Lathosterolosis: an inborn error of human and murine cholesterol

Lathosterolosis: an inborn error of human andmurine cholesterol synthesis due to lathosterol5-desaturase deficiency

Patrycja A. Krakowiak1, Christopher A. Wassif1, Lisa Kratz2, Diana Cozma1,

Martina Kovarova3,4, Ginny Harris1, Alexander Grinberg5, Yinzi Yang6,

Alasdair G.W. Hunter7, Maria Tsokos8, Richard I. Kelley2 and Forbes D. Porter1,*

1Unit on Molecular Dysmorphology, Heritable Disorders Branch, National Institute of Child Health and Human

Development, National Institutes of Health, Bethesda, MD 20892, USA, 2The Johns Hopkins University, Kennedy

Krieger Institute, Baltimore, MD 21205, USA, 3Institute of Molecular Genetics, Academy of Sciences of the Czech

Republic, 14220 Prague, Czech Republic, 4Molecular Inflammation Section, National Institute of Arthritis and

Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA, 5Laboratory of

Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes

of Health, Bethesda, MD 20892, USA, 6National Human Genome Research Institute, National Institutes of Health,

Bethesda, MD 20892, USA, 7Genetics Patient Service Unit, Children’s Hospital of Eastern Ontario, Ottawa, Ontario

K1H 8L1, Canada and 8National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

Received March 31, 2003; Revised and Accepted May 7, 2003

Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholesterol in the next to laststep of cholesterol synthesis. Inborn errors of cholesterol synthesis underlie a group of human malformationsyndromes including Smith–Lemli–Opitz syndrome, desmosterolosis, CHILD syndrome, CDPX2 andlathosterolosis. We disrupted the lathosterol 5-desaturase gene (Sc5d ) in order to further our understandingof the pathophysiological processes underlying these disorders and to gain insight into the correspondinghuman disorder. Sc5d�/� pups were stillborn, had elevated lathosterol and decreased cholesterol levels, hadcraniofacial defects including cleft palate and micrognathia, and limb patterning defects. Many of themalformations found in Sc5d�/� mice are consistent with impaired hedgehog signaling, and appear to be aresult of decreased cholesterol rather than increased lathosterol. A patient initially described as atypical SLOSwith mucolipidosis was shown to have lathosterolosis by biochemical and molecular analysis. We identified ahomozygous mutation of SC5D (137A>C, Y46S) in this patient. An unique aspect of the lathosterolosisphenotype is the combination of a malformation syndrome with an intracellular storage defect.

INTRODUCTION

Inborn errors of cholesterol synthesis have been shown to causea number of human malformation syndromes. The prototypicalexample is the Smith–Lemli–Opitz syndrome (SLOS) due toa deficiency of 3b-hydroxysterol D7-reductase (DHCR7)activity (1). Other malformation syndromes due to cholesterolbiosynthesis defects include desmosterolosis (3b-hydroxysterolD24-reductase), X-linked dominant chondrodysplasia punctatatype 2 (3b-hydroxysterol D8,D7-isomerase), Greenberg skeletaldysplasia (3b-hydroxysterol D14-reductase activity of the lamin

B receptor), and CHILD syndrome (3b-hydroxysterol D8,D7-isomerase and 3b-hydroxysterol dehydrogenase). Although themolecular defect has not been reported, impaired lanosterol-14-a-demethylase activity has been associated with some cases ofAntley–Bixler syndrome. The clinical, biochemical andmolecular aspects of these syndromes have been reviewed(2–5).

Lathosterol 5-desaturase (SC5D) catalyzes the conversion oflathosterol to yield 7-dehydrocholesterol (7DHC) in thecholesterol synthetic pathway (Fig. 1A). In the subsequentenzymatic step, 7DHC is reduced by DHCR7 to yield

*To whom correspondence should be addressed at: HDB, NICHD, NIH, Bld. 10, Rm 9S241, 10 Center Dr., Bethesda, MD 20892, USA.Tel: þ1 3014354432; Fax: þ1 3014805791; Email: [email protected]

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cholesterol. Mutations of DHCR7 cause SLOS, which is anautosomal-recessive, multiple malformation/mental retardationsyndrome with an estimated birth prevalence of about 1/25 000to 1/40 000 in North America (3,6). We previously reported thedevelopment and characterization of a mouse model for SLOS(Dhcr7�/�) (7). Dhcr7�/� pups demonstrated growth retarda-tion, craniofacial abnormalities including cleft palate, poorfeeding and an uncoordinated suck, hypotonia and decreasedmovement. To understand the pathophysiological processesthat underlie the genesis of the malformations found in SLOSand the Dhcr7�/� mouse, one needs to consider the potentialdetrimental effects of decreased cholesterol levels versus theteratogenic effects of increased 7DHC. To help distinguishbetween these two possibilities, we produced a mouse model inwhich Sc5d was disrupted. Sc5d mutant embryos would beexpected to have decreased cholesterol levels similar to Dhcr7

mutant embryos; however, lathosterol rather than 7DHC wouldbe the accumulating intermediate. A second goal of producinga Sc5d mutant mouse was to aid in the recognition of a humanlathosterolosis syndrome.

We report the development and characterization of alathosterolosis mouse model. Sc5d�/� pups are stillborn,demonstrate intrauterine growth retardation, have craniofacialabnormalities including cleft palate and micrognathia, and limbpatterning defects. Many of the malformations are consistentwith impaired hedgehog functioning during development dueto decreased cholesterol levels. We also report the identificationof a human lathosterolosis patient with an SLOS-likephenotype and mucolipidosis.

RESULTS

Disruption of the lathosterol 5-desaturase gene

Sc5d was disrupted in mouse embryonic stem cells usingtargeted homologous recombination. Recombination betweenthe targeting vector and the endogenous Sc5d allele resultedin the insertion of the neomycin phosphotransferase gene(PGK-neor) and deletion of exon 5 and part of exon 6 of Sc5d(Fig. 1B). Southern blot analysis of EcoRI-digested genomicDNA from 129 G418-resistant embryonic stem cell clonesidentified two clones (1.5%) in which homologous recombina-tion occurred between the targeting vector and a Sc5d allele.Proper targeting between both flanks of the targeting vector andthe endogenous allele was initially detected by Southern blotanalysis using probe A, and confirmed by PCR amplification ofboth flanks (Fig. 1C). Clones 48 and 83 were used to producegermline transmitting chimeric founders, and an identicalphenotype was observed with both lines.

Heterozygous mice appeared phenotypically normal. Thus, weintercrossed Sc5dþ/� mice to determine if a recessive phenotypewas present. No Sc5d�/� mice were identified at weaning(n¼ 322); however, when all pups and embryos were genotyped(n¼ 426) we found a close to expected Mendelian ratio foran autosomal-recessive trait of 27.5% þ/þ, 50.2% þ/� and22.3% �/� (Fig. 1D).

Phenotypic characterization of Sc5d�/� mice

Homozygous mutant pups were stillborn, growth retarded, hadcraniofacial malformations, short limbs, autopod patterningdefects and kinked tails (Fig. 2A–O). Cardiac activity wasdetected in Sc5d�/� embryos just prior to birth, thus mutantpups died during or immediately after birth. Intrauterinegrowth retardation was present with mutant pups having abirth weight of 1.10� 0.01 g (n¼ 30) compared with a birthweight of 1.38� 0.12 (n¼ 86) for controls (P< 0.001,unpaired t-test).

Craniofacial abnormalities included micrognathia, cleftpalate, a narrow frontonasal process and calvarial defects.Micrognathia was present in all Sc5d�/� mice (Fig. 2A–D).Alizarin red and alcian blue stained skeletal preparationsdemonstrated that the micrognathia was due to hypoplasia ofthe distal mandibular arch (Fig. 2C, D, F and G). Cleft palate(Fig. 2E) was observed in 14/16 (88%) of the Sc5d�/� pups but

Figure 1. Targeted disruption of Sc5d. (A) Lathosterol 5-desaturase (Sc5d)catalyzes the conversion of lathosterol to 7-dehydrocholesterol in the next tothe last step of cholesterol synthesis by the Kandutsch-Russell pathway.This enzymatic reaction is impaired in lathosterolosis. 3b-Hydroxysterol D7-reductase (Dhcr7) reduces 7-dehydrocholesterol to form cholesterol, and thisis the enzymatic step impaired in Smith–Lemli–Opitz syndrome. (B) Sc5dgenomic structure, targeting vector and resulting mutant allele. Solid boxeslabeled with Roman numerals represent exons. Coding regions and non-cod-ing regions are filled with black and gray, respectively. Arrows represent thelocation of PCR primers. Endonuclease restriction sites: E, EcoRI; H,HindIII; RV, EcoRV; P, PstI; Bg, BglII. PGKneo is the neomycin phospho-transferase gene used for positive selection, and DTA is the diphtheria toxinA subunit used for negative selection. (C) PCR analysis of untargeted R1embryonic stem cells and two targeted clones (48 and 83). Homologousrecombination between both flanks of the targeting vector and the endogenousallele was confirmed by PCR amplification of the 50-flank (primers A/B) andthe 30-flank (primers C/D). (D) PCR genotyping of embryos from an Sc5dþ/�

intercross. Primer pair E/F amplifies a 200 bp portion of exon 6 which isdeleted in the mutant allele. Primer pair G/H amplifies a 600 bp portion ofthe PGKneo insertion present in the mutant allele.

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was not observed among 38 controls (P< 0.0001, Fisher’sexact test). In mutant pups the nose was narrower than normal(Fig. 2A, B and H), and the mineralized portion of theinterparietal bone was hypoplastic (Fig. 2C, D and H).

Limb patterning defects were observed in both theproximal–distal as well as the preaxial–postaxial axes.Postaxial polydactyly, which consisted of a pedunculated post-minimus with no skeletal elements (Fig. 2I), was identified onthe forelimbs of 7/26 (32%) of Sc5d7 /� and 4/76 (5%)littermate controls (P< 0.01, Fisher’s exact test). Skeletalpreparations demonstrated short, malformed fore and hindlimbs (Fig. 2J–L) and variable expression of a bifurcation of

the fourth medial phalanges of either the fore or hind limbs(Fig. 2M and N). The bifurcation of the fourth medialphalanges was identified in at least one limb, in 56% (5/9) ofthe Sc5d7 /� embryos but in none of 10 control embryos(P¼ 0.01, Fisher’s exact test). Other phenotypic findingsincluded gracile ribs (data not shown) and kinked tails(Fig. 2O). Histological examination was notable for decreasedglycogen in the liver, decreased zymogen granules in thepancreas, increased vacuolation in brown fat, subcutaneousedema and diffuse atelectasis in the lung. No consistentabnormalities of the spleen, thymus, adrenal, testis/ovary orspinal cord were observed.

Figure 2. Phenotypic characterization of Sc5d�/� pups. Sc5dþ/þ (A) and Sc5d�/� (B) neonatal pups. Alizarin red and alcian blue stained skeletal preparationsdemonstrated that the micrognathia is due to hypoplasia of the distal mandibular arch (C, D, F, G). The posterior fontanelle appears enlarged in the mutant pups,and the mineralized portion of the interparietal bone (arrowheads) is smaller (C, D, H). Cleft palate (E) was found in 88% of the Sc5d�/� pups. The frontonasalstructure is narrower in Sc5d�/� pups compared with controls (H). Limb patterning defects included postaxial polydactyly of the forelimb (I) found in 32% of themutant animals, shortening of both the fore (J, K) and hind limb (L), bowing of the radius, ulna, tibia and fibia (J, K, L), and a bifurcation of the fourthmiddle phalangeal bone (M, N). The proximal phalangeal bones in the Sc5d �/� pups have a more rectangular shape compared with the proximal phalangeal bonesin the Sc5dþ/þ pups, and the middle phalangeal bones appear hypoplastic (M). Kinked tails (O) were present in most of the Sc5d�/� pups.

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Identification of a human lathosterolosis patient

The Sc5d�/� mouse has many phenotypic features found inSLOS. Thus, we obtained a sterol profile on fibroblasts from anatypical SLOS patient who died at 18 weeks of age withintracellular storage (8). This patient resembled SLOS in thathe had growth failure, microcephaly, ptosis, cataracts, shortnose, micrognathia, prominent aveolar ridges, ambiguousgenitalia, 2–3 toe syndactyly and postaxial hexadactyly of thefeet. This patient differed from SLOS in that histopathologicalexamination showed intracellular accumulation of mucopoly-saccharides and lipids in tissue macrophages, liver Kupffercells and in non-neuronal cells of the central nervous system.Compared with a control cell line, GC/MS analysis of sterolsfrom patient fibroblasts grown in cholesterol-deficient mediashowed the presence of an additional sterol peak (Fig. 3Aand B). The retention time of this peak matched that of alathosterol standard. The mass spectrum of this peak matchedboth that of a lathosterol standard and published spectra forlathosterol (NIST). Consistent with a deficiency of SC5Dactivity, patient fibroblasts progressively accumulated lathos-terol when grown in cholesterol-deficient medium (Fig. 3C).After 6 days in culture, lathosterol accounted for 35.0� 1.3%(mean� SD, n¼ 3) of total sterols in mutant fibroblastscompared with 6.9� 0.8% (mean� SD, n¼ 3) in controlfibroblasts (P< 0.001, unpaired t-test). To establish thatdeficient SC5D enzymatic activity in patient fibroblasts wasdue to mutation of the SC5D gene, the SC5D transcript frompatient fibroblasts was amplified by RT–PCR and sequenced. Asingle mutation, 137A>C (Y46S), was identified (Fig. 4A).This single mutation was confirmed by genomic sequencing.Both of the proband’s parents were heterozygous for thismutation (Fig. 4A). No consanguinity was present; however,both parents are of French Canadian ancestry. The 137A>Callele was not detected in 116 normal chromosomes, and theY46 position corresponds to a conserved amino acid (Fig. 4B)in the first coding exon. SC5D is encoded on chromosome 11.

Biochemical characterization of Sc5d�/� miceand fibroblasts

Sterol analysis, using gas chromatography/mass spectroscopy(GC/MS), showed that tissues from E18.5 Sc5d�/� embryoshad markedly increased levels of lathosterol (Fig. 5A) anddecreased levels of cholesterol (Fig. 5B) compared with tissuesfrom either Sc5dþ/þ or Sc5dþ/� embryos. In various tissueslathosterol represented 39% (serum), 48% (cortex), 53%(midbrain), 62% (liver), 52% (kidney) or 55% (skeletal muscle)of total sterols. In Sc5dþ/þ tissues, lathosterol accounted for0.1–1.1% of total sterols, with the highest fractions in cortex(0.9%) and midbrain (1.1%). In cortex and midbrain fromE18.5 Sc5dþ/� embryos, lathosterol represented 3.5 and 4.2%of total sterols respectively, and in peripheral tissues rangedfrom 0.2 to 1.9% of total sterols. We characterized theaccumulation of lathosterol during embryonic development.In liver tissue from Sc5d�/� embryos the fraction of lathosterolincreased from 32% at E12.5 to 61% at E18.5 with acorresponding decrease in cholesterol (Fig. 5C). Analysis ofsterol content from E12.5 to E18.5 embryonic brain tissuedemonstrated increased lathosterol and markedly decreased

cholesterol levels in Sc5d�/� embryos compared with Sc5dþ/þ

embryos (Fig. 5D). Decreased cholesterol levels in the olderSc5d�/� embryos probably represent a dilution of maternallyderived cholesterol from the yolk sac as endogenous synthesisresults in accumulation of lathosterol.

Figure 3. Biochemical characterization of the human lathosterolosis fibroblasts.Gas chromatography/mass spectrometry sterol profiles from control (A) andpatient (B) fibroblasts showed the presence of an abnormal sterol peak (3) aftergrowth for three days in cholesterol deficient media. Retention time of this peakwas 16.47 min, which matched the retention time of a lathosterol standard. IS,internal standard (coprostanol); 1, cholesterol; 2, cholesta-8(9)-dien-3b-ol; 3,lathosterol. (C), When cultured in cholesterol deficient media, the patient fibro-blasts (solid bars) progressively accumulated lathosterol compared to a control(open bars) fibroblast culture. Bars on this graph depict the mean and the stan-dard error of the mean for three samples.


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Desmosterol is a major sterol present in the central nervoussystem prior to the onset of myelination (9). In the synthesis ofdesmosterol, Sc5d catalyzes the conversion of cholesta-7,24-diene-3b-ol to yield 7-dehydrodesmosterol which is thenreduced by Dhcr7 to form desmosterol. In cortex tissue fromE18.5 Sc5d�/� embryos desmosterol levels were markedlydecreased, and there was a 35-fold accumulation of cholesta-7,24-diene-3b-ol in addition to a 63-fold accumulation oflathosterol (Fig. 5E). Cortex tissue from heterozygous embryosshowed a slight increase in both lathosterol (4.2-fold) andcholesta-7,24-diene-3b-ol (2.2-fold).

The malformations found in the Sc5d�/� mice are moresevere than those observed in Dhcr7�/� mice (7,10). We thuscompared a number of biochemical parameters betweenSc5d�/� and Dhcr7�/� embryos to determine what mayunderlie the increased severity in the lathosterolosis mousemodel. To exclude either a greater total cholesterol or totalsterol deficit in the lathosterolosis mouse model, we comparedtotal cholesterol and total sterol levels in Dhcr7�/� andSc5d�/� mouse tissues. We confirmed that total sterol levels aredecreased in the serum from Dhcr7�/� E18.5 embryos as

previously reported by Fitzky et al. (10); however, both totalcholesterol and total sterol levels are similar in cortex and liverfrom Dhcr7�/� and Sc5d�/� E18.5 embryos (Fig. 5F). Totalcholesterol measurements do not distinguish between unester-ified and esterified cholesterol. Although total cholesterol levelswere similar between the two mouse models, a greater deficit of

Figure 4. Mutation analysis of the human lathosterolosis patient. (A) DNAsequencing showed the presence of an A to C transversion at nucleotide posi-tion 137 in the proband. Both the mother and father were found to be hetero-zygous at this position. (B) Comparison of amino acid sequences from sterol5-desaturase enzymes shows that the Y46 position is highly conserved.Identical residues are shaded in blue and similar residues are shaded in green.H.s, Homo sapiens (XP_033679); M.m., Mus musculus (BAA33730.1); R.n.,Rattus norvegicus (BAB19798.1); S.p. Schizosaccharomyces pombe(CAA22610.1); S.c., Saccharomyces cerevisiae (AAA34594); C.g., Candidaglabrata (AAB02330.1); A.t., Arabidopsis thaliana (AAF32465.1); N.t.,Nicotiana tabacum (AAD04034.1).

Figure 5. Biochemical characterization of Sc5d �/� embryos. Serum and tissuelathosterol (A) levels and cholesterol (B) levels were determined by gas chro-matography/mass spectrometry analysis. Increased lathosterol and decreasedcholesterol levels were present in tissue and serum from Sc5d �/� E18.5 dayembryos compared to Sc5dþ/þ and þ/� littermates. Heterozygous embryoshad mildly elevated lathosterol levels. (C), Cholesterol (solid squares, solid cir-cles) and lathosterol (open squares, open circles) levels in liver, during deve-lopment, were determined in Sc5d þ/þ (squares) and Sc5d �/� (circles)embryos of the indicated gestational age. Values are expressed as a fractionof total sterols. (D) Cholesterol (solid symbols) and lathosterol (open symbols)levels in whole brains from developing embryos were determined in Sc5d þ/þ

(squares) and Sc5d �/� (circles) embryos of the indicated gestational age. (E)Cholesterol, lathosterol, desmosterol and cholesta-7,24-dien-3b-ol levels inthe cortex of E18.5 embryos. (F) Cholesterol and total sterol levels in control(Dhcr7þ/þ : Sc5dþ/þ), Dhcr7 �/� and Sc5d �/� serum and tissues. Serum andtissue is from E18.5 embryos. Total sterol levels were significantly decreasedin the serum from Dhcr7 �/� embryos compared with control (P< 0.001,unpaired t-test). Total sterol levels in serum, cortex and liver from Sc5d�/�

embryos were not significantly different than control levels (P> 0.10, unpairedt-test). (G) A significant decrease (P< 0.001, ANOVA) in the fraction of unes-terified cholesterol was observed for both E12.5 Dhcr7�/� and Sc5d�/�

embryos compared with controls. Graph shows the mean and standard devia-tion for seven embryos of each genotype.


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unesterified or free cholesterol could potentially explain theincreased phenotypic severity observed in the lathosterolosismouse model. In E12.5 Sc5d�/� embryos the fraction of freecholesterol was significantly decreased compared to controls;however, a similar free cholesterol deficit was also observed inE12.5 Dhcr7�/� embryos (Fig. 5G). Cholesterol-rich raftfractions play a major role in signal transduction. We thuscompared the partitioning of lathosterol and 7DHC into raftfractions. The ratio of either lathosterol or 7DHC to cholesterolin caveolin 1 positive raft fractions from either mouseembryonic fibroblasts or human skin fibroblasts grown inlipoprotein deficient medium was similar to the correspondingwhole cell ratio of either lathosterol or 7DHC to cholesterol.Thus, we found no evidence for a difference in theincorporation of either lathosterol or 7DHC in cholesterol rafts.

Intracellular storage defect in lathosterolosis

A unique aspect of the described patient’s phenotype was thefinding of mucolipidosis. Therefore, we were thereforeinterested in determining if this aspect of the disorder wasreplicated in the mouse model. Although liver size wasrelatively increased in the Sc5d�/� pups (7% of body weightcompared with 4% in controls), lysosomal storage was notevident upon histological examination of either liver or braintissue. The lack of overt lysosomal accumulation may havebeen due to the early perinatal death of Sc5d�/� animals. Wethen tested whether this defect could be found in fibroblasts.When human SC5DY46S/Y46S and mouse Sc5d�/� fibroblastswere grown in cholesterol deficient media, enlarged membrane-bound cytoplasmic vacuoles with lamellar inclusions (arrowsand insets) were present in cells from the lathosterolosis patientand the Sc5d�/� mouse (Fig. 6A–D). Similar intracellularinclusions were not found in control cell lines. By morpho-logical criteria, the intracellular inclusions appeared to be inlysosomes.

DISCUSSION

In this paper we report the development and characterization ofa mutant mouse in which we disrupted Sc5d, and theidentification of a human lathosterolosis patient. The develop-ment of this mouse model and the identification of a humanpatient provide further insight into the biological processeswhich underlie the malformations and clinical problems foundin this distinct group of human malformation syndromes.

This is the second lathosterolosis patient to be identified (11).Although the phenotypic spectrum of lathosterolosis is notdefined by two cases, similarities between these patients andSLOS led to their identification. Phenotypic findings of ptosis,cataracts, micrognathia with prominent alveolar ridges, ambi-guous genitalia, 2–3 toe syndactyly and postaxial polydactylyfound in the patient described in this paper are frequentlydescribed in SLOS patients (12). Phenotypic similaritiesbetween our patient and the previous patient include micro-cephaly, high arched palate, postaxial hexadactyly of the feetand toe syndactyly. Although liver disease was present inboth patients, no intracellular storage was observed in a liverbiopsy of the patient described by Brunetti-Pierri et al. (11).Hepatomegally due to intracellular storage is not reported in

SLOS and may help in some cases to clinically separate the twodisorders. Sterol analysis by GC/MS, which is the diagnostictest for SLOS, can distinguish between these two inborn errorsof cholesterol synthesis. Fibroblasts from both patientsaccumulate lathosterol when grown in delipidated media.Consistent with the more severe phenotype found in ourpatient, lathosterol accounted for 35% of the total sterols infibroblasts after 6 days in culture; whereas, lathosterolaccounted for 12.5% of total sterols after 15 days in culturein the initial case.

The Sc5d mutant mouse model replicates many aspects of thehuman disorder. Biochemically, both the human patient andSc5d�/� mice have a sterol profile characterized by a markedelevation of lathosterol and decreased cholesterol levels.Fibroblasts from both the human patient and the mouse modeldeveloped enlarged membrane-bound cytoplasmic vacuoleswith inclusions when grown in cholesterol-deficient medium.Phenotypical similarities include growth failure, abnormalnasal structure, abnormal palate, micrognathia and postaxialpolydactyly.

To understand the etiological processes underlying mal-formations found in the inborn errors of cholesterol biosyn-thesis and to understand the phenotypic differences observed inSc5d�/� embryos compared to Dhcr7�/� embryos, one needsto consider a number of possible mechanisms. First, specificmalformations could arise due to a cholesterol or steroldeficiency. Second, specific malformations could arise due toa specific teratogenic effect of bioactive cholesterol precursors.One goal of producing the lathosterolosis mouse modeldescribed in this manuscript was to compare the Sc5d�/�

phenotype to the Dhcr7�/� phenotype. Both mutants havedecreased tissue cholesterol levels; however, the accumulatingsterol intermediates differ. A third mechanism that needs to beconsidered is that phenotypic differences could arise due to afunctional sterol deficiency. In this case the ability of theaccumulating sterol intermediate to functionally substitute forcholesterol could influence phenotypic expression or pene-trance due to structural differences between the precursorsterols. Alternatively, cholesterol homeostasis could be per-turbed in a manner that would decrease the amount ofcholesterol available for biological processes. Specifically,alterations in cholesterol esterification or subcellular localiza-tion could lead to a functional cholesterol deficiency. It isunlikely that one of these postulated mechanisms will provide aunifying explanation to explain all of the phenotypic findings.The availability of both genetic and teratogenic mouse modelswhich disrupt cholesterol synthesis at different enzymatic stepsprovide the tools to address this question. The teratogenicmodel systems are useful, but are limited by the lack ofenzymatic specificity (13–15). This paper reports the develop-ment of a lathosterolosis mouse model which can be directlycompared with our previously reported SLOS mouse model (7).

Decreased cholesterol levels likely underlie some of thedevelopmental malformations found in both lathosterolosis andSLOS. In rats, many of the teratogenic effects of AY9944, aninhibitor of both Dhcr7 and the sterol D8-isomerase, can beprevented by provision of cholesterol (15–18). Furthermore,Gaoua et al. (18) showed that 7DHC, unlike cholesterol, doesnot prevent the teratogenic effects of AY9944. Impairedhedgehog function has been proposed as a mechanism


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underlying malformations found in SLOS (19). Hedgehogfamily members play diverse roles in embryonic development,and cholesterol is necessary for maturation of these morpho-gens (20–22). Initially it was proposed that the presence ofabnormal sterols would inhibit Shh autoprocessing; however,Cooper et al. (23) showed that 27-carbon cholesterol precursorsincluding 7DHC and lathosterol can substitute for cholesterolin this reaction. Recent work has shown that hedgehogsignaling is impaired in embryonic fibroblasts derived fromboth the Sc5d mutant mouse described in this paper and theDhcr7 mutant mouse. Specifically, hedgehog signaling isimpaired at the level of Smoothened, and this inhibition isdue to decreased sterol levels (24).

Some of the craniofacial and limb anomalies in the Sc5d�/�

mouse and the lathosterolosis patient are consistent with thehypothesis that hedgehog function is impaired during deve-lopment. Hedgehog signaling is mediated by the receptorPatched, and binding of hedgehog to Patched relieves inhibitionof Smoothened by Patched. Activation of Smoothenedmodulates activity of Gli proteins which regulate transcriptionof hedgehog related genes. Sonic hedgehog (Shh) is involved incraniofacial morphogenesis, and loss of Shh function results in

a narrow frontonasal process, cleft palate and decreased distaldevelopment of the mandible (25,26). Consistent with a defectin Shh function, the Sc5d�/� embryos described in this paperhad a narrow frontonasal process, a significant incidence ofcleft palate, and micrognathia due to hypoplasia of the distalmandible. Mutations in GLI3 cause Greig cephalopolysyndac-tyly (27), postaxial polydactyly type-A/B (28), and PallisterHall syndrome (29). These human syndromes include postaxialpolydactyly. Postaxial polydactyly was observed in 32% of theSc5d�/� pups, and in the human lathosterolosis patientidentified in this report. Similarities between craniofacial andlimb anomalies (post-axial polydactyly and 2–3 toe syndac-tyly) found in both SLOS and lathosterolosis are consistentwith the hypothesis that hedgehog signaling is impaired due tolow cholesterol levels.

Cleft palate is found in 88% of the Sc5d �/� pups. Thismalformation is also present in 9% of Dhcr7�/� pups (7), andhemizygous male Tattered mouse embryos with mutation of the3b-hydroxysterol D7,D8-isomerase gene (30). 3b-HydroxysterolD7,D8-isomerase catalyzes the isomerization of cholesta-8(9)-en-3b-ol to yield lathosterol in the enzymatic stepimmediately preceding Sc5d action. In the human syndromes

Figure 6. Electron micrographs show intracellular membrane-bound inclusions in human and mouse lathosterolosis fibroblasts. Skin fibroblasts from a controlindividual (A), and the lathosterolosis patient (B) were grown in cholesterol deficient media for six days. Magnification for (A) and (B) are 5200-fold and6610-fold, respectively. Embryonic fibroblasts from Sc5d þ/þ (C) and Sc5d�/� (D) mice were grown in cholesterol-deficient media for 4 days. Magnificationsfor (C) and (D) are 6610-fold and 8900-fold respectively. Inset magnifications are 11 500-fold.


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due to inborn errors of cholesterol synthesis, cleft palate isreported in about a third of SLOS patients and has beendescribed in desmosterolosis. Desmosterolosis is due todeficiency of 3b-hydroxysterol D24-reductase, which catalyzesthe reduction of desmosterol to yield cholesterol (31). Thepresence of cleft palate in two different human inborn errors ofcholesterol synthesis and three distinct mouse mutants of thisenzymatic pathway suggests that the mechanism underlying thecleft palate phenotype is probably due to decreased cholesterolduring development rather than a consequence of a specificteratogenic effect of a precursor sterol. The higher frequency ofcleft palate found in Sc5d�/� mice compared with Dhcr7�/�

mice cannot be explained based solely on cholesterol or totalsterol levels. Both total cholesterol and total sterol levels aredecreased to a similar extent in embryonic tissues from thesetwo mouse models. We also measured unesterified cholesterollevels in E12.5 day embryos to determine if a functionalcholesterol deficiency could possibly explain differencesbetween the Sc5d�/� and Dhcr7�/� mice. Although the fractionof total cholesterol which was unesterified was significantlydecreased in both Sc5d�/� and Dhcr7�/� embryos comparedwith control embryos, this decrease was similar in both mutants.Decreased unesterified or free cholesterol levels may exacerbatethe functional cholesterol deficit found in these two disorders.The increased frequency of cleft palate observed in the Sc5d�/�

mice (88%) compared with Dhcr7�/� mice (9%) may be due todifferences in the ability of lathosterol compared with 7DHC tosubstitute for cholesterol during development. Few studies havecompared the biophysical properties of both 7DHC andlathosterol to those of cholesterol. Although both 7DHC andlathosterol are less efficient than cholesterol in condensingartificial membranes, the delta 5(6) bond of cholesterol, which ispresent in 7DHC but absent in lathosterol, is thought to optimizeinteraction between cholesterol and phospholipids (32). 7DHChas been reported to strongly promote the formation ofsphingolipid/sterol raft domains (33); however, similar data onlathosterol is not published. Since both Smoothened andPatched localize to raft domains (34,35), we investigated thesterol composition of caveolin positive raft fractions from bothSLOS and lathosterolosis fibroblasts. Although we cannotexclude a functional defect due to differences in sterolcomposition, both lathosterol and 7DHC are present in caveolinpositive raft fractions. Further work is necessary to define thefunctional ability of 7DHC versus lathosterol to substitute forcholesterol.

Some malformations found in the inborn errors of cholesterolsynthesis are probably due to specific teratogenic effects ofbioactive precursor sterols. Precursor sterols may havebioactive properties themselves or could give rise to bioactiveproducts. 4,4-dimethyl-5a-cholesta-8,24-diene-3b-ol and 4,4-dimethyl-5a-cholesta-8,14,24-triene-3b-ol are meiosis activat-ing sterols (36) and are able to activate LXRa nuclearreceptors (37). Both 7DHC and 8-DHC give rise to unsaturatedbile acids (38) and unsaturated steroid hormone precursors(39,40). It is yet to be determined whether the resultingaberrant unsaturated steroids have agonistic or antagonisticproperties; however, it is plausible that their presence mayaffect developmental processes. Chevy et al. (41) have recentlypostulated that limb abnormalities not found in AY9944treated, but found in triparanol treated rat embryos are likely

due to effects of desmosterol, D8-cholesten-3b-ol and zymos-terol rather than decreased cholesterol levels. Triparanolinhibits both sterol D24-reductase and sterol D8-isomerase. Invitro culture of rat embryos with photooxidized 7DHC impairsdevelopment of the embryos (42). 7DHC has been shown todisturb cholesterol homeostasis. Fitzky et al. (10) have shownthat 7DHC induces the degradation of HMG-CoA reductase,and have proposed that this mechanism underlies the decreasedtotal sterol levels found in SLOS. Wassif et al. (43) have shownthat 7DHC impairs LDL-cholesterol metabolism in SLOSfibroblasts. Hemizygous male tattered mice (Td ), which have amutation of the 3b-hydroxysterol D7,D8-isomerase gene, haveagenesis of the intestines (30). Hemizygous male bare patches(Bpa) and striated mice (Str), which have a mutation of the3b-hydroxysterol dehydrogenase gene, die early in gestation(44). These abnormalities are not observed in either Sc5d�/� orDhcr7�/� embryos, and thus may represent a specificteratogenic defect due to accumulation of cholesta-8(9)-en-3b-ol or 4,4-dimethylcholesta-8-en-3b-ol in Td or Bpa/Strembryos, respectively. The bifurcation of the fourth medialphalanges found in Sc5d�/� embryos has not been reported inother inborn errors of cholesterol synthesis, and thus may bedue to a specific teratogenic effect of lathosterol accumulation.

The multiple malformations and clinical problems encoun-tered in SLOS and other inborn errors of cholesterol synthesisprobably arise due to a combined effect of increased precursorlevels, decreased cholesterol levels and variable ability of theprecursor sterols to functionally substitute for cholesterol.Comparison of phenotypic findings found in the humandisorders, the teratogenic rat models, and genetic mousemodels should provide insight into the genesis of a specificmalformation.

Histochemical staining showed the storage of both lipid andmucopolysaccharides and lysosomal inclusions were identifiedby electron microscopy in tissue samples from the lathostero-losis patient (8). Intracellular LDL cholesterol transport isperturbed in SLOS fibroblasts (43). In both DHCR7 and SC5Dmutant fibroblasts we have observed transient increased filipinstaining. This suggests that LDL-cholesterol metabolism maybe impaired in both disorders. Altered intracellular cholesterollevels have been shown to modulate membrane and lipidtrafficking in normal and sphingolipid-storage disease fibro-blasts (45,46). Based on the histochemical findings reported forthe human patient and the replication of the intracellularstorage defect in both human and murine fibroblasts whengrown in cholesterol deficient medium, we postulate thataccumulation of lathosterol may disrupt normal intracellularmembrane and lipid trafficking.

This paper describes the identification of a human lathostero-losis patient and characterization of a lathosterolosis mousemodel. The lathosterolosis mouse which replicates many of thedefects described in the lathosterolosis patient will permitfurther investigation of the pathophysiological processes thatunderlie this human malformation syndrome. The lysosomalstorage defects reported in the human lathosterolosis patientand the identification of an intracellular storage defect inSc5d�/� fibroblasts raises the question of whether otherunclassified ‘mucolipidosis’ or lysosomal storage patients havemutations in SC5D. Identification of additional patients will beimportant to delineate the full phenotypic spectrum of


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lathosterolosis and to determine if less severely affectedpatients exist. Based on the clinical experience with SLOS,dietary cholesterol supplementation may benefit these patients.

MATERIALS AND METHODS

Targeting of embryonic stem cells, generation ofchimeric mice and mouse manipulation

Two bacterial artificial chromosomes (BAC) containing Sc5dwere identified by hybridization to a mouse EST (AA245978)(Genome Systems). Prior to constructing the targeting vector, a14.5 kb HindIII/EcoRI fragment containing exons 1–6 wascloned into pBS (Stratagene), a restriction endonuclease mapwas obtained, and the intron–exon structure of the gene wasdelineated. The targeting vector was constructed in pBS andconsisted of a 4.3 kb EcoRV/PstI50-flank, a 2.5 kb BglII/HindIII30-flank and a PGKneo1 cassette (Stratagene). A 2.2 kbPstI/BglII fragment encoding exon 5 and most of exon 6 wasdeleted. The negative selectable marker, DTA, was cloned into aSalI site. The targeting vector was linearized on the 50 end of thetargeting construct with NotI. Electroporation and cell cultureconditions were as previously described (7). Screening of G418-resistant clones was performed by Southern blot of an EcoRIgenomic digest probed with a 600 bp PstI/HindIII genomicfragment ( probe A). The endogenous fragment was 7 kb and thetargeted allele fragment was 13 kb. Positive clones wereconfirmed by long-range PCR (Expand kit, Roche) with primersinternal to the neomycin resistance gene and external to thetargeting flanks: 50-flank—primer A-50-CAGCTGGACAG-CCGCGAGTG-30 and primer B-50-TGACGAGTTCTTC-TGAGGGG-30; 30-flank—primer C-5-0CCCCTCAGAAGAA-CTCGTCA-30 and primer D-50-GGTTTGACTTAGGAGTTC-ACTGC-30. Four PCR primers were used in a combined reactionfor genotyping. These were mutant—G-50-CTGTGCTCGAC-GTTGTCACTG-30 and H-50-GATCCCCTCAGAAGAACTC-GT-30, which amplify a 600 bp fragment of the neomycinphosphotransferase gene—and wild-type—E-50-ATGCTTTTC-ACCCTGTGGAC-30 and F-50-GTGGTGGTCTGTGTGGT-GAG-30, which amplify a 200 bp product of exon 6. PCR cyclingconditions consisted of 5 min denaturation at 94�C followed by35 cycles of 30 s at 94�C, 60 s at 63�C, 60 s at 72�C and a finalextension of 10 min at 72�C. For timed matings, the identificationof a copulatory plug was considered to be E0.5 and embryonicage was confirmed by inspection. Animal work was performedunder an NICHD approved animal study protocol.

Sterol analysis

For sterol analysis, cell pellets were homogenized and saponifiedfor 1 h with 4% KOH in ethanol at 60�C. Five micrograms ofcoprostanol were added as an internal standard. The sampleswere then extracted in an equal volume of ethyl acetate, driedunder nitrogen and derivitized with BSFTA plus 1% TMCS(Pierce). Samples were analyzed by both gas chromatography/flame ionization detection (Agilent 6890) and gas chromato-graphy/mass spectrometry (Trace Thermo Finnigan) using aPhenomenex ZB-1701 column (30 m� 0.32 mm� 0.25 mm).Cholesterol, coprostanol, 7-dehydrocholesterol, and lathosterol

standards were obtained from Sigma. For free sterol analysis,the sample was divided and half was analyzed withoutsaponification.

Histological analysis

Tissues were fixed using buffered formalin (Sigma), paraffin-embedded, and stained with hematoxylin and eosin. Skeletalstaining with alcian blue and alizarin red S was performed asdescribed by McLeod (47). For electron microscopy, cellpellets or tissues were fixed in 2.5% glutaraldehyde in PBS( pH 7.4), postfixed in OsO4, and embedded in Maraglas 655(Ladd Research Industries). Sections were stained with uranylacetate–lead citrate and examined in a Philips CM10 electronmicroscope.

Cell culture

Mouse and human fibroblasts were grown (37�C, 5% CO2) inDMEM supplemented with 10% fetal bovine serum. 3T3-likemouse embryo fibroblasts were derived as previously described(48). Cholesterol-deficient culture was done in McCoy’s 5Amedia supplemented with 7.5% lipoprotein deficient serum (49).Caveolin-positive membrane fractions were purified fromembryonic fibroblasts as previously described (50).

Mutation and molecular biology analysis

RNA for RT–PCR was isolated using an RNeasy mini kit(Qiagene), and DNAwas isolated using a Gentra kit. Sequencingwas performed using Beckmann Coulter’s CEQ2000 sequencerand reagents. Primers used for RT–PCR were: C5h1-50-GGGCTAAGTGATGGATCTTG-30 and C5h6-50-CCACG-ATGCTGATTTCCAA-30 ( product size, 1 kb). RT–PCR wasperformed using SuperScript RT–PCR One Step (Invitrogen)with the following cycling conditions: 30 min at 50�C, 2 min at94�C, 35 cycles of 94�C for 15 s, 60�C for 30 s and 72�C for 90 swith a final extension at 72�C for 10 min. The RT–PCR productwas gel-purified (GeneClean II) and sequenced using primersC5h1, C5h6, C5h2-50-ATGAAGGCCTCTGTGAATCC, C5h3-50-TGATGACCTAGGAGAGTTTCCA-30, C5h4-50-GCCAA-TCCTATCCCACAAAG-30 and C5h5-50-TCATGACGGTGA-TTTTCGTG-30. The SC5D coding sequence was identified inGenbank (XP_033679) and used to search the human genomesequence (www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F¼HsBlast.html&&ORG¼Hs). Individual exons were amplified byPCR, gel purified, and sequenced. Genomic DNAwas sequencedusing intronic primers: exon 1 (405 bp) C5he1F (-54)50-GCCTGGAAAAATAGAGACATGG-30, C5he1R (þ131)50-ACCCATTTGTGGTTGGTCTC-30, exon 2 (410 bp) C5he2Fa(�119) 50-GTTTGGAGGTAA-GCCCCTTC-30, C5he2Ra(þ155) 50-GCAAGACATG-AACACATGAAAA-30, exon 3(246 bp) C5he3F (�47) 50-CCCAGGAGCTGAGTTTTGAT-30, C5he3R (þ97) 50-TGGGTAGAGGAAATTCTTGGAA-30,exon 4 (390 and 401 bp) C5he4Fa (�80) 50-TCCAACATGAG-TGTGAGAAGC-30, C5he4Ra (c834as) 50-GCCAATCCTAT-CCCACAAA-G-30, C5he4Fb (c717as) 50-ATTTTCGTGT-CCCCCAAATC-30, C5he4Rb (c1116as) 50-CCACGATGC-TGATTTCCAA. Genomic PCR was performed usingInvitrogen’s Platinum Taq DNA polymerase with a final


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magnesium concentration of 2.0 mM. Cycling conditions were:94�C for 2 min followed by 35 cycles of 94�C for 30 s, 62�Cfor 30 s, 72�C for 30 s and a final extension at 72�C for 10 min.Homology alignments were performed using BoxShade (http://molbio.info.nih.gov/molbio/gcglite/boxshade.html).

ACKNOWLEDGEMENTS

We would like to thank Sing-Ping Huang, Dr Heiner Westphal,Dr Diana Hanes and Dr Jerrold Ward for their assistance withthis project. We would like to acknowledge the contributionof Peter Carolan in performing the whole mount in situexperiments and the efforts of Mones Abu-Asab. The8(9)-cholestenol was a gift from the Schroepfer laboratory.We would like to thank Brooke Wright, Dr Lina Cerro-Correa,Dr Janice Chou and Dr William Gahl for their input on thismanuscript, and acknowledge the assistance of Todd Hunter forsearching through dusty attic files to track down patientrecords. Finally, we would like to express our gratitude to theparents of the child described in this paper for their continuedhelp and understanding.

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Lathosterolosis: an inborn error of human and murine cholesterol