ACADSB Human

What is the ACADSB gene and its encoded protein?

The ACADSB gene encodes for the mitochondrial short/branched-chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase family of enzymes. This protein catalyzes two critical reactions: (1) the third reaction in the L-isoleucine degradation pathway, specifically the conversion of 2-methylbutyryl-CoA to tiglyl-CoA, and (2) the first oxidative step of short straight-chain acyl-CoAs, including butyryl-CoA and hexanoyl-CoA . Additionally, SBCAD can utilize valproyl-CoA as a substrate, suggesting a potential role in valproate metabolism .

Research methodology: To characterize ACADSB protein function, researchers typically employ enzyme activity assays using purified recombinant protein with various acyl-CoA substrates. Spectrophotometric methods measuring the reduction of electron acceptors can quantify dehydrogenase activity, while mass spectrometry can identify reaction products and intermediates.

What is the genomic location and structure of human ACADSB?

The ACADSB gene is located on chromosome 10, specifically within the chromosomal band 10q25-q26 . The gene has been characterized at the molecular level with a genomic reference sequence of NG_008003.1 and transcript reference of NM_001609.3 .

Research methodology: Chromosomal localization was initially determined through Southern blot analysis using human/rodent somatic cell hybrids and further refined through fluorescence in situ hybridization (FISH) . Current mapping approaches include next-generation sequencing and bioinformatic analysis of reference genomes.

How is ACADSB deficiency identified and monitored clinically?

SBCAD deficiency (SBCADD, OMIM# 600301/610006) is identified through metabolite analysis of blood, urine, and fibroblast samples . The primary diagnostic markers include:

• Elevated C5-carnitine levels detected through newborn screening

• Elevated 2-methylbutyrylglycine (2MBG) in urine

Long-term monitoring involves regular assessment of:

• DBS (dried blood spot) or serum C5 concentration

• Urine 2MBG concentration

• Serum glucose, ammonia, and CK concentrations

• Liver function tests

• Blood gases

• Cardiac function via heart ultrasound and electrocardiogram

• Growth parameters and psychomotor development

Research methodology: Acylcarnitine analysis is performed using tandem-mass spectrometry (LC/MS-MS) on either DBS or serum samples, with established intra-day precision of approximately 0.7% coefficient of variation (CV) and inter-day precision of approximately 5% CV . Urine organic acid profiles are assessed via gas chromatography-mass spectrometry (GC-MS) .

What methods are used to identify and characterize ACADSB gene mutations?

Molecular characterization of ACADSB variants involves multiple steps:

• DNA extraction from EDTA peripheral venous blood samples

• PCR amplification of all exons and parts of flanking intron regions

• Sequencing according to standard procedures

• Variation reporting following the Human Genome Variation Society (HGVS) nomenclature

• Annotation according to:

  • NCBI SNPs Database

  • ClinVar database

  • Human Gene Mutation Database (HGMD) Professional

  • Classification based on American College of Medical Genetics and Genomics (ACMG) guidelines

Research methodology: While standard PCR and Sanger sequencing remain valid, next-generation sequencing approaches including targeted gene panels, whole-exome sequencing, and whole-genome sequencing offer higher throughput for variant identification. Functional validation of variants can be performed through expression studies in cellular models or enzyme activity assays with purified recombinant proteins containing the specific mutations.

How does ACADSB expression correlate with cancer progression?

ACADSB has been found to be down-regulated in multiple cancers, with decreased expression correlating with poor prognosis in certain cancer types . Specific findings include:

• ACADSB plays important roles in glioma, colorectal cancer (CRC), and hepatocellular carcinoma (HCC)

• In clear cell renal cell carcinoma (ccRCC), decreased ACADSB expression predicts poor prognosis

Research methodology: To study ACADSB expression in cancer, researchers employ:

• RNA-sequencing or qRT-PCR for transcriptional analysis

• Western blotting or immunohistochemistry for protein expression

• Cancer tissue microarrays for high-throughput expression profiling

• Kaplan-Meier survival analysis to correlate expression levels with patient outcomes

• Gene knockdown or overexpression experiments to assess functional consequences of altered ACADSB expression

What is the prevalence and phenotypic spectrum of ACADSB deficiency?

SBCADD shows variable clinical presentation. The current understanding is:

• Most individuals with SBCADD, including those identified through newborn screening, show no health problems

• A small percentage develop symptoms shortly after birth or later in childhood

• Initial symptoms may include poor feeding, lethargy, vomiting, and irritability

• Severe manifestations include dyspnea, seizures, and coma

• Other features can include poor growth, muscle weakness, delay in motor skills, and intellectual disability

• A founder mutation exists in the Hmong Chinese population, where affected individuals have remained largely asymptomatic

Research methodology: Population studies utilize newborn screening data analysis, with biochemical confirmation of suspected cases. Case-control studies examining genotype-phenotype correlations help elucidate the clinical spectrum. Long-term follow-up studies track developmental outcomes and metabolic stability.

What experimental approaches are used to study ACADSB function in metabolic pathways?

Research on ACADSB's metabolic roles employs multiple experimental approaches:

• Metabolomic Profiling: Using LC-MS/MS or GC-MS to identify metabolic perturbations in ACADSB-deficient cells or tissues

• Stable Isotope Tracing: Employing 13C-labeled isoleucine or other substrates to track metabolic flux through ACADSB-dependent pathways

• CRISPR-Cas9 Gene Editing: Creating cellular models with ACADSB knockout or specific mutations

• Recombinant Enzyme Assays: Assessing substrate specificity and kinetic parameters of wild-type and mutant ACADSB proteins

• Mitochondrial Functional Assays: Measuring oxygen consumption rates and mitochondrial membrane potential in ACADSB-deficient models

These approaches provide complementary insights into ACADSB's role in branched-chain amino acid metabolism and fatty acid oxidation.

How do researchers distinguish pathogenic from benign ACADSB variants?

Determining ACADSB variant pathogenicity involves multiple lines of evidence:

• Population Frequency Analysis: Evaluating variant prevalence in population databases like gnomAD

• In Silico Prediction Tools: Using algorithms like SIFT, PolyPhen-2, and CADD to predict functional impact

• Conservation Analysis: Assessing evolutionary conservation of affected amino acid residues

• Functional Assays: Measuring enzyme activity of recombinant proteins containing variants

• Clinical Correlation: Analyzing biochemical profiles of patients carrying specific variants

• Segregation Analysis: Examining variant co-segregation with disease in affected families

The ACMG guidelines provide a standardized framework for integrating these evidence types to classify variants as pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign .

What are the current methods for monitoring metabolic stability in SBCADD patients?

Long-term monitoring of SBCADD patients employs standardized approaches:

• Biomarker Trend Analysis: Tracking serum C5 and urine 2MBG trends, classified as:

  • Increased: >10% increase from baseline

  • Stable: between -10% and +10% change

  • Decreased: >10% decrease from baseline

• Biochemical Surveillance: Regular assessment of metabolic parameters including:

  • Serum glucose levels

  • Ammonia concentration

  • Creatine kinase levels

  • Liver function tests

  • Blood gas analysis

• Clinical Monitoring: Tracking growth parameters, developmental milestones, and system-specific assessments (cardiac, neurological)

The 10% threshold for trend classification was established based on analytical performance characteristics, specifically setting the threshold at approximately 10 times the intraday CV and twice the interday CV of the analytical methods .

How is ACADSB being investigated as a potential cancer biomarker?

Recent research has identified ACADSB as a potential biomarker in multiple cancer types:

• Expression Profiling: Decreased ACADSB expression predicts poor prognosis in clear cell renal cell carcinoma (ccRCC)

• Functional Studies: ACADSB plays important roles in glioma, colorectal cancer, and hepatocellular carcinoma

• Metabolic Reprogramming: ACADSB alterations may contribute to cancer-specific metabolic phenotypes through effects on branched-chain amino acid metabolism

Research methodology: Biomarker validation studies typically employ multi-cohort designs with discovery and validation phases. Techniques include immunohistochemistry on tissue microarrays, transcriptomic analysis of patient samples, and correlation with clinical outcomes. Mechanistic studies investigate how ACADSB affects cancer cell proliferation, migration, and response to therapy.

What computational approaches are being used to understand ACADSB structure-function relationships?

Computational biology has become increasingly important for ACADSB research:

• Homology Modeling: Utilizing structural information from related acyl-CoA dehydrogenases to predict ACADSB tertiary structure

• Molecular Dynamics Simulations: Investigating how mutations affect protein stability and substrate binding

• Systems Biology Approaches: Integrating ACADSB into metabolic network models to predict systemic effects of altered function

• Machine Learning Applications: Developing improved algorithms for variant pathogenicity prediction based on multiple data types

These computational methods complement experimental approaches and can generate hypotheses for further laboratory investigation.