Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1

A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC–mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography–MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway.
Introduction
During the past decades, widespread application of fluoroaromatic compounds as agrochemicals and pharmaceuticals has lead to an increased occurrence of environmental contaminants containing fluorine (Key et al. 1997). Fluorinated compounds are rare in nature (Harper and O’Hagan 1999). The stability of the carbon–fluorine bond (116 kcal/mol in CH3F, compared to 81 kcal/mol for the carbon-chlorine bond in CH3Cl) makes most fluorine-containing compounds much more resistant to biodegradation than their unsubstituted analogs (Key et al. 1997). Furthermore, the van der Waals radius of fluorine is small (1.47 Å, in between that of a hydrogen and an oxygen). Yet, the electronegativity of fluorine causes strong polarization of C–F bonds, and fluorine substituents may be involved in biological interactions (Howard et al. 1996).
Regardless of the recalcitrance of most fluoroaromatics to biodegradation, several bacterial cultures have been described that aerobically degrade fluorobenzoic acids (Oltmanns et al. 1989; Engesser et al. 1980; Harper and Blakley 1971; Schlomann et al. 1990). Research on bacterial fluorophenol degradation has been limited to studies with whole cells, cell extracts, or purified enzymes from Rhodococcus species that were obtained by enrichment with other aromatic compounds as a growth substrate (Boersma et al. 1998, 2001; Bondar et al. 1998, 1999; Finkelstein et al. 2000). The fluorobenzene-degrading organism Rhizobiales F11 could grow on 4-fluorophenol (4-FP), but information on the pathway of 4-FP metabolism is lacking.
Cometabolic degradation of difluorophenols and trifluorophenols by several Rhodococcus species is initiated by a phenol hydroxylase that catalyzes ortho-hydroxylation, resulting in the formation of the respective fluorocatechol, which is then cleaved by an intradiol dioxygenase to produce fluoromuconate (Bondar et al. 1998). Conversion of 4-FP by whole cells of the phenol-degrading organism Rhodococcus opacus 1cp resulted in the formation of 4-fluorocatechol, 1,2,3-trihydroxy-5-fluorobenzene, and fluoromuconates (Finkelstein et al. 2000). Yeasts and fungi that are able to cometabolically transform fluorinated phenols have also been described. Whole cells of Exophiala jeanselmei transformed 4-FP into 4-fluorocatechol and 3-fluoromuconate (Boersma et al. 1998). Penicillium frequentans metabolized monofluorophenols in the presence of glucose or phenol. The metabolism of meta-or para-fluorophenols yielded the corresponding catechol and 4-carboxymethylenebut-2-en-4-olide (Hofrichter and Schreibner 1993; Hofrichter et al. 1994). None of these organisms could utilize fluorophenols as a growth substrate.
To our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. In the present paper, we describe the isolation and characterization of a bacterial strain growing on 4-FP as the sole source of carbon and energy. Based on the identification of several intermediates, a metabolic route for the degradation of 4-FP by this strain is proposed.
Materials and methods
Media and growth conditions NB medium contained 8 g of Nutrient Broth (Difco) per liter. Mineral salts medium (MM) contained per liter 5.3 g Na2HPO4 12H2O, 1.4 g KH2PO4, 0.2 g MgSO4 7H2O, 0.5 g (NH4)2SO4, 5 ml trace metals solution (Janssen et al. 1985), and 10 mg of yeast extract (Difco Laboratories). When required, the solid medium was obtained by adding 15 g/l of Difco agar. Strain IF1 was grown at 30°C on a rotary shaker (180 rpm). Cultures were grown in 100-ml flasks filled to 25% of their volume and were closed with Teflon-lined screw caps. E. coli cells were grown in Luria–Bertani medium (LB) at 37°C on a rotary shaker.
Enrichment and isolation of 4-FP-degrading cultures A variety of soil samples, collected from different sites in The Netherlands that are contaminated with halogenated aliphatic compounds (such as monochlorobenzene, hexachlorobenzene, and trichloropropane), were used as the initial inoculum for the 4-FP enrichments. The soil samples were used to inoculate flasks containing 30 ml of sterile minimal salts medium and 1 mM of 4-FP, supplied in the liquid phase as the sole carbon and energy source. The cultures were incubated at room temperature on a rotary shaker (150 rpm), and 40% of the suspension was transferred to a new flask containing fresh medium every 15 days. During this time, growth (optical density at 600 nm) and liberation of fluoride were monitored. Samples of the enrichment culture were periodically spread onto minimal salts agar plates containing 1 mM 4-FP and onto NB plates as soon as growth on 4-FP was established. Pure cultures were obtained by repetitive streaking onto solid MM containing 4-FP and tested separately for growth on 1 mM 4-FP liquid medium. Growth and fluoride release were again monitored to verify 4-FP degradation. Strains capable of 4-FP degradation as a sole source of carbon and energy were used for further experiments. Strain IF1 was deposited at Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, under accession number NCCB 100218.
Sequencing of the 16S rRNA gene For cloning of the 16S ribosomal ribonucleic acid (rRNA) gene, a single colony of strain IF1 was directly used for polymerase chain reaction (PCR) amplification. The primers 63f (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387r (5′-GGGCGGWGTGTACAAGGC-3′; Marchesi et al. 1998) were used for PCR amplification. The PCR reaction mixture (50 μl) contained Taq PCR buffer, 2.5 mM MgCl2, 20 pmol of each appropriate primer, 200 mM of each deoxyribonucleotide triphosphate, 1 U Taq DNA polymerase, and biomass of strain IF1. The PCR conditions were 94°C for 10 min followed by 1 min at 95°C, 1 min at 55°C, 1.5 min at 72°C, and 5 min at 72°C. The resulting fragments were cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into E. coli TOP10 cells. The transformed cells were plated on LB plates containing 0.5 mg/ml of ampicillin, and the positive colonies were used for plasmid isolation and sequencing.
Phylogenetic analysis Alignments of the 16S rRNA gene were made using sequences downloaded from the Ribosomal database project II (RDP II; Cole et al. 2005), after searching for nearest neighbors using the sequence match tool. Further searches were conducted using BLAST and FASTA of the European Molecular Biology Laboratory (EMBL) database for 16S rRNA gene sequences that are closely related to the 16S rRNA gene of strain IF1 but not available from the RDP II. Alignments were subsequently made using the profile alignment option in ClustalX (Thompson et al. 1997), refined using BioEdit ver 7.0.5.2 (Hall 1999), and subsequently parsed through Gblock (Castresana 2000) to remove ambiguously aligned sections and increase the robustness of the data. Phylogenetic trees were determined using the neighbor-joining (NJ) method. Evolutionary distances for the global tree were calculated using the Kimura-2-parameter model with a transition/transversion ratio of 2 (Fig. 1). Further trees were constructed in Phylip version 3.6a3 (J. Felsenstein), using maximum parsimony and maximum likelihood methods. All NJ trees were tested statistically by means of bootstrap analysis.
Fig. 1Phylogenetic tree of the 16S rRNA gene sequence of strain IF1. The scale bar represents 0.1 fixed mutation per site. Bootstrap values were derived from 1,000 analyses. The DNA sequences were aligned using ClustalX, and the tree was constructed by the neighbor-joining program from a similarity matrix of pairwise comparisons made by using the Kimura-2-parameter algorithm. Tree a shows the global position of the strain IF1 while tree b shows the Bayesian tree and the more precise relationship of strain IF1 to other members of the genus Arthrobacter. The values in parenthesis in this tree are the posterior probabilities for the nodes that show where IF1 resides
Preparation of cell extracts Cells were grown in MM with 4 mM 4-FP, harvested by centrifugation at 5,000 × g for 15 min, washed with ice-cold TD buffer (0.1 M Tris–HCl, pH 7.5, and 0.1 M 1,4-dithiothreitol), and stored at −20°C until further use. The frozen cells were thawed, resuspended in TD buffer, and incubated with lysozyme (50 mg/ml) for 1 h at 30°C. A French press was used to disrupt the cells, and the crude extracts were centrifuged at 10,000 × g for 15 min to separate the soluble from the particulate fraction. The supernatant was used for further experiments. Protein concentrations were determined with the Biorad protein assay kit.
Enzyme assays Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were measured spectrophotometrically at 260 and 375 nm, respectively. The reaction mixtures contained 0.1 mM catechol, TD buffer, and cell-free extract (0.1 mg of protein) in a final volume of 1 ml. 4-Fluorocatechol 1,2-dioxygenase was measured as catechol 1,2-dioxygenase but with 4-fluorocatechol instead of catechol as the substrate.
The 4-FP monooxygenase activity was measured spectrophotometrically by following the consumption of NADH at 340 nm in a reaction mixture containing cell-free extract (0.1 mg protein), 0.1 mM 4-FP, 0.1 mM NADH, and buffer in a total volume of 1 ml. The observed rates were corrected for substrate-independent NADH oxidation.
Hydroxymuconic semialdehyde dehydrogenase was measured at 375 nm. Reaction mixtures contained (in a final volume of 1 ml) about 0.05 mM freshly prepared hydroxymuconic semialdehyde, TD buffer, 0.1 mM NAD, and cell-free extract (0.1 mg of protein). Hydroxymuconic semialdehyde was obtained by incubation of catechol with the cell-free extract of Pseudomonas putida mt-2 as described previously (Mars et al. 1998).
Hydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between 230 and 330 nm in a reaction mixture of 1 ml final volume containing 0.1 mM hydroquinone, TD buffer, and cell-free extract (0.1 mg of protein). Hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. Reaction mixtures contained 1 mM of substrate, MM, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml.
Oxygen uptake measurements Strain IF1 was grown with glucose or 4-FP as the sole carbon source and harvested by centrifugation at 6,000 × g for 15 min at 4°C. Cells were resuspended in MM, and O2 consumption was measured with a fiber optic oxygen sensor (MOPS-1, ProSense BV, Hannover, Germany). All reactions were performed in a stirred vessel at room temperature. The reaction mixtures contained 1 mM of substrate (4-FP, hydroquinone, 4-fluorocatechol, hydroxyquinol, or catechol), MM, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml.
Analytical methods For capillary gas chromatography (GC), 4 ml samples were extracted with 1 ml of diethyl ether. A model 6890 gas chromatograph (Hewlett-Packard) equipped with a flame ionization detector and a HP-5 column (Agilent 19091J-413, 30 m × 0.25 mm × 0.25 μm) were used for the analysis. GC-mass spectrometry (MS) analysis was carried out with a model 5973 mass selective detector (Hewlett-Packard) coupled to a HP 6890 series injector and a HP1 column (Agilent 19091Z-433; 30 m × 0.25 mm × 0.25 μm). High-performance liquid chromatography (HPLC) was carried out using a Chrompack C18 column (10 cm × 5 mm) connected to a Jasco UV-1575 detector, which monitored absorbance at 214 and 254 nm, and operated with Jasco PU-980 pumps and a Jasco AS-1555 sampler. The mobile phase was water/acetonitrile (70:30), 5 mM potassium phosphate (pH = 3), and 340 mg/l sodium dodecyl sulfate, and the flow rate was 1 ml/min.
Liquid chromatography (LC)-MS was carried out with a ZMD Micromass spectrometer, equipped with a XTerra MS, Symmetry Shield C8 column (4.6 × 150 mm), a Waters 996 photodiode array detector, and a Waters 2690 separations module. Samples of 20 μl were analyzed, and compounds were isocratically eluted at a flow rate of 1 ml/min with a solution of water/acetonitrile (80:20) and 10 mM formic acid.
Concentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model 96-09, Thermo Russell, Scotland). Fresh sodium fluoride standards were prepared for calibration curves.
Chemicals 4-FP (>98%) was obtained from Sigma-Aldrich (Steinheim, Germany). All chemicals were of the highest purity grade available (Sigma-Aldrich; Acros Organics, Geel, Belgium). The compound 4-fluorocatechol was kindly provided by Dr. Erik de Vries. Purity of 4-FP, 4-fluorocatechol, and hydroquinone was checked by HPLC.
Nucleotide sequence accession numbers The 16S rRNA sequence of strain IF1 was deposited at GenBank with the accession no. DQ425093.
Results
Isolation of a 4-FP-degrading bacterium To obtain a bacterial culture that is able to use 4-FP as carbon and energy source for growth, enrichments were performed and followed over time. Two months of selective enrichment by repeated transfer of samples from a culture that displayed growth on 4-FP to fresh 4-FP containing media resulted in a microbial consortium that was capable of growth on 4-FP as a sole source of carbon and energy. Samples of the consortium were repeatedly plated onto NB agar plates and MM agar plates containing 4-FP. This procedure resulted in the isolation of three pure strains named IF1, IF2, and IF3. The strains were restreaked on MM supplemented with 4-FP and inoculated in liquid cultures with 1 mM 4-FP. Fluoride liberation was observed for all three strains but not in control incubations to which no bacterial inoculum was added. All the three strains showed growth, as monitored at 600 nm, and thus were able to use 4-FP as a sole carbon and energy source. After 5 days of incubation in liquid culture supplied with 1 mM 4-FP, strain IF1 reached 100% fluoride release and an optical density (OD) of 0.119 at 600 nm. Strain IF2 released 76% of the fluorine and reached an OD600 of 0.09, while strain IF3 reached an OD of 0.076 and released 54% of the theoretical amount of fluoride ions. When a mixed culture of the three strains in liquid media was used, an OD600 of 0.217 and 100% fluorine release were measured after 4 days. Apparently, the mixed culture contained, even after prolonged adaptation, organisms with varying efficiencies of 4-FP utilization. Because strain IF1 showed the highest degradation rates combined with stoichiometric release of fluoride, this organism was chosen for further studies.
Microbiological characterization Strain IF1 is a Gram-positive motile bacterium with a rhodococcus lifecycle. The optimal temperature for growth is 30°C. The 16S rRNA gene sequence was determined. Initial searches against the RDP II and EMBL rRNA databases resulted in very close associations with members of the genus Arthrobacter, the closest of which were the quinaldine-degrading strain Arthrobacter sp. KA1-1 (Overhage et al. 2005) and the 4-nitroguaiacol-degrading actinobacterium Arthrobacter nitroguajacolicus sp. nov (Kotouckova et al. 2004). The 16S rRNA gene sequences of these organisms were greater than 99% identical to IF1’s. The phylogenetic analysis places the isolates in the phylum Actinobacteria and the genus Arthrobacter (Fig. 1). The subtree that were obtained shows the detailed relationship of isolate IF1 to other members of the genus Arthrobacter. The topologies of all the trees that were obtained during statistical analysis were in agreement and clearly placed this isolate in the genus Arthrobacter (data not shown). Furthermore, the change in form during the growth cycle between rod and coccus is typical of the lifecycle of the Arthrobacter genus.
Catabolic activities of Arthrobacter sp. strain IF1 To study the degradation potential of strain IF1, a range of organic compounds were tested as growth substrates. Cells were inoculated into MM, and different organic substrates were added at a concentration of 1 mM. After 72-h incubation growth, substrate disappearance and halide release were measured.
Growth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, 4-fluorocinnamic acid, and 4-nitrophenol were used as substrates. The organism did not grow on 2-fluorophenol, 3-fluorophenol, 4-chlorophenol, 4-bromophenol, 4-iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on 2-bromoethanol. The fact that strain IF1 is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on 4-fluorocatechol indicates that 4-fluorocatechol is not the most likely intermediate in the 4-FP pathway, although toxic effects could also play a role.
To test the range of 4-FP concentrations tolerated by strain IF1, experiments were conducted in sealed flasks with 4-FP at concentrations of 1 to 7 mM as a sole carbon and energy source. Control assays without 4-FP showed no growth or release of fluoride, and sterile controls showed no abiotic loss of 4-FP. Between 1 and 4 mM 4-FP, the substrate was completely consumed, stoichiometric release of fluoride was observed, and biomass increased linearly with the amount of 4-FP added (Fig. 2a). This indicates that the degradation of 4-FP by strain IF1 does not give large amounts of fluorinated dead-end products. Concentrations of 4-FP above 4 mM caused a toxic effect on the growth of IF1 (Fig. 2b). The use of 5 mM 4-FP promoted growth, but a longer lag time was observed. At 7 mM 4-FP, no growth occurred even after 15 days of incubation.
Fig. 2Fluoride release and biomass formation by strain IF1 with different concentrations of 4-FP. a Stoichiometric fluoride release. Symbols: squares, optical density at 600 nm; circles, fluoride concentration. b Growth in the presence of different levels of 4-FP. The initial concentrations used were: triangles, 1 mM 4-FP; circles, 4 mM 4-FP; diamonds, 5 mM 4-FP; squares, 7 mM 4-FP
Growth on 4-FP and formation of metabolites A batch culture of strain IF1 supplied with 1 mM 4-FP as the only source of carbon and energy was monitored in time. Growth was accompanied by an increase in biomass, decrease in 4-FP, and formation of fluoride (Fig. 3). After 120 h, there was complete conversion of 1 mM 4-FP, and 1 mM fluoride was formed, indicating that there was no transient accumulation of fluorinated intermediates over the whole growth period. When cells grown on 4-FP were incubated in the presence of the iron chelator 2,2′-dipyridyl, no 4-FP degradation occurred, and no fluoride was released in the medium. This indicates that initial or further enzymes involved in 4-FP metabolism require ferrous ions for activity.
Fig. 3Biodegradation of 4-FP and fluoride liberation in a batch culture of strain IF1. The carbon source used is 1 mM 4-FP. Symbols: triangles, 4-FP concentration; diamonds, optical density; squares, fluoride concentration
To isolate intermediates of the degradation of 4-FP, samples from a batch culture containing 4 mM 4-FP were taken at appropriate intervals and analyzed by GC, HPLC, and LC-MS. Metabolites that were detected were numbered in order of time of appearance in the culture. GC analysis indicated that at least four metabolites were formed and degraded over time (Table 1). Metabolite I, the earliest product that was observed, had the same retention time as an authentic hydroquinone standard and metabolite II had the same retention time as a standard of hydroxyquinol. GC-MS analysis showed that the mass spectrum of metabolite I was indeed similar to that of the hydroquinone standard, with a molecular ion peak at 110 and at 55 m/z. Metabolite II gave a molecular ion peak at 126 and 63 m/z, which is typical for hydroxyquinol, and the spectrum coincided with that of a standard. Metabolites V and VI were detected by GC but did not ionize in GC-MS under the conditions tested and were not identified.
Table 1Retention times in GC and HPLC analyses of metabolic intermediates formed by cells of strain IF1 growing on 4-FP and of some authentic standardsCompoundGC retention time (min)HPLC retention time (min)am/z of observed fragment ions detected by GC-MSMetabolitesI20.71.3110, 81, 54II24.31.1126, 80, 52III–c0.75–IV–2.6–V16.5––VI19.4––VII–3.1b159, 80dAuthentic standards4-FP15.73112, 83, 574-Fluorocatechol14.42128, 82, 51Hydroquinone20.71.3110, 81, 54Hydroxyquinol24.31.1126, 80, 52Catechol19.1––aElution followed at 254 nmbAnalyzed after separation on a Symmetry Shield C8 column (4.6 mm × 150 mm) as described in “Materials and methods” for LC-MS analysisc– Not determineddLC-MS analysis
When the supernatant of IF1 cells growing on 4 mM 4-FP were subjected to HPLC analysis, four peaks were detected (Table 1). Metabolite I had the same retention time as the hydroquinone standard, and metabolite II coeluted with an authentic standard of hydroxyquinol. Hydroquinone was detected in culture supernatants by HPLC between 7.5 and 51 h (Fig. 4). LC-MS analysis of the extracted samples of the supernatant revealed the presence of a compound (VII) of which the negative ion spectrum shows a molecular ion peak at 159 m/z and a peak at 80 m/z, which are expected for the negative ionization of 3-oxoadipate.
Fig. 4Accumulation of hydroquinone during growth of strain IF1 on 4 mM 4-FP. The optical density (diamonds), 4-FP concentration (squares), and hydroquinone concentration (x marks) are indicated. Hydroquinone was identified by GC-MS and quantified by GC-FID
The above results suggest that 4-FP is initially converted to hydroquinone. The most likely enzyme involved in such a conversion is a 4-FP monooxygenase.
Enzyme activities To test inducibility of enzymes involved in 4-FP degradation, cells of Arthrobacter sp. strain IF1 were grown on glucose or 4-FP, washed, and tested for oxygen consumption in the presence of different substrates. Washed suspensions of cells grown on 4-FP rapidly oxidized 4-FP, hydroquinone, and 1,2,4-benzenetriol (hydroxyquinol) without a lag. Catechol and 4-fluorocatechol did not stimulate oxygen uptake (Table 2). This result indicates that 4-fluorocatechol and catechol are not likely intermediates and that hydroquinone and hydroxyquinol are. Glucose-grown cells did not oxidize any of the aromatic substrates tested with the exception of hydroxyquinol. This observation implies that hydroxyquinol may be converted by a constitutive oxygenase, whereas most other enzymes seem inducible. Cells grown with hydroxyquinol showed complete conversion of benzenetriol in the presence or absence of 2,2′-dipyridyl, indicating that the putative hydroxyquinol oxygenase does not require ferrous ions to be active and that oxidation beyond this compound is not inhibited by the chelator. The formation of hydroquinone, which can be readily oxidized by 4-FP-grown cells, is in agreement with the involvement in 4-FP degradation of a 4-FP monooxygenase that is induced by 4-FP.
Table 2Substrates oxidized by 4-FP and glucose grown cells of Arthrobacter sp. strain IF1aAssay substrateO2 uptake (μmol/min/mg of protein) by whole cells after growth onFluorophenolGlucose4-Fluorophenol0.356<0.001Hydroquinone0.313<0.001Hydroxyquinol0.1950.1564-Fluorocatechol<0.001<0.001Catechol<0.001<0.001a Oxygen consumption was measured with an oxygen sensor as described in Materials and Methods. All substrate were used at a concentration of 1 mM
To study in more detail the degradation pathway of 4-FP by strain IF1, cell-free extracts of IF1 grown on 4-FP were investigated for the presence of several enzyme activities (Table 3). When catechol 1,2- and 2,3-dioxygenases were tested for, no activity was found. Furthermore, no activity was detectable for 4-fluorocatechol dioxygenase. Consequently, the pathway does not proceed through catechol or a substituted catechol. When cell extracts were assayed for 4-FP monooxygenase, activity could be found in the presence of NADH but not in the presence of NADPH as the reducing cosubstrate. This observation indicates that the cells contain an NADH-dependent 4-FP monooxygenase activity. HPLC assays of incubations of cell extracts with 4-FP showed that substrate conversion was complete. The formation of the putative 4-FP monooxygenase was induced by 4-FP because no activity was found in extracts of cells grown in the presence of glucose.
Table 3Specific activities of enzymes in crude extracts of strain IF1 pregrown in 4-FP or glucoseAssay substrateEnzyme testedSpecific activity (U/mg of protein) after growth on4-FluorophenolGlucoseCatechol1,2-Dioxygenase<0.01<0.012,3-Dioxygenase<0.01<0.014-FluorocatecholDioxygenase<0.01<0.014-FluorophenolMonooxygenase0.12<0.01HydroquinoneDioxygenase<0.01<0.01Monooxygenase0.313<0.01Hydroxymuconic semialdehydeDehydrogenase<0.01<0.01HydroxyquinolDioxygenase0.1950.156
When hydroquinone was used as an assay substrate, its typical spectrophotometric peak at 288 nm was not replaced by a peak absorbing at 290 to 320 nm, which would have pointed to formation of hydroxymuconic semialdehyde. Thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. The latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain IF1, indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of 4-FP. These results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the 4-FP pathway by strain IF1.
When cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with HPLC. When the reaction was monitored spectrophotometrically, the peak at 287 nm, typical of hydroxyquinol, was substituted by a peak at 245 nm, typical of maleylacetate. This indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into 3-oxoadipate.
Discussion
This study reports the isolation and characterization of Arthrobacter strain IF1, an organism capable of growth with 4-FP as a sole source of carbon and energy. The biodegradation of 4-FP by strain IF1 was analyzed by GC, GC-MS, HPLC, LC-MS, oxygen uptake experiments, and measurements of enzymatic activities. A metabolic pathway is proposed on the basis of the results of this study (steps 1 and 2, ortho-cleavage) and by analogy with other systems (Fig. 5). We suggest that the degradation of 4-FP in strain IF1 starts with the conversion by a monooxygenase to benzoquinone, which is immediately reduced to hydroquinone. Hydroquinone then undergoes a further hydroxylation to form hydroxyquinol. This benzenetriol is the ring fission substrate, which is transformed by ortho-cleavage and yields maleylacetate, which is further converted to 3-oxoadipate.
Fig. 5Proposed pathway for 4-fluorophenol degradation by Arthrobacter sp. strain IF1. Hydroquinone and hydroquinole were identified as intermediates by GC-MS
Up to now, two main metabolic routes for the aerobic degradation of halogenated phenols have been described in the literature. In bacteria that degrade mono- and dichlorophenols, a degradation pathway is usually observed in which the substituted phenol is hydroxylated to the corresponding catechol, which is followed by ortho-cleavage of the aromatic ring (Haggblom 1992; Hollender et al. 1997; Wieser et al. 1994). On the other hand, biodegradation pathways in which the substituted phenol is converted via hydroquinone to maleylacetate have been found, mainly in organisms that grow on polyhalophenols or 4-nitrophenol (Kiyohara et al. 1992; Xun et al. 1992; Kadiyala and Spain 1998; Nordin et al. 2005; Perry and Zylstra 2007). Most studies have focused on the organisms and pathways that involve degradation via catechols.
A clear indication for the first step in the 4-FP catabolic pathway of strain IF1 was obtained by mass spectroscopic analysis of culture supernatants, which indicated the formation of hydroquinone as an early nonfluorinated intermediate. This result, in combination with the enzyme assays and oxygen uptake experiments, suggests the involvement of an inducible 4-FP monooxygenase that is NADH dependent, although the first expected product of this reaction, i.e., benzoquinone, was not detected. Monooxygenation of an aromatic substrate carrying an electron-withdrawing halogen group or a nitro-substituent can result in simultaneous hydroxylation and dehalogenation or nitrite removal. This has, for example, been described for the conversion of tetrafluoro-p-hydroxybenzoate by para-hydroxybenzoate hydroxylase (Husain et al. 1980) and the oxidation of 2,4-6-trifluorophenol by a monooxygenase from Ralstonia eutropha JMP134 (Xun and Webster 2004). The quinone that is produced is chemically reduced by NADH to a hydroxyquinol, leading to a net stoichiometry of two NADH oxidized per halide or nitrite that is released (Fig. 5). As in our case, no transient accumulation of a benzoquinone is usually observed, indicating that benzoquinone reduction is rapid. Other examples of halophenol degradation with formation of hydroquinone derivatives are the degradation of trichlorophenol to 2,6-dichlorohydroquinone by a strain of Pseudomonas pickettii (Kiyohara et al. 1992), the degradation of pentachlorophenol by a Sphingobium sp. through hydroxylation by a flavoprotein monooxygenase (Xun and Orser 1991), and the degradation of 4-chlorophenol by an Arthrobacter sp. (Bae et al. 1996).
Our results further indicate that hydroquinone is not the ring fission substrate because no enzymatic activities for hydroquinone dioxygenase or hydroxymuconic semialdehyde dehydrogenase were found. Instead, upon conversion of hydroquinone by strain IF1, we observed the formation of a transient metabolite that was identified as hydroxyquinol, indicating that a second hydroxylation takes place before ring fission. The production of hydroxyquinol could be due to the action of a separate hydroquinone monooxygenase, or it could be caused by a second hydroxylation step by the 4-FP monooxygenase. Conversion of hydroquinone to hydroxyquinol before ring fission was also suggested for strains Arthrobacter that degrade 4-nitrophenol (Perry and Zylstra 2007) and 4-chlorophenol (Nordin et al. 2005). Ring fission of hydroxyquinol would produce maleylacetate, which we did not detect as an intermediate in 4-FP degradation, but a transient metabolite was found with mass properties identical to those of 3-oxoadipate, which is expected to be the next intermediate in hydroxyquinol degradation.
Most cometabolic transformations of fluorophenols described in the literature involve the initial action of a phenol hydroxylase that results in the formation of fluorocatechols, which are subsequently transformed into fluoromuconates through a catechol 1,2-dioxygenase. The conversion usually proceeds with lactonization and elimination of fluoride (Boersma et al. 2001; Bondar et al. 1999; Finkelstein et al. 2000; Boersma et al. 1998; Bondar et al. 1998). Our results suggest that Arthrobacter strain IF1, which grows on 4-FP, possesses a new pathway for the degradation of 4-FP that involves immediate defluorination by a monooxygenase. Strain IF1 avoids the accumulation of possible toxic metabolites such as 3- or 4-fluorocatechol by this initial dehalogenation.

4-fluorophenol biodegradation fluoride hydroquinone monooxygenase


Foramen magnum meningiomas: detailed surgical approaches and technical aspects at Lariboisière Hospital and review of the literature

Foramen magnum meningiomas are challenging tumors, requiring special considerations because of the vicinity of the medulla oblongata, the lower cranial nerves, and the vertebral artery. After detailing the relevant anatomy of the foramen magnum area, we will explain our classification system based on the compartment of development, the dural insertion, and the relation to the vertebral artery. The compartment of development is most of the time intradural and less frequently extradural or both intraextradural. Intradurally, foramen magnum meningiomas are classified posterior, lateral, and anterior if their insertion is, respectively, posterior to the dentate ligament, anterior to the dentate ligament, and anterior to the dentate ligament with extension over the midline. This classification system helps to define the best surgical approach and the lateral extent of drilling needed and anticipate the relation with the lower cranial nerves. In our department, three basic surgical approaches were used: the posterior midline, the postero-lateral, and the antero-lateral approaches. We will explain in detail our surgical technique. Finally, a review of the literature is provided to allow comparison with the treatment options advocated by other skull base surgeons.
Introduction
Meningiomas are common neoplasms representing 14.3 to 19% of all intracranial tumors [63]. Among all the meningiomas, only 1.8 to 3.2% arises at the foramen magnum (FM) level [3]. Nevertheless, meningiomas are the most commonly observed FM tumors, representing 70% of all benign tumors [13, 15–19, 55, 64]. Most of the time, these are strictly intradural. Ten percent have an extradural extension: Most are intra- and extradural, and a few may be entirely extradural [9, 13, 15–19, 24, 25, 56, 60, 61].
The lesion is often large when discovered because of their slow-growing rate, their indolent development, the difficulty of the diagnosis leading to a long interval since the first symptom, and the wide subarachnoid space at this level [7].
The prerequisite for treating FM meningiomas (FMMs) is the perfect knowledge of the surgical anatomy. We will then first detail a comprehensive review of the relevant anatomy of the FM area with special emphasis on the vertebral artery (VA) V3 and V4 segments. Second, we will describe our classification system. This classification system helps for determining the best surgical approach and for anticipating the position of lower cranial nerves. The technical aspects of our surgical approaches will then be detailed extensively. Finally, we provide a summary of the relevant literature, detailing surgical results and surgical approaches advocated by other skull base surgeons and discussing the extent of bone resection and the need for fusion.
Surgical anatomy
Limits of the FM
A meningioma is considered to be located into the FM region if its insertion zone is mainly situated into the FM area. According to the FM limits that we have previously used, the FM area is defined by these landmarks [13, 18, 19] (Fig. 1):
Anterior border: lower third of the clivus and upper edge of the body of C2Lateral borders: jugular tubercles and upper aspect of C2 laminasPosterior border: anterior edge of the squamous occipital bone and C2 spinous processFig. 1Illustration of the foramen magnum anatomy through a postero-lateral approach. The skin incision (dotted line) extends on the midline just upper to the occipital protuberance and curves laterally toward the pathological side. The right vertebral artery has been elevated from the C1 posterior arch. The C1 posterior arch has been resected on the pathological side, and a suboccipital craniectomy has been performed. The dura matter has been opened. 1 CN IX–X–XI, 2 PICA, 3 CN XII, 4 vertebral artery V4 segment, 5 C1, 6 dentate ligament, 7 vertebral artery V3 segment
The VA V3 and V4 segments
The VA V3 and V4 segments are important vascular structures whose anatomy must be perfectly known when approaching FMMs.
In fact, after piercing the lateral aspect of the occipito-atlantal dura mater, the VA V3 segment becomes the V4 segment and then joins the contralateral one to form the basilar artery [8].
The VA V3 segment anatomy and techniques of approach, exposure, and transposition have been extensively described [8]. In summary, the VA V3 segment, also called the “suboccipital segment,” extends from the C2 transverse process to the FM dura mater. Its course is divided in three portions: a vertical portion, between the transverse processes of C2 and C1, a horizontal portion in the groove of the posterior arch of the atlas, and an oblique portion on leaving this groove up to the dura mater [8].
Along its course, this VA V3 segment can be at the origin of small collateral branches: in the middle of the posterolateral aspect of the C1–C2 portion, corresponding to the C2 radicular artery, a small muscular branch coursing posteriorly at the upper exit of the C1 transverse foramen, the posterior meningeal artery, and the posterior spinal artery, just before its dural penetration. This last branch enters the dural foramen where the C1 nerve root exits the spinal canal [50] and can also be bound with the VA by fibrous dural bands. The posterior spinal artery may also arise from the initial intradural part of the VA V4 segment or from the posteroinferior cerebellar artery (PICA) [11, 33, 43, 50]. Then, this branch runs medially behind the most rostral attachment of the dentate ligament [50]. On reaching the lower medulla, it divides into ascending and descending branches, providing, respectively, the vascular supply to a part of the medulla oblongata and the spinal cord [50]. The VA V3 segment is surrounded by a periosteal sheath that invaginates into the dura when piercing the FM lateral dura, thus creating a double furrow for 3–4 mm. In fact, the periosteal sheath is in continuity with the outer layer of the dura. At this level, the VA is attached to the periosteal sheath, and the adventitia is adherent to the double furrow forming a sort of distal fibrous ring [8]. In such a way, despite being the most mobile segment, the VA V3 segment is fixed at its extremities.
Anatomical relations of the VA are modified by head movements of rotation, as well as during surgical positioning. In neutral position, the vertical and horizontal portions of the V3 segment are perpendicular. On the contrary, after head rotation, as required during an anterolateral approach, both segments are stretched and run parallel, only separated by the posterior arch of the atlas, because the C1 transverse process is pushed anteriorly by this movement, away from the C2 transverse process [8].
Abnormalities of the VA V3 segment are important to know and to identify preoperatively:
In 40% of the subjects, the VAs size is different: One side is dominant, and the other is hypoplastic or atretic [8].The VA V3 segment can end at the PICA or at the occipital artery [12]. In 20% of the cases, the PICA originates extracranially, arising from the VA above C1, between C1 and C2, or even in the V2 segment [29, 36].The VA V3 segment can have an intradural course that corresponds to a VA duplication: One atretic portion follows the normal course, but the main portion pierces the dura between C1 and C2 [23, 26, 30, 40, 53].A proatlantal artery consists of a persistent congenital anastomosis between the carotid artery and the VA; it is often associated with an atretic proximal VA and an extracranial origin of the PICA [12, 36, 48].Finally, the groove of the arch of the atlas can be turned into a tunnel if the occipitoatlantal membrane is calcified or ossified, raising some difficulties to expose the horizontal portion at this level [23, 47].
The VA V4 segment ascends from the dura up to the anterior aspect of the pontomedullary sulcus where it joins the controlateral one to form the basilar artery. Initially, the VA V4 segment faces posteriorly and medially the occipital condyle, the hypoglossal canal, and the jugular tubercle. Later, the VA V4 segment lies on the clivus and runs in front of the hypoglossal and the lower cranial nerves rootlets.
At the FM level, the VA V4 segment is at the origin of several branches: the PICA, the anterior spinal artery, the anterior and posterior meningeal arteries.
The PICA is the main VA branch and originates at, above, or below the FM level [56]. The anterior spinal artery starts near the vertebrobasilar junction. Most of the time, the anterior spinal arteries of both sides joins together above the FM level near the lower end of the olives, before descending through the FM and running on the anterior midline [56]. In some cases, one artery is dominant, or no fusion exists between both arteries, only one of these arteries forming the anterior spinal artery.
The vascular supply of the FM dura originates from the anterior and posterior VA meningeal branches and the meningeal branches of the ascending pharyngeal and occipital arteries [11, 42, 50]. Infrequently, meningeal branches are coming from the PICA, the posterior spinal artery, and the VA V4 segment. The anterior meningeal branch arises from the VA at the level of the third interspace. The posterior meningeal artery originates from the VA posterosuperior aspect when turning around the lateral mass of the atlas, above the posterior arch of the atlas, just before penetrating the dura, or just at the beginning of the V4 segment [8, 11, 50]. The ascending pharyngeal artery, a branch of the external carotid artery, gives meningeal branches passing into the hypoglossal canal and the jugular foramen. The meningeal branch of the occipital artery is inconstant and runs through the mastoid emissary foramen [50].
Lower cranial nerves
The glossopharyngeal (CN IX), vagus (CN X), and spinal accessory (CN XI) nerves arise from rootlets of the postolivary sulcus and join the jugular foramen, passing ventral to the choroid plexus protruding from the foramen of Luschka and dorsal to the VA [49].
The accessory nerve is composed of rootlets originating from the spinal cord and the medulla. Spinal rootlets join together to form the main trunk, which ascends through the FM running behind the dentate ligament and unites with the upper medullary rootlets. Anastomoses with the dorsal roots of the upper cervical nerves are frequent, that with the C1 nerve root being the most common and largest one [49]. The upper medullary rootlets penetrate directly the jugular foramen [49].
The hypoglossal nerve (CN XII) arises from rootlets of the preolivary sulcus. This nerve runs anterolateral through the subarachnoid space and pass behind the VA to reach the hypoglossal canal. Rarely, the VA separates the CN XII rootlets [49].
The dentate ligament
The dentate ligament is a white fibrous sheet that extends from the pia mater, medially, to the dura mater, laterally. It forms arches leaving passage to the VA for the first one and the second cervical nerve root for the second one.
Our classification system of foramen magnum meningiomas
Meningiomas are considered to be located in the FM area if their base of insertion is mainly located within the FM limits. This definition excludes tumors invading secondarily the FM region (Figure 2).
Fig. 2Classification of foramen magnum meningiomas. Foramen magnum meningiomas are classified according to their compartment of development, their dural insertion, and their relation to the vertebral artery. The relation to the vertebral artery permits to anticipate the displacement of the lower cranial nerves. Tumors growing below the vertebral artery push the lower cranial nerves at the superior aspect of the lesion. On the other hand, tumors developed above or on both sides of the vertebral artery displace the lower cranial nerves in all directions and their position can then not be anticipated. Three basic surgical approaches are used. The extent of bone removal is delimited by the dotted lines
The definitive objective of a classification system is to define preoperatively the surgical strategy based on preoperative imaging characteristics of the lesion. The surgical strategy in cases of FMMs is the surgical approach but also the anticipation of modified vital structure position.
In our classification system, FMMs can be classified according to their compartment of development, their dural insertion, and to their relation to the VA [18].
According to the compartment of development, FMMs can be subdivided in:
IntraduralExtraduralIntra- and extradural
Intradural meningiomas are the most commonly observed. Extradural meningiomas like at any other locations are very invasive, into the bone, the nerves and vessels sheaths, and soft tissues. The VA sheath and even its adventitia can also be infiltrated. This raises some difficulties and explains the higher incidence of incomplete removal as compared to intradural meningiomas [31, 32, 39, 56, 60, 62].
According to the insertion on the dura, FMMs can be defined in the antero-posterior plane as:
Anterior, if insertion is on both sides of the anterior midlineLateral, if insertion is between the midline and the dentate ligamentPosterior, if insertion is posterior to the dentate ligament
Anterior meningiomas push the spinal cord posteriorly. Therefore, the surgical opening between the neuraxis and the FM lateral wall is narrow, and the drilling must extend to the medial part of the FM lateral wall to improve the access. In almost every case, no drilling of the lateral mass of the atlas and occipital condyle is necessary. Exceptionally, anterior meningiomas of small size without anterior compartment enlargement need more bone resection but never includes more than one fifth of these elements. On the other hand, lateral meningiomas displace the neuraxis posterolaterally and widely open the surgical access; therefore, the drilling has never to be extended into the lateral mass of the atlas or the occipital condyle.
Finally, surgical strategies vary according to the relation to the VA, FMM having the possibility to develop:
Above the VABelow the VAOn both sides of the VA
Meningiomas are more often located below the VA. In this case, the lower cranial nerves are always pushed cranially and posteriorly. There is no need to look for them. They will come into view on reaching the superior tumoral part. On the other hand, if the lesion grows above the VA, the position of the lower cranial nerves cannot be anticipated; the nerves may be displaced separately in any direction. After partial debulking of the tumor, one has to look for them so as to identify and protect them during the tumor resection. In case of tumoral development on both sides of the VA, a similar problem may exist with the position of the lower cranial nerves. Moreover, the dura around the VA penetration may be infiltrated by the tumor. As previously mentioned, the dura is normally adherent to the adventitia, and complete resection of the tumor at this level is hazardous. In this case, which is rarely observed, it may be safer to leave a cuff of infiltrated dura around the VA and to coagulate this zone.
Surgical aspects
Preoperative and perioperative considerations
Standard preoperative workup includes magnetic resonance imaging (MRI), computed tomography (CT) scan, and sometimes angiography.
On MRI, gadolinium-enhanced sequences help to precisely delimit the dural attachment zone, the tumor, and its relation to neural and vascular structures. On T2-weighted images, the presence of an arachnoid plane between the tumor and the neuraxis is sometimes visible.
Bone windows CT scan is helpful in case of extradural extension to investigate bone erosion and to schedule preoperatively the need for fusion.
Conventional angiography is generally useless. There are only two indications for preoperative angiography:
If a highly vascularized tumor is suspected and embolization is contemplatedTo perform a balloon occlusion test in case of VA encasement (extradural or recurrent meningioma and meningioma inserted around the VA). In our experience, it has never been necessary to occlude the VA.
Intraoperative neurophysiological monitorings have been used by several surgeons [3, 7] and includes somatosensory-evoked potentials, brainstem auditory-evoked potentials, and electromyographic monitoring of lower cranial nerves, by recordings through an endotracheal tube (CN X) and with a needle in the sterno-mastoid (SM) muscle (CN XI) and the tongue (CN XII).
Surgical approaches and techniques at Lariboisière Hospital
The midline posterior approach
The midline posterior approach is indicated for posterior meningiomas, whatever their intra- and extradural extension, if they remain posterior to the plane of the dentate ligament and medial to the VA [15–17]. In such cases, the neuraxis is pushed anteriorly.
The patient may be in the sitting, ventral, or lateral position. To decrease venous bleedings, the sitting position is preferred at Lariboisière Hospital as far as there is no contraindication such as a patent foramen ovale; air embolism is prevented by hypervolemia and G-suit.
The skin is incised on the midline from the occipital protuberance down to the upper cervical region. The midline avascular plane is opened between the posterior muscles, up to the occipital protuberance and down to the spinous process of C2. Bone opening is performed using a drill and Kerrison rongeurs and is always limited to the lower part of the occipital bone and the posterior arch of the atlas. The dura is then incised in a T- or Y-shaped fashion and retracted with stitches.
Postero-lateral approach
The postero-lateral approach is preferred for any intradural process located laterally and/or anteriorly to the neuraxis and for extradural lesions developed on the posterior part of the lateral FM wall [15–17]. For tumors extending far beyond the anterior midline, the postero-lateral approach has the advantage to allow a bilateral approach in the same stage (Fig. 3, 4, and 5).
Fig. 3a–c Preoperative MRI. A large lateral foramen magnum meningioma displaces the neuraxis. d, e Postoperative CT scan. The meningioma has been completely resected. The spinal cord has regained a normal shape. f Reconstructed 3D CT scan after contrast administration. The resection of the posterior arch of the atlas is visible on the right side. The lateral mass of the atlas (star) was left intact. The vertebral artery (arrow) has been elevated from the C1 posterior arch (compare with the left side) during the dissectionFig. 4a, b Preoperative MRI. A large anterior foramen magnum meningioma severely compresses the neuraxis, which is reduced to a crescent (star). c, d Postoperative MR images confirm the complete resection of the tumorFig. 5Surgical steps during a postero-lateral approach. a The left vertebral artery V3 segment (black arrow) has been elevated from the lateral part of the C1 posterior arch (white arrowhead). The medial portion has been resected up to the midline (black arrowhead). The dural entrance of the vertebral artery, where the V3 segment becomes the V4 one, is delineated by the dotted line. b The dura matter has been incised. The inferior contraincision extends inferiorly to the site of entrance of the vertebral artery into the dura matter. The C1 posterior rootlets are identified (black arrowhead). The inferior portion of the meningioma (white star) severely compresses the spinal cord (black star). c The vertebral artery V4 segment (white arrow), the spinal accessory nerve (black arrowheads), and the XIIth cranial nerve (white arrowhead) have been controlled. Of note, the dura matter (black star) is stretched over the left C1 lateral mass by a stitch to enlarge the lateral access and after the occipital craniotomy, the fall of the left cerebellum is prevented by a blade (white star). d After the complete removal of the meningioma, both vertebral arteries are visible. On the left side, we observe the section of a feeding vessel (white arrow). The right PICAs is visible (black arrow) as well as the Xth (black star) and the XIIth (black arrowhead) cranial nerves
The postero-lateral approach is a lateral extension of the midline posterior approach. The patient must be carefully positioned in the same position as during a posterior midline approach. The head must be placed in neutral position. Any flexion must be avoided because it decreases the space in front of the neuraxis and therefore may worsen the compression and the neurological condition. The vertical midline skin incision is identical, but the incision is curved laterally on the tumoral side just below the occipital protuberance toward the mastoid process. The posterior muscles are divided along the occipital crest and retracted laterally to expose the occipital bone, the posterior arch of the atlas, and the C2 lamina, if required. The exposure may be extended on a limited way on the contralateral side.
VA exposure At this step, the VA running above C1 needs to be exposed to safely resect the C1 posterior arch up to the C1 lateral mass. The exposure of the horizontal segment of the VA V3 segment progresses from the midline of the posterior arch of the atlas laterally toward the atlas groove. The medial border of the groove is clearly marked by a step with a decrease in the height of the posterior arch. The safest way to expose the VA is to dissect strictly in the subperiosteal plane. The periosteum of the C1 posterior arch needs to be elevated from medial to lateral and inferiorly to superiorly. Working in this manner permits to expose the posterior aspect of the posterior arch of the atlas, to bring into view the step at the medial end of the groove and then to elevate the VA from the atlas groove. In fact, the VA V3 segment is surrounded by a venous plexus; both artery and veins are enclosed in a periosteal sheath. Both provide protection against VA damage. If tearing occurs, venous bleedings can be controlled by direct bipolar coagulation on the VA periosteal sheath. Dissection at the superior aspect of the VA is more difficult because there is no good landmark and the periosteal sheath is in continuity with the ligaments covering the occipital condyle. Cutting of the ligaments must proceed a few millimeters above the VA on the occipital condyle by following the superior aspect of the VA sheath. The occipitoatlantal membrane covering the groove of the posterior arch of the atlas can be calcified or ossified, turning the groove into a tunnel, raising some difficulties to expose the horizontal portion at this level [23, 47]. After being fully exposed, the VA can be gently displaced superiorly to show the lateral mass of the atlas.
Bone opening The posterior arch of the atlas is resected with rongeurs from the midline toward the transverse process. To obtain a decompression of the neuraxis before the tumoral resection, the posterior arch of the atlas must also be resected beyond the midline toward the controlateral side. By this way, hyperpressure induced even by gentle manipulation during resection are not transmitted to the neuraxis. The lower part of the occipital bone is drilled or resected with rongeurs laterally toward the sigmoid sinus and also beyond the midline.
The lateral extension of the bone opening is established according to the position of the meningioma. In case of lateral meningiomas, the spinal cord is displaced toward the contralateral side, and then any resection of the FM lateral wall is not necessary. In case of anterior meningiomas, the spinal cord is pushed posteriorly, and the surgical corridor has to be enlarged laterally by drilling up to the medial side of the C1 lateral mass and/or the occipital condyle. Drilling further the lateral mass of the atlas or the occipital condyle is exceptionally necessary. In the exceptional case where the opening must be enlarged, no more than 20% of the medial part of the FM lateral wall has to be drilled. In such a way, stabilization is never required.
The cranio-caudal bone resection is scheduled according to the position of the tumor regarding to the VA. The drilling has to be extended toward the lateral mass of the atlas if the tumor grows below the VA, to the jugular tubercle and the occipital condyle if it grows above, and toward both parts of the FM lateral wall, if it encircles the VA on both sides.
Dura opening The most adequate dural incision is a curvilinear incision starting at the inferolateral corner then running vertically at a paramedian level (5 to 10 mm from the midline) and curving toward the superolateral corner. This type of incision permits to take the maximum benefit of the bone opening without contraincision and is generally easy to close. Keeping the dura over the neuraxis provides protection during the surgical dissection. Moreover, arachnoid connections between the dura and the medulla are left undissected to prevent an anterior fall of the neuraxis during tumor resection.
Intradural step Lateral meningiomas are directly brought into view and their dural attachment directly accessible. Contrarily, anterior meningiomas are partially hidden by the neuraxis. The first step of the intradural stage, before beginning tumor resection, is to identify several important structures and to open the surgical access to the lesion by completely releasing the neuraxis. Important structures are the VA, the accessory nerve, and the dentate ligament. The VA is identified by following the course of the V3 segment where it pierces the dura matter. The first two digits of the dentate ligament have to be divided. To further improve the surgical access, the first cervical nerve root can be divided distal to its connections with the accessory nerve.
At this step, the surgical technique must be individualized according to the tumor location: above, below, or on both sides of the VA.
As previously mentioned, in cases of tumor developed below the VA, the lower cranial nerves are always pushed cranially and posteriorly by the tumor. These nerves will then be found at the superior pole of the lesion at the end of the surgery. The resection must start at the caudal aspect of the meningioma with the goal to release the dural attachment and to suppress the vascular supply first; then, the tumor is debulked in a dry surgical field with a sucker, an ultrasound aspirator, or a laser, according to the tumor consistency. When liberating the dural insertion, it is important to keep undetached a small part of the base, at the side of the neuraxis, to avoid free movement of the lesion, which can be responsible for inadvertent damage to the neuraxis during the remnant resection. When hollowing the tumor, a small layer is also kept with its capsule against the neuraxis. This part will be resected at the last surgical step and under better conditions when the meningioma is completely devascularized and the surgical field widely open.
If the tumor is developed above the VA, two special points must be taken into consideration: the displacement of the lower cranial nerves and the dissection of the VA branches. Indeed, in such location, the displacement of the lower cranial nerves cannot be anticipated. To prevent damage, the rootlets must be under control on the side of the jugular foramen and then followed along their courses more or less adherent to the meningioma. With the lesion being progressively debulked, the nerve rootlets can be more easily mobilized, often inferiorly, to allow a more confident tumor resection at some distance from fragile nervous structures. The tumor dissection from the VA branches, especially the PICA, is another difficulty encountered only with tumors developed above the VA. Precise knowledge of the patient anatomy based on preoperative investigation is mandatory.
If the meningioma encases the VA, the technique is identical as described above. Special consideration is nevertheless required if the meningioma has its insertion on the dura surrounding the VA penetration. The dural resection is better achieved by progressing from the extradural side toward the intradural aspect, along the VA because the VA invaginates into the dura with its periosteal sheath. This furrow can be resected as much as the VA adventitia is not invaded.
Dural closure A watertight dural closure is required to prevent a postoperative cerebrospinal fluid (CSF) leakage. The closure is generally easy with the curvilinear incision. If necessary, a dural patch using the suboccipital aponeurosis achieves a perfect closure. In any case, the muscular and aponeurosis layers must be tightly closed.
Antero-lateral approach
The antero-lateral approach is rarely used in our department as we consider that it is a good choice only in some meningiomas with extradural extension through the bony structures [15–17].
The antero-lateral approach to the craniocervical junction has been extensively described in the article focusing on the surgical exposure of the VA V3 segment and will not be detailed again for this reason [8]. Some points required nevertheless special consideration. The head has to be slightly extended with a rotation of 60° toward the opposite side. Because of this rotation, VA segments above and below the C1 posterior arch run parallel, separated by this bone. Opening the C1 transverse foramen is mandatory for VA transposition.
Bone opening When completely liberated, the VA can be pulled out and transposed, bringing into view the FM lateral wall as the occipital condyle, the anterior and posterior arches of the atlas, the lateral mass of the atlas, and the C1–C2 joint become visible.
The exposure must now be adapted to each case, depending on the location and extension of the meningioma. The odontoid process can be reached by passing over the C1–C2 joint. The mastoid process can be resected and small bridging bone removed to open completely the jugular foramen from the end of the sigmoid sinus, to the jugular tubercle just underneath the junction with the jugular bulb, up to the beginning of the internal jugular vein.
Dura opening The dural opening is most of the time out of concern because the antero-lateral approach is mainly indicated for extradural meningiomas. When lesions are developed in both extradural and intradural compartment, the dura is already opened by the lesion and must only be enlarged.
Dural closure Dural closure can be a considerable problem in cases where the dural defect is anteriorly located at the level of the clivus, the anterior arch of the atlas, and the odontoid process.
Postoperative complications
The antero-lateral approach could induce a transient dysfunction of the accessory nerve by the nerve manipulation, responsible for pain along the trapezius muscle and weakness of the trapezius muscle and/or the SM muscle. During this approach, manipulation of the sympathetic chain could also be at the origin of a transient Horner’s syndrome.
VA damage has never been observed. In case of tumoral encasement, a balloon occlusion test must be realized.
Preservation of the lower CNs can be hazardous. If CN IX and X are damaged, postoperative swallowing problems must be anticipated.
Review of the literature
Published series
Yasargil has reviewed series published from 1924 to 1976 and counted 114 cases of FMMs. Since then, more than 400 cases of FMMs have been reported in the literature [2–7, 10, 18–22, 27, 35, 37, 38, 41, 44–46, 52, 54, 55, 57, 58]. George et al. [16, 19] reported on a series in the French literature of 106 craniocervical meningiomas from 21 hospitals. The largest single-center series have been published by Meier et al. [38] in 1984, Samii et al. [55] in 1996, and George et al. [18] in 1997. Table 1 summarizes published series over the last 20 years in the English literature.
Table 1Review of the literature of published series of FMMs over the last 20°years in the English literatureAuthorYearNb ptFMM location (%)Recurrence (%)Va encasement (%)ApproachVA transpResection JTPartial mastNb CR (%)Extent CRInstability (%)Outcome (%)Resection (%)Transient morbidity (%)Permanent morbidity (%)Mortality (%)FURecurr (%)AnteriorLateralPosteriorImprovedUnchangedWorsenedTotalSubtotalGilsbach19875100–FLY1/3100.00.0200.0Guidetti19881782.40100.01211.0Sen, Sekhar19905802080–ELYYY1001/3–1/2020206060.040.06020.0Crockard199131003333TONo00331000.066.0100.010066.020.633.0Kratimenos1993810012.50–FLY1/3087.512.5025.0––Babu19949100ELYYY1001/3–1/2088.811.278.05611.19.40.0Akalan1994812.5087.50PM008812100.000.0Bertalanffy199619100–FL SO TCY1001/30100.00.000.0––Samii199638955540PM, LSO17.501/3063.030.037.056.021.05.0George1997404552.502.5038100Partial0902.507.5087.510.00.007.557.60.0Pirotte19986100–Y1001/2–1/30100.00.01717.0Sharma1999105050PM, FL00Yes100.015.0Salas199924100–TC/ELTJYY1001/3066.033.0–0.014.8–Arnautovic20001810011.10–TCY1001/2–1/30891175.012.55511.116.640.05.5Roberti200121EL TC76.024.021.509.5Goel20011710059SO11.801/3–1/4010082.018.060.0Nanda20026100–FL00100100.00.000.043.00.0Marin Sanabria2002772.5028.50TO, SO, TC291/3–1/2080.0020100.00.072.5514.0Parlato20037–Y<1/2086.0%14.00.024.00.0Boulton2003106010300070201090.010.040.0100.0Pamir20042291940FL951/3095.54.5274.500.040.00.0Margalit200518100Lat50Partial (9/18)0.0Bassiouni200625325711443FL0096.04.040.084.073.20.0CR Condyle resection, EL extreme-lateral, FMM foramen magnum meningioma, FL far lateral/postero-lateral, FU follow-up, JT jugular tubercle, mast mastoidectomy, Lat lateral, Nb number, pt patient, Recurr recurrence, SO suboccipital, TC transcond dylar, TO transoral, transp transposition, VA vertebral artery, Y yes
Heterogenicity of published series
A heterogenous group of patients constituted the published studies. Several studies included not only FMMs but also other FM pathologies such as neurofibromas, schwannomas, as well as other various lesions leading result analysis precarious [2, 4, 10, 35, 38, 41, 45, 57, 58]. Table 1 summarizes also the high variability of tumor location, rate of VA encasement, rate of tumor recurrence, and surgical approaches. The proportion of anterior FMMs is comprised between 12.5 [2] and 100% [3, 27, 41]. The subdivision between anterior and lateral FMM is not always detailed in studies including 90 to 100% of antero-lateral FMMs [4, 6, 20, 21, 35, 44, 46, 54, 55, 57]. Posterior FMMs were always included in studies with anterior or antero-lateral lesions with a variable proportion from 2.5, 5, 9, 11, 28.5, 30, up to 50% [5, 7, 18, 37, 44, 55, 58]. The rate of VA encasement can be higher than 59% and the rate of tumor recurrence up to 80% [21, 57] (Table 1).
Surgical approaches, extent of bone resection, and need for fusion
FMMs are undoubtedly challenging tumors, requiring special considerations because of the vicinity of the brainstem, medulla oblongata, lower cranial nerves, and the VA. Several approaches have been advocated. The definite goals are to achieve the largest tumor removal and the lowest morbidity rate as possible. Minimizing the morbidity is obtained by choosing the appropriate exposure. The approach must allow adequate controls of important neurovascular structures, without exposing to unnecessary risks.
There is no discussion about the best surgical approach of posterior FMMs. The posterior approach is the best option, is well known by neurosurgeons, and is associated with a low morbidity rate.
The debate about the best surgical approach is more open for lateral and mainly anterior FMMs.
The transoral approach has been reported sporadically [10, 39]. Despite providing access to the anterior part of the craniocervical junction, this approach has several drawbacks in case of intradural lesions: increased risk of CSF fistula and meningitis after crossing of the contaminated oral cavity, poor access to laterally extending tumors resulting in a low rate of complete resection, and increased risk of posteroperative instability and velopalatine insufficiency [10, 39].
The two main surgical approaches reported in the literature are the far-lateral approach [28], also called postero-lateral approach or lateral suboccipital approach, and the extreme-lateral approach, also named antero-lateral approach. As detailed by Rhoton [51], the far-lateral approach is a lateral suboccipital approach directed behind the sternocleidomastoid muscle and the VA and just medial to the occipital and atlantal condyles and the atlanto-occipital joint. The extreme lateral approach is a direct lateral approach deep to the anterior part of the sternocleidomastoid muscle and behind the internal jugular vein along the front of the VA. In fact, both approaches permit drilling of the occipital condyle but provide a different exposure because of the differences in the approach direction.
The extreme-lateral transcondylar approach for FMMs was first reported by Sen and Sekhar [57] in 1990. Further publications of the same experienced skull base surgeons followed [4, 52, 54]. Salas et al. [54] reported in 1999 several variations of the extreme-lateral approach based on a series of 69 patients, including 24 meningiomas. The lesions were removed most of the time through a partial transcondylar approach and more rarely through transfacetal, retrocondylar, and extreme transjugular approaches. During the partial transcondylar approach, the posterior one third of occipital condyle and superior facet of C1 were drilled away. These authors also performed a partial mastoidectomy [4, 54, 57]. Arnautovic et al. [3] published their experience in 2000 on a series of 18 ventral FMMs. They used also the extreme-lateral transcondylar approach. The condyle drilling ranged approximately from one third to one half of the condyle, without causing craniocervical instability. The extreme-lateral trancondylar approach requires the VA transposition to reach and drill the occipital condyle [3, 4, 27, 45, 54, 57].
As others [5, 6, 20, 21, 27, 35, 41, 44, 55, 58], we advocate rather the postero-lateral approach, also called far-lateral approach, even for anterior intradural FMMs. During this approach, the VA is controlled in the horizontal portion of the V3 segment, above the C1 posterior arch. VA transposition is rarely performed and has only been reported in one series [46]. The extent of FM lateral wall drilling is variable, in fact directly proportional to the tumor extension to the contralateral side. In the literature, the occipital condyle resection varies between 0 and 66% [27, 41, 44, 46, 55]. In a recent publication, Bassiouni [5] classified judiciously surgical approaches in two groups: transcondylar or retrocondylar if the occipital condyle is not drilled. In our review of the literature (Table 1), we found nine series in which the retrocondylar approaches was used to reach anterior or anterolateral meningiomas [2, 5, 7, 21, 35, 37, 41, 55, 58]. In four of these series, a retrocondylar approach was not used in all cases but in 89.2, 82.5, 71.5, and 50% [21, 35, 37, 55]. The five other series are homogeneous and only included patients treated through a retrocondylar approach [2, 5, 7, 41, 58]. Of these, three studies reported complete resection in 100% of the cases [2, 41, 58]. In the two others, complete resection was noted in 90 and 96% of the cases, and the remaining lesions were resected subtotally [5, 7]. Surgical results were good, and surgical morbidity and mortality rates low [2, 5, 7, 41].
Extradural lesions can be treated either by posterolateral or anterolateral approach (Fig. 2). The extent of drilling during this procedure can be larger but is only dictated by the tumoral invasion and must be limited to the destroyed or invaded bone. In such a way, the question of instability is only a preoperative concern. In our experience of tumors located at the craniocervical junction, we have not observed any instability if less than half of the C0–C1 and C1–C2 joints were resected [15]. In our experience, VA transposition was merely performed in selected cases of FMMs with extradural extension and never for intradural lesions.
Surgical results
Morbidity–mortality–prognosis factors
In the Yasargyl’s review of the literature of series published before 1976, the overall mortality rate was approximately 13% but could be as high as 45% in some series [34].
Over the last 20 years, the overall mortality is 6.2%. The mortality rate is comprised between 0 and 25% (Table 1). Mortality rates higher than 10% were mainly observed in small series [4, 27, 37, 46, 57, 58].
In the Yasargil’s review, a good outcome was noted in 69% and a fair and poor outcome, respectively, in 8 and 10%. In series larger than ten patients published over the last 20  years (Table 1), neurological improvement, stability, and worsening were noted, respectively, in 70–100, 2.5–20, and 7.5–10% of the cases. The permanent morbidity rate is comprised between 0 and 60%. The permanent morbidity rate is lower through a far-lateral approach (0–17%), either transcondylar or retrocondylar, than through an extreme-lateral transcondylar approach (21%–56%, considering series without recurrent tumor). Lower cranial nerves dysfunctions are the most frequently encountered preoperative deficits. These deficits have the propensity to recover even completely postoperatively [3–5], except in cases of en plaque meningiomas or recurrent tumor [55].
Several factors lead the surgical procedure still more difficult, then influencing negatively the morbidity rate: anterior tumor location [18, 55], tumor size (smaller lesions are more difficult to resect because the surgical corridor is small), tumor invasiveness, extradural extension [18], VA encasement [22], absence of arachnoidal sheath [5, 14, 65], and adherences in recurrent lesions [4, 5, 55, 57].
Rate of tumoral resection
Based on a multicentric study from 21 hopitals, George et al. [19] reported 77, 16, and 7%, respectively, of complete, subtotal, and partial removals. Over the last 20 years, most of the studies reported complete or subtotal removal of the tumor [2, 4–7, 10, 18–22, 27, 35, 37, 41, 44–46, 52, 54, 57, 58]. Factors limiting the resection completeness are adherences of the lesion to vital structures, VA encasement and invasiveness of the lesion. Adherences are observed during repeated surgery and explain the lower rate of complete tumor resection (60–75% of Simpson grade 1) in surgical series in which a high rate of recurrent tumors are included [3, 55, 57]. Eventually, in recurrent tumors, some authors advocate leaving a small tumor remnant to preserve a low morbidity rate [5, 18]. On the other hand, Arnautovic et al. [3] favored radical removal of recurrent tumors with the goal of providing a relatively long and stable postoperative course, even at the price of frequent but transient morbidity induced by lower cranial nerves dysfunction. Arnautovic et al. [3] have also demonstrated that the rate of complete removal is higher at first surgery than when treating recurrence, advising then to be more aggressive at the first surgical presentation. VA encasement was noted in 38 to 59% in some series [5, 18, 21, 44, 55]. This factor was recognized as an independent factor of incomplete removal [55]. The location of the meningioma, either intraextradural or extradural, reflects the tumoral invasiveness. These tumors are less favorable to be completely resected than pure intradural lesions [19, 39]. In the French cooperative study [19], the rate of complete removal of intradural, extradural, and intraextradural meningiomas was, respectively, 83, 50, and 45%.
Our experience
Our experience has been published in 1997 after the treatment of 40 FMMs operated in the period 1980–1993 [18]. Twenty-four lesions were intradural, two were extradural, and four were intraextradural. Eighteen were considered anterior, 21 lateral, and one was posterior. The tumor was above the VA in four cases, below in 20 cases, and on both sides in 16 cases. The posterolateral approach was used in 31 cases, the anterolateral one in five cases, and the posterior midline in four cases. The rates of complete resection for intradural and extradural lesions were, respectively, 94 and 50%. Postoperatively, the clinical condition improves in 90% of patients, remains stable in 2.5%, and worsened in 7.5%. Our present experience over the last 25 years is now based on 97 FMMs (unpublished data). Complete removal was achieved in 86% and subtotal removal in 11%. Subtotal removals were due to extradural extension or to recurrent cases. The rate of complete removal increased up to 94% when selecting only intradural lesions treated at first presentation. We mainly use the far-lateral retrocondylar approach for intradural FMMs. In some cases, the drilling of the FM lateral wall has to be performed for intradural anterior meningiomas but remains limited at worst to the medial 20% of the FM lateral wall. Whatever, this extent of drilling has to be tailored according to the tumor characteristics. We consider nevertheless that more bone resection is never necessary because of the anatomical anterior position of the lateral mass of the atlas and the occipital condyle. Our attitude has been reinforced by cadaveric study, which has demonstrated that increasing the bone drilling is not associated with a significant widening of the surgical corridor [41]. In fact, resecting one third and one half of the occipital condyle increases the visibility respectively by 15.9 and 19.9°. Two anatomic reports have demonstrated that condyle resection allows a wider angle of exposure to gain the anterior foramen area [1, 59]. However, these studies did not consider the fact that in surgical approaches to anterior lesions, space-occupying lesions can enlarge the surgical corridor. We consider that the extreme-lateral approach could be associated with a higher morbidity rate than the far-lateral approach. The exposure allowed by the far-lateral retrocondylar or partial transcondylar approach is adequate for resecting even anterior intradural FMMs. The supposed benefit in term of exposure provided by the extreme transcondylar approach is counterbalanced by the risks associated with the CN XI dissection, the VA transposition, and the condyle drilling.
Conclusions
FMMs are challenging tumors in the vicinity of the brainstem, the VA, and lower cranial nerves. Several surgical approaches are possible, each one with specific indications. The drilling of the FM lateral wall required during the approaches is always limited and by itself should not be at the origin of any instability. Postoperative complications can be dramatic and must be anticipated.

foramen magnum
meningioma
surgical approach
tumor
vertebral artery


Brief Report: The Use of WAIS-III in Adults with HFA and Asperger Syndrome

The WAIS III was administered to 16 adults with high functioning autism (HFA) and 27 adults with Asperger syndrome. Differences between Verbal Intelligence (VIQ) and Performance Intelligence (PIQ) were not found. Processing Speed problems in people with HFA appeared. At the subtest level, the Asperger syndrome group performed weak on Digit Span. Comprehension and Block Design were relative strengths. In the HFA group, performance on Digit-Symbol Coding and Symbol Search was relatively poor. Strengths were found on Information and Matrix Reasoning. The results suggest that the VIQ-PIQ difference cannot distinguish between HFA and Asperger syndrome. WAIS III Factor Scale and Subtest patterning provides a more valid indicator.
Introduction
Over the past few years, interest in HFA and Asperger syndrome in adults with normal intelligence has increased markedly. However, not much is known about the cognitive profiles of these groups. Only a few studies exist about adults who function relatively well in society and have been diagnosed late in life (Howlin 2004; Vermeulen 2002). The present study aims to assess the cognitive profiles of this relatively well-functioning subgroup by means of the Wechsler Intelligence Scale III (WAIS III, Wechsler 1997).
In WAIS III, the intelligence pattern is described at three levels: The first level contains Performance Intelligence and Verbal Intelligence. The second level consists of the four factor scales: Verbal Comprehension, Perceptual Organization, Freedom from Distractibility and Processing Speed. The third level contains the subtests. The following paragraphs summarizes research on the intelligence profiles of adults with the autistic disorder or Asperger syndrome on the basis of these three levels.
The Performance IQ (PIQ)––Verbal IQ (VIQ) dichotomy has been incorrectly used for years to underpin the diagnosis of autistic disorder or Asperger syndrome. It is questionable whether the two constructs should even be applied in general, because research did not support the construct validity of the VIQ-PIQ dichotomy (Taub 2001).
For adults with HFA, studies on WAIS-R have yielded contradictory results (Minshew et al. 1992; Siegel et al. 1996; Vermeulen 2002), which may reflect the validity problems of the VIQ-PIQ dichotomy (Arnau and Thompson 2000; Taub 2001).
The factor scale level is of great importance in assessing cognitive abilities because factor analytic studies indicate that the factor scales give the best estimates of the four factors underlying intelligence (Arnau and Thompson 2000; Ryan and Paolo 2001).
No studies have been performed on WAIS-III profiles for adults with HFA or Asperger syndrome as far as we know. Therefore no information is available on the results of factor scales in these groups. This leads to the conclusion that the most important factors of the intelligence pattern for adults with HFA or Asperger syndrome are still unknown.
On subtest level, some studies on WAIS or WAIS-R found low Comprehension versus high Block Design scores (Goldstein et al. 2001; Rumsey and Hamburger 1988). A relatively high variability between the subtests scores in adults with HFA has also been reported (Siegel et al. 1996).
In summary, research shows that among adults with HFA or Asperger syndrome, results of VIQ-PIQ differences vary and may be influenced by the validity problems of the VIQ-PIQ dichotomy. The factor scale scores and the subtest patterns provide a better representation of the intelligence pattern.
Aims of the Present Study
The present study aims to acquire insight into the WAIS III profiles of normal intelligent adults with HFA and Asperger syndrome. Profiles in the total group and differences between the two diagnostic groups will be examined.
Methods
Procedure
All participants were recruited from the GGZ (Mental Health Center) Eindhoven and Oost-Brabant. The participants met the criteria for Asperger syndrome or HFA. Participants with relevant neurodevelopmental conditions and genetic conditions were excluded, as were institutionalized patients and patients with a Full Scale IQ below 80.
Subjects
The mean Full Scale IQ of the participants was 110.16, individual scores varied between 83 and 145 (see Table 1).
Table 1Characteristics of participantsIQ and ageMSDRange    Full scale IQ110.1616.0583–145    Mean age41.9310.6720–60Diagnosisn%    Autistic disorder1637.2     Asperger syndrome2762.8 Gender    Male3990.7     Female49.3 Education    Lower/middle education1841.9     Higher education2558.1 Employment status    Employed or retired3069.8     Studying12.3    Unemployed1227.9 Current living circumstances    Lives with partner2353.5     Lives independently 1227.8     Sheltered living24.7     Lives with parents 614.0
All individuals ranged in age from 18 to 60 years. The mean age was 41.93. Of all participants, 25 finished higher education and 30 individuals had work. 23 participants lived together with a partner. The relatively large number of participants who had a relationship, worked and were well educated emphasizes the relatively high level of functioning in this group.
Assessment of Disorder
Hetero-anamnestic information was gathered using the Dutch version of the Autistic Disorder Diagnostic Interview, revised version (ADI-R, Lord et al. 1994), administered by psychologists who were officially trained in the administration and scoring of the instrument. To gather anamnestic information, a semi-structured interview was used to assess presence of the DSM-IV criteria of HFA and Asperger syndrome (APA 1994). Because of the controversial nature of the DSM-IV criteria (Ghaziuddin et al. 1992; Mayes et al. 2001), additional questions were used to differentiate between HFA and Asperger syndrome, based on the diagnostic criteria of Gillberg and Gillberg (1989) and ICD-10 (WHO 1993).
Assessment of Intelligence
The intelligence profile was assessed using the Dutch translation of the WAIS III (Wechsler 1997). The WAIS-III has excellent psychometric properties (Sattler and Ryan 1999) and has been validated for the Dutch population (Wechsler 1997).
Results
Analyses were done at the three WAIS-III levels: VIQ versus PIQ, the four factor scales and all subtests. Preliminary analysis included checks for normality, linearity, influential data points and assumptions of repeated measures. No serious deviations were found. T-tests showed that both diagnosis groups were comparable in education and work status, as well as in gender distribution.
Differences Between WAIS III VIQ and PIQ
Differences between VIQ and PIQ for all participants and both diagnostic groups were analyzed by means of paired t-tests. No statistically significant effects were found for any of the investigated groups (see Table 2).
Table 2VIQ and PIQ differences in the total group and in diagnostic groupsVIQPIQMean difference nMSDMSDTotal group110.3013.83108.4218.211.8843Asperger111.41 13.57112.5217.281.1127Autistic disorder108.4414.49101.50 18.136.9416
Differences Between Factor Scale Scores
Factor Scale profiles were studied within the total group and between the two diagnostic subgroups by means of repeated measures analysis of variance. Mauchly’s test indicated that the assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh–Feldt correction (ε = .89). Post-hoc comparisons using the Sidak adjustment for multiple comparisons showed that the main effect of the WAIS III Factor Scale was statistically significant (F(2.7,109.7) = 7.0, p < 0.001). An interaction effect of differences in Factor Scale mean by diagnostic group was also found (F(2.7, 109.7) = 2.7, p = 0.05). To find out which differences in WAIS III Factor Scale means added to the significant main effect, post hoc pairwise comparisons were done. This showed that the main effect in the total group can be attributed to Processing Speed being significantly lower than Verbal Comprehension (p < .01) and Perceptual Organization (p < .005).
Post hoc pairwise comparisons were done for the two diagnostic groups to analyse the ‘within group’ effect. In the Asperger group, no significant differences in Factor Scale mean scores were found. The HFA group however, showed a significant lower Processing Speed compared to Verbal Comprehension (p < .01), Perceptual Organization (p < .01) and Freedom from Distractibility (p < .05) (see Table 3).
Table 3Factor scale scores for the total group and the diagnostic groupsFactor scaleMSDNVerbal comprehensionAutistic disorder107.5*12.116Asperger syndrome110.811.927Total109.6*12.043Perceptual organizationAutistic disorder105.0*18.716Asperger syndrome111.813.027Total109.3*15.543Freedom from distractibilityAutistic disorder105.1*18.216Asperger syndrome107.215.427Total106.416.343Processing speedAutistic disorder91.8*17.416Asperger syndrome106.519.427Total101.0*19.843*p < .05.
Differences Between WAIS III Subtest Scores
The Subtest profiles were explored within the total group and between the two diagnostic subgroups by means of a repeated measures analysis of variance. The assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh–Feldt correction (ε = .82). Post-hoc comparisons were performed using the Sidak adjustment for multiple comparisons. The results (see Table 4) showed a significant main effect of the type of Subtest (F(10.7,438.7) = 4.8, p < 0.001).
Table 4Mean standardized subtest scores for the total groupSubtest scores MSDnVocabulary11.63*2.56443Similarities11.422.49043Arithmetic11.77*3.04643Digit span10.723.26843Information12.42*2.77943Comprehension12.53*2.77243Letter-number sequencing10.982.95643Picture completion10.883.25343Digit-Symbol Coding9.81*3.43843Block design12.02*3.56243Matrix reasoning11.98*2.45443Picture arrangement11.533.73143Symbol search10.37*3.97043Object assembly11.163.08643*p < .05.
An interaction effect of Subtest by diagnosis was also found (F(10.7, 438.7) = 2.1, p < 0.05), indicating that the patterning of the WAIS III subtest mean scores for the two diagnostic groups differs. Table 5 and 6 show the mean Subtest scores and standard deviations for the HFA group and the Asperger syndrome group.
Table 5Mean standardized subtest scores for the autistic disorder groupSubtest Scores MSDNVocabulary11.312.49616Similarities10.941.76916Arithmetic11.443.70516Digit span11.313.40016Information12.13*3.28416Comprehension11.752.17616Letter-number sequencing10.253.15216Picture completion10.814.07016Digit-Symbol Coding8.38*3.03016Block design10.563.44416Matrix reasoning11.44*2.82816Picture arrangement10.193.67416Symbol search8.44*3.48316Object assembly9.883.32416*p < .05.Table 6Mean standardized subtest scores for the Asperger syndrome groupSubtest scoresMSDnVocabulary11.812.63227Similarities11.702.82627Arithmetic11.962.63827Digit Span10.37*3.20027Information12.592.48527Comprehension13.00*3.01327Letter-number sequencing11.412.80527Picture completion10.932.74527Digit-Symbol Coding10.673.43127Block design12.89*3.40127Matrix reasoning12.302.19827Picture arrangement12.333.59527Symbol search11.523.84727Object assembly11.932.71627*p < .05.
Post hoc pair wise comparisons showed that the main effect in the total group can be attributed to the fact that Digit-Symbol Coding was significantly lower than Vocabulary (p < .05), Arithmetic (p < .05), Information (p < .005), Comprehension (p < .005), Block Design (p < .05) and Matrix Reasoning (p < .005). Furthermore, Symbol Search was lower than Information (p < .05) and Comprehension (p < .05).
Post hoc pair-wise comparisons were also performed for the two diagnostic groups to analyze the ‘within group’ effect. The two groups showed significant differences in Subtest scores. In the Asperger syndrome group, Digit Span was lower than Comprehension (p = .005) and Block Design (p < .05).
In the HFA group performance was significantly higher in Information compared to Digit-symbol Coding (p < .05) and Symbol Search (p < .05). Furthermore, Digit-Symbol Coding was lower than Matrix Reasoning (p < .05).
Discussion
WAIS VIQ Versus PIQ
No significant differences were found between VIQ and PIQ in the total group nor in the two diagnostic subgroups. The results are in line with factor analytic studies showing that the VIQ-PIQ dichotomy is not valid for general populations (Arnau and Thompson 2000; Taub 2001).
WAIS III Factor Scale Level
The Asperger syndrome group was characterized by a flat Factor Scale profile in the Asperger syndrome group, while the HFA group performed significant low in Processing Speed. A low Processing Speed indicates problems in speed of processing visual information (Wechsler 1997). Adults with HFA apparently need more time than other people to process and integrate visual information and to act on this information.
The Processing Speed performance of the HFA group might be influenced by problems with top-down processing and ignoring irrelevant details, which are characteristic of people with HFA (Happé 2005; Shah and Frith 1993). In order to maintain an overview of what they are doing, they work slowly.
WAIS III Subtest Level
Analyses showed different Subtest patterns in the HFA and the Asperger syndrome groups. The HFA group performed significantly low in Digit-Symbol Coding and Symbol Search. These two subtests together form the Processing Speed Factor. The low scores for these subtests represent the problems in speed of processing visual information as described in the preceding paragraph.
The HFA group showed significantly high performance in Information and Matrix Reasoning. High scores for Information are in line with the fact that people with autism usually acquire much factual knowledge (Happé 1999).
Matrix Reasoning taps nonverbal perceptual reasoning. Matrix Reasoning is the only Perceptual Organization subtest without a time limit and is possibly not influenced by low Processing Speed performance scores. The good performance of the HFA group can probably be attributed to their visual-spatial strengths (Lincoln et al. 1995; Tsatsanis 2005) and to the absence of a time limit for this subtest.
In the Asperger group, scores for Digit Span were relatively low. Digit Span taps working memory capabilities (Wechsler 1997), which can been defined as ‘the ability to hold in mind past states of the environment and past actions while currently performing an action’ (Russell 1997). People with autism or Asperger syndrome tend to store information in details instead of using strategies, which often leads to problems in retaining information (Happé 2005; Minshew et al. 1992; Tsatsanis 2005). Low Digit Span scores in the Asperger group may reflect problems in applying strategies to retain information.
The Asperger syndrome group performed significantly well on Comprehension. High scores on Comprehension in this group seem to contradict former research results (Klin et al. 2005; Mayes and Calhoun 2003; Siegel et al. 1996). However, people with Asperger syndrome often try to function in society by analyzing social situations at a cognitive level, which has been described as using an ‘explicit theory of mind’ (Frith and Happé 1999). A extremely well developed explicit theory of mind may have caused the Asperger syndrome group to have such high scores on Comprehension.
The Asperger Syndrome group also performed significantly well on Block Design. Strengths in Block Design have often been reported in studies of people with HFA or Asperger syndrome (Happé 2005; Shah and Frith 1993). This has been attributed to strengths in processing unconnected stimuli outside a meaningful context, which go together with the central coherence problems seen in people with autistic impairment (Shah and Frith 1993).
Conclusions
The present study found people with Asperger syndrome to differ significantly from people with HFA in WAIS III Factor Scale profiles and WAIS III Subtest patterning. In people with HFA Processing Speed problems were found. Further, the HFA and Asperger syndrome group showed different subtest patterns. The present study supports the idea that HFA and Asperger syndrome can be differentiated empirically at the level of intellectual functioning. This lends support to the hypothesis that HFA and the Asperger syndrome are two separate disorders.

wais-iii
asperger syndrome
high functioning autism
intelligence
processing speed