Abstract
Background
Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans. The aetiology is heterogeneous and remains unexplained in approximately 75% of patients.
Objective
To examine cholesterol biosynthesis in lymphoblastoid cell lines of 228 patients with HPE, since perturbations of cholesterol homeostasis are an important model system to study HPE pathogenesis in animals.
Methods
An in vitro loading test that clearly identifies abnormal increase of C27 sterols in lymphoblast‐derived cells was developed using [2‐14C] acetate as substrate.
Results
22 (9.6%) HPE cell lines had abnormal sterol pattern in the in vitro loading test. In one previously reported patient, Smith–Lemli–Opitz syndrome was diagnosed, whereas others also had clearly reduced cholesterol biosynthesis of uncertain cause. The mean (SD) cholesterol levels were 57% (15.3%) and 82% (4.7%) of total sterols in these cell lines and controls, respectively. The pattern of accumulating sterols was different from known defects of cholesterol biosynthesis. In six patients with abnormal lymphoblast cholesterol metabolism, additional mutations in genes known to be associated with HPE or chromosomal abnormalities were observed.
Conclusions
Impaired cholesterol biosynthesis may be a contributing factor in the cause of HPE and should be considered in the evaluation of causes of HPE, even if mutations in HPE‐associated genes have already been found.
Holoprosencephaly (HPE) is a common congenital malformation characterised by incomplete cleavage of the embryonic forebrain.1 HPE occurs in approximately 1 in 10 000 births and in 1 in 250 embryos,1 and ranges from cyclopia with a proboscis and a single prosencephalic vesicle to obligate HPE carriers with a normal central nervous system by MRI and normal facial appearance. Multiple genetic causes of HPE have been identified, the most common being cytogenetic abnormalities.2 Approximately 20% of infants with normal karyotypes have missense mutations in one of the four genes currently linked to isolated, sporadic and familial, non‐syndromic HPE: Sonic Hedgehog (SHH3), Zinc finger transcription factor 2 (ZIC24), Sine oculis homeobox (Drosophila) homologue 3 (SIX35), and TG‐interacting factor (TGIF6). Microdeletions in these genes occur in another 5%.7 However, the underlying cause of HPE remains unknown in most cases.
Among a variety of embryological toxins that can elicit abnormal craniofacial development including HPE,8 inhibitors and other perturbations of cholesterol biosynthesis have been shown to be important factors, most likely because cholesterol is required for SHH signal response.9 For example, HPE is induced by exposure of rat embryos to inhibitors of the distal steps of cholesterol biosynthesis (AY9944, BM15766 and triparanol; fig 1), causing accumulation of cholesterol precursors and decreased cholesterol concentrations.10,11 HPE‐spectrum malformations also occur in about 5% of patients with Smith–Lemli–Opitz syndrome (SLOS), a defect in the last step of cholesterol biosynthesis catalysing the conversion of 7‐dehydrocholesterol to cholesterol,12 and in mouse embryos deficient in megalin, a member of the low‐density lipoprotein receptor family that is expressed in embryonic neuroectoderm.13 Finally, certain plant alkaloids (cyclopamine, jervine) that inhibit intracellular cholesterol transport promote HPE in chick and mouse embryos.14 Although these compounds were first thought to cause HPE by reducing SHH signalling, a study demonstrated that cyclopamine directly inhibits Smoothened, a transmembrane protein involved in embryonic signalling distal to SHH.15
Figure 1 Pathway of distal cholesterol biosynthesis. Enzyme defects known for specific human diseases are indicated by a bar. The inhibitors are printed in bold, below the enzymes they inhibit.
Many studies have shown that the expressed HPE phenotype is extremely variable even in families segregating a heterozygous HPE‐causing mutation. Preliminary evidence suggests that this phenotypic variability may reflect a combination of genetic, epigenetic and environmental influences,16 including anomalies in cholesterol metabolism.17 Since no systematic investigations of cholesterol metabolism in patients with HPE have been reported, we examined cholesterol biosynthesis in lymphoblastoid cell lines of a large cohort of patients with HPE with a newly developed, sensitive test system.
Materials and methods
Materials
[2‐14C]acetic acid (56 mCi/mmol) was purchased from Hartmann Analytic, Braunschweig, Germany. Cholesterol, desmosterol, lathosterol, 7‐dehydrocholesterol, lanosterol, 1,4‐bis(2‐chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), cabosil M‐5, insulin‐transferrin‐sodium selenite media supplement and sodium acetate were purchased from Sigma Aldrich, Taufkirchen, Germany. 5α‐Cholestane was from Merck, Darmstadt, Germany. N‐Methyl‐N‐trimethylsilylheptaflour(o)butyramide (MSHFBA) was from Macherey‐Nagel, Düren, Germany.
Hexane, ethanol, benzene, ethylacetate, NaOH, Na2CO3 and KOH were purchased from Roth, Karlsruhe, Germany. Silica gel 250 μm AgNO3 thin‐layer chromatography (TLC) plates (10% silver nitrate) were from Analtech, Newark, Delaware, USA. Bromothymol blue was purchased from Alltech Grom, Rottenburg, Germany. Dulbecco's modified Eagle medium and phosphate‐buffered saline (PBS) were obtained from Cell Concepts, Umkirch, Germany. RPMI 1640, MEM‐NEAA (non‐essential amino acids), fetal calf serum, amphotericin B, penicillin/streptomycin were from PAA Laboratories, Cölbe, Germany. Gentamycin was obtained from Invitrogen, Karlsruhe, Germany and the mycoplasma test was from Cambrex BioScience, Verviers, Belgium.
Patients
Lymphoblastoid cell (LC) cultures from 214 patients with various forms of HPE, 13 parents and one sibling were included in this study. In all, 100 individuals were male, 125 female, and in 3 patients the gender was unknown because of early fetal death. All cell lines and submicroscopic deletions in four genes known to be associated with HPE (SHH, ZIC2, SIX3 and TGIF) were analysed for mutations. Control human LC lines were obtained from nine healthy individuals. Blood sampling and experiments in LC cultures were performed after informed consent was obtained. These studies were approved by the Institutional Review Board of the National Human Genome Research Institute, NIH‐1195‐1(8‐98), and by the ethics committee of the medical faculty, University of Heidelberg (No. 211/2002), Germany.
Lymphoblastoid cell culture
For generation of LC lines, B‐lymphocytes of 0.5–10 ml venous EDTA blood samples from patients with HPE and healthy volunteers were isolated using Ficoll density gradient centrifugation, immortalised by exposure to Epstein–Barr virus (EBV) supernatant and cultivated for at least 6 weeks before application.18
LCs were cultivated to saturation density at 37°C in RPMI 1640+l‐glutamine supplemented with penicillin, streptomycin, amphotericin B, gentamycin and MEM‐NEAA with 100 ml/l fetal calf serum. Each LC culture was tested for contamination by Mycoplasma species before the experiments.
In vitro [2‐14C]acetate loading test
LCs were preincubated in lipid‐depleted cell culture medium, prepared according to a published procedure,19 for 24 h to activate endogenous cholesterol synthesis. Briefly, lipid depletion was achieved by mixing fetal calf serum with Cabosil M5. The mixture was stirred overnight, centrifuged and the supernatant was filtered sterile. The cholesterol content of the lipid‐depleted medium was 0.12 mg/dl. Cell viability was determined by trypan blue exclusion, and 4×106 viable cells were washed in PBS, incubated with lipid‐depleted medium containing 2.5 µCi [2‐14C]acetate for 2 h and chased in lipid‐depleted medium containing 144 mM sodium acetate for another 2 h. Cells were washed, harvested by centrifugation, and homogenised in 1000 µl of a solution containing 200 mM sodium carbonate and 100 mM NaOH. The homogenate was stored at −80°C until extraction. 500 µl of the sample was hydrolysed at 60°C for 30 min with 1 ml degassed 4% (weight/volume (w/v)) KOH in ethanol, followed by extraction with n‐hexane. Neutral sterols were separated on a silver nitrate silica gel TLC plate with benzene/ethylacetate (9/1 v/v) as mobile phase. The plates were thoroughly dried and development in the same solvent mixture repeated. Quantification of sterols was achieved by phosphoimaging (phosphoimager FLA 3000, Fujifilm, Tokyo, Japan) and subsequent densitometric analysis.
Differentiation and quantification of sterols by gas chromatography‐mass spectrometry
LCs were cultured in lipid‐depleted medium for 72 h. A total of 4×106 viable cells were harvested by centrifugation, washed with PBS and hydrolysed with ethanolic KOH as described above. 5α‐Cholestane was added as internal standard. After extraction with water and n‐hexane, the sample was derivatised with N‐methyl‐N‐trimethylsilylheptaflour(o)butyramide. For GC‐MS analysis, the quadrupole mass spectrometer MSD 5972A (Agilent, Santa Rosa, California, USA) was run in the selective ion‐monitoring mode. The following characteristic mass fragment pairs were used for quantification: m/z 217/357 (5α‐cholestane, internal standard), m/z 329/368 (cholesterol), m/z 325/351 (7‐ and 8‐dehydrocholesterol (DHC)), m/z 343/372 (desmosterol), m/z 255/458 (lathosterol), m/z 393/498 (lanosterol), and m/z 213/229 (8(9)‐cholestenol).
Gas chromatography separation was achieved on a capillary column (DB‐5MS, 30 m×0.25 mm; film thickness: 0.25; J&W Scientific, Folsom, California, USA) using helium as a carrier gas. A volume of 1 µl of the derivatised sample was injected in splitless mode.
Mutation analysis by direct sequencing
Genomic DNA was isolated from LC using standard methods. All coding exons and adjacent intron regions of the 7‐dehydrocholesterol recuctase (DHCR7) gene were sequenced with a capillary fluorescent sequencer (Applied Biosystems, Darmstadt, Germany). DHCR7 genomic primer sequences are available on request.
Statistical analysis
To assess significant differences between sterol patterns in control and patient LC, non‐parametric exact permutation tests were applied. Permutation tests are especially useful for small sample sizes (total number of observations <50) or single case designs. They have a relative asymptotic efficiency of one compared with parametric analogues (eg, t tests). Permutation tests incorporate all available numerical information (measurement values and sample sizes), but do not make any assumptions about the underlying distribution of the variables.20,21
Results
Separation of sterol standards
We developed a test system that separates C27–C30 sterols using TLC. Unmarked sterol standards (lanosterol, cholesterol, desmosterol, lathosterol and 7‐dehydrocholesterol) were dissolved in ethanol, spotted on a 10% silver nitrate silica gel TLC plate with benzene/ethylacetate as mobile phase. Plates were dried and developed a second time in the same solvent, resulting in a higher retention fraction (RF) and a better separation. To visualise the sterol bands, the plate was sprayed with bromothymol blue. The smallest amount of sterol that could be visualised was 10 µg, which exceeds the usual physiological concentrations of cholesterol precursor sterols. Because 8‐DHC is not available commercially, we extracted the plasma sterols of a patient biochemically severely affected by SLOS (7‐DHC 665 µmol/l, 8‐DHC 373 µmol/l, cholesterol 367 µmol/l by GC‐MS analysis) and separated the dehydrosterols by TLC. The spots were scraped from the plate, extracted and analysed by gas chromatography‐mass spectrometry (GC‐MS). 7‐DHC and cholesterol were detected in the positions known from the standards, while 8‐DHC was found in the same position as desmosterol and lathosterol.
Influence of different organic solvents on sterol separation
Different ratios of benzene:ethylacetate were tested to identify the one allowing optimal separation of sterols, especially of cholesterol from desmosterol. Figure 2 shows that a sufficient separation of all sterols apart from cholesterol and desmosterol was obtained by benzene/ethylacetate 9/1 v/v and benzene/ethylacetate 5/1 v/v. Cholesterol and desmosterol could not be separated by any combination of the organic solvents. Higher concentrations of benzene caused lower RFs and poorer separation of lathosterol and desmosterol. Chloroform/acetone 27.6/1 for primary and diethyl ether/n‐hexane 5/1 used for secondary development, which are described in the literature,22 resulted in the highest RF values, but adequate separation of cholesterol, desmosterol and lathosterol could not be obtained.
Figure 2 Influence of different organic solvents on the sterol separation. 10 µM of unmarked sterol standards (lanosterol, cholesterol, desmosterol, lathosterol and 7‐dehydrocholesterol (DHC)) was applied on a 10% silver nitrate silica plate and resolved with different mobile phases (two developments each). (A) Benzene/ethylacetate 5/1 v/v, (B) benzene/ethylacetate 9/1 v/v, (C) benzene/ethylacetate 19/1 v/v, and (D) primary development: chloroform/acetone 27.6/1 v/v; RF, retention fraction; secondary development: dimethyl ether/n‐hexane 5/1 v/v.
Inhibitors of cholesterol biosynthesis demonstrate sufficient separation of most [2‐14C]sterols
The amount of sterol required to visualise non‐radioactive sterols on the TLC plate greatly exceeds physiological concentrations. We therefore investigated whether a better separation could be achieved with the lower sterol concentrations used in our radioactive test system. To produce specific patterns of cholesterol precursors, we incubated control LC with three different inhibitors of cholesterol synthesis, AY9944, triparanol and progesterone, followed by [2‐14C]acetate loading (fig 3). The resulting sterol patterns without radioactive labelling were analysed in parallel by GC‐MS.
Figure 3 Effect of cholesterol biosynthesis inhibitors on control lymphoblastoid cells (LCs) in the [2‐14C]acetate loading test. Control LCs were preincubated on lipid‐depleted cell culture medium for 24 h, and exposed to 1 µM AY9944, 1 µM triparanol, or 10 µM progesterone for 30 min before 2.5 µCi [2‐14C]acetate was added for 2 h. The LCs were then chased in lipid‐depleted cell culture medium containing 144 mM sodium acetate for another 2 h. DHC, dehydrocholesterol.
Triparanol is an inhibitor mainly of sterol‐Δ24 reductase, causing an accumulation of desmosterol, which was demonstrated in both test systems. At higher concentrations of triparanol, sterol‐Δ8‐isomerase is also blocked,23 causing an increase in 8(9)‐cholestenol and 8‐DHC levels. This inhibition was only seen in the more sensitive [2‐14C]acetate loading test, with the corresponding bands located directly above 7‐DHC and directly below desmosterol/lathosterol. A sufficient separation of cholesterol and desmosterol and of desmosterol and 8‐DHC in the radioactive test, when the amounts were not much greater than physiological concentrations, was therefore confirmed.
AY9944 inhibits sterol‐Δ7 reductase. The resulting accumulation of 7‐DHC and, to a far lesser degree, 8‐DHC11 as well as reduced cholesterol synthesis were clearly shown in both test systems, but were more evident in the loading test. Higher concentrations of AY9944 inhibit sterol‐Δ8‐isomerase,24 which could be shown in both systems.
Progesterone inhibition, expected to block sterol‐Δ24 reductase at a concentration of 10 µM,25 caused diminished cholesterol synthesis and moderate increases in desmosterol, which were not as prominent as with triparanol. Slight increases in all identified sterols and the occurrence of additional unknown bands are in accordance with a possible role of progesterone in the inhibition of intracellular sterol transport.26
Because the [2‐14C]acetate loading test labels only newly synthesised sterols, the sterol quantification differs from the GC‐MS analysis, which represents all sterols in the cells (table 1). The location of the radioactive sterol bands corresponds to those of the non‐radioactive standards. However, [2‐14C]desmosterol and [2‐14C]lathosterol could not be separated by the system. Because an increase of each component would result in an abnormal pattern and differentiation could be achieved later by GC‐MS, we considered this sufficient for a screening test.
Table 1 Quantification of sterols after exposure of control cells to inhibitors of cholesterol biosynthesis: comparison between gas chromatography‐mass spectrometry and the [2‐14C]acetate loading test.
| Controls (n = 9) | Controls+inhibitors (n = 2) | ||||||
|---|---|---|---|---|---|---|---|
| Median | Range | Mean (SD) | Triparanol 1 µM | AY9944 1 µM | Progesterone 10 µM | ||
| GC‐MS | |||||||
| Cholesterol | 94.3 | 92.5–97.5 | 94.7 (1.6) | 94.3 | 74.7 | 92.9 | |
| Desmosterol | 0.79 | 0.32–1.72 | 0.84 (0.4) | 3.25 | 0.45 | 4.45 | |
| Lathosterol | 1.42 | 0.34–1.99 | 1.36 (0.5) | 1.40 | 3.76 | 1.76 | |
| 7‐DHC | 0.41 | 0.16–0.55 | 0.41 (0.1) | 0.60 | 18.95 | 0.57 | |
| 8‐DHC | 0.2 | 0.13–0.46 | 0.24 (0.1) | 0.20 | 1.31 | 0.21 | |
| 8(9)‐Cholestenol | <dl | <dl | <dl | <dl | 1.34 | < dl | |
| Lanosterol | 0.13 | 0.01–1.72 | 0.32 (0.5) | 0.18 | 0.08 | 0.10 | |
| [2‐14C]acetate loading | |||||||
| Cholesterol | 82 | 74–90 | 82 (5) | 28 | 5 | 19 | |
| Desmosterol/lathosterol | 8.2 | 4.4–10.6 | 7.8 (1.9) | 22 | 16 | 16 | |
| 7‐DHC | 1.9 | 1.0–3.4 | 2.0 (0.8) | 12 | 39 | 34 | |
| 8‐DHC | <dl | <dl | <dl | 9 | 14 | 9 | |
| 8(9)‐Cholestenol | <dl | <dl | <dl | 21 | 21 | 11 | |
| Lanosterol | 4.7 | 2.3–7.9 | 4.7 (1.9) | 7 | 6 | 10 | |
DHC, dehydrocholesterol; dl, detection limit; GC‐MS, gas chromatography‐mass spectrometry.
GC‐MS analysis was performed after cultivation of the cells on lipid‐depleted culture medium for 48 h and exposure to each inhibitor for 72 h. The [2‐14C]acetate loading test was performed as described in the legend of fig 3. Data are expressed as percentage of total sterols.
Cholesterol metabolism in human LC lines
We next investigated the sterol pattern in LC lines from 228 patients with HPE using the [2‐14C]acetate loading test. The amount of newly synthesised radioactive sterols was compared with controls (n = 9, table 1). An abnormal sterol pattern was defined if the percentage of total sterols exceeded (cholesterol precursors) or fell below (cholesterol) 2 SD of the control mean. Because 8(9)‐cholestenol is undetectable in controls, any amount was considered abnormal. GC‐MS sterol analysis was performed in all cell lines with an abnormal sterol pattern in the [2‐14C]acetate loading test. Experiments were performed either in triplicate ([2‐14C]acetate loading test) or in duplicate (GC‐MS).
Abnormal sterol patterns were found in 22 patients (9.6%) by [2‐14C]acetate loading (table 2). Mean (SD) cholesterol levels as a fraction of total sterols in these patients were 57% (15.3%), whereas they were 82% (4.7%) in controls (p<0.001). Mean (SD) 7‐DHC levels were 11.3% (8.9%) in patients and 2% (0.8%) in controls (p<0.001). Mean (SD) desmosterol/lathosterol/8‐DHC levels were 18% (7.5%) in patients and 7.8% (1.9%) in controls (p<0.001). Mean (SD) lanosterol levels were not significantly different in patients and controls: patients 6.8% (4.0%) and controls 4.7% (1.9%, p = 0.16). Notably, nine of the patients with abnormal loading test results had normal sterol profiles by GC‐MS.
Table 2 Incorporation of [2‐14C]acetate into cellular sterols and gas chromatography–mass spectrometry sterol analysis of lymphoblastoid cell lines from a patient with known Smith–Lemli–Opitz syndrome and from 22 patients with holoprosencephaly showing an abnormal sterol pattern in the [2‐14C]acetate loading test.
| Cholesterol | Desmosterol | Lathosterol | 7‐DHC | 8‐DHC | Lanosterol | DHCR7 mutation | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 14C acetate loading | GC‐MS | 14C acetate loading | GC‐MS | 14C acetate loading | GC‐MS | 14C acetate loading | GC‐MS | 14C acetate loading | GC‐MS | 14C acetate loading | GC‐MS | ||
| Controls | 72–91 | 91–98 | 11.7 | 1.7 | 11.7 | 2.4 | 3.5 | 0.64 | 11.7 | 0.4 | 8.5 | 1.3 | |
| Patient | |||||||||||||
| SLOS | 4.9 | 60 | 25 | 0.1 | 25 | 4.7 | 61 | 32 | 25 | 3.7 | 8.0 | 0.1 | R352W/IVS8‐1G>C |
| 144 | 63 | 91 | 15 | 2.3 | 15 | 2.1 | 4.8 | 0.6 | 15 | 0.3 | 8.9 | 1.9 | ND |
| 329 | 60 | 95 | 16 | 0.9 | 16 | 1.9 | 9.6 | 0.4 | 16 | 0.2 | 7.4 | 0.3 | −/− |
| 331 | 36 | 94 | 37 | 0.7 | 37 | 3.6 | 13 | 0.9 | 37 | 0.4 | 8.4 | 0.1 | −/− |
| 383 | 68 | 93 | 13 | 0.4 | 13 | 5.1 | 6.9 | 0.8 | 13 | 0.4 | 3.3 | < dl | −/− |
| 613 | 19 | 72 | 23 | 0.1 | 23 | 3 | 47 | 22 | 23 | 1.8 | 6.3 | 0.2 | T154R/G410S |
| 646 | 34 | 97 | 38 | < dl | 38 | 1.6 | 11 | 0.8 | 38 | 0.2 | 8.6 | < dl | ND |
| 719 | 62 | 95 | 17 | 0.3 | 17 | 2.0 | 5.4 | 0.5 | 17 | 0.3 | 8.0 | < dl | −/− |
| 811 | 76 | 94 | 11 | 0.4 | 11 | 1.8 | 5.8 | 1.4 | 11 | 0.2 | 3 | < dl | −/− |
| 859 | 37 | 92 | 21 | 5.4 | 21 | 1 | 11 | 0.7 | 21 | 0.7 | 22 | < dl | ND |
| 887 | 56 | 94 | 19 | 0.8 | 19 | 2.1 | 6.2 | 0.4 | 19 | 0.2 | 7 | 0.4 | −/− |
| 893 | 64 | 91 | 18 | 0.3 | 18 | 5.8 | 2.8 | 0.9 | 18 | 0.5 | 9.2 | 0.1 | ND |
| 1265 | 63 | 93 | 14 | 0.2 | 14 | 0.9 | 14 | 0.9 | 14 | 1.5 | 6.9 | < dl | −/− |
| 6006 | 48 | 94 | 18 | 0.4 | 18 | 1.3 | 19 | 1.6 | 18 | 0.4 | 3.9 | < dl | −/− |
| 6833 | 75 | 95 | 11 | 0.4 | 11 | 2.2 | 7.9 | 0.4 | 11 | 0.2 | 3.2 | 0.4 | −/− |
| 7304 | 58 | 94 | 21 | 0.9 | 21 | 1.9 | 11 | 0.5 | 21 | 0.2 | 5.4 | 0.5 | −/− |
| 7334 | 77 | 94 | 7.6 | 0.4 | 7.6 | 2.1 | 5.6 | 0.5 | 7.6 | 0.4 | 5.9 | 0.1 | R242H/− |
| 7835 | 66 | 96 | 14 | 0.6 | 14 | 1.6 | 12 | 0.9 | 14 | 0.3 | 4.9 | 0.4 | −/− |
| 7858 | 74 | 93 | 10 | 0.4 | 10 | 0.8 | 11 | 0.3 | 10 | 0.2 | 2.6 | 0.2 | −/− |
| 7880 | 51 | 91 | 18 | 0.5 | 18 | 1.7 | 12 | 0.3 | 18 | 0.2 | 5.1 | 0.3 | −/− |
| 8064 | 61 | 94 | 18 | 0.9 | 18 | 1.5 | 5.9 | 0.7 | 18 | 0.4 | 8.6 | 0.5 | ND |
| 8103 | 42 | 97 | 22 | 1.1 | 22 | 0.7 | 14 | 0.3 | 22 | 0.2 | 8.6 | 0.3 | ND |
| 8121 | 66 | 93 | 14 | 0.3 | 14 | 1.3 | 13 | 0.3 | 14 | 0.1 | 2.7 | 0.3 | −/− |
DHC, dehydrocholesterol; DHCR, 7‐dehydrosterol reductase; GC‐MS, gas chromatography‐mass spectrometry; ND, not detected; SLOS, Smith–Lemli–Opitz syndrome.
Indicated is the percentage of total sterols, mean of three independent experiments ([2‐14C]acetate‐loading) or two independent experiments (GC‐MS). The control values represent mean (2 SD) of nine controls (cholesterol) or mean (2 SD) (cholesterol precursors). Data are printed in bold if they fell below this range (cholesterol) or exceeded this range (cholesterol precursors).
Patient 613 displayed the typical SLOS pattern both by [2‐14C]acetate loading and by GC‐MS, notably a marked increase in 7‐DHC. GC‐MS analysis additionally showed a moderate increase in 8‐DHC and decrease in cholesterol. There was a mild, possibly non‐specific, increase in lathosterol. In the [2‐14C]acetate loading test, there was nearly no newly synthesised cholesterol, reflecting the severity of biochemical impairment. Surprisingly, in this patient and in a patient with known SLOS (SLOS‐positive control in table 2), 8‐DHC could not be separated from the desmosterol/lathosterol band. The moderately increased desmosterol/lathosterol/8‐DHC band therefore likely reflects an accumulation of 8‐DHC. Patient 613 had two different mutations in DHCR7, confirming the diagnosis of SLOS. Because 7‐DHC was the prominent sterol in 14 other cell lines, we excluded SLOS in them by mutation analysis (table 2). In the remaining patients, [2‐14C]acetate loading revealed an abnormal sterol pattern with equal increases in several sterols and a reduction in cholesterol in most patients. Table 3 lists the clinical features of all patients with an abnormal sterol pattern.
Table 3 Clinical characterisation of patients with an abnormal sterol pattern in lymphoblastoid cells.
| Patient | Age at diagnosis | Gender | Mutation | Cerebral malformation | Facial malformation | Additional clinical features | Severity | Reference |
|---|---|---|---|---|---|---|---|---|
| 144 | 1 day | F | Alobar HPE, ACC, arrhinencephaly, Chiari I malformation | Dysplastic ears, wide nasal bridge, high narrow palate | Lumbosacral MMC | Severe | ||
| 329 | 3 months | F | PTC: T728M | Partial ACC | Midline CLP, SCI, hypotelorism, flat midface | Omphalocele, ambiguous genitalia, panhypopituitarism, PDA | Severe | Ming 200227 |
| 331 | 3 days | F | Semilobar HPE, hypoplastic CC, pituitary gland absent | Microcephaly, midline CLP, hypotelorism, up‐slanted palpebral fissures | ASD, PDA, vascular malformation, talipes equinovarus, NEC, anal malformation, epilepsy | Moderate–severe | ||
| 383 | 1 month | F | Semilobar HPE | Microcephaly, Robin sequence, preauricular tag | VSD, ASD, partial syndactyly toes 2,3,4 r | Moderate | ||
| 613 | 2 months | F | DHCR7: T154R/G410S | Semilobar HPE, cebocephaly | Microcephaly, median CLP, hypotelorism, ptosis, single nostril | Partial bilateral syndactyly toes 2+3, polydactyly | Moderate–severe | Kelley 199612 |
| 646 | Adult | F | 46, XX, t(10;13) (p15;q23) | Normal brain | Normal face | Two sons with alobar HPE | Asymptomatic | |
| 719 | 2½ years | F | Lobar HPE, partial ACC, singular ventricle | Microcephaly, hypotelorism, SCI, microphthalmia | DI, infantile spasms, spastic tetraparesis | Mild–moderate | ||
| 811 | Adult | M | Normal brain | Normal face | Son with semilobar HPE | Asymptomatic | ||
| 859 | 1 day | M | 46, XY, r (7); del SHH | Semilobar HPE | Microcephaly, median CLP, hypotelorism, ptosis, single nostril | Micropenis, rugate scrotum, hypoplastic nipples, intractable seizures | Severe | Sawyer 199628 |
| 887 | 7 years | F | Semilobar HPE, partial ACC, hydrocephalus, IH cyst | Mental retardation | Mild–moderate | |||
| 893 | 2½ months | F | dup ZIC2 | Semilobar HPE | Normal face | None | Not known | |
| 1265 | 20 months | F | SIX3: L226V | Normal brain | Microcephaly, hypotelorism, midface hypoplasia, absence of nasal bones, bilateral CLP | None | Mild | Wallis 19995 |
| 6006 | 1 month | F | Alobar HPE, hydrocephalus | Hypotelorism, midface hypoplasia, small nose, submucous CP | Seizures | Moderate | ||
| 6833 | 3 months | M | ZIC2: 552‐568 dup16 | Semilobar HPE | Microbrachycephaly, narrrow palate | DI, muscular hypertonia, severe psychomotor retardation, recurrent aspiration | Moderate–severe | |
| 7304 | F | |||||||
| 7334 | 4 months | F | HPE | Not known | ||||
| 7835 | 9 months | M | HPE | SCI, nasal pyriform aperture stenosis | Mild | |||
| 7858 | 5 months | F | HPE | Not known | ||||
| 7880 | 20 months | F | Semilobar HPE | Mild | ||||
| 8064 | 5 months | F | Semilobar HPE | Mild | ||||
| 8103 | F | |||||||
| 8121 | 4 months | F | Normal brain | Microcephaly, hypotelorism, SCI, low‐set ears, bilateral cataracts | IUGR, oligohydramnios, 2/3 toe syndactyly, hypopituitarism, mental retardation | Mild |
ACC, agenesis of corpus callosum; ASD, atrial septal defect; CC, corpus callosum; CLP, cleft lip palate; CP, cleft palate; DI, diabetes insipidus; HPE, holoprosencephaly; IH, intrahemispheric; IUGR, intrauterine growth retardation; MMC, myelomeningocele; NEC, necrotising enterocolitis; PDA, patent ductus arteriosus; SCI, single central incisor; VSD, ventricular septal defect.
Discussion
In an important investigation into the biochemical basis of HPE in SLOS, Cooper et al9 showed that abnormal SHH signalling in SLOS was caused by decreased cellular levels of cholesterol, not by the increase of any cholesterol precursor. Because SLOS represents only one of many genetic disorders that should manifest decreased tissue levels of cholesterol, we developed a sensitive sterol‐labelling assay to detect both mild enzymatic defects in post‐squalene cholesterol biosynthesis and disorders in which the dominant abnormality will be only a decreased rate of cholesterol synthesis. We previously described this assay as a sensitive and rapid method for diagnosis of SLOS in human fibroblasts even in biochemically mildly affected patients.29 In lymphoblasts of a patient with known moderately affected SLOS, the assay shows an accumulation of 7‐DHC and desmosterol/lathosterol/8‐DHC and a massively reduced cholesterol synthesis (table 2; patient with SLOS). We then applied this new assay to 228 HPE cell lines, in which we expected to find a variety of abnormalities of cholesterol homeostasis.
In 22 of 228 patients with HPE, we found impaired cholesterol biosynthesis. In all, 18 of these 22 patients had reduced incorporation of [2‐14C]acetate into cholesterol, and all had abnormal levels of one or more different cholesterol precursors (table 2). Notably, nine of these patients had normal GC‐MS results, which we interpret as a greater sensitivity of the [2‐14C]acetate loading test, which specifically displays newly synthesised sterols under unfavourable metabolic conditions, whereas the GC‐MS method measures total cellular sterols. Nevertheless, GC‐MS is indispensable to clearly identify the accumulated sterols, because desmosterol, lathosterol and 8‐DHC cannot be separated by the TLC method.
In addition to one patient with a SLOS pattern and confirmed mutations in DHCR7 (T154R/G410S), we found an isolated accumulation of 7‐DHC in four patients and a combination of increased 7‐DHC and desmosterol/lathosterol/8‐DHC in 11 others. Because we could not determine which of the three latter sterols was increased, SLOS was at first a possible diagnosis in all patients. However, SLOS was excluded in all by molecular analysis (table 2).
Five patients had alterations in known HPE genes in addition to their sterol abnormalities. Patient 859 had a midline cleft palate and a ring chromosome 7 with 7q36.3 deletion including SHH and an accumulation of lanosterol, a C30 sterol, in the [2‐14C]acetate loading test. This observation is consistent with possible haploinsufficiency for INSIG1, which is located at 7q36.3, close to SHH. Insig‐deficient mouse embryos have midline facial clefting and accumulate lanosterol and other cholesterol precursors,30 presumably on the basis of enhanced degradation rates for enzymes distal to lanosterol synthase. In patient 329, a PTC T728M mutation was found, which is located between transmembrane domains 6 and 7. This domain is predicted to be a cytoplasmic loop with yet unknown functional relevance. It is not associated with the sterol‐sensing domain (SSD_5TM; IPR000731) in the patched protein, so a primary effect of this mutation on sterol levels is unlikely. In patient 646, a t(10;13)(p15;q23) translocation was identified. Even though we have not studied the translocation breakpoints in detail, it is of theoretical interest that IDI1 is located in 10p15. IDI1 encodes a peroxisomally localised enzyme that catalyses the interconversion of isopentenyl diphosphate to dimethylallyl diphosphate, an early step in cholesterol biosynthesis. Although a defect in the pre‐squalene pathway could impair overall cholesterol synthesis and cause the reduced incorporation of [2‐14C]acetate into cholesterol, the observed accumulation of cholesterol precursors could not be explained. There is no evidence for sterol regulatory genes in 13q22.3–13q32.
In the remaining five patients (144, 646, 893, 8064 and 8103), nearly all cholesterol precursors were increased to the same amount, whereas cholesterol synthesis was variably reduced, so that a single enzyme block was unlikely. In earlier studies, it was reported that the cholesterol precursors desmosterol, lathosterol, lanosterol, zymosterol and 7‐DHC are transported from their site of synthesis in the endoplasmic reticulum (ER) to the cell membrane.31,32,33,34 There is evidence that the transport process of these sterols differs from that of newly synthesised cholesterol and from each other.34 After arrival at the cell membrane, sterol precursors are either effluxed to high‐density lipoprotein and phosphatidylcholine vesicles,31 or transported back to the ER for conversion to cholesterol.35 Disruption of sterol transport from the ER to the plasma membrane or back to the ER will cause an accumulation of one or more cholesterol precursors within the cell and in a reduced availability of cholesterol. We therefore speculate that an impaired intracellular transport of cholesterol precursors and the resulting decrease in cholesterol could alter SHH signalling in these patients.
Taken together, our data suggest that an accumulation of sterol intermediates caused either by defective regulation of cholesterol biosynthesis or by defects of intracellular sterol transport could further aggravate impaired SHH signalling, leading to a severe malformation syndrome. A detailed analysis of cholesterol biosynthesis and homeostasis should therefore be considered in all patients with HPE.
Electronic database information
Bioinformatic Harvester (http://harvester.embl.de); InterPro (http://www.ebi.ac.uk/interpro); Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = gene); UCSC Proteome Browser (http://genome.ucsc.edu/cgi‐bin/pbGateway).
Acknowledgements
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to DH and GFH (HA 2853/2‐1) and by the Division of Intramural Research, National Genome Research Institute, NIH, to MM. We thank E Strohmaier and B Janssen for DHCR7 mutation analysis and T Paulik‐Rebe, P Feyh, A Hinz, S Exner‐Camps and M Herm for excellent technical assistance.
Abbreviations
DHC - dehydrocholesterol
ER - endoplasmic reticulum
GC‐MS - gas chromatography‐mass spectrometry
HPE - holoprosencephaly
PBS - phosphate‐buffered saline
LC - lymphoblastoid cell
RF - retention fraction
SHH - Sonic Hedgehog
SLOS - Smith–Lemli–Opitz syndrome
TLC - thin‐layer chromatography
Footnotes
Competing interests: None.
References
- 1.Muenke M, Beachy P A. Holoprosencephaly. In: Scriver CR, Beaudet AL, eds. The metabolic and molecular bases of inherited disease. 8th edn. New York: McGraw‐Hill, 2001
- 2.Roessler E, Muenke M. Holoprosencephaly: a paradigm for the complex genetics of brain development. J Inherit Metab Dis 199821481–497. [DOI] [PubMed] [Google Scholar]
- 3.Roessler E, Belloni E, Gaudenz K, Jay P, Berta P, Scherer S W, Tsui L C, Muenke M. Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat Genet 199614357–360. [DOI] [PubMed] [Google Scholar]
- 4.Brown S A, Warburton D, Brown L Y, Yu C Y, Roeder E R, Stengel‐Rutkowski S, Hennekam R C, Muenke M. Holoprosencephaly due to mutations in ZIC2, a homologue of Drosophila odd‐paired. Nat Genet 199820180–183. [DOI] [PubMed] [Google Scholar]
- 5.Wallis D E, Roessler E, Hehr U, Nanni L, Wiltshire T, Richieri‐Costa A, Gillessen‐Kaesbach G, Zackai E H, Rommens J, Muenke M. Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nat Genet 199922196–198. [DOI] [PubMed] [Google Scholar]
- 6.Gripp K W, Wotton D, Edwards M C, Roessler E, Ades L, Meinecke P, Richieri‐Costa A, Zackai E H, Massague J, Muenke M, Elledge S J. Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet 200025205–208. [DOI] [PubMed] [Google Scholar]
- 7.Bendavid C, Haddad B H, Griffin A, Huizing M, Dubourg C, Gicquel I, Cavalli L R, Pasquier L, Long B, Ouspenskaia M, Odent S, Lacbawan F, David V, Muenke M. Multicolor FISH and quantitative PCR and detect submicroscopic deletions in holoprosencephaly patients with a normal karyotype. J Med Genet 200643496–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Cohen M M, Jr, Shiota K. Teratogenesis of holoprosencephaly. Am J Med Genet 20021091–15. [DOI] [PubMed] [Google Scholar]
- 9.Cooper M K, Wassif C A, Krakowiak P A, Taipale J, Gong R, Kelley R I, Porter F D, Beachy P A. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 200333508–513. [DOI] [PubMed] [Google Scholar]
- 10.Roux C. Teratogenic action of triparanol in animals. Arch Fr Pediatr 196421451–464. [PubMed] [Google Scholar]
- 11.Roux C, Aubry M. Teratogenic action in the rat of an inhibitor of cholesterol synthesis, AY 9944. C R Seances Soc Biol Fil 19661601353–1357. [PubMed] [Google Scholar]
- 12.Kelley R L, Roessler E, Hennekam R C, Feldman G L, Kosaki K, Jones M C, Palumbos J C, Muenke M. Holoprosencephaly in RSH/Smith‐Lemli‐Opitz syndrome: does abnormal cholesterol metabolism affect the function of Sonic Hedgehog? Am J Med Genet 199666478–484. [DOI] [PubMed] [Google Scholar]
- 13.Willnow T E, Hilpert J, Armstrong S A, Rohlmann A, Hammer R E, Burns D K, Herz J. Defective forebrain development in mice lacking gp330/megalin. Proc Natl Acad Sci USA 1996938460–8464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Binns W, James L F, Shupe J L, Everett G. A congenital cyclopian‐type malformation in lambs induced by maternal ingestion of a range plant, veratum californicum. Am J Vet Res 1963241164–1175. [PubMed] [Google Scholar]
- 15.Chen J K, Taipale J, Cooper M K, Beachy P A. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 2002162743–2748. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ming J E, Muenke M. Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J Hum Genet 2002711017–1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Edison R, Muenke M. The interplay of genetic and environmental factors in craniofacial morphogenesis: holoprosencephaly and the role of cholesterol. Congenit Anom (Kyoto) 2003431–21. [DOI] [PubMed] [Google Scholar]
- 18.Ventura M, Gibaud A, Le Pendu J, Hillaire D, Gerard G, Vitrac D, Oriol R. Use of a simple method for the Epstein‐Barr virus transformation of lymphocytes from members of large families of Reunion Island. Hum Hered 19883836–43. [DOI] [PubMed] [Google Scholar]
- 19.Haas D, Armbrust S, Haas J P, Zschocke J, Mühlmann K, Fusch C, Neumann L M. Smith‐Lemli‐Opitz syndrome with a classical phenotype, esophageal achalasia and borderline sterol plasma levels. J Inherit Metab Dis 2005281191–1196. [DOI] [PubMed] [Google Scholar]
- 20.Edgington E S.Randomization tests. New York: Dekker, 1995
- 21.Streitberg B, Röhmel J. Exact distributions for permutations and rank tests: an introduction to some recently published algorithms. Stat Softw Newsl 19861210–17. [Google Scholar]
- 22.Brunetti‐Pierri N, Corso G, Rossi M, Ferrari P, Balli F, Rivasi F, Annunziata I, Ballabio A, Russo A D, Andria G, Parenti G. Lathosterolosis, a novel multiple‐malformation/mental retardation syndrome due to deficiency of 3beta‐hydroxysteroid‐delta5‐desaturase. Am J Hum Genet 200271952–958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Chevy F, Illien F, Wolf C, ROUX C Limb malformations of rat fetuses exposed to a distal inhibitor of cholesterol biosynthesis. J Lipid Res 2002431192–1200. [PubMed] [Google Scholar]
- 24.Rahier A, Taton M. Sterol biosynthesis: strong inhibition of maize delta 5,7‐sterol delta 7‐reductase by novel 6‐aza‐B‐homosteroids and other analogs of a presumptive carbocationic intermediate of the reduction reaction. Biochemistry 1996357069–7076. [DOI] [PubMed] [Google Scholar]
- 25.Lindenthal B, Holleran A L, Aldaghlas T A, Ruan B, Schroepfer G J, Jr, Wilson W K, Kelleher J K. Progestins block cholesterol synthesis to produce meiosis‐activating sterols. FASEB J 200115775–784. [DOI] [PubMed] [Google Scholar]
- 26.Metherall J E, Waugh K, Li H. Progesterone inhibits cholesterol biosynthesis in cultured cells. Accumulation of cholesterol precursors. J Biol Chem 19962712627–2633. [DOI] [PubMed] [Google Scholar]
- 27.Ming J E, Kaupas M E, Roessler E, Brunner H G, Golabi M, Tekin M, Stratton R F, Sujansky E, Bale S J, Muenke M. Mutations in patched‐1, the receptor for Sonic Hedgehog, are associated with holoprosencephaly. Hum Genet 2002110297–301. [DOI] [PubMed] [Google Scholar]
- 28.Sawyer J R, Lukacs J L, Hassed S J, Arnold G L, Mitchell H F, Muenke M. Sub‐band deletion of 7q36. 3 in a patient with ring chromosome 7: association with holoprosencephaly, Am J Med Genet 199665113–116. [DOI] [PubMed] [Google Scholar]
- 29.Haas D, Morgenthaler J, Neumann L M, Armbrust S, Zschocke J, Okun J G, Hoffmann G F. In vitro 14C acetate loading of human fibroblasts as a sensitive method to identify Smith‐Lemli‐Opitz patients with a mild biochemical phenotype. J Inherit Metab Dis 200427(Suppl 1)211 [Google Scholar]
- 30.Engelking L J, Evers B M, Richardson J A, Goldstein J L, Brown M S, Liang G. Severe facial clefting in Insig‐deficient mouse embryos caused by sterol accumulation and reversed by lovastatin. J Clin Invest 20061162356–2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Johnson W J, Fischer R T, Phillips M C, Rothblat G H. Efflux of newly synthesized cholesterol and biosynthetic sterol intermediates from cells. Dependence on acceptor type and on enrichment of cells with cholesterol. J Biol Chem 199527025037–25046. [DOI] [PubMed] [Google Scholar]
- 32.Lange Y, Muraski M F. Cholesterol is not synthesized in membranes bearing 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. J Biol Chem 19872624433–4436. [PubMed] [Google Scholar]
- 33.Echevarria F, Norton R A, Nes W D, Lange Y. Zymosterol is located in the plasma membrane of cultured human fibroblasts. J Biol Chem 19902658484–8489. [PubMed] [Google Scholar]
- 34.Lusa S, Heino S, Ikonen E. Differential mobilization of newly synthesized cholesterol and biosynthetic sterol precursors from cells. J Biol Chem 200327819844–19851. [DOI] [PubMed] [Google Scholar]
- 35.Lange Y, Echevarria F, Steck T L. Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts. J Biol Chem 199126621439–21443. [PubMed] [Google Scholar]