Pre-symptomatically detected novel variant of CPT1A deficiency: a case report

INTRODUCTION

Carnitine palmitoyltransferase 1A (CPT1A) is an enzyme which plays a role in hepatic mitochondrial fatty acid oxidation (FAO).¹ CPT1A deficiency (OMIM ID #255120) is a rare autosomal recessive disorder (as low as 1:750,000 to 1:2,000,000) with impaired mitochondrial long-chain fatty acid beta-oxidation, resulting in accumulation of high levels of long chain fatty acids in the liver. Impaired mitochondrial FAO leads to typical presentations of hypoketotic hypoglycemia and hepatic encephalopathy.^2,3 However, patients with CPT1A deficiency do not present with symptoms until there is increased energy demands as noted in fasting or febrile illness, thus increasing the chances of delayed diagnosis and management.⁴ However, it can be detected pre symptomatically by tandem mass spectrometry.^2,5 Hence, to detect pre-symptomatically and start early treatment, CPT1A is included in newborn screening programs in several countries. In this report, we present a case of pre-symptomatic CPT1A deficiency with a novel c.232G>A variant of uncertain significance, detected through newborn screening and later confirmed by exome sequencing. This emphasizes importance of pre-symptomatic detection of CPT enzyme deficiency through newborn screening. Evaluation, clinical assessment and management of disorder has been discussed.

CASE REPORT

Conceived naturally after 5 years of married life, by parents who had 3^rd degree consanguinity. Born to primi gravida with gestational diabetes mellitus and was delivered by emergency caesarean section (Indication: Meconium-stained liquor/fetal distress) at 36 weeks and 3 days (Late preterm) with a birth weight of 3.17 kilograms (Appropriate for gestational age). Baby cried immediately after birth. APGAR score was 7/10 and 8/10 at 1^st and 5^th minute respectively. She was treated in NICU for transient tachypnea of newborn. However, baby had lability in oxygen saturation and required prolonged oxygen support by nasal prongs with minimal FiO₂ requirement until 3 days of life. Echocardiogram ruled out persistent pulmonary hypertension. Baby received antibiotics empirically which were discontinued as septic work up including blood culture was negative. Baby received nutrition through IV fluids until distress subsided and later switched to exclusive breastfeeding. Sugar monitoring was done which was normal throughout the hospital stay. Baby required repeated double surface phototherapy for neonatal hyperbilirubinemia (O-A setting). Baby was discharged on day 7 of life. The diagnosis was arrived at through newborn screening done on day 4 of life, when the baby was stable and on direct breastfeeding, showed ‘slightly high’ concentration of free carnitine (C0) in the plasma sample (85.55 µmol/L; ref range 18.5-71.15 µmol/L) with normal C0/C16+C18 ratio. As per American college of Medical Genetics guidelines, clinical exome sequencing was done on DNA extracted from blood.⁶ Clinically relevant mutations were annotated using published variants in literature and a set of diseases databases-ClinVar, OMIM, GWAS, HGMD (v2020.2) and SwissVar. Common variants are filtered based on allele frequency in 1000genome phase 3, gnomAD (v2.1), EVS, dbSNP (v151), 1000 Japanese genome and internal Indian population database. Only non-synonymous and splice site variants consisting of 6120 genes were used for clinical interpretation. Silent variations that do not result in any change in amino acid in the coding region were excluded. In this case, A homozygous missense variation in exon 3 of the CPT1A gene (chr11:g.68812486C>T; depth: 152x) that results in the amino acid substitution of asparagine for aspartic acid at codon 78 (p.Asp78Asn; ENST00000265641.10) was detected, which is a novel c.232G>A variant of uncertain significance.

Further plan of action as per American college of medical genetics recommendation (ACT sheet) was initiated.⁶ Family was informed and counselled, baby’s clinical status ascertained, and a management strategy formulated. Enzyme activity assays, sequencing of variants in parents and periodic follow up of child to assess clinically for appearance of any related or new symptoms are recommended to ascertain clinical significance of this variant. Baby has been on periodical follow up and has remained asymptomatic on exclusive breastfeeds. Family screening of these mutations for patient’s parents was not performed yet, as the child has remained asymptomatic thus far. Genetic counseling through family screening of identified mutations is necessary if the parents want to have another baby.

DISCUSSION

The CPT1 enzyme has three tissue specific isoforms: the liver isoform (CPT1A), the muscle isoform (CPT1B), and the brain isoform (CPT1C) which are encoded by different genes. Mutations in the CPT1A gene (11q13.1-q13.2) results in deficiency of the liver isoform of carnitine palmitoyl transferase 1A. The CPT1A enzyme is located in the outer mitochondrial membrane where it converts the long chain fatty acyl-CoA molecules to their respective acyl-carnitine molecules. The carnitine acylcarnitine translocase system then transports these fatty acyl co-A molecules, across the inner mitochondrial membrane into the mitochondria to undergo beta-oxidation.⁷ In the absence of this system, long chain fatty acids cannot be transported into mitochondria. Hepatic mitochondrial fatty acid oxidation is an important alternative source of energy when glycogen reserves are used up during phases of increased energy demands like fasting or a febrile illness. The reduced ability to utilize long-chain fatty acids for ketogenesis leads to clinical presentation of hepatomegaly and hypoketotic hypoglycaemia, which can lead to seizures and coma.⁸

To reduce the risk of neurological damage and sequelae, one must prevent hypoglycemia. Hence, it is important to diagnose these patients early. CPT1A deficiency has been included in newborn screening programs using tandem mass spectrometry to detect an abnormal carnitine profile.^3,5,9 TMS usually uses a few indicators such as C0 level, C16 level, C18 level, or C0/ (C16+C18) ratio to diagnose CPT 1A deficiency. In a Japanese report of pre-symptomatic detection of CPT1A deficiency by TMS newborn screening, the patient did not show hypoglycemia or jaundice.¹⁰ However, elevation of free carnitine level (C0) was noticed by newborn screening. Fingerhut et al reported 2 false negative cases in the method using C16 level.⁵ Although, some consider C0/ (C16+C18) ratio to be a very reliable diagnostic marker for CPT1A deficiency, even when patient is stable, a vice versa of C0 / (C16+C18) being in normal range in patients has also been observed.^5,9 In fact, in our case the C0/C16 + C18 ratios were low as opposed to studies which reported 6-60 fold increase.⁵ The data is attributable to variant of unknown significance with possible lesser clinical severity as reflected in a case report by Borch et al.¹¹

The reliability of urine organic acids is also debatable. Studies have reported that C12 dicarboxylic acids and 3-OH glutaric acids are elevated in acute cases of CPT1A deficiency.^12-15 However, a few studies have also reported urine organic acids to be normal when the patient is stable.^8,10

American college of medical genetics recommends timely confirmatory testing. Molecular genetic analysis for the CPT1A gene is the confirmative test for diagnosis Targeted gene sequencing is a cost-effective approach to detect variants present in multiple/large genes in an individual, in which selective capture and sequencing of the protein coding regions of the genome/genes is performed. Approximately 30 kinds of mutations have been reported till date (the Human Gene Mutation Database, ttp://www.hgmd.cf.ac.uk). Some well-known founder mutations include c.1436C>T (p.Pro479Leu) in Inuit population and c.2129G>A (p.Gly710Glu) in Hutterite populations.^16-18 In our case, a homozygous missense variation in exon 3 of the CPT1A gene that results in the amino acid substitution of Asparagine for Aspartic Acid at codon 78 (p.Asp78Asn; ENST00000265641.10) was detected. The p.Asp78Asn variant has not been reported in the 1000 genomes database and has a minor allele frequency of 0.0006% and 0.002% in the gnomAD and Indian database respectively, making it a novel variant of uncertain significance (difficult to classify as either pathogenic (disease causing) or benign (non-disease causing) based on current available scientific evidence).^19-22

CPT1 deficiency can have a variable presentation. Newborns may appear asymptomatic but can progress to fasting hypoketotic hypoglycemia, lethargy, hepatomegaly, elevated transaminases, elevated creatine kinase and cardiac rhythm disturbances and seizures, usually precipitated by fasting or acute illness. Critical hypoketotic hypoglycemia is a common presentation with most patients having recurrent hypoglycemic events as the first manifestation especially in the event of prolonged fasting or infection where glycogen reserves are exhausted soon.²³ However, in the present case, the patient did not demonstrate any hypoglycemic events before diagnosis. After diagnosis, infant did not show symptoms such as hypoglycemia and encephalopathy and showed a normal weight gain pattern during follow up.

Atypical presentations in published literature include hepatosplenomegaly, nephromegaly and cholestatic jaundice as the first manifestation of CPT1A deficiency.^24,25

In the case report of a patient with cholestatic jaundice along with CPT1A deficiency, it is postulated that cholestatic jaundice is due to impaired adenosine triphosphate (ATP)-dependent bile acid secretion from lack of energy.²⁶ However milder forms of CPT1A deficiency might be able to supply glucose through mitochondrial FAO.²⁷ Early diagnosis is difficult in these patients before progression of hepatic dysfunction. Hence in any patient showing an unusual course of jaundice, screening for metabolic disorders including TMS analysis and carnitine assay should be considered. No features were suggestive of cholestasis jaundice in our case.

Renal tubular acidosis in CPT1A deficient patients has been reported.²⁸ The kidney is rich in mitochondria with fatty acids being an important source of energy. Therefore, RTA can be due to lack of energy for the kidney from impaired fatty acid oxidation, which can be treated with medium chain triglyceride supplement. A study also reported nephromegaly with significant proteinuria due to accumulation of long chain fatty acid and dyslipidemic changes in kidney.²⁴ No symptoms or clinical findings were suggestive if RTA or nephromegaly in our case.

Key to management is in prevention of hypoglycemia. A sufficient level of glucose should be constantly supplied to patients with CPT1A deficiency during long time of fasting. Higher glucose infusion rates are essential in CPT1A deficient patients along with mandatory blood glucose level monitoring during stressful conditions including major operation and severe infection.²³ The long-term treatment is to provide a steady diet which is low in fat, high in carbohydrate and a supply of medium-chain fatty acids. Fasting periods should be minimalized and avoided.

An essential component of neonatal screening for inherited diseases is to have a diagnostic algorithm for each disorder to be able to establish accuracy of diagnosis. In cases of variant of unknown significance, the diagnostic or management algorithm from American College of Medical genetics can be followed. Enzyme activity assays, sequencing of variants in parents and periodic follow up of child to assess clinically for appearance of any related or new symptoms are recommended to ascertain clinical significance of this variant and plan management accordingly. More such reports can be encouraging to detect the prevalence of such disorders in Indian population which remains largely undiscovered. Careful follow-up programs and evaluation of new genetic variants are required to strike a balance between the psychosocial risks and the clinical benefits of screening.

CONCLUSION

We conclude that Newborn screening is essential to detect CPT1A deficiency. Exome sequencing was helpful in clinching the diagnosis in this case pre-symptomatically and institute management for the same in a timely manner.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: Not required

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