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ECHS1 disease in two unrelated families of Samoan descent: Common variant ‐ rare disorder

Abstract Mutations in the short‐chain enoyl‐CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT‐PCR, RNA‐Seq, RNA‐Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p. Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.  Yamada et al., 2015). Insufficient conversion of methacrylyl-CoA into 3-OH-isobutyryl-CoA and of acryloyl-CoA into 3-OH-propionyl-CoA, leads to accumulation of these highly reactive and toxic precursors. This then results in the inhibition of mitochondrial electron transport chain (ETC) as well as pyruvate dehydrogenase complex (PDHC) activities, leading to decreased ATP generation (Peters et al., 2014). Thus, deficiencies in SCEH can mimic metabolic defects in energy production with a clinical picture resembling PDHC deficiency or mitochondrial disease (Bedoyan et al., 2017;Ferdinandusse et al., 2015;Peters et al., 2014). Inheritance of ECHS1 deficiency is autosomal recessive and onset typically occurs during infancy or childhood (Al Mutairi, Shamseldin, Alfadhel, Rodenburg, & Alkuraya, 2017). Most patients present with developmental delay, regression, dystonia, feeding difficulties, cardiomyopathy, and brain MRI changes consistent with Leigh syndrome. Since Sharpe et al., reviewed the mutational, clinical, and biochemical spectrum of 42 patients from 33 families with ECHS1 disease, an additional nine new cases have been reported, none of which are due to synonymous mutations (Aretini et al., 2018;Carlston, Ferdinandusse, Hobert, Mao, & Longo, 2019;Pajares et al., 2020;Ronchi et al., 2020;Sharpe & McKenzie, 2018;Uchino et al., 2019;Wu et al., 2020;Yang & Yu, 2020). Here we report clinical, biochemical, molecular, and functional data on four patients from two unrelated families, presenting with two novel mutations in ECHS1. The missense mutation c.832G > A (p.Ala278Thr) decreases ECHS1 protein stability while the silent c.489G > A (p.Pro163=) mutation is associated with decreased levels of normally spliced mRNA.

| Editorial policies and ethical considerations
All patients were followed at the Division of Metabolic Disorders, CHOC Children's hospital. Written consent was obtained from all study participants and/or their legal guardians via CHOC approved IRB# 130990.

| Biochemical testing
In Family 1, acryloyl and methacrylyl metabolites were measured in urine blotters via LC/MS-MS at the Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, Australia, as published (Peters et al., 2015). In Family 2, measurements were performed in urine at Laboratory Genetic Metabolic Diseases, Amsterdam UMC, Academic Medical Center, and The Netherlands with similar methodology.

| Whole exome sequencing
Whole exome sequencing was performed on a clinical basis by Baylor Genetics (Family 1, Patient 2), GeneDx (Family 1, Patients 1, 3 and parents), and Ambry Genetics (Family 2, Trio) and on a research basis by Omega Bioservices for the unaffected brother of Family 1. All data was obtained under IRB# 130990 and reanalyzed at CHOC using CLC-Bio Genomics Workbench and variants were filtered and annotated using Annovar as published (Wang, Li, & Hakonarson, 2010).

| Whole genome sequencing
Whole genome sequencing was performed at Rady Children's Institute for Genomic Medicine on DNA extracted from whole blood for Family 1, (Patient 1, 3, the unaffected brother and both parents) via published method (Kingsmore et al., 2019). Alignments against Hg19 were exported as BAM files and haplotypes were established using Integrative Genomics Viewer (IGV) tools as published (Thorvaldsdóttir, Robinson, & Mesirov, 2013).
2.6 | Nonsense mediated decay experiment Cells were cultured as described above. Emetine (Sigma) was diluted to 100 mg/ml in H 2 O and used as a 1,000× stock at a final concentration of 100 μg/ml in sterile filtered growth medium. Exposure time prior to harvest for RNA generation was 5 hr.

| Immunoblotting
Protein was extracted from fibroblast cell pellets by incubation with RIPA buffer (Pierce) and HALT protease/phosphatase inhibitor cocktail (Thermo-Fisher) for 30 min at 4 C and then centrifuged at 16,000 rpm for 20 min at 4 C. DCA Assay (Biorad) was performed in triplicate to determine concentration of lysates. Lysates were stored at −80 C in NuPage LDS buffer (LifeTechnologies). Sample Reducing Agent (LifeTechnologies) was added to the lysates and antioxidant was added to the running buffer per the manufacturer recommendations. SDS-PAGE was run on 1.5 mm NuPage 4-12% Bis-Tris Gels with 1× NuPage MES Running Buffer (LifeTechnologies) for 1 hr at 200 V. The PVFD membrane was incubated with methanol and transfer buffer. The protein was transferred to the PVFD membrane using Trans-Blot Turbo RTA Blotting System run at the Mixed MW protocol: 1.3 A, 25 V for 7 min (Biorad). The membrane was washed with fast wash buffer (FWB) for 10 min and the incubated in Fast Blocking Buffer (FBB) (Pierce) for 30 min at RT. The membrane was incubated with ECHS1 antibody 1:10,000 (Abcam, ab174312) and beta-Actin antibody 1:5,000 (Abcam, 119716) overnight at 4 C. The next day, the membrane was washed with FWB 3 × 5 min at RT and then incubated with the Goat Anti-Rabbit IgG H&L (HRP) secondary antibody 1:20,000 (Abcam, ab6721). The protein was detected using SuperSignal Chemi-luminescent substrate (LifeTechnologies) and imaged on CL-Xposure film (LifeTechnologies).
The results were analyzed using ImageJ and Microsoft Excel.

| RNA extraction
RNA was extracted from fibroblast cell pellets using PureLink RNA Mini Kit (Ambion) including on column DNAse treatment per manufacturer recommendations. Quality control of RNA samples was performed using a Bioanalyzer system (Agilent).

| Reverse transcriptase PCR
Reverse transcriptase PCR (RT-PCR) was performed using 300 ng of RNA and the SuperScript III one step RT-PCR kit with platinum Taq.
2.10 | Quantification of ECHS1 expression ECHS1 RNA expression levels were measured using a CFX Connect Real-Time PCR Detection System (Biorad). Two microgram of RNA extracted from fibroblast pellets were reverse transcribed using Superscript VILO cDNA synthesis kit (Invitrogen) according to manufacturer's recommendation. Quantitative PCR (qPCR) was performed via TaqMan gene expression assay (Applied Biosystems) using TaqMan Fast Advanced Master Mix and ECHS1 assay Hs00929130_m1 spanning the exon 5-6 boundary. GAPDH assay Hs02786624_g1 and beta actin (ACTB) assay Hs99999903_m1 were used as the reference genes. Samples were run in triplicates and expression including SEM was generated via the ΔΔCt method using Microsoft Excel.

| RNA-Seq
Stranded libraries were constructed using 1 μg of total RNA as the input for the Illumina TruSeq total RNA kit in conjunction with the gold ribozero rRNA depletion kit. Quality control was performed via Agilent Bioanalyzer. Hundred cycle paired end sequencing was performed on a HiSeq 4000 by the UCI High Throughput Facility. Trimmed reads were aligned to GRCh38 using CLC Genomics Workbench. Alignments were exported as BAM files and Sashimi plots were established using IGV. 2.13 | SCEH enzyme activity SCEH Activity was measured in fibroblasts for Family 1 and for Patient 4 from Family 2, as published .

| Clinical findings
A detailed clinical description of Family 1 that includes the response to a valine restricted diet has been recently reported (Abdenur et al., 2020). Briefly, Family 1 consists of 4 boys with nonconsanguineous parents. The mother is of Samoan descent and the father is of European derived (Portuguese) ethnicity. Of the four children, three are affected (Patient 1-3) and one of the affected (Patient 2) has passed away ( Figure 1). Starting in infancy, the patients presented with developmental delay, regression, hypotonia in the trunk with rigidity and dystonic posturing in extremities. They all developed nystagmus and demonstrated changes in brain MRI (bilateral, symmetrical basal ganglia hyperintensities in T2) consistent with Leigh syndrome. Intermittent episodes of metabolic acidosis and ketosis with normal acylcarnitines were documented during intercurrent illnesses.
The oldest brother (Patient 1) had a less severe clinical course. By 13 years of age he had developed spasticity and mild joint contractures and was only able to walk with assistance. He had dysarthria, was able to comprehend, read, and write at a second-grade level, and was still able to eat by mouth. Patient 2 had a more severe phenotype.
He died at age 9 years after an intercurrent illness, complicated by respiratory insufficiency. By that time, he had little interaction, no head control, was unable to sit unsupported and had developed dysphagia necessitating gastrostomy tube feedings. The youngest brother (Patient 3) had a severe presentation, similar to Patient 2 and was alive at 6 years. None of the patients were on a valine restricted diet during the observation period of this study. deficiency. Subsequently, SCEH activity was also measured in fibroblasts for all family members revealing even more reduced SCEH activity in the other 2 affected brothers (6% of controls in Patient 1 and 5% of controls in Patient 3), confirming the enzymatic diagnosis for this family. A reduction was also seen, but to a lesser extent, in samples from the parents as well as the unaffected brother ( Figure 2a). Diagnostic markers for ECHS1 disease in urine blotters revealed elevated S-(2-carboxypropyl)cysteamine, S-(2-carboxyethyl) cysteamine, N-acetyl-S-(2-carboxypropyl)cysteine and 2,3-dihydroxy-2-methylbutyric acid in the severely affected Patient 3 while the more mildly affected Patient 1 showed only mild elevations in N-acetyl-S-(2-carboxypropyl)cysteine (Table 1). A sample from the index patient was not available, while both parents and the unaffected brother had normal metabolite levels in urine blotters.

| Molecular and biochemical findings
Immunoblot analysis with antibodies against ECHS1 demonstrated reduced protein levels for all samples from affected family members, which were as low as 21% of control. The sample from the father, who is a carrier for c.832G > A (p.Ala278Thr) had protein levels reduced to 51% of control (Figure 2b) while the mother who was not reported to be a carrier of any nonsense variants in ECHS1 had a ECHS1 protein level at 41% of control, indicating that she is likely a carrier of another, not previously reported variant in ECHS1. Samples from their unaffected son, who is not a carrier of the father's mutation, were reduced by 20%. Since we had previously excluded a maternal deletion or rearrangement in ECHS1 via array CGH, these results indicated that the mother carried a variant either in a deep intronic or regulatory region of the gene. To identify this second variant, we started our investigations by amplifying the entire ECHS1 cDNA from Patient 2 via RT-PCR using a forward primer in the 5 0 UTR and a reverse primer in the 3 0 UTR. Only one single band, representing the full-length ECHS1 transcript (873 bp), was observed. We also per- (rs140410716) with a peak height < 50% for the A base in exon 4 ( Figure S2), for which we found the patients' mother to be homozygous. The reported overall population frequency (gnomAD v2.1.1) is 0.001 for the variant and further specifies an increased frequency in East Asians at 0.01 which, includes 2 homozygotes (accessed December 2, 2019) (Karczewski et al., 2020).
To assess whether ECHS1 mRNA levels were significantly reduced in this family we performed transcriptome array analysis using the human ClariomD assay. We performed comparative analysis of one array each from the three patients of Family 1, their unaffected brother, their mother as well as the patient of Family 2 (n = 6) with arrays from five unrelated healthy controls including a total of four biological replicates for the controls (n = 9). We filtered for all significantly down regulated genes (p < .05, fold change <−1.5) of the 1,158 mitochondrial protein compendium Mitocarta 2.0 genes (Calvo et al., 2016). We then prioritized the resulting list of 22 genes by their p values and found ECHS1 as the fourth most statistically significantly downregulated gene at p = .0008 and a fold change of −1.78 (Table S1).
We also used RNA-Array results to choose glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the most appropriate housekeeping gene for our confirmatory results via qPCR due to its stable expression.
Quantitative PCR was performed using a TaqMan assay spanning ECHS1 exon 5-6 junction and GAPDH as an internal reference gene.
Results showed ECHS1 mRNA to be decreased in all affected boys, their unaffected brother as well as their mother. The mother had the lowest relative ECHS1 expression at 52% of control followed by the deceased patient at 56% of control. The other two patients and their unaffected brother all had levels approximating 60% of control, while the father's relative ECHS1 mRNA level was slightly increased at 114% of control (Figure 2c) indicating no effect of c.832G > A (p. Ala278Thr) on ECHS1 RNA stability for his sample gene ( Figure S2).
Trends were similar when using ACTB as the internal reference gene ( Figure S3).
To assess whether there was a correlation between c.489G > A (p.Pro163=) and the observed decrease in ECHS1 mRNA we interrogated the effect of the variant on splicing via Human Splice Finder (HSF) 3.1 (Desmet et al., 2009). Three HSF algorithms predict c.489G > A (p.Pro163=) to result in weakening of an exonic splicing enhancer, therefore suggesting the inefficient splicing of exon 4. We also interrogated c.489G > A (p.Pro163=) with SpliceAI, which predicted partial skipping of exon 4 with a splice donor loss score of Δ 0.16 and a splice acceptor loss of Δ 0.17 (Jaganathan et al., 2019). RT-PCR for control and maternal fibroblast RNA using a forward primer overlapping the exon 2-3 junction and a reverse primer in exon 6 of ECHS1 however was essentially normal and a decrease in intensity of the PCR product was not observed. Skipping of exon 4 would have shortened the RT-PCR amplicon by 100 bp resulting in a frameshift after exon 3 with a stop codon in exon 6 ( Figure 3a). Since we did not observe this shortened band, we reasoned that the frameshift could have led to nonsense mediated decay with insufficient template to produce the mutant amplicon. To investigate this possibility we cultured the cells from Patient 2, his mother and two age matched controls with Emetine, a known nonsense mediated decay inhibitor (Boichard et al., 2008). RT-PCR now revealed a shortened band in RNA from maternal fibroblasts, but findings were not significantly different in patient versus control (Figure 3b). This finding is consistent with the fact that the mother is homozygous for c.489G > A (p. Pro163=) and abnormal splicing would affect transcripts from both alleles. Our findings therefore indicate that c.489G > A (p.Pro163=) reduces normally spliced ECHS1 mRNA resulting in nonsense mediated decay.
To further exclude whether there may have been additional variants from the mother which contributed to the reduction in mRNA we performed whole genome sequencing on all family members T A B L E 1 Metabolic markers characteristic of ECHS1 disease in patient urine and/or urine blotters To find additional evidence that the exon 4, c.489G > A (p. Pro163=) variant is indeed pathogenic we set out to find additional patients with this alteration.

| Family 2 3.2.1 | Clinical findings
The second family was ascertained by a database search for c.489G > A (p.Pro163=) (rs140410716) in patients with suspected mitochondrial disease and negative WES via Ambry Genetics. The database search resulted in a hit for an unrelated patient who curiously was also compound heterozygous for the same two variants as Family 1. In Family 2, c.489G > A (p.Pro163=) was inherited from the heterozygous father and the c.832G > A (p.Ala278Thr) mutation was inherited from the mother (Figure 1). The patient of Family 2 was found to not carry the intron 6 variant described for Family 1. We now had two completely unrelated families with the same primary variants of interest in ECHS1. The affected daughter of Family 2 was born without complications at 40 weeks of gestation after normal pregnancy via planned repeat cesarean section. Her birth weight was 3.17 kg. Parents are nonconsanguineous with the mother being of Chinese, Portuguese, Filipino, German, and American Indian descent and her father of Samoan descent. She has an 11 years old full brother as well as two older half-brothers. The patient developed normally until 8 months of age when she was admitted to the ED for fever and refusal to eat, leading to dehydration. She was treated with IV fluids and was discharged after 1 night. Few weeks thereafter, the patient was noted to have bilateral nystagmus and regression of motor skills.
Ophthalmology examination revealed no retinal abnormalities and MRI of the brain, obtained at 10 months, showed hyperintense signal abnormalities on T2, T2-flair and diffusion weighted images in bilateral globus pallidus. A mitochondrial disorder was highly suspected. Lactic acid in blood was reportedly elevated, while amino acids, acylcarnitines, F I G U R E 3 (a) Schematic of proposed impact of c.489G > A (p.Pro163=) on splicing. (b) Cells from adult and child control as well as the mother and Patient 2 of Family 1 were cultured with (+) and without (−) Emetine to halt nonsense mediated decay at 0.1 mg/ml for 5 hr prior to RNA extraction. RT-PCR was performed using a forward primer overlapping the exon 2/3 junction and a reverse primer in exon 6 (primers are denoted by arrows). PCR products were run on a 2% agarose gel. (c) RT-PCR to study the impact of the intron 6 variant on splicing using a forward primer in exon 6 and a reverse primer overlapping the exon 7-8 junction, a hypothetical band of 1,094 bp, representing abnormal splicing, was not observed carnitine, CK, urine organic acids, and Electron Transport Chain (ETC) activity in fibroblasts were normal. The patient had a "dual genome panel" testing via Baylor Genetics which did not reveal a potential gene candidate. This panel included full sequence of the mtDNA, as well as a genetic panel for 162 nuclear genes associated with mitochondrial disorders which did not include ECHS1.
The patient eventually regained the ability to crawl, was able to feed herself, and acquired some language. However, over time she Upon identification of the variants in ECHS1 the patient was referred to CHOC Children's for further evaluation and management.
She was 8 years old and her weight, height and HC were below the third percentile with a BMI at −1.21 SD. The patient was not dysmorphic, was aware of her surroundings but had limited interaction. She was able to track and follow with eyes but had intermittent nystagmus. Hearing appeared normal but she was only able to communicate with sounds and some word approximations. The patient was fed small pieces of food with care to avoid gagging or choking. A neurological exam revealed proximal hypotonia, no head control, and appendicular hypertonia with dystonic movements and brisk deep tendon reflexes.

| Molecular and biochemical findings
Patient fibroblast culture was established to investigate the pathogenicity of the variants and for comparison to Family 1. Enzyme activity measurements revealed a residual SCEH activity at 16% of controls (n = 6) ( Figure 2a). N-Acetyl-S-(2-carboxypropyl) cysteine, a metabolite characteristic for ECHS1 disease was mildly elevated at 4.95 μmol/ mmol creatinine (< 3.0) in patient's urine (Table 1) can be detected in urine, have been suggested to inhibit ETC and PDHC activity, which may explain the clinical presentation with developmental delay, regression, dystonia, feeding abnormalities and brain MRI abnormalities consistent with Leigh syndrome Haack et al., 2015). Patients with ECHS1-related disease therefore mimic classical mitochondrial disease with a variable phenotype ranging from mild paroxysmal exercise intolerance to neonatal lethal Leigh syndrome (Bedoyan et al., 2017;Olgiati et al., 2016).
For the families reported here, ECHS1 disease was difficult to diagnose, despite their classic mitochondrial phenotype and the use of WES as well as WGS performed by multiple laboratories. One reason was that the c.832G > A (p.Ala278Thr) mutation had not been mentioned in the various clinical reports even though it is rare and predicted to be pathogenic. Most autosomal recessive disease variant filtering algorithms require two putative pathogenic alleles in the same gene to make the list. The reporting of one single heterozygous allele in an autosomal recessive disorder is generally limited to known/ expected disease-causing variant(s) which ECHS1 was not at the time.
The reporting of single variants for recessive disorders, which fit the phenotype or suspected cellular functions, however, can be extremely helpful to the clinician. These variants provide important clues to prompt the physician to order additional biochemical or enzymatic analyses which may otherwise not have been clearly indicated. Our delineation of ECHS1-related disease was made possible via segregation analysis with three affected and one unaffected sibling, pointing to a shared paternal ECHS1 mutation. The clinical course in the family was also highly concordant with published ECHS1 cases including regression, developmental delay and dystonia (Fitzsimons et al., 2018).
The clinical and molecular findings presented a strong indication for assessment of SCEH activity on fibroblasts for Family 1. SCEH enzyme activity was decreased in fibroblasts (5-16% of controls), confirming the diagnosis and showing that the c.489G > A (p.Pro163=) mutation affects enzyme activity Peters et al., 2014). Metabolite levels from urine blotters for the two living affected brothers in Family 1 were modestly increased and appeared to correlate with the disease severity in concordance with prior reports (Haack et al., 2015). Of note, biochemical markers were measured in samples obtained out of crisis which, in conjunction with the hypomorphic second mutation, may explain why levels were only mildly increased. Although the biochemical phenotype and residual enzyme activity could predict a "mild disorder," our patients, with exception of Patient 1 in Family 1, had a severe phenotype. This is consistent with a previous report of a patient with a severe phenotype and mild increase in metabolite levels (Huffnagel et al., 2018).  (Strauss et al., 2015). High carrier frequencies of 20% are also known for sickle cell disease and beta thalassemia attributed to increased fitness against Malaria infection (Ferreira et al., 2011;Lazarin et al., 2013). It might be speculated that c.489G > A (p. Pro163=) in ECHS1 provides some sort of advantage with respect to fitness to have become that enriched in this population. Synonymous variants have also been reported in other inborn metabolic diseases (Ohlsson et al., 2019;Salomons et al., 2007). They produce different levels of both, normal and aberrant transcripts from the same allele.
Thus, the resulting enzymatic activity will be higher or lower depending on levels of the normal or aberrant RNA transcribed. We did not detect aberrant ECHS1 RNA species for the mother and so despite the fact that the mother has two alleles of the "hypomorphic" mutation she is not affected as protein levels are decreased but are representing functionally normal enzyme above a threshold levels which would cause pathogenicity.
Ultimately, the patient's phenotype will depend not only on the severity of the silent variant, but also on the presence of a pathogenic alteration on the second allele. In our patients, the mild silent variant in combination with the severe c.832G > A (p.Ala278Thr) mutation resulted in severe disease in three patients and a milder phenotype in the remainder. As severe disease was seen in two different families, and both phenotypes were present within the same family, it is unlikely that environmental factors were determinants of the variability in disease expression. It is possible that the severity of the metabolic crises and/or the age when they occurred may have played a role in the different degree of brain damage observed. Alternatively, it is possible that the four patients described in this study may have VOUS in modifier genes involving related metabolic pathways affecting expression of the disease. In fact, c.489G > A (p.Pro163=) (rs140410716) has been revealed as a disease modifier itself and has been associated with worse cardiac outcomes in a genome study of Han Chinese patients who had undergone STENT for acute coronary syndromes. Additionally, the study found lowered ECHS1 expression to be associated with a more severe clinical phenotype (Liu et al., 2018). Another study interested in genomic modifiers of coronary disease found a clear correlation of drug refractory dilated cardiomyopathy and several polymorphisms in ECHS1 (Campbell et al., 2018). Interestingly, cardiac involvement in ECHS1 deficiency has been described in patients with severe disease presentation, the patients reported in this study, however, did not show any cardiac involvement (Haack et al., 2015). ECHS1 has also been suggested as a nutrient sensor which bridges the gene's involvement in metabolism to obesity and heart disease. The investigations by Zhang et al. show that high nutrient states result in the acetylation of ECHS1 protein at p.Lys101 leading to impaired binding of its substrate enoyl-CoA followed by ubiquitination of the protein. Ubiquitinated ECHS1 protein however is resistant to mitochondrial import and is prematurely degraded. (Zhang et al., 2017). Further investigations in the Samoan population correlating increased incidence of common, multigenic disease with variants in ECHS1 may shed light on the interplay of this gene with metabolism in the future.

ACKNOWLEDGMENTS
We thank the families for their commitment to participation in these studies and all health care providers who cared for the patients and families. We gratefully acknowledge the studies and participants who provided biological samples and data for TOPMed. The authors acknowledge the contribution from the Samoan Obesity, Lifestyle, and Genetic Adaptations (OLaGA) Study Group.

CONFLICT OF INTEREST
The authors have no conflict of interest to declare.