3-Methylcrotonyl-CoA carboxylase deficiency: Mutational spectrum derived from comprehensive newborn screening

Highlights

• Updated mutational spectra of MCCC1 and MCCC2 genes

• 3D predicted model of the human proteins based on the Pseudomonas aeruginosa homologues

• Understanding the phenotype-genotype relationship

• Discussion on the maintenance of 3MCG in newborn screening programs

Abstract

The deficiency of 3-methycrotonyl-CoA carboxylase (3-MCC; EC 6.4.1.4) is an autosomal recessive organic aciduria that is included in the newborn screening programs of several countries. This study reports data mainly obtained from the Portuguese newborn screening program collected over a ten-year period. Analysis of the MCCC1 and MCCC2 genes yielded 26 previously unreported mutations and a variant of clinically unknown significance. These mutations are discussed in the context of their likely impact on the function of the 3-MCC enzyme, with a view to exploring whether a phenotype-genotype correlation might be discerned. Further, these mutations were analysed in the context of what is known of the MCCC1 and MCCC2 mutational spectra, information that will be useful in both clinical and laboratory practice.

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.