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One potential hotspot ACADVL mutation in Chinese patients with very-long-chain acyl-coenzyme A dehydrogenase deficiency
Journal Pre-proofs One Potential Hotspot ACADVL Mutation in Chinese Patients with Verylong-chain Acyl-coenzyme A Dehydrogenase Deficiency Xiyuan Li, Rui Ma, Yi Liu, Lulu Kang, Ruxuan He, Jinqing Song, Jing Ren, Yang Li, Min Huang, Jianlong Men, Yanling Yang PII: DOI: Reference:
S0009-8981(19)32156-4 https://doi.org/10.1016/j.cca.2019.11.034 CCA 15944
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Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
26 September 2019 18 November 2019 26 November 2019
Please cite this article as: X. Li, R. Ma, Y. Liu, L. Kang, R. He, J. Song, J. Ren, Y. Li, M. Huang, J. Men, Y. Yang, One Potential Hotspot ACADVL Mutation in Chinese Patients with Very-long-chain Acyl-coenzyme A Dehydrogenase Deficiency, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.11.034
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One Potential Hotspot ACADVL Mutation in Chinese Patients with Very-long-chain Acyl-coenzyme A Dehydrogenase Deficiency Running title: Three novel and one common mutations of VLCAD deficiency Xiyuan Lia,b1, Rui Maa1, Yi Liub, Lulu Kangb, Ruxuan Heb, Jinqing Songb, Jing Rena, Yang Lia, Min Huangc, Jianlong Mena*, and Yanling Yangb* a
Precision Medicine Center, General Hospital of Tianjin Medical University, Tianjin
300020, China b
c
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
Department of Pediatrics, Similan Clinic, Beijing 100070, China
1
The authors contributed equally to this work.
*Corresponding author: Jianlong Men, Ph.D. No.154 Anshan Road, Heping District, Precision Medicine Center, General Hospital of Tianjin Medical University, Tianjin 300020, China Email: [email protected] Yanling Yang, M.D., Ph.D. No.1 Xianmen Street, Xicheng District, Department of Pediatrics, Peking University First Hospital, Beijing 100034, China Email: [email protected]
1
Abstract Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD deficiency), a rare autosomal recessive disorder, is characterized by hypoketotic hypoglycemia, cardiomyopathy, liver damage, and myopathy. VLCAD deficiency is caused by defects of ACADVL gene, which encodes VLCAD protein. The aim of this study was to determine the clinical, biochemical, prognosis and mutation spectrum of patients with VLCAD deficiency in mainland China. A total of Six families visited us, four patients (2 boys and 2 girls) were admitted in hospital due to liver dysfunction, hypoglycemia, and positive newborn screen result. The parents of the other two patients (2 girls) visited us for genetic consultation after their children's death. All the six patients had elevated level of serum tetradecenoylcarnitine (C14:1-carnitine), four of them showed decreased free carnitine (C0) level, and three had dicarboxylic aciduria. Eight types of mutations of the ACADVL gene were detected, three of them are
novel,
including
c.563G>A
(p.G188D)
c.1387G>A
(p.G463R)
and
c.1582_1586del (p.L529Sfs*31). The p.R450H mutation accounts for 9/52 alleles (5/40 in previous study of 20 unrelated patients, and 4/12 in this study) of genetically diagnosed Chinese VLCAD deficiency cases. The four alive patients (Patient 1-4) responded well to diet prevention and drug therapy with stable hepatic dysfunction condition. In conclusion, we describe three novel mutations of the ACADVL gene among six unrelated families with VLCAD deficiency. Moreover, we suggest that the p.R450H may be a potential hotspot mutation in the Chinese population. Keywords: ACADVL; Fatty acid oxidation dysfunction; gene; Very-long-chain acyl-coenzyme A dehydrogenase deficiency; VLCAD deficiency
1. Introduction Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD deficiency; MIM# 201475), a rare autosomal recessive disorder that affects mitochondrial fatty acid β-oxidation, is caused by defects in the acyl-CoA dehydrogenase very long chain (ACADVL) gene (MIM# 609575). VLCAD deficiency is a severe form of fatty acid oxidation defects (FAODs) that are responsible for 5% of all sudden and unexpected deaths in infants [1; 2]. The ACADVL gene encodes very long-chain acyl-coenzyme A dehydrogenase (VLCAD, 140 kDa, EC 1.3.99.13), the first enzyme involved in fatty acid β oxidation. This enzyme is located on the inner mitochondrial membrane and is responsible for β oxidation of 14–20 carbon fatty acids [3]. Accumulation of toxic long chain acylcarnitine causes organ dysfunction of the heart, skeletal muscle and liver due to fatty acid degeneration and energy production deficit. Further, since long-chain fatty acid serves as the main source of energy in human body, the blockage of VLCAD activity results in energy depletion, specifically hurting organs which have high energy consumption. According to the clinical phenotype, VLCAD deficiency is divided into three subtypes, (1) the severe neonatal-onset myocardial form which is responsible for cardiomyopathy and high mortality rate; (2) the infantile-onset hepatic form manifested with non-ketotic hypoglycemia and liver damage; and (3) the adolescent or adult-onset myopathy form typically characterized by fasting or exercise-induced muscle weakness, pain, spasms and rhabdomyolysis [4]. To the best of our knowledge, 22 genetically diagnosed patients with VLCAD deficiency have been reported in the Chinese population [5; 6; 7; 8; 9]. Datasets on VLCAD
deficient
patients
(https://www.ncbi.nlm.nih.gov/pubmed/)
were by
retrieved searching
from the
PubMed
keywords:
Very
long-chain acyl-coenzyme A dehydrogenase deficiency, or VLCAD deficiency; China, or Chinese; and ACADVL. The VLCAD deficiency pathogenic mutations 2
spectrum in Chinese population has not been established. Here, we report the clinical, biochemical, and potentially causative mutations in six Chinese VLCAD deficiency patients, including three novel mutations and a mutation at a putative hotspot of the ACADVL gene, thus expanding the variant spectrum of the disease. 2. Materials and Methods 2.1 Patients Six patients (two boys and four girls) from six unrelated Chinese families were diagnosed between March 2015 and November 2018 at the Department of Pediatrics, Peking University First Hospital, China. The clinical onset of the symptoms was between the age of 2 days and 1 year. The patients were admitted with indications of liver dysfunction and hypoglycemia, and the parents of the other two patients (Patient 5 and 6) visited us for genetic consultation (Table 1). The parents of all patients were healthy and non-consanguineous. This study was approved by the institutional review board of the Peking University First Hospital, and it was conducted as per the Declaration of Helsinki. 2.2 Metabolic studies The blood amino acids and acylcarnitines were analyzed using tandem mass spectrometry (MS/MS). The tests were measured in dried blood spots by API 3200 MS/MS analyzer (SHIMADZU Asia Pacific Pte. Ltd, Japan) [10]. 2.3 ACADVL analysis An informed consent was obtained from the parents of the patients for genetic analysis. Peripheral blood samples were collected from all the parents and Patient 1-4, blood and urine samples had been collected before Patient 5 and 6’s death. Lymphocytes genomic DNA was extracted using a TIANamp blood DNA kit (Tiangen Biotech, China). ACADVL gene was amplified by polymerase chain 3
reaction (PCR) and sequenced. The sequence data were compared with the reference sequence of ACADVL (NM_000018) to detect the variants. Afterwards, variants were
compared
to
http://www.1000genomes.org),
1000
Genomes and
Project
ExAC
data browser
(1000G, data
(http://exac.broadinstitute.org). PolyPhen-2 (http://genetics.bwh.harvard.edu/pph) and MutationTaster (http://www.mutationtaster.org) software were used to predict the impact of variations alterations on the protein function. 3. Results The clinical data and results of laboratory examination are presented in Table 1. All patients were born at term. Four patients (Patient 2, 3, 5 and 6) had acute metabolic crisis attack, three patients (Patient 2, 5, and 6) presented with acute metabolic crisis triggered by infection, vomiting and fasting but one patient (Patient 5) did not have obvious trigger events. Elevated plasma tetradecenoylcarnitine (C14:1-carnitine) level, as well as transaminase were detected in all six patients. Decreased free carnitine (C0-carnitine) found in four patients (Patient 2, 3, 5 and 6) and dicarboxylic aciduria was detected in three patients (Patient 2, 5 and 6). Two patient died before diagnosis (Patient 5 and 6), four patients (Patient 1-4) responded well to diet prevention and drug therapy with stable hepatic dysfunction condition. 3.1 Patients Patient 1, 2, 3 and 4 Patient 1, a 3 years and 9 months old girl was diagnosed during newborn screen; she was advised at 3 days to avoid fasting, and take low-fat and high-carbohydrate meals. Elevation of plasma C14:1 and transaminases was observed at 25 days after birth. Transaminase level was about 15-35 U/L above the normal limits and she had no other obvious symptoms of VLCAD deficiency. Patient 2, a 2 years and 9 months old boy presented with drowsiness and weakness during pneumonia attack at 2 months of age; he soon went into coma after vomiting two times. Laboratory tests 4
showed very low blood glucose levels, elevated serum transaminase levels, an increased C14:1-carnitine and decreased C0-carnitine levels. This indicated impaired liver function, VLCAD deficiency, and secondary carnitine deficiency as well as hepatomegaly were later detected. Patient 3, a 8 years and 9 months old girl, first showed VLCAD deficiency symptoms (liver dysfunction, hepatomegaly, hypoglycemia) during fasting at the age of 1 year. She had high serum transaminase, low C0-carnitine and high C14:1-carnitine levels. Patient 4 who was a 10 months old boy, presented with drowsiness, weakness and feeding difficulty at 4 months old. He was found to have liver dysfunction, hepatomegaly, and hypoglycemia. Normal C0-carnitine with elevated C14:1-carnitine were also detected. All the four patients were recommended to avoid fasting, and observe low-fat intake and medium-chain triglycerides (MCTs) formula or MCTs oil depending on age. The ones who had very low levels of C0-carnitine (Patient 2 and 3) were treated with L-carnitine, and those who had relatively severe phenotype like severe liver dysfunction (Patient 3 and 4) received bezafibrate treatment. Closed biochemical and imaging examination was performed to all the four patients to control disease development and monitor the side effect of L-carnitine. After treatment, three patients (Patient 2-4) showed a mildly elevated serum transaminase levels (about 10-35 U/L above the normal limits). Patient 1’s condition was better, the only symptom was occasionally liver dysfunction with a mildly elevated serum transaminase level (about 0-10 U/L above the normal limits). All the four patients had no additional metabolic attacks up to this point. Patient 5 and 6 Patients 5 and 6, both girls died at the age of 40 and 2 days, respectively. They had similar medical histories and their families had a positive history. In fact the previous child (a boy) in Patient 5's family showed cyanosis and died 4 days after birth from heart failure. The previous child (a boy) of Patient 6's family presented with hypotonia and hypoglycemia (1.9mmol/L) in 2 days post birth. Later, the boy 5
had cough and a fever when he was 2 months old, hypoglycemia, hepatomegaly, and was later diagnosed with liver dysfunction. Eventually, the boy died from multiple organ failure four days after rescue. Patient 5 firstly presented with feeding difficulty in 40 days, and then she developed breathing difficulties, seizures and later went into a coma. She would later be found to have hypoglycemia, hepatomegaly and liver dysfunction, and she soon died due to respiratory and heart failure. Similarly, Patient 6 showed feeding difficulties in 2 days, and was detected with cyanosis after two times of vomiting. Hypoglycemia, hyperlipidemia, hepatomegaly and liver dysfunction were detected. She eventually died due to respiratory failure. The autopsy showed hepatic congestion with adipohepatic changes, focal myocardial hemorrhage and pneumorrhagia. Their parents came to our hospital with the patients’ dried blood spots for biochemical and genetic diagnosis. 3.2 Features of the identified mutations Eight mutations were identified from all the six patients (Table 2). Three of them
[c.563G>A (p.G188D),
c.1387G>A (p.G463R)
and
c.1582_1586del
(p.L529Sfs*31)] are novel (Figure 1), these variation were neither found in 1000 Genomes Project nor the ExAC program. All the three novel mutations are conserved among species (Table 3). The two missense mutations (p.G188D and p.G463R) were predicted to be "probably damaging" and "disease-causing” by PolyPhen-2.0 and MutationTaster software, respectively. The frameshift mutation, c.1582_1586del, was predicted to be "disease-causing" by MutationTaster software. The potential hotspot mutation, c.1349G>A (p.R450H, Figure 1), was identified three times from infantile-onset hepatic form patients (Patient 2-4) and was reported in this population before [8]. 4. Discussion VLCAD deficiency was first identified in 1993 by Aoyama et al., and was considered to cause the most severe type of FAODs [3]. In this study, we present six 6
Chinese patients with VLCAD deficiency and describe their symptoms, clinical findings, treatment, and response to the treatment. We also describe the mutations in the ACADVL gene, including three novel mutations and one potential population-specific hotspot mutation. VLCAD deficiency is initially diagnosed based on clinical features, biomarkers, liver enzyme analysis and genetic analysis. Metabolic crisis is the major reason for consultation which can be triggered by periods of fasting, illness and exercise [4]. Likewise, vomiting and diarrhea, which lead to fasting, are common trigger events of the metabolic crisis in patients with VLCAD deficiency [11]. In our patients, two neonatal-onset myocardial type patients (Patient 5 and 6) and four infantile-onset hepatic type patients (Patient 1-4) were found. The triggers of acute metabolic crisis included pneumonia, fasting and vomiting. Once patients are hospitalized, elevated C14:1-carnitine level by MS/MS serves as the biochemical diagnosis marker [4]. However, the positive predictive value of this test for VLCAD deficiency is about 3.1% [12], false positive and false negative results occasionally occur. Schymik et al. reported a VLCAD deficiency case with normal blood C14:1-carnitine levels which were elevated at one time, meanwhile some healthy infants and fasting babies show positive MS/MS test results probably due to active fatty acid β-oxidation, increased lipolysis and enhanced β-oxidation [13, 14]. In this study, we repeated the blood C14:1-carnitine level test to avoid false results. Therefore, further diagnostic methods are involved for VLCAD deficiency such as fibroblast fatty acid β-oxidation, VLCAD enzyme activity, immunoreactive VLCAD protein antigen expression and the ACADVL gene analysis [4]. Dicarboxylic aciduria helps the diagnosis, especially in acute episodes, reflects fatty acids metabolic dysfunction. In this study, all the six patients had significantly elevated blood C14:1-carnitine, four patients showed decreased blood C0-carnitine, and dicarboxylic aciduria was found in three patients (Patient 2, 5 and 6). The 7
enzyme activity test was not performed because of the typical clinical phenotypes and pathogenic ACADVL gene mutations. To
date,
about
283
variants
have
been
reported
in
ACADVL
(http://www.hgmd.cf.ac.uk/ac/index.php). According to previous reports, c.848T>C (p.V283A), was found in ~10% of all individuals with a positive NBS in the United States [15]. None of the six patients examined here carried this variant. As we mentioned before, a total 22 genetically diagnosed patients with VLCAD deficiency were reported in Chinese population, among them, two pairs of siblings (4 patients) are included [8]. Thus, there are 20 unrelated patients reported in the previous studies and 6 patients in the present study. Consequently, there are 52 mutant alleles of the ACADVL gene in Chinese population. Incidentally, the mutation, c.1349G>A (p.R450H), was found in the three patients in the present study (4/12 alleles, see Table 2). This mutation was also found in five patients reported in previous studies of Chinese population. Each of them is a compound heterozygote of c.1349G>A, either c.1795G>A (p.E599K) [8], c.1273 G>A [6], c.65C>A (p.S22X), c.1396G>T, or c.215C>T [7; 9]. Thus, among the 52 mutant alleles, p.R450H accounts for 8 alleles (9/52 alleles, 5/40 in previous reports and 4/12 in this study), suggesting that the p.R450H mutation could be a specific common variant Chinese population. Although this mutation was found in other East Asia patients, it is not recognized as a potential hotspot one in any of the East Asia countries. A low residual VLCAD activity of 5% normal was detected at 30°C although significant activity was absent at 37°C of p.R450H mutation, indicating that homozygous p.R450H mutation is a temperature-sensitive mild mutation in vitro, which may related to the mildest phenotype of VLCAD deficiency [16]. However, patients harboring heterozygous p.R450H have all the three sub-types of VLCAD deficiency, though the individuals in Chinese population only had the infantile-onset subtype [7; 9]. The clinical symptoms of heterozygous p.R450H affected ones including hypoglycemia, hepatomegaly, developmental delay, cardiomyopathy [7; 9], rhabdomyolysis, myalgia [16], and asymptomatic individuals [17]. As the patient 8
with homozygous p.R450H mutation was not reported, and there is a contradiction of the phenotype in vitro and in clinical, we suspect that a heterozygous p.R450H mutation cannot determine the VLCAD deficiency phenotype on its own. On the other hand, two mutations, c.664G>A and c.664G>C lead to a same amino acid change (p.G222R), and have been proposed to be relatively common mutations in the Chinese population [8]. There were two Chinese sibling patients reported to have carried a heterozygote mutation of c.664G>C (p.G222R) (the other heterozygote mutation was not detected); a boy carrying a heterozygote c.664G>A (p.G222R) (the other heterozygote mutation was not detected); a boy who carried a compound heterozygote for c.664G>C (p.G222R) and c.896A>T (p.K299M) [8]; and a boy who was heterozygotic for c.664G>C (p.G222R) and c.1605+1 G>T [7; 9]. However, the p.G222R mutation was not detected in the later reports or in our six patients. Thus, this mutation accounts for 4/52 alleles in the Chinese population, which may be special but not as common as c.1349G>A (p.R450H) in this population. Andresen et al. reported a clear phenotype–genotype relationship in VLCAD deficiency patients: nonsense or truncating mutations that resulted in no residual enzyme activities always lead to neonatal phenotypes, while missense mutations with residual enzyme activities cause milder childhood or adult phenotypes [18]. However, the phenotype–genotype relationship among missense mutations is still not clear [19]. In our study, admittedly, truncating mutations were detected in neonatal-onset patients (p.Q100Vfs*3 of Patient 5 and p.Q562Vfs*29 of Patient 6), whereas this type of mutations was found in infant-onset patients (p.W427X of Patient 1 and p.L529Sfs*31 of Patient 2), whose symptoms are relatively mild. Thus, the type of mutation cannot be the only criterion of VLCAD deficiency disease severity. Treatments of VLCAD deficiency generally include: 1) Avoid long fasting, 2) drug therapy, high carbohydrate/low fat diet which is often not so strict, and early 9
infusion of glucose in sick times such as pyrexia, infection, or diarrhea. L-carnitine therapy for FAOD patients is still controversial [4], as a matter of fact, two siblings with VLCAD deficiency experienced more frequent episodes of rhabdomyolysis after L-carnitine supplementation [20]. However, many VLCAD deficiency patients present with very low C0-carnitine levels which is also fatal, because of the consequent oxidation defects of the majority of fatty acids [8]. In our study, all the four alive patients were advised to take high carbohydrate diet instead of high-fatty foods, as well as to avoid long fasting and excessive exercise. Further, all of the individuals with low free carnitine levels (Patient 2-4) were given oral L-carnitine supplement with close biochemical and iconography monitoring. According to the patient's condition and an experimental protocol [21], MCTs oil was also given to all patients. As the condition of Patient 3 and 4 is was more severe than the first two patients, they were given bezafibrate. Our patients responded well to the treatment, as evidenced by stable liver function and reduced metabolic crisis attack frequency. Considering that the follow-up time is limited in our study, the treatment responses requires long-time observation. In conclusion, we report one common mutation, p.R450H, in the Chinese population and three novel mutations, p.G188D, p.G463R, and p.L529Sfs*31. These results provide a framework for future genetic diagnosis of VLCAD deficiency. Declaration of interest statement None. Acknowledgments This work was supported by the grants from National Key Research and Development Program of China (Grant No. 2017YFC1001700), National Natural Science Foundation of China (Grant No. 81801638), and the National Key Research and Development Program of China-Precision Medicine Research Project (Grant No.
10
2016YFC0905600-2016YFC0905601). We would like to thank all patients, and the contributions from Similan Clinic. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the insti-tutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. References: [1] T. Yamamoto, H. Tanaka, H. Kobayashi, K. Okamura, T. Tanaka, Y. Emoto, K. Sugimoto, M. Nakatome, N. Sakai, H. Kuroki, S. Yamaguchi, and R. Matoba, Retrospective review of Japanese sudden unexpected death in infancy: the importance of metabolic autopsy and expanded newborn screening, Mol. Genet. Metab.102 (2011) 399-406. https://doi.org/10.1016/j.ymgme.2010.12.004. [2] M.J. Bennett, and S. Powell, Metabolic disease and sudden, unexpected death in infancy, Hum. Pathol. 25 (1994) 742-6.10. https://doi.org/1016/0046-8177(94)90241-0. [3] T. Aoyama, M. Souri, S. Ushikubo, T. Kamijo, S. Yamaguchi, R.I. Kelley, W.J. Rhead, K. Uetake, K. Tanaka, and T. Hashimoto, Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients, J. Clin. Invest. 95 (1995) 2465-73. https://doi.org/10.1172/JCI117947. [4] N.D. Leslie, C.A. Valencia, A.W. Strauss, J. Connor, and K. Zhang, Very Long-Chain Acyl-Coenzyme
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https://doi.org/10.1016/j.ymgme.2016.05.012.
13
Figure 1. Novel and hotspot ACADVL mutations in this study. (a) the heterozygous c.563G>A (p.G188D) mutation; (b) the heterozygous c.1349G>A (p.R450H) (hotspot); (c) the heterozygous c.1387G>A (p.G463R) mutation; (d) the heterozygous c.1582_1586del (p.L529Sfs*31) mutation. Red square frame: missence mutations changes; red line: bases deletion.
14
Table 1. Clinical and laboratory data of six Chinese patients with VLCAD deficiency Patients
1
2
3
4
5
6
Gender
F
M
F
M
F
F
Age of onset
25d
2m
1y
4m
40d
2d
Age of diagnosis
3d
2m
5y
7m
after death
after death
Present Age
3y9m
2y9m
8y9m
10m
died at 42d
died at 3d
drowsiness
-
+
+
+
+
+
seizures
-
-
-
-
+
-
cyanosis
-
-
-
-
+
-
coma
-
+
-
-
+
+
pneumonia, vomiting
fasting
N/A
N/A
vomiting
Normal range
Units
Symptoms and signs
Trigger
events (acute N/A
crisis) Positive family history
-
-
-
-
+
-
Liver dysfunction
+
+
+
+
+
+
Hepatomegaly
-
+
+
+
+
+
Hypoglycemia
-
+
+
+
+
+
Cardiomyopathy
-
-
-
-
+
+
Myopathy
-
-
-
-
+
+
Serum CK
18
45
102
63
8954 ↑
9907 ↑
26 - 164
U/L
Serum CK-MB
4.5
3.1
12.5
7.3
455.3 ↑
573.9
0 - 30
U/L
Blood C14:1-carnitine
2.8 ↑
2.6 ↑
3.1 ↑
2.2 ↑
4.3 ↑
6.3 ↑
0.01 - 0.5
μmol/L
Blood C0-carnitine
21.5
14.0 ↓
9.7 ↓
22.9
3.6 ↓
4.1 ↓
20 - 60
μmol/L
Dicarboxylic aciduria
-
+
-
-
+
+
15
Outcome
mild liver dysfunction mild liver dysfunction
mild liver dysfunction
mild liver dysfunction
dead
dead
M, male; F, female; y, years; m, months; d, days; CK, creatine kinase; CK-MB, creatine kinase MB.
16
Table 2. Mutations detected in ACADVL gene among six Chinese patients with VLCAD deficiency Patients Mutation at the
Mutation type Mutation at the PolyPhen-2.0 prediction
nucleotide level 1.
Mutationtaster prediction Conservation
protein level
and score
and score
Frequency
c.563G>A
heterozygous
p.G188D
Probably damaging (1)
Disease-Causing (1)
Conserved
2/12
c.1280G>A
heterozygous
p.W427X
N/A
Disease-Causing (1)
Conserved
1/12
c.1349G>A
heterozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
c.1582_1586del
heterozygous
p.L529Sfs*31
N/A
Disease-Causing (1)
Conserved
1/12
c.1349G>A
heterozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
c.563G>A
heterozygous
p.G188D
Probably damaging (1)
Disease-Causing (1)
Conserved
2/12
4
c.1349G>A
homozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
5
c.295_296del
heterozygous
p.Q100Vfs*3
N/A
Disease-Causing (1)
Conserved
1/12
c.896A>T
heterozygous
p.K299M
Probably damaging (0.99)
Disease-Causing (0.99)
Conserved
1/12
c.1387G>A
heterozygous
p.G463R
Probably damaging (1)
Disease-Causing (0.99)
Conserved
1/12
c.1683_1684del
heterozygous
p.Q562Vfs*29 N/A
Disease-Causing (1)
Conserved
1/12
2
3
6
N/A: Not Applicable.
17
Table 3. Amino acid alignment in VLCAD residues (highlighted in bold). Species
p.G188
Humans
KELGAFGLQVPSEL RIFRIFEGTNDILRL
Pan troglodytes
KELGAFGLQVPSEL RVFRIFEGTNDILRL SGLV - PELSRSGELA
M. mulatta
KELGAFGLQVPSEL - - - RIFEGTNDILRL
SGIVHPELSRSGELA
Mus musculus
KELGAFGLQVPSEL RIFRIFEGANDILRL
SGIVHPELSRSGELA
Trubripes
- - - - A F GLQVPSEL RIFRIFEGTNDILRL
QGSVHPELAQSGDLT
Drosophila melanogaster WELGAFG IQVPSEF RIFRIFEGTNDILRL
SGHVVGELLPYAKKT
C. elegans
AELGTFGVLVPPEL
p.G463
p.L529 SGLVHPELSRSGELA
RIFRIFEGANDVLRL GQVVDASLQDSAKVL
-: the amino acid is not existed in the certain species.
18
Highlights: Eight mutations of the ACADVL gene were identified from six Chinese VLCAD deficiency patients. Three novel mutations [c.563G>A (p.G188D), c.1387G>A (p.G463R) and c.1582_1586del (p.L529Sfs*31)] were found. The mutation p.R450H is firstly regarded as the hotspot mutation in the Chinese population.
Author Contributions Yanling Yang and Jianlong Men designed and directed the study; Xiyuan Li and Rui Ma performed genetic testing and wrote the manuscript; The other authors collected clinical data and assisted genetic testing. All authors critically reviewed and approved the final manuscript.
S0009-8981(19)32156-4 https://doi.org/10.1016/j.cca.2019.11.034 CCA 15944
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
26 September 2019 18 November 2019 26 November 2019
Please cite this article as: X. Li, R. Ma, Y. Liu, L. Kang, R. He, J. Song, J. Ren, Y. Li, M. Huang, J. Men, Y. Yang, One Potential Hotspot ACADVL Mutation in Chinese Patients with Very-long-chain Acyl-coenzyme A Dehydrogenase Deficiency, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.11.034
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One Potential Hotspot ACADVL Mutation in Chinese Patients with Very-long-chain Acyl-coenzyme A Dehydrogenase Deficiency Running title: Three novel and one common mutations of VLCAD deficiency Xiyuan Lia,b1, Rui Maa1, Yi Liub, Lulu Kangb, Ruxuan Heb, Jinqing Songb, Jing Rena, Yang Lia, Min Huangc, Jianlong Mena*, and Yanling Yangb* a
Precision Medicine Center, General Hospital of Tianjin Medical University, Tianjin
300020, China b
c
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
Department of Pediatrics, Similan Clinic, Beijing 100070, China
1
The authors contributed equally to this work.
*Corresponding author: Jianlong Men, Ph.D. No.154 Anshan Road, Heping District, Precision Medicine Center, General Hospital of Tianjin Medical University, Tianjin 300020, China Email: [email protected] Yanling Yang, M.D., Ph.D. No.1 Xianmen Street, Xicheng District, Department of Pediatrics, Peking University First Hospital, Beijing 100034, China Email: [email protected]
1
Abstract Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD deficiency), a rare autosomal recessive disorder, is characterized by hypoketotic hypoglycemia, cardiomyopathy, liver damage, and myopathy. VLCAD deficiency is caused by defects of ACADVL gene, which encodes VLCAD protein. The aim of this study was to determine the clinical, biochemical, prognosis and mutation spectrum of patients with VLCAD deficiency in mainland China. A total of Six families visited us, four patients (2 boys and 2 girls) were admitted in hospital due to liver dysfunction, hypoglycemia, and positive newborn screen result. The parents of the other two patients (2 girls) visited us for genetic consultation after their children's death. All the six patients had elevated level of serum tetradecenoylcarnitine (C14:1-carnitine), four of them showed decreased free carnitine (C0) level, and three had dicarboxylic aciduria. Eight types of mutations of the ACADVL gene were detected, three of them are
novel,
including
c.563G>A
(p.G188D)
c.1387G>A
(p.G463R)
and
c.1582_1586del (p.L529Sfs*31). The p.R450H mutation accounts for 9/52 alleles (5/40 in previous study of 20 unrelated patients, and 4/12 in this study) of genetically diagnosed Chinese VLCAD deficiency cases. The four alive patients (Patient 1-4) responded well to diet prevention and drug therapy with stable hepatic dysfunction condition. In conclusion, we describe three novel mutations of the ACADVL gene among six unrelated families with VLCAD deficiency. Moreover, we suggest that the p.R450H may be a potential hotspot mutation in the Chinese population. Keywords: ACADVL; Fatty acid oxidation dysfunction; gene; Very-long-chain acyl-coenzyme A dehydrogenase deficiency; VLCAD deficiency
1. Introduction Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD deficiency; MIM# 201475), a rare autosomal recessive disorder that affects mitochondrial fatty acid β-oxidation, is caused by defects in the acyl-CoA dehydrogenase very long chain (ACADVL) gene (MIM# 609575). VLCAD deficiency is a severe form of fatty acid oxidation defects (FAODs) that are responsible for 5% of all sudden and unexpected deaths in infants [1; 2]. The ACADVL gene encodes very long-chain acyl-coenzyme A dehydrogenase (VLCAD, 140 kDa, EC 1.3.99.13), the first enzyme involved in fatty acid β oxidation. This enzyme is located on the inner mitochondrial membrane and is responsible for β oxidation of 14–20 carbon fatty acids [3]. Accumulation of toxic long chain acylcarnitine causes organ dysfunction of the heart, skeletal muscle and liver due to fatty acid degeneration and energy production deficit. Further, since long-chain fatty acid serves as the main source of energy in human body, the blockage of VLCAD activity results in energy depletion, specifically hurting organs which have high energy consumption. According to the clinical phenotype, VLCAD deficiency is divided into three subtypes, (1) the severe neonatal-onset myocardial form which is responsible for cardiomyopathy and high mortality rate; (2) the infantile-onset hepatic form manifested with non-ketotic hypoglycemia and liver damage; and (3) the adolescent or adult-onset myopathy form typically characterized by fasting or exercise-induced muscle weakness, pain, spasms and rhabdomyolysis [4]. To the best of our knowledge, 22 genetically diagnosed patients with VLCAD deficiency have been reported in the Chinese population [5; 6; 7; 8; 9]. Datasets on VLCAD
deficient
patients
(https://www.ncbi.nlm.nih.gov/pubmed/)
were by
retrieved searching
from the
PubMed
keywords:
Very
long-chain acyl-coenzyme A dehydrogenase deficiency, or VLCAD deficiency; China, or Chinese; and ACADVL. The VLCAD deficiency pathogenic mutations 2
spectrum in Chinese population has not been established. Here, we report the clinical, biochemical, and potentially causative mutations in six Chinese VLCAD deficiency patients, including three novel mutations and a mutation at a putative hotspot of the ACADVL gene, thus expanding the variant spectrum of the disease. 2. Materials and Methods 2.1 Patients Six patients (two boys and four girls) from six unrelated Chinese families were diagnosed between March 2015 and November 2018 at the Department of Pediatrics, Peking University First Hospital, China. The clinical onset of the symptoms was between the age of 2 days and 1 year. The patients were admitted with indications of liver dysfunction and hypoglycemia, and the parents of the other two patients (Patient 5 and 6) visited us for genetic consultation (Table 1). The parents of all patients were healthy and non-consanguineous. This study was approved by the institutional review board of the Peking University First Hospital, and it was conducted as per the Declaration of Helsinki. 2.2 Metabolic studies The blood amino acids and acylcarnitines were analyzed using tandem mass spectrometry (MS/MS). The tests were measured in dried blood spots by API 3200 MS/MS analyzer (SHIMADZU Asia Pacific Pte. Ltd, Japan) [10]. 2.3 ACADVL analysis An informed consent was obtained from the parents of the patients for genetic analysis. Peripheral blood samples were collected from all the parents and Patient 1-4, blood and urine samples had been collected before Patient 5 and 6’s death. Lymphocytes genomic DNA was extracted using a TIANamp blood DNA kit (Tiangen Biotech, China). ACADVL gene was amplified by polymerase chain 3
reaction (PCR) and sequenced. The sequence data were compared with the reference sequence of ACADVL (NM_000018) to detect the variants. Afterwards, variants were
compared
to
http://www.1000genomes.org),
1000
Genomes and
Project
ExAC
data browser
(1000G, data
(http://exac.broadinstitute.org). PolyPhen-2 (http://genetics.bwh.harvard.edu/pph) and MutationTaster (http://www.mutationtaster.org) software were used to predict the impact of variations alterations on the protein function. 3. Results The clinical data and results of laboratory examination are presented in Table 1. All patients were born at term. Four patients (Patient 2, 3, 5 and 6) had acute metabolic crisis attack, three patients (Patient 2, 5, and 6) presented with acute metabolic crisis triggered by infection, vomiting and fasting but one patient (Patient 5) did not have obvious trigger events. Elevated plasma tetradecenoylcarnitine (C14:1-carnitine) level, as well as transaminase were detected in all six patients. Decreased free carnitine (C0-carnitine) found in four patients (Patient 2, 3, 5 and 6) and dicarboxylic aciduria was detected in three patients (Patient 2, 5 and 6). Two patient died before diagnosis (Patient 5 and 6), four patients (Patient 1-4) responded well to diet prevention and drug therapy with stable hepatic dysfunction condition. 3.1 Patients Patient 1, 2, 3 and 4 Patient 1, a 3 years and 9 months old girl was diagnosed during newborn screen; she was advised at 3 days to avoid fasting, and take low-fat and high-carbohydrate meals. Elevation of plasma C14:1 and transaminases was observed at 25 days after birth. Transaminase level was about 15-35 U/L above the normal limits and she had no other obvious symptoms of VLCAD deficiency. Patient 2, a 2 years and 9 months old boy presented with drowsiness and weakness during pneumonia attack at 2 months of age; he soon went into coma after vomiting two times. Laboratory tests 4
showed very low blood glucose levels, elevated serum transaminase levels, an increased C14:1-carnitine and decreased C0-carnitine levels. This indicated impaired liver function, VLCAD deficiency, and secondary carnitine deficiency as well as hepatomegaly were later detected. Patient 3, a 8 years and 9 months old girl, first showed VLCAD deficiency symptoms (liver dysfunction, hepatomegaly, hypoglycemia) during fasting at the age of 1 year. She had high serum transaminase, low C0-carnitine and high C14:1-carnitine levels. Patient 4 who was a 10 months old boy, presented with drowsiness, weakness and feeding difficulty at 4 months old. He was found to have liver dysfunction, hepatomegaly, and hypoglycemia. Normal C0-carnitine with elevated C14:1-carnitine were also detected. All the four patients were recommended to avoid fasting, and observe low-fat intake and medium-chain triglycerides (MCTs) formula or MCTs oil depending on age. The ones who had very low levels of C0-carnitine (Patient 2 and 3) were treated with L-carnitine, and those who had relatively severe phenotype like severe liver dysfunction (Patient 3 and 4) received bezafibrate treatment. Closed biochemical and imaging examination was performed to all the four patients to control disease development and monitor the side effect of L-carnitine. After treatment, three patients (Patient 2-4) showed a mildly elevated serum transaminase levels (about 10-35 U/L above the normal limits). Patient 1’s condition was better, the only symptom was occasionally liver dysfunction with a mildly elevated serum transaminase level (about 0-10 U/L above the normal limits). All the four patients had no additional metabolic attacks up to this point. Patient 5 and 6 Patients 5 and 6, both girls died at the age of 40 and 2 days, respectively. They had similar medical histories and their families had a positive history. In fact the previous child (a boy) in Patient 5's family showed cyanosis and died 4 days after birth from heart failure. The previous child (a boy) of Patient 6's family presented with hypotonia and hypoglycemia (1.9mmol/L) in 2 days post birth. Later, the boy 5
had cough and a fever when he was 2 months old, hypoglycemia, hepatomegaly, and was later diagnosed with liver dysfunction. Eventually, the boy died from multiple organ failure four days after rescue. Patient 5 firstly presented with feeding difficulty in 40 days, and then she developed breathing difficulties, seizures and later went into a coma. She would later be found to have hypoglycemia, hepatomegaly and liver dysfunction, and she soon died due to respiratory and heart failure. Similarly, Patient 6 showed feeding difficulties in 2 days, and was detected with cyanosis after two times of vomiting. Hypoglycemia, hyperlipidemia, hepatomegaly and liver dysfunction were detected. She eventually died due to respiratory failure. The autopsy showed hepatic congestion with adipohepatic changes, focal myocardial hemorrhage and pneumorrhagia. Their parents came to our hospital with the patients’ dried blood spots for biochemical and genetic diagnosis. 3.2 Features of the identified mutations Eight mutations were identified from all the six patients (Table 2). Three of them
[c.563G>A (p.G188D),
c.1387G>A (p.G463R)
and
c.1582_1586del
(p.L529Sfs*31)] are novel (Figure 1), these variation were neither found in 1000 Genomes Project nor the ExAC program. All the three novel mutations are conserved among species (Table 3). The two missense mutations (p.G188D and p.G463R) were predicted to be "probably damaging" and "disease-causing” by PolyPhen-2.0 and MutationTaster software, respectively. The frameshift mutation, c.1582_1586del, was predicted to be "disease-causing" by MutationTaster software. The potential hotspot mutation, c.1349G>A (p.R450H, Figure 1), was identified three times from infantile-onset hepatic form patients (Patient 2-4) and was reported in this population before [8]. 4. Discussion VLCAD deficiency was first identified in 1993 by Aoyama et al., and was considered to cause the most severe type of FAODs [3]. In this study, we present six 6
Chinese patients with VLCAD deficiency and describe their symptoms, clinical findings, treatment, and response to the treatment. We also describe the mutations in the ACADVL gene, including three novel mutations and one potential population-specific hotspot mutation. VLCAD deficiency is initially diagnosed based on clinical features, biomarkers, liver enzyme analysis and genetic analysis. Metabolic crisis is the major reason for consultation which can be triggered by periods of fasting, illness and exercise [4]. Likewise, vomiting and diarrhea, which lead to fasting, are common trigger events of the metabolic crisis in patients with VLCAD deficiency [11]. In our patients, two neonatal-onset myocardial type patients (Patient 5 and 6) and four infantile-onset hepatic type patients (Patient 1-4) were found. The triggers of acute metabolic crisis included pneumonia, fasting and vomiting. Once patients are hospitalized, elevated C14:1-carnitine level by MS/MS serves as the biochemical diagnosis marker [4]. However, the positive predictive value of this test for VLCAD deficiency is about 3.1% [12], false positive and false negative results occasionally occur. Schymik et al. reported a VLCAD deficiency case with normal blood C14:1-carnitine levels which were elevated at one time, meanwhile some healthy infants and fasting babies show positive MS/MS test results probably due to active fatty acid β-oxidation, increased lipolysis and enhanced β-oxidation [13, 14]. In this study, we repeated the blood C14:1-carnitine level test to avoid false results. Therefore, further diagnostic methods are involved for VLCAD deficiency such as fibroblast fatty acid β-oxidation, VLCAD enzyme activity, immunoreactive VLCAD protein antigen expression and the ACADVL gene analysis [4]. Dicarboxylic aciduria helps the diagnosis, especially in acute episodes, reflects fatty acids metabolic dysfunction. In this study, all the six patients had significantly elevated blood C14:1-carnitine, four patients showed decreased blood C0-carnitine, and dicarboxylic aciduria was found in three patients (Patient 2, 5 and 6). The 7
enzyme activity test was not performed because of the typical clinical phenotypes and pathogenic ACADVL gene mutations. To
date,
about
283
variants
have
been
reported
in
ACADVL
(http://www.hgmd.cf.ac.uk/ac/index.php). According to previous reports, c.848T>C (p.V283A), was found in ~10% of all individuals with a positive NBS in the United States [15]. None of the six patients examined here carried this variant. As we mentioned before, a total 22 genetically diagnosed patients with VLCAD deficiency were reported in Chinese population, among them, two pairs of siblings (4 patients) are included [8]. Thus, there are 20 unrelated patients reported in the previous studies and 6 patients in the present study. Consequently, there are 52 mutant alleles of the ACADVL gene in Chinese population. Incidentally, the mutation, c.1349G>A (p.R450H), was found in the three patients in the present study (4/12 alleles, see Table 2). This mutation was also found in five patients reported in previous studies of Chinese population. Each of them is a compound heterozygote of c.1349G>A, either c.1795G>A (p.E599K) [8], c.1273 G>A [6], c.65C>A (p.S22X), c.1396G>T, or c.215C>T [7; 9]. Thus, among the 52 mutant alleles, p.R450H accounts for 8 alleles (9/52 alleles, 5/40 in previous reports and 4/12 in this study), suggesting that the p.R450H mutation could be a specific common variant Chinese population. Although this mutation was found in other East Asia patients, it is not recognized as a potential hotspot one in any of the East Asia countries. A low residual VLCAD activity of 5% normal was detected at 30°C although significant activity was absent at 37°C of p.R450H mutation, indicating that homozygous p.R450H mutation is a temperature-sensitive mild mutation in vitro, which may related to the mildest phenotype of VLCAD deficiency [16]. However, patients harboring heterozygous p.R450H have all the three sub-types of VLCAD deficiency, though the individuals in Chinese population only had the infantile-onset subtype [7; 9]. The clinical symptoms of heterozygous p.R450H affected ones including hypoglycemia, hepatomegaly, developmental delay, cardiomyopathy [7; 9], rhabdomyolysis, myalgia [16], and asymptomatic individuals [17]. As the patient 8
with homozygous p.R450H mutation was not reported, and there is a contradiction of the phenotype in vitro and in clinical, we suspect that a heterozygous p.R450H mutation cannot determine the VLCAD deficiency phenotype on its own. On the other hand, two mutations, c.664G>A and c.664G>C lead to a same amino acid change (p.G222R), and have been proposed to be relatively common mutations in the Chinese population [8]. There were two Chinese sibling patients reported to have carried a heterozygote mutation of c.664G>C (p.G222R) (the other heterozygote mutation was not detected); a boy carrying a heterozygote c.664G>A (p.G222R) (the other heterozygote mutation was not detected); a boy who carried a compound heterozygote for c.664G>C (p.G222R) and c.896A>T (p.K299M) [8]; and a boy who was heterozygotic for c.664G>C (p.G222R) and c.1605+1 G>T [7; 9]. However, the p.G222R mutation was not detected in the later reports or in our six patients. Thus, this mutation accounts for 4/52 alleles in the Chinese population, which may be special but not as common as c.1349G>A (p.R450H) in this population. Andresen et al. reported a clear phenotype–genotype relationship in VLCAD deficiency patients: nonsense or truncating mutations that resulted in no residual enzyme activities always lead to neonatal phenotypes, while missense mutations with residual enzyme activities cause milder childhood or adult phenotypes [18]. However, the phenotype–genotype relationship among missense mutations is still not clear [19]. In our study, admittedly, truncating mutations were detected in neonatal-onset patients (p.Q100Vfs*3 of Patient 5 and p.Q562Vfs*29 of Patient 6), whereas this type of mutations was found in infant-onset patients (p.W427X of Patient 1 and p.L529Sfs*31 of Patient 2), whose symptoms are relatively mild. Thus, the type of mutation cannot be the only criterion of VLCAD deficiency disease severity. Treatments of VLCAD deficiency generally include: 1) Avoid long fasting, 2) drug therapy, high carbohydrate/low fat diet which is often not so strict, and early 9
infusion of glucose in sick times such as pyrexia, infection, or diarrhea. L-carnitine therapy for FAOD patients is still controversial [4], as a matter of fact, two siblings with VLCAD deficiency experienced more frequent episodes of rhabdomyolysis after L-carnitine supplementation [20]. However, many VLCAD deficiency patients present with very low C0-carnitine levels which is also fatal, because of the consequent oxidation defects of the majority of fatty acids [8]. In our study, all the four alive patients were advised to take high carbohydrate diet instead of high-fatty foods, as well as to avoid long fasting and excessive exercise. Further, all of the individuals with low free carnitine levels (Patient 2-4) were given oral L-carnitine supplement with close biochemical and iconography monitoring. According to the patient's condition and an experimental protocol [21], MCTs oil was also given to all patients. As the condition of Patient 3 and 4 is was more severe than the first two patients, they were given bezafibrate. Our patients responded well to the treatment, as evidenced by stable liver function and reduced metabolic crisis attack frequency. Considering that the follow-up time is limited in our study, the treatment responses requires long-time observation. In conclusion, we report one common mutation, p.R450H, in the Chinese population and three novel mutations, p.G188D, p.G463R, and p.L529Sfs*31. These results provide a framework for future genetic diagnosis of VLCAD deficiency. Declaration of interest statement None. Acknowledgments This work was supported by the grants from National Key Research and Development Program of China (Grant No. 2017YFC1001700), National Natural Science Foundation of China (Grant No. 81801638), and the National Key Research and Development Program of China-Precision Medicine Research Project (Grant No.
10
2016YFC0905600-2016YFC0905601). We would like to thank all patients, and the contributions from Similan Clinic. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the insti-tutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. References: [1] T. Yamamoto, H. Tanaka, H. Kobayashi, K. Okamura, T. Tanaka, Y. Emoto, K. Sugimoto, M. Nakatome, N. Sakai, H. Kuroki, S. Yamaguchi, and R. Matoba, Retrospective review of Japanese sudden unexpected death in infancy: the importance of metabolic autopsy and expanded newborn screening, Mol. Genet. Metab.102 (2011) 399-406. https://doi.org/10.1016/j.ymgme.2010.12.004. [2] M.J. Bennett, and S. Powell, Metabolic disease and sudden, unexpected death in infancy, Hum. Pathol. 25 (1994) 742-6.10. https://doi.org/1016/0046-8177(94)90241-0. [3] T. Aoyama, M. Souri, S. Ushikubo, T. Kamijo, S. Yamaguchi, R.I. Kelley, W.J. Rhead, K. Uetake, K. Tanaka, and T. Hashimoto, Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients, J. Clin. Invest. 95 (1995) 2465-73. https://doi.org/10.1172/JCI117947. [4] N.D. Leslie, C.A. Valencia, A.W. Strauss, J. Connor, and K. Zhang, Very Long-Chain Acyl-Coenzyme
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13
Figure 1. Novel and hotspot ACADVL mutations in this study. (a) the heterozygous c.563G>A (p.G188D) mutation; (b) the heterozygous c.1349G>A (p.R450H) (hotspot); (c) the heterozygous c.1387G>A (p.G463R) mutation; (d) the heterozygous c.1582_1586del (p.L529Sfs*31) mutation. Red square frame: missence mutations changes; red line: bases deletion.
14
Table 1. Clinical and laboratory data of six Chinese patients with VLCAD deficiency Patients
1
2
3
4
5
6
Gender
F
M
F
M
F
F
Age of onset
25d
2m
1y
4m
40d
2d
Age of diagnosis
3d
2m
5y
7m
after death
after death
Present Age
3y9m
2y9m
8y9m
10m
died at 42d
died at 3d
drowsiness
-
+
+
+
+
+
seizures
-
-
-
-
+
-
cyanosis
-
-
-
-
+
-
coma
-
+
-
-
+
+
pneumonia, vomiting
fasting
N/A
N/A
vomiting
Normal range
Units
Symptoms and signs
Trigger
events (acute N/A
crisis) Positive family history
-
-
-
-
+
-
Liver dysfunction
+
+
+
+
+
+
Hepatomegaly
-
+
+
+
+
+
Hypoglycemia
-
+
+
+
+
+
Cardiomyopathy
-
-
-
-
+
+
Myopathy
-
-
-
-
+
+
Serum CK
18
45
102
63
8954 ↑
9907 ↑
26 - 164
U/L
Serum CK-MB
4.5
3.1
12.5
7.3
455.3 ↑
573.9
0 - 30
U/L
Blood C14:1-carnitine
2.8 ↑
2.6 ↑
3.1 ↑
2.2 ↑
4.3 ↑
6.3 ↑
0.01 - 0.5
μmol/L
Blood C0-carnitine
21.5
14.0 ↓
9.7 ↓
22.9
3.6 ↓
4.1 ↓
20 - 60
μmol/L
Dicarboxylic aciduria
-
+
-
-
+
+
15
Outcome
mild liver dysfunction mild liver dysfunction
mild liver dysfunction
mild liver dysfunction
dead
dead
M, male; F, female; y, years; m, months; d, days; CK, creatine kinase; CK-MB, creatine kinase MB.
16
Table 2. Mutations detected in ACADVL gene among six Chinese patients with VLCAD deficiency Patients Mutation at the
Mutation type Mutation at the PolyPhen-2.0 prediction
nucleotide level 1.
Mutationtaster prediction Conservation
protein level
and score
and score
Frequency
c.563G>A
heterozygous
p.G188D
Probably damaging (1)
Disease-Causing (1)
Conserved
2/12
c.1280G>A
heterozygous
p.W427X
N/A
Disease-Causing (1)
Conserved
1/12
c.1349G>A
heterozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
c.1582_1586del
heterozygous
p.L529Sfs*31
N/A
Disease-Causing (1)
Conserved
1/12
c.1349G>A
heterozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
c.563G>A
heterozygous
p.G188D
Probably damaging (1)
Disease-Causing (1)
Conserved
2/12
4
c.1349G>A
homozygous
p.R450H
Probably damaging (1)
Disease-Causing (1)
Conserved
4/12
5
c.295_296del
heterozygous
p.Q100Vfs*3
N/A
Disease-Causing (1)
Conserved
1/12
c.896A>T
heterozygous
p.K299M
Probably damaging (0.99)
Disease-Causing (0.99)
Conserved
1/12
c.1387G>A
heterozygous
p.G463R
Probably damaging (1)
Disease-Causing (0.99)
Conserved
1/12
c.1683_1684del
heterozygous
p.Q562Vfs*29 N/A
Disease-Causing (1)
Conserved
1/12
2
3
6
N/A: Not Applicable.
17
Table 3. Amino acid alignment in VLCAD residues (highlighted in bold). Species
p.G188
Humans
KELGAFGLQVPSEL RIFRIFEGTNDILRL
Pan troglodytes
KELGAFGLQVPSEL RVFRIFEGTNDILRL SGLV - PELSRSGELA
M. mulatta
KELGAFGLQVPSEL - - - RIFEGTNDILRL
SGIVHPELSRSGELA
Mus musculus
KELGAFGLQVPSEL RIFRIFEGANDILRL
SGIVHPELSRSGELA
Trubripes
- - - - A F GLQVPSEL RIFRIFEGTNDILRL
QGSVHPELAQSGDLT
Drosophila melanogaster WELGAFG IQVPSEF RIFRIFEGTNDILRL
SGHVVGELLPYAKKT
C. elegans
AELGTFGVLVPPEL
p.G463
p.L529 SGLVHPELSRSGELA
RIFRIFEGANDVLRL GQVVDASLQDSAKVL
-: the amino acid is not existed in the certain species.
18
Highlights: Eight mutations of the ACADVL gene were identified from six Chinese VLCAD deficiency patients. Three novel mutations [c.563G>A (p.G188D), c.1387G>A (p.G463R) and c.1582_1586del (p.L529Sfs*31)] were found. The mutation p.R450H is firstly regarded as the hotspot mutation in the Chinese population.
Author Contributions Yanling Yang and Jianlong Men designed and directed the study; Xiyuan Li and Rui Ma performed genetic testing and wrote the manuscript; The other authors collected clinical data and assisted genetic testing. All authors critically reviewed and approved the final manuscript.