The Invitae Elevated Leucine Panel analyzes five genes that are associated with elevated leucine on newborn screening (NBS) or plasma amino acid analysis. This panel is indicated for any individual in whom a diagnosis of maple syrup urine disease (MSUD), or variant MSUD, is suspected due to a positive newborn screen for MSUD, elevated branched-chain amino acids (especially elevated leucine) on plasma amino acid analysis, the presence of alloisoleucine on plasma amino acid analysis, or clinical presentation. Newborn screening may miss intermittent MSUD, so any individual with a clinical or biochemical phenotype suggestive of MSUD should be tested, regardless of a prior negative newborn screen. Age of diagnosis and subsequent metabolic control are the greatest determinants of long-term outcome.
BCKDHA BCKDHB DBT DLD PPM1K
BCKDHA BCKDHB DBT DLD PPM1K
The Invitae Organic Acidemias Panel has been designed to provide a broad genetic analysis of this class of disorders. Depending on the individual’s clinical and family history, this broader panel may be appropriate. It can be ordered at no additional cost.
Elevated leucine on newborn screening (NBS) or plasma amino acid analysis may be the result of maple syrup urine disease (MSUD) or dihydrolipoamide dehydrogenase (DLD) deficiency. MSUD is an inborn error of metabolism caused by an inability to completely break down the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine. Inability to breakdown the BCAAs leads to their accumulation in blood and tissue, causing the characteristic maple-syrup urine odor that is often detectable in the urine or cerumen of affected individuals. Dihydrolipoamide dehydrogenase (DLD) deficiency affects several metabolic pathways including the ability to metabolize branched- chain amino acids and other pathways involved in energy production. It results in elevated branched- chain amino acids with lactic acidosis.
MSUD has a broad phenotypic spectrum that includes classic, intermediate, intermittent, and thiamin-responsive forms. Classic cases generally have a residual enzyme activity of less than 3% whereas intermediate, intermittent, and thiamin-responsive MSUD cases have variable residual enzyme activity ranging from 3%–40%.
Classic MSUD represents most known cases and presents in the neonatal period, following protein catabolism. Affected neonates present within the first 48 hours of life with irritability, poor feeding and rapid onset of ketoacidosis. If untreated, the patients rapidly deteriorate and may become lethargic with intermittent apnea, neurologic abnormalities, and even coma and respiratory failure. Intermediate MSUD has greater enzymatic activity and may present in infancy with feeding problems, growth delays, and developmental delay or in later childhood with developmental delay. Most individuals with intermediate MSUD are diagnosed between five to seven years of age. Individuals with intermittent MSUD are clinically well during infancy and childhood but present with clinical and biochemical abnormalities during times of illness or physiologic stress. Patients with thiamin-responsive MSUD also present later in life; their enzymatic activity can be increased with thiamin supplementation because thiamin is a necessary cofactor. Defects in the enzymatic regulator PP2Cm leads to an inactive BCKHD complex and results in a milder, variant form of MSUD, but minimal data exists regarding this condition. All forms of MSUD are susceptible to periods of acute metabolic decompensation due to BCAA build-up. Such episodes are frequently precipitated by infection, injury, or physiologic stress.
MSUD is treatable by lifetime dietary restriction of branched-chain amino acids and aggressive intervention during metabolic crisis. Dietary therapy must be managed by a nutritionist to prevent malnutrition. Some cases are responsive to pharmacologic doses of thiamin. Thiamin treatment does not eliminate the need for dietary intervention, but it reduces the degree of restriction. Some affected individuals have been successfully treated by liver transplant.
MSUD is genetically heterogeneous (see the table below). An affected individual is homozygous or compound heterozygous for pathogenic variants in the same gene. No cases have been identified with pathogenic variants in different genes. Biallelic variants in any of the three genes can cause any of the four clinical phenotypes. Pathogenic variants are most common in BCKDHA and BCKDHB. Thiamin responsive patients have been found to have a greater incidence of missense DBT variants that result in a full-length mutant protein.
|BCKDHA||E1α||Branched-chain keto acid dehydrogenase E1, alpha subunit|
|BCKDHB||E1β||Branched-chain keto acid dehydrogenase E1, beta subunit|
|DBT||E2||Dihydrolipoamide branched-chain transacylase E2|
|PPM1K||Regulatory E2 phosphatase||Protein phosphatase 2Cm (PP2Cm)|
DLD deficiency affects multiple metabolic pathways as dihydrolipoamide dehydrogenase serves as the E3 subunit in three enzyme complexes: BCKDH complex, pyruvate dehydrogenase complex and alpha ketoglutarate dehydrogenase complex. There is a broad phenotypic spectrum ranging from an early-onset infantile presentation to adults with isolated liver involvement. Most affected individuals are symptomatic by two years of age and common features include a Reye-like presentation with encephalopathy and seizures. Prognosis in early-onset cases is generally poor with death typically occurring by early childhood. Individuals with the isolated hepatic presentation often experience nausea, emesis, hepatomegaly, muscle cramping and occasionally behavior and/or vision disturbances. Biochemical findings include elevations of branched- chain amino acids including allo-isoleucine, lactate, urine alpha ketoglutarate, hypoglycemia and ketoacidosis.
For patients with a clinical and biochemical diagnosis of MSUD, approximately 100% will have two pathogenic variants in one of these three genes. DLD is the only known gene associated with dihydrolipoic dehydrogenase deficiency. Approximately 98% of individuals with a biochemical diagnosis of DLD will have two pathogenic variants in DLD.
|Gene||Proportion of MSUD cases|
MSUD and DLD deficiency are inherited in an autosomal recessive manner.
MSUD and DLD deficiency are fully penetrant, with a broad phenotypic spectrum.
The general population incidence of MSUD has been reported as 1 in 120,000–500,000. This may be underestimated due to intermittent biochemical presentations and a mild disease that results in a subclinical phenotype.
MSUD has a much higher incidence in the Old Order Mennonite population of southeastern Pennsylvania due to a BCKDHA founder mutation (p.Y393N). Incidence has been reported as high as 1 in 176 live births.
There is an increased carrier frequency of 1 in 113 among the Ashkenazi Jewish population for the p.R133P mutation in BCKDHB.
The incidence and prevalence of DLD in the general population is not known, but there is an increased incidence in the Ashkenazi Jewish population. The estimated incidence of DLD deficiency among the Ashkenazi Jewish population is 1:35,000 – 1:48,000. The carrier frequency of the c.685G>T (p.Gly229Cys) pathogenic variant is believed to be 1:94 – 1:110.
This test is indicated for any individual with elevated branched-chain amino acids on plasma amino acid analysis. The presence of allo-isoleucine is pathognomonic for MSUD.
Newborn screening has been documented to miss intermittant forms of MSUD as biochemical abnormalities, biochemical abnormalities are generally only unmasked during times of physiologic stress.
For considerations for testing please refer to:
For management guidelines please refer to:
Invitae is a College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified clinical diagnostic laboratory performing full-gene sequencing and deletion/duplication analysis using next-generation sequencing technology (NGS).
Our sequence analysis covers clinically important regions of each gene, including coding exons, +/- 10 base pairs of adjacent intronic sequence in the transcript listed below. In addition, analysis covers the select non-coding variants specifically defined in the table below. Any variants that fall outside these regions are not analyzed. Any specific limitations in the analysis of these genes are also listed in the table below.
Based on validation study results, this assay achieves >99% analytical sensitivity and specificity for single nucleotide variants, insertions and deletions <15bp in length, and exon-level deletions and duplications. Invitae's methods also detect insertions and deletions larger than 15bp but smaller than a full exon but sensitivity for these may be marginally reduced. Invitae’s deletion/duplication analysis determines copy number at a single exon resolution at virtually all targeted exons. However, in rare situations, single-exon copy number events may not be analyzed due to inherent sequence properties or isolated reduction in data quality. Certain types of variants, such as structural rearrangements (e.g. inversions, gene conversion events, translocations, etc.) or variants embedded in sequence with complex architecture (e.g. short tandem repeats or segmental duplications), may not be detected. Additionally, it may not be possible to fully resolve certain details about variants, such as mosaicism, phasing, or mapping ambiguity. Unless explicitly guaranteed, sequence changes in the promoter, non-coding exons, and other non-coding regions are not covered by this assay. Please consult the test definition on our website for details regarding regions or types of variants that are covered or excluded for this test. This report reflects the analysis of an extracted genomic DNA sample. In very rare cases, (circulating hematolymphoid neoplasm, bone marrow transplant, recent blood transfusion) the analyzed DNA may not represent the patient's constitutional genome.
|Gene||Transcript reference||Sequencing analysis||Deletion/Duplication analysis|