Do renal and cardiac malformations in the fetus signal carnitine palmitoyltransferase II deficiency? A rare lethal fatty acid oxidation defect

Abstract

The neonatal form of carnitine palmitoyltransferase II (CPT II) deficiency is a rare lethal inherited disorder of fatty acid oxidation. Carnitine essentially transfers long-chain fatty acids across the mitochondrial membranes for β-oxidation, where CPT II plays a key role. CPT II deficiency phenotypical forms include lethal neonatal, severe infantile and myopathic forms. We present a term small-for-gestational-age neonate with hypoglycaemia, seizures, refractory cardiac arrhythmias and intracranial haemorrhage. Plasma acylcarnitine profile and the genetic study confirmed CPT II deficiency. Additionally, likely pathogenic variants in the SLC22A5 gene point to primary carnitine deficiency. Antenatal findings of polycystic kidney disease and cardiomegaly were confirmed postnatally. All supportive measures, including extracorporeal life support, failed to improve the clinical course, and the baby succumbed. Major renal, cerebral and cardiac anomalies were reported with CPT II deficiency. In our case, fetal polycystic nephromegaly and cardiomegaly with parental consanguinity should have signalled the possibility of this disorder.

Background

Carnitine is derived from the diet or biosynthesised from lysine and methionine in the liver and excreted in the urine.¹ The key step in the fatty acid oxidation (FAO) process is the entry of long-chain fatty acids (LCFAs) into the mitochondria through the carnitine shuttle. In the fasting state, the carnitine palmitoyltransferase I (CPT I) enzyme in the outer mitochondrial membrane converts LCFA from their coenzyme A (CoA) ester to long-chain acylcarnitines (LCACs), allowing its entry into the intermembranous space. They are then translocated through the inner mitochondrial membrane in the presence of transporter carnitine-acylcarnitine translocase (CACT) and then reactivated to long-chain acyl-CoA for β-oxidation in the mitochondrial matrix. The CPT II enzyme encoded by the CPT2 gene converts acylcarnitine back to fatty acyl-CoA. The regenerated carnitine is pumped back to the cytosol by CACT to be recycled again in the carnitine shuttle (figure 1).²

Figure 1.

Carnitine transport and fatty acid oxidation. CACT, carnitine-acylcarnitine translocase; CoA, coenzyme A; CPT I, carnitine palmitoyltransferase I; CPT II, carnitine palmitoyltransferase II.

In autosomal recessively inherited CPT II deficiency, the LCACs accumulate inside the mitochondria and plasma, failing to generate energy. In neonatal form, infants present early in life with signs of postnatal energetic failure leading to an early demise.³ Many fetuses with CPT II deficiency were reported to have severe forms of renal, cerebral and cardiac anomalies.⁴ However, major anomalies often warrant termination, or they die early before a metabolic screening could confirm the diagnosis, missing the window to link CPT II deficiency. We highlight the significance of antenatally detected major renal and cardiac anomalies, which may hint at the possibility of CPT II deficiency, especially with parental consanguinity.

Case presentation

A term female baby was born by normal vaginal delivery to a first-degree consanguineous couple with two healthy living children and one early pregnancy loss. The neonate was symmetrically small-for-gestational-age (SGA) with a birth weight of 2545 g (6th percentile, −1.57 SDS), an occipitofrontal circumference of 32 cm (6th percentile, −1.54 SDS) and a length of 49 cm (41st percentile, −0.23 SDS). There was no family history of neonatal or unexplained childhood deaths.

Fetal ultrasound (US) scans at 20 weeks’ gestation reported bilateral nephromegaly with echogenic kidneys and cardiomegaly. Follow-up scans at 29 weeks identified left renal cysts. The amniotic fluid index was normal. This case was discussed in the weekly birth defect-fetal medicine meeting and made care plans with the multidisciplinary team for postnatal management.

The baby was born with Apgar scores of 9 at 1 and 5 min of life, respectively. She was transferred to the special care neonatal unit to evaluate fetal scan abnormalities and for close vital signs and capillary blood glucose (CBG) monitoring. She was non-dysmorphic, and her general physical and systemic examinations were unremarkable. Breast feeding with top-up milk was continued while on CBG monitoring. She developed hypoglycaemia at 18 hours of life, which resolved with buccal glucose gel and milk feeds. However, she continued to have recurrent hypoglycaemia with a nadir of 1.8 mmol/L, needing a 9 mg/kg/min glucose infusion rate to achieve normoglycaemia. She had normal bowel and bladder output.

Due to temperature instability, antibiotics were initiated for presumed sepsis. Screening for infective markers was unremarkable, and the blood culture was sterile. The postnatal US confirmed bilateral polycystic kidney disease. An echocardiogram showed a structurally normal heart with biventricular hypertrophy. At 55 hours of life, she developed recurrent apnoeas. CBG levels were normal. She was stabilised with nasal oxygen and transferred to the neonatal intensive care unit for respiratory support. Soon she developed generalised seizures requiring anticonvulsants and ventilatory support. Hyperkalaemia was noted (8.1 mmol/L) and was treated with intravenous calcium gluconate, insulin-glucose infusion and nebulised salbutamol. With an episode of hyperkalaemia, she developed circulatory collapse with pulseless electrical activity and ventricular tachycardia, which required cardiopulmonary resuscitation and defibrillation (figure 2A–C). Hypotension was refractory to saline boluses and required inotrope support. Soon after resuscitation, blood gases became acidotic, needing bicarbonate correction.

Figure 2.

(A) ECG features of hyperkalaemia with widened QRS complex and tall symmetrical T waves. (B) ECG showing ST elevation in V1–2, with ST depression in leads V5–6. (C) Refractory ventricular fibrillation.

Investigations

A tandem mass spectrometry (TMS) screen was done on day 3, and the results returned on day 6 of life, with low carnitine and raised LCFA, especially C16,18 and C18:1-octadecanoylcarnitine and an abnormal (C16+C18:1)/C2 ratio of 5.03 (normal <0.3). This profile is highly suggestive of CPT II/CACT deficiency. Day 4 plasma acylcarnitine profile also showed persistent elevation of LCFA, consistent with the diagnosis. On day 7, a repeat acylcarnitine profile following carnitine therapy showed high carnitine levels and a significant rise in LCFA (table 1).

Table 1.

Acylcarnitine profile in newborn screening and plasma

Acetylcarnitine profile	Plasma acylcarnitine (µmol/L) Day 4 of life	Plasma acylcarnitine (µmol/L) Day 7 of life	Reference (µmol/L)	Newborn screening (µmol/L) Day 3 of life
C0—free carnitine	4 L	96	15–40	3 L (abnormal <8)
C2—acetylcarnitine	3 L	2 L	4–22	2.8 L (abnormal <7)
C3—propionylcarnitine	0.06 L	0.06 L	0.18–0.73	–
C3DC—malonylcarnitine	0.01 L	0.01 L	0.02–0.12	–
C4—n-butyryl-/isobutyrylcarnitine	0.02 L	0.05 L	0.09–0.36	–
C4OH—3-Hydroxy-butyrylcarnitine	0.01 L	0.01 L	0.02–0.15	–
C5—isovaleryl-/2-Methylbutyrylcarnitine	0.01 L	0.02 L	0.04–0.30	–
C5:1—tiglylcarnitine	0.01	0.01	0.01–0.11	–
C5OH—3-Hydroxy-isovalerylcarnitine	0.02	0.02	0.01–0.11	–
C5DC—glutaryl/3-Hydroxydecanolycarnitine	0.03	0.05	0.03–0.19	–
C6—hexonylcarnitine	0.02 L	0.03	0.03–0.11	–
C8—octanoylcarnitine	0.01 L	0.05	0.05–0.23	–
C10—decanoylcarnitine	0.04 L	0.22	0.05–0.45	–
C10:1—decenoylcarnitine	0.04 L	0.14	0.06–0.23	–
C10:2—decadienoylcarnitine	0.01 L	0.02	0.02–0.15	–
C12—dodecanoylcarnitine	0.13	0.55 H	0.02–0.33	–
C12:1—dodecanoylcarnitine	0.05	0.21	0.02–0.25	–
C14—tetradecanoylcarnitine	0.44 H	1.69 H	0.02–0.22	–
C14:1—tetradecadienoylcarnitine	0.25	0.67 H	0.02–0.39	–
C14:2—tetradecadienoylcarnitine	0.04	0.18 H	0.01–0.11	–
C16—hexadecanoylcarnitine	4.35 H	18.68 H	0.03–0.60	9.53 H (abnormal >7)
C16:1—hexadecenoylcarnitine	0.8 H	4.28 H	0.01–0.23
C18—octadecanoylcarnitine	0.92 H	2.45 H	0.02–0.17	3.27 H (abnormal >2.5)
C18:1—octadecanoylcarnitine	2.89 H	10.58 H	0.04–0.58	4.57 H (abnormal >3)
C18:2—linoleylcarnitine	0.32 H	1.41 H	0.01–0.16	–
C16OH—3-Hydroxy-hexadecanoylcarnitine	0.14 H	0.38 H	0.00–0.05	–
C16:1OH—3-Hydroxy-hexadecenoylcarnitine	0.48 H	0.3 H	0.00–0.03	–
C18OH—3-Hydroxy-steaeroylcarnitine	0.03 H	0.05 H	0.00–0.02	–
C18:1OH—3-Hydroxy-linoleylcarnitine	0.04 H	0.13 H	0.00–0.02	–
(C16+C18:1)/C2 ratio	–	–	–	5.03 H (normal <0.3)

Molecular testing done at Invitae diagnostics (San Francisco, California, USA) found a novel homozygous pathogenic variant, c.63dup (p.Ser22GInfs*37), in CPT2, confirming the diagnosis of CPT II deficiency. Homozygous likely pathogenic variants were identified in SLC22A5 (c.641C>T(p.Ala214val), in keeping with a concurrent diagnosis of primary carnitine deficiency (table 2).

Table 2.

Genetic test results

Gene	Variant	Zygosity	Variant classification
CPT2	c.63dup(p.Ser22Glnfs*37	Homozygous	Pathogenic
SLC22A5	c.641C>T(p.Ala214Val)	Homozygous	Likely pathogenic

Her haemogram revealed anaemia, thrombocytopenia and coagulopathy, requiring multiple blood product transfusions. Biochemical analysis showed hyperammonaemia, transaminitis, raised creatine kinase (CK), CK-MB and serum creatinine. Cardiac enzymes were markedly elevated, with ST-elevation on a 12-lead ECG, suggesting myocardial ischaemia in the background of possible cardiomyopathy (table 3 and figure 2B).

Table 3.

Haematological and biochemical parameters during the illness

Day of life	2	3	4	5	7	8	9	10	12	13
Full blood count
Haemoglobin  (g/L)	190	170	159	142	111	104	96	93	86	94
WCC (109/L)	33.91	12.99	16.89	15.84	5.27	6.81	6.93	5.48	2.54	2.49
Platelet (109/L)	326	386	414	235	146	112	69	52	39	37
Biochemistry
ALT (U/L)			60	74	36	28		18
AST (U/L)			701	609	475	187		63
Ammonia (µmol/L)				113		44
Lactate (mmol/L)				3.5
Troponin I (ng/L)				>50 000	>45 699	>288 70	>41 698	>28 399
CK (U/L)				4074	3125	1195	513	221
CK-MB (U/L)				295.3	110.1	19.8	12	8.2
Creatinine (µmol/L)		93	114	102	105	123	160	210	45	39
Coagulation profile
PT (s)				18.2	18.6	17.8	17.8	22	22.6	37
APTT (s)				48.7	122.9	118.3	148.8	163.9	121	180
INR				1.59	1.63	1.54	1.54	2.04	2.1	4.1

ALT, alanine transaminase; APTT, activated partial thromboplastin time; AST, aspartate aminotransferase; CK, creatine kinase; INR, international normalised ratio; PT, prothrombin time; WCC, white cell count.

Day 3 cranial US scan was normal except for a left germinal matrix haemorrhage. A repeat scan on day 12 showed severe right cerebral parenchymal haemorrhage with cerebral oedema, causing a mass effect on the right lateral ventricle with a midline shift to the left. Avascular echogenic regions were noted in the right frontal and temporal lobes, measuring 2.5 cm.

Differential diagnosis

CACT deficiency: differentiating CACT deficiency from the neonatal form of CPT II deficiency is difficult using acylcarnitine profiling. Molecular genetic testing is diagnostic with compound heterozygous or homozygous pathogenic variants in SLC25A20.⁵

Glutaric acidaemia type II: these patients have raised acylcarnitines from C4 to C18. A rise in a range of urine organic acids differentiates glutaric aciduria from CPT II deficiency. Molecular genetic testing confirms the diagnosis.⁶

Treatment

Her management included optimising calories, supplementing carnitine and using medium-chain triglycerides (MCTs). On day 4, her cardiorespiratory status improved briefly before deteriorating on day 5 of life, in which she developed further two episodes of circulatory collapse with recurrent ventricular tachycardia and fibrillation. Further cardiac imaging showed biventricular hypertrophy with reduced contractility. Subsequently, she developed cardiorespiratory failure with refractory ventricular arrhythmias and was placed on inhaled nitric oxide and later extracorporeal membrane oxygenation (ECMO) support. Intravenous amiodarone infusion was started, and defibrillation was attempted again. However, she remained in ventricular fibrillation.

Outcome and follow-up

Her clinical course was complicated by hypotension and acute renal failure requiring haemodialysis. She subsequently developed a severe intracranial bleed in the right cerebral hemisphere, which led to the decision for comfort care. ECMO support was withdrawn, and the child demised on day 14 of life (figure 3). The parents did not consent to an autopsy.

Figure 3.

Timeline depicting the sequence of events from fetal to day 14 of postnatal life. CPR, cardiopulmonary resuscitation; ECMO, extracorporeal membrane oxygenation; HFOV, high-frequency oscillatory ventilation; ICH, intracranial haemorrhage; iNO, inhaled nitric oxide; TMS, tandem mass spectrometry; US, ultrasound; VF, ventricular fibrillation, VT, ventricular tachycardia.

Discussion

We have portrayed a classic case of the lethal neonatal form of CPT II deficiency in a child born to a consanguineous couple. Onset was typical with hypoglycaemia on day 1 of life, followed by seizures, hypotension, refractory cardiac arrhythmias, renal failure and intracerebral haemorrhage, leading to the withdrawal of supportive care and subsequent demise. TMS and plasma acylcarnitine profile suggested CPT II deficiency, confirmed with molecular testing. Two likely pathogenic variants in the SLC22A5 gene point to primary carnitine deficiency. Antenatal findings of fetal polycystic kidney disease and cardiomegaly were confirmed postnatally.

Inherited mitochondrial carnitine-acylcarnitine cycle disorders include CPT I, CACT and CPT II deficiency. CPT II has three phenotypical presentations: lethal neonatal, severe infantile and adult myopathic forms, the latter being the most common. Symptoms appear on fasting or with intercurrent illness.^{3 4}

The lethal neonatal form has been described in at least 20 families worldwide, while the severe infantile form has been identified in approximately 30 families. The most frequent myopathic form has been reported in more than 300 cases.^{4 5 7}

The antenatal phenotype includes facial dysmorphism, cerebral malformations, cystic dysplastic kidneys and diffuse fatty infiltration. Pregnancies complicated by oligohydramnios, fetal cerebral ventriculomegaly, corpus callosum agenesis, vermian hypoplasia, pachygyria, polymicrogyria and intraventricular calcifications have been described.⁸ Boemer et al reported the recurrence of severe cerebral dysgenesis with Dandy-Walker malformation in three successive pregnancies with fetuses having CPT II deficiency. Authors reviewed 19 reported cases with malformations associated with CPT II deficiency and found 14 of 19 (74%) with major cerebral dysgenesis, 11 of 19 (57%) with renal cysts/nephromegaly and 3 of 19 (15%) with cardiomegaly.⁴ Oey et al studied the expression of the CPT2 gene in the mitochondrial oxidation of LCFA during early human development. Authors demonstrated high CPT II enzyme activity in the heart, liver and brain in a 6-week-old embryo, explaining the severity and multiorgan involvement in CPT II deficiency.⁹ In retrospect, fetal enlarged cystic kidneys and cardiomegaly in our case should have signalled the need to make an early postnatal diagnosis.

The neonatal form of CPT II deficiency often becomes symptomatic soon after birth. The affected infant may have dysmorphic features such as microcephaly, a sloping forehead, overfolded helices, long and tapered fingers and toes, contractures and hypoplastic toenails; however, none of which were found in our case.¹⁰ In CPT II deficiency, the body cannot use LCFA as an energy source and must rely only on glucose. Once glucose has been used up and the body fails to use fat, they develop hypoketotic hypoglycaemia. Other manifestations of this multisystemic disease include respiratory distress and cardiac arrhythmias. These patients may have cystic dysplastic kidneys, cardiomyopathy,^{4 11} structurally abnormal brain and neuronal migration defects⁸ due to defective mitochondrial β-oxidation causing impaired phospholipid synthesis and accumulation of intermediary metabolites in utero, which are toxic to the developing fetus.^{9 12} Our patient, too, developed hypoglycaemia, seizures and cardiovascular collapse following recurrent arrhythmias.

The patient deteriorated very rapidly, and the sequence of events was unexplainable until the acylcarnitine profiles and TMS returned abnormal. Infants with the neonatal form of CPT II deficiency have been reported to live for a few days to a few months,³ and our patient died at 2 weeks of life despite maximum therapy.

Among other phenotypical forms of CPT II deficiency, the severe infantile hepatocardiomuscular type presents with hypoketotic hypoglycaemia, liver failure and cardiomyopathy in the first year of life. It could potentially cause fatality even if treated early. The more common myopathic form typically presents with myoglobinuria in adults precipitated by exercise, fasting or infection.^{5 13}

A novel pathogenic homozygous variant of the CPT2 gene was identified in our case. The sequence change in exon 1 creates a premature translational stop signal (p.Ser22GInfs*37) in the CPT2 gene, resulting in an absent or disrupted protein causing loss-of-function variants, which are known to be pathogenic.¹⁴

Our patient also had primary carnitine deficiency, with homozygous likely pathogenic variants found in SLC22A5 (c.641C>T(p.Ala214Val)). The missense change in alanine with valine in the SLC22A5 gene has been reported with primary carnitine deficiency.¹⁵ Carnitine biosynthetic disorders are rare as most carnitine is from the diet. The biosynthetic component of carnitine is small; hence, these disorders may remain asymptomatic if dietary intake is sufficient.¹⁶ Due to defective LCFA transport and oxidation, primary carnitine deficiency presents with hypoglycaemia due to increased glucose consumption without regeneration via gluconeogenesis during fasting. Decreased LCFA utilisation leads to fat accumulation in the liver, skeletal muscle and heart resulting in organ dysfunction. A broad spectrum of signs and symptoms of primary carnitine deficiency appears during infancy or early childhood, which includes severe brain dysfunction, lethargy, cardiomyopathy, vomiting, confusion and muscle weakness. Some affected infants develop hypoketotic hypoglycaemia, hepatomegaly with transaminitis and hyperammonaemia. The concurrent presence of a primary carnitine uptake defect and recycling in this patient could have an exacerbating factor in the fatty acid oxidation cycle impairment. Primary carnitine deficiency is diagnosed by TMS, showing low free carnitine levels and molecular testing for SLC22A5 gene. If molecular testing is inconclusive, a skin biopsy can be done to assess carnitine transport in cultured fibroblasts.¹⁷

To the best of our knowledge, a combined genetic metabolic disorder of this kind was not reported in the literature.

With the advancement in laboratory technology, TMS can be used for neonatal metabolic screening for more than 40 inborn errors of metabolism (IEM) conditions, including FAO disorders.¹⁸ Significant elevations of C16 and C18-acylcarnitine with decreased free carnitine in the TMS profile indicate CPT II/CACT deficiency. A (C16+C18)/C2 ratio of >0.3 helps discriminate false-positive results, increasing the diagnostic specificity of the screening, as noted in our case and others.¹⁹ For a definitive diagnosis, rapid molecular genetic testing is recommended.²⁰ More than 90 pathogenic mutations have been reported in the CPT2 gene with consistent genotype–phenotype correlation. Protein truncation and mRNA degradation have been found in the neonatal form of this disorder.^{4 5} Alternatively, this diagnosis can be made by detecting decreased CPT II enzyme activity in cultured fibroblasts or skeletal muscles.⁵ Other biochemical findings such as very high serum CK concentration and transaminitis of 20–400 times higher have been observed.²¹

If parents are carriers of CPT II deficiency, prenatal and pre-implantation genetic testing can be offered for future pregnancies.²² Antenatal diagnosis of CPT II deficiency using TMS in amniotic fluid was unsuccessful, probably due to slow excretion and poor solubility of acylcarnitine in fetal urine.⁴ Fetal diagnosis can be made by analysing CPT II enzyme activity in cultured amniocytes or chorionic villi samples (CVS).²³

Once the diagnosis is established, the treatment principles include nutrition by providing high carbohydrate (70%) and low fat (<20%) as MCT to provide fuel for glycolysis, avoidance of known triggers like illness, extreme cold, extreme heat and drugs (ibuprofen, diazepam and sodium valproate),^{8 24 25} frequent feeding to avoid fasting and the use of carnitine supplements.^{3 5} Use of glucose infusion to slow down catabolism and reduce fat utilisation is suggested to prevent recurrent hypoglycaemia, affecting the long-term neurodevelopmental outcome.²⁶ Adequate hydration is essential in those with cystic dysplastic kidneys, curtailing the progression of renal failure.

Carnitine use in CPT II deficiency remains controversial and is expected to convert potentially toxic long-chain acyl-CoA to acylcarnitine and eliminate it in the urine.¹³ Carnitine supplementation remains the mainstay in primary carnitine deficiency. Cochrane review concluded that there is a lack of evidence-based data on the efficacy and safety of carnitine supplements in IEM.²⁷ In mouse models, there was inhibition of oxidative phosphorylation in the mitochondria of the ischaemic myocardium due to LCAC accumulation.²⁸ The hERG (human ether-a-go-go-related gene) channel, which is essential for cardiac repolarisation, is regulated by acylcarnitine, which can contribute to the development of arrhythmias.²⁹ The risk increases in phases of acute metabolic crises, restricting the use of carnitine.²⁴ On initiation of carnitine supplements in our reported case, the patient remained in refractory arrhythmias. Hence, carnitine was discontinued on recovery of carnitine levels.

Conclusion

Paediatricians should familiarise themselves with early red flags such as lethargy, vomiting, poor feeding, altered sensorium and seizures as symptomatology of IEM. TMS screening is a sensitive tool to pick up FAO defects. The neonatal form of CPT II deficiency has a poor prognosis. It is essential to recognise this disorder as early as possible and ensure frequent feedings with strict avoidance of fasting, a low-fat, high-carbohydrate diet, and supplementation with MCT. Neonatal management can be more streamlined if an antenatal diagnosis can be made using CVS. Antenatal detection of polycystic kidney disease should raise the suspicion of CPT II deficiency, especially with a history of parental consanguinity or recurrent fetal losses, as noted in our case. Unfortunately, most fetuses with major malformations, especially of the brain and kidneys, are terminated, missing the opportunity for metabolic screening, foiling the establishment of coexisting IEM.

Patient’s perspective.

Parent’s perspective: It was shocking news for the family to hear about the severe medical condition of our daughter. We lost her in 2 weeks. Our family is grateful to the intensive care team for the treatment given to our daughter during this short period.

Learning points.

• The neonatal form of carnitine palmitoyltransferase II (CPT II) deficiency is a rare and lethal form of fatty acid oxidation defect.

• Antenatal detection of cerebral dysgenesis, polycystic kidney disease or cardiomyopathy may be a clue to CPT II deficiency in the background of parental consanguinity or recurrent fetal losses/neonatal deaths.

• Tandem mass spectrometry screening of newborn infants helps make early diagnosis and confirm using acylcarnitine profile and molecular testing.

• Hypoglycaemia and arrhythmia remain the most common presentation.

• Supportive therapy is needed in newborn infants initially with glucose infusion and later with milk feeds having 70% carbohydrate and 20% fat as medium-chain triglycerides. The use of carnitine remains controversial.

Ethics statements

Patient consent for publication

Parental/guardian consent obtained.