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Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

The congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG), the most common N-glycosylation disorder, is a multisystem disease for which no effective treatment is available. The recent functional characterization of disease-causing mutations described in patients with PMM2-CDG led to the idea of a therapeutic strategy involving pharmacological chaperones (PC) to rescue PMM2 loss-of-function mutations. The present work describes the high-throughput screening, by differential scanning fluorimetry, of 10,000 low-molecular-weight compounds from a commercial library, to search for possible PCs for the enzyme PMM2. This exercise identified eight compounds that increased the thermal stability of PMM2. Of these, four compounds functioned as potential PCs that significantly increased the stability of several destabilizing and oligomerization mutants and also increased PMM activity in a disease model of cells overexpressing PMM2 mutations. Structural analysis revealed one of these compounds to provide an excellent starting point for chemical optimization since it passed tests based on a number of pharmacochemical quality filters. The present results provide the first proof-of-concept of a possible treatment for PMM2-CDG and describe a promising chemical structure as a starting point for the development of new therapeutic agents for this severe orphan disease.

Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957–898A>G and c.1957–920C>A identified in the methylmalonyl–coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957–898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957–920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation. Hum Mutat 30:1–7, 2009. © 2009 Wiley-Liss, Inc.

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B 12 metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B12. The mutational spectrum includes nine previously described mutations: c.3G>A(p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1 +2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients. © 2009 Wiley-Liss, Inc.

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.

When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11G>A and L311P). All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes.

Antisense oligonucleotide therapy to modulate splicing mutations in inherited diseases is emerging as a treatment option also for metabolic defects. In this article, we report the effect of cellular antisense therapy to suppress pseudoexon activation in primary dermal fibroblasts from patients with mutations in the PTS gene encoding 6-pyruvoyltetrahydropterin synthase (PTPS), which leads to tetrahydrobiopterin and monoamine neurotransmitter deficiency. Pathogenic inclusion of SINE or LINE-derived cryptic exons in different PTPS patients due to the intronic mutations c.84-322A>T, c.163 + 695_163 + 751del57, or c.164-712A>T was demonstrated by transcript analysis in fibroblasts and minigene ex vivo assays. Antisense morpholino oligonucleotides (AMOs) directed to the pseudoexons 3' or 5' splice sites were designed with the aim of preventing the pathological pseudoexon inclusion. At the time of AMO transfection, we investigated patients' cells for correct PTS-mRNA splicing and functional recovery of the PTPS protein. Transcriptional profiling after 24 hr posttransfection revealed a dose- and sequence-specific recovery of normal splicing. Furthermore, PTPS enzyme activity in all three patients' fibroblasts and the pterin profile were close to normal values after antisense treatment. Our results demonstrate proof-of-concept for pseudoexon exclusion therapy using AMO in inherited metabolic disease. Hum Mutat 32:1-9, 2011. © 2011 Wiley-Liss, Inc.

Hereditary tyrosinemia type I (HTI) is an autosomal recessive disease characterized by a deficiency in fumarylacetoacetate hydrolase (FAH) activity. In this work, the FAH genotype was established in a group of 29 HTI patients, most of them from the Mediterranean area. We identified seven novel mutations-IVS8-1(G>A, IVS10-2(A>T), 938delC, E6/I6del26, W78X, Q328X, and G343W-and two previously described mutations-IVS6-1(G>T) and IVS12+5(G>A). Fully 92.8% of the patients were carriers of at least one splice site mutation, with IVS6-1(G>T) accounting for 58.9% of the total number of alleles. The splice mutation group of patients showed heterogeneous phenotypic patterns ranging from acute forms with severe liver malfunction to chronic forms with renal manifestations and slow progressive hepatic alterations. Qualitative FAH cDNA expression was the same in all IVS6-1(G>T) homozygous patients regardless of their clinical picture. One patient with a heterozygous combination of a nonsense (Q328X) and a frameshift (938delC) mutation showed an atypical clinical picture of hypotonia and repeated infections. Despite the high prevalence of IVS12+5(G>A) in the northwestern European population, we found only two patients with this mutation in our group.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl-CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd-numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of alpha and beta subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of alpha and beta subunits by Western blot of many PCCA patients, as well as conventional mRNA-minus result of Northern blots for either gene or beta subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT-PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene-no mutation is predominant in the populations studied-while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense-mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1-10, 2011. © 2011 Wiley-Liss, Inc.

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K(M) for ATP and K(D) for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations.

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG—type Ia—is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256–1G>C and c.640–9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640–15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256–1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640–9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640–15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen. Hum Mutat 0, 1–9, 2009. © 2009 Wiley-Liss, Inc.

Homocystinuria is an autosomal recessive disease most commonly caused by mutations in cystathionine β-synthase (CBS). In this study we present the mutation analysis of 36 Colombian individuals from 10 unrelated kindred, with 11 individuals clinically classified as homocystinuric. Mutation analysis of the CBS gene revealed p.T191M, a prevalent mutation in Spain and Portugal, in the homozygous state in seven of the unrelated patients. Genotype-phenotype assessment of the p.T191M homozygous patients showed a high level of variability, including different severity in one pair of affected siblings. None of the patients responded biochemically to treatment with pharmacological doses of pyridoxine and folic acid as revealed by essentially unchanged homocysteine levels. This study offered a unique opportunity to study 18 heterozygous (p.T191M/wt) relatives of the homocystinuric patients. One atypical finding was that many of them presented with above average total homocysteine levels, putting them at an increased risk for vascular disease. Cryptorchidism was present in three of the cases, one of which presented also with Klinefelter syndrome. In addition to the previously described p.T191M mutation, a new mutation, p.A288T, was identified in a single individual. In this paper we present the first characterization, at a molecular level, of patients with homocystinuria from Colombia. © 2006 Wiley-Liss, Inc.

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194TC (p.I65T), c.204AT (p.R68S), c.731CT (p.P244L), c.782GA (p.R261Q), c.926CT (p.A309V), c.1162GA (p.V388M), and c.1162GA (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.

When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11GA and L311P). All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes.

Hereditary tyrosinemia type I (HTI) is an autosomal recessive disease characterized by a deficiency in fumarylacetoacetate hydrolase (FAH) activity. In this work, the FAH genotype was established in a group of 29 HTI patients, most of them from the Mediterranean area. We identified seven novel mutations-IVS8-1(GA, IVS10-2(AT), 938delC, E6/I6del26, W78X, Q328X, and G343W-and two previously described mutations-IVS6-1(GT) and IVS12+5(GA). Fully 92.8% of the patients were carriers of at least one splice site mutation, with IVS6-1(GT) accounting for 58.9% of the total number of alleles. The splice mutation group of patients showed heterogeneous phenotypic patterns ranging from acute forms with severe liver malfunction to chronic forms with renal manifestations and slow progressive hepatic alterations. Qualitative FAH cDNA expression was the same in all IVS6-1(GT) homozygous patients regardless of their clinical picture. One patient with a heterozygous combination of a nonsense (Q328X) and a frameshift (938delC) mutation showed an atypical clinical picture of hypotonia and repeated infections. Despite the high prevalence of IVS12+5(GA) in the northwestern European population, we found only two patients with this mutation in our group.

In this study we report the mutation analysis performed in Cuban PKU patients using DGGE and direct sequencing. Sixteen different mutations have been detected, which account for 91% of the total mutant alleles. Haplotype analysis and genealogical data support the European (mainly Spanish) origin of the mutations. Two mutations were found at unexpectedly high frequencies, E280K and R261Q, possibly due to consanguinity and genetic drift, among other factors. Hum Mutat 18:252, 2001.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl-CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd-numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of alpha and beta subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of alpha and beta subunits by Western blot of many PCCA patients, as well as conventional mRNA-minus result of Northern blots for either gene or beta subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT-PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene-no mutation is predominant in the populations studied-while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Propionyl-CoA carboxylase (PCC) is a biotin-dependent enzyme located in the mitochondrial matrix. Mutations in the PCCA and PCCB genes, which encode the a and b subunits of this heteropolymer, result in propionic acidemia (PA). We report the molecular analysis of b-deficient patients from Spain and Austria. Subjects were screened for defects affecting the PCCB gene by direct sequencing from genomic PCR products, restriction digests and mRNA analysis by RT-PCR. Study by western blot of the presence of immunoreactive b-PCC protein was also performed. A total of four novel sequence variations were found including the point mutations V205D, and M442T, and the frameshift mutation 790-791insG. Additionally, a new point change, L17M, was identified on the same allele as 790-791insG. The missense changes described above were not found in at least 40 control chromosomes analyzed. The Austrian patients were homozygous for V205D. One of the Spanish subjects was heterozygous for M442T and the known mutation c1170insT. The other Spanish patient carried L17M+790-791insG on one allele, and the described mutation E168K on the other mutant chromosome. The mutations V205D and M442T were confirmed at RNA level and also we have detected the presence of immunoreactive b-PCC protein translated from these mutant alleles. The patient having L17M+790-791insG and E168K also presented immunoreactive b-PCC protein. However, no cDNA product was obtained from the chromosome carrying L17M+790-791insG. We propose that 790-791insG, which causes a frameshift and a premature stop codon, is responsible for this finding. In any case, the translation from this mutant cDNA would produce a severity truncated peptide and, in consequence, a non-functional protein. Expression analysis of all these changes will help us to clarify their structural/functional consequences.

This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela. In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe. In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3. The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems. This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients. Hum Mutat 11:354–359, 1998. © 1998 Wiley-Liss, Inc.

Propionyl-CoA carboxylase (PCC) is a biotin-dependent enzyme located in the mitochondrial matrix. Mutations in the PCCA and PCCB genes, which encode the a and b subunits of this heteropolymer, result in propionic acidemia (PA). We report the molecular analysis of b-deficient patients from Spain and Austria. Subjects were screened for defects affecting the PCCB gene by direct sequencing from genomic PCR products, restriction digests and mRNA analysis by RT-PCR. Study by western blot of the presence of immunoreactive b-PCC protein was also performed. A total of four novel sequence variations were found including the point mutations V205D, and M442T, and the frameshift mutation 790-791insG. Additionaly, a new point change, L17M, was identified on the same allele as 790-791insG. The missense changes described above were not found in at least 40 control chromosomes analyzed. The Austrian patients were homozygous for V205D. One of the Spanish subjects was heterozygous for M442T and the known mutation c1170insT. The other Spanish patient carried L17M+790-791insG on one allele, and the described mutation E168K on the other mutant chromosome. The mutations V205D and M442T were confirmed at RNA level and also we have detected the presence of immunoreactive b-PCC protein translated from these mutant alleles. The patient having L17M+790-791insG and E168K also presented immunoreactive b-PCC protein. However, no cDNA product was obtained from the chromosome carrying L17M+790-791insG. We propose that 790-791insG, which causes a frameshift and a premature stop codon, is responsible for this finding. In any case, the translation from this mutant cDNA would produce a severily truncated peptide and, in consequence, a non-functional protein. Expression analysis of all these changes will help us to clarify their structural/functional consequences. Hum Mutat 14:89–90, 1999. © 1999 Wiley-Liss, Inc.