Reduced half-life of holocarboxylase synthetase from patients with severe multiple carboxylase deficiency

Multiple carboxylase deficiency is a clinical condition caused by defects in the enzymes involved in biotin metabolism, holocarboxylase synthetase (HLCS) or biotinidase. HLCS deficiency is a potentially fatal condition if left untreated, although the majority of patients respond to oral supplementation of 10-20 mg/day of biotin. Patients who display incomplete responsiveness to this therapy have a poor long-term prognosis. Here we investigated cell lines from two such HLCS-deficient patients homozygous for the c.647T>G p.L216R allele. Growth of the patients' fibroblasts was compromised compared with normal fibroblasts. Also the patient cells were not sensitive to biotin-depletion from the media, and growth rates could not be restored by re-administration of biotin. The molecular basis for the HLCS deficiency was further investigated by characterisation of the p.L216R protein. The HLCS mRNA was detected in MCD and normal cell lines. However, protein and enzyme activity could not be detected in the patients' cells. In vitro kinetic analysis revealed that enzyme activity was severely compromised for recombinantly expressed p.L216R and could not be increased by additional biotin. Furthermore, the turn-over rate for the mutant protein was double that of wildtype HLCS. These results help provide a molecular explanation for the incomplete biotin-responsiveness of this p.L216R form of HLCS.     

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.     

Clinical findings and biochemical and molecular analysis of four patients with holocarboxylase synthetase deficiency

Holocarboxylase synthetase (HLCS) deficiency (HLCSD) is a rare autosomal recessive disorder of biotin metabolism. HLCS catalyzes the biotinylation of the four human biotin-dependent carboxylases. Using the newly available human genomic sequence, we report the map of HLCS genomic structure and the predicted exon/intron boundaries. Moreover, the molecular studies of four patients (two Italians, one Iranian, and one Australian) affected by HLCS deficiency are here reported. The clinical findings, the age of onset, and response to biotin treatment differed between our patients. The diagnosis was made by organic acid analysis and confirmed by enzymatic analysis in three patients. Six mutations in the HLCS gene were identified, including two novel (N511K and G582R) and four known missense mutations (L216R, R508W, V550M, and G581S). Five of the mutations are localized within the HLCS biotin-binding domain, whereas the L216R amino acid change is located in the N-terminal region outside of the putative biotin-binding domain. This mutation, previously reported in a heterozygous state, was detected for the first time in a patient with homozygous status. The patient's severe clinical phenotype and partial responsiveness to biotin support a genotype-phenotype correlation through the involvement of residues of the N-terminal region in a substrate specificity recognition or regulation of the HLCS enzyme.     

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.     

Holocarboxylase synthetase deficiency: novel clinical and molecular findings

Tammachote R, Janklat S, Tongkobpetch S, Suphapeetiporn K and Shotelersuk V. Holocarboxylase synthetase deficiency: novel clinical and molecular findings. Multiple carboxylase deficiency (MCD) is an autosomal recessive metabolic disorder caused by defective activity of biotinidase or holocarboxylase synthetase (HLCS) in the biotin cycle. Clinical symptoms include skin lesions and severe metabolic acidosis. Here, we reported four unrelated Thai patients with MCD, diagnosed by urine organic acid analysis. Unlike Caucasians, which biotinidase deficiency has been found to be more common, all of our four Thai patients were affected by HLCS deficiency. Instead of the generally recommended high dose of biotin, our patients were given biotin at 1.2 mg/day. This low‐dose biotin significantly improved their clinical symptoms and stabilized the metabolic state on long‐term follow‐up. Mutation analysis by polymerase chain reaction‐sequencing of the entire coding region of the HLCS gene revealed the c.1522C>T (p.R508W) mutation in six of the eight mutant alleles. This suggests it as the most common mutation in the Thai population, which paves the way for a rapid and unsophisticated diagnostic method for the ethnic Thai. Haplotype analysis revealed that the c.1522C>T was on three different haplotypes suggesting that it was recurrent, not caused by a founder effect. In addition, a novel mutation, c.1513G>C (p.G505R), was identified, expanding the mutational spectrum of this gene.     

一例多种羧化酶缺乏症的HLCS基因变异分析

目的对1例临床诊断为多种羧化酶缺乏症(multiple carboxylase deficiency,MCD)的患儿及其父母进行相关致病基因的变异分析,为临床诊断及遗传咨询提供依据。方法应用PCR技术和DNA测序技术对患儿的MCD致病基因BT和HLCS编码区进行变异检测,并对患儿父母进行相应基因变异分析。在80名正常人中对未报道过的基因变异进行PCR-限制性片段长度多态性分析。结果患儿的BT基因编码区未发现碱基改变,HLCS基因存在c.286delG(p.Val96Leufs*162)和c.1648G>A(p.Val550Met)复合杂合变异,其中c.286delG(p.Val96Leufs*162)经PCR-限制性片段长度多态性分析验证为新变异。结论HLCS基因c.286delG(p.Val96Leufs*162)和c.1648G>A(p.Val550Met)变异可能为患儿的致病原因,致病基因的检出为临床诊断及遗传咨询提供了依据,同时丰富了HLCS基因变异谱。     

Ligase IV syndrome

Ligase IV (LIG4) syndrome belongs to the group of hereditary disorders associated with impaired DNA damage response mechanisms. Subjects affected with this rare autosomal recessive disease exhibit microcephaly, unusual facial features, growth retardation, developmental delay, skin anomalies, and are typically pancytopenic. The disease is characterized by pronounced radiosensitivity, genome instability, malignancy, immunodeficiency, and bone marrow abnormalities. LIG4 syndrome results from mutations in the DNA ligase IV gene encoding an enzyme that plays a pivotal role in repairing double strand DNA breaks and V(D)J recombination. Since LIG4 null-mutant mice are embryonic lethal and biallelic null mutations have not been described to date in LIG4-deficient patients, viability of the DNA ligase IV deficiency syndrome appears to require at least one allele with a hypomorphic mutation. Mutations R278H, Q280R, H282L, M249E located in the vicinity of the active site are typical hypomorphic because they do not affect ligase expression and retain residual albeit reduced activity of the enzyme at levels of 5–10% of that for the wild-type ligase. Carriers heterozygous for those mutations usually develop moderate defects in V(D)J recombination, mild immune abnormalities and malignancy. In contrast, mutations resided in OBD, i.e. in the C-terminal subdomain of the catalytic domain, and in XRCC4-binding domain more dramatically inhibit the ligase function and also greatly decrease its expression. A truncating mutation R580X and a frameshift mutation K424FS resulting in loss of the C-terminal XRCC4-binding domain have deleterious effect on both expression and function of LIG4 and represent a null allele.     

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P≤0.01), the specific effective airway resistance (P≤0.005) and the forced expiratory volume in 1 s (P≤0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.     

Functional characterization of nonsynonymous single nucleotide polymorphisms in the electrogenic Na+�CHCO 3 ? cotransporter NBCe1A

The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1 encoded by SLC4A4 plays essential roles in the regulation of intracellular/extracellular pH. Homozygous mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. In the present study, we tried to perform functional characterization of the four nonsynonymous single nucleotide polymorphisms (SNPs), E122G, S356Y, K558R, and N640I in NBCe1A. Functional analysis in Xenopus oocytes revealed that while the K558R variant had a significantly reduced transport activity corresponding to 47% of the wild-type activity, the remaining variants E122G, S356Y, and N640I did not change the NBCe1A activity. Apparent Na(+) affinity of K558R was not different from that of wild-type NBCe1A. Immunohistological analyses in HEK293 cells and MDCK cells indicated that none of these SNPs changed the trafficking behaviors of NBCe1A. Functional analysis in HEK293 cells also revealed that only the K558R variant had a reduced transport activity, corresponding to 41-47% of the wild-type activity. From these results, we conclude that among four SNPs, only the K558R variant, which is predicted to lie in transmembrane segment 5, significantly reduces the NBCe1A activity without changing the trafficking behavior or the apparent extracellular Na(+) affinity.     

Identification and functional characterization of CYP4V2 genetic variants exhibiting decreased activity of lauric acid metabolism

The objectives of the present study were to identify CYP4V2 genetic variants and characterize their functional consequences. A total of 26CYP4V2 genetic variants were identified, including seven novel variants in 60 randomly selected healthy subjects. Six protein-coding variants were studied, including three novel variants (L22V, R287T, and G410C) and three previously reported variants (R36S, Q259K, and H331P). The cDNA sequences encoding each amino acid variant and the wild-type CYP4V2 protein were cloned into the pcDNA/PDEST40 expression vector and transfected into eukaryotic 293T cells for overexpression of the CYP4V2 coding variants. CYP4V2 H331P and CYP4V2 G410C exhibited significant decreases in activity for lauric acid oxidation (20–30% of wild-type activity), when compared to the wildtype, which was correlated with low expression of CYP4V2 H331P and G410C substituted proteins. The other four CYP4V2 amino variants were comparable to wild-type CYP4V2 for lauric acid metabolism. The CYP4V2 H331P and G410C substitutions were predicted to cause a structural change through in silico analysis. In conclusion, the present study provides functional information about CYP4V2 genetic variants. These findings will be valuable for interpreting individual variations in phenotypes associated with CYP4V2 function in the clinical setting.     

Mutational studies in a patient with the hydrops fetalis form of mucopolysaccharidosis type VII

Four prior mutations have been reported in three patients with β-glucuronidase deficiency mucopolysaccharidosis (MPS VII), none of whom had the severe, infantile, hydropic form of the disease. We identified two mutations in the first reported case of nonimmune hydropic MPS VII whose cultured fibroblasts had <1% of residual activity. The first mutation was a C→T transition at position 1061 of the cDNA in exon 6 that gave rise to an Ala→Val substitution in codon 354 (A354V). The second was a C→T transition at position 1831 in exon 12 that produced an Arg→Trp substitution in codon 611 (R611W). Transient expression in COS-7 cells revealed that both mutant enzymes were synthesized as normal-size precursors in normal quantities, but both exhibited accelerated turnover. The expressed A354V enzyme had a t_0.5 (half-life) of 33 hr (wild-type t_0.5 >60 hr) and a specific activity 35% of wild-type enzyme. The R611W enzyme had a t_0.5 of 20 hr and no detectable catalytic activity. The t_0.5 of enzyme produced on cotransfection with A354V and R611W was nearly identical to that of A354V alone. Mutant enzyme expressed in transfected murine MPS VII cells gave similar residual activities relative to the wild-type enzyme. In COS cells, the A354V monomers formed mixed tetramers with coexpressed rat monomers, but the product of R611W did not. The higher than expected activity, both in COS cells and in murine MPS VII cells expressing A354V, provides further evidence that overexpression can partially correct some β-glucuronidase mutations, apparently by driving the folding reaction of monomers or the assembly into tetramers by mass action. © 1993 Wiley-Liss, Inc.     

12例多种羧化酶缺乏症的基因突变分析

目的 旨在从基因水平证实多种羧化酶缺乏症(multiple carboxylase deficiency,MCD)的诊断,探讨我国MCD患儿的基因突变情况.方法 12例MCD患儿接受基因诊断.采用PCR及直接测序法分别对4例生物素酶(biotinidase,BT)缺乏症和8例全羧化酶合成酶(holocarboxylase synthetas,HLCS)缺乏症进行BT基因和HLCS基因突变分析,对基因新突变通过限制性片段长度多态性分析及患儿父母和50名正常对照者基因检测以证实.结果 12例患儿基因突变检出率100%.4例BT缺乏症中发现BT基因突变6种:c.98-104del7ins3,c.1369G>A(V457M),c.1157G>A(W386X),c.1284C>A(Y428X),c.1384delA,c.1493_1494insT,后4种为新突变.8例HLCS缺乏症中发现HLCS基因突变4种:c.126G>T(E42D),c.1994G>C(R665P),c.1088T>A(V363D),c.1522C>T(R508W),后两种为热点突变[75%(12/16)],c.1994G>C为新突变.结论 本研究从基因水平上证实了12例MCD的诊断.共发现了6种BT基因突变,4种HLCS基因突变,其中5种为新突变;得出2种HLCS基因的热点突变.     

Structure and function of mammalian DNA ligases

DNA joining events are required for the completion of DNA replication, DNA excision repair and genetic recombination. Five DNA ligase activities, I–V, have been purified from mammalian cell extracts and three mammalian LIG genes, LIG1, LIG3 and LIG4, have been cloned. During DNA replication, the joining of Okazaki fragments by the LIG1 gene product appears to be mediated by an interaction with proliferating cell nuclear antigen (PCNA). This interaction may also occur during the completion of mismatch, nucleotide excision and base excision repair (BER). In addition, DNA ligase I participates in a second BER pathway that is carried out by a multiprotein complex in which DNA ligase I interacts directly with DNA polymerase β. DNA ligase IIIα and DNA ligase IIIβ, which are generated by alternative splicing of the LIG3 gene, can be distinguished by their ability to bind to the DNA repair protein, XRCC1. The interaction between DNA ligase IIIα and XRCC1, which occurs through BRCT motifs in the C-termini of these polypeptides, implicates this isoform of DNA ligase III in the repair of DNA single-strand breaks and BER. DNA ligase II appears to be a proteolytic fragment of DNA ligase IIIα. The restricted expression of DNA ligase IIIβ suggests that this enzyme may function in the completion of meiotic recombination or in a postmeiosis DNA repair pathway. Complex formation between DNA ligase IV and the DNA repair protein XRCC4 involves the C-terminal region of DNA ligase IV, which contains two BRCT motifs. This interaction, which stimulates DNA joining activity, implies that DNA ligase IV functions in V(D)J recombination and non-homologous end-joining of DNA double-strand breaks. At the present time, it is not known whether DNA ligase V is derived from one of the known mammalian LIG genes or is the product of a novel gene.     

Asthma and IL‐4 receptor alpha gene variants

Linkage of allergy to chromosome 16 has been described in several studies, together with a positive association with interleukin 4 receptor α gene variants. Our aim was to replicate these findings in a sample of German and Swedish families recruited through sib‐pairs affected by bronchial asthma. None of the markers showed linkage with the main phenotype of asthma or with total serum IgE. Seropositivity to D. pteronyssinus showed borderline significance in a region flanking the IL4Rα location. Single nucleotide polymorphisms (SNPs) leading to the protein exchanges I50V, E375A, C406R, S478P and Q551R in the IL‐4 receptor α were examined for allele sharing in sibs with asthma. Multiple regression analysis was performed for association with total serum IgE and specific IgE. Allele sharing of IL4Rα SNPs in asthmatic children was not significantly increased for any of the examined SNPs except for the intracytoplasmatic polymorphism 551R (0.79 vs. 0.84 expected, P = 0.044). The variants 50V, 478P and 551R were associated with slightly increased, and 375A and 406R with decreased total IgE levels, all at a non‐significant level. None of the examined IL4Rα variants were correlated to asthma severity. In summary, a single gene effect of IL4Rα variants or any other gene on chromosome 16 could not be shown in this selected population of children with asthma. As there could be interactions with multiple genetic and environmental factors, IL4Rα could still be involved in asthma pathogenesis.     

Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical,biochemical, and molecular genetic aspects

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.     

An ethnic-specific polymorphism in the catalytic subunit of glutamate-cysteine ligase impairs the production of glutathione intermediates in vitro

Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy–Weinberg disequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.     

New case of mitochondrial HMG-CoA synthase deficiency. Functional analysis of eight mutations

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).     

Comparison of in vivo and in vitro evolution of CCR5 to CXCR4 coreceptor use of primary human immunodeficiency virus type 1 variants

During the course of at least 50% of HIV-1 subtype B infections, CCR5-using (R5) viruses evolve towards a CXCR4-using phenotype. To gain insight in the transition from CCR5 to CXCR4 coreceptor use, we investigated whether acquisition of CXCR4 use in vitro of R5 viruses from four patients resembled this process in vivo. R5 variants from only one patient acquired CXCR4 use in vitro. These variants had envelopes with higher V3 charge and higher number of potential N-linked glycosylation sites when compared to R5 variants that failed to gain CXCR4 use in vitro. In this patient, acquisition of CXCR4 use in vitro and in vivo was associated with multiple mutational patterns not necessarily involving the V3 region. However, changes at specific V3 positions were prerequisite for persistence of CXCR4-using variants in vivo, suggesting that positive selection targeting the V3 loop is required for emergence of CXCR4-using variants during natural disease course.     

The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase

The molecular mechanism underlying the metabolic defect in phenylketonuria (PKU) patients carrying the V388M missense mutation of the phenylalanine hydroxylase (PAH) gene has been characterized. An in vitro prokaryotic expression system has been used to produce both the wild-type and the mutant form of the human PAH (hPAH) protein. The recombinant enzymes, obtained as fusion proteins, were purified by immobilized metal affinity chromatography and recovered in high yields. The wild-type hPAH possessed a high specific activity and its kinetic properties were the same as those reported for the enzyme isolated from human liver and other recombinant wild-type hPAH enzymes. The recombinant V388M mutant form exhibited a reduced specific activity equivalent to 30% of the wild-type hPAH enzyme when assayed using the synthetic cofactor (6-methyltetrahydropterin). Lower values were obtained (23 and 19%) when the mutant enzyme was assayed with the natural cofactor ((6R)-tetrahydrobiopterin) and different concentrations of l-phenylalanine. The enzyme kinetic studies of the V388M mutant protein revealed that this enzyme was a kinetic variant form of hPAH with a reduced affinity for l-phenylalanine and for the natural cofactor ((6R)-tetrahydrobiopterin). The residual activities determined for the V388M form of hPAH were compatible with the phenotype presented by the PKU patients harboring the V388M mutation in the PAH gene.