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Human Molecular Genetics Advance Access originally published online on April 2, 2007
Human Molecular Genetics 2007 16(10):1176-1187; doi:10.1093/hmg/ddm065
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Published by Oxford University Press 2007. For Permissions, please email: journals.permissions@oxfordjournals.org

HEM dysplasia and ichthyosis are likely laminopathies and not due to 3ß-hydroxysterol {Delta}14-reductase deficiency

Christopher A. Wassif1, Kirstyn E. Brownson1, Allison L. Sterner1, Antonella Forlino3, Patricia M. Zerfas2, William K. Wilson4, Matthew F. Starost2 and Forbes D. Porter1,*

1 Heritable Disorders Branch, NICHD and 2 Diagnostic and Research Services Branch, OD, NIH, DHHS, Bethesda, MD 20892, USA 3 Department of Biochemistry, Section of Medicine and Pharmacy, University of Pavia, Italy 4 Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA

* To whom correspondence should be addressed at: HDB, NICHD, NIH, DHHS Bld. 10, Room 9D42 10 Center Dr, Bethesda, MD 20892-1830, USA. Tel: +1 3014354432; Fax: +1 3014805791; Email: fdporter{at}mail.nih.gov

Received January 30, 2007; Revised March 5, 2007; Accepted March 13, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosis in mice. LBR is a bifunctional protein with both a lamin B binding and a sterol {Delta}14-reductase domain. It previously has been proposed that LBR is the primary sterol {Delta}14-reductase and that HEM dysplasia and ichthyosis are inborn errors of cholesterol synthesis. However, DHCR14 also encodes a sterol {Delta}14-reductase and could provide enzymatic redundancy with respect to cholesterol synthesis. To test the hypothesis that LBR and DHCR14 both function as sterol {Delta}14-reductases, we obtained ichthyosis mice (Lbr–/–) and disrupted Dhcr14. Heterozygous Lbr and Dhcr14 mice were intercrossed to test for a digenic phenotype. Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mutant mice have distinct physical and biochemical phenotypes. Dhcr14{Delta}4-7/{Delta}4-7 mice are essentially normal, whereas Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice are growth retarded and neurologically abnormal. Neither of these mutants resembles the ichthyosis mouse and biochemically, no sterol abnormalities were detected in either liver or kidney tissue. In contrast, relatively small transient elevations of {Delta}14-sterols were observed in Lbr–/– and Dhcr14{Delta}4-7/{Delta}4-7 brain tissue, and marked elevations were seen in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 brain. Pathological evaluation demonstrated vacuolation and swelling of the myelin sheaths in the spinal cord of Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice consistent with a demyelinating process. This was not observed in either Lbr–/– or Dhcr14 {Delta}4-7/{Delta}4-7 mice. Our data support the conclusions that LBR and DHCR14 provide substantial enzymatic redundancy with respect to cholesterol synthesis and that HEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The synthesis of cholesterol from lanosterol requires removal of the 14{alpha}-methyl group from either lanosterol or dihydrolanosterol which results in the formation of a C14–C15 double bond. This C14–C15 double bond must subsequently be reduced by a sterol {Delta}14-reductase (1) (Fig. 1A). Two different genes, DHCR14 and LBR, have been shown to encode proteins with sterol {Delta}14-reductase activity. DHCR14 (TM7SF2, SR-1) was initially cloned from bovine liver, and expression in COS-7 cells confirmed that it has sterol {Delta}14-reductase activity (2). The lamin B receptor (LBR) has two functional domains: the N-terminal domain projects into the nucleoplasm and binds to both B-type lamins and chromatin proteins (3), whereas the C-terminus of LBR is highly homologous to sterol reductases and has been shown to encode sterol {Delta}14-reductase activity (4).


Figure 1
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  		Figure 1. (A) During cholesterol synthesis, the C14–C15 double bond found in 4,4-dimethylcholesta-8(9),14-dien-3ß-ol is reduced by a sterol {Delta}14-reductase. Both DHCR14 and LBR have been shown to have sterol {Delta}14-reductase activity. If reduction of the C14-C15 double bond is impaired, 4,4-dimethylcholesta-8(9),14-dien-3ß-ol is metabolized to yield cholesta-8(9),14-dien-3ß-ol. Sterol {Delta}24-reduction can occur at various points in the cholesterol synthetic pathway, thus although not depicted, a similar mechanism explains the accumulation of cholesta-8(9),14,24-trien-3ß-ol. (B) The Dhcr14 genomic structure, targeting vector and resulting mutant allele. Filled boxes with roman numerals represent the exons. The relative positions of PCR primers used for identifying properly targeted ES cell clones are labeled (A–D). (C) PCR analysis of untargeted R1 ES cell and two targeted ES cell clones (c129 and c162). Proper homologous recombination between the targeting vector and the endogenous allele was confirmed by amplification of the 5' and 3' flanks using primer pairs A/B and C/D, respectively. (D) PCR genotyping of progeny from a heterozygous Dhcr14 intercross. A 248 bp PCR product corresponds to the control Dhcr14+ allele, and a 602 bp PCR product corresponds to the mutant Dhcr14{Delta}4-7 allele.

 
Over the past 14 years, a number of human malformation syndromes have been shown to be due to inborn errors of cholesterol synthesis (5,6). Hydrops-Ectopic calcification-Moth-eaten skeletal dysplasia (HEM dysplasia, Greenberg dysplasia, OMIM #215140) is a lethal, autosomal recessive, skeletal dysplasia initially described by Greenberg et al. (7). Clinical manifestations include short-limb dwarfism, hydrops and polydactyly. Radiographic findings include ectopic ossification, fragmented (moth-eaten) long bones, platyspondyly and deficient skull ossification. Sterol analysis of tissues from four HEM dysplasia fetuses showed normal cholesterol levels and abnormal, but minor (<1% of total sterols), elevations of cholesta-8(9),14-dien-3ß-ol and cholesta-8(9),14,24-trien-3ß-ol consistent with impaired sterol {Delta}14-reduction (5). Given the quantitatively minor sterol abnormality, it was hypothesized that the major dysmorphic features found in HEM dysplasia cases might be secondary to hormonal like effects of the accumulating 14-dehydrosterols (5). In 2003, Waterham et al. (8) reported an apparent homozygous mutation of the LBR gene in a HEM dysplasia patient, and this group concluded that LBR functions as the primary sterol {Delta}14 reductase. Homozygous mutations of Lbr in the mouse are associated with the ichthyosis phenotype (9). Sterol analysis of the ichthyosis mouse has not been reported. Mutations of LBR also underlie autosomal dominant Pelger–Huët anomaly (10). The Pelger–Huët anomaly consists of hyposegmentation of the nuclei of polymorphonuclear leukocytes. Although it may be only an issue of null versus hypomorphic alleles, the relationship between homozygous Pelger–Huët anomaly (which has not been associated with major malformations in humans) and HEM dysplasia is not yet clear (11).

Given the minor sterol abnormality reported in HEM dysplasia, we hypothesized that LBR and DHCR14 are redundant sterol {Delta}14-reductases and that both human HEM dysplasia and mouse ichthyosis result from impaired LBR function rather than impaired sterol synthesis. Herein, we report the development and characterization of a Dhcr14 mutant mouse strain, the biochemical characterization of ichthyosis mice (Lbr mutant), and characterization of compound Lbr and Dhcr14 mutant mice. Our findings support the hypothesis that LBR and DHCR14 provide substantial redundancy for sterol {Delta}14-reduction and strongly suggest that HEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Disruption of Dhcr14
Dhcr14 was disrupted in mouse embryonic stem cells using targeted homologous recombination. Recombination between the targeting vector and the endogenous Dhcr14 allele results in the insertion of the neomycin phosphotransferase gene (PGKneo) and disruption of exons 4–7 (Fig. 1B). PCR analysis identified 12/171 (7%) embryonic stem cell clones that underwent homologous recombination between the endogenous gene and both flanks of the targeting vector (Fig. 1C). Clone c129 was used to produce a germline-transmitting chimeric founder.

Phenotypic characterization
Heterozygous Dhcr14+/{Delta}4-7 mice appeared phenotypically normal. Thus, Dhcr14+/{Delta}4-7 mice were intercrossed to obtain homozygous Dhcr14 mutant mice (Fig. 1D). Dhcr14{Delta}4-7/{Delta}4-7 mice were identified after weaning in the expected Mendelian ratio (27, 50 and 23% for Dhcr14+/+, Dhcr14+/{Delta}4-7 and Dhcr14{Delta}4-7/{Delta}4-7, respectively, n = 168, P = 0.90). Dhcr14{Delta}4-7/{Delta}4-7 mice appeared normal (Fig. 2) and histopathological analysis revealed no significant differences between control and mutant mice. Dhcr14{Delta}4-7/{Delta}4-7 mutant mice were fertile and produced normal size litters. Eight female Dhcr14{Delta}4-7/{Delta}4-7 mice were followed for over 1 year, and no age-dependent problems were identified.


Figure 2
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  		Figure 2. Dhcr14{Delta}4-7/{Delta}4-7, Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 phenotypes. Photograph of 10-day-old live (A) and euthanized (B) Dhcr14{Delta}4-7/{Delta}4-7 (left), Lbr–/– (middle) and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 (right) mice. Dhcr14{Delta}4-7/{Delta}4-7 resemble normal control mice, whereas Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 have distinct phenotypes.

 
Lbr+/1088insCC mice were obtained from Jackson Laboratories and intercrossed to obtain Lbr1088insCC/1088insCC (Lbr–/–) offspring. The icJ mutation in Lbr, 1088CC, results in a frame shift that alters 21 amino acids (365–385) and introduces a stop codon at position p.386 (9). Lbr–/– mice could be identified on the basis of sparse hair by 9 days of age. Phenotypic findings in Lbr–/– mice were consistent with those previously described for these mice (9) and included growth retardation, ichthyosis, Pelger–Huët anomaly and syndactyly.

Dhcr14+/{Delta}4-7 and Lbr+/– heterozygous mice were interbred to test for a digenic phenotype. Lbr+/–:Dhcr14+/{Delta}4-7 compound heterozygous mice were viable, phenotypically normal and fertile. These mice were intercrossed to obtain Lbr–/–:Dhcr14+/{Delta}4-7, Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. In conjunction with the single mutant mice, Lbr–/–:Dhcr14+/+ and Lbr+/+:Dhcr14{Delta}4-7/{Delta}4-7, these three compound mutant mice allow one to test for a digenic phenotype. None of these compound mutant genotypes were found after weaning 137 mice from 19 separate litters. We thus investigated whether these compound mutant mice could be identified at earlier ages.

Both Lbr–/–:Dhcr14+/{Delta}4-7 and Lbr–/–:Dhcr14{Delta}4-7/{Delta}4-7 embryos died in utero, whereas Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 pups were viable. Lbr–/–:Dhcr14{Delta}4-7/{Delta}4-7 embryos appear to die during early embryogenesis. At E11.5, we could find reabsorbing embryonic tissue corresponding to this genotype. Lbr–/–:Dhcr14+/{Delta}4-7 results in a prenatal lethal phenotype with a variable age of in utero death. Four Lbr–/–:Dhcr14+/{Delta}4-7 embryos were recovered soon after birth. Evaluation of these embryos showed cleft palate (1/4), variable autopod defects including fusion of the distal phalanges of the second and third digits, and decreased hepatic extramedullary hematopoiesis. Histopathological examination of the eye, ear, esophagus, salivary glands, lung, heart, kidney, adrenal gland, spleen, small intestine, large intestine, ovary, pancreas, skin and skeletal muscle was unremarkable.

Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7, Lbr–/–, and Dhcr14{Delta}4-7/{Delta}4-7 mice are phenotypically distinct. Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice appeared normal at birth, and no significant weight differences were observed when 1-day-old Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 or Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 pups were compared to control littermates (data not shown). However, by 10 days of age, the mutant phenotypes could be differentiated. Dhcr14{Delta}4-7/{Delta}4-7 mice appeared normal; however, both Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice demonstrated impaired growth (Fig. 2). In contrast to Lbr–/– mice, both Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice had normal appearing fur (Fig. 2) and skin histology (Fig. 3A). In addition to these physical findings, the most notable difference was that by 10 days of age Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice developed ataxia and tremor (Supplementary Material, Video). These neurological findings were not observed in either Dhcr14{Delta}4-7/{Delta}4-7 or Lbr–/– mice. Consistent with the neurological phenotype, pathological examination of the spinal cord of Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice showed vacuolization consistent with a demyelinating process (Fig. 3B). This pathological finding was not observed in either Lbr or Dhcr14 mutant mice (Fig. 3B). The Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice die by 14 days of age, whereas both Lbr–/– and Dhcr14{Delta}4-7/{Delta}4-7 mice survive beyond weaning. Because of the growth retardation observed in Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice, we evaluated tibial growth plates (Fig. 3C). Although no growth retardation was observed in Dhcr14{Delta}4-7/{Delta}4-7 mice, the growth plate proliferative and hypertrophic zones were slightly less organized in comparison to the control. Lbr–/– growth plates demonstrated marked disorganization of the hypertrophic zone and mild disorganization of the proliferative zone. In addition, the trabecular bone appeared immature with residual cartilage. Bone development in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 was markedly abnormal. Both the proliferative and hypertrophic zones are markedly shorter and appear more like the growth plate expected in a young adult (1–2 months old) animal. Bone trabeculae are less interconnected and less abundant. This suggests that the bone may be osteoporotic in these animals. Consistent with what has previously been reported for Lbr mutant and heterozygous mice, chromatin clumping was observed by electron microscopy in spleen cells from both Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice (Fig. 3D). This was not observed in Dhcr14 mutant mice.


Figure 3
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  		Figure 3. Histological analysis of Dhcr14{Delta}4-7/{Delta}4-7, Lbr–/–, and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. (A) Photomicrograph of skin from control and mutant mice. Skin from Lbr–/– mice is characterized by epidermal hyperplasia with orthokeratotic hyperkeratosis. In contrast, skin from Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–: Dhcr14{Delta}4-7/{Delta}4-7 was normal appearing. (B) Histological analysis of spinal cord showed swelling of the myelin sheaths and vacuolization consistent with a demyelinating process in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7. This was not observed in either Dhcr14{Delta}4-7/{Delta}4-7 or Lbr–/– mice. (C) Although both mice demonstrated growth retardation, histological analysis of tibial growth plates demonstrated different and distinct abnormalities in Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. (D) Electron microscopy of splenic tissue showed chromatin clumping in both Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7. Chromatin clumping is consistent with Pelger–Huët anomaly and is known to occur in both homozygous and heterozygous Lbr mutant mice. This was not observed in Dhcr14{Delta}4-7/{Delta}4-7 mice.

 
Biochemical analysis
Gas chromatography/mass spectrometry (GC/MS) was used to perform sterol analysis on liver, kidney and brain cortex tissue obtained from 10-day-old control, Dhcr14{Delta}4-7/{Delta}4-7, Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. Abnormal sterol precursors were below our limit of detection in both liver and kidney tissue (data not shown). However, abnormal sterols were detected in brain cortex tissue from Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice (Fig. 4). On the basis of their chromatographic properties and mass spectra, the accumulating sterols were tentatively identified to be cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol. Since standards for these sterols were not readily available, their identity was confirmed by NMR analysis (Table 1).


Figure 4
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  		Figure 4. GC/MS sterol analysis. Brain sterol chromatograms from 10-day-old control (A), Dhcr14{Delta}4-7/{Delta}4-7 (B), Lbr–/– (C) and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 (D) mice. Sterols were identified by both retention time and mass spectra. Labeled peaks correspond to coprostanol (1, internal standard), cholesterol (2), desmosterol (3), cholesta-8,14-dien-3ß-ol (4), cholesta-8,14,24-trien-3ß-ol (5) and 4,4-dimethylcholesta-8,14-dien-3ß-ol (6).

 


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  		Table 1. Percent of unsaturated sterolsa in brain cortex tissue from control and mutant mice

 
Quantification of sterol levels showed that brain cortex cholesterol levels were significantly decreased (P < 0.01) to ~68 and 67% of control values in Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/– mice, respectively, and were markedly decreased to 27% of normal (P < 0.001) in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice (Fig. 5A). Total sterol levels showed a trend toward being decreased in both Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/– mice compared with control values; however, this was not quite significant (0.05 < P < 0.10) if a multiple comparison statistical test was applied (data not shown). Desmosterol levels were normal in Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/– mice. However, desmosterol was markedly reduced in brain cortex from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice (Fig. 5A). Although not significant, a variable and minor (0–3% of total sterols) elevation of cholesta-8,14-dien-3ß-ol was observed in brain cortex tissue from 10-day-old Lbr–/– mice (Fig. 5B). In contrast, Dhcr14{Delta}4-7/{Delta}4-7 mice showed a moderate increase in cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol (13 and 1.9% of total sterols, respectively), and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice showed a major elevation of 47 and 11%, respectively, of these same two sterols (Fig. 5B).


Figure 5
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  		Figure 5. Sterol analysis of brain cortex. (A) Cholesterol and desmosterol levels were quantified in brain cortex tissue from 10-day-old mice of the indicated genotype. Compared with control and compound heterozygous mice, cholesterol was significantly decreased in brain cortex tissue from both Lbr–/– and Dhcr14{Delta}4-7/{Delta}4-7 mice. Both cholesterol and desmosterol, a sterol normally present in the central nervous system at this age, were dramatically decreased in cortex tissue from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. (B) In brain cortex from 10-day-old mice, cholesta-8(9),14-dien-3ß-ol was significantly increased in both Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice and cholesta-8(9),14,24-trien-3ß-ol was significantly increased in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. Although the increase was not statistically significant, cholesta-8(9),14,24-trien-3ß-ol could clearly be detected in Dhcr14{Delta}4-7/{Delta}4-7 brain cortex whereas it was not detected in tissue from either control or Lbr mutant mice. (C) Relatively small and transient increases in {Delta}14-sterols were observed in both Lbr (filled circle) and Dhcr14 (triangle) mutant mice. Compared with control brain cortex, {Delta}14-sterols were elevated in 1-day-old Lbr–/– tissue (P < 0.001); however, precursor levels were normalizing by 10 days and not significantly different from control values. Dhcr14{Delta}4-7/{Delta}4-7 mice showed a different temporal pattern in that {Delta}14-sterol levels were minimally elevated at birth (0.75 ± 0.24% of total sterols), significantly elevated at 10 days (P < 0.01) and returned to below our limit of detection by 21 days. Brain cortex {Delta}14-sterol levels were significantly different in Lbr–/– and Dhcr14{Delta}4-7/{Delta}4-7 tissues both at 1 day of age (P < 0.001) and 10 days of age (P < 0.05). In Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 (open circle) brain cortex {Delta}14-sterol levels increase dramatically from 2.8 to 58% of total sterols. (D) GC/MS sterol profile from a stillborn Lbr–/–:Dhcr14+/{Delta}4-7 fetus. In addition to elevated cholesta-8(9),14-dien-3ß-ol (peak 3) and cholesta-8(9),14,24-trien-3ß-ol (peak 4), an abnormal peak (5) corresponding to 4,4-dimethyl-cholesta-8,14,-dien-3ß-ol was clearly detected. Peak 2 is cholesterol and peak 1 corresponds to the internal standard coprostanol. (A–C) Three to six samples were analyzed and the mean and standard deviation are represented. In (C), error bars are not depicted if they are smaller than the symbol.

 
In order to determine whether there was a temporal dependence of precursor accumulation, sterol analysis was performed on brain cortex, kidney and liver tissue from both 1-day and 21-day-old mice. Identical to what was observed at 10 days of age, liver and kidney sterols appeared normal in all three mouse mutants at both of these additional time points (data not shown). Analysis of brain cortex tissue showed transient elevations of {Delta}14-sterols in Lbr–/– and Dhcr14{Delta}4-7/{Delta}4-7 mice (Fig. 5C). In brain cortex from 1-day-old Lbr–/– mice, cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol accounted for 12.4% of total sterols. However, these levels decreased to below our limit of detection by 10–21 days of age. Analysis of Dhcr14{Delta}4-7/{Delta}4-7 brain cortex tissue showed a transient increase in {Delta}14-sterols at 10 days of age that resolved by 21 days of age. In contrast, cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol increased dramatically from 2.8 to 57.8% of total sterols between 1 and 10 days of age in brain samples from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice.

Because of technical constraints in producing a Dhcr14 mutation and breeding to obtain compound mutant mice, all of the sterol data reported above are from mice on a mixed C57Bl/6 and 129/Sv genetic background. We initially studied the Lbr mutation on an isogenic C57Bl/6 genetic background, and we were unable to detect accumulation of {Delta}14-sterols by GC/MS. Thus, the transient accumulation of {Delta}14-sterols in Lbr–/– mice appears to be significantly influenced by genetic background. No phenotypic differences were appreciated on these two genetic backgrounds.

We were able to obtain tissue from a single stillborn Lbr–/–:Dhcr14+/{Delta}4-7 pup for GC/MS analysis. The liver sterol profile was normal; however, cholesterol synthesis in brain cortex was markedly impaired. In addition to elevations of cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol, GC/MS analysis also showed an accumulation of 4,4-dimethyl-cholesta-8,14,-dien-3ß-ol, an earlier cholesterol precursor (Fig. 5D).

Given the absence of a physical phenotype and a relatively minor biochemical phenotype in Dhcr14{Delta}4-7/{Delta}4-7 mice, we considered the possibility that the Dhcr14 mutation did not produce a null allele. By quantitative PCR analysis, Dhcr14 expression was undetectable in both brain cortex and liver tissue (Fig. 6C), and reverse transcriptase–PCR analysis confirmed the absence of a detectable Dhcr14 transcript in the mutant mice (data not shown).


Figure 6
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  		Figure 6. Dhcr14 and Lbr expression analysis. DHCR14/Dhcr14 (black bars) and LBR/Lbr (gray bars) expression was quantified using real-time PCR in both human skin fibroblasts (A) and mouse embryonic fibroblasts (B). In both cases, cells were grown in medium supplemented with cholesterol containing serum (FBS) and cholesterol-depleted serum (LPDS). Endogenous cholesterol synthesis was inhibited by culturing cells with 5 mcg/ml (human cell lines) or 1 mcg/ml (mouse cell lines) simvastatin (simv.). Data are expressed as fold-increase over control (FBS), and as the mean ± standard deviation of three samples assayed in triplicate (*P < 0.05, **P < 0.001). LBR and Lbr expression changes were not significant. (C) Dhcr14 (black bars) and Lbr (gray bars) expression in liver and brain tissue from control and mutant mice was measured by real-time PCR. Data are expressed as fold-increase over control (Lbr+/+:Dhcr14+/+) and as the mean ± standard deviation of three samples assayed in triplicate. ND, not detected. Residual mutant transcripts are present in Lbr–/– tissues, thus accounting for the low signal present in these tissues.

 
NMR analysis was also used to characterize the sterol abnormality in Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice. Twenty different sterols were identified and quantified from the NMR spectra of non-saponifiable lipids from 10-day-old brain cortex (Table 1). Sterol content of the compound heterozygous brains was normal. The major precursor sterols accumulating in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mouse brains were C27 {Delta}8,14 and C27 {Delta}8,14,24 species. In addition, there was a significant accumulation (0.8 and 1.5% of total sterols, respectively) of the corresponding {Delta}8,14 and {Delta}8,14,24 4{alpha}-methyl and 4,4-dimethyl sterols. A similar accumulation of sterol intermediates was observed in brain tissue from Dhcr14{Delta}4-7/{Delta}4-7 mice. However, the accumulation of C27 {Delta}8,14 and {Delta}8,14,24 species was more modest.

Expression analysis
Biochemical analysis suggested that Lbr and Dhcr14 are partially redundant with respect to sterol {Delta}14-reduction. Thus, we investigated whether this redundancy was reflected at the level of gene expression. Control human skin fibroblasts grown in cholesterol-depleted medium (LPDS), both with and without the addition of simvastatin to block endogenous cholesterol synthesis, showed significantly increased DHCR14 expression in response to LPDS (13-fold, P < 0.05), and LPDS plus simvastatin (58-fold, P < 0.001) compared with control levels (Fig. 6A). In contrast, LBR expression was not significantly increased in response to cholesterol depletion (Fig. 6A). Mouse embryonic fibroblasts showed similar results. Although the response to LPDS was not significant, Dhcr14 expression increased 10-fold (P < 0.001) in response to treatment with LPDS plus simvastatin, whereas Lbr expression was not significantly increased (Fig. 6B).

We also studied Dhcr14 and Lbr expression in liver and brain tissue from 1-day-old Dhcr14 and Lbr mutant mice (Fig. 6C). In liver tissue, there appeared to be compensatory expression of Dhcr14 and Lbr. In Lbr mutant mice, Dhcr14 expression increased significantly (6.2-fold, P < 0.01), relative to control livers. Conversely, Lbr expression increased significantly (3.6-fold, P < 0.001) in Dhcr14 mutant mice. This apparent compensatory increase is much less apparent in brain cortex. Dhcr14 expression is 1.1-fold (not significant) of normal in Lbr–/– brain tissue, and Lbr shows a significant (P < 0.001) but relatively minor (1.7-fold) increase in expression in Dhcr14{Delta}4-7/{Delta}4-7 brains. Dhcr14 expression could not be detected in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 tissues. Lbr expression was increased 1.9-fold in liver tissue and 1.2-fold in brain tissue from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Inborn errors of postsqualene cholesterol synthesis underlie a number of human multiple malformation/mental retardation syndromes. These include Smith–Lemli–Opitz syndrome (SLOS) and the SLOS-like disorders of desmosterolosis and lathosterolosis, as well as two skeletal dysplasias, CHILD syndrome (Congenital Hemidysplasia with Ichthyosiform erythroderma and Limb Defects) and X-linked dominant chondrodysplasia punctata type 2 (CDPX2) (5,6). In screening different skeletal dysplasias for inborn errors of cholesterol synthesis, Kelley (5) found minor elevations of cholesta-8(9),14-dien-3ß-ol and cholesta-8(9),14,24-trien-3ß-ol consistent with impaired sterol {Delta}14-reduction in HEM dysplasia fetuses. Reduction of the C14–C15 double bond introduced by removal of the 14{alpha}-methyl group is an early step in the synthesis of cholesterol from lanosterol. Two different proteins, LBR and DHCR14, have been shown to have sterol {Delta}14-reductase activity (2,4). Molecular analysis of an HEM dysplasia patient revealed no DHCR14 mutations, but showed an apparent homozygous mutation of LBR (8). On the basis of these findings, it was proposed that HEM dysplasia is due to an inborn error of cholesterol synthesis and that LBR functions as the primary sterol {Delta}14-reductase (8). In addition, because the sterol defect was relatively minor, it was also previously proposed that the severe HEM dysplasia phenotype results from hormonal-like effects of the accumulating precursor sterols (5).

LBR is a bifunctional protein: in addition to functioning as a sterol {Delta}14-reductase, LBR functions, as its name implies, as a lamin B receptor in the inner nuclear membrane. Thus, LBR is involved in chromatin binding and organization. The abnormal nuclear morphology and chromatin organization seen in Pelger–Huët anomaly is consistent with a defect in LBR function that impairs lamin B and chromatin binding. Human HEM dysplasia or mouse ichthyosis, which result from homozygous mutations of the LBR gene, could plausibly result from impaired lamin B binding, impaired sterol {Delta}14-reduction, or a combination of these two factors. Because DHCR14 is also a sterol {Delta}14-reductase, and the sterol defect reported in HEM dysplasia was relatively mild, we hypothesized that LBR and DHCR14 provide functional redundancy with respect to cholesterol synthesis and thus HEM dysplasia and ichthyosis are, at the mechanistic level, more likely to be laminopathies rather than inborn errors of cholesterol synthesis.

To test the hypothesis that Dhcr14 and Lbr provide functional redundancy with respect to sterol {Delta}14-reduction, we obtained ichthyosis (Lbr–/–) mice and produced a Dhcr14 mutant mouse model. These mutant mice were then bred to test for a digenic phenotype. Characterization of Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice showed that they have distinct physical and biochemical phenotypes and that the ichthyosis phenotype is not concordant with a defect in sterol {Delta}14-reduction. The data reported in this paper show that Dhcr14 and Lbr provide significant enzymatic redundancy with respect to sterol {Delta}14-reductase activity, that accumulation of {Delta}14-sterols does not cause the mouse ichthyosis phenotype and that accumulation of {Delta}14-sterols does not appear to cause a hormonal-like disruption of development. From this cumulative evidence, we propose that HEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis.

Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mutant mice have distinct phenotypes. Whereas Lbr–/– mutant mice had the expected ichthyosis phenotype, Dhcr14{Delta}4-7/{Delta}4-7 mice were essentially normal and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice had a unique phenotype. Neither Dhcr14{Delta}4-7/{Delta}4-7 nor Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice had the ichthyotic skin lesions typical of the Lbr mutants. Consistent with a defect in lamin B and chromatin binding, both Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 showed chromatin abnormalities. This has previously been shown for both Lbr+/– and Lbr–/– mice (9). Neither abnormalities of chromatin nor Pelger–Huët anomaly were observed in Dhcr14{Delta}4-7/{Delta}4-7 mice. Both Lbr–/– and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice demonstrated marked growth retardation and growth plate abnormalities. In Lbr–/– mice, growth plate abnormalities primarily affected the hypertrophic zone and the bone trabeculae appeared immature. In contrast, growth plates from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice were markedly shortened and had an adult-type appearance suggestive of an advanced bone age. Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice demonstrated a unique neurological phenotype consisting of progressive ataxia and tremors. Pathologically, this neurological phenotype is associated with abnormal myelin in the spinal cord. In contrast, neither the Lbr–/– nor Dhcr14{Delta}4-7/{Delta}4-7 mice had these findings. The difference in postnatal survival found in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/–:Dhcr14+/{Delta}4-7 is likely related to complete loss of the lamin B/chromatin binding function of Lbr in Lbr–/–:Dhcr14+/{Delta}4-7 embryos. Increased in utero death has previously been reported in Lbr–/– mice (9).

Sterol analysis of Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 strongly supports the idea that Lbr and Dhcr14 provide significant functional redundancy with respect to sterol {Delta}14-reduction. No significant accumulation of {Delta}14-sterols was observed in liver or kidney tissue from any of these three mutant mice or in liver tissue from an Lbr–/–:Dhcr14+/{Delta}4-7 embryo. This result demonstrates that in liver tissue a single Lbr or Dhcr14 allele is sufficient for cholesterol synthesis. It is plausible that maternal milk could have provided a source of cholesterol for peripheral tissues prior to weaning. However, precursor sterols were undetectable by GC/MS in peripheral tissues and absent in neonatal tissue obtained prior to feeding, thus provision of cholesterol via maternal milk is an unlikely explanation of our results. In contrast, variable defects in cholesterol synthesis were observed in brain cortex tissue. Sterol analysis of brain cortex tissue from Dhcr14{Delta}4-7/{Delta}4-7 mice showed a transient increase of {Delta}14-sterols in brain tissue at 10 days of age. Sterol analysis of Lbr mutant tissues has not previously been reported. A transient and genetic background-dependent elevation of {Delta}14-sterols was observed at birth in Lbr–/– brain tissue. Brain cortex tissue from an Lbr–/–:Dhcr14+/{Delta}4-7 embryo also showed marked accumulation of {Delta}14-sterols. Sterol analysis of brain tissue from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice showed a major postnatal accumulation of {Delta}14-sterols. The time course of this marked accumulation {Delta}14-sterols is concordant with the time course of myelination. Thus, it is plausible that the increased need for cholesterol synthesis during myelin formation accounts for both the transient increase in {Delta}14-sterols in the Dhr14 mutant mice and the marked accumulation in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 brains. The transient elevation of {Delta}14-sterols in brain from 1-day-old Lbr–/– pups and the markedly abnormal accumulation of {Delta}14-sterols brain tissue from an Lbr –/–:Dhcr14+/{Delta}4-7 embryo suggests that Lbr may be more critical with respect to sterol {Delta}14-reduction in the prenatal central nervous system. The relatively small, tissue limited and transient elevations of {Delta}14-sterols found in Dhcr14{Delta}4-7/{Delta}4-7 and Lbr–/– mice argue strongly for significant enzymatic redundancy of Dhcr14 and Lbr with respect to sterol {Delta}14-reduction. This conclusion is further supported by the tissue-limited sterol synthetic defect observed in Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice, and the finding that complete inhibition of sterol {Delta}14-reduction, as presumably occurs in Lbr–/–:Dhcr14{Delta}4-7/{Delta}4-7 embryos, results in embryonic death soon after implantation.

Gene expression studies support the hypothesis that both LBR and DHCR14 are involved in cholesterol synthesis. In human and mouse fibroblasts, expression of DHCR14 and Dhcr14, respectively, is markedly increased in response to cholesterol depletion. In contrast, the apparent minor increase in LBR and Lbr expression in response to cholesterol depletion is not significantly different than control values. This suggests that expression of LBR and Lbr is not regulated in response to cholesterol levels. These data are similar to what has recently been reported for COS-1 cells (12). These results are also consistent with the identification of Dhcr14, but not Lbr, as an SREBP2-regulated transcript in mouse liver (13). In contrast to these observations, there appears to be a reciprocal increase in expression of these two genes in liver tissue from mutant animals. Dhcr14 expression is increased 6.2-fold in Lbr mutant liver and Lbr expression is increased 3.6-fold in Dhcr14 mutant liver. This result is consistent with the hypothesis that Lbr and Dhcr14 provide redundant sterol {Delta}14 reductase activity and likely explains why a biochemical defect is not observed in either liver or kidney tissue. Given the lack of a response to cholesterol depletion in normal cells by Lbr and considering the fact that Lbr was not identified as a target of SREBP2, it is possible that Lbr expression is increased in response to elevated levels of {Delta}14-sterols. Neither Dhcr14 nor Lbr showed this reciprocal increase in expression in brain tissue. This suggests alternative regulation of these genes in the central nervous system and may explain why we observed sterol abnormalities only in brain tissue.

Sterol analyses of brain tissue from Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice raises a number of issues with respect to cholesterol synthesis in general. The ratio of 24,25-dihydrosterols to {Delta}24 sterols was approximately 10 for {Delta}5 sterol species and approximately 3 for {Delta}8,14 species. Thus, impaired sterol {Delta}14-reduction does not appear to impair Dhcr24 activity, and, as expected, reduction of the {Delta}24 bond occurs mainly at or near the stage of 4- and 14-demethylation. The major accumulating precursor sterols are cholesta-8,14-dien-3ß-ol and cholesta-8,14,24-trien-3ß-ol rather than the corresponding 4{alpha}-methyl and 4,4-dimethyl sterols. This finding supports the idea that 4-demethylation can occur at either the {Delta}8 or {Delta}8,14 stage and occurs prior to isomerization to lathosterol ({Delta}7). Furthermore, it shows that sterol {Delta}8-isomerase (Ebp) does not efficiently catalyze isomerization of {Delta}8,14 sterols. Although trace levels of {Delta}5,8(14) have been detected in blood from SLOS patients (14), this work provides strong evidence for the accumulation of {Delta}5,8(14), {Delta}6,8,14, {Delta}7,9(11) and {Delta}7,14 sterols in a mammalian genetic disorder. The accumulation of {Delta}5,8(14) is an interesting finding since it is not obvious how this sterol is synthesized. Its presence raises the possibility of an alternative synthetic pathway in which a {Delta}8(14) sterol is the initial product of C14 demethylation. Further work is definitely necessary to test this speculative hypothesis of altered cholesterol synthesis and to further characterize the biology of the accumulating sterol precursors. This new mouse model will provide a unique tool to advance our understanding of the postsqualene cholesterol biosynthetic pathway.

The presence of redundant enzymes for sterol {Delta}14-reduction is unique in the scheme of postsqualene cholesterol biosynthesis. It is not clear why such redundancy would be necessary. Although these enzymes appear completely redundant in liver tissue, redundancy in brain tissue is substantial but not complete. In addition, there also may be a temporal component involved. Additionally, regulation of Dhcr14 and Lbr expression appears to differ between the two genes and between liver and brain cortex tissue. Further work is necessary to completely define these issues. Again, these new mouse models will serve as unique tools for future investigations of cholesterol synthesis.

The laminopathies represent a diverse group of human genetic syndromes due to mutations of lamin A and a protease, ZMPSTE24, involved in the processing of lamin A. These human disorders result in premature aging syndromes (Hutchinson–Gilford progeria and atypical Werner syndrome), myopathies (Emery–Dreifuss muscular dystrophy type 2 and type 3, limb girdle muscular dystrophy and dilated cardiomyopathy, type 1A), neuropathies (Charcot–Marie–Tooth disease, type 2B1), lipodystrophies (Dunnigan type, generalized lipoatrophy/lipodystrophy and mandibuloacral dysplasia) and restrictive dermopathy (15). LBR localizes to the inner nuclear membrane and binds both chromatin and lamin B as part of a meshwork of intermediate filament proteins known as the nuclear lamina (3). Given the distinct physical and biochemical phenotypes observed in Lbr–/–, Dhcr14{Delta}4-7/{Delta}4-7 and Lbr+/–:Dhcr14{Delta}4-7/{Delta}4-7 mice, it is most plausible that HEM dysplasia is a laminopathy and may represent the first human disorder associated with disturbance of lamin B function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of the Dhcr14{Delta}4-7 allele
We identified the mouse mRNA, genomic sequence and genomic organization of Dhcr14 using NCBI databases (mRNA accession AF480070 [GenBank] .1 and genomic AC131114 [GenBank] .3). These sequences were used to design PCR primers to amplify both the 4.4 kb 5' flank and the 2.8 kb 3' flank from mouse 129Sv genomic DNA derived from J1 embryonic stem cells. Restriction endonuclease sequences were incorporated into the 5' end of these primers to facilitate subsequent cloning. Roche Taq polymerase and dNTPs were used for PCR amplification. The 5' flank was amplified using primers {Delta}14-5'B (5'-gagaagctggcagcctttgc-3') and {Delta}14-5'D XbaI (5'-tctagaccacctacctcatccctacc-3'). The resulting PCR product was cloned as a NotI/XbaI fragment into the targeting vector pALS-4. The endogenous NotI restriction site is 11 bp internal to the {Delta}14-5'B primer. The 3' flank was amplified utilizing primers {Delta}14-3'B EcoRI (5'-gaattcggcttcatgctggtctttgg-3') and {Delta}14-3'D SalI (5'-gtcgactggagacacgaaagttaccg-3'). This fragment was cloned as an EcoRI/SalI fragment into pALS-4. For positive selection, the neomycin phosphotransferase gene (PGK-neo) was cloned into this construct between the two targeting flanks as an XbaI/EcoRI fragment. The negative selectable marker, diphtheria toxin A, was cloned into a SalI site at the end of the 3' flank. Coding regions of the targeting vector were sequenced to confirm the construct.

The targeting vector (Fig. 1A) was linearized with NotI and electroporated into J1 mouse embryonic stem cells, and selection for G418-resistant colonies was as previously described (16). PCR amplifications using primer A (5'-gagctgtttgctgctcaggg-3') and primer B (5'-ttaagggccagctcattcctcc-3') were used to identify embryonic stem cells that underwent homologous recombination with the 5' flank of the targeting vector and the endogenous allele. Homologous recombination between the 3' flank and the endogenous gene was then confirmed using primers D (5'-gagtttggacgacttctgcgc-3') and C (5'-gccagaggccacttgtgtagc-3'). PCR cycling conditions consisted of 5 min of denaturation at 94°C followed by 30 cycles of 30 s at 94°C, 45 s at 62°C, 180 s at 72°C and a final extension of 5 min at 72°C. The PCR products were then separated on a 1% agarose gel (Fig. 1B).

Mouse embryonic stem cell clone c129 was then injected into C57/B6 blastocysts to obtain chimeric animals. Chimeric founders were mated with C57/B6 mice to establish germline transmission of the Dhcr14 mutant allele (Dhcr14{Delta}4-7). Dhcr14+/{Delta}4-7 mice were intercrossed to obtain Dhcr14{Delta}4-7/{Delta}4-7 mice. Genotyping of the Dhcr14 allele was performed by PCR. Four primers were used in a single assay. Primers C14-S1 (5'-gatgcaggaggcagagcttcg-3') and C14-S3 (5'-ccaaagaccagcatgaagcc-3') produce a 248 bp band corresponding to the control allele. Primers Neo 5' (5'-ctgtgctcgacgttgtcactg-3') and Neo 3' (5'-gatcccctcagaagaactcgt-3') produce a 602 bp fragment corresponding to the mutant allele. PCR cycling conditions were 5 min at 94°C, followed by 30 cycles of 60 s at 94°C, 60 s at 63°C and 60 s at 72°C followed by a final incubation at 72°C for 10 min.

For timed matings, the identification of a copulatory plug was considered to be E0.5 and embryonic age was confirmed by inspection. Animal work was performed under an NICHD approved animal study protocol.

Sterol analysis
Neutral sterols were extracted from tissue and analyzed by GC/MS as previously described (17). The non-saponifiable lipids from 10-day-old mouse brain cortex tissue were analyzed by 1D and 2D NMR. Spectra were measured in CDCl3 solution at 25°C on Varian Inova 600 and 800 MHz NMR spectrometers equipped with a cold probe. The half-height linewidth was < 0.5 Hz. The detection limit for most minor sterol components ranged from 0.0005 to 0.01% of the amount of cholesterol. Sterols were identified by precise comparisons against spectral data reported for unsaturated sterols (18), with confirmation by heteronuclear 2D NMR for {Delta}5,7, {Delta}5,8, {Delta}5,8(14), {Delta}5,7,9(11), {Delta}6,8, {Delta}7, {Delta}7,9(11), {Delta}8, {Delta}8(14) and {Delta}8,14 sterols. Because of the high reproducibility of NMR data reported for unsaturated sterols (18), no authentic sterol standards were needed. Sterols were quantified from signal intensities in the methyl, allylic, hydroxylmethine and olefinic regions of the proton spectrum. Disparities in the sterol profile between mutants were confirmed by examination of difference spectra. The extent of C4 demethylation and {Delta}24 reduction was gauged from 1H, 13C and 2D spectra. Direct 1H NMR quantification from upfield methyl signals provides for each biosynthetic intermediate the sum of {Delta}24 and 24,25-dihydro species. This methodological limitation conveniently removes extraneous detail about the status of the side-chain double bond. The data obtained from Lbr+/–:Dhcr14+/{Delta}4-7 compound heterozygous brain were not significantly different than control mouse brain (data not shown).

Histological analysis and electron microscopy
Tissues were fixed using buffered formalin (Sigma), paraffin-embedded, sectioned and stained with hematoxylin and eosin using standard procedures. Tissue sections were viewed and photographed using a Zeiss Axioscope with a Zeiss Axiocam.

For electron microscopy, splenic tissue was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. The tissues were washed with cacodylate buffer and postfixed with 1% OsO4 for 2 h. The tissue was washed again with 0.1 M cacodylate buffer, serially dehydrated in ethanol and embedded in Eponate 12 resin (Ted Pella, Redding, CA, USA). Thin sections, ~80 nm, were obtained by utilizing the Leica ultracut-UCT ultramicrotome (Leica, Deerfield, IL, USA) and placed onto 300 mesh copper grids and stained with saturated uranyl acetate in 50% methanol and then with lead citrate. The grids were viewed in the Philips 410 electron microscope (FEI, Hillsboro, OR, USA) at 80 kV and images were recorded on Kodak SO-163 film (Rochester, NY, USA).

Cell culture
3T3-like mouse embryonic fibroblasts were derived as previously described (19). The human skin fibroblast cell line (GM05659C) was obtained from the NIGMS Human Genetic Mutant Cell Repository, and this work was performed under an NICHD IRB approved protocol. Fibroblasts were grown (37°C, 5% CO2) in Dulbecco's modified Eagle's Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini, Calabasas, CA, USA). Cholesterol-deficient culture was performed in McCoy's 5A medium (Invitrogen) supplemented with 7.5% lipoprotein-deficient serum (LPDS). LPDS was prepared using organic extraction as previously described (20). Simvastatin (R.J. Chemicals, Inc., Pompano Beach, FL, USA) was dissolved in ethanol and added to the medium to give a final concentration of 5 mcg/ml for human cell lines and 1 mcg/ml for mouse cell lines.

Expression analysis
For quantitative PCR, RNA was extracted from tissues and cells using an RNAeasy Mini Kit (Qiagen, Santa Clarita, CA, USA). RNA (100 ng) was reverse transcribed using a High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) as per manufacturer's protocol. Quantitative PCR assays were performed using DHCR14, Dhcr14, LBR, Lbr, HMGR and Hmgr Assays on Demand from Applied Biosystems. Analysis was performed on an ABI Prism 7000. All assays were validated, performed in triplicate and normalized to either GAPDH or Gapdh. A minimum of three separate specimens were analyzed for each data point. Fold-change relative to control levels was determined using the {Delta}{Delta}Ct method.

Statistical analysis
Data are reported as mean ± standard deviation. For statistical analysis of sterol and expression differences, an ANOVA Tukey–Kramer multiple comparisons test was used. A chi-squared test was used to evaluate for deviation from the expected Mendelian ratio. Unless otherwise specified, P < 0.05 was considered significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
This work was supported by the intramural research programs of the National Institute of Child Health and Human Development, National Institutes of Health, and by the Office of Research Services, National Institutes of Health. We would also like to express our appreciation to Dr Steven Fliesler for his critical review of this manuscript.

Conflict of Interest statement. The authors report no conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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  8. Waterham H.R., Koster J., Mooyer P., Noort Gv G., Kelley R.I., Wilcox W.R., Wanders R.J., Hennekam R.C., Oosterwijk J.C. Autosomal recessive HEM/Greenberg skeletal dysplasia is caused by 3 beta-hydroxysterol delta 14-reductase deficiency due to mutations in the lamin B receptor gene. Am. J. Hum. Genet. (2003) 72:1013–1017.[CrossRef]