Identification of Four New Mutations in Chilean Patients with Various Forms of Maple Syrup Urine Disease and Genotype-Phenotype Correlations

Background: Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disease caused by deficient activity of the branched-chain α-keto acid dehydrogenase (BCKD) enzymatic complex. BCKD is a mitochondrial complex encoded by four genes: BCKDHA, BCKDHB, DBT, and DLD. MSUD is predominantly caused by mutations in the BCKDHA, BCKDHB, and DBT genes encoding the E1α, E1β, and E2 subunits of the BCKD complex, respectively. The aim of this study was to characterize the genetic basis of MSUD by identifying point mutations in the BCKDHA, BCKDHB, and DBT genes in a cohort of Chilean MSUD patients, and to describe their phenotypic heterogeneity. It is a descriptive cross-sectional study with 18 MSUD patients was carried out using PCR and DNA sequencing. Results: Four new pathogenic mutations were identified in the 18 patients that were analyzed: one in BCKDHA (p.Thr338Ile); two in BCKDHB (p.Gly336Ser e p.Pro240Thr); and one in DBT (p.Gly406Asp). Four additional pathogenic mutations found in the analyzed patients have been described previously. Conclusion: There were no correlations observed between genotypes and phenotypes. Thus, if MSUD is diagnosed more promptly, such as in neonatal screening, it might be possible to establish genotype-phenotype relationships more efficiently.

Many mutations that cause MSUD have already been described involving the catalytic subunits of the branched chain α-keto acid dehydrogenase (BCKD) complex, and, according to altered loci, three genetic subtypes have been proposed: type Ia (MIM # 608348) for mutations found in BCKDHA (subunit E1α), type Ib (MIM #248.611) involving mutations found in BCKDHB (subunit E1β), and type II (MIM # 248610), with mutations in the DBT gene (subunit E2) (5,6). BCKDHA has been mapped to human chromosome 19, at 19q13.2. The gene encompasses approximately 27.2 kb of DNA, with coding sequence distributed as 9 exons and involving 1791 bp. BCKDHB is situated at 6q14.1, spans approximately 240 kb of genomic DNA, and has 11 exons encoding 1572 bp. DBT, at 1p31, has 63 kb of DNA, also has 11 exons and coding sequence of 10831 pb (3,7). Considering these three determinate disease genes, more than 140 mutations are known, and have already been described in the literature. (8).
MSUD is inherited in a recessive autosomal fashion, and its global incidence is estimated at 1 in 185,000 newborns. Although it is a rare defect, in some Mennonite populations settled in Pennsylvania, and in some cities of the United States, the estimated elevated incidence is 1 in 200 live births (3). A study carried out in Portugal by Quental et al. (2010), based on an analysis of cases diagnosed by mass spectrometry, found an incidence of 1 per 86,800 newborns (9). In Brazil, a study conducted by Margutti (2015) identified 11 new mutations in 25 patients with the disease, with three in the BCKDHA gene (p. Pro39Leu, p. Gly56Arg, and p. Tyr120Ter), six in BCKDHB (p. Arg63Pro, p.
The clinical manifestations of patients with MSUD are varied, and depend on the levels of residual enzyme activity. The clinical phenotypes associated are classified as classic, intermediate, intermittent, responsive to thiamine therapy, and lipoamida dehydrogenase deficiency (E3 subunit), depending on, among others, the age of onset and severity of the disease. In the classical form of MSUD, with less than 3% residual enzyme activity, symptoms appear soon after birth. Patients typically have ketosis and plasma concentrations of LEU over 2,000 μmol/l. In untreated newborns, maple syrup odor can be detected in earwax within the first 12-24 h, and in urine 48-72 h after birth.
Elevated plasma concentrations of BCAA, as well as widespread disturbances to plasma concentrations of amino acids are present at 12-24 h of age; elevation of keto acids and ketonuria, irritability, are observed 24-72 hours after delivery; encephalopathy manifesting as lethargy and intermittent breathing difficulties are seen after 4 to 5 days of life; and coma and central respiratory failure can occur between 7 to 10 days (1).
The intermediate form of MSUD features in infancy and childhood, being characterized by psychomotor developmental delay, failure to thrive, seizures and walking difficulty. Ketosis and plasma concentrations of LEU less than 2,000 μmol/l are typical. Although BCAA elevation is persistent, and there is neurological impairment, it is not seen in severe newborn organ decompensation of the classical form. Enzymatic activity in these cases is between 3 and 30% (2).
The intermittent form appears during infancy or childhood, and the patient usually has normal growth and development, with, bouts of ataxia that may be accompanied by ketoacidosis. The increase in BCAA only happens during decompensations.
The sensitivity to thiamine form has a clinical presentation similar to intermediate or intermittent cases, without acute decompensation. Thiamine is a subunit E1 cofactor, regulating the activity of all of the enzyme complex. Thus, the administration of thiamine decreases serum levels of BCAA. The doses of thiamine used may vary from 10 to 1,000 mg per day (11).
The E3 subunit deficiency form is very rare, having been reported in approximately 20 cases from around the world. The prognosis for this form of the disease seems to be associated with residual enzymatic activity between 0% and 25% (11).
There are no literature studies that describe genotypic profiles of MSUD patients in the Chilean population.

Objectives
We aimed to identify mutations in the genes BCKDHA, BCKDHB and DBT in a cohort of Chilean patients clinically diagnosed with MSUD, and to analyze the clinical characteristics of patients, in order to identify possible genotype-phenotype correlations.

Patients
The nutrition team from the Genetics and Metabolic Diseases Laboratory of the Nutrition and Food

Molecular Analysis
DNA was extracted from peripheral blood leukocytes for the molecular analysis of the three genes involved in MSUD (BCKDHA, BCKDHB, and DBT).

Mutation Analysis
Sequencing results were visualized using FinchTV® version 1.4.0 software (Geospiza, Seattle, WA, USA) and compared with the relevant reference sequences from the GenBank® database (12). The nomenclature for the description of sequence variants detected was derived from the recommendations of the Human Genome Variation Society (http://www.hgvs.org/mutnomen) (13).
Sequence variants were also evaluated for their disease-causing potential using the Mutation Taster application (16).

Clinical Data
The nutrition team from INTA, Chile University, provided clinical data regarding the patients studied.
The clinical data provided were anthropometry at birth, complications during pregnancy, birth date, age of diagnosis, hospitalization, and clinical and laboratory tests for leucine, valine and isoleucine.

Statistical Analysis
Fisher's exact test was applied in a sample of patients with the more prevalent mutation, p.Ile214K, in an attempt to assess the degree of correlation between clinical and genetic variables, and to check the possibility of establishing genotype/phenotype relationships. Fisher's exact test is a statistical significance test generally used in contingency table analysis of small sample sizes. The probability of significance was adopted with p-values < 0.05.

Molecular Analysis
Of the 18 patients 88% presented with mutations in the BCKDHB gene, 1 patient had a mutation in [1217 G>A] in DBT (Table 1).
This study was able to identify regions with the highest incidence of mutations in exon 6 of BCDKHB.
Exons 4, 5, 9 and 10 exhibited 1 mutation in each, as depicted in the schematic representation of BCKDHB in Figure 1.

Mutation Pathogenicity
Among the mutations already described in the literature, the Ile214Lys mutation of Spanish origin had the highest incidence, totaling 77 % of the patients, followed by mutation Pro200*, also of Spanish origin in 33% (Table 2). Of these mutations, those found in the heterozygous state were p.Gly131Val, p.Pro200Stop, and p.Ile214Lys. Homozygous mutations identified were p.Gly131Val, and p.Ile214lys. The new mutation Pro240Thr was found in 16% of the samples; it was also located in exon 6 of BCKDHB, and was the most prevalent of the new mutations. After being subjected to in silico analysis, the new mutations were all considered pathogenic (Table 3). Of these new mutations, the ones found in the homozygous state were p.Thr338Ile, p.Gly406Asp and p.Pro240Thr. Those found as heterozygous were p.Pro240Thr and p.Gly336Ser.

Discussion
In this study, 5% of patients presented with the intermediate form of MSUD, and the majority, 95 percent, had the classic form, mainly due to early onset of symptoms, with prevalence of homozygous mutations observed in 61% of cases. It is interesting that in none of the patients had consanguinity been reported involving the parents, and in two cases there were historical reports of illness in the family. This fact might be linked to greater genetic homogeneity in the Chilean population. MSUD is initially diagnosed through clinical examinations, and by finding the peculiar odor of maple syrup in urine. BCAA serum analysis is the most convenient method, primarily in neonatal screening. If a patient has high dosages of leucine, valine, isoleucine, or alloisoleucine (>5 mm/L), MSUD must be considered (17). In addition, levels of leucine greater than 1,000 µmol/L are considered critical, as they can produce irrevocable damage, or even lead to death (18). Levels of leucine at diagnosis in our study participants ranged from 440 to 3962 µmol/L, and most children (72%) presented with more than 1,000 µmol/L. BCKDHB harbored the great majority of mutations, 6 of the 8 mutations found in this cohort are located on the E1b subunit. Among the mutations already described in the literature, Ile214Lys was the one here with the highest incidence, totaling 77% of the patients. The new mutation with high incidence was Pro240Thr, found in 16% of samples, and also located in BCKDHB This fact can also be verified with the p.Pro240Thr variant, a new mutation found, which, when homozygous, manifested in patient 8 as serious NPMDD, and also in patient 15 with light NPMDD.
High levels of leucine are seen at diagnosis in most critical patients, and this can produce permanent damage or even death (19). The results of a Brazilian study (18) on the spectrum of MSUD over the last two decades found no significant association between the severity of mental development delay and 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 can be considered a determining factor more important in cognitive and psychomotor development than initial levels of leucine (19). Early diagnosis and treatment in MSUD are crucial to reduce the severity of brain damage (1).
MSUD treatment includes diets low in protein, supplementation with BCAA-free formula, and symptomatic treatment during metabolic crises, such as ingestion of mannitol to treat brain edema, injection of insulin to lower blood glucose, and use of N-carbamylglutamate to reduce serum levels of ammonia (22).
As the liver is responsible for 15% of the production of BCKD, liver transplantation can restore enzyme activity in patients with MSUD (23). Liver transplantation may benefit patients with the classical form; however, it does not reverse the disease process. (24).
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 MSUD, with BCKDHA gene mutations; prenatal diagnosis for the fetus was performed (25). In a study by Li et al. (2015), prenatal diagnosis for a mutation in BCKDHB was also made (26). In both cases, treatment was started at birth, and diagnosis was possible due to awareness of an existing mutation in the family.

Conclusions
In this study, we found a total of eight mutations, of which four are not described in the literature: p.Thr338Ile, p.Gly336Ser, p.Gly406Asp, and p.Pro240Thr. Six of the eight identified mutations are located in exon 6 of BCKDHB, and this gene was the locus that presented the most frequent mutations in this population, with the I214K variant present in 77% of the patients.
Considering the age at disease diagnosis, among the new mutations, patients with p.Thr338Ile, p.Gly336Ser, and p.Gly406Asp exhibited a classic manifestation, with early onset of symptoms: only p.Pro240Thr presented diagnosis at a later time, manifesting as an intermediate phenotype in one patient. The biochemical values for BCAA levels and clinical evolution in those patients were not consistent with those described in the literature for these clinical phenotypes.
We found no association between initial levels of leucine and the severity of MSUD. This can be attributed to the fact that long-term metabolic control can be considered a determining factor that is more important in cognitive and psychomotor development than initial levels of leucine.
If MSUD were to be diagnosed more promptly by neonatal screening, perhaps it would be possible to establish genotype-phenotype associations more efficiently.  Correlation between leucine values in μmol/L and age at diagnosis in days, with a p value of 0.989.