Research paper3-Methylcrotonyl-CoA carboxylase deficiency: Mutational spectrum derived from comprehensive newborn screening
Introduction
The introduction of tandem mass spectrometry (MS/MS) in newborn screening (NBS) has allowed the detection of various organic acidurias, including the deficiency of 3-methycrotonyl-CoA carboxylase (3-MCC; EC 6.4.1.4). Methylcrotonylglycinuria (MCG; MIM 210200 and MIM 210210) is an autosomal recessive disorder first identified by Eldjarn et al. (1970). In MCG, the catabolism of leucine is blocked at the fourth step owing to the deficiency of the 3-MCC enzyme which catalyzes the conversion of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA, a reversible reaction dependent upon ATP and using bicarbonate as a source of carboxyl groups. The enzymatic impairment of 3-MCC leads to the accumulation of 3-methylcrotonyl CoA inside the mitochondria where alternative pathways form 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). The latter, after conjugation with carnitine, and having been coupled with 3-MCG, is readily excreted in the urine (de Kremer et al., 2002).
3-MCC is one of the four biotin-dependent carboxylases known in humans; the other three are acetyl-CoA carboxylase (ACC; EC: 6.4.1.2), propionyl-CoA carboxylase (PCC; EC: 6.4.1.3) and pyruvate carboxylase (PC; EC: 6.4.1.1). Of these four biotin-dependent enzymes, only ACC is cytosolic whereas the others are active in the mitochondrial matrix.
3-methylcrotonyl-CoA carboxylase deficiency is the most frequent organic aciduria detected in screening programs from European, American and Australian populations (Baumgartner et al., 2001, Frazier et al., 2006, Gibson et al., 1998, Koeberl et al., 2003, Stadler et al., 2006, Wilcken et al., 2003). Patients with 3-MCC deficiency experience normal growth and development until the emergence of an acute episode of metabolic decompensation, occurring typically between 6 months and 3 to 5 years of age (Sweetman, 2001). This episode is usually triggered by an infection or the introduction of high protein foods in the diet. The biochemical diagnosis of 3–MCC deficiency is made by means of the detection of organic acids in the urine, through gas chromatography/mass spectrometry (GC/MS), and the blood profile of acylcarnitines performed by MS/MS. The organic acid profile is characterized by a marked increase of 3-MCG and 3-HIVA acids, whilst the acylcarnitines profile reveals a highly elevated concentration of 3-hydroxyisovalerylcarnitine (C5-OH) and an increased ratio of this compound to propionylcarnitine (C3) (Holzinger et al., 2001). 3-Methylcrotonylcarnitine (C5:1) may or may not be present. It is common for these patients to have a secondary carnitine deficiency due to its combination with 3-HIVA for subsequent urinary excretion.
3-MCC comprises two hetero-subunits assembled into a α6β6 dodecamer. The larger α subunit contains the biotin carboxylase (BC) domain and the biotin carboxyl carrier protein domain covalently bound with a biotin prosthetic group and the binding site for the two substrates (bicarbonate and ATP). The smaller β-subunit has the carboxyltransferase (CT) domain and is essential for binding to 3-methylcrotonyl CoA which is characterized by its highly conserved functional domains (Chu and Cheng, 2007, Grunert et al., 2012, Pasquali et al., 2006, Stadler et al., 2005).
The α-subunits of 3-MCC are encoded by the MCCC1 gene, which is located on chromosome 3q27 and spans about 70 kb of genomic DNA (Obata et al., 2001). The MCCC1 gene encodes a protein of 725 amino acids which has a molecular weight of approximately 80 kDa (Holzinger et al., 2001). The β-subunit is a protein of 563 amino acids, with a molecular weight of approximately 61.8 kDa encoded by the MCCC2 gene, which is located on chromosome 5q12-q13 (Baumgartner et al., 2001, Holzinger et al., 2001, Gallardo et al., 2001).
Individuals with 3-MCC deficiency harbor mutations in either MCCC1 or MCCC2. Currently, the Human Gene Mutation Database (HMGD) (Stenson et al., 2014) records a total of 81 mutations associated to the MCCC1 and 89 to the MCCC2 gene, most of which are missense mutations although small insertions/deletions, nonsense, frameshift, and splice site mutations are also identified. The mutations identified to date are evenly distributed throughout the entire sequences of the two genes, without any evidence of mutational hot-spots (Stadler et al., 2006). Previous studies have been unable to establish a genotype-phenotype correlation since no mutations appear to be exclusively associated with symptomatic or asymptomatic cases, and no specific mutations have been associated with either milder or more severe phenotypes (Baumgartner et al., 2001, Stadler et al., 2006, Holzinger et al., 2001, Gallardo et al., 2001, Dantas et al., 2005). In this regard, it is important to note that the clinical, biochemical and genetic data appear to support the conclusion that factors other than the genotypes of the MCCC1 and MCCC2 loci can influence the phenotypic consequences of 3-MCC deficiency, including modifying genes and, perhaps more importantly, the extent to which the pathway is stressed by dietary or other environmental factors such as excessive protein breakdown associated with infections (Wolfe et al., 2007).
In this study, we document the molecular data covering a ten-year period of MCG identification in the Portuguese NBS program. All the mutations identified in MCCC1 and MCCC2 genes were analysed in the context of what is currently known of the MCC mutational spectra.
Section snippets
Biochemical findings
The study presented here was based upon data obtained from extended neonatal screening, through the analysis of 903,528 newborns, corresponding to 99.8% of all births in Portugal (including Azores and Madeira) between March 2005 and December 2015. A total of 36 newborns were identified, from dried blood spots, with high values of C5OH, the primary biochemical marker of this disease. The cutoff value for a positive C5OH measure (≥ 1 μmol/L) was as established by the Portuguese Newborn Screening
MCCC1 and MCCC2 mutational spectrum
The MCCC1 and MCCC2 genes of a total of 41 cases (36 Portuguese and five Spanish) were analysed by DNA sequencing: one or both mutant alleles were found in 39 patients (Table 1). No mutations were found in either the MCCC1 or MCCC2 in the remaining two patients (cases 15 and 16). In total, 37 different mutations were found in our cohort including nonsense, missense, microdeletion, microinsertion and splice site mutations; 27 of these mutations were novel (Fig. 1A and B). The mutations were
Concluding remarks
Several studies have shown that in a few cases of 3-MCC deficiency, a phenotype comprising metabolic decompensation with hypoglycaemia, ketonaemia and severe metabolic acidosis develops. The diagnosis is therefore relevant as this information can avoid or greatly shorten any decompensation during intercurrent illness. The proportion of individuals with 3-MCC deficiency who will come to clinical attention is not known but it could be around 4 to 5% of cases. Wilcken posed the question: “if 3-MCC
Conflicts of interest statement
The authors declare no conflict of interest.
Acknowledgments
IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute
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2020, Experimental and Molecular PathologyCitation Excerpt :Affected children are also thought to be at risk for suffering from hypotonia, seizures and varying degrees of developmental delay (Van Dyk et al., 2016; Ficicioglu and Payan, 2006). The disorder is connected with the mutations of MCCC1 and MCCC2 genes (Baumgartner et al., 2001; Stadler et al., 2006; Yang et al., 2015; Fonseca et al., 2016; Cozzolino et al., 2018). Human MCCC1 is located to chromosome region 3q25-q27 and has 19 exons, while MCCC2 is located to chromosome region 5q12-q13 and consists of 17 exons (Gallardo et al., 2001; Holzinger et al., 2001).
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Both authors contributed equally to this work.