Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA: carboxylase deficiency.

3-Methylcrotonyl-CoA: carboxylase (EC 6.4.1.4; MCC) deficiency is an inborn error of the leucine degradation pathway (MIM *210200) characterized by increased urinary excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. The clinical phenotypes are highly variable ranging from asymptomatic to profound metabolic acidosis and death in infancy. Sequence similarity with Glycine max and Arabidopsis thaliana genes encoding the two subunits of MCC permitted us to clone the cDNAs encoding the alpha- and beta-subunits of human MCC. The 2580 bp MCCA cDNA encodes the 725 amino acid biotin-containing alpha-subunit. The MCCA gene is located on chromosome 3q26-q28 and consists of 19 exons. The 2304 bp MCCB cDNA encodes the non-biotin-containing beta-subunit of 563 amino acids. The MCCB gene is located on chromosome 5q13 and consists of 17 exons. We have sequenced both genes in four patients with isolated biotin-unresponsive deficiency of MCC. In two of them we found mutations in the MCCA gene. Compound heterozygosity for a missense mutation (S535F) and a nonsense mutation (V694X) were identified in one patient. One heterozygous mutation (S535F) was found in another patient. The remaining two patients had mutations in the MCCB gene. One consanguineous patient was homozygous for a missense mutation (R268T). In the other we identified a missense mutation in one allele (E99Q) and allelic loss of the other. Mutations were correlated with an almost total lack of enzyme activity in fibroblasts. These data provide evidence that human MCC deficiency is caused by mutations in either the MCCA or MCCB gene.     

3-Methylcrotonyl-CoA carboxylase deficiency: mutation analysis in 28 probands, 9 symptomatic and 19 detected by newborn screening

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder that appears to be the most frequent organic aciduria detected in tandem mass spectrometry (TMS)-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. We report mutation analysis in 28 MCC-deficient probands, 19 of whom were asymptomatic newborns detected by TMS newborn screening, and nine presented with clinical symptoms. Ten have mutations in MCCA, and 18 in MCCB. We identified 10 novel MCCA and 14 novel MCCB mutant alleles including missense, nonsense, frameshift and splice site mutations, and show that three of the missense mutations result in severely decreased MCC activity when expressed in MCC-deficient cell lines. Our data demonstrate no clear correlation between genotype and phenotype suggesting that factors other than the genotype at the MCC loci have a major influence on the phenotype of MCC deficiency.     

Novel mutations in the human MCCA and MCCB gene causing methylcrotonylglycinuria

Methylcrotonylglycinuria (MCG) is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The clinical phenotypes are highly variable ranging from neonatal onset with severe neurological involvement to asymptomatic adults. Here we identified two novel MCCA (exon 3: c.137G>A; p.46G>E), (IVS7-1G>A splice site mutation), and four novel MCCB (exon 11: c.1065A>T; p.355L>F), (exon 15: c.1430A>G; p.477Q>R), (exon 16: c.1549G>A; p.517G>R), (exon 16: c.1559A>C; p.520Y>S) mutant alleles from five MCC-deficient patients.     

Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency appears to be the most frequent organic aciduria detected in tandem mass spectrometry (MS/MS) screening programs in the United States, Australia, and Europe. A pilot study of newborn screening using MS/MS has recently been commenced in Japan. Our group detected two asymptomatic MCC deficiency patients by the pilot screening and collected data on another three MCC deficiency patients to study the molecular bases of the MCC deficiency in Japan. Molecular analyses revealed novel mutations in one of the causative genes, MCCA or MCCB, in all five of the patients: nonsense and frameshift mutations in MCCA (c.1750C > T/c.901_902delAA) in patient 1, nonsense and frameshift mutations in MCCB (c.1054_1055delGG/c.592C > T) in patient 2, frameshift and missense mutations in MCCB (c.1625_1626insGG/c.653_654CA > TT) in patient 3, a homozygous missense mutation in MCCA (c.1380T > G/ 1380T > G) in patient 4, and compound heterozygous missense mutations in MCCB (c.569A > G/ c.838G > T) in patient 5. No obvious clinical symptoms were observed in patients 1, 2, and 3. Patient 4 had severe neurological impairment and patient 5 developed Reye-like syndrome. The increasing use of MS/MS newborn screening in Japan will further clarify the clinical and genetic heterogeneity among patients with MCC deficiency in the Japanese population.     

Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic,clinical, and biological implications

Mucopolysaccharidosis (MPS) types IIIA, B, C, and D are a group of autosomal recessive lysosomal storage diseases caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. The progressive lysosomal storage of heparan sulfate eventually results in the clinical onset of disease, which is predominantly characterized by severe central nervous system degeneration. MPS‐IIIA and MPS‐IIIB involve deficiencies of heparan sulfate sulfamidase (SGSH) and α‐N‐acetylglucosaminidase (NAGLU), respectively. Both the SGSH and NAGLU genes have been cloned and characterized, thereby permitting mutation analysis of MPS‐IIIA and MPS‐IIIB patients. A total of 62 mutations have now been defined for MPS‐IIIA consisting of 46 missense/nonsense mutations, 15 small insertions/deletions, and one splice site mutation. A total of 86 mutations have been identified in the NAGLU gene of MPS‐IIIB patients; 58 missense/nonsense mutations, 27 insertions/deletions, and one splice site mutation. Most of the identified mutations in the SGSH and NAGLU genes are associated with severe clinical phenotypes. Many of the missense, nonsense, and insertion/deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on SGSH and NAGLU activity and intracellular processing and trafficking. For MPS‐IIIA and MPS‐IIIB many of the reported mutations are unique making screening the general population difficult. However, molecular characterization of MPS‐IIIA patients has revealed a high incidence of particular mutations of different geographical origins, which will be beneficial for the molecular diagnosis of MPS‐IIIA. Hum Mutat 18:264–281, 2001. © 2001 Wiley‐Liss, Inc.     

Structural insights on pathogenic effects of novel mutations causing pyruvate carboxylase deficiency

Pyruvate carboxylase (PC), a key enzyme for gluconeogenesis and anaplerotic pathways, consists of four domains, namely, biotin carboxylase (BC), carboxyltransferase (CT), pyruvate carboxylase tetramerization (PT), and biotin carboxyl carrier protein (BCCP). PC deficiency is a rare metabolic disorder inherited in an autosomal recessive way. The most severe form (form B) is characterized by neonatal lethal lactic acidosis, whereas patients with form A suffer chronic lactic acidosis with psychomotor retardation. Diagnosis of PC deficiency relies on enzymatic assay and identification of the PC gene mutations. To date, six mutations of the PC gene have been identified. We report nine novel mutations of the PC gene, in five unrelated patients: three being affected with form B, and the others with form A. Three of them were frameshift mutations predicted to introduce a premature termination codon, the remaining ones being five nucleotide substitutions and one in frame deletion. Impact of these mutations on mRNA was assessed by RT‐PCR. Evidence for a deleterious effect of the missense mutations was achieved using protein alignments and three‐dimensional structural prediction, thanks to our modeling of the human PC structure. Altogether, our data and those previously reported indicate that form B is consistently associated with at least one truncating mutation, mostly lying in CT (C‐terminal part) or BCCP domains, whereas form A always results from association of two missense mutations located in BC or CT (N‐terminal part) domains. Finally, although most PC mutations are suggested to interfere with biotin metabolism, none of the PC‐deficient patients was biotin‐responsive. Hum Mutat 0:1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Propionic acidemia: identification of twenty-four novel mutations in Europe and North America

Propionic acidemia is an inherited metabolic disease caused by the deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC), one of the four biotin-dependent enzymes. PCC is a multimeric protein composed of two different alpha- and beta-PCC subunits, nuclearly encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause the clinically heterogeneous disease propionic acidemia. In this work we describe the mutational analysis of PCCA and PCCB deficient patients from different European countries (Spain, Italy, Belgium, Croatia, and Austria) and from America (mainly USA). We report 24 novel PA mutations, nine affecting the PCCA gene and 15 affecting the PCCB gene. They include six missense mutations, one nonsense mutation, one point exonic mutation affecting splicing, seven splicing mutations affecting splice sequences, and nine short insertions or deletions, only two in-frame. We have found a highly heterogenous spectrum of PCCA mutations, most of the PCCA deficient patients are homozygous carrying a unique genotype. The PCCA mutational spectrum includes a high proportion of short insertions or deletions affecting one nucleotide. In the PCCA mutant alleles analyzed we have also found one single nucleotide change, a novel nonsynonymous SNP. On the other hand, the PCCB deficient patients carry a more reduced spectrum of mutations, 50% of them are missense. This work represents an extensive update of the mutational study of propionic acidemia providing important information about the worldwide distribution of PA mutations and representing another essential part in the study of the phenotype-genotype correlations for the prediction of the metabolic outcome and for the implementation of treatments tailored to each PA patient.     

Systemic lupus erythematosus in a man with Noonan syndrome

Hereditary multiple exostoses (EXT) is an autosomal dominant bone disease characterized by the formation of cartilage-capped prominences. EXT is genetically heterogeneous with at least four chromosomal loci. Among the four loci, the exostosis type 1 gene (EXT1) and type 2 gene (EXT2) have been cloned. Previous studies have shown that disease-type-specific frequency of mutations is different among various ethnic populations. To determine those frequencies in the Japanese, we conducted a large-scale mutation screening on both genes. In 23 of 43 Japanese families examined, we found 21 different mutations, of which 18 are novel. Seventeen (40%) of the 23 families had a mutation in EXT1 and six (14%) had a mutation in EXT2, suggesting that the former mutations are more frequent than the latter in Japanese EXT families. Of the 17 families with EXT1 mutations, 13 had those causing premature termination of the EXT1 protein and four showed missense mutations, whereas five of the six families with EXT2 mutations had those causing premature termination and one showed missense mutation. Interestingly, all four EXT1 missense mutations occurred in an arginine residue at codon 340 (R340) that is known as a critical site for expression of heparan sulfate glycosaminoglycans, suggesting that the region encompassing the arginine residue may play an important role in the function of the EXT1 protein. These results expand our knowledge of the ethnic difference of EXT and the structure-function relationship of the EXT genes. Copyright Wiley-Liss. Inc.     

High frequency of ENG and ALK1/ACVRL1 mutations in German HHT patients

Morbus Osler or HHT (hereditary hemorrhagic telangiectasia) is a disorder of the fibrovascular tissue that is inherited in an autosomal dominant way with frequency rates between 1:2,500 and 1:40,000. The disease provokes malformations of the blood vessels sometimes resulting in life-threatening complications. Presently, two genes involved in the development of HHT have been identified: ACVRL1 and ENG. Both of them encode proteins that belong to the TGF-beta receptor complex family and play an essential role in the formation of the vascular system. Recently, several mutations in ACVRL1 and ENG have been described in other European populations. However, no data concerning mutation frequencies in the German population have been reported so far. Therefore, we screened our collective of German HHT patients (28 single cases and 11 familial cases) for mutations in both genes by direct sequencing. We detected 11 mutations already described elsewhere and 19 novel ones. Furthermore, evidence for the pathogenic role of four new missense mutations was collected by screening a healthy control collective using RFLP analysis. Interestingly, the majority of ACVRL1 mutations represented missense mutations, whereas mutations in ENG mostly resulted in a shortened protein. Our results demonstrate the importance of ACVRL1 and ENG mutations in German HHT patients displaying mutation frequencies over 80%.     

Identification of twelve novel mutations in patients with classic and variant forms of maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder of panethnic distribution caused by a deficiency of the activity of branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in the human BCKD genes E1alpha (BCKDHA), E1beta (BCKDHB) and E2 (DBT) are known to result in MSUD, referred to as type Ia, Ib and II mutations respectively. In this study 16 patients with the classic severe form of MSUD and three patients with milder variant forms of the disease were investigated for mutations in the E1alpha-, E1beta- and E2-gene by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The patients' clinical and biochemical phenotypes were well characterized. One novel type Ia missense mutation, eight novel type Ib (three missense, two nonsense, two small deletions, one small duplication) and three novel type II (two missense, one splice site) mutations were identified in patients. Moreover, eleven previously described mutations were detected: five type Ia (four missense, one nonsense), three type Ib mutations (two missense, one nonsense) and three type II mutations (two missense, one small deletion). Fourteen patients are homozygous for one single mutation, five patients are compound-heterozygous for two different mutations affecting one of the three genes. Thus, in all 19 patients the identified mutations can most probably be considered the molecular basis of the disease.     

Mutations in GABRA1, GABRA5, GABRG2 and GABRD receptor genes are not a major factor in the pathogenesis of familial focal epilepsy preceded by febrile seizures

GABA(A) receptors mutations have been reported in few epilepsy families with febrile seizures (FS) followed by generalized epilepsy. It is not known if such mutations may underlie FS followed by partial epilepsy, which is a more common type of epilepsy. We searched for disease-causing mutations in the genes of the alpha1, alpha5, gamma2 and delta subunits of the GABA-A receptor that were previously shown to contain epilepsy-causing mutations or epilepsy susceptibility polymorphisms. All coding and untranslated exons of these four GABA(A) subunit genes were screened in 74 unrelated patients with familial partial epilepsy preceded by FS. Most patients had temporal lobe epilepsy (TLE). We did not detect any disease-causing mutations that would be consistent with missense, nonsense or splice site mutations in any of the four analyzed genes. We conclude that these genes are not a major genetic factor in familial TLE preceded by FS.     

Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2 genes

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of exostoses, which are cartilage-capped bony protuberances mainly located on long bones. Two genes, EXT1 and EXT2, and at least one other unidentified gene, are known to be involved in the formation of exostoses. To date, 49 different EXT1 and 25 different EXT2 mutations have been found in EXT patients, and there is evidence that mutations in these two genes are responsible for over 70% of the EXT cases. Among the 49 EXT1 mutations there are 9 nonsense, 21 frameshift, and 5 splice site mutations; 2 in-frame deletions of 1 and 5 amino acids respectively; and 12 missense mutations. For EXT2, 8 nonsense, 11 frameshift, 3 splice site and 3 missense mutations are described. The majority of these mutations are mutations causing loss of function, which is consistent with the presumed tumor suppressor function of the EXT genes.     

Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome

Jagged1 (JAG1) is a cell surface ligand in the Notch signaling pathway and mutations in this gene cause Alagille syndrome (AGS). JAG1 mutations have been identified in 60-70% of AGS patients studied, and these include total gene deletions ( approximately 6%), protein-truncating mutations (insertions, deletions and nonsense mutations) (82%) and missense mutations (12%). Based on the finding that total JAG1 deletions cause AGS, haploinsufficiency has been hypothesized to be a mechanism for disease causation; however, the mechanism by which missense mutations cause disease is not understood. To date, 25 unique missense mutations have been observed in AGS patients. Missense mutations are non-randomly distributed across the protein with clusters at the 5' end of the protein, in the conserved DSL domain, and two clusters within the EGF repeats. To understand the effect of the missense mutations on protein localization and function, we have studied four missense mutations (R184H, L37S, P163L and P871R). In two assays of JAG1 function, R184H and L37S are associated with loss of Notch signaling activity relative to wild-type JAG1. Neither R184H or L37S is present on the cell surface and both are abnormally glycosylated. Furthermore, these mutations lead to abnormal accumulation of the protein, possibly in the endoplasmic reticulum. Both P163L and P871R are associated with normal levels of Notch signaling activity and are present on the cell surface, consistent with these changes being polymorphisms rather than disease-causing mutations.     

Novel missense and frameshift mutations in the activin receptor-like kinase-1 gene in hereditary hemorrhagic telangiectasia. Mutations in brief no. 164. Online

Hereditary hemmorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dyplasia and recurrent hemorrhage. One of the causative genes is the activin receptor-like kinase-1 (ALK-1) gene located on chromosome 12q13. ALK-1 is an endothelial cell type I receptor for the TGF-beta superfamily of ligands. As a number of mutations have been identified in the kinase domain of ALK-1, we initiated a mutation analysis specifically targeting the first four coding exons of ALK-1 in order to determine if mutations in the extracellular and transmembrane domains are also present in HHT. Six new mutations have been identified. Three frameshift mutations were identified in exons encoding the extracellular and transmembrane domains. These mutations would grossly truncate the ALK-1 protein and are thus classic null alleles. Three new missense mutations within the exons encoding the extracellular domain, in addition to two previously described missense mutations, are located at or near highly conserved cysteines. These mutations may disrupt intra- or inter-molecular disulfide bridges required for ligand binding. The combined data suggest that both severe and subtle changes in the ALK-1 amino acid sequence can lead to receptor dysfunction and result in the HHT disease phenotype.     

Coding sequence mutations in the alpha subunit of propionyl-CoA carboxylase in patients with propionic acidemia.

Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, alpha and beta. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the alpha subunit. We investigated coding sequence mutations in the alpha subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in alpha subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the alpha subunit. We analyzed fibroblast extracts for the presence of a biotinylated alpha subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated alpha subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in an Escherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the alpha subunit.     

The FANCA gene in Japanese Fanconi anemia: reports of eight novel mutations and analysis of sequence variability

Fanconi anemia (FA), an autosomal recessive disorder characterized by a progressive pancytopenia associated with congenital anomalies and high predisposition to malignancies, is a genetically and clinically heterogeneous disease. At least eight complementation groups (FA-A to FA-H) have been identified with their relative prevalence varying among the ethnical backgrounds. Recently, responsible genes, FANCA and FANCC, have been cloned. This report describes mutations of the FANCA gene, which we studied by direct sequencing of cDNA with confirmation on genomic DNA in 15 unclassified Japanese FA patients. A total of 19 sequence alterations were identified, of which 10 (six missense and four silent alterations) were likely to be nonpathogenic polymorphism. The remaining nine alterations, of which eight were novel mutations, were assumed to be pathogenic and consisted of two missense mutations and seven mutations resulting in truncation of gene product, demonstrating a wide allelic heterogeneity. The pathogenic mutations were found in 12 patients (80%); they were either homozygous or compound heterozygous in 10 patients, apparently heterozygous in two patients and none in three patients. We conclude that the sequence variability is intrinsic to the FANCA gene and that the relative prevalence of the FA-A subtype is unusually high in Japanese FA patients.