Keywords FBXL4 - hypoglycemia - mitochondrial DNA - mitochondrial diseases - very long-chain
acyl-CoA dehydrogenase deficiency - pediatric genetics
Introduction
An inherited metabolic disorder (IMD) of fatty acid oxidation called very long-chain
acyl-coenzyme A dehydrogenase (VLCAD; OMIM #201475) deficiency is brought on by pathogenic
variants in the ACADVL gene. The first stage of mitochondrial oxidation of long-chain
fatty acids with chain lengths of 14 to 20 carbons is catalyzed by the VLCAD enzyme.
These long-chain fatty acids cannot be digested in VLCAD, which might result in metabolic
crises brought on by an insufficient supply of energy.
There is a spectrum of VLCAD phenotypes based on clinical presentation, biochemical
tests, and/or genetic analysis. Three subgroups of VLCAD are frequently distinguished:
severe, moderate, and mild.[1 ] The severe, or early-onset, type of the disease often manifests within the first
months of life and is characterized by cardiomyopathy, arrhythmias, hypotonia, hepatomegaly,
and hypoglycemia. The mild type often manifests as periods of hypoketotic hypoglycemia
and hepatomegaly linked to a catabolic load during late infancy or early adolescence.
Compared with severe VLCAD, cardiomyopathy is substantially less likely in mild VLCAD.
Usually beginning at adolescence, the mild or late variant is characterized by episodic
myopathy coupled with exercise intolerance and rhabdomyolysis.
The second disorder found in our case is a rare autosomal recessive disorder called
encephalomyopathic mtDNA depletion syndrome 13 (MTDPS13; OMIM # 615471), which is
caused by biallelic variants in the FBXL4 gene (MIM 605654). The FBXL4 (F-box and
leucine-rich repeat protein 4) gene has a crucial role in preserving the stability
and integrity of mitochondrial DNA (mtDNA). Worldwide, different breeds are affected
by defects in mtDNA maintenance by variants of the FBXL4 gene. Clinically, MTDPS13
often exhibits encephalopathy, hypotonia, failure to thrive, prolonged lactic acidosis,
and developmental delays,[2 ]
[3 ]
[4 ] among others. More than half of the patients have feeding difficulties, and some
show microcephaly and have characteristic facial features, for example, elongated,
protruding ears, epicanthal folds, downslanting palpebral fissures, thick eyebrows,
and cataracts. Neuroimaging often reveals white matter abnormalities.[5 ] To our knowledge, no cases of FBXL4 deficiency and VLCAD deficiency have been documented
in the literature. Consequently, we chose to make public the first case of encephalomyopathic
MTDPS13 associated with VLCAD deficiency.
Methods
Peripheral blood samples were collected from the patient and his parents. Written
consent was taken before the sample was collected. The exome of the index patient
was sequenced at CENTOGENE (Rostock, Germany).
Genomic DNA is enzymatically fragmented, and the target regions are enriched using
DNA capture probes. These regions include approximately 41 Mb of the human coding
exome (targeting >98% of the coding RefSeq from the human genome build GRCh37/hg19),
as well as the mitochondrial genome. The generated library is sequenced on an Illumina
platform to obtain at least 20 times coverage depth for greater than 98% of the targeted
bases. An in-house bioinformatics pipeline, including read alignment to the GRCh37/hg19
genome assembly and the revised Cambridge Reference Sequence (rCRS) of the Human Mitochondrial
DNA (NC_012920), variant calling, annotation, and comprehensive variant filtering,
is applied. All variants with a minor allele frequency (MAF) of less than 1% in the
gnomAD database and disease-causing variants reported in HGMD, ClinVar, or CentoMD
are evaluated. The investigation for relevant variants is focused on coding exons
and flanking ± 10 intronic nucleotides of genes with clear gene-phenotype evidence
(based on OMIM information). The American College of Medical Genetics and Genomics
(ACMG) guidelines for classification of variants and all relevant variants related
to the phenotype of the patient are reported.
Clinical Description
A 33-day-old Saudi male infant was referred to a genetic physician as a case of severe
failure to thrive, recurrent hypoglycemia, and positive newborn screening for VLCAD
on special formula. He was born to consanguineous, healthy parents after a full-term,
uneventful pregnancy. His birth weight, height, and head circumference were 2.10 kg
(third percentile), 31.5 cm (third percentile), and 44.05 cm (50th percentile), respectively.
His Apgar score was unknown. He looked dysmorphic with a small head, an elongated
face, bitemporal narrowing, epicanthal folds, downslanting palpebral fissures, thick
eyebrows, micrognathia, and a low-set ears. In his postnatal history, he was kept
in the neonatal intensive care unit (NICU) due to poor sucking and severe hypoglycemia.
He was managed accordingly and discharged home after 10 days with encouragement on
a special formula and mitochondrial cocktails. The patient was readmitted 1 week ago
with a history of lethargy, hypoglycemia, and persistent lactic acidosis. The patient
was admitted to the ICU, and dichloroacetic acid and sodium bicarbonate were used
to treat metabolic acidosis and hyperlactatemia. Dextrose 10%, riboflavin, thiamine,
and biotin were added to rule out other possible causes of mitochondrial disorders.
But his condition deteriorated. He needed mechanical ventilator support and he expired.
There was no family history of genetic or metabolic disease; there was no history
of death, miscarriage, or brain atrophy. The patient had a healthy 4-year-old sister.
Laboratory investigations revealed venous blood gas with pH 7.37, pCO2 1.5 mm Hg, and HCO3 15.4 mmol/L, lactate of 6.9 mmol/L (normal 2 mmol/L), ammonia of 81.6 mol/L (normal
40 mmol/L), elevated alanine at 721 U/L (normal 41 U/L), and aspartate aminotransferase
of 515.2 U/L (normal 40 U/L). The tandem mass spectrometry showed significant elevation of the biochemical marker C14:1 (3.744 µmol/L), which indicated VLCAD , and urine analysis by gas chromatography/mass spectrometry (GC-MS) revealed significant
lactic acid and acetoacetate levels that were all elevated (higher than the normal
reference values). Chest radiography demonstrated an increased cardiothoracic ratio
and mild cardiomegaly, and echocardiographic evaluation showed a small size atrial
septal defect (ASD) with increased left ventricular thickness and normal systolic
function in favor of hypertrophic cardiomyopathy. Transcranial ultrasound was unremarkable.
Whole-exome sequencing detected two different variant variants: a homozygous pathogenic
variant in ACADVL c.134C > A p. (Ser45*) and a homozygous pathogenic variant in the
FBXL4 c.1698A > G p. (Ile566Me). Genetic testing and target variants were done for
the parents, and genetic counseling was performed ([Fig. 1 ]).
Fig. 1 Chromatogram of the patient.
Discussion
Clinical and genetic diversities characterize inherited metabolic diseases, which
are rare disorders. Atypical features are usually described as an expansion of the
established phenotype. In this study, we describe the case of a newborn from Saudi
Arabia who had unique characteristics that were later explained by a specific combination
of two metabolic disorders, MTDPS13 and VLCAD deficits, which led to a reduction in
the availability of energy in the skeletal muscle or brain. In such cases, energy
demand is raised, and both situations are known to be triggered by fasting, infection,
and other catabolic circumstances.
In this report, we present a genetic case of two different diseases with an unusual
phenotype and two different gene variants. This is an interesting presentation, as
VLCAD deficiency in itself is a rare metabolic disease, and FBXL4 -related encephalomyopathy MTDPS represents an even rarer occurrence. Two different
gene variants were found: a homozygous FBXL4 variant c.1698A > G and an ACADVL variant
c.134C > A. The occurrence of these variants in isolation have been described, but,
to the best of our knowledge, both these gene variants occurring together in one patient
to cause two different metabolic diseases have not yet been described in the literature.
A defect of FBXL4 protein function results from FBXL4 gene variants that cause FBXL4-related
encephalomyopathic MTDPS13. It is known as mtDNA depletion when there are abnormalities
in the maintenance of mtDNA as a result of lack activity of this protein. Many of
the body cells have impaired mitochondrial activity as a result of mtDNA depletion.
Cell dysfunction brought on by diminished mitochondrial function eventually manifests
most visibly in the brain, muscles, and other tissues with high energy requirements.
Encephalomyopathy and other symptoms of the FBXL4-related encephalomyopathic MTDPS
are caused by this cell malfunction.
Most of the affected patients with FBXL4 -related encephalomyopathic MTDPS13 usually present with global developmental delay, hypotonia, and persistent lactic
acidosis resulting in early death, as Bonnen et al[2 ] reported in three unrelated consanguineous families. Microcephaly, craniofacial
abnormalities, and congenital cataracts have also been documented. Gai et al[4 ] also reported nine children with early-onset mitochondrial encephalomyopathy. Most
of them presented with lactic acidosis, neutropenia, and hyperammonemia. All the patients
had severe psychomotor retardation, hypotonia, seizures, and failure to thrive, and
reported three infant deaths from metabolic decompensation related to a severe illness.
Our patient had clinical phenotypes similar to those of the patients reported in these
studies, but he did not show hyperammonemia or seizures. During the physical examinations,
most of the previous patients showed scoliosis, small feet, hypospadias, and dysmorphic
features. Facial features included microcephaly, malformed protruding ears, cataracts,
a narrow, elongated face, thick eyebrows, epicanthal folds, downslanting of the space
between the eyelids, and short, upturned nose. Unfortunately, magnetic resonance imaging
(MRI) of the brain was not done due to the patient's critical and unstable condition,
but the majority of brain MRI scans done in previous cases have demonstrated generalized
cerebral atrophy, cerebellar hypoplasia, dilated ventricles, a thin corpus callosum,
and altered signals in the supratentorial and infratentorial white matter with involvement
of the basal ganglia and thalami. An echocardiogram documented hypertrophic cardiomyopathy
in two patients with encephalomyopathy due to MTDPS13. Therefore, the involvement
of hypertrophic cardiomyopathy in our patient can be due to VLCAD deficiency by itself,
MTDPS13, or both. The FBXL4 variant c.1698A > G p. (Ile566Met) causes an amino acid
change from Ile to Met at position 566. According to HGMD Professional 2022.1, this
variant has previously been described as a disease causing developmental delay and
lactic acidosis.[3 ]
[6 ]
[7 ]
In our patient, recurrent hypoglycemia was the most clinical sign of a metabolic disease.
A lengthy sequence of biochemical and genetic investigations led to the identification
of VLCAD deficiency as a second disease, explaining this “challenging” phenotype in
inherited metabolic diseases.[8 ]
Even if the diagnosis requires ACADVL genetic analysis and/or functional tests on
fibroblasts or lymphocytes, the development of newborn screening programs has increased
the frequency of VLCAD cases. ACADVL has so far been reported to have more than 300
known variants (
http://www.hgmd.cf.ac.uk/ac/gene.php?gene=ACADVL ). The herein-described patient carried the ACADVL variant c.134C > A p. (Ser45*),
which creates a premature stop codon. According to HGMD Professional 2022.1, this
variant has previously been described as causing a VLCAD deficiency.[7 ]
The most prevalent clinical phenotype for VLCAD deficiency is a severe early-onset
variant of VLCAD associated with significant mortality and high incidence of cardiomyopathy.
Compared with encephalomyopathic MTDPS13, an uncommon cardiomyopathy has been reported
in only two patients so far.[4 ] The outcome of VLCAD deficiency in Saudi Arabia remains poor, with recurrent admission
for metabolic crisis despite early diagnosis detected by newborn screening and immediate
treatment with a special formula containing medium chain triglyceride (MCT) and carnitine.
So preventive methods like prenatal diagnosis and preimplantation genetic diagnosis
are considered important to reduce the incidence of inherited diseases,[9 ] especially the incidence of more than just metabolic disorders, which is the most
probable explanation for the high rate of consanguinity in the Saudi Arabian population.
Conclusion
In this particular infant, the occurrence of these two uncommon metabolic disorders
resulted in quite an unusual clinical presentation. The rise in “double trouble” instances
shows that it is worthwhile to look for a more accurate diagnosis when an already
established phenotype is compounded by unusual traits and difficult diagnosis.
