ECHDC1 Human

What is ECHDC1 and what is its primary enzymatic function?

ECHDC1 (Ethylmalonyl-CoA decarboxylase) is a metabolite repair enzyme that primarily functions to decarboxylate ethylmalonyl-CoA, which is an intermediate in the formation of ethylmalonic acid (EMA) . The enzyme catalyzes the conversion of ethylmalonyl-CoA to butyryl-CoA, effectively preventing the accumulation of ethylmalonic acid in cellular metabolism . ECHDC1 belongs to the family of lyases and specifically acts on carbon-carbon bonds, with EC number 4.1.1.94 . The enzyme plays a crucial role in maintaining metabolic homeostasis by preventing the accumulation of potentially harmful metabolites.

Experimental approaches to study ECHDC1 activity typically involve measuring the decrease in acid-stable [14C]ethylmalonyl-CoA in vitro, as described in Linster et al. (2011) . This methodology provides a direct assessment of the enzyme's capacity to decarboxylate its substrate under controlled conditions.

How does ECHDC1 deficiency potentially contribute to human disease?

ECHDC1 deficiency may contribute to disease through multiple mechanisms, primarily by allowing increased accumulation of ethylmalonic acid (EMA), which has been demonstrated to have cytotoxic effects . When ECHDC1 function is compromised, particularly in combination with common ACADS variants (c.625G>A and c.511C>T), elevated EMA levels can potentially lead to a pathological state .

Research has shown that EMA can inhibit electron transport chain and creatine kinase activity in human skeletal muscle, promote lipid and protein oxidative damage in the brain, and disturb energy and redox homeostasis . These cellular disturbances may contribute to neurological symptoms observed in some individuals with elevated EMA levels.

What experimental models are suitable for studying ECHDC1 function?

Several experimental models have proven effective for investigating ECHDC1 function in research settings:

• Human fibroblast cultures: Patient-derived fibroblasts with different ACADS c.625G>A genotypes provide an invaluable biological system for studying the effects of ECHDC1 variants on cellular metabolism . These cell lines allow researchers to examine the consequences of ECHDC1 deficiency in a human cellular context with relevant physiological backgrounds.

• Lentiviral knockdown systems: Research has successfully employed shRNA-mediated knockdown of ECHDC1 in fibroblasts to simulate haploinsufficiency and study the resulting metabolic effects . This approach enables precise control over ECHDC1 expression levels and assessment of downstream consequences.

• Recombinant protein expression: Wild-type and mutant ECHDC1 proteins can be expressed in E. coli (BL21) using expression plasmids, purified, and subjected to enzymatic activity assays to evaluate the functional consequences of specific variants . The approach typically involves cloning ECHDC1 cDNA into vectors like pET28a and inducing expression with IPTG.

• qPCR analysis: Quantitative PCR using TaqMan probes targeting the exon 5/6 boundary of ECHDC1 enables precise quantification of gene expression levels across different experimental conditions . This technique allows researchers to correlate ECHDC1 expression with metabolic phenotypes.

Table 1: Experimental Models for ECHDC1 Research

How do ECHDC1 variants interact with ACADS variants to influence ethylmalonic aciduria?

The interaction between ECHDC1 and ACADS variants represents a potential example of synergistic heterozygosity affecting metabolic pathways. Research indicates that ECHDC1 haploinsufficiency combined with homozygosity for the ACADS c.625G>A variant has a synergistic effect on cellular EMA excretion . This biochemical interaction provides insight into why some individuals with common ACADS variants develop symptoms while others remain asymptomatic.

The mechanism appears to involve two converging metabolic pathways:

• ACADS variants (particularly c.625G>A) may lead to reduced enzymatic activity of short-chain acyl-CoA dehydrogenase, resulting in increased levels of butyryl-CoA substrates that can be converted to ethylmalonyl-CoA .

• ECHDC1 deficiency reduces the cell's capacity to decarboxylate ethylmalonyl-CoA, preventing its conversion back to butyryl-CoA and allowing increased accumulation of ethylmalonic acid .

Experimental evidence supporting this interaction comes from knockdown experiments of ECHDC1 in healthy human cells with different ACADS c.625G>A genotypes. These studies demonstrated that ECHDC1 haploinsufficiency combined with homozygosity for the ACADS c.625G>A variant synergistically increased cellular EMA excretion . This finding suggests that the two genetic variants together have a greater impact than would be expected from their individual effects.

To investigate such interactions, researchers should consider:

• Metabolic flux analysis to track carbon flow through affected pathways

• Isotope tracing experiments to quantify the contribution of each pathway to EMA production

• Creation of cellular models with controlled expression of both genes to establish dose-response relationships

What are the molecular consequences of specific ECHDC1 variants identified in humans?

Research has identified several functionally significant ECHDC1 variants in humans, with distinct molecular consequences:

To comprehensively characterize ECHDC1 variants, researchers should employ multiple complementary approaches:

• In silico prediction tools: Tools such as SpliceSiteFinder-like, MaxEntScan, NNSplice, GeneSplicer, and Human Splicing Finder can predict the effects of variants on splicing .

• Protein structure/function prediction: PolyPhen-2 and SIFT can assess the potential impact of missense variants on protein structure and function .

• Conservation analysis: ConSurf can evaluate the evolutionary conservation of specific amino acid residues, providing insight into their functional importance .

• Experimental validation: Direct measurement of enzymatic activity, protein stability, and expression levels is essential for confirming the functional consequences of variants.

What is the emerging role of ECHDC1 in cancer biology?

Recent research has identified ECHDC1 as a potential tumor suppressor gene, particularly in breast cancer . Multi-modal meta-analysis of cancer cell line omics profiles has revealed that ECHDC1 may play a previously unrecognized role in tumor biology .

The tumor suppressive function of ECHDC1 may be related to its role in metabolic regulation. Cancer cells often exhibit altered metabolism, including changes in fatty acid synthesis and utilization. ECHDC1's function in preventing the formation of methyl- and ethyl-branched fatty acids could potentially interface with cancer-specific metabolic programs. Decreased ECHDC1 expression or function might contribute to metabolic adaptations that support tumor growth.

Research approaches to investigate ECHDC1's role in cancer should include:

• Expression profiling: Analysis of ECHDC1 expression across different cancer types and correlation with clinical outcomes

• Functional studies: Knockdown and overexpression experiments in cancer cell lines to assess effects on proliferation, migration, and invasion

• Metabolomics analysis: Comprehensive profiling of metabolites in cells with altered ECHDC1 expression to identify cancer-relevant metabolic changes

• In vivo studies: Animal models with tissue-specific ECHDC1 modulation to evaluate effects on tumor initiation and progression

What methodologies are optimal for measuring ECHDC1 enzymatic activity in biological samples?

Measuring ECHDC1 enzymatic activity in biological samples requires specialized techniques that directly assess the enzyme's capacity to decarboxylate ethylmalonyl-CoA. The following methodologies have been established:

• Radiometric assays: The gold standard approach involves measuring the decrease in acid-stable [14C]ethylmalonyl-CoA in reaction mixtures containing purified enzyme or cell lysates . This method provides direct quantification of decarboxylase activity but requires specialized facilities for handling radioactive materials.

• Mass spectrometry-based assays: LC-MS/MS can be used to directly measure the conversion of ethylmalonyl-CoA to butyryl-CoA, providing a non-radioactive alternative with high sensitivity and specificity. This approach enables simultaneous measurement of multiple related metabolites.

• Indirect assessment through metabolite profiling: Measuring cellular or urinary EMA levels provides an indirect assessment of ECHDC1 function. Typically, EMA is quantified in urine and reported as mmol/mol creatinine, with values >150 mmol/mol creatinine considered elevated .

For accurate activity measurements, researchers should consider:

• Using appropriate controls including wild-type recombinant ECHDC1

• Including samples with known ECHDC1 variants as reference points

• Normalizing activity to protein concentration or expression level

• Verifying assay conditions including pH, temperature, and substrate concentrations for optimal enzyme function

How can researchers effectively study the potential digenic or oligogenic inheritance patterns involving ECHDC1?

Studying complex inheritance patterns involving ECHDC1 requires sophisticated genetic and functional approaches:

• Comprehensive sequencing: Next-generation sequencing of multiple genes in affected individuals and family members can identify potential interacting variants . Panel-based approaches targeting ECHDC1, ACADS, and other genes involved in related metabolic pathways provide efficient screening.

• Family-based studies: Segregation analysis across multiple generations can reveal patterns consistent with digenic or oligogenic inheritance. Mapping phenotype severity against variant combinations helps establish genotype-phenotype correlations.

• Functional validation: Cell-based models expressing different combinations of variants can demonstrate synergistic effects on metabolic pathways . Measurement of relevant metabolites (like EMA) under various genotype combinations provides evidence for gene-gene interactions.

• Statistical approaches: Specialized statistical methods for evaluating epistasis and genetic interactions are essential for distinguishing true interactions from coincidental co-occurrence of variants.

• Animal models: Generation of transgenic models carrying human variants in orthologous genes can validate hypothesized interactions in an in vivo context.

The analysis of digenic inheritance involving ECHDC1 builds on established frameworks from other conditions where this pattern has been observed, including examples from Parkinson's disease (PINK1 and DJ-1), congenital hypogonadotropic hypogonadism (GNRHR), and recurrent exertional rhabdomyolysis . These precedents provide methodological guidance for investigating similar patterns with ECHDC1.