Case reports: three novel variants in PCCA and PCCB genes in Chinese patients with propionic acidemia

Background Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by the deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC) and is associated with pathogenic variants in either of the two genes PCCA or PCCB. The present study aimed to identify the genetic cause of three Chinese patients with PA. Case presentation Three Chinese PA patients were diagnosed by using gas chromatography-mass spectrometry(GC-MS), tandem mass spectrometry (MS/MS) and molecular diagnostic methods. All patients had onset in the neonatal period. One patient died of infection and metabolic decompensation, and the other two had mild to moderate developmental delay/mental retardation. Mutation analysis of the PCCA gene identified that patient 1 carried the compound heterozygous c.1288C > T(p.R430X) and c.2002G > A(p.G668R), and patient 2 was homozygous for the c.1426C > T(p.R476X) mutation. Mutation analysis of the PCCB gene identified that patient 3 harbored the compound heterozygous mutations c.359_360del AT(p.Y120Cfs*40) and c.1398 + 1G > A. Among these mutations, three (c.1288C > T, c.359_360del AT and c.1398 + 1G > A) are novel. Conclusions We reported three Chinese PA patients who had PCCA or PCCB mutants. Among them, in the PCCA gene, c.1288C > T(p.R430X) was a nonsense mutation, resulting in a truncated protein. c.359_360del AT was a frameshift mutation, leading to a p.Y120Cfs*40 change in the amino acid sequence in the PCCB protein. c.1398 + 1G > A was a splicing mutation, causing skipping of the exons 13–14. In conclusion, the novel mutations uncovered in this study will expands the mutation spectrum of PA.

localized on chromosomes 13q32 and 3q21-q22, respectively [5,6]. To date, more than 200 mutations in both the PCCA and PCCB genes have been reported (www. hgmd.cf.ac.uk/ac/gene.). There is a large degree of genetic heterogeneity in the PCCA gene, and no prevalent mutations have been observed in any population studied. On the contrary, in the PCCB gene, there was a limited number of mutations accounting for most of the patients in different ethnic groups. Among the Japanese, c.923-924insT, c.1644-6C > G and R399Q are the most common mutations [7]. There are two main types of PCCB gene mutations in patients with PA in Latin America, c.1218-1231dell4insl2 and E168K, accounting for more than 60% of cases [8]. Here we reported three patients with PA who were diagnosed using gas chromatographymass spectrometry (GC-MS) and tandem mass spectrometry (MS/MS) [9]. We then identified three novel mutations in the PCCA and PCCB genes by Sanger sequencing.

Affected individuals
Three unrelated patients are from Guangxi China, and were clinically diagnosed with PA using via urine organic acid analysis by gas chromatography-mass spectrometry (GC-MS) and carnitine analysis by tandem mass spectrometry (MS/MS) ( Table 1).

Clinical phenotype
All patients were born full term with normalbirth weight. Patient 1 was a boy and Patient 3 was a girl both of non-consanguineous parents. Patient 2 was a boy of first-degree-cousin parents. All patients were screened in the Genetic and Metabolic Central Laboratory affiliated to Guangxi Maternal and Child Health Hospital. The results are shown in Table 1. Propionyl glycine, 3hydroxypropionate and methyl citrate were increased in urine excretion. MS/MS showed increased of acylcarnitine profile revealed and increased C3-acylcarnitin, C3/ C2 and C3/C0 acylcarnitine ratio. Patient 1 was 3 weeks old when he presented with tachypnea and malignant hyperthermia, and was diagnosed with presumed sepsis. He was admitted to the hospital three times due to recurrent andrespiratory tract infections. At 6 months of age, he developed generalized tonic clonic seizures, metabolic acidosis (bicarbonate, 15.4 mmol/L) and hyperammonemia (ammonia, 516 μmol/L). Only then did he undergo the screening by MS/MS and GC-MS, and diagnosed with PA. Unfortunately, he died at six and a half months of age from sudden cardiac arrest. Patient 2 had suffered from recurrent vomiting, lethargy and hyperventilation at the age of 1 month. Fortunately, the patient was diagnosed with PA and treated with low-isoleucine, −methionine, −threonine, and-valine diet, L-carnitine, and biotin at the first time. Thereafter, he experienced a few metabolic crises; and the result of MS/MS and GC/MS detection showed an improvement in metabolic level (Table 1). He underwent fundoplication and gastrostomy tube placement at age of 1.5 years for caloric intake. He is now 3 years old and has shown mild mental retardation, and his weight has increased from the 5th percentile to the 20th percentile.
Patient 3 was screened on the third day after birth. She initially received an intravenous infusion, low isoleucine, methionine, threonine and proline special formulations, as well as oral carnitine and biotin treatment. Thereafter, she had several episodes of infections, diarrhea, metabolic acidosi; and the result of MS/MS and GC/MS detection are shown in Table 1. She is now 5 years old with moderate mental retardation. In the past few years, she has been admitted to the hospital several times due to episodes of infections,diarrhea, metabolic acidosis and generalized tonic-clonic seizures. She also had poor weight gain (6th percentile). Her recent heart assessment is normal.

Mutation analysis
Peripheral blood was obtained from the patients and their parents. DNA was isolated from peripheral blood using the Lab-Aid DNA kit ( To evaluate whether novel variants were diseasecausing mutations or polymorphisms, PolyPhen 2.0 and Mutation Taster tools were performed to analyze the functional effects of novel variants. Variants were further evaluated according to the ACMG and AMP standards and guidelines [10]. Three PCCA mutations and two PCCB mutations were identified. Patient 1 was compound heterozygous for c.1288C > T and c.2002G > A changes in the PCCA gene. Heterozygous c.1288C > T and c.2002G > A mutations were identified in the father and mother, respectively. Patient 2 was homozygous for c.1426C > T(p.R476X) in the PCCA gene, and his parents were heterozygous. Patient 3 was compound heterozygous for c.359_360del AT and c.1398 + 1G > A changes in the PCCB gene. The heterozygous c.359_360del AT and c.1398 + 1G > A mutations were identified in the father and mother, respectively. The novel mutations of the PCCA gene(c.1288C > T) and PCCB gene(c.359_360del AT and c.1398 + 1G > A) were not detected in any of the 200 normal controls (600 alleles) enrolled in this study. According to the ACMG standards and guidelines for the interpretation of sequence variants, these novel mutations are pathogenic. Clinical findings and genotypes are summarized in Table 4 and Fig. 1.

Discussion and conclusions
In China, increasing numbers of patients with genetic metabolic deficiencies are being diagnosed and treated in the neonatal period [11]. However, in terms of managing PA patients, it is different from those countries where newborn screening for these disorders is freely available. Some cases will only seek medical attention if intoxication has appeared. In this study, although all PA cases presented in an acute neonatal form, patient 1 and 2 were diagnosed with PA, after presenting clinical symptoms, and not through neonatal screening. Patient 1 initially presented with tachypnea and malignant hyperthermia, and was diagnosed with presumed sepsis. At that time, the patient's relevant metabolic levels (such as blood ammonia level) were not measured. This resulted in a delay in reaching until an accurate diagnosis, during which the patient developed generalized tonic clonic seizures, metabolic acidosis and hyperammonemia. The patient died at six and a half months of age from sudden cardiac arrest. Patient 2 was diagnosed by metabolite screening at the first time after symptoms appeared. After treatment, the prognosis was good.
Newborn screening is a very important means of reducing the burden and the mokrtality rate of the disease [12].
In this study, we identified three different mutations in the PCCA gene and two different mutations in the PCCB gene. Patient 1 was compound heterozygous for c.1288C > T and c.2002G > A changes in the PCCA gene. The c.2002G > A(p.G668R) mutation has been previously reported [13], that is maps to the biotinylation domain, and it is defective in biotin binding [14]. The other mutation c.1288C > T has been reported in the Genome Aggregation Database as a SNP (rs776821944); however, the frequency of the variant only in genome aggregation was 0.000008/1, and was reported to be of no clinical significance. The mutation of c.1288C > T (p.R430X) is located in biotin carboxylation domain of the PCC enzyme and causes a premature termination codon, resulting in a truncated protein that undergoes nonsense-mediated decay (NMD) [15,16]. According to the ACMG standards and guidelines for the interpretation of sequence variants, the mutation of c.1288C > T(p.R430X) is pathogenic. The homozygous mutation c.1426C > T(p.R476X) in the PCCA gene identified in Patient 2 was firstly reported in Indian patients [2]. Deepti Gupta et al. demonstrated that the nonsense mutation c.1426C > T(p.R476X) could form truncated proteins that undergo NMD [17]. The mutations of c.2002G > A(p.G668R), c.1288C > T (p.R430X) and c.1426C > T(p.R476X) impact the structure of the α subunit and reduced the activity of the PCC enzyme to varying degrees, leading to a variable phenotypes in patients. Patient 3 was compound heterozygous for To date, of all mutations in PCCB described in patients, phenotypic severity has been observed to differ in patients with biallelic nonsense, deletion, or null mutations, which may be related to differences between individuals in NMD activity and NMD efficiency. Splice site variants are also seen, and, in general, result in milder disease [18]. Therefore, our patients exhibit a varying degrees of phenotype, which may be due to differences in protein activity, expression, and timely treatment.
In summary, we report 3 PA patients and the molecular basis of their disease were identified by PCRsequencing of all coding exons of the PCCA and PCCB genes. Three novel mutations, c.1288C > T (p.R430X) in the PCCA gene, c.359_360delAT(p.Y120Cfs*40) and c.1398 + 1G > A in the PCCB gene, were identified. The present study will expand the mutation spectrum of PA.