Viktor Kožich

Jitka Sokolova

Veronika Klatovska

Jakub Krijt

Miroslav Janošík

Karel Jelínek

Jan P Kraus

Jitka Sokolová

Veronika Klatovská

synthase mutations: effect of mutation topology on folding and activity

Keywords: Homocysteine, cystathionine beta-synthase deficiency, homocystinuria, folding, misfolding, topology, mutations, activity, E.coli, expression CBS, cystathionine beta-synthase PAGE, polyacrylamide gel electrophoresis PLP, pyridoxal 5´-phosphate SDS, sodium dodecylsulfate Homocysteine, cystathionine beta-synthase deficiency, homocystinuria, folding, misfolding, topology, mutations, activity, E.coli, expression

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Introduction

Cystathionine beta-synthase (CBS) deficiency (OMIM 236200, also known as homocystinuria due to CBS deficiency) is a well known multisystemic inborn error of metabolism with widely varying estimates of frequency. Although the numbers of patients with clinically ascertained CBS deficiency is rather low and the prevalence is only 1:330,000 worldwide [START_REF] Mudd | Disorders of transsulfuration[END_REF] the molecular epidemiological studies suggest an incidence of around 1:10,000 in several European populations [START_REF] Gaustadnes | Prevalence of congenital homocystinuria in Denmark[END_REF][START_REF] Janosik | Birth prevalence of homocystinuria in Central Europe: frequency and pathogenicity of mutation c.1105C>T (p.R369C) in the cystathionine beta-synthase gene[END_REF][START_REF] Refsum | Birth prevalence of homocystinuria[END_REF]. Classical forms of this disease manifest by combination of neurological, connective tissue and vascular involvement in childhood while milder forms of the disease may be diagnosed later in adulthood with only vascular manifestation. Severity of the untreated disease is determined mostly by the underlying genetic defect and patients with milder forms of CBS deficiency usually respond to pharmacological doses of pyridoxine by a marked decrease of their total homocysteine plasma levels.

More than 150 mutant alleles have been described in patients with CBS deficiency with a representation of all types of genetic variants (http://www.uchsc.edu/cbs/cbsdata/cbsmain.htm). Missense mutations are the most common variants described in the CBS gene representing 87% of the 753 analyzed patient-derived CBS alleles as of April 2010. In a previous study we have proposed that missense CBS mutant enzymes may misfold and that this behavior may be responsible for their pathogenicity (Janosik, et al., 2001b). This observation was later confirmed by other groups and also by expressing mutants in systems other than E.coli (for references see Table 1). The finding that misfolding and decreased activities of some mutant proteins may be corrected with the help of chemical chaperones further supports the role of misfolding in the pathogenesis of CBS deficiency [START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF]. Despite many years of research novel data on structural and biochemical properties of CBS mutant proteins are scattered in the literature and a comprehensive analysis of the folding-related findings is missing.

In this study, we examined systematically catalytic and folding-related structural properties of a panel of 27 mutants located in different functional domains of the CBS molecule. The panel of mutations contained among others the most prevalent mutant CBS alleles to represent well the molecular mechanisms relevant for the majority of patients suffering from CBS deficiency. Since purification of CBS mutant proteins for further studies is not universally achievable we decided to study properties of mutant proteins in crude E.coli extracts obviating thus any possible loss of mutants during the purification procedure. We studied the solubility of the mutant proteins in SDS, their oligomeric structure, activity, response to Sadenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) and changes of these parameters after expression at a decreased temperature. We subsequently examined whether the location of mutant residues in the three dimensional CBS structure in respect to solvent accessibility is a determinant of their properties. We show that mutations of residues buried in protein globule appear to have more severe effect on folding than those exposed to the solvent.

Methods

Nucleotide and codon numbering

Nucleotide numbering reflects the cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference GenBank sequence NM_000071.2, according to the Journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon number 1.

Molecular dynamics simulation

In silico analyses presented in this paper can be divided in two parts. The first part presents an analysis of mutant structures of catalytic domain of CBS and the second part presents analysis of CBS regulatory domain. X-ray structure of the active core of the CBS protein (PDB code 1JBQ; [START_REF] Meier | Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein[END_REF]) was utilized as the starting model for the catalytic domain. 1JBQ X-ray structure contains the first 413 residues of the protein and covers the whole catalytic domain. Firstly we optimized the native structure in program Modeller (http://salilab.org/modeller; [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF]) which combines empirical spatial restraints and CHARMM force field into an objective function and employs conjugate gradients and molecular dynamics with simulated annealing methods for energy optimization.

Model refinement level property of the Modeller procedure was set to very low. In the next step the optimized native structure was mutated in silico and mutant structures were optimized in the same way.

Although a model of the regulatory domain of human CBS (residues 415-543) has been published a few years ago [START_REF] Sen | Mapping peptides correlated with transmission of intrasteric inhibition and allosteric activation in human cystathionine beta-synthase[END_REF] the X-ray structure of this part of the CBS molecule is as yet unknown. We generated our own model of this portion of the wild type CBS using the comparative structure modeling. As the initial model we used X-ray structure of the conserved CBS domain of the protein TA0289 from Thermoplasma acidophilum (PDB code 1PVM; [START_REF] Proudfoot | Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain[END_REF]). Sequence of the CBS regulatory domain was aligned with the TA0289 sequence and coordinates of its native structure were derived from the 1PVM coordinates (data not shown). The native structure was then optimized and mutant structures were computed as in case of the active core. For the native as well as all computed mutated structures of both domains, the surface accessible area of amino acid residues was calculated and normalized by maximum accessible surface area for the corresponding residue type by the program Modeller [START_REF] Sali | Derivation of rules for comparative protein modeling from a database of protein structure alignments[END_REF]. A residue was considered exposed if its accessible surface area was larger than 40 A 2 and/or if its relative accessible surface was larger than 9%, while considered buried otherwise [START_REF] Mirkovic | Structure-based assessment of missense mutations in human BRCA1: implications for breast and ovarian cancer predisposition[END_REF].

Expression in E.coli

The wild type CBS and mutant constructs were derived from the pHCS3 expression plasmid [START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency[END_REF]. Seven mutant plasmids, namely the c.341C>T (p.A114V), c.442G>A (p.G148R), c.526G>A (p.E176K), c.1224-2A>C (r.1224_1358del; p.W409_G453del), c.1265C>T (p. P422L), c.1304T>C (p.I435T) and c.1397C>T (p.S466L) were prepared using the previously described procedure of replacing a fragment of wild-type CBS expression plasmid with the equivalent restriction fragment derived from the mutant patient-derived cDNA [START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency[END_REF]. The remaining mutations were introduced into the wild-type expression plasmid by help of the GeneTailor Site-directed mutagenesis kit (Invitrogen, Carlsbad, California) according to the manufacturer´s procedure. The sequences of mutagenic primers are given in Supp. Table S1. The authenticity of all constructs was verified by dideoxy sequencing using ALF sequencer (Amersham Pharmacia Biotech, Piscataway, New Jersey).

The expression plasmids were used to transform E.coli DH5α cells (Gibco BRL, Carlsbad, California). The cells were grown at 37°C or 18°C in SOB (Super Optimal Broth) media with 100 µg/ml ampicilin to OD 600 of ~0.5, expression of CBS was carried out in the presence of isopropyl β-D-1-thiogalactopyranoside (final concentration 0.33 mmol/l) at 37°C or 18°C for 3 hours or overnight, respectively. Each mutant protein was expressed in at least five independent experiments on separate days (i.e. 3 and 2 expression at 37°C and 18°C, respectively). Each series of mutant proteins contained also bacteria transformed with the pKK388.1 vector lacking the CBS insert as a negative control, and the wild type CBS that was used to normalize data.

Lysates from E.coli cell pellets were prepared by sonication in lysis buffer (50 mmol/l Tris-Cl pH 8, 10 mmol/l EDTA, 1 mmol/l DTT, 0.1% lysozyme) with the addition of commercial Protease Inhibitor Cocktail (Sigma Aldrich, St. Luis, Missouri) according to manufacturer´s protocol. A 50 uL aliquot of each sonicate was saved for the analysis of CBS in the particulate and non-particulate fraction, the remaining sonicate was centrifuged at 15,000 g for 30 minutes at 4° C, supernatant was aliquoted and stored for subsequent analyses at -85°C.

Protein concentration in the supernatant was determined by the method of Lowry with bovine serum albumin as a standard [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Optimizing extraction of aggregated CBS

We tested several methods that have been previously used by other authors to extract aggregated proteins-such as amyloid-from bacterial lysates [START_REF] Carra | HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells[END_REF].

Optimization was carried out with the wild type CBS and four mutant enzymes (p.A114V, p.V180A, p.K102N, p.I278T). We have compared the effects of the following denaturing reagents: a) 1.5% SDS in the presence of 0.1M beta-mercaptoethanol (without preincubation), b) 3% SDS in the presence of 0.1M beta-mercaptoethanol (without preincubation), c) 6M urea, 1.5% SDS and 0.1M beta-mercaptoethanol (1 hour incubation at room temperature) d) 6M urea/thiourea (v/v 2:1) containing 1.5% SDS and 0.1M beta-mercaptoethanol (1 hour incubation at room temperature) and e) 100% formic acid (30 minutes incubation at 37 ºC) F o r P e e r R e v i e w followed by a lyophilization and dissolution in denaturating solution containing 1.5% SDS and 0.1M beta-mercaptoethanol. After incubation of uncentrifuged sonicates with different denaturing reagents for the indicated time the samples were boiled for 10 minutes at 100 °C and analyzed by SDS-PAGE and western blotting as described below. These experiments revealed, that methods b) and e) provided a higher yield of immuno-reactive CBS signal compared to the other methods. Owing to a simplicity and a sufficient efficacy of method b) we subsequently used the extraction of insoluble CBS from pelets and supernatants by 3% SDS.

Analysis of particulate and non-particulate fraction

50 µL of sonicated cell extracts were centrifuged at 4°C at 15,000g for 30 minutes, the supernatants were saved and the pellets were resuspended in 50µL of denaturation buffer (50 mmol/l Tris-Cl pH8, 0.1 mol/l beta-mercaptoethanol, 3% SDS). To solubilize the CBS present in the samples 5 µL of either the resuspended particulate fraction or of the supernatant were mixed with 15 µL of the denaturation buffer and boiled at 100°C for 10 minutes, subsequently 4 µL of electrophoresis loading buffer supplemented with 0.1 mmol/l dithiothreitol were added and the solution was heated for 10 minutes at 100° prior to loading on the SDS-PAGE gel. Samples of the supernatant and particulate fraction of each mutant protein were loaded next to each other on the gel and subjected to electrophoresis and western blot analysis (see below the section Electrophoresis and Western Blot Analysis), each gel contained also the supernatant and particulate fraction of a negative control and of bacteria expressing wild type CBS. The sharply demarcated signal of CBS in each fraction was quantified by chemiluminescence after subtracting the background of the negative control. For each mutant the signal in the supernatant and the particulate fractions were added yielding thus the total CBS antigen of the respective mutant, this total signal was subsequently normalized to the total signal of wild type CBS enzyme after correction for the protein concentration.

Electrophoresis and Western Blot Analysis

Separation of CBS under denaturing conditions was carried out in 4-15% gradient native polyacrylamide gels (Criterion Precast Gel, BioRad) with standard Laemmli SDS-containing buffer system [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. For the analysis of CBS under native conditions cell lysates containing 10 µg of total protein were separated using the same precast polyacrylamide gels with standard Laemmli buffer system without SDS [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Proteins separated by either SDS-PAGE or native PAGE were transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, Massachusetts) using semi-dry blotting transfer technique. After transfer nonspecific binding sites were blocked by overnight incubation with 5% non-fat dry milk in 1 x phosphate-buffered saline solution (PBS), pH 7.0 and 0.2% Tween 20. The membrane was subsequently incubated with immunopurified anti-CBS antibody diluted 1:5000 in 1 x PBS containing 3% bovine serum albumin for 1 hour and after a series of washes with secondary anti-rabbit IgG antibody conjugates with HRP (Pierce, Rockford, Illinois) for 30 minutes. The secondary antibody was diluted 1:30 000 in 1 x PBS/ 0.2% Tween 20 containing 5% non-fat dry milk. After a second series of washes the signal was visualised using the West Pico Super Signal system (Pierce Biotechnology, Rockford, Illinois) followed by bioimaging system ChemiGenius-Q (Syngene Inc., Federick, Maryland) with cooled CCD camera. The amount of sharply demarcated fractions of tetramers and oligomers and of fuzzy high-molecular weight aggregates were quantified with Gene Tools software (Syngene Inc.) after the subtraction of background on an E.coli extract lacking CBS that was analyzed in each series.

Estimation of molecular weight by Ferguson plot

Molecular weight of CBS fractions with varying mobility was estimated using a modification of the method published by Ferguson [START_REF] Ferguson | Starch-Gel Electrophoresis--Application to the Classification of Pituitary Proteins and Polypeptides[END_REF][START_REF] Hedrick | Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis[END_REF]. The original method is based on electrophoresing the analysed protein together with markers in a series of polyacrylamide gels with increasing concentration of acrylamide. We have determined the relative mobilities normalised to the dye front (R f ) of commercial molecular mass markers (Blue Range, Pierce) ferritin (440 kDa), catalase (232kDa), urease (272 and 545 kDa), BSA (66 and 132 kDa) and egg albumin (45kDa) using 10 cm long 5%, 6%, 7%, 8%, 9% and 10% native polyacrylamide gels and Laemmli electophoresis buffers without SDS [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] after staining of the gels by Coomassie blue R The E.coli extracts containing mutant and wild type CBS were separated by the same series of polyacrylamide gels, the CBS fractions were visualized by western blotting and immunodetection as described above and the relative mobilities normalizes to the dye front have been determined.

The logarithms R f of each band of the standard proteins were plotted against the acrylamide gel concentrations and slope of regression line of each standard protein was calculated. The calibration line was derived as a function of the logarithms of negative slopes and the molecular weights of the standard. The negative slopes of all oligomeric forms of wild type CBS and of selected mutant proteins were determined by the same method and these values were used for calculation of molecular weight of the different CBS fractions. The bacterial extracts used for this analysis contained the wild type CBS, the p.K102N, p.S466L, p.E302K, p.G305R, p.G307S and p.R125Q variant proteins expressed at 18°C.

CBS enzymatic assay

The CBS activity in E.coli lysates was assayed by modification of the previously published procedure [START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency[END_REF]. The assay mixture contained 100 mM Tris-HCl (pH 8.6), 0.5 mg/mL BSA, 0.5 mM pyridoxal 5´-phosphate, 1mM dithiothreitol, 10 mM homocysteine, 10 mM 2,3,3-2 H-labeled serine (Cambridge Isotope Laboratories) and 27.5 µl of E.coli extract in lysis buffer containing 75 µg of bacterial protein. The assay mixture was incubated at 37°C for 1 hour in a total volume of 50 µL. For measurement of responsivity to adenosyl compounds the assay mixture contained either 0.5 mM AdoMet or AdoHcy. The activity was determined by the LC-MS/MS measurement of the product of enzyme reaction, 3,3-2 H-labeled cystathionine, using the commercially available kit for amino acid analysis (EZ:faast, Phenomenex, Torrance, USA) (Krijt et al, unpublished).

Statistical analyses

Means, standard deviations, and medians were determined using the spreadsheet Microsof Office Excel 2003 (Microsoft, Czech Republic), linear regression analysis was carried out using the same software.

Results

Selection of mutations and in silico analysis

In order to gain clinically as well as mechanistically relevant insight into the role of misfolding in CBS deficiency we first selected a series of naturally occurring CBS mutations for further laboratory studies. The set of nine most prevalent mutations having a frequency of at least 10 alleles in the CBS Mutation Database was selected; these included the p.A114V, p.R125Q, p.E144K, p.T191M, p.R266K, p.I278T, p.G307S, p.W409_G453del and p.D444N. This set was expanded by additional 18 less frequent mutations known to be localized in different domains of the CBS protein [START_REF] Meier | Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein[END_REF][START_REF] Meier | Structural insights into mutations of cystathionine beta-synthase[END_REF]. For the list of 1, location of the mutations in the CBS active core and in the modelled carboxyterminal domain is shown in Figure 1. The final series reflects well the molecular pathology of CBS deficiency as these 27 mutations are present on about 70% of all pathogenic CBS alleles known in humans to date.

Based on our previous work [START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF] we hypothesized that CBS mutations of residues buried in protein globule may have more severe effect on folding than those exposed to the solvent. After selecting the mutants we performed an in silico analysis with calculation of the solvent-accessible surface area. Based on previously published criteria we considered mutant residues with relative and absolute solvent-accessible surface area higher than 9% and/or 40 A 2 , respectively, as being solvent exposed while the rest of the mutations were considered being buried in the globule [START_REF] Mirkovic | Structure-based assessment of missense mutations in human BRCA1: implications for breast and ovarian cancer predisposition[END_REF]. Table 1 shows the predicted solvent accessibility and demonstrates that mutations belonging to these two classes are represented almost equally in the series of 27 variant proteins.

Total amount of CBS mutant proteins expressed in E.coli and their solubility

It has been shown previously that some CBS mutant proteins expressed in bacteria misassemble and aggregate [START_REF] De Franchis | Four novel mutations in the cystathionine beta-synthase gene: effect of a second linked mutation on the severity of the homocystinuric phenotype[END_REF]Gaustadnes, et al., 2002;Janosik, et al., 2001b;[START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF]Yamanishi, et al., 2006). It is conceivable that aggregated mutant CBS polypeptides will form inclusion bodies and that a substantial fraction of misfolded CBS proteins may thus escape detection if the commonly used supernatant fraction of bacterial extracts is utilized for western blotting analysis.

To test this assumption we first optimized a method for extracting water-soluble as well as aggregated and water-insoluble CBS from bacteria expressing wild type and four mutant enzymes (for details see the Methods section). Boiling in 3% SDS was subseqeuntly used for the entire series of 27 mutant proteins to determine the amount of CBS antigen in both the particulate (i.e. pellet after centrifugation) and non-particulate (i.e.supernatant) fractions of sonicated bacterial extracts. The sum of CBS signal in both fractions is referred to as the total SDS-soluble CBS antigen.

Using the SDS extraction we observed that all 27 mutant proteins were present in detectable amounts not only in the supernatants but more importantly also in the particulate fractions of the bacterial extracts. The presence of CBS antigen in both fractions is shown in Figure 2; however, this publication gel does not allow inferring on relative proportions of the mutant proteins in the supernatant and particulate fractions neither on their relation to the wild type enzyme (for details see the Methods section and legend to Figure 2). The signal of total SDSsoluble CBS antigen of the mutant proteins was generally somehow decreased with a median of 66% of the wild type CBS (see Supp. Table S2; gels used for quantification are not shown) although some variant proteins were present in increased quantities. These data suggest that compared to the wild type enzyme the majority of mutant CBS proteins are less stable in E.coli; however, we cannot exclude that some of them are also less soluble in 3% SDS.

Proportion of the SDS-soluble CBS antigen which is present in the particulate fraction indicates the propensity of the mutant protein to form high molecular weight aggregates and inclusion bodies. The particulate fraction of the wild type CBS lysates contained only 12% of antigen of the total SDS-soluble signal while more of the signal in the pellet was observed for the mutant proteins (median value was 18 % of the total SDS-soluble antigen with a range of 5 to 50%; see Supp. Table S2). These data lend support to our hypothesis that the mutant proteins in this study were in general more prone to formation of large molecular weight aggregates and/or inclusion bodies. This part of our study further indicates that the previously reported analyses of mutants CBS in the supernatants of centrifuged bacterial extracts (Kozich, et al., 1993) may have underestimated the steady state amounts of mutant proteins present in bacteria and that extraction by 3% SDS is needed to recover the insoluble aggregated molecular species.

Impaired quaternary structure of mutant proteins as a measure of their misfolding

The published data strongly support the hypothesis that CBS deficiency may be a conformational disease caused by misfolding of mutant enzymes. Detailed studies of folding, however, require protein purification which would be especially difficult to accomplish for the most severely affected CBS enzymes. Since misfolded polypeptide chains cannot be correctly assembled and tend to form aggregates we examined the quaternary structure of the mutant proteins in the non-particulate fraction of crude bacterial extracts as a surrogate marker of their folding status. To assess the oligomeric structure of the mutant CBS enzymes we used electrophoresis in gradient polyacrylamide gels under non-denaturing conditions followed by western blotting. Visual inspection of blots showed variable amounts of fuzzy and putatively misassembled CBS, and largely differing amounts of clearly demarcated CBS tetramers or higher order oligomers as shown in Figure 2. Subsequent quantitative analysis of the blots revealed that 88% of the total water-soluble antigen of the wild type CBS is present as tetramers/oligomers.

In contrast these correctly assembled fractions were substantially decreased in our set of mutant proteins (median tetrameric/oligomeric signal was only 31% of the total water-soluble CBS antigen, range 1%-92%; data are given in Supp. Table S2). Since the CBS antigen in the non-particulate fraction of mutant proteins was also decreased to a median 60% compared to the wild type the resulting net yield of tetramers/oligomers of the mutant proteins was severely reduced (median 12% of tetramers compared to the wild type CBS). There was no clear correlation between the location of mutant proteins in different domains and their misfolding with the exception of mutant proteins in the carboxyterminal domain that were mostly assembled correctly. In summary the above analyses demonstrated that a substantial proportion of CBS mutant proteins formed severely reduced amounts of correctly assembled tetramers; these findings add further support to the notion that misfolding is an important pathogenic mechanism in CBS deficiency.

Different mobility of CBS mutant proteins in native westerns

The above mentioned western blot analysis revealed slight but consistent differences in migration distance of the putative tetramers/oligomers of many mutant proteins in comparison to the wild type CBS tetramer (see Figure 2). These mobility alterations may have resulted from charge differences, conformational changes of tetramers or even different number of subunits in the differently migrating fractions. To explore the latter hypothesis we used the Ferguson plot to determine the molecular weight of four fractions seen for wild type enzyme, and the molecular weight of fast migrating (K102N, S466L), slow migrating (E302K, G305R and G307S) and fast/slow migrating (R125Q) mutant proteins. For results see Supp. Figure S1.

The apparent molecular weights of sharply demarcated and thus presumably correctly assembled oligomer fractions seen for the wild type enzyme were 177, 398, 512 and 724 kDa.

Considering the limitations of the technique employing crude extracts with subsequent western blotting and keeping in mind that CBS is known to form tetramers, the results suggest that in our electrophoretic system the wild type CBS is present predominantly in the form of tetramers, and in small amounts also as octamers, dodecamers and hexadecamers. Two fractions of the R125Q mutant protein were predicted to be composed of tetramers and octamers, respectively. There was only a small difference in slopes and thus in estimated molecular weights of the remaining fast (162 and 166 kDa) and slow (199)(200)(201)(202)(203)(204)(205)(206)(207)(208)(209)(210)(211)(212)(213) migrating mutant proteins as demonstrated in Supp. Figure S1. Consequently, the estimated number of CBS subunits ranged between 2.7 and 3.5 per fraction which is congruent with tetrameric composition of these fractions. In summary, the results of Ferguson plot do not support a hypothesis that different mobility of mutant proteins compared to wild type CBS is caused by changes in the number of enzyme subunits. Thus the observed varying mobility of CBS mutant proteins results most likely from subtle changes in the shape and/or charge of the assembled tetramers.

Catalytic activity of mutant proteins

In order to enable measurements of low residual activities of mutant proteins we firstly developed a sensitive method for the analysis of the CBS reaction product -cystathionine.

The radioactive substrate used in previous studies -14 C labeled serine-was replaced by 2,3,3-2 H-labeled serine and the amount of 3,3-2 H-labeled cystathionine produced was determined using LC-MS/MS (Krijt et al, unpublished). Using this technique, we were able to measure reproducibly activities as low as 0.3 nmol cystathionine/hour/mg of protein, which represents about 0.2% of the average wild type CBS activity in E.coli extracts.

Using the sensitive new assay we observed an extreme variation of activities ranging from 0 to 245% of the wild type enzyme with a median activity of 3% of the wild type CBS (for details see Table 2). Only three mutant proteins-p.G305R, p.T262R and p.N228K-did not exhibit any measurable residual activity above 0.3 nmol/mg/hour while the remaining 24 mutant proteins had above-threshold activity in at least two of the three independent expression experiments. The activities determined in the present study are for the majority of mutant proteins congruent with the previously published values listed in Table 1. In contrast to effects on folding location of the mutant proteins appears to play a more important role in affecting their catalytic activity. There was a tendency for mutations located in the active site, heme binding pocket, and elsewhere in the active core to exhibit rather low activities while mutations inside or on the surface of the first CBS domain and at the dimer-dimer interface were in many cases as active as the wild type enzyme (for details see Table 2). These data suggest that mutations in different domains of the enzyme may have substantially variable impact on its catalytic activity and that they may not affect folding and activity simultaneously.

Effect of AdoMet and AdoHcy on the catalytic activity of mutant proteins

To get an insight into the possible pathophysiological and therapeutic implications of AdoHcy and AdoMet in CBS deficiency we studied the effect of these two compounds on the activity of mutant proteins in non-particulate fractions of bacterial extracts. Blood concentration of AdoHcy is increased in patients with CBS deficiency (Orendac, et al., 2004) due to its reverse synthesis from homocysteine and adenosine with the help of AdoHcy hydrolase, an enzyme which can be in vitro blocked with different inhibitors. AdoMet is an allosteric activator of CBS that can possibly stimulate the residual activity of mutant proteins and that can be administered as a drug in the form of tosylate.

Activity of the wild type CBS enzyme increased 3.9x and 1.3x in the presence of 0.5mM AdoMet and 0.5mM AdoHcy, respectively (see Table 2). Since activation of CBS by AdoHcy

has not yet been reported we explored whether it could not have been caused by contamination of this compound by AdoMet. However, LC-MS/MS analysis showed that the AdoHcy standard contained less than 1% of AdoMet indicating that the 1.3x increase in wild type CBS activity in crude bacterial extracts can be attributed to AdoHcy itself.

We classified without formal statistical testing the response of mutant proteins to AdoMet and AdoHcy in this simple screening system as follows: clear activation similar to the wild type enzyme, clear inhibition, or absence of expected activation. About one half of the mutant enzymes in the panel exhibited an unusual response to either one of the adenosyl compounds: the p.E176K was inhibited by AdoMet, the p.E302K was inhibited by AdoHcy, and the p.H65R, p.T191M and p.C165Y appeared to be inhibited by both adenosyl compounds. In addition, mutations p.E302K, p.W409_G453del, p.I435T, p.L539S, p.E144K, p.G148R, p.G307S, p.I278T and p.S466L were not activated by AdoMet to an extent that was usually observed for the wild type CBS (see Table 2). These data show that responses of about half of mutant proteins to AdoMet and AdoHcy may differ substantially from those of the wild type CBS. The relevance of these findings for regulation of the activity of mutant proteins and possibly for treatment of CBS deficiency is further explored in the Discussion section. Low temperature during expression has been shown to enhance folding of various mutant protein proteins that are prone to misfolding. To examine whether any of the CBS mutations under study may be rescued by more folding-permissive conditions we expressed all 27 mutant proteins at 18°C. Compared to expression at 37°C the lower temperature has not dramatically changed the total amount of SDS-soluble mutant CBS enzymes and only slightly decreased the proportion of signal in the particulate fraction (median of 18% at 37°C versus 15% of total signal at 18°C; see Supp. Tables S2 andS3). However, the median amount of correctly folded oligomers in the water-soluble non-particulate fraction detected on native-PAGE increased about 2-times from 31% of total signal at 37°C to 68% of total signal at 18°C (Supp. Table S2 andS3). Several mutant proteins responded to low expression temperature with a dramatic (i.e. more than approximately 3-fold) increase of the proportion of tetramers compared to the proportion of tetramers/oligomers of the wild type CBS, namely the p.R266K, p.R369C, p.R125Q, p.P78R, p.V180A and p.I435T. However, this folding promoting effect of low temperature was not universal and some mutant proteins produced conversely less tetramer. All these observations indicate that for some mutant proteins more folding-permissive conditions facilitate the attainment of correct tertiary structure which subsequently increases the assembly of tetramers.

Effect of low temperature on properties of mutant proteins

In addition, the low expression temperature increased the median catalytic activity of mutant proteins from 3% of wild type at 37°C to a median of 13% of wild type at 18°C (see Table 2).

The mutant proteins p.R266K, p.R125Q, p.R369C, p.V180A and p.P78R exhibited more than approximately 2-fold increase in activity relative to the wild type which correlated well with the increased amount of tetramer. These five mutant proteins may be considered true folding mutants as their residual activity directly correlates with the amount of tetramer. In summary, the lower expression temperature substantially increased activity of five mutant proteins further supporting the hypothesis that deficient CBS activity may be in some instances caused by misfolding that may be alleviated by folding permissive conditions.

Solvent exposure of mutant residues as a determinant of folding and activity

We hypothesized that topology of mutations in respect to solvent exposure/buriedness may have an impact on folding and assembly of mutant CBS molecules. Therefore we compared in the next step the typical properties of CBS enzymes carrying buried versus solvent accessible mutations by comparing median values in Table 2 and in Supp. Table S2 andS3 identical amounts of total SDS-soluble antigen. However, the propensity of buried mutations to correctly fold and assemble is impaired as clearly demonstrated by the lower proportion of tetrameric/oligomeric fractions in the total CBS antigen in native gels with a median of only 14% of total antigen for buried mutations versus 36% for the solvent accessible ones. More importantly the buried mutations are about 23-times less active with median activity of only 0.4% of the wild type CBS versus 9.4% of the wild type enzyme for solvent exposed mutations. In addition, the buried mutations are not activated by AdoMet (the median activation of buried mutations of 1.0x contrasts with the 2.5x activation of solvent exposed mutation) and seem to be inhibited by AdoHcy (median activation of buried mutations is 0.8x while it is 1.3x for solvent exposed mutations). These differences between buried and solvent exposed mutations appear to be further augmented by the expression at 18°C. In summary these comparisons strongly suggest that buried mutations may lead to much severe impairment of CBS structure and function than the solvent exposed ones.

Expression of several mutant proteins at 18°C resulted in an increase in their catalytic activity as well as in the amount of tetramers/oligomers; we therefore proposed that these variant proteins may be considered true folding mutations. To evaluate this phenomenon in a more systematic way we have plotted the normalized activity of mutant proteins against the normalized amount of tetramers/oligomers using the combined data from five independent expression experiments. Supp. Figure S2 demonstrates that among the solvent exposed mutations there are indeed examples of such increase of activities mirroring the increased amounts of tetramers (e.g. p.A114V, p.H65R, p.K102N, p.P78R, p.R125Q and p.V180A).

However, changes in amounts of tetramers of buried mutations were not usually accompanied by corresponding differences in activities. That led us to speculate that the solvent exposure of mutations may be indeed an important determinant of their propensity to misfold and to lose the catalytic activity. We have next performed a regression analysis in which we combined data for all buried and all solvent exposed mutant proteins, respectively. This analysis has demonstrated that the activity of solvent exposed mutations correlates quite strongly with the amount of oligomers (r 2 = 0.53; see Figure 3,Panel B) and suggests that for this type of mutations enhanced folding achieved by e.g. low temperature or chaperones may result in increased residual activity. In contrast the mutations buried in the enzyme globule did not exhibit such properties as there was only a weak correlation between the activity and the amount of oligomers (r 2 =0.11; see Figure 3, panel A). The above described regression

Discussion

This paper focused on testing in a systematic way the hypothesis that misfolding plays an important role in CBS deficiency. To examine the hypothesis we selected a representative set of mutants located in different functional domains of the CBS molecule. Several lines of evidence suggest that misfolding is an important pathogenic mechanism in CBS deficiency:

(i) the amount of SDS-soluble mutant CBS polypeptides in particulate fraction was increased by 50% compared to the wild type possibly due to inclusion body formation, (ii) the net yield of mutant tetramers/oligomers in the non-particulate water-soluble fractions was severely reduced to only 12% of the wild type enzyme, and (iii) more folding permissive conditions (i.e. expression at 18°C) led to a doubling of tetramer amounts and partial restoration of activity. The mutant proteins studied in our paper represent about 70% of known patient derived CBS alleles, and 18 of these 27 mutations clearly result into moderate to severe misfolding of the CBS enzyme (i.e. formation of less than 25% of tetramer of the wild type). This is an important finding which is relevant to 362 of 753 mutant alleles deposited in the CBS Mutation Database as of April 2010; in other words, about one half of all known chromosomes affected by pathogenic mutations code for mutant polypeptides that misfold and misassemble. Taken together all these data suggest that abnormal folding is an important and common molecular mechanism in CBS deficiency.

An important finding of our study is the more important role of solvent accessibility of mutations rather than their location in enzyme domains in predicting properties of mutant enzymes. In general, buried mutations exhibited much more profound detrimental effect on enzyme folding and activity compared to the solvent accessible mutations. Appearance of sufficient amounts of tetramers in native gels did not predict enzyme activity for the buried mutants, however, increased tetramer formation in surface mutants elicited by low expression temperature led to an increase in residual activity. The plot of activity versus amount of tetramer (see Figures 3 andSupp In this study we observed clearly abnormal response to AdoMet and AdoHcy for about one half of mutant proteins: (i) several previously characterized mutants in the COOH-terminal regulatory region were hyperactive and were not further activated by AdoMet [START_REF] Dawson | Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria[END_REF][START_REF] Kluijtmans | The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment[END_REF][START_REF] Kluijtmans | Defective cystathionine beta-synthase regulation by Sadenosylmethionine in a partially pyridoxine responsive homocystinuria patient[END_REF]; these mutants were previously shown to have similar conformation to that of the activated wild type CBS with bound AdoMet, (ii) in contrast, another class of mutant proteins with low activity was either not stimulated or was even inhibited by AdoMet, and (iii) several mutant proteins were inhibited by AdoHcy. It has been shown previously that whole blood or plasma concentrations of AdoHcy are grossly elevated and that the AdoMet levels are either normal or increased in patients with CBS deficiency [START_REF] Heil | DNA methylation status is not impaired in treated cystathionine beta-synthase (CBS) deficient patients[END_REF][START_REF] Orendac | Homocystinuria due to cystathionine beta-synthase deficiency: novel biochemical findings and treatment efficacy[END_REF]. It is at present unknown whether the disturbed AdoMet and AdoHcy metabolism in CBS deficiency may modulate the residual activity of mutant enzymes and whether these observations may have any therapeutic implications.

The major limitation of our work relates to the different intracellular milieu of prokaryotic and eukaryotic cells, and especially to the differences in folding machineries and in protein surveillance/degradation systems. Therefore the conclusions from our study have to be taken with care and have to be validated by analyses of mutant proteins that will be expressed in eukaryotic cells or obtained from tissues of animal models of CBS deficiency. Despite these limitations, utility of the simple bacterial expression systems has been appreciated for their ability to demonstrate the inherent predisposition of other mutant enzymes such as phenylalanine hydroxyls towards aberrant folding and misassembly (Waters, 2003).

The above described observations may lead to the development of new strategies for treating patients with CBS deficiency. The most important implication is the potential of chaperones to rescue to a varying extent the misfolding and decreased activity of a substantial proportion of mutant proteins. We have shown previously in a selected set of mutant proteins that chemical chaperones may restore biological function of CBS mutants [START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF]. In addition, we examined the effect of six chemical chaperones or CBS ligands on a set of 27 mutant proteins presented in this paper and we have observed an effect of some of these compounds (Kopecka, et al., in press ). However, the practical utility of chemical chaperones for the treatment of misfolding disorders is modest and a more promising modality would be the use of a specific pharmacological CBS chaperone that awaits its discovery. The second group of treatment options implied theoretically from our present study are the AdoHcy hydrolase inhibitors that may decrease the concentrations of AdoHcy and alleviate thus inhibitory effect of AdoHcy on some mutant proteins, or the administration of AdoMet tosylate to stimulate the residual activity of other mutant CBS enzymes. AdoMet tosylate in doses of several hundreds of milligrams per day has been used in treatment of liver disease, myopathies and depression without reports on adverse effects [START_REF] Mato | Biosynthesis of S-adenosylmethionin[END_REF]. Whether or not chaperones and adenosyl compounds will be of any use in patients with CBS deficiency and will impove quality of their lives is presently unknown and needs to be explored in the future. ECE: tetramer, 0-5.82%WT activity [START_REF] Katsushima | Expression study of mutant cystathionine beta-synthase found in Japanese patients with homocystinuria[END_REF][START_REF] Orendae | Identification and functional analysis of two novel mutations in the CBS gene in Polish patients with homocystinuria[END_REF][START_REF] Urreizti | Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations[END_REF] [PLP] : 10x of WT [START_REF] Chen | Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response[END_REF] p.E144K c.430 G>A 0.10 0.20 B 12 ECE: <1%WT activity [START_REF] Dawson | Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria[END_REF]Janosik, et al., 2001b) p.C165Y c.494 G>A 1.85 3.00 B 8 ECE: 0-3%WT activity [START_REF] Gordon | Mutational analysis of the cystathionine beta-synthase gene: a splicing mutation, two missense mutations and an insertion in patients with homocystinuria. Mutations in brief no. 120[END_REF][START_REF] Kluijtmans | The molecular basis of homocystinuria due to cystathionin beta-synthase deficiency in Dutch homocystinuria patients[END_REF][START_REF] Kluijtmans | The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment[END_REF] Other locations in active core p.N228K c.684 C>G/A 0.06 0.10 B 4 ECE: aggregates, 0% WT activity (Gaustadnes, et al., 2002) Page 1.4x of WT [START_REF] Chen | Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response[END_REF]; ECE: aggregates, 0-2%WT activity (Janosik, et al., 2001b;[START_REF] Kluijtmans | The molecular basis of homocystinuria due to cystathionin beta-synthase deficiency in Dutch homocystinuria patients[END_REF][START_REF] Kluijtmans | The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment[END_REF][START_REF] Kozich | Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency[END_REF]) SCE: WT activity, tetramer, <2%-5% WT activity, K m [PLP] : 0.7x of WT [START_REF] Chen | Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response[END_REF][START_REF] Kruger | Cystathionine beta-synthase deficiency in Georgia (USA): correlation of clinical and biochemical phenotype with genotype[END_REF][START_REF] Shan | Mutations in the regulatory domain of cystathionine beta synthase can functionally suppress patient-derived mutations in cis[END_REF][START_REF] Singh | Chemical chaperone rescue of mutant human cystathionine beta-synthase[END_REF]; MLE: ~2% WT activity (Wang, et al [START_REF] Marble | Characterization of a cystathionine beta-synthase allele with three mutations in cis in a patient with B6 nonresponsive homocystinuria[END_REF][START_REF] Sebastio | The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations[END_REF] p.T191M (Janosik, et al., 2001b), 46.3-50%WT activity (de Franchis, et al., 1999;[START_REF] Sebastio | The molecular basis of homocystinuria due to cystathionine beta-synthase deficiency in Italian families, and report of four novel mutations[END_REF] p [START_REF] Kluijtmans | The molecular basis of homocystinuria due to cystathionin beta-synthase deficiency in Dutch homocystinuria patients[END_REF][START_REF] Kluijtmans | The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment[END_REF] 62x of WT [START_REF] Evande | Alleviation of intrasteric inhibition by the pathogenic activation domain mutation, D444N, in human cystathionine betasynthase[END_REF]; ECE: 96-100% WT activity [START_REF] Kluijtmans | The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment[END_REF][START_REF] Kluijtmans | Defective cystathionine beta-synthase regulation by Sadenosylmethionine in a partially pyridoxine responsive homocystinuria patient[END_REF] F o r P e e r R e v i e w [START_REF] Maclean | High homocysteine and thrombosis without connective tissue disorders are associated with a novel class of cystathionine beta-synthase (CBS) mutations[END_REF]; CHFE: ~300% WT acivity [START_REF] Maclean | High homocysteine and thrombosis without connective tissue disorders are associated with a novel class of cystathionine beta-synthase (CBS) mutations[END_REF]; MLE: tetramer, ~400% WT activity [START_REF] Gupta | Cystathionine beta-synthase p.S466L mutation causes hyperhomocysteinemia in mice[END_REF] [START_REF] Meier | Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein[END_REF][START_REF] Meier | Structural insights into mutations of cystathionine beta-synthase[END_REF]. Nucleotide numbering reflects the cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference GenBank sequence NM_000071.

  . We have observed similar stability of these two classes of mutant proteins in E.coli as evidenced by

  solvent exposed CBS mutations may be more likely to respond to interventions aimed at correcting their misfolding.

  .

  FigureS2) shows a good correlation for solvent exposed mutants implying that enzyme activity of these genetic variants is determined in part by the and in part by the precise location of the mutant residue and the character of the change.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .Figure 3 .

 23 Figure 2. Amounts of mutant CBS antigen in fractions of bacterial extracts. Top part of

  plot. The slopes of regression lines obtained by plotting the relative mobilities (R f ) of markers against the gel concentrations in logarithmic scale were used to calculate the molecular weight of CBS fractions. The tables on the bottom show the values of calculated molecular weights and the deduced oligomeric structure of the four fractions of the wild type CBS enzyme (Panel A) and of six selected mutants (Panel B).

  of activity and amounts of tetramer. For each mutant data on activity in relation to the amount of tetramers/oligomers from five independent expression experiments are plotted; Ο and ∆ indicate expression at 37°C and 18°C, respectively. All values are normalized to the amounts of tetramers and activities of the wild type CBS, data are shown on a logarithmic scale. Panel A, buried mutations; panel B

29 of 42 John Wiley & Sons, Inc.

		Human Mutation
		PE: V max 0.8-1.4% of WT, K m [PLP] :
	p.I278T	F o r P e e r R e v i e w c.833 T>C 0.00 0.00 B 146

Table 1 .

 1 Panel of studied mutations. Mutations selected for this study are grouped according to their location in different domains of the CBS 3-dimensional structure

	)

Table 2 . Activities of mutant enzymes in non-particulate water-soluble fractions of bacterial extracts.

 2 Each mutant was expressed at 37°C and 18°C in three and two independent experiments, respectively. The numbers in the table represent means and standard deviations of activities determined by LC-MS/MS method with the exception of three bottom lines, which show medians of groups. The effect of AdoMet and AdoHcy on activities of mutants expressed at 37°C was determined in at least 2 experiments, effect on mutants expressed at 18°C was examined only in selected cases. n.d., not detected (activity lower than the limit of quantifications, i.e. 0.3 nmol cystathionine/mg protein/hour); B, buried mutations; S, solvent exposed mutations; N/A, not analyzed

	Page 32 of 42

2, according to the Journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon number 1. Solvent accessibility has been calculated using the program Modeller, mutations are classified based on predicted solvent accessibility as solvent exposed (S, accessible surface area larger than 40A 2 and/or relative accessible surface area larger than 9%) or buried (B). Number of alleles carrying particular mutation has been taken from the CBS mutation database (http://www.uchsc.edu/cbs/cbsdata/cbsmain.htm) as of April 7, 2010. Data on properties of mutant enzymes has been extracted from literature, expression systems are abbreviated as follows: ECE, E.coli crude extracts; SCE, S.cerevisiae crude extracts; CHFE, Chinese hamster fibroblasts crude extracts; CHO, Chinese hamster ovary cells crude extract; MLE, mouse liver extracts; PE, purified enzymes.

Temperature of expression 37°C SDS-soluble CBS antigen in fractions Water-soluble CBS antigen in supernatant Solvent exposure Mutation Particulate fraction of the total antigen of each mutant Total antigen of mutants relative to wild type

					Tetrameric fraction	Tetramers of mutants
					of the total mutant	relative to tetramer of
					antigen	wild type
		B p.G148R	8.8 ± 2.7	52.8 ± 29.5	3.0 ± 4.3	1.4 ± 2.0
	Active site	B p.G305R	7.7 ± 3.5	65.8 ± 9.1	83.4 ± 8.3	98.0 ± 24.0
		B p.G307S	21.2 ± 2.3	67.9 ± 14.3	66.7 ± 9.3	111.7 ± 46.1
		S p.H65R	17.7 ± 1.0	91.2 ± 12.5	31.4 ± 16.6	23.1 ± 9.5
	Heme binding pocket	B p.T262R	16.0 ± 5.4	49.6 ± 3.8	2.4 ± 2.3	1.7 ± 1.4

John Wiley & Sons, Inc.Human Mutation

Acknowledgement

The authors would like to thank Ms.Alena Dutá and Kateřina Raková for technical help and Aleš Hnízda,MSc. and Jana Kopecká, MSc. for critical reading of the manuscript. This work was supported by the Wellcome Trust International Senior Research Fellowship in Biomedical Science in Central Europe (Reg. No. 070255/Z/03/Z), institutional support was provided by the Research Project from the Ministry of Education of the Czech Republic (Reg.

No. MSM0021620806).

John Wiley & Sons, Inc. S3. Amounts of mutant CBS antigen in fractions of bacterial extracts expressed at 18°C. The SDS-soluble antigen was determined by boiling the particulate and non-particulate fractions of bacterial extracts in 3% and by SDS-PAGE followed by western blotting; the signal was quantified by chemiluminescence; the percentual proportion of antigen in the pellets is shown in the first column, total CBS antigen normalized to the wild type CBS is given in the second column. The ability of mutants to fold and assemble was examined by electrophoresis of water soluble non-particulate fractions under non-denaturing conditions and western blotting; percentual proportion of tetramers of the total signal is shown in the third column, yield of tetramers/oligomers in comparison to the amount of wild type tetramers is shown in the fourth column. All values with the exception of last 3 lines are means and standard deviations from 2-3 determinations.

Human Mutation

F o r P e e r R e v i e w

n.d., not detected


Caroline Michot

Laurence Hubert

Michele Brivet

Linda De Meirleir

Vassili Valayannopoulos

Wolfgang Müller-Felber

Ramesh Venkateswaran

Helene Ogier De Baulny

Isabelle Desguerre

Cécilia Altuzarra

Angela ; Hübner

Michèle Brivet

Hélène Ogier

Elizabeth Thompson

Martin Smitka

Angela Huebner

Marie Husson

Rita Horvath

Patrick Chinnery

Frederic M Vaz

Arnold Munnich

Orly Elpeleg

Agnès Delahodde

Yves De Keyzer

Pascale De Lonlay
email: pascale.delonlay@nck.aphp.fr.

Prof Pascale

LPIN1 gene mutations: a major

Keywords: rhabdomyolysis, LPIN1, founder effect, intragenic deletion Rhabdomyolysis, LPIN1, intragenic deletion, founder effect

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Myoglobinuria is a rare condition resulting from the destruction of skeletal muscle fibers (rhabdomyolysis). Hereditary myoglobinurias have been ascribed to mitochondrial fatty acid ß-oxidation defects (FAO), mitochondrial respiratory chain (RC) deficiency and inborn errors of glycogenolysis [START_REF] Dubowitz | Proceedings of the 27th ENMC sponsored workshop on congenital muscular dystrophy. 22-24 April 1994, The Netherlands[END_REF][START_REF] Tein | Neonatal metabolic myopathies[END_REF][START_REF] Tonin | Metabolic causes of myoglobinuria[END_REF]. Metabolic investigations include plasma carnitine and acylcarnitine profiles, urinary organic acids analysis and in vitro studies of FAO in fresh lymphocytes or in cultured fibroblasts. When the latter are normal, common practice recommends performing a skeletal muscle biopsy for histological and enzymatic studies. Despite these investigations, the disease mechanism remains unknown in at least half of the patients [START_REF] Ohkuma | Clinical and genetic analysis of lipid storage myopathies[END_REF].

Recently, LPIN1 mutations (MIM *605518) have been reported as a novel cause of rhabdomyolysis [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF]. Lipin-1 is a 890 amino acid protein predominantly expressed in muscle and adipose tissue [START_REF] Donkor | Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns[END_REF][START_REF] Reue | Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism[END_REF][START_REF] Reue | The lipin protein family: dual roles in lipid biosynthesis and gene expression[END_REF], initially identified by positional cloning in the fatty liver dystrophy mouse (fld) [START_REF] Peterfy | Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin[END_REF]. It exhibits a dual role, as a phosphatidate phosphatase 1 (PAP) for triacylglycerol and phospholipid biosynthesis [START_REF] Donkor | Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns[END_REF][START_REF] Han | The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme[END_REF], and as a transcriptional co-activator through its association with PPARα and PGC-1α to regulate the expression of genes encoding FAO and RC enzymes [START_REF] Donkor | Adipose tissue lipin-1 expression is correlated with peroxisome proliferator-activated receptor alpha gene expression and insulin sensitivity in healthy young men[END_REF][START_REF] Finck | Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway[END_REF][START_REF] Reue | The lipin protein family: dual roles in lipid biosynthesis and gene expression[END_REF][START_REF] Sugden | PPAR control: it's SIRTainly as easy as PGC[END_REF]. Expression of LPIN1 is also required for adipocyte differenciation and function [START_REF] Phan | Biphasic expression of lipin suggests dual roles in adipocyte development[END_REF]. Lipin-1 contains two highly conserved domains, a N-LIP domain (residues 1-114) of unknown function, and a C-LIP domain (residues 673-830) which contains the canonical DXDXT motif for PAP activity and the LXXIL motif for nuclear receptor binding [START_REF] Reue | Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism[END_REF][START_REF] Reue | The lipin protein family: dual roles in lipid biosynthesis and gene expression[END_REF].

Studying a series of 29 cases of unexplained myoblobinuria, we found two LPIN1 mutations in 59% of the patients. Moreover, a prevalent intragenic deletion was observed, prompting to screen for this frequent LPIN1 deletion prior to other tests in the investigation of myoglobinuric patients.

MATERIALS AND METHODS.

Patients.

A total of 29 patients from 23 families (4 consanguineous families; 22 Eurocaucasians, 1 African, 5 Maghrebis and 1 Asian) were included in the study. Patients 1, 2 and 3 have been previously described [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF]. Inclusion criteria were: i) episode(s) of rhabdomyolysis, ii) since early infancy (< 5 years), iii) plasma creatine phosphokinase (CK) levels above 10 000 UI/L during bouts of myoglobinuria.

Metabolic investigations.

Extensive metabolic work-up excluded primary FAO disorders, carnitine palmitoyl transferase 2 (CPT2) deficiency, glycogen storage diseases and dystrophinopathies.

In patients 1, 3 and 4, serum leptin and adiponectin levels were determined using a commercial radioimmunoassay kit (RIA, LINCO Research Inc., Saint Louis, MI, USA).

Glucose metabolism was evaluated by HbA1c level and an oral glucose tolerance test (1.75 g/kg dextrose orally administrated, dosage of blood glucose and insulin at 0, 30, 60 and 120 min) in patients 1, 3 and 4.

Body composition measurement was assessed by dual-absorptiometry (Hologic 4500W instrument) in patients 3 and 4 [START_REF] Ellis | The reference child and adolescent models of body composition. A contemporary comparison[END_REF].

Phospholipids were assayed on muscle biopsies of patient 1 (previously described, [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF]) and patient 4 and on plasma samples of patients 1, 3, 4, by lipid extraction and HPLC-MS quantification, and compared to three control samples [START_REF] Valianpour | Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis[END_REF].

RC activities were measured spectrophotometrically in skeletal muscle and cultured skin fibroblasts [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF]. CPT2 activity was studied in skeletal muscle [START_REF] Isackson | Identification of 16 new disease-causing mutations in the CPT2 gene resulting in carnitine palmitoyltransferase II deficiency[END_REF]. Informed consent was obtained from the patients using a form approved by the Hospital Necker ethics board committee.

Genomic DNA and total RNA were obtained from peripheral blood leukocytes and/or cultured fibroblasts using standard procedures. cDNA synthesis was performed using High capacity cDNA reverse transcription kit (Applied Biosystems).

The coding sequence of the LPIN1 gene (GenBank NM_145693.1) was entirely determined from cDNAs or genomic DNA. Sequencing reactions were performed with the Big Dye Terminator Cycle Sequencing kit v.3.1 and analyzed on an ABI3100 sequencer (Applied Biosystems). Sequence analyses were performed using Seqscape software v2.5 (Applied Biosystems).

Parents' genotype was established to determine the allelic transmission of the LPIN1 mutations.

Mutations numbering is based on cDNA sequence, with +1 corresponding to the A of ATG translation initiation codon (codon 1) in the reference sequence.

For the diagnosis of the intragenic deletion, a genomic DNA fragment extending from exon 17 to exon 20 was amplified by long range PCR, using specific primers (5': GCTGCTGAGTCCCAGCAGCCTCTTCTCTGC 3'; 5': GTGGTCGACTACTTCACAGAGTCTCACATA 3') with 2.5 U LA-Takara DNA polymerase (TaKaRa Bio Inc. Japan) for 30 cycles (10 sec 94°C, 30 sec 65°C and 14 min at 68°C).

The precise localization of the deletion breakpoints was established by sequencing a genomic DNA fragment amplified with the primers (5': AAAGGTCTGGCACATCTTCTGTT 3' (intron 17) ; 5': AATCCCATTTAGC CCACCGACTCAG 3' (intron 19)), with Taq DNA polymerase (Roche) for 35 cycles (30 sec 95°C, 30 sec 65°C and 2 min at 72°C).

Haplotype studies were performed in all patients carrying the genomic deletion and in their parents. We used the intragenic polymorphic markers D2S328, located in intron 16, and D2S168, D2S2377, D2S2200, W437 and D2S2199 flanking the LPIN1 gene.

The presence of sequences known to participate in genomic rearrangements was searched for using bioinformatic tools (http://www.repeatmasker.org) as well as analysis of splicing consensus motifs and splicing enhancers/silencers (http://spliceport.cs.umd.edu/, http://genes.mit.edu/burgelab/rescue-ese/, http://rulai.cshl.edu/ tools/ESE2/).

The deleterious nature of the intragenic deletion on PAP activity was examined in a yeast complementation assay, using the ∆pah1 strain deleted for the yeast ortholog of the LPIN1 gene [START_REF] Han | The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme[END_REF]. LPIN1 cDNAs, normal and lacking exons 18 and 19, were obtained by RT-PCR and subcloned in the XhoI/BamHI sites of the BFG-I shuttle vector. The yeast ∆pah1 cells were transformed with the BFG-I plasmid encoding the normal human lipin-1 or the deleted human lipin-1 (lipin-1∆). After selection of yeast cells bearing plasmids, serial dilutions (1:10) of the cells were spotted onto YPD (complete medium, 2% glucose) and YPG (complete medium, 2% glycerol) and growth was scored after 3 days of incubation at 28 and 36°C.

RESULTS.

Molecular characterization of the patients.

More than half of our myoglobinuric patients (17/29) were homozygous or compound heterozygous for LPIN1 mutations or intragenic deletion (Table 1). These molecular abnormalities were confirmed both on cDNA and genomic DNA and not found in 250 control chromosomes. *: patients previously described [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF]. CK = creatine phosphokinase. Patients of the same family share a fused box for mutation description. The patients 3, 5 and 9 had a sibling who died in the same condition (bout of rhabdomyolysis). Thirteen different mutations were discovered scattered throughout the LPIN1 coding region, including 12 nonsense mutations (either direct at residues 19, 315, 388, 801 or as a frameshift consequence) and 1 intragenic deletion (Figure 1). 9/12 nonsense mutations lead to a predicted protein totally lacking the C-LIP domain, and the 3 remaining stop mutations are located within the C-LIP domain, but do not remove the DXDXT and LXXIL motifs. The last mutation, c.2295-866_2410-30del (p.Glu766_Ser838del), is an in-frame intragenic deletion, removing a large part (residues 766-838) of the C-LIP domain, but preserves both DXDXT and LXXIL motifs. Unexpectedly, this intragenic deletion was present in 8/17 patients, all Caucasians, who displayed an abnormal cDNA lacking exons 18 and 19. Long range PCR of their genomic DNA encompassing exons 17 to 20 generated an abnormal amplification product, approximately 2 kb shorter than that of controls. The breakpoints of the deletion were identical in all patients. The deletion was 1763 bp long and extended from intron 17 (nt +3377) to intron 18 (nt + 782, 30 bp upstream to exon 19), encompassing exon 18, but not exon 19.

In silico analysis of splicing enhancers and branch-point within the deleted sequence did not bring clues to understand the absence of exon 19 at the cDNA level. Similarly, no repeated elements susceptible to mediate a recurrent chromosomal rearrangement were detected by sequence analysis in the vicinity of the breakpoints. A common minimal haplotype (markers D2S328, D2S2200 and D2S2199) segregated with the intragenic deletion in the 8 children. Because all these patients were of Caucasian origin, a founder effect is highly probable.

De novo mutations were not observed in our series as all 26 parents were heterozygous for one LPIN1 mutation, except the symptomatic father of patients 11 and 12 who was homozygous due to his parent's consanguinity. Unaffected children were homozygous or heterozygous for the wild type allele.

The functional consequences of the absence of exons 18 and 19 in the LPIN1 mRNA were examined in a yeast complementation assay as previously reported [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF]. The ∆pah1 yeast strain was transformed by a plasmid encoding the human lipin-1 or its lipin-1∆ mutant. As described [START_REF] Santos-Rosa | The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth[END_REF], the ∆pah1 mutant strain grew more slowly than wild type cells (W303) at 28°C on glycerol and exhibited a temperaturesensitive phenotype at 36°C on glucose and glycerol (Figure 2). This phenotype was complemented by expressing the human LPIN1 cDNA, as cells recovered a growth rate comparable to wild type cells at both 28 and 36°C. In contrast, ∆pah1 cells expressing the human lipin-1∆ mutant (figure 2) did not grow on glycerol at 28°C and glucose at 36°C indicating that the c.2295-866_2410-30del mutation of LPIN1 alters lipin-1 activity and is of functional significance.

Clinical investigations of patients with LPIN1 mutations.

LPIN1 mutations were detected in patients of various ethnic origins: 12/22 Caucasian, 1/1 African, 1/1 Asian and 3/5 Maghrebi patients with a sex ratio M/F=0.89.

Patients underwent their first bout of rhabdomyolysis before five years (median age = 21 months). Bouts were almost invariably precipitated by febrile illnesses and occasionally by anaesthesia or fasting (Table 1). The number of acute episodes ranged from 1 to 10 per patient. Five patients died during myoglobinuric bouts, and three siblings (not included in the study) died from similar episodes. One sibling of patient 10 died at 3 weeks of life from pneumonia. He also presented with cardiomegaly and hepatomegaly at the time of his death.

Between episodes, all patients but one (patient 4) had normal physical examination including normal muscular testing. Their basal plasma CK levels were normal or subnormal as well. The oldest patient is now 23 years old. Patient 4 exhibited writing cramps and permanent myalgia increasing with effort and requiring wheel-chair. Abnormal muscular testing was noted. Another patient (patient 1) presented with recurrent episodes of severe atopic dermatitis.

Most heterozygous parents (23/25) were asymptomatic. The two moderately symptomatic heterozygous carriers (cramps or moderate myalgias) harboured two different point mutations (mother of patient 2, c.2401C>T; and mother of patient 3, c.1441+2T>C). The healthy sister of patient 3, harbouring the LPIN1 intragenic deletion, reported exercise-induced muscle pain. The father of patients 11 and 12, homozygous for his children's mutation, suffered of numerous bouts of myoglobinuria.

Metabolic investigations of patients with LPIN1 mutations.

All patients including the oldest one had normal plasma levels of total, LDL and HDL cholesterol, triglycerides, and lactate. Electromyography and abdominal and heart ultrasounds were also normal; no liver steatosis was noted. Brain MRI was normal in all patients but one (patient 4) who had an arachnoid cyst. Muscle histology was either normal or showed moderate abnormalities, including lipid inclusions, predominance of type I muscle fibers, atrophy of type II fibers and rarely subsarcolemmal aggregates of mitochondria with ragged-red fibers. Histochemical staining for cytochrome c oxidase (COX) activity and respiratory chain activities in skeletal muscle were normal in all patients but one : patient 2, whose muscle fiber staining revealed homogeneously weak COX activity (de [START_REF] De Lonlay-Debeney | Respiratory chain deficiency presenting as recurrent myoglobinuria in childhood[END_REF].

Although a complete analysis of lipid metabolism could not be performed for every patient, partial data were available for some of them. The father of patients 11 and 12 had partial CPT2 deficiency in skeletal muscle (38 mU/mg protein, controls 90 mU/mg protein), while CPT2 activity in fibroblasts and lymphocytes as well as CPT2 gene sequencing were normal (data not shown).

Analysis of phospholipids content in muscle tissues of patients 1 [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF] and 4 and in plasma of patients 1, 3 and 4 showed no difference in major phospholipid species compared to controls, including phosphatidic acid and lysophosphatidic acid (data not shown).

Average weight percentiles and normal fat distribution were found in all patients, confirmed by dualabsorptiometry in patients 3 and 4 (data not shown). Adiponectine and leptine plasma levels were normal in patients 1, 3 and 4, as well as oral glucose tolerance test and HbA1c level (data not shown).

DISCUSSION

Here, we report on a very high incidence of LPIN1 mutations in a large series of young patients with severe rhabdomyolysis. Because it accounted for 59% of cases in our study and 56% of tested families, LPIN1 mutations appear as the second cause of rhabdomyolysis of early-onset, after primary FAO defects as a whole. Moreover, since 8/17 mutated patients carried an intragenic deletion with identical breakpoints, on the background of a common haplotype, a founder effect of Caucasian origin is highly likely. The deleterious nature of this deletion, homozygous in some patients, was supported by yeast complementation assay.

The outcome of the disease was severe, as five patients died during a myoglobinuric episode, as well as three siblings. Episodes of myoglobinuria were precipitated by febrile illnesses and in a few cases by fasting. Various inflammatory inducers including lipopolysaccharides, Zymosan and proinflammatory cytokines have been recently shown to repress LPIN1 expression, leading to decreased expression of PPARα, PPARγ and genes involved in energy metabolism [START_REF] Feingold | Infection decreases fatty acid oxidation and nuclear hormone receptors in the diaphragm[END_REF][START_REF] Lu | LPS and proinflammatory cytokines decrease lipin-1 in mouse adipose tissue and 3T3-L1 adipocytes[END_REF][START_REF] Tsuchiya | A Jak2 inhibitor, AG490, reverses lipin-1 suppression by TNF-alpha in 3T3-L1 adipocytes[END_REF]. It is conceivable therefore that the acute-phase response induced by inflammation could cause dramatic alterations in lipid and lipoprotein metabolism [START_REF] Gabay | Acute-phase proteins and other systemic responses to inflammation[END_REF][START_REF] Kishimoto | Cytokine and murine coxsackievirus B3 myocarditis. Interleukin-2 suppressed myocarditis in the acute stage but enhanced the condition in the subsequent stage[END_REF] and eventually trigger episodes of myoglobinuria in lipin-1 deficient patients.

While lipodystrophy is a major feature in the natural Lpin1 mutant mouse strain fld [START_REF] Peterfy | Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin[END_REF][START_REF] Reue | Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism[END_REF][START_REF] Reue | Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene[END_REF], neither insulin resistance, nor dyslipidemic signs were observed in our lipin-1 deficient children. The explanation of the different phenotypes presented by lipin-1 deficient mice and humans remains largely unknown, as well as the physiopathology of the muscular symptoms in man. Skeletal muscle is one of the tissues expressing the highest levels of lipin-1, which may explain why muscle is particularly affected by the LPIN1 mutations in human. Because of the multiple roles played by lipin-1, several mechanisms leading to rhabdomyolysis, alone or combined, may be considered.

On the one hand, lipin-1 has an enzymatic PAP activity and is involved in glycerolipid biosynthesis. As it has been suggested [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF], lipin-1-related rhabdomyolysis could result from lyso-phosphatidate accumulation and the subsequent remodelling of membranes induced by phospholipid imbalances [START_REF] Farooqui | Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders[END_REF], as proposed in Barth syndrome [START_REF] Chicco | Role of cardiolipin alterations in mitochondrial dysfunction and disease[END_REF][START_REF] Mckenzie | Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients[END_REF]. The phospholipid content in skeletal muscle was abnormal in one previously described patient [START_REF] Zeharia | Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood[END_REF] but not in two others, one already reported and another. Similarly, plasma phospholipid concentrations were in the normal range in three tested patients. The preliminary data available at this step do not support an important role of phospholipid imbalance alone as a trigger for massive rhabdomyolysis, also it could not be excluded. Moreover, there is growing evidence showing that triacylglycerol (TG) synthesis is coupled to fatty acid oxidation [START_REF] Liu | Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1[END_REF]. Fatty acids may have to be incorporated into TG and then turned over before they can be efficiently oxidized. Thus, low rates of TG synthesis resulting from mutations of LPIN1 could produce a defect in fatty acid oxidation. In this view, it should be noted that the ∆pah1 yeast complementation assay reflects PAP activity. Also bouts of rhabdomyolysis may result from the association of various functional defects linked to the PAP activity deficiency, but further studies are clearly required to determine how alterations in these pathways participate to the disease. On the other hand, lipin-1 also plays a role of co-activator in PPARα and PGC-1α-mediated transcription, which stimulates the expression of genes related to mitochondrial energy pathways [START_REF] Donkor | Adipose tissue lipin-1 expression is correlated with peroxisome proliferator-activated receptor alpha gene expression and insulin sensitivity in healthy young men[END_REF][START_REF] Finck | Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway[END_REF][START_REF] Higashida | Potential role of lipin-1 in exercise-induced mitochondrial biogenesis[END_REF]. Besides, lipin-1 has been clearly demonstrated to act in exercise-induced adaptation and oxidative gene expression in muscle [START_REF] Higashida | Potential role of lipin-1 in exercise-induced mitochondrial biogenesis[END_REF]. Alteration of this co-activator activity could down-regulate simultaneously several metabolic pathways important for the energetic supply of muscle cells [START_REF] Phan | Biphasic expression of lipin suggests dual roles in adipocyte development[END_REF]. The isolated metabolic data currently available in our patients (abnormal oxidative phosphorylation activity, CPT2 activity defect) might be compatible with the hypothesis of an energy-dependent muscle disease, but need to be confirmed in more extensive studies. On this line, it should be noted that defective growth on glycerol medium of the ∆pah1 yeast strain suggests a global impairment of aerobic/oxidative metabolism, even though it does not assess lipin-1 transcriptional activity.

Further investigations will help understand the links between deficiency of each of lipin-1 activities and the disease.

In conclusion, lipin-1 deficiency should be regarded as a major cause of severe myoglobinuria in early childhood. Since 59% of our cohort display deleterious LPIN1 mutations, it may prompt clinicians to include the screening for LPIN1 mutations at early stage of the metabolic work up of myoglobinurias, after exclusion of FAO defects, but certainly prior to muscle biopsy. The high frequency of the intragenic LPIN1 deletion in the Caucasian patients provides a valuable screening test to diagnose these patients, before sequencing the full LPIN1 sequence. Finally, we believe that metabolic investigations of severe rhabdomyolysis in young patients should include successively i) metabolic investigation of FAO, ii) research of the intragenic LPIN1 deletion in blood samples by long range PCR, iii) full coding sequence analysis of LPIN1 gene in blood, iv) only if all these results are normal, proceed to skeletal muscle biopsy.

Figure 1 .

 1 Figure 1. Schematic diagram of the mutations reported on the lipin-1 protein. The highly conserved domains are shown as shaded boxes. Mutations (as amino acid changes) are indicated with vertical lines.

Figure 2 .

 2 Figure 2. Functional analysis of LPIN1 intragenic deletion by functional complementation of the yeast pah1 null mutant. Growth of the yeast wild type strain (W303) and ∆pah1 derivatives on either glucose (YPD) or glycerol (YPG) medium at 28 (left panels) and 36°C (right panels). ∆pah1 was transformed with the human wild-type LPIN1 (LPIN1) or mutant (lpin1∆) cDNAs. The five spots for each experiment correspond to decreasing dilutions of transformed yeast cells.

Table 1 . Clinical and molecular characterization of patients with LPIN1 mutations.

 1

<LPIN1 mutations and severe rhabdomyolysis of early childhood> 5

			Patient		Clinical presentation	Mutation
	N° Age	S e x	Ethnic origin	Con nity san gui	(CK: UI/L)	Myolysis episodes (triggering event)	Nucleotide	Amino Acid
	1*	5y	M Mauritania Yes	200 000	2 at 27 and 30m (fever)	c.192+2T>C c.192+2T>C	p.Cys30LeufsX3 p.Cys30LeufsX3
	2*	died at 6y	F	France	No	100-150 000	4 between 15m and 6y (fever)	c.2401C>T c.2295-866_2410-30del	p.Arg801X p.Glu766_Ser838del
	3 * 10y F	France	No	120 000	3 between 18m and 10y (fever)	c.1441+2T>C c.2295-866_2410-30del	p.Asn417LysfsX22 p.Glu766_Ser838del
	4 5 18m M Belgium 10y M France F No No o 120 000 900 000	2 at 16m and 8y (fever) 1 at 1y	c.377_380dup c.2295-866_2410-30del c.2295-866_2410-30del c.2295-866_2410-30del	p.Met128GlnfsX45 p.Glu766_Ser838del p.Glu766_Ser838del p.Glu766_Ser838del
	6	3y	F	France-Asia	No	300-500 000 r	2 at 2y and 2,5y (fever)	c.944C>G c.1162C>T	p.Ser315X p.Arg388X
	7 8 9	died at 2.5y died at 4y died <1y	M Belgium F Belgium M Egypt	No No No	57 000 > 20 000 P > 20 000 e 1 at 2,5y (fever) 1 at 4y (fasting) 1 at 5m (fasting) e r	c.2295-866_2410-30del c.2295-866_2410-30del c.2513+1G>A c.2513+1G>A	p.Glu766_Ser838del p.Glu766_Ser838del p.Asp804ValfsX6 p.Asp804ValfsX6
	10 11 12 13 14 23y F died at 4y F England 6y M North africa 8y F North africa 8y F Germany France	No Yes Yes No No	10-450 000 50 000 300 000 1 000 000 > 100 000	2 at 14m and 4y (fever) 1 at 5y (general anesthesia) 1 at 2y (fasting) 10 between 2 and 8y (fever) 5 between 4 and 16 y (fever, fasting with local anaesthetic) R e c.2295-866_2410-30del c.921delT c.1162C>T / c.1162C>T c.2295-866_2410-30del c.946_952del v i e w	p.Glu766_Ser838del p.Gln308ArgfsX36 p.Arg388X p.Arg388X p.Glu766_Ser838del p.Asp316LeufsX26
	15 15y M France	No	> 100 000	7 between 4 and 16 y (fever, fasting with local anaesthetic)	c.1259delC c.2513+1G>A	p.Pro420LeufsX39 p.Asp804ValfsX6
							6 between 4 and 16 y
	16 18y M France	No	> 100 000	(fever, fasting with local
							anaesthetic)
	17	4y	F Germany	No	450 000	2 at 3y and 4 y	c.2253_2254del c.57C>A	p.Leu752AlafsX17 p.Tyr19X

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ACKNOWLEDGMENTS

We thank Norma Romero, Vincent Frochot, Jean-Louis Bresson and Jean-Jacques Robert for their help in metabolic investigations, and Marc Jeanpierre for genetic analyses.

This work was supported by AFM (grants 13864 and 13988), Fondation de l'Avenir (grant 09071), Fondation pour la Recherche Médicale (fellowship CM), the Prinses Beatrix Fonds (n° WAR05-0126), the Barth syndrome foundation. Patrick Chinnery is Wellcome Trust Senior Fellow in Clinical Science. No conflict of interests has to be disclosed, nor commercial considerations.