Novel mutation in carnitine palmitoyltransferase 1A detected through newborn screening for a presymptomatic case in China：A case report and literature review


 Background：Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a rare mitochondrial fatty acid oxidation (FAO) disorder that results in hypoketotic hypoglycemia and hepatic encephalopathy. It is caused by mutation in CPT1A. To date, only two symptomatic cases of CPT1A deficiency have been reported in China.Case presentation: A newborn male, without any disease-related clinical manifestations, was diagnosed with CPT1A deficiency through newborn screening. Increased free carnitine levels and a significantly increased C0/(C16+C18) ratio were detected at 3 days of age, and subsequently, mutations in CPT1A were found by gene sequence analysis. The patient was advised a low-fat, high-protein diet and followed up regularly. During three-years of follow-up since, the patient showed normal growth velocity and developmental milestones. Whole-exome sequencing identified two mutations, c.2201T >C (p.F734S) and c.1318G>A (p.A440T), in the patient. The c.2201T >C mutation, which has been reported previously, was inherited from his father, while the c.1318G>A, a novel mutation, was inherited from his mother. The amino acid residues encoded by original sequences are highly conserved across different species. These mutations slightly altered the three-dimensional structure of the protein, as analyzed by molecular modeling, suggesting that they may be pathogenic.Conclusion: This is the first case of CPT1A deficiency detected through newborn screening based on diagnostic levels of free carnitine, in China. We identified two missense mutations, c.2201T >C and novel c.1318G>A, in the patient. Our findings have expanded the gene spectrum of this rare condition and provided a basis for family genetic counseling and prenatal diagnosis.

A male child was born of a normal pregnancy and natural delivery in our hospital and is the only child of his parents. His gestational age was 39 weeks, Apgar score 10/1, 10/5, 10/10, birth weight 3,500 g. When the boy was 44 days old, a newborn screening sample obtained at 3 days of age showed increased blood free carnitine(C0) level of 128.1 mmol/L (ref < 50 mmol/L) and increased C0/(C16 + C18) ratio of 512.4 (ref < 42). These abnormal results were con rmed by testing again on day 51 after birth, the results showed 65.86 mmol/L free carnitine (ref < 100 mmol/L) and a signi cantly increased C0/(C16 + C18) ratio of 1423.97 (ref < 100), which were consistent with CPT1A de ciency. [3] Based on these results, the boy was thoroughly examined. Cranial MRI showed no signi cant abnormalities echocardiogram was normal. Laboratory ndings for blood sample were: pH ,7.4; base excess, 4. normalized after treatment. The patient was discharged when the urine level of amino acids and organic acids became normal. The diagnosis of CPT1A de ciency was considered and con rmed by gene sequencing.
The patient was advised a low-fat, high-protein diet and followed-up regularly. On earlier occasions when he fell sick, hypoglycemia was prevented by early intervention with glucose infusion.
Every three months, the patient was examined by a specialist to evaluate if he suffered any neurologic damage due to possible episodes of hypoketotic hypoglycemia that are associated with CPT1A de ciency. There were no motor retardation and hypotonia. At 12 months of age, the boy could walk and talk. During the 3 years of follow-up since, his psychomotor development has been appropriate for his age.

Molecular Genetic Findings
CPT1A (Ensemble gene: ENST00000110090) was sequenced for the patient after obtaining written informed consent of his parents. The results showed two mutations: c.2201T > C (p.F734S) and c.1318G > A (p.A440T) in exons 18 and 11, respectively. Then family screening of these mutations for patient's parents was performed. Results showed that the c.2201T > C mutation was transmitted from his father, while c.1318G > A mutation was transmitted from his mother (Fig. 1B, C-H). These variations were not listed in the SNP database(http://www.ncbi.nlm.nih.gov/projects/SNP/) Human Gene Mutation Database Professional(http://www.hgmd.cf.ac.uk/ac/index.php). However, c.2201T > C has been reported once in one case of CPT1A de ciency in China, [4] while c.1318G > A has not been previously reported. According to PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/), the c.2201T > C mutation was predicted to be "probably damaging" (score, 0.995) and c.1318G > A was predicted to be "possibly damaging" (score, 0.875), suggesting that both mutations may cause disease. The sequencing data revealed that both the mutations detected in our patient were missense mutations causing p.F734S and p.A440T replacement. Further, we conducted molecular modeling to predict the effect of these mutations on the protein structure of CPT1A (Fig. 2). We found that replacement of Thr440 with Ala440 results in an additional hydrogen bond between Thr440-MET436, and replacement of Ser734 with Phe734 results in the loss of hydrophobic bond force between Ser734-Phe549. As hydrogen and hydrophobic bond play an important role in maintaining protein spatial conformation and stability, these subtle changes in spatial structure may affect protein function. These data suggest that the mutation causing these substitutions may not be polymorphisms, but disease-causing mutations.

Discussion And Conclusion
CPT1A de ciency is a rare metabolic disease that affects fatty acid oxidation (FAO), and in the majority of cases, patients are diagnosed only after the appearance of clinic symptoms. Most patients present these symptoms by the age of 2 years with hypoketotic hypoglycemia induced by fasting or illness. [5] This is usually accompanied by liver dysfunction; transient lipemia and renal tubular acidosis may also be present. [1] As our patient underwent the newborn screening at the age of 3 days, indicators of CPT1A de ciency, namely, increased free carnitine and a signi cantly increased C0/(C16 + C18) ratio, were detected early. The diagnosis of CPT1A de ciency was con rmed by gene sequence analysis. Dietetic management and avoidance of prolonged fasting were recommended to improve the patient's clinical outcome. [5] Therefore, our patient developed normally, without severe metabolic crisis, till date.
Analysis of CPT1A is necessary for accurate diagnosis. So far, more than 30 mutations in CPT1A, responsible for the CPT1A de ciency, have been identi ed. [6] Our patient carried two missense mutations c.2201T > C (p.F734S), previously reported in one Chinese patient, [4] and c.1318G > A (p.A440T), a novel mutation. The encoded amino are highly conserved across species. The glycine residue at 734 and 440 in CPT1A is highly conserved in bovine, chicken, chimpanzee, goat, horse, pig, rat, and macaque (Fig. 1I), suggesting that these loci play key roles in CPT1A normal function. According to prediction software analysis these mutations do not appear to be polymorphisms,but are more likely to be disease-causing mutations. As p.F734S mutation was only reported in a Chinese patient with heterozygous gene mutation, it may be a unique to Chinese lineage.

*Inuit mutation #Hutterite mutation
Newborn screening programs, which allow early detection of metabolic markers in dried whole blood spots when the newborn is catabolic, are therefore very important. [3] However, screening for CPT1A de ciency is not included in the newborn screening program in every province in China. Since 2016, Central China (Hubei Province) newborn screening program has included screening for disorders of fatty acid oxidation using tandem mass spectrometry (MS/MS), and more than 120,000 newborn children have been tested so far. The MS/MS is used to detect elevated free carnitine to C16 + C18 ratio, which is characteristic of CPT1A patient [3] . According to the data from newborn screening programs in Australia, Germany, and the USA the incidence of CPT1A de ciency may be as low as 1:750,000 to 1:2,000,000. [12] The present case is the rst presymptomatic CPT1A de ciency case detected through newborn screening in China. A previous reports has indicated that following a strict dietary regimen allows the CPT1A-de cient infant to lead a healthy life with normal growth and development. [3] Consistent with this reports, our patient was diagnosed at an early age, received timely intervention, and showed a normal growth trend. Therefore, the newborn metabolic screen is important for early diagnosis and treatment.
Considering the simplicity of this method, it can be implemented across the country. Moreover, in the present cases, genetic counseling was recommended for the parents, should they wish to have another baby.
In conclusion, we used neonatal screening using MS/MS to diagnose CPT1A de ciency in presymptomatic newborn. The early diagnosis and diet management improved the prognosis in our patient. Further, we identi ed a novel mutation c.1318G > A in CPT1A, which is probably disease-causing. Thus, our nding has expanded the gene spectrum of this rare condition and provided a basis for genetic counseling of the family and prenatal diagnosis. This study was approved by the Ethics Committee of Maternal and Child Health Hospital of Hubei Province. All participants gave their written informed consent to take part in the present study.

Consent for publication
This family have given their written consents for the case report to be published.

Availability of data materials
The datasets used and analyzed during the current study are available from the corresponding author on the reasonable request.
YG wrote the manuscript and researched data. HF researched data and contributed to the manuscript. FY instructed and supervised this study. All authors read and approved the nal manuscript.