A case study of lethal neonatal CPT II deficiency: Novel insights from genetic analysis

Abstract

Introduction

Carnitine Palmitoyltransferase II (CPT II) deficiency encompasses a spectrum of disorders, with the lethal neonatal form (LNF) representing the rarest and most severe. While there are numerous CPT2 gene variants that can cause CPT II deficiency, only 16 variants of these are known to be associated with LNF. This report presents the case of a neonatal male diagnosed with lethal CPT II deficiency, characterized by the presence of two heterogeneous variants. Additionally, we provide a comprehensive review of all clinical symptoms, biochemistry, and reported pathogenic variants associated with LNF CPT II deficiency.

Case presentation

A neonatal male exhibited typical symptoms and biochemical features of CPT II deficiency, along with abnormal long-chain fatty acid profiles, notably an exceptionally high level of C18OH. Genetic analysis of the dried blood spot (DBS) sample revealed two heterozygous variants: CPT2 p.E174K and p.R554X. Both the healthy father and mother carried heterozygous variants, p.R554X and p.E174K, respectively.

Discussion

The symptoms of the LNF CPT II deficiency are characterized by the unavailability of fatty acids for energy production and the accumulation of lipids in tissues, primarily due to the extremely low activity of CPT II. The genetic variants associated with these cases are notably limited, and all of them are classified as ‘severe’ variants. In the presented case, the co-occurrence of p.R554X with another severe variant, p.E174K, manifests as LNF, this compelling evidence strongly supports the assertion that p.R554X is a potentially severe pathogenic variant contributing to CPT II deficiency.

Conclusion

This report represents the initial documentation of a LNF CPT II deficiency case characterized by the presence of two heterozyous CPT2 variants: p.E174K and p.R554X. As a result, the p.R554X variant is potentially classified as a severe pathogenic variant. It further emphasizes the significance of early detection and precise mutation classification for effective disease.

1. Introduction

Carnitine palmitoyltransferase II (CPT II) deficiency is a disorder of mitochondrial fatty acid β-oxidation, classified as a type of inborn error of metabolism. The disorder can manifest as myopathic form (also called stress-induced myopathic form or milder adult muscular form) (86 % of cases), infantile or severe infantile hepato-cardio-muscular form (8 % of cases), and lethal neonatal form (LNF) (6 % of cases) [1]. LNF is the rarest and most severe form. The LNF form is rapidly fatal and presents shortly after birth with respiratory distress, seizures, altered mental status, hepatomegaly, cardiomegaly, cardiac arrhythmia, and renal dysgenesis. The disease is caused by CPT2 mutation (OMIM # 608836). To our knowledge, at least 26 lethal neonatal cases have been reported [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. Symptoms present quickly after birth as hypoketonic hypoglycemia, liver failure, neuronal damage, and cystic renal dysplasia. Only 16 variants are known to be associated with LNF [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. These variants are known to cause severely reduced activity of CPT II, contributing to the lethal phenotype. Variants can result in truncated proteins, decreased protein stability, inactive enzymes or destruction of the splice recognition site. The variants may occur as either heterozygous or homozygous forms [17].

Herein, we present a case exhibiting typical clinical symptoms of LNF CPT II deficiency, which was identified through newborn screening by tandem mass spectrometry (MS/MS) and genetic analysis of CPT2 gene.

2. Case presentation

A full-term 2500-g male baby was born by vaginal delivery to a mother with a normal pregnancy. He is the second child, and his sibling is normal. The patient experienced cardiac arrest, apnea, and convulsion. The symptoms appeared 24 h after birth and he died 5 h later. The biochemistry analysis revealed metabolic acidosis, hypoglycemia, hyperammonemia, increased liver enzymes, and low prothrombin level. Tandem mass spectrometry (MS/MS) analysis of acylcarnitines in a dried blood spot card revealed an extreme increase in C18OH acylcarnitines, approximately 23 times higher than the upper limit. Furthermore, other long-chain fatty acids also increased significantly such as C14, C16, C16:1, C18, C18:1. Finally, the results indicated a decrease of C2 and C3 levels, 2 to 4 times lower than the lower limits of the reference range.

Because the clinical symptoms and newborn screening results cannot distinguish between CPT II deficiency and carnitine-acylcarnitine translocase deficiency [18,19], we analyzed both CPT2 and SLC25A20 genes. The results showed that there was no variant detected in the SLC25A20 gene, but there were two heterozygous variants in the CPT2 gene: p.E174K in exon 4 and p.R554X in exon 5. In addition to the pathogenic variant CPT2 p.E174K causing CPT II deficiency, the CPT2 p.R554X variant is only known in a patient with milder form of myoglobinuria [20]. Therefore, we continued to analyze CPT2 gene in the healthy parent, who presents without symptoms of CPT II deficiency and abnormal long-chain fatty acid levels. The mother was found to have a heterozygous p.E174K variant, and the father had a heterozygous p.R554X variant. The results suggest that p.R554X is also a pathogenic variant. Considering these results, the presence of two pathogenic variants, p.E174K and p.R554X, confirmed the diagnosis of LNF CPT II deficiency.

3. Discussion

LNF CPT II deficiency is the rarest form of the disorder [16]. The lethal neonatal form (LNF) of CPT II deficiency is a rapidly fatal condition caused by mutations in the CPT2 gene. It presents shortly after birth with severe clinical symptoms, including respiratory distress, seizures, altered mental status, hepatomegaly, cardiomegaly, cardiac arrhythmia, and renal dysgenesis. Biochemical findings in LNF cases often reveal a increase in levels of long-chain fatty acids, such as C18, C16, or C14 carnitines, due to defective CPT II enzyme activity, which impairs β-oxidation of these fatty acids. The reported case consistent with the diagnosis of LNF, as evidenced by typical clinical symptoms, biochemical indications of β-oxidative failure, and the presence of two heterozygous CPT2 mutations.

There are at least 26 cases that have been reported in the literature (Table 1) [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. CPT II deficiency affects both males and females equally, and the patients' ethnic backgrounds are varied. The onset time may occur as early as the 2nd trimester of pregnancy or at birth, and the symptoms can vary. The most common symptoms include cardiomyopathy, respiratory distress, and liver damage, with the possibility of renal cysts and brain damage. In our case, the patient exhibited symptoms consistent with cardiac arrest and convulsions. The biochemistry testing shows the same results between the reports and our case, including hypoglycemia and metabolic acidosis. Liver damage can manifest through elevated liver enzymes and low prothrombin activity. In all cases, the carnitine and fatty acid profiles showed reduced levels of total and free carnitine, including C2, C3; alongside increased long-chain fatty acylcarnitine, such as C16, C16:1, C18, and C18:1. In our case, C18OH level increased markedly increase, reaching up to 23 times the upper limit. The symptoms and abnormal biochemistry arise from the inefficient utilization of fatty acids to produce energy for metabolism and muscular action. As a result, lipid accumulation occurs in multiple tissues, including the liver, kidney, and skeletal muscle, which may potentially exacerbate the symptoms. In the LNF, CPT II activity is almost ceased, with activity in cultured fibroblast cells approximating only 7 % compared to the control [[10], [11], [12], [13], [14], [15]]. The enzyme activity in LNF is significantly lower than the milder adult muscular form, which exhibits 20 % to 46 % residual CPT II activity, without any consequences on long-chain fatty acid oxidation in normal circumstances [[21], [22], [23]]. However, the level of CPT II deficiency in the LNF and the infantile form is comparable, with CPT II activity also low, ranging from 2.5 to 10 % compared to the control [5,24,25].

Table 1.

Total reported cases of LNF CPT II dificiency.

No.	CPT2 variant	CPT II activity vs friboblast control	Sex, onset time – death time/outcomes	Symptoms and biochemistry
1	11-BP DUP, NT997	8 %	N/A-25 days [2]	Renal cyst at autopsy
2	c.1237delAG p.F448L	Below detection limit	Male, 23 weeks of gestration, at birth – 34 days Female, 22 weeks of gestration - terminated [3]	Absence of the corpus callosum, ventriculomegaly, intracerebral periventricular calcifications, renal cyst, cardiomegaly. Renal insufficiency, hepatoseatosis, feeding difficultie. Metabolic acidosis, increased serum liver enzyme. Severe nonketotic dicarboxylic aciduria
3	p.Q413fs p.S38fs	6 %	Male, 17 h - 3 days [4]	Weak cry, decrease tone, tachypnea, sepsis, hyperkalemia, hypoglycemia, renal insuffiency, elevated transaminases. Bradycardia, seizures, liver calcification, dysplatic kidney, unstable cardiac function.
4	p.P227L	N/A	Female, N/A - 14 days [5]	Normal at birth but developed hypoglycemia and hyperammonemia in the nursery. Increased liver enzyme. Heart block, polycystic kidneys, and seizures.
5	c.534Tinsdel25† IVS2-1G-A	2 %	1 female and 3 males, between day 1 and day 10 – died [7]	Seizures, lethargy, hypotonia, respiratory insufficiency, cardiac arrhythmias, cardiomegaly, and hepatomegaly
6	p.P50H p.P595fs	N/A	Male 2nd trimester, 24 h - 6 days [5]	Renal syst and enlarged kidney, periventricular calcification Deteriorated respiratory, left ventricular hypertrophy
7	p.E641fs	N/A	Female, prenatal screening, at birth - 30 h [5]	Enlarged kidney due to cyst, enlarged heart, partial absent of corpus callosum in the brain Apnea Kidney failure, increase C16, C18:2, C18:1,C18
8	p.R579* p.E174K†	N/A	Male, extremely early - 2 days [6]	Severe hepatocardiomuscular. Autopsy: massive pulmonary atelectasis, cardiomegaly and hepatomegaly with intracytoplasmic steatosis
9	p.R296Q c.534Tinsdel25†	7 %	Male, At birth [8]	Respiratory distress, pulmonary hypertension, Dandy-Walker Syndrome Metabolic acidosis and hypotension. Bradycardia, hepatic dysfunction. Low total carnitine, low free carnitine and elevated acylcarnitine to precarnitine ratio, increased long-chain fatty acid: C16, C18:1
10	c.534Tinsdel25†	N/A	Female, 12 days [9]	Dandy-Walker syndrome, metabolicacidosis, cardiorrespiratory failure. Increased C16, C16:1, C18, C18:1, decreased C2.
11	c.38delG c.1369 A > T	N/A	2 male, N/A – shortly after birth. Pregnancy screening – terminated [16]	Multicystic kidneys and oligohydramnios Cystic renal dysplasia, pulmonary hypoplasia, and Potter sequence. Cardiomegaly, hepatomegaly hepatostetosis
12	p.R554X p.E174K†	N/A	Male, 24 h - 29 h (our case)	Cyanosis, cardiac arrest, apnea, convulsion. Metabolic acidosis, hyperammonia, hypoglycemia, increased liver enzyme, low prothrombin. Decreased C2, C3, increased C14, C16, C16:1, C18, C18:1, C18OH, C18:1OH
13	N/A	2.5–10 %	5 female, 2 male, 3 N/A, arly after birth - 34 days a [10–2] [14,15]	Cardiomegaly, hepatomegaly hepatostetosis, renal cysts, seizures. Lipid accumulation was evident at autopsy, including in the liver, heart, kidney, adrenal cortex, skeletal muscle, and lungs. Nonketotic hypoglycemia, free carnitine decreased in the urine and plasma, elevation of plasma long-chain acylcarnitines

N/A: not available.

a: One patient was treated with l-carnitine and a high-carbohydrate diet at 6 months, but the abdominal ultrasonographic were unchange, hypertrophic concentric cardiomyopathy.

† The variants present in more than one family.

Patients with LNF CPT II deficiency typically die within several days after birth. In some cases, including our patient and the one reported by Isackson P.J. et al. [5], death occurred as early as 29 h after birth. Although the patient was treated with l-carnitine and a high-carbohydrate diet, the symptoms did not improve, and the disease continued to progress [11]. Furthermore, LNF and the infantile forms exhibit very low CPT II activity, but the infantile form can result in either death or recovery with proper treatment [10]. This suggests that the onset time and the circumstances of the onset are crucial, as they determine the body's ability to compensate.

Sixteen variants have been reported in 26 lethal neonatal cases relating to 11 different families [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. All variants are severe and present either in homozygous form or as two heterozygous variants (Table 1). These variants may lead to either truncated proteins, decreased protein stability, inactive enzymes or destruction of the splice recognition site. Our LNF case, p.E174K (OMIM 600650.0006) and p.R554X was found. The p.R554X variant introduces a premature stop codon, which is predicted by Mutation taster software, resulting in a truncated protein and likely severely reduced enzyme activity. Interestingly, the p.R554X variant has previously been reported in a male patient, 30-year-old with myoglobinuria, a milder adult muscular CPT II deficiency. This patient carried two heterozygous variants, the “mild” variant p.S113L and the new variant p.R554X [20]. Although our patient has LNF CPT II deficiency, both parents are normal. We continued to analyze the CPT2 gene in healthy parents. The result showed that the mother carried the p.E174K heterozygous and the father carried the p.R554X heterozygous. Therefore, to manifest the clinical symptoms, p.R554X has to combine with another variant such as mild p.S113L causing milder form - myoglobinuria or severe p. E714K causing LNF. Taken together, we can suggest that p.R554X is a severe truncate pathogenic variant. Furthermore, the combination of p.E174K with another truncated variant p.R579X also causes LNF manifestation [6].

Effectively managing this highly lethal disorder necessitates early diagnosis and appropriate treatment. The recent classification of p.R554X as a severe pathogenic variant, coupled with the identification of the inaugural LNF case resulting from the combination of p.E714K and p.R554X, enriches our comprehension of the spectrum of severe variants linked to LNF and CPT II deficiency. This enhanced knowledge holds the potential to improve early diagnostics and precise classification, which is helpful for clinical decision-making and family counselling.

4. Conclusions

We report a case with typical clinical symptoms of CPT II deficiency and carnitine/acylcarnitine profiles by newborn screening using MS/MS and confirmed by genetic analysis This is the first time CPT II deficiency and lethal neonatal form is reported in Vietnam. The patient harbors a combination of two heterozygous variants, CPT2 p.E174K and p.R554X. The p.R554X can be classified as a severe variant. The newfound could significantly contribute to the understanding of genetic variants associated with LNF CPT II deficiency.

CRediT authorship contribution statement

Van-Thao Ta: Writing – review & editing, Data curation, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Data availability

Data will be made available on request.