DBT Gene Mutation Among Maple Syrup Urine Disease (MSUD) In Malaysian Population

—Maple syrup urine disease (MSUD) is an autosomal recessive genetic disease and an inherited autosomal recessive trait. It is characterised by a deficiency of an enzyme complex, resulting in an excess of branched-chain amino acids (BCAAs), which are toxic to the nervous system. Our four cases generally presented with lethargy and poor feeding weeks after birth. They were all treated for sepsis until the laboratory results showed high levels of BCAAs, which indicated MSUD. Genetic analysis showed that the four cases were homozygous for the DBT gene mutation c.1196C>G (p.S399C), a possible founder mutation. All of our cases were managed accordingly, with regular monitoring of the BCAA levels. Dietary support, with infant formulas free of BCAAs, was provided to all four cases with regular follow-up at the paediatric genetic clinic. All cases had spastic diplegic and developmental delays.


I. INTRODUCTION
Maple syrup urine disease (MSUD) is an inherited autosomal recessive genetic disorder.It was first described in 1954 in a family with four successive affected new-borns who developed progressive neurological disease within the first week of life.MSUD is characterised by a deficiency of a branched-chain alpha-keto acid dehydrogenase (BCKAD) complex, which is required to metabolise the three branchedchain amino acids (BCAAs): leucine, isoleucine, and valine.
The worldwide incidence has been reported to be one in 185,000, and the incidence and prevalence vary among countries.MSUD affects about one in every 180,000 newborns in the United States [1].In Karachi, Pakistan, the incidence is 9.1% among aminoacidopathies [2].It is more common in populations with a high frequency of consanguinity, and there is no sex predilection.
MSUD is classified into five clinical subtypes: classical, intermediate, intermittent, thiamine responsive, and dihydrolipoyl dehydrogenase deficient.Patients appear normal at birth; however, they may present with feeding difficulties, with progression to lethargy and unresponsiveness.The classical subtype is the most common and severe type, manifesting as dystonia, vomiting, poor feeding, and seizures during the new-born period [3].
MSUD is diagnosed by the presence of clinical features with a high index of suspicion.The biochemical hallmarks of the disorder are an increase in serum BCAAs, alloisoleucine in plasma, and BCAAs and branched chain ketoacids (BCKAs) in urine.Accumulation of these three types of amino acids and their corresponding alpha-keto acids leads to progressive neurodegeneration and encephalopathy in untreated infants.Without early intervention and treatment, these infants will develop severe and permanent brain damage.They can succumb to death within the first months of life.
Early diagnosis is critical, and dietary intervention should be started immediately to manage MSUD.The two most important aspects of MSUD treatment are long-term management and the treatment of acute metabolic crisis episodes to prevent complications.The goal of treatment is to immediately reduce plasma BCAA and BCKA levels.Dietary restriction of BCAAs is necessary to maintain plasma BCAA concentrations at levels as close to normal as possible.This is essential for restoring and maintaining metabolic homeostasis in patients with MSUD.Here, we present four patients with MSUD who were diagnosed, treated, and monitored in our teaching hospital.

A. Case 1
A 13-day-old baby boy was admitted to the neonatal intensive care unit with a history of fever, lethargy, poor feeding, and fits.He is a product of a non-consanguineous marriage, and there is no family history of similar problems.On examination, the baby was in respiratory distress.He had choreoathetoid movements.The baby was treated for sepsis with antibiotics.The results of routine laboratory investigations were normal.A computed tomography (CT) scan of the brain showed cerebral oedema.The plasma amino acid profile showed valine (627 µmol/L), isoleucine (459 µmol/L), and leucine (3,602 µmol/L).Alloisoleucine was detected.Urine organic acid analysis showed marked elevation of 2-ketoisocaproate, 2-keto-3 methyl valerate, and 2-hydroxy isovalerate, with moderate elevation of lactate.The patient was diagnosed with MSUD, and treatment was started immediately.At the time of writing, the patient is undergoing regular BCAA monitoring.Genetic analysis was conducted and showed the homozygous DBT gene mutation c.1196C>G (p.S399C).Both parents were tested for the genetic mutation and confirmed as carriers.

B. Case 2
An eight-day-old baby girl was admitted to the neonatal intensive care unit with a history of lethargy, poor feeding, and fits.On examination, the baby was not in respiratory distress.She had opisthotonos and dystonic movements.Her reflexes were brisked.She is the product of a nonconsanguineous marriage.There is no family history of similar problems, and the patient was on mixed feeding.She was treated for sepsis with antibiotics.The results of routine laboratory investigations were normal.The plasma amino acid profile showed valine (1,039 µmol/L), isoleucine (686 µmol/L), and leucine (3,714 µmol/L).Alloisoleucine was detected.Urine organic acid analysis showed marked elevation of 2-ketoisocaproate, 2-keto-3 methyl valerate, and 2-hydroxy isovalerate, with moderate elevation of 4-hydroxy phenyl lactate and 2-hydroxy-3 methyl valerate.She was discharged well with regular BCAA monitoring.Genetic analysis was conducted and showed the homozygous DBT gene mutation c.1196C>G (p.S399C).Both parents were tested for the genetic mutation and confirmed as carriers.

C. Case 3
A 30-day-old baby boy presented with poor weight gain, irritability, and upper motor neuron signs.He was treated for meningitis and to rule out inborn metabolic errors.He is a product of a non-consanguineous marriage and the youngest of five siblings.His second brother died at the age of 28 days after being admitted for feeding intolerance and unexplained neurological problems.His other siblings were well.On examination, the baby was in respiratory distress and under nourished.His weight placed him in <3rd percentile, his length placed him in the 3rd-15th percentile, and his head circumference placed him in the 3rd-10th percentile.He was hypertonic, with brisk deep tendon reflexes and absent primitive reflexes.The baby was treated for sepsis with antibiotics.The results of routine laboratory investigations were normal.A CT scan of the brain showed a generalised hypodense appearance of the cerebral white matter, both thalamus, internal capsules, and brainstem, possibly due to ischemic changes.The plasma amino acid profile showed valine (804 µmol/L), isoleucine (385 µmol/L), and leucine (2,959 µmol/L).Alloisoleucine was detected.He was diagnosed with MSUD, and treatment was started immediately.At the time of writing, the patient is undergoing regular BCAA monitoring.Genetic analysis was conducted and showed the homozygous DBT gene mutation c.1196C>G (p.S399C).Both parents were tested for the genetic mutation and confirmed as carriers.

D. Case 4
A three-day-old baby boy was admitted to the neonatal intensive care unit with a history of fever, lethargy, and poor feeding.He is a product of a consanguineous marriage; his parents are cousins.There is no family history of similar problems.The patient was on mixed feeding.He was treated for sepsis with antibiotics.The results of routine laboratory investigations were normal.The plasma amino acid profile showed valine (718 µmol/L), isoleucine (804 µmol/L), and leucine (3127 µmol/L).Urine organic acid analysis showed marked elevation of 2-ketoisocaproate, 2-keto-3 methyl valerate, and 2-hydroxy isovalerate, with moderate elevations of acetoacetate, 4-hydroxy phenyl lactate, and 3hydroxy butyrate.He was diagnosed with MSUD, and treatment was started immediately.At the time of writing, the patient is undergoing regular BCAA monitoring.Genetic analysis was conducted and showed the homozygous DBT gene mutation c.1196C>G (p.S399C).Both parents were tested for the genetic mutation and confirmed as carriers.

III. DISCUSSION
MSUD is a potentially life-threatening metabolic disorder.The presentation begins with non-specific symptoms of neurological dysfunction, seizures, irritability, and poor feeding.These are followed by focal neurological signs, such as abnormal movements, increasing spasticity, and coma.If left untreated, patients can succumb to brain damage and death within weeks.
MSUD is inherited in an autosomal recessive manner.It results from mutations in one of three different genes: BCKDHA, BCKDHB, and DBT [4].These mutations result in the absence or decreased activity of BCKAD enzymes, which are responsible for breaking down BCAAs.The accumulated BCAAs produce toxic by-products (ketoacids), resulting in metabolic acidosis (Figure 1).All four cases presented to our hospital in infancy (Table 1).Based on the onset and presentation of these cases, they were categorised as classical MSUD.At birth, patients with classical MSUD seem normal; they develop symptoms weeks after birth.Generally, the clinical manifestation of MSUD depends on the severity of the BCKAD deficiency.Classical MSUD usually involves less than 2% enzyme activity [5].Patients present with lethargy, inactivity, poor feeding, and irritability.Their urine smells like maple syrup or burnt sugar due to ketonuria in the first weeks of life.
In Malaysia, clinical suspicion and the detection of inborn errors of metabolism play an important role in diagnosis.The initial step for the detection of BCAAs or any aminoacidopathies is urine sample analysis.In the past, urine samples were analysed using thin-layer chromatography, which was tedious and laborious.What's more, the results were often inconclusive because of the presence of other substances in the urine (e.g., drugs), and alloisoleucine (the pathognomonic marker for MSUD) cannot be detected using this method [5].
New methods have been developed for screening and confirmation.The introduction of new-born screening using tandem mass spectrometry (TMS) in the early 1990s improved the sensitivity and specificity of the detection of inborn metabolic errors [6].TMS is used to analyse dry blood spotted onto filter paper, and it is one of the most effective MSUD detection methods.Several inherited disorders can now be identified via new-born screening, and with this screening the numbers have increased more than five-fold [7,8].
In all our cases, a high index of suspicion of a genetic disease led to the submitting blood samples for testing to rule out inborn errors of metabolism.Blood was spotted on filter paper and sent to a centre that specialises in screening for inborn errors of metabolism using TMS.The results of all four cases suggested MSUD.Further quantification via high-performance liquid chromatography showed elevated levels of BCAAs (Figure 3) compared to the normal range (Figure 2) and the presence of alloisoleucine (Figure 4).Work-up for mutational analysis was performed for our four cases and both their parents.
Mutational analysis for MSUD among the Malaysian population has shown the BCKDHB gene to be the most affected gene compared to the BCKDHA and DBT genes [9].In all of our cases, the patients had a similar mutation in the DBT gene.They were all found to have a homozygous c.1196C>G (p.S399C) genotype.In all cases, both parents were carriers of the same mutation.
Physiologically, the DBT gene encodes the dihydrolipoyl transacylase (E2) subunit, an important specific protein in the enzyme complex.This enzyme complex is organised as a cubic core consisting of 24 identical E2 subunits with E1 and E3 (a specific kinase and a specific phosphatase, respectively) attached through ionic interaction.These enzymes are responsible for the regulation of the BCKD complex.Mutations found in these genes are thought to cause disease via the creation of a premature termination codon.Silico analysis has suggested that this mutation results in damage to protein function [9].Mutations in the DBT gene affect the production of E2 and regulation of the BCKD enzyme complex, resulting in the inability to metabolise the essential BCAAs leucine, isoleucine, and valine [15].
The mutation detected in our cases is a new gene mutation that has been observed among ethnic Malays.This finding suggests the possibility of a founder mutation in the DBT gene; however, further tests need to be conducted to confirm the founder effect among this population [9].Founder mutations in the DBT gene have been reported among the Filipino population [10] and in the general population of the Paiwan Austronesian Aboriginal tribe in southern Taiwan [11].In addition to the founder mutations in the DBT gene, founder mutations have also been identified in the BCKDHA and BCKDHB genes in three different populations [12][13][14].
A founder mutation is a mutation that appears in the DNA of one or more individuals who are founders of a distinct population.This mutation is initiated by changes in the DNA that can be passed down to subsequent generations.The mutations originate in long stretches of DNA on a single chromosome; indeed, the original haplotype is the whole chromosome.As generations progress, the proportion of the haplotype that is common to all carriers of the mutation is shortened (due to genetic recombination).This shortening allows scientists to roughly estimate the age of the mutation [12].There is an apparent correlation between DBT gene mutations and thiamine-responsive phenotype.The biochemical mechanism of the thiamine response in this type of mutation is currently under investigation [15].A study in South India, however, reported that patients with DBT gene mutations were non-responsive to thiamine supplementation and had worse prognoses than patients who did not have such mutations [16].
Based on the diagnosis and acute condition presented, all our cases were attended to and managed immediately.In the management of the acute state, the aim was to remove the BCAAs and their BCKAs.Peritoneal dialysis and hemofiltration are two of the most effective methods for critically ill infants; however, none of our cases underwent either of these procedures.Instead, they were all treated for sepsis with antibiotics, followed by dietary restriction of BCAAs to achieve metabolic homeostasis.BCAA levels were repeatedly measured and monitored during the crisis, with strict monitoring of other biochemical parameters.
Our cases were discharged well, and they were started on specialised milk and thiamine supplements.Support and counselling sessions were provided to the parents.However, despite the restrictions on the consumption of certain proteins and the specialised diet, the risk of metabolic crisis still exists for our cases due to the inadequate supply of specialised milk and co-existing infections.They have been managed accordingly during each crisis, with regular follow-ups at our genetic clinics.Since diagnosis of MSUD is time sensitive, a delay in diagnosis is invariably associated with learning disability.Correlation between phenotype and genotype does not depend on the clinical situation; however, neurological deterioration is directly associated with early diagnosis and treatment [17].All of our cases had some form of mild-tomoderate learning difficulties, and this disability was further enhanced with episodes of crisis that had caused global developmental delay.
IV. CONCLUSION Several types of mutations associated with MSUD have been reported in Malaysia.All of our patients were in the category of classical MSUD with confirmed DBT gene mutation and the possibility of founder mutation.They presented early during infancy and were treated for sepsis.Despite the administration of aggressive treatment to prevent complications, all cases had developmental delay.Protein restrictions via a specialised diet and artificial formulas were provided to all our cases to ensure they received the necessary nutrients for growth and development.Their BCAA levels are being evaluated regularly, and their growth and development are being closely monitored.

Figure 1 .
Figure 1.Metabolic pathway of BCAA (valine, leucine, and isoleucine) degradation.The degradation of the essential BCAAs is impaired by BCKAD deficiency.

Figure 2 .
Figure 2. Plasma amino acid levels of a normal person.

Figure 3 .
Figure 3. Plasma amino acid levels of one of our patients with MSUD showed high leucine, isoleucine, and valine levels.

Figure 4 .
Figure 4. Chromatogram showing alloisoleucine detection in one of our patients.Alloisoleucine is a by-product of isoleucine transamination.

TABLE 1 .
Summary of the four cases diagnosed with MSUD: Plasma BCAA values and organic acid analysis findings at presentation.