hem dysplasia and ichthyosis are likely laminopathies and not due to 3 -hydroxysterol...

HEM dysplasia and ichthyosis are likelylaminopathies and not due to 3b-hydroxysterolD14-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,*

1Heritable Disorders Branch, NICHD and 2Diagnostic and Research Services Branch, OD, NIH, DHHS, Bethesda,

MD 20892, USA, 3Department of Biochemistry, Section of Medicine and Pharmacy, University of Pavia, Italy and4Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA

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

Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosisin mice. LBR is a bifunctional protein with both a lamin B binding and a sterol D14-reductase domain. It pre-viously has been proposed that LBR is the primary sterol D14-reductase and that HEM dysplasia and ichthyo-sis are inborn errors of cholesterol synthesis. However, DHCR14 also encodes a sterol D14-reductase andcould provide enzymatic redundancy with respect to cholesterol synthesis. To test the hypothesisthat LBR and DHCR14 both function as sterol D14-reductases, we obtained ichthyosis mice (Lbr2/2) anddisrupted Dhcr14. Heterozygous Lbr and Dhcr14 mice were intercrossed to test for a digenic phenotype.Lbr2/2, Dhcr14D4-7/D4-7 and Lbr1/2:Dhcr14D4-7/D4-7 mutant mice have distinct physical and biochemical pheno-types. Dhcr14D4-7/D4-7 mice are essentially normal, whereas Lbr1/2:Dhcr14D4-7/D4-7 mice are growth retardedand 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 transientelevations of D14-sterols were observed in Lbr2/2 and Dhcr14D4-7/D4-7 brain tissue, and marked elevationswere seen in Lbr1/2:Dhcr14D4-7/D4-7 brain. Pathological evaluation demonstrated vacuolation and swellingof the myelin sheaths in the spinal cord of Lbr1/2:Dhcr14D4-7/D4-7 mice consistent with a demyelinating pro-cess. This was not observed in either Lbr2/2 or Dhcr14 D4-7/D4-7 mice. Our data support the conclusions thatLBR and DHCR14 provide substantial enzymatic redundancy with respect to cholesterol synthesis and thatHEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis.

INTRODUCTION

The synthesis of cholesterol from lanosterol requires removalof the 14a-methyl group from either lanosterol or dihydrola-nosterol which results in the formation of a C14–C15double bond. This C14–C15 double bond must subsequentlybe reduced by a sterol D14-reductase (1) (Fig. 1A). Two differ-ent genes, DHCR14 and LBR, have been shown to encode pro-teins with sterol D14-reductase activity. DHCR14 (TM7SF2,SR-1) was initially cloned from bovine liver, and expressionin COS-7 cells confirmed that it has sterol D14-reductaseactivity (2). The lamin B receptor (LBR) has two functional

domains: the N-terminal domain projects into the nucleoplasmand binds to both B-type lamins and chromatin proteins (3),whereas the C-terminus of LBR is highly homologous tosterol reductases and has been shown to encode sterolD14-reductase activity (4).

Over the past 14 years, a number of human malformationsyndromes have been shown to be due to inborn errors ofcholesterol synthesis (5,6). Hydrops-Ectopic calcification-Moth-eaten skeletal dysplasia (HEM dysplasia, Greenbergdysplasia, OMIM #215140) is a lethal, autosomal recessive,skeletal dysplasia initially described by Greenberg et al. (7).Clinical manifestations include short-limb dwarfism, hydrops

Published by Oxford University Press 2007.For Permissions, please email: [email protected]

*To whom correspondence should be addressed at: HDB, NICHD, NIH, DHHS Bld. 10, Room 9D42 10 Center Dr, Bethesda, MD 20892-1830, USA.Tel: þ1 3014354432; Fax: þ1 3014805791; Email: [email protected]

Human Molecular Genetics, 2007, Vol. 16, No. 10 1176–1187doi:10.1093/hmg/ddm065Advance Access published on April 2, 2007

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Figure 1. (A) During cholesterol synthesis, the C14–C15 double bond found in 4,4-dimethylcholesta-8(9),14-dien-3b-ol is reduced by a sterol D14-reductase.Both DHCR14 and LBR have been shown to have sterol D14-reductase activity. If reduction of the C14-C15 double bond is impaired,4,4-dimethylcholesta-8(9),14-dien-3b-ol is metabolized to yield cholesta-8(9),14-dien-3b-ol. Sterol D24-reduction can occur at various points in the cholesterolsynthetic pathway, thus although not depicted, a similar mechanism explains the accumulation of cholesta-8(9),14,24-trien-3b-ol. (B) The Dhcr14 genomicstructure, targeting vector and resulting mutant allele. Filled boxes with roman numerals represent the exons. The relative positions of PCR primers used foridentifying properly targeted ES cell clones are labeled (A–D). (C) PCR analysis of untargeted R1 ES cell and two targeted ES cell clones (c129 andc162). Proper homologous recombination between the targeting vector and the endogenous allele was confirmed by amplification of the 50 and 30 flanksusing primer pairs A/B and C/D, respectively. (D) PCR genotyping of progeny from a heterozygous Dhcr14 intercross. A 248 bp PCR product correspondsto the control Dhcr14þ allele, and a 602 bp PCR product corresponds to the mutant Dhcr14D4-7 allele.

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and polydactyly. Radiographic findings include ectopic ossifi-cation, fragmented (moth-eaten) long bones, platyspondylyand deficient skull ossification. Sterol analysis of tissuesfrom four HEM dysplasia fetuses showed normal cholesterollevels and abnormal, but minor (,1% of total sterols),elevations of cholesta-8(9),14-dien-3b-ol andcholesta-8(9),14,24-trien-3b-ol consistent with impairedsterol D14-reduction (5). Given the quantitatively minorsterol abnormality, it was hypothesized that the major dys-morphic features found in HEM dysplasia cases might besecondary to hormonal like effects of the accumulating14-dehydrosterols (5). In 2003, Waterham et al. (8) reportedan apparent homozygous mutation of the LBR gene in aHEM dysplasia patient, and this group concluded that LBRfunctions as the primary sterol D14 reductase. Homozygousmutations of Lbr in the mouse are associated with the ichthyo-sis phenotype (9). Sterol analysis of the ichthyosis mouse hasnot been reported. Mutations of LBR also underlie autosomaldominant Pelger–Huet anomaly (10). The Pelger–Huetanomaly consists of hyposegmentation of the nuclei of poly-morphonuclear leukocytes. Although it may be only an issueof null versus hypomorphic alleles, the relationship betweenhomozygous Pelger–Huet anomaly (which has not beenassociated with major malformations in humans) and HEMdysplasia is not yet clear (11).

Given the minor sterol abnormality reported in HEM dys-plasia, we hypothesized that LBR and DHCR14 are redundantsterol D14-reductases and that both human HEM dysplasia andmouse ichthyosis result from impaired LBR function ratherthan impaired sterol synthesis. Herein, we report the develop-ment and characterization of a Dhcr14 mutant mouse strain,the biochemical characterization of ichthyosis mice (Lbrmutant), and characterization of compound Lbr and Dhcr14mutant mice. Our findings support the hypothesis that LBRand DHCR14 provide substantial redundancy for sterolD14-reduction and strongly suggest that HEM dysplasia andichthyosis are laminopathies rather than inborn errors ofcholesterol synthesis.

RESULTS

Disruption of Dhcr14

Dhcr14 was disrupted in mouse embryonic stem cells usingtargeted homologous recombination. Recombination betweenthe targeting vector and the endogenous Dhcr14 alleleresults in the insertion of the neomycin phosphotransferasegene (PGKneo) and disruption of exons 4–7 (Fig. 1B). PCRanalysis identified 12/171 (7%) embryonic stem cell clonesthat underwent homologous recombination between theendogenous gene and both flanks of the targeting vector(Fig. 1C). Clone c129 was used to produce a germline-transmitting chimeric founder.

Phenotypic characterization

Heterozygous Dhcr14þ/D4-7 mice appeared phenotypicallynormal. Thus, Dhcr14þ/D4-7 mice were intercrossed to obtainhomozygous Dhcr14 mutant mice (Fig. 1D). Dhcr14D4-7/D4-7

mice were identified after weaning in the expected Mendelianratio (27, 50 and 23% for Dhcr14þ/þ, Dhcr14þ/D4-7 andDhcr14D4-7/D4-7, respectively, n ¼ 168, P ¼ 0.90).Dhcr14D4-7/D4-7 mice appeared normal (Fig. 2) and histopatho-logical analysis revealed no significant differences betweencontrol and mutant mice. Dhcr14D4-7/D4-7 mutant mice werefertile and produced normal size litters. Eight femaleDhcr14D4-7/D4-7 mice were followed for over 1 year, and noage-dependent problems were identified.

Lbrþ/1088insCC mice were obtained from Jackson Labora-tories and intercrossed to obtain Lbr1088insCC/1088insCC

(Lbr2/2) offspring. The icJ mutation in Lbr, 1088CC, resultsin a frame shift that alters 21 amino acids (365–385) andintroduces a stop codon at position p.386 (9). Lbr2/2 micecould be identified on the basis of sparse hair by 9 days ofage. Phenotypic findings in Lbr2/2 mice were consistentwith those previously described for these mice (9) andincluded growth retardation, ichthyosis, Pelger–Huetanomaly and syndactyly.

Figure 2. Dhcr14D4-7/D4-7, Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7 phenotypes. Photograph of 10-day-old live (A) and euthanized (B) Dhcr14D4-7/D4-7 (left), Lbr2/2

(middle) and Lbrþ/2:Dhcr14D4-7/D4-7 (right) mice. Dhcr14D4-7/D4-7 resemble normal control mice, whereas Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7 have distinctphenotypes.

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Dhcr14þ/D4-7 and Lbrþ/2 heterozygous mice were interbredto test for a digenic phenotype. Lbrþ/2:Dhcr14þ/D4-7 com-pound heterozygous mice were viable, phenotypicallynormal and fertile. These mice were intercrossed to obtainLbr2/2:Dhcr14þ/D4-7, Lbrþ/2:Dhcr14D4-7/D4-7 and Lbr2/2:Dhcr14D4-7/D4-7 mice. In conjunction with the single mutantmice, Lbr2/2:Dhcr14þ/þ and Lbrþ/þ:Dhcr14D4-7/D4-7, thesethree compound mutant mice allow one to test for a digenicphenotype. None of these compound mutant genotypes werefound after weaning 137 mice from 19 separate litters. Wethus investigated whether these compound mutant micecould be identified at earlier ages.

Both Lbr2/2:Dhcr14þ/D4-7 and Lbr2/2:Dhcr14D4-7/D4-7

embryos died in utero, whereas Lbrþ/2:Dhcr14D4-7/D4-7 pupswere viable. Lbr2/2:Dhcr14D4-7/D4-7 embryos appear to dieduring early embryogenesis. At E11.5, we could find reabsorb-ing embryonic tissue corresponding to this genotype. Lbr2/2:Dhcr14þ/D4-7 results in a prenatal lethal phenotype with a vari-able age of in utero death. Four Lbr2/2:Dhcr14þ/D4-7 embryoswere recovered soon after birth. Evaluation of these embryosshowed cleft palate (1/4), variable autopod defects including

fusion of the distal phalanges of the second and third digits,and decreased hepatic extramedullary hematopoiesis. Histo-pathological examination of the eye, ear, esophagus, salivaryglands, lung, heart, kidney, adrenal gland, spleen, small intes-tine, large intestine, ovary, pancreas, skin and skeletal musclewas unremarkable.

Lbrþ/2:Dhcr14D4-7/D4-7, Lbr2/2, and Dhcr14D4-7/D4-7 miceare phenotypically distinct. Lbrþ/2:Dhcr14D4-7/D4-7 miceappeared normal at birth, and no significant weight differenceswere observed when 1-day-old Lbr2/2, Dhcr14D4-7/D4-7 orLbrþ/2:Dhcr14D4-7/D4-7 pups were compared to control litter-mates (data not shown). However, by 10 days of age, themutant phenotypes could be differentiated. Dhcr14D4-7/D4-7

mice appeared normal; however, both Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7 mice demonstrated impaired growth (Fig. 2).In contrast to Lbr2/2 mice, both Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 mice had normal appearing fur (Fig. 2)and skin histology (Fig. 3A). In addition to these physical find-ings, the most notable difference was that by 10 days of ageLbrþ/2:Dhcr14D4-7/D4-7 mice developed ataxia and tremor(Supplementary Material, Video). These neurological findings

Figure 3. Histological analysis of Dhcr14D4-7/D4-7, Lbr2/2, and Lbrþ/2:Dhcr14D4-7/D4-7 mice. (A) Photomicrograph of skin from control and mutant mice.Skin from Lbr2/2 mice is characterized by epidermal hyperplasia with orthokeratotic hyperkeratosis. In contrast, skin from Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 was normal appearing. (B) Histological analysis of spinal cord showed swelling of the myelin sheaths and vacuolization consistent with a demye-linating process in Lbrþ/2:Dhcr14D4-7/D4-7. This was not observed in either Dhcr14D4-7/D4-7 or Lbr2/2 mice. (C) Although both mice demonstrated growthretardation, histological analysis of tibial growth plates demonstrated different and distinct abnormalities in Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7 mice. (D) Elec-tron microscopy of splenic tissue showed chromatin clumping in both Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7. Chromatin clumping is consistent with Pelger–Huetanomaly and is known to occur in both homozygous and heterozygous Lbr mutant mice. This was not observed in Dhcr14D4-7/D4-7 mice.




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were not observed in either Dhcr14D4-7/D4-7 or Lbr2/2 mice.Consistent with the neurological phenotype, pathologicalexamination of the spinal cord of Lbrþ/2:Dhcr14D4-7/D4-7

mice showed vacuolization consistent with a demyelinatingprocess (Fig. 3B). This pathological finding was not observedin either Lbr or Dhcr14 mutant mice (Fig. 3B). The Lbrþ/2:Dhcr14D4-7/D4-7 mice die by 14 days of age, whereas bothLbr2/2 and Dhcr14D4-7/D4-7 mice survive beyond weaning.Because of the growth retardation observed in Lbr2/2 andLbrþ/2:Dhcr14D4-7/D4-7 mice, we evaluated tibial growthplates (Fig. 3C). Although no growth retardation wasobserved in Dhcr14D4-7/D4-7 mice, the growth plate prolifera-tive and hypertrophic zones were slightly less organizedin comparison to the control. Lbr2/2 growth platesdemonstrated marked disorganization of the hypertrophiczone and mild disorganization of the proliferative zone.In addition, the trabecular bone appeared immaturewith residual cartilage. Bone development in Lbrþ/2:Dhcr14D4-7/D4-7 was markedly abnormal. Both the prolifera-tive and hypertrophic zones are markedly shorter andappear more like the growth plate expected in a youngadult (1–2 months old) animal. Bone trabeculae are lessinterconnected and less abundant. This suggests that thebone may be osteoporotic in these animals. Consistent withwhat has previously been reported for Lbr mutant and hetero-zygous mice, chromatin clumping was observed by electronmicroscopy in spleen cells from both Lbr2/2 and Lbrþ/2:Dhcr14D4-7/D4-7 mice (Fig. 3D). This was not observed inDhcr14 mutant mice.

Biochemical analysis

Gas chromatography/mass spectrometry (GC/MS) was used toperform sterol analysis on liver, kidney and brain cortex tissueobtained from 10-day-old control, Dhcr14D4-7/D4-7, Lbr2/2

and Lbrþ/2:Dhcr14D4-7/D4-7 mice. Abnormal sterol precursorswere below our limit of detection in both liver and kidneytissue (data not shown). However, abnormal sterols weredetected in brain cortex tissue from Dhcr14D4-7/D4-7 andLbrþ/2:Dhcr14D4-7/D4-7 mice (Fig. 4). On the basis of theirchromatographic properties and mass spectra, the accumulat-ing sterols were tentatively identified to becholesta-8,14-dien-3b-ol and cholesta-8,14,24-trien-3b-ol.Since standards for these sterols were not readily available,their identity was confirmed by NMR analysis (Table 1).

Quantification of sterol levels showed that brain cortexcholesterol levels were significantly decreased (P , 0.01) to�68 and 67% of control values in Dhcr14D4-7/D4-7 andLbr2/2 mice, respectively, and were markedly decreased to27% of normal (P , 0.001) in Lbrþ/2:Dhcr14D4-7/D4-7 mice(Fig. 5A). Total sterol levels showed a trend toward beingdecreased in both Dhcr14D4-7/D4-7 and Lbr2/2 mice comparedwith control values; however, this was not quite significant(0.05 , P , 0.10) if a multiple comparison statistical testwas applied (data not shown). Desmosterol levels werenormal in Dhcr14D4-7/D4-7 and Lbr2/2 mice. However, des-mosterol was markedly reduced in brain cortex from Lbrþ/2:Dhcr14D4-7/D4-7 mice (Fig. 5A). Although not significant, avariable and minor (0–3% of total sterols) elevation of

Figure 4. GC/MS sterol analysis. Brain sterol chromatograms from 10-day-old control (A), Dhcr14D4-7/D4-7 (B), Lbr2/2 (C) and Lbrþ/2:Dhcr14D4-7/D4-7 (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-3b-ol (4), cholesta-8,14,24-trien-3b-ol (5) and 4,4-dimethylcholesta-8,14-dien-3b-ol (6).




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cholesta-8,14-dien-3b-ol was observed in brain cortex tissuefrom 10-day-old Lbr2/2 mice (Fig. 5B). In contrast,Dhcr14D4-7/D4-7 mice showed a moderate increase incholesta-8,14-dien-3b-ol and cholesta-8,14,24-trien-3b-ol(13 and 1.9% of total sterols, respectively), and Lbrþ/2:Dhcr14D4-7/D4-7 mice showed a major elevation of 47 and11%, respectively, of these same two sterols (Fig. 5B).

In order to determine whether there was a temporal depen-dence of precursor accumulation, sterol analysis was per-formed on brain cortex, kidney and liver tissue from both1-day and 21-day-old mice. Identical to what was observedat 10 days of age, liver and kidney sterols appeared normalin all three mouse mutants at both of these additional timepoints (data not shown). Analysis of brain cortex tissueshowed transient elevations of D14-sterols in Lbr2/2 andDhcr14D4-7/D4-7 mice (Fig. 5C). In brain cortex from1-day-old Lbr2/2 mice, cholesta-8,14-dien-3b-ol andcholesta-8,14,24-trien-3b-ol accounted for 12.4% of totalsterols. However, these levels decreased to below our limitof detection by 10–21 days of age. Analysis of Dhcr14D4-7/

D4-7 brain cortex tissue showed a transient increase inD14-sterols at 10 days of age that resolved by 21 days ofage. In contrast, cholesta-8,14-dien-3b-ol andcholesta-8,14,24-trien-3b-ol increased dramatically from 2.8to 57.8% of total sterols between 1 and 10 days of age inbrain samples from Lbrþ/2:Dhcr14D4-7/D4-7 mice.

Because of technical constraints in producing a Dhcr14mutation and breeding to obtain compound mutant mice, allof the sterol data reported above are from mice on a mixedC57Bl/6 and 129/Sv genetic background. We initiallystudied the Lbr mutation on an isogenic C57Bl/6 genetic

background, and we were unable to detect accumulation ofD14-sterols by GC/MS. Thus, the transient accumulation ofD14-sterols in Lbr2/2 mice appears to be significantly influ-enced by genetic background. No phenotypic differenceswere appreciated on these two genetic backgrounds.

We were able to obtain tissue from a single stillborn Lbr2/2:Dhcr14þ/D4-7 pup for GC/MS analysis. The liver sterol profilewas normal; however, cholesterol synthesis in brain cortexwas markedly impaired. In addition to elevations ofcholesta-8,14-dien-3b-ol and cholesta-8,14,24-trien-3b-ol,GC/MS analysis also showed an accumulation of4,4-dimethyl-cholesta-8,14,-dien-3b-ol, an earlier cholesterolprecursor (Fig. 5D).

Given the absence of a physical phenotype and a relativelyminor biochemical phenotype in Dhcr14D4-7/D4-7 mice, weconsidered the possibility that the Dhcr14 mutation did notproduce a null allele. By quantitative PCR analysis, Dhcr14expression was undetectable in both brain cortex and livertissue (Fig. 6C), and reverse transcriptase–PCR analysis con-firmed the absence of a detectable Dhcr14 transcript in themutant mice (data not shown).

NMR analysis was also used to characterize the sterolabnormality in Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7

mice. Twenty different sterols were identified and quantifiedfrom the NMR spectra of non-saponifiable lipids from10-day-old brain cortex (Table 1). Sterol content of the com-pound heterozygous brains was normal. The major precursorsterols accumulating in Lbrþ/2:Dhcr14D4-7/D4-7 mouse brainswere C27 D8,14 and C27 D8,14,24 species. In addition, therewas a significant accumulation (0.8 and 1.5% of totalsterols, respectively) of the corresponding D8,14 and D8,14,24

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

Genotype Lbrþ/2:Dhcr14þ/D4-7 Lbrþ/þ:Dhcr14D4-7/D4-7 Lbrþ/2:Dhcr14D4-7/D4-7

C27sterolsD5 96.7 80.6 42.7D8,14 0.02 8.2 45.8D5,7 0.1 2.7 2.1D5,8(14) 0.0 0.6 3.4D5,7,9(11) 0.0 0.5 0.9D7 1.3 2.0 0.6D8 0.5 0.9 0.5D5,8 0.2 0.4 0.4D8(14) 0.1 0.1 0.1D7,9(11) 0.00 0.08 0.21D6,8 0.05 0.20 0.13D6,8,14 0.00 0.01 0.11D0 0.12 0.08 0.09D7,14 0.00 0.02 0.09D6,8(14) 0.01 0.01 0.024a-Methyl sterolsD8 0.31 0.20 0.04D8,14 0.01 0.6 0.84,4-Dimethyl sterolsD8 0.27 0.16 0.04D8,14 0.01 2.0 1.5Lanosterol 0.2 0.3 0.3

aSterols are designated according to their position of unsaturation, excluding any D24 unsaturation. For example, D5,8(14) denotescholesta-5,8(14)-dien-3b-ol and cholesta-5,8(14),24-trien-3b-ol. Similarly, D5 denotes cholesterol (D5) and desmosterol (D5,24). Values are from800 MHz 1H NMR signal intensities from single mouse samples, with relative uncertainties of +5% for major sterols (20–50% levels), +10% forminor sterols (0.4–4% levels) and +30% for trace sterols.




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4a-methyl and 4,4-dimethyl sterols. A similar accumulation ofsterol intermediates was observed in brain tissue fromDhcr14D4-7/D4-7 mice. However, the accumulation of C27

D8,14 and D8,14,24 species was more modest.

Expression analysis

Biochemical analysis suggested that Lbr and Dhcr14 are par-tially redundant with respect to sterol D14-reduction. Thus, weinvestigated whether this redundancy was reflected at the levelof gene expression. Control human skin fibroblasts grown incholesterol-depleted medium (LPDS), both with and withoutthe addition of simvastatin to block endogenous cholesterolsynthesis, showed significantly increased DHCR14 expressionin response to LPDS (13-fold, P , 0.05), and LPDS plus

simvastatin (58-fold, P , 0.001) compared with controllevels (Fig. 6A). In contrast, LBR expression was not signifi-cantly increased in response to cholesterol depletion(Fig. 6A). Mouse embryonic fibroblasts showed similarresults. Although the response to LPDS was not significant,Dhcr14 expression increased 10-fold (P , 0.001) in responseto treatment with LPDS plus simvastatin, whereas Lbrexpression was not significantly increased (Fig. 6B).

We also studied Dhcr14 and Lbr expression in liver andbrain tissue from 1-day-old Dhcr14 and Lbr mutant mice(Fig. 6C). In liver tissue, there appeared to be compensatoryexpression of Dhcr14 and Lbr. In Lbr mutant mice, Dhcr14expression increased significantly (6.2-fold, P , 0.01), rela-tive to control livers. Conversely, Lbr expression increasedsignificantly (3.6-fold, P , 0.001) in Dhcr14 mutant mice.

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 indicatedgenotype. Compared with control and compound heterozygous mice, cholesterol was significantly decreased in brain cortex tissue from both Lbr2/2 andDhcr14D4-7/D4-7 mice. Both cholesterol and desmosterol, a sterol normally present in the central nervous system at this age, were dramatically decreased incortex tissue from Lbrþ/2:Dhcr14D4-7/D4-7 mice. (B) In brain cortex from 10-day-old mice, cholesta-8(9),14-dien-3b-ol was significantly increased in bothDhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 mice and cholesta-8(9),14,24-trien-3b-ol was significantly increased in Lbrþ/2:Dhcr14D4-7/D4-7 mice. Althoughthe increase was not statistically significant, cholesta-8(9),14,24-trien-3b-ol could clearly be detected in Dhcr14D4-7/D4-7 brain cortex whereas it was not detectedin tissue from either control or Lbr mutant mice. (C) Relatively small and transient increases in D14-sterols were observed in both Lbr (filled circle) and Dhcr14(triangle) mutant mice. Compared with control brain cortex, D14-sterols were elevated in 1-day-old Lbr2/2 tissue (P , 0.001); however, precursor levels werenormalizing by 10 days and not significantly different from control values. Dhcr14D4-7/D4-7 mice showed a different temporal pattern in that D14-sterol levelswere 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 by21 days. Brain cortex D14-sterol levels were significantly different in Lbr2/2 and Dhcr14D4-7/D4-7 tissues both at 1 day of age (P , 0.001) and 10 days ofage (P , 0.05). In Lbrþ/2:Dhcr14D4-7/D4-7 (open circle) brain cortex D14-sterol levels increase dramatically from 2.8 to 58% of total sterols. (D) GC/MSsterol profile from a stillborn Lbr2/2:Dhcr14þ/D4-7 fetus. In addition to elevated cholesta-8(9),14-dien-3b-ol (peak 3) and cholesta-8(9),14,24-trien-3b-ol(peak 4), an abnormal peak (5) corresponding to 4,4-dimethyl-cholesta-8,14,-dien-3b-ol was clearly detected. Peak 2 is cholesterol and peak 1 correspondsto the internal standard coprostanol. (A–C) Three to six samples were analyzed and the mean and standard deviation are represented. In (C), error bars arenot depicted if they are smaller than the symbol.




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This apparent compensatory increase is much less apparent inbrain cortex. Dhcr14 expression is 1.1-fold (not significant) ofnormal in Lbr2/2 brain tissue, and Lbr shows a significant(P , 0.001) but relatively minor (1.7-fold) increase inexpression in Dhcr14D4-7/D4-7 brains. Dhcr14 expressioncould not be detected in Lbrþ/2:Dhcr14D4-7/D4-7 tissues. Lbrexpression was increased 1.9-fold in liver tissue and 1.2-foldin brain tissue from Lbrþ/2:Dhcr14D4-7/D4-7 mice.

DISCUSSION

Inborn errors of postsqualene cholesterol synthesis underlie anumber of human multiple malformation/mental retardationsyndromes. These include Smith–Lemli–Opitz syndrome(SLOS) and the SLOS-like disorders of desmosterolosis andlathosterolosis, as well as two skeletal dysplasias, CHILD syn-drome (Congenital Hemidysplasia with Ichthyosiform erythro-derma and Limb Defects) and X-linked dominantchondrodysplasia punctata type 2 (CDPX2) (5,6). In screeningdifferent skeletal dysplasias for inborn errors of cholesterolsynthesis, Kelley (5) found minor elevations ofcholesta-8(9),14-dien-3b-ol and cholesta-8(9),14,24-trien-3b-ol consistent with impaired sterol D14-reduction in HEMdysplasia fetuses. Reduction of the C14–C15 double bond

introduced by removal of the 14a-methyl group is an earlystep in the synthesis of cholesterol from lanosterol. Two differ-ent proteins, LBR and DHCR14, have been shown to havesterol D14-reductase activity (2,4). Molecular analysis of anHEM dysplasia patient revealed no DHCR14 mutations, butshowed an apparent homozygous mutation of LBR (8). Onthe basis of these findings, it was proposed that HEM dyspla-sia is due to an inborn error of cholesterol synthesis and thatLBR functions as the primary sterol D14-reductase (8). Inaddition, because the sterol defect was relatively minor, itwas also previously proposed that the severe HEM dysplasiaphenotype results from hormonal-like effects of the accumu-lating precursor sterols (5).

LBR is a bifunctional protein: in addition to functioning as asterol D14-reductase, LBR functions, as its name implies, as alamin B receptor in the inner nuclear membrane. Thus, LBR isinvolved in chromatin binding and organization. The abnormalnuclear morphology and chromatin organization seen inPelger–Huet anomaly is consistent with a defect in LBR func-tion that impairs lamin B and chromatin binding. Human HEMdysplasia or mouse ichthyosis, which result from homozygousmutations of the LBR gene, could plausibly result fromimpaired lamin B binding, impaired sterol D14-reduction, ora combination of these two factors. Because DHCR14 isalso a sterol D14-reductase, and the sterol defect reported in

Figure 6. Dhcr14 and Lbr expression analysis. DHCR14/Dhcr14 (black bars) and LBR/Lbr (gray bars) expression was quantified using real-time PCR in bothhuman 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 intriplicate (�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 andbrain tissue from control and mutant mice was measured by real-time PCR. Data are expressed as fold-increase over control (Lbrþ/þ:Dhcr14þ/þ) and as themean + standard deviation of three samples assayed in triplicate. ND, not detected. Residual mutant transcripts are present in Lbr2/2 tissues, thus accountingfor the low signal present in these tissues.




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HEM dysplasia was relatively mild, we hypothesized thatLBR and DHCR14 provide functional redundancy withrespect to cholesterol synthesis and thus HEM dysplasia andichthyosis are, at the mechanistic level, more likely to be lami-nopathies rather than inborn errors of cholesterol synthesis.

To test the hypothesis that Dhcr14 and Lbr provide func-tional redundancy with respect to sterol D14-reduction, weobtained ichthyosis (Lbr2/2) mice and produced a Dhcr14mutant mouse model. These mutant mice were then bred totest for a digenic phenotype. Characterization of Lbr2/2,Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 mice showedthat they have distinct physical and biochemical phenotypesand that the ichthyosis phenotype is not concordant with adefect in sterol D14-reduction. The data reported in thispaper show that Dhcr14 and Lbr provide significant enzymaticredundancy with respect to sterol D14-reductase activity, thataccumulation of D14-sterols does not cause the mouse ichthyo-sis phenotype and that accumulation of D14-sterols does notappear to cause a hormonal-like disruption of development.From this cumulative evidence, we propose that HEM dyspla-sia and ichthyosis are laminopathies rather than inborn errorsof cholesterol synthesis.

Lbr2/2, Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7

mutant mice have distinct phenotypes. Whereas Lbr2/2

mutant mice had the expected ichthyosis phenotype,Dhcr14D4-7/D4-7 mice were essentially normal and Lbrþ/2:Dhcr14D4-7/D4-7 mice had a unique phenotype. NeitherDhcr14D4-7/D4-7 nor Lbrþ/2:Dhcr14D4-7/D4-7 mice had theichthyotic skin lesions typical of the Lbr mutants. Consistentwith a defect in lamin B and chromatin binding, both Lbr2/2

and Lbrþ/2:Dhcr14D4-7/D4-7 showed chromatin abnormalities.This has previously been shown for both Lbrþ/2 and Lbr2/2

mice (9). Neither abnormalities of chromatin nor Pelger–Huetanomaly were observed in Dhcr14D4-7/D4-7 mice. Both Lbr2/2

and Lbrþ/2:Dhcr14D4-7/D4-7 mice demonstrated markedgrowth retardation and growth plate abnormalities. InLbr2/2 mice, growth plate abnormalities primarily affectedthe hypertrophic zone and the bone trabeculae appeared imma-ture. In contrast, growth plates from Lbrþ/2:Dhcr14D4-7/D4-7

mice were markedly shortened and had an adult-type appear-ance suggestive of an advanced bone age. Lbrþ/2:Dhcr14D4-7/

D4-7 mice demonstrated a unique neurological phenotype con-sisting of progressive ataxia and tremors. Pathologically, thisneurological phenotype is associated with abnormal myelinin the spinal cord. In contrast, neither the Lbr2/2 norDhcr14D4-7/D4-7 mice had these findings. The difference inpostnatal survival found in Lbrþ/2:Dhcr14D4-7/D4-7 andLbr2/2:Dhcr14þ/D4-7 is likely related to complete loss ofthe lamin B/chromatin binding function of Lbr in Lbr2/2:Dhcr14þ/D4-7 embryos. Increased in utero death has pre-viously been reported in Lbr2/2 mice (9).

Sterol analysis of Lbr2/2, Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 strongly supports the idea that Lbr andDhcr14 provide significant functional redundancy withrespect to sterol D14-reduction. No significant accumulationof D14-sterols was observed in liver or kidney tissue fromany of these three mutant mice or in liver tissue from anLbr2/2:Dhcr14þ/D4-7 embryo. This result demonstrates thatin liver tissue a single Lbr or Dhcr14 allele is sufficient forcholesterol synthesis. It is plausible that maternal milk could

have provided a source of cholesterol for peripheral tissuesprior to weaning. However, precursor sterols were undetect-able by GC/MS in peripheral tissues and absent in neonataltissue obtained prior to feeding, thus provision of cholesterolvia maternal milk is an unlikely explanation of our results.In contrast, variable defects in cholesterol synthesis wereobserved in brain cortex tissue. Sterol analysis of braincortex tissue from Dhcr14D4-7/D4-7 mice showed a transientincrease of D14-sterols in brain tissue at 10 days of age.Sterol analysis of Lbr mutant tissues has not previously beenreported. A transient and genetic background-dependentelevation of D14-sterols was observed at birth inLbr2/2 brain tissue. Brain cortex tissue from an Lbr2/2:Dhcr14þ/D4-7 embryo also showed marked accumulation ofD14-sterols. Sterol analysis of brain tissue from Lbrþ/2:Dhcr14D4-7/D4-7 mice showed a major postnatal accumulationof D14-sterols. The time course of this marked accumulationD14-sterols is concordant with the time course of myelination.Thus, it is plausible that the increased need for cholesterolsynthesis during myelin formation accounts for both thetransient increase in D14-sterols in the Dhr14 mutant miceand the marked accumulation in Lbrþ/2:Dhcr14D4-7/D4-7

brains. The transient elevation of D14-sterols in brain from1-day-old Lbr2/2 pups and the markedly abnormalaccumulation of D14-sterols brain tissue from an Lbr 2/2:Dhcr14þ/D4-7 embryo suggests that Lbr may be more criticalwith respect to sterol D14-reduction in the prenatal centralnervous system. The relatively small, tissue limited and tran-sient elevations of D14-sterols found in Dhcr14D4-7/D4-7 andLbr2/2 mice argue strongly for significant enzymatic redun-dancy of Dhcr14 and Lbr with respect to sterol D14-reduction.This conclusion is further supported by the tissue-limited sterol synthetic defect observed in Lbrþ/2:Dhcr14D4-7/D4-7 mice, and the finding that complete inhibitionof sterol D14-reduction, as presumably occurs in Lbr2/2:Dhcr14D4-7/D4-7 embryos, results in embryonic death soonafter implantation.

Gene expression studies support the hypothesis that bothLBR and DHCR14 are involved in cholesterol synthesis. Inhuman and mouse fibroblasts, expression of DHCR14 andDhcr14, respectively, is markedly increased in response tocholesterol depletion. In contrast, the apparent minor increasein LBR and Lbr expression in response to cholesterol depletionis not significantly different than control values. This suggeststhat expression of LBR and Lbr is not regulated in response tocholesterol levels. These data are similar to what has recentlybeen reported for COS-1 cells (12). These results are also con-sistent with the identification of Dhcr14, but not Lbr, as anSREBP2-regulated transcript in mouse liver (13). In contrastto these observations, there appears to be a reciprocal increasein expression of these two genes in liver tissue from mutantanimals. Dhcr14 expression is increased 6.2-fold in Lbrmutant liver and Lbr expression is increased 3.6-fold inDhcr14 mutant liver. This result is consistent with the hypoth-esis that Lbr and Dhcr14 provide redundant sterol D14

reductase activity and likely explains why a biochemicaldefect is not observed in either liver or kidney tissue. Giventhe lack of a response to cholesterol depletion in normalcells by Lbr and considering the fact that Lbr was not ident-ified as a target of SREBP2, it is possible that Lbr expression




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is increased in response to elevated levels of D14-sterols.Neither Dhcr14 nor Lbr showed this reciprocal increase inexpression in brain tissue. This suggests alternative regulationof these genes in the central nervous system and may explainwhy we observed sterol abnormalities only in brain tissue.

Sterol analyses of brain tissue from Lbrþ/2:Dhcr14D4-7/D4-7

mice raises a number of issues with respect to cholesterol syn-thesis in general. The ratio of 24,25-dihydrosterols to D24

sterols was approximately 10 for D5 sterol species andapproximately 3 for D8,14 species. Thus, impaired sterolD14-reduction does not appear to impair Dhcr24 activity,and, as expected, reduction of the D24 bond occurs mainly ator near the stage of 4- and 14-demethylation. The majoraccumulating precursor sterols are cholesta-8,14-dien-3b-oland cholesta-8,14,24-trien-3b-ol rather than the corresponding4a-methyl and 4,4-dimethyl sterols. This finding supports theidea that 4-demethylation can occur at either the D8 or D8,14

stage and occurs prior to isomerization to lathosterol (D7). Fur-thermore, it shows that sterol D8-isomerase (Ebp) does notefficiently catalyze isomerization of D8,14 sterols. Althoughtrace levels of D5,8(14) have been detected in blood fromSLOS patients (14), this work provides strong evidence forthe accumulation of D5,8(14), D6,8,14, D7,9(11) and D7,14 sterolsin a mammalian genetic disorder. The accumulation ofD5,8(14) is an interesting finding since it is not obvious howthis sterol is synthesized. Its presence raises the possibilityof an alternative synthetic pathway in which a D8(14) sterolis the initial product of C14 demethylation. Further work isdefinitely necessary to test this speculative hypothesis ofaltered cholesterol synthesis and to further characterize thebiology of the accumulating sterol precursors. This newmouse model will provide a unique tool to advance our under-standing of the postsqualene cholesterol biosynthetic pathway.

The presence of redundant enzymes for sterol D14-reductionis unique in the scheme of postsqualene cholesterol biosyn-thesis. It is not clear why such redundancy would be necess-ary. Although these enzymes appear completely redundantin liver tissue, redundancy in brain tissue is substantial butnot complete. In addition, there also may be a temporal com-ponent involved. Additionally, regulation of Dhcr14 and Lbrexpression appears to differ between the two genes andbetween liver and brain cortex tissue. Further work is necess-ary to completely define these issues. Again, these new mousemodels will serve as unique tools for future investigations ofcholesterol synthesis.

The laminopathies represent a diverse group of humangenetic syndromes due to mutations of lamin A and a protease,ZMPSTE24, involved in the processing of lamin A. Thesehuman disorders result in premature aging syndromes(Hutchinson–Gilford progeria and atypical Werner syn-drome), myopathies (Emery–Dreifuss muscular dystrophytype 2 and type 3, limb girdle muscular dystrophy anddilated cardiomyopathy, type 1A), neuropathies (Charcot–Marie–Tooth disease, type 2B1), lipodystrophies (Dunnigantype, generalized lipoatrophy/lipodystrophy and mandibuloa-cral dysplasia) and restrictive dermopathy (15). LBR localizesto the inner nuclear membrane and binds both chromatin andlamin B as part of a meshwork of intermediate filament pro-teins known as the nuclear lamina (3). Given the distinctphysical and biochemical phenotypes observed in Lbr2/2,

Dhcr14D4-7/D4-7 and Lbrþ/2:Dhcr14D4-7/D4-7 mice, it is mostplausible that HEM dysplasia is a laminopathy and may rep-resent the first human disorder associated with disturbanceof lamin B function.

MATERIALS AND METHODS

Generation of the Dhcr14D4-7 allele

We identified the mouse mRNA, genomic sequence andgenomic organization of Dhcr14 using NCBI databases(mRNA accession AF480070.1 and genomic AC131114.3).These sequences were used to design PCR primers toamplify both the 4.4 kb 50 flank and the 2.8 kb 30 flank frommouse 129Sv genomic DNA derived from J1 embryonicstem cells. Restriction endonuclease sequences were incorpor-ated into the 50 end of these primers to facilitate subsequentcloning. Roche Taq polymerase and dNTPs were used forPCR amplification. The 50 flank was amplified using primersD14-50B (50-gagaagctggcagcctttgc-30) and D14-50D XbaI(50-tctagaccacctacctcatccctacc-30). The resulting PCR productwas cloned as a NotI/XbaI fragment into the targeting vectorpALS-4. The endogenous NotI restriction site is 11 bp internalto the D14-50B primer. The 30 flank was amplified utilizingprimers D14-30B EcoRI (50-gaattcggcttcatgctggtctttgg-30) andD14-30D SalI (50-gtcgactggagacacgaaagttaccg-30). This frag-ment was cloned as an EcoRI/SalI fragment into pALS-4.For positive selection, the neomycin phosphotransferasegene (PGK-neo) was cloned into this construct between thetwo targeting flanks as an XbaI/EcoRI fragment. The negativeselectable marker, diphtheria toxin A, was cloned into a SalIsite at the end of the 30 flank. Coding regions of the targetingvector were sequenced to confirm the construct.

The targeting vector (Fig. 1A) was linearized with NotIand electroporated into J1 mouse embryonic stem cells,and selection for G418-resistant colonies was as previouslydescribed (16). PCR amplifications using primer A (50-gagctgtttgctgctcaggg-30) and primer B (50-ttaagggccagctcattcctcc-30) were used to identify embryonic stem cells that under-went homologous recombination with the 50 flank of thetargeting vector and the endogenous allele. Homologousrecombination between the 30 flank and the endogenous genewas then confirmed using primers D (50-gagtttggacgacttctgcgc-30) and C (50-gccagaggccacttgtgtagc-30). PCR cyclingconditions consisted of 5 min of denaturation at 948C followedby 30 cycles of 30 s at 948C, 45 s at 628C, 180 s at 728C and afinal extension of 5 min at 728C. The PCR products were thenseparated on a 1% agarose gel (Fig. 1B).

Mouse embryonic stem cell clone c129 was then injectedinto C57/B6 blastocysts to obtain chimeric animals. Chimericfounders were mated with C57/B6 mice to establish germlinetransmission of the Dhcr14 mutant allele (Dhcr14D4-7).Dhcr14þ/D4-7 mice were intercrossed to obtain Dhcr14D4-7/D4-7

mice. Genotyping of the Dhcr14 allele was performed byPCR. Four primers were used in a single assay. PrimersC14-S1 (50-gatgcaggaggcagagcttcg-30) and C14-S3 (50-ccaaagaccagcatgaagcc-30) produce a 248 bp band corresponding tothe control allele. Primers Neo 50 (50-ctgtgctcgacgttgtcactg-30) and Neo 30 (50-gatcccctcagaagaactcgt-30) producea 602 bp fragment corresponding to the mutant allele. PCR




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cycling conditions were 5 min at 948C, followed by 30 cyclesof 60 s at 948C, 60 s at 638C and 60 s at 728C followed by afinal incubation at 728C for 10 min.

For timed matings, the identification of a copulatory plugwas considered to be E0.5 and embryonic age was confirmedby inspection. Animal work was performed under an NICHDapproved animal study protocol.

Sterol analysis

Neutral sterols were extracted from tissue and analyzed byGC/MS as previously described (17). The non-saponifiablelipids from 10-day-old mouse brain cortex tissue were ana-lyzed by 1D and 2D NMR. Spectra were measured inCDCl3 solution at 258C on Varian Inova 600 and 800 MHzNMR spectrometers equipped with a cold probe. The half-height linewidth was ,0.5 Hz. The detection limit for mostminor sterol components ranged from 0.0005 to 0.01% ofthe amount of cholesterol. Sterols were identified by precisecomparisons against spectral data reported for unsaturatedsterols (18), with confirmation by heteronuclear 2D NMRfor D5,7, D5,8, D5,8(14), D5,7,9(11), D6,8, D7, D7,9(11), D8, D8(14)

and D8,14 sterols. Because of the high reproducibility ofNMR data reported for unsaturated sterols (18), no authenticsterol standards were needed. Sterols were quantified fromsignal intensities in the methyl, allylic, hydroxylmethine andolefinic regions of the proton spectrum. Disparities in thesterol profile between mutants were confirmed by examinationof difference spectra. The extent of C4 demethylation and D24

reduction was gauged from 1H, 13C and 2D spectra. Direct 1HNMR quantification from upfield methyl signals provides foreach biosynthetic intermediate the sum of D24 and24,25-dihydro species. This methodological limitation con-veniently removes extraneous detail about the status of theside-chain double bond. The data obtained from Lbrþ/2:Dhcr14þ/D4-7 compound heterozygous brain were not signifi-cantly 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 hematoxylinand eosin using standard procedures. Tissue sections wereviewed and photographed using a Zeiss Axioscope with aZeiss Axiocam.

For electron microscopy, splenic tissue was fixed in 2% glu-taraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at48C. The tissues were washed with cacodylate buffer and post-fixed with 1% OsO4 for 2 h. The tissue was washed again with0.1 M cacodylate buffer, serially dehydrated in ethanol andembedded in Eponate 12 resin (Ted Pella, Redding, CA,USA). Thin sections, �80 nm, were obtained by utilizingthe Leica ultracut-UCT ultramicrotome (Leica, Deerfield, IL,USA) and placed onto 300 mesh copper grids and stainedwith saturated uranyl acetate in 50% methanol and then withlead citrate. The grids were viewed in the Philips 410 electronmicroscope (FEI, Hillsboro, OR, USA) at 80 kV and imageswere recorded on Kodak SO-163 film (Rochester, NY, USA).

Cell culture

3T3-like mouse embryonic fibroblasts were derived as pre-viously described (19). The human skin fibroblast cell line(GM05659C) was obtained from the NIGMS HumanGenetic Mutant Cell Repository, and this work was performedunder an NICHD IRB approved protocol. Fibroblasts weregrown (378C, 5% CO2) in Dulbecco’s modified Eagle’sMedium (Invitrogen, Carlsbad, CA, USA) supplementedwith 10% fetal bovine serum (FBS) (Gemini, Calabasas,CA, USA). Cholesterol-deficient culture was performed inMcCoy’s 5A medium (Invitrogen) supplemented with 7.5%lipoprotein-deficient serum (LPDS). LPDS was preparedusing organic extraction as previously described (20).Simvastatin (R.J. Chemicals, Inc., Pompano Beach, FL,USA) was dissolved in ethanol and added to the medium togive a final concentration of 5 mcg/ml for human cell linesand 1 mcg/ml for mouse cell lines.

Expression analysis

For quantitative PCR, RNA was extracted from tissues andcells using an RNAeasy Mini Kit (Qiagen, Santa Clarita,CA, USA). RNA (100 ng) was reverse transcribed using aHigh-Capacity cDNA Archive kit (Applied Biosystems,Foster City, CA, USA) as per manufacturer’s protocol. Quan-titative PCR assays were performed using DHCR14, Dhcr14,LBR, Lbr, HMGR and Hmgr Assays on Demand fromApplied Biosystems. Analysis was performed on an ABIPrism 7000. All assays were validated, performed in triplicateand normalized to either GAPDH or Gapdh. A minimum ofthree separate specimens were analyzed for each data point.Fold-change relative to control levels was determined usingthe DDCt method.

Statistical analysis

Data are reported as mean + standard deviation. For statisticalanalysis of sterol and expression differences, an ANOVATukey–Kramer multiple comparisons test was used. Achi-squared test was used to evaluate for deviation from theexpected Mendelian ratio. Unless otherwise specified,P , 0.05 was considered significant.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

This work was supported by the intramural research programsof the National Institute of Child Health and Human Develop-ment, National Institutes of Health, and by the Office ofResearch Services, National Institutes of Health. We wouldalso like to express our appreciation to Dr Steven Flieslerfor his critical review of this manuscript.

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




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hem dysplasia and ichthyosis are likely laminopathies and not due to 3 -hydroxysterol...

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