Mutations in the holocarboxylase synthetase gene HLCS

Holocarboxylase synthetase (HLCS) deficiency is an autosomal recessive disorder. HLCS is an enzyme that catalyzes biotin incorporation into carboxylases and histones. Since the first report of the cDNA sequence, 30 mutations in the HLCS gene have been reported. Mutations occur throughout the entire coding region except exons 6 and 10. The types of mutations are one single amino acid deletion, five single nucleotide insertions/deletions, 22 missense mutations, and two nonsense mutations. The only intronic mutation identified thus far is c.1519+5G>A (also designated IVS10+5G>A), which causes a splice error. Several lines of evidence suggest that c.1519+5G>A is a founder mutation in Scandinavian patients. Prevalence of this mutation is about 10 times higher in the Faroe Islands than in the rest of the world. The mutations p.L237P and c.780delG are predominant only in Japanese patients. These are probably founder mutations in this population. Mutations p.R508W and p.V550M are identified in several ethic groups and accompanied with various haplotypes, suggesting that these are recurrent mutations. There is a good relationship between clinical biotin responsiveness and the residual activity of HLCS. A combination of a null mutation and a point mutation that shows less than a few percent of the normal activity results in neonatal onset. Patients who have mutant HLCS with higher residual activity develop symptom after the neonatal period and show a good clinical response to biotin therapy.     

Deficiency of Biotinyl-AMP Synthetase Activity in Fibroblasts of Patients with Holocarboxylase Synthetase Deficiency

A simple, rapid assay was developed to diagnose holocarboxylase synthetase deficiency. Holocarboxylase synthetase first catalyzes the formation of biotinyl-AMP from biotin and ATP, an activity designated as biotinyl-AMP synthetase. In the second step of the reaction, biotin is transferred from biotinyl-AMP to the enzymatically inactive apocarboxylase to form an active holocarboxylase. The assay for holocarboxylase synthetase activity therefore requires a protein apocarboxylase substrate which is not readily available. In the assay for biotinyl-AMP synthetase, hydroxylamine reacts nonenzymatically with the product of the enzymatic reaction, biotinyl-AMP, to form biotinylhydroxamate. At the end of the reaction, unreacted radioactive biotin substrate, which is negatively charged at neutral pH, is bound to an anion-exchange resin and a neutral radioactive biotinylhydroxamate product in the supernatant is counted. In fibroblasts from 11 patients with proven holocarboxylase synthetase deficiency, the mean biotinyl-AMP synthetase activity at 25 nM biotin was 4% of the control mean with a range of 0.2 to 8%. This is an improved assay because it does not require preparation of an apocarboxylase substrate and is suitable for the diagnosis of patients with holocarboxylase synthetase deficiency.     

Clinical, biochemical, and molecular characterization of patients with glutathione synthetase deficiency

Pyroglutamic aciduria (5-oxoprolinuria) is a rare autosomal recessive disorder caused by either glutathione synthetase deficiency (GSSD) or 5-oxoprolinase deficiency. GSSD results in low glutathione levels in erythrocytes and may present with hemolytic anemia alone or together with pyroglutamic aciduria, metabolic acidosis, and CNS damage. Five patients with pyroglutamic aciduria were studied. All presented with hemolytic anemia and metabolic acidosis. Two (brothers) also had Fanconi nephropathy, which is not seen in pyroglutamic aciduria. Molecular analyses of the GSS gene was performed in 3 patients. RT-PCR and heteroduplex analysis identified a homozygous deletion in 1 patient and a homozygous mutation in 2 others (brothers with Fanconi nephropathy). Sequencing of glutathione synthetase (GSS) cDNA from the first patient showed a 141-bp deletion corresponding to the entire exon 4, whilst the corresponding genomic DNA showed a G491 --> A homozygous splice site mutation. Sequencing of GSS cDNA from the Fanconi nephropathy patients showed a C847 --> T [ARG283 --> CYS] mutation in exon 9.     

Biotinidase deficiency: novel mutations and their biochemical and clinical correlates

Biotinidase deficiency is a defect in the recycling of the vitamin biotin. Biotin supplementation can markedly improve the neurological and cutaneous symptoms of affected children and prevent symptoms in children identified by newborn screening or treated since birth. We have determined thirteen novel mutations in children with the disorder. Two nonsense mutations, eight single missense mutations, three allelic double missense mutations, and two are polymorphisms were identified in the biotinidase gene (BTD). One of the missense mutations, c.734G>A (p. C245Y), is the first to be reported that alters the cysteine in the putative location crucial for ester formation and binding of the biotinyl-moiety in the active site of the enzyme. These mutations add to the growing list of mutations that are helping to delineate structure/function relationships of the enzyme.     

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.     

Liver pathology in a new congenital disorder of urea synthesis: N-acetylglutamate synthetase deficiency

Summary Detoxification through the urea cycle is the means by which mammalian organisms dispose of their excess ammonia. Within this cycle, N-acetylglutamate (NAG) is the most important cofactor for optimal enzyme activity. It is formed from acetyl CoA and glutamate through the action of N-acetylglutamate synthetase (NAGS). Recently, a congenital deficiency of NAGS has been reported. In this communication, we present results of structural investigations on liver tissue of the index patient with NAGS defect.Light microscopy revealed small, eosinophilic inclusions in some of the hepatocytes. Electron microscopy showed vesicular SER and fibrillar material in expanded cisterns of the RER, presumably corresponding to the inclusions seen in light microscopy. Immunofluorescence of liver tissue uncovered a discrete distribution of intracellular albumin in the form of small deposits. We suggest that in NAGS deficiency, some secretory proteins might be incompletely processed due to the lack of a protease activator, NAG.     

Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families

Ornithine carbamoyltransferase deficiency is the most common inherited defect of the urea cycle. We examined 28 male and 9 female patients from 29 families and identified 25 distinct mutations in OTC, 14 of which were novel. Three novel missense mutations (p.Ala102Pro, p.Pro158Ser, p.Lys210Glu) and a novel deletion of the Leu43 are not directly involved either in the enzyme active site or in the intersubunit interactions; however, the mutations include conserved residues involved in intramolecular interaction network essential for the function of the enzyme. Three novel large deletions – a 444 kb deletion affecting RPGR, OTC and TSPAN7, a 10 kb‐deletion encompassing OTC exons 5 and 6 and a 24.5 kb‐deletion encompassing OTC exons 9 and 10 – have probably been initiated by double strand breaks at recombination‐promoting motifs with subsequent non‐homologous end joining repair. Finally, we present a manifesting heterozygote carrying a hypomorphic mutation p.Arg129His in combination with unfavorably skewed X‐inactivation in three peripheral tissues.     

Familial X-linked mental retardation and isolated growth hormone deficiency: Clinical and molecular findings

We report on several members of a family with varying degrees of X-linked mental retardation (XLMR), isolated growth hormone deficiency (IGHD), and infantile behaviour but without other consistent phenotypic abnormalities. Male patients continued to grow until well into their twenties and reached a height ranging from 135 to 159 cm. Except one, all female carriers were mentally normal; their adult height ranged from 159 to 168 cm. By linkage studies we have assigned the underlying genetic defect to the Xq24–q27.3 region, with a maximum lod score of Z = 3.26 at θ = 0.0. for the DXS294 locus. The XLMR-IGHD phenotype in these patients may be due to pleiotropic effects of a single gene or it may represent a contiguous gene syndrome. © 1996 Wiley-Liss, Inc.     

Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles

Canals I, Elalaoui SC, Pineda M, Delgadillo V, Szlago M, Jaouad IC, Sefiani A, Chabás A, Coll MJ, Grinberg D, Vilageliu L. Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles. The Sanfilippo syndrome type C [mucopolysaccharidosis IIIC (MPS IIIC)] is caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. We report the first molecular study on several Spanish Sanfilippo syndrome type C patients. Seven Spanish patients, one Argentinean and three Moroccan patients were analysed. All mutant alleles were identified and comprised nine distinct mutant alleles, seven of which were novel, including four missense mutations (p.A54V, p.L113P, p.G424V and p.L445P) and three splicing mutations due to two point mutations (c.633+1G>A and c.1378‐1G>A) and an intronic deletion (c.821‐31_821‐13del). Furthermore, we found a new single nucleotide polymorphism (SNP) (c.564‐98T>C). The two most frequent changes were the previously described c.372‐2A>G and c.234+1G>A mutations. All five splicing mutations were experimentally confirmed by studies at the RNA level, and a minigene experiment was carried out in one case for which no fibroblasts were available. Expression assays allowed us to show the pathogenic effect of the four novel missense mutations and to confirm that the already known c.710C>A (p.P237Q) is a non‐pathogenic SNP. Haplotype analyses suggested that the two mutations (c.234+1G>A and c.372‐2A>G) that were present in more than one patient have a common origin, including one (c.234+1G>A) that was found in Spanish and Moroccan patients.     

Molecular and clinical analyses of cystic fibrosis in the South of Spain

We report molecular and clinical analyses in 71 unrelated patients with cystic fibrosis (CF) from Andalusia (South of Spain). Direct mutation analysis of six mutations of the CFTR gene (ΔF508, G542X, R1162X, N1303K, W1182X and 1949del84) was performed. The proportion of CF chromosomes with the above-mentioned mutations was 58.5%. Haplotype analysis was performed with the marker/enzyme pairs XV2C/ TaqI and KM19/ PstI . A particular haplotype has been found associated with each of the studied mutations, while the pooled data for the unknown mutations are not associated with any particular haplotype. This lack of association indicates that there will not be a single predominant mutation amongst the other CF chromosomes. To assess the relationship between genotype and phenotype in these patients, we correlated the pancreatic status and the occurrence of chronic Pseudomona aeruginosa infection with the observed genotype. Pancreatic insufficiency was present in all patients in whom the analyzed mutations were found to be homozygous or compound heterozygous. We also found a higher rate of Pseudomonas colonization in the group of patients in whom the genotype was homozygous or compound heterozygous for the analysed mutations when compared with the group of patients with a different genotype, but the difference was not statistically significant.     

Molecular and clinical analyses of Japanese patients with carbamoylphosphate synthetase 1 (CPS1) deficiency

Carbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan.     

Functional and molecular studies in primary carnitine deficiency

Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported (n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen‐2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency.     

Pyruvate carboxylase deficiency type A and type C: Characterization of five novel pathogenic variants in PC and analysis of the genotype–phenotype correlation

Pyruvate carboxylase deficiency (PCD) is caused by biallelic mutations of the PC gene. The reported clinical spectrum includes a neonatal form with early death (type B), an infantile fatal form (type A), and a late‐onset form with isolated mild intellectual delay (type C). Apart from homozygous stop‐codon mutations leading to type B PCD, a genotype–phenotype correlation has not otherwise been discernible. Indeed, patients harboring biallelic heterozygous variants leading to PC activity near zero can present either with a fatal infantile type A or with a benign late onset type C form. In this study, we analyzed six novel patients with type A (three) and type C (three) PCD, and compared them with previously reported cases. First, we observed that type C PCD is not associated to homozygous variants in PC. In silico modeling was used to map former and novel variants associated to type A and C PCD, and to predict their potential effects on the enzyme structure and function. We found that variants lead to type A or type C phenotype based on the destabilization between the two major enzyme conformers. In general, our study on novel and previously reported patients improves the overall understanding on type A and C PCD.     

Ultrastructural pathology in congenital defects of the urea cycle: Ornithine transcarbamylase and carbamylphosphate synthetase deficiency

Summary Inborn defects of urea synthesis, leading to hyperammonemia, are complex inherited disorders, whose structural sequelae in different tissues and organs have not yet been studied in detail. Ultrastructural investigations have been performed on two cases of deficiencies of two consecutive enzymes of the urea cycle, carbamylphosphate synthetase and ornithine transcarbamylase, and the findings are compared with previously reported results. With regard to liver pathology it appeared that 1). Hepatocytes in CPS deficiency mainly exhibited changes of SER and mitochondrial compartments, whereas 2). OTC deficiency was characterized by regressive liver cell change, with abnormal configuration of the RER, formation of telolysosomes and peribiliary vesiculation. It is suggested that the mitochondrial disorder in the CPS defect is directly related to the lack of a major enzyme protein in this organelle, resulting in structural damage. The leading renal change in CPS deficiency is foot process fusion of glomerular podocytes. Brain alteration in this disorder is similar to that reported for other hyperammonaemic urea cycle defects.     

Independent mutational events are rare in the ATM gene: haplotype prescreening enhances mutation detection rate

Mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). Many different mutations have been identified using various techniques, with detection efficiencies ranging from 57 to 85%. In this study, we employed short tandem repeat (STR) haplotypes to enhance mutation identification in 55 unrelated A-T families of Iberian origin (20 Spanish, 17 Brazilian, and 18 Hispanic-American); we were able to identify 95% of the expected mutations. Allelic sizes were standardized based on a reference sample (CEPH 1347-2). Subsequent mutation screening was performed by PTT, SSCP, and DHPLC, and abnormal regions were sequenced. Many STR haplotypes were found within each population and six haplotypes were observed across several of these populations. Single nucleotide polymorphism (SNP) haplotypes further suggested that most of these common mutations are ancestrally related, and not hot spots. However, two mutations (8977C>T and 8264_8268delATAAG) may indeed be recurring mutational events. Common haplotypes were present in 13 of 20 Spanish A-T families (65%), in 11 of 17 Brazilian A-T families (65%), and, in contrast, in only eight of 18 Hispanic-American families (44%). Three mutations were identified that would be missed by conventional screening strategies. In all, 62 different mutations (28 not previously reported) were identified and their associated haplotypes defined, thereby establishing a new database for Iberian A-T families, and extending the spectrum of worldwide ATM mutations.     

Selective IgG subclass deficiency: quantification and clinical relevance

Each of the four human IgG subclasses exhibits a unique profile of effector functions relevant to the clearance and elimination of infecting microorganisms. The quantitative response within each IgG subclass varies with the nature of the antigen, its route of entry and, presumably, the form in which it is presented to the immune system. This results in antibody responses to certain antigens being predominantly or exclusively of a single IgG subclass. An inability to produce antibody of the optimally protective isotype can result in a selective immunodeficiency state. This is particularly apparent for responses to certain bacterial carbohydrate antigens that are normally of IgG2 isotype. A failure to produce the appropriate specific antibody response may result in recurrent upper and/or lower respiratory tract infection. Careful patient investigation can identify such deficiencies and suggest appropriate clinical management. In this review we outline the biology and clinical relevance of the IgG subclasses and summarize current rational treatment approaches.     

Expanding the clinical spectrum of mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase deficiency with Turkish cases harboring novel HMGCS2 gene mutations and literature review

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHS) deficiency is a very rare autosomal recessive inborn error of ketone body synthesis and presents with hypoketotic hypoglycemia, metabolic acidosis, lethargy, encephalopathy, and hepatomegaly with fatty liver precipitated by catabolic stress. We report acute presentation of two patients from unrelated two families with novel homozygous c.862C>T and c.725‐2A>C mutations, respectively, in HMGCS2 gene. Affected patients had severe hypoketotic hypoglycemia, lethargy, encephalopathy, severe metabolic and lactic acidosis and hepatomegaly after infections. Surprisingly, molecular screening of the second family showed more affected patients without clinical findings. These cases expand the clinic spectrum of this extremely rare disease.     

The molecular basis of cystathionine beta-synthase deficiency in Australian patients: genotype-phenotype correlations and response to treatment

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. It is inherited as an autosomal recessive trait and common clinical features are: dislocation of the optic lens, osteoporosis, mental retardation, and thromboembolism. We determined the molecular basis of CBS deficiency in 36 Australian patients from 28 unrelated families, using direct sequencing of the entire coding region of the CBS gene. The G307S and I278T mutations were the most common mutations. They were present in 19% and 18% of independent alleles, respectively. In total, seven novel and 20 known mutations were detected. Of those, the two novel missense mutations (C109R and G347S), as well as two known missense mutations (L101P and N228K), were expressed in E. Coli. All mutant proteins completely lacked catalytic activity. Furthermore, we studied the correlation between genotype and the biochemical response to pyridoxine treatment in the patients of whom 13 were pyridoxine responsive, 21 were non-responsive, and two were partially responsive. The G307S mutation always resulted in a severe non-responsive phenotype, whereas I278T resulted in a milder B6 responsive phenotype. From our results, we were also able to establish three other mild mutations: P49L, R369C, and V371M.     

Triosephosphate isomerase deficiency: biochemical and molecular genetic analysis for prenatal diagnosis

Inherited deficiency of the glycolytic enzyme triosephosphate isomerase leads to a multisystem disorder characterized by progressive neuromuscular dysfunction, chronic nonspherocytic hemolytic anemia and increased susceptibility to severe infections. Most patients die within the first 6 years. We examined a family with severe triosephosphate isomerase deficiency. The 1-year-old index patient suffered from hemolytic anemia, neuromuscular impairment and pneumonias, with the necessity of intermittent mechanical ventilation. Triosephosphate isomerase activity in erythrocytes was reduced to about 20% of normal. Heat stability of the enzyme was strongly reduced; concentration of the physiological substrate, dihydroxyacetone phosphate was increased 20-fold due to the metabolic block. Direct sequencing of the triosephosphate isomerase gene revealed homozygosity for the formerly described GAGGA C -mutation changing 104 GluAsp. During a 2nd pregnancy we examined a cord blood sample obtained in the 19th gestational week. The biochemical data on enzyme activity, heat stability of the enzyme and concentration of dihydroxyacetone phosphate were in the normal range. The molecular genetic analysis confirmed the presence of the normal triosephosphate isomerase alleles. Pregnancy was continued, resulting in the delivery of an unaffected, healthy newborn.     

IgA deficiency: clinical correlates and responses to pneumococcal vaccine

We surveyed historical and laboratory data for 127 IgA-deficient patients (ages 2-67), referred to an immunology clinic; the commonest medical history was recurrent respiratory infections (50%), followed by autoimmunity (28%) asthma and allergy (13%). Fifty-two subjects have been given a pneumococcal vaccination; vaccine responses to 12 serotypes were significantly related to serum IgG2 levels (P = 0.004). Six immunized IgA/IgG2-deficient subjects produced insignificant amounts of antibodies to these pneumococcal serotypes; 10 others with normal IgG2 levels also had subnormal vaccine responses. IgA-deficient patients who had at least one B8 allele (n = 19) had a significantly greater response to this vaccine than the HLA-B8-negative subjects (n = 24) (P = 0.024). There was no relationship between a history of recurring infections and pneumococcal vaccine responses; HLA status was not related to a history of autoimmunity.