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The effect of homozygous deletion of the BBOX1 and Fibin genes on carnitine level and acyl carnitine profile
© Rashidi-Nezhad et al.; licensee BioMed Central Ltd. 2014
Received: 12 October 2013
Accepted: 26 June 2014
Published: 1 July 2014
Carnitine is a key molecule in energy metabolism that helps transport activated fatty acids into the mitochondria. Its homeostasis is achieved through oral intake, renal reabsorption and de novo biosynthesis. Unlike dietary intake and renal reabsorption, the importance of de novo biosynthesis pathway in carnitine homeostasis remains unclear, due to lack of animal models and description of a single patient defective in this pathway.
We identified by array comparative genomic hybridization a 42 months-old girl homozygote for a 221 Kb interstitial deletions at 11p14.2, that overlaps the genes encoding Fibin and butyrobetaine-gamma 2-oxoglutarate dioxygenase 1 (BBOX1), an enzyme essential for the biosynthesis of carnitine de novo. She presented microcephaly, speech delay, growth retardation and minor facial anomalies. The levels of almost all evaluated metabolites were normal. Her serum level of free carnitine was at the lower limit of the reference range, while her acylcarnitine to free carnitine ratio was normal.
We present an individual with a completely defective carnitine de novo biosynthesis. This condition results in mildly decreased free carnitine level, but not in clinical manifestations characteristic of carnitine deficiency disorders, suggesting that dietary carnitine intake and renal reabsorption are sufficient to carnitine homeostasis. Our results also demonstrate that haploinsufficiency of BBOX1 and/or Fibin is not associated with Primrose syndrome as previously suggested.
Carnitine (L-3-hydroxy-4-N,N,N-trimethylaminobutyrate) is crucial for energy metabolism. It is a conditional essential nutrient found in animals, numerous microorganisms and plants [1–3]. It allows the transport of activated fatty acids from the cytosol to the mitochondria, where they are beta-oxidized. Other functions of carnitine include peroxisome fatty acid oxidation, modulating intracellular coenzyme A homeostasis and removal of excess acyl groups from the body via the preferential renal excretion of acylcarnitines [4–7].
In mammals, carnitine homeostasis is achieved and maintained by a combination of absorption from dietary sources, de novo biosynthesis and efficient, renal tubular reabsorption [4, 8]. Diet is the primary source of carnitine and dietary bioavailability of L-carnitine can vary considerably because of the broad range of nutritional choice. Meat, fish and dairy products are main sources in human, so vegetarians obtain very low amount of carnitine from their diet. However, compensatory mechanisms, including renal reabsorption in conjugation with de novo biosynthesis, are proficient in conserving carnitine homeostasis when dietary L-carnitine consumption is low [4, 8, 9].
L-carnitine is synthesized from the amino acid precursors lysine and methionine via four enzymatic reactions. These enzymes are found in all the human cells, with the exception of butyrobetaine-gamma 2-oxoglutarate dioxygenase 1(BBOX1) that is only expressed in the liver, kidneys and brain [5, 10]. In contrast to the diet and renal reabsorption, the significance of carnitine de novo biosynthesis for energy homeostasis remains unclear , as no animal model and a single patient were described. While this patient was deficient for trimethyllysine hydroxylase epsilon (TMLHE), the first enzyme of the carnitine biosynthesis pathway [11, 12], we report on the first instance of homozygous deletion of BBOX1, the last enzyme of that pathway.
Proband’s anthropometric measurements
Body Mass Index (BMI)
Proband’s metabolic investigations
< 5 nmol/L
< 4 mU/L
Amino-acid TLC (U)
Ferric Chloride Test (U)
DNPH Test (U)
Na-Nitropruside Test (U)
Amino-acids (DBS) (TMS)
Acylcarnitines (DBS) (TMS)*
< 18 mg/dL
> 20% act
Glutaric acid (DBS)
Proband’s carnitine and acylcarnitine profile
Carnitine is a critical molecule in the transport of fatty acids to the mitochondria and their subsequent beta-oxidation. Its homeostasis is maintained through oral intake, renal reabsorption and de novo biosynthesis. Although, the importance of carnitine dietary intake and renal reabsorption were thoroughly studied [9, 20–23], the impact of de novo biosynthesis remains unclear as we lack animal models and a single patient deficient in enzymes involved in the carnitine biosynthesis pathway was described [8, 11, 12].
The female patient described here was referred to our genetic counseling and cytogenetic service because of microcephaly, dysmorphic features and IUGR. Array CGH revealed a homozygous deletion within chromosomal band 11p14.2 that deletes both the Fibin and the BBOX1 genes, probably resulting in absence of de novo carnitine biosynthesis. The evaluation of metabolites involved directly or indirectly in the fatty acid b-oxidation pathway showed an increased ratio of AC/FC in the proband at 42 but not at 60 months of age (Table 3). Previous studies showed that such an increased ratio is an indicator of carnitine insufficiency; a situation in which there is inadequate free carnitine in response to an increase in metabolic needs [24–26]. It was further postulated that the AC/FC ratio reflects the intramitochondrial acyl-CoA/CoA ratio and thus that its alteration could be indicative of mitochondrial dysfunction . The clinical and metabolic findings in the proband are not compatible with the described carnitine deficiency disorders [27–32] even if her levels of free carnitine were close to the lower limit of normal range demonstrating that the hypothesis that a defect in carnitine biosynthesis will not manifest itself as a systemic carnitine deficiency in omnivorous humans . Corroboratingly, the TMLHE-deficient patients showed normal plasma carnitine level [11, 12].
It is unclear if the clinical features shown by the proband, such as microcephaly for example, are associated with her minor carnitine insufficiency. The etiology of the different observed phenotypes could be associated to the absence of the Fibin gene, the second gene encompassed within the 11p14.2 deletion. Fibin is a secreted protein identified in zebrafish, mice and humans potentially acting downstream of retinoic acid and wnt signaling. It is essential for pectoral fin bud initiation and tbx5 expression in zebrafish . Alternatively they could be triggered by the reported perturbation of expression of copy-normal genes that neighbor structural rearrangement [33–37] or an unidentified recessive mutation inherited from both of her parents. Further studies are required to discriminate these different possibilities. Of note, consanguineous marriage is common practice and increasing in frequency in Iran [38, 39]. It was suggested, however, that carnitine biosynthesis may be a risk factor for nondysmorphic autism as the TMLHE-deficient patient was identified in an autism spectrum disorder (ASD) cohort [11, 12]. While our patient showed some dysmorphisms and global developmental delay she did not present autistic traits. Correspondingly she was microcephalic, a feature generally associated with schizophrenia rather than ASD that is generally associated with the mirroring macrocephaly [40–42].
In conclusion, we present to our knowledge, the first patient with homozygous deletion of BBOX1, the second individual with a complete defect in carnitine de novo biosynthesis. She presents a mild decrease in free carnitine level but no clinical manifestations of carnitine deficiency disorders, suggesting that dietary carnitine intake and renal reabsorption are sufficient for carnitine homeostasis in omnivorous individuals.
All samples used in this study were collected with the approval of the local ethics committee (“Commission cantonale vaudoise d'éthique de la recherché sur l'être humain”). Written informed consent was obtained from the patient’s parents for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal and appropriate informed consent. The latter signed by the parents includes the permission to publish pictures of the proband.
We thank the patient and her parents for their participation and members of the Lausanne Genomic Technologies Facility for technical help. ARN is recipient of a scholarship from the Iran Ministry of Health. This work was supported by Iran National Science Foundation (INSF) (grant 87042100), Tehran University of Medical Sciences (TUMS) (grant 8999), Genetic Office of Iran Ministry of Health, and grants from the European Commission anEUploidy Integrated Project (037627), the Swiss National Science Foundation and a Swiss National Science Foundation Sinergia grant to AR. This study makes use of data generated by the DECIPHER Consortium. A full list of centres that contributed to the generation of the data is available from http://decipher.sanger.ac.uk and via email from firstname.lastname@example.org. Funding for the latter project was provided by the Wellcome Trust.
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