MSUD meets most of the criteria from Wilson and Jungner (1968) for neonatal screening (23). In countries where MSUD is included in neonatal screening, patients are usually diagnosed before the 10th day of life. While in countries where it is not included in public neonatal screening programs, such as Chile, diagnosis is usually delayed.
MSUD treatment includes the application of low protein diets, supplementation with BCAA-free formula and symptomatic treatment during metabolic crises, including ingestion of mannitol to treat brain edema, injection of insulin to lower blood glucose, and use of N-carbamylglutamate to reduce serum ammonia levels (24).
As the liver is responsible for 15% of BCKD production, liver transplantation can restore enzyme activity in patients with MSUD (25). Liver transplantation may benefit patients with the classical form of the disease; however, it does not reverse the disease process (26).
Prenatal diagnosis is important to identify defects before birth, especially with difficult to treat conditions. Accurate genetic analysis of probands has allowed DNA-based prenatal diagnosis of single gene disorders. You et al. (2014) reported a case of a Chinese family with a BCKDHA gene mutation; where the fetus underwent prenatal diagnosis (27). In a study by Li et al. (2015), they were also able to identify a BCKDHB mutation in a prenatal screen (28). In both cases, treatment was started at birth, and diagnosis was possible as a result of the increased awareness of the care givers and family.