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Mutations in the 7-Sterol Reductase Gene in Patients with the Smith

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Mutations in the 7-Sterol Reductase Gene in Patients with the Smith Proc. Natl. Acad. Sci. USA Vol. 95, pp. 8181–8186, July 1998 Genetics Mutations in the D7-sterol reductase gene in patients with the Smith–Lemli–Opitz syndrome BARBARA U. FITZKY*, MARTINA WITSCH-BAUMGARTNER†,MARTIN ERDEL†,JOON NO LEE‡,YOUNG-KI PAIK‡, HARTMUT GLOSSMANN*, GERD UTERMANN†, AND FABIAN F. MOEBIUS*§ *Institut fu¨r Biochemische Pharmakologie, Universita¨tInnsbruck, Peter Mayr Strasse 1, A-6020 Innsbruck, Austria; †Institut fu¨r Medizinische Biologie und Humangenetik, Scho¨pfstrasse 41, A-6020 Innsbruck, Austria; and ‡Department of Biochemistry and Bioproducts Research Center Yonsei University, Seoul 120–749, Korea Edited by Michael Brown, University of Texas Southwestern, Dallas, TX, and approved May 7, 1998 (received for review April 9, 1998) ABSTRACT The Smith–Lemli–Opitz syndrome (SLOS) ICRFP700C2137Q06) was isolated by hybridizing a 32P- is an inborn disorder of sterol metabolism with characteristic labeled human DHCR7 cDNA probe to prespotted filters congenital malformations and dysmorphias. All patients suf- obtained from the Resource CentreyPrimary Database (Ber- fer from mental retardation. Here we identify the SLOS gene lin, Germany). Intron–exon boundaries were identified by as a D7-sterol reductase (DHCR7, EC 1.3.1.21) required for PCR amplification of intron sequences by using cDNA se- the de novo biosynthesis of cholesterol. The human and murine quence-derived oligonucleotides and DNA sequencing. Sites genes were characterized and assigned to syntenic regions on for restriction enzymes were mapped by Southern hybridiza- chromosomes 11q13 and 7F5 by fluorescense in situ hybrid- tion of exon-specific probes to P1 clone digests and digestion ization. Among the mutations found in patients with the of PCR-amplicons, respectively. SLOS, are missense (P51S, T93M, L99P, L157P, A247V, Cloning of the Murine cDNA and Gene. Mouse Dhcr7 V326L, R352W, C380S, R404C, and G410S), nonsense cDNAs were identified in the expressed sequence tag database (W151X), and splice site (IVS8–1G>C) mutations as well as with the TBLASTN algorithm. The clone with the longest an out of frame deletion (720–735 del). The missense muta- 59-sequence (GenBank accession no. AA003001) was se- tions L99P, V326L, R352W, R404C, and G410S reduced quenced entirely and found to contain a 262-bp deletion. heterologous protein expression by >90%. Our results Another clone (GenBank accession no. AA475208) as well as strongly suggest that defects in the DHCR7 gene cause the two reverse transcription-PCR products from mouse liver SLOS. contained the deleted region that was replaced with a BamHI– XbaI fragment from clone AA475208. Vectors for heterolo- The autosomal recessive Smith–Lemli–Opitz syndrome gous expression in yeast (mD7-ORF and mD7-myc) were (SLOS, MIM 270400) is a remarkable example of an inborn prepared with oligonucleotides CCGCTCGAGAAAATG- error of metabolism that impairs morphogenesis. Phenotypic GCTTCGAAATCCCAGC (introducing a XhoI site upstream expression is variable and comprises congenital anomalies of the start codon) and CCGCTCGACAGATCTTGCTTC- such as microcephaly, cleft palate, syndactyly of toes 2 and 3, GAAATCCCAGCAC (introducing a 59 BglII site in frame polydactyly, and visceral malformations as well as postnatal with the 9E10 c-myc epitope cassette, ref. 4). For Northern blot failure to thrive and mental retardation (1). Genitalia in analysis a 32P-labeled Dhcr7 cDNA was hybridized to mouse severely affected boys are either ambiguous or fail to become multiple tissue Northern blots (CLONTECH). A cosmid clone masculinized (1). A deficiency of the ultimate step of choles- (clone I.D. MPMGc121P1353Q3) was isolated from a genomic terol biosynthesis is suspected to cause the SLOS because a library (strain 129Ola) by hybridizing the 32P-labeled Dhcr7 decrease of plasma cholesterol and the accumulation of its cDNA probe to prespotted filters obtained from the Resource precursor 7-dehydrocholesterol are always observed (2). Sterol CentreyPrimary Database (Berlin, Germany). metabolism depends on the tight regulation of endogenous Genetic Analysis. All patients were diagnosed with SLOS by biosynthesis and receptor mediated transport (3). Failure to their physicians who provided DNA or blood samples for remove the D7-bond in de novo-synthesized sterols could be caused by a deficiency of the catalytic subunit, a regulatory molecular analysis. The clinical diagnosis was biochemically protein, or of a cofactor of the D7-sterol reductase (DHCR7). confirmed by identification of 7-dehydrocholesterol in the We recently cloned the cDNA encoding the catalytic subunit serum by gas chromatography and mass spectrometry. of the enzyme (4). Using this cDNA, we now isolated and Genomic DNA was prepared from cultured fibroblasts (SLO5 characterized the corresponding human and mouse genes. and SLO9) or peripheral lymphocytes. Exons 3–9 were am- Among the mutations found in patients with biochemically plified by using PCR (annealing temperature 65°C) with the oligonucleotides shown in Table 2. For single strand confor- confirmed diagnosis of SLOS were ten missense, one non- m sense, and one splice acceptor site mutation as well as a 16-bp mation polymorphism (SSCP) analysis, 7 l of a PCR reaction deletion. Several of the missense mutations reduced heterol- was subjected to electrophoresis on a Mutation Detection ogous protein expression by .90%. This result strongly sug- Enhancement (FMC) gel that was stained with silver (5). PCR gests that DHCR7 defects cause the SLOS. products were purified on QIAquick columns (Qiagen) and cycle sequenced on an ABI377 automated sequencer (Perkins– Elmer). PCR analysis of cDNA from fibroblasts with cDNA EXPERIMENTAL PROCEDURES sequence-derived oligonucleotides was performed by isolating Isolation and Mapping of the Human DHCR7 Gene. AP1 mRNA with RNAzol B (Molecular Research Center, Cincin- clone from a human genomic library (clone I.D. This paper was submitted directly (Track II) to the Proceedings office. D The publication costs of this article were defrayed in part by page charge Abbreviations: DHCR7, 7-sterol reductase; SLOS, Smith–Lemli– Opitz syndrome; wt, wild type; TMS, transmembrane segment; SSCP, payment. This article must therefore be hereby marked ‘‘advertisement’’ in single strand conformation polymorphism. accordance with 18 U.S.C. §1734 solely to indicate this fact. Data deposition: The sequence reported in this paper has been © 1998 by The National Academy of Sciences 0027-8424y98y958181-6$2.00y0 deposited in the GenBank database (accession no. AF057368). PNAS is available online at http:yywww.pnas.org. §To whom correspondence should be addressed. 8181 Downloaded by guest on September 23, 2021 8182 Genetics: Fitzky et al. Proc. Natl. Acad. Sci. USA 95 (1998) nati, OH), and transcribing cDNA with a 1st strand cDNA clone in a filter-spotted human genomic library. Intron–exon synthesis kit (Pharmacia). boundaries and partial intron sequences were determined by Fluorescence in Situ Hybridization. Fluorescence in situ PCR amplification of introns and sequencing. The isolated hybridization was performed as described (6) by using DNA clone contained the entire ORF and the 59- and 39- from the genomic human P1 and the murine cosmid clone, nontranslated regions of the cDNA. Restriction enzyme di- respectively. Chromosomal in situ suppression hybridization gests of the P1 clone and subsequent Southern hybridization and detection by immunofluorescence was carried out on with exon-specific probes identified the restriction sites shown metaphase chromosome preparations from phytohemaggluti- in Fig. 1A. The gene contains nine exons and eight introns and nin-stimulated normal human lymphocytes and normal mouse spans ;14 kilobases (Fig. 1A, Table 1). Introns 4, 7, and 8 embryo fibroblasts with biotinylated (human) or digoxigenin- interrupt putative transmembrane segments (Fig. 2). The two labeled (mouse) probes. Fifty of 50 human and 66 of 80 mouse first exons are noncoding, and the start codon is localized in metaphase cells exhibited specific labeling of one or both exon 3. Exon 9 is the largest exon and contains the stop codon homologs of chromosomes 11 and 7, respectively. and the two polyadenylation sites recently described (4). The In Vitro Mutagenesis and Heterologous Expression of Mu- P1 clone was hybridized in situ to human metaphase chromo- tated cDNAs. A modified wild-type (wt) DHCR7 cDNA was somes and detected by fluorescence on chromosomal region constructed from mycD7-ORF (4) in pBluescriptKSII (Strat- 11q13 (data not shown). agene) by introducing a SpeI restriction site with oligonucle- Assignment of the Murine Gene to Chromosome 7. To otide GGAATTCACTAGTCCTTAGAAGATTCCAGGC. confirm the chromosomal assignment and as a prerequisite for Mutations were introduced in AccI–SacI (296T.C) and SacI– future in vivo studies, we cloned the murine ortholog by SpeI (976G.T, 1054C.T, 1210C.T, 1228G.A, 1158C.T, screening the expressed sequence tag database for sequences 1272T.C, and 976T.G) cassettes by two-step mutagenesis as with homology to the human DHCR7. The protein is 89% described (7). Second site mutagenesis was ruled out by DNA sequencing of both strands. wt and mutated cDNAs were identical and 98% similar with the human enzyme and lacks subcloned into pCIneo (Promega) with XhoI–NotI restriction four amino acid residues in the N terminus (Fig. 2). In contrast sites. Cultivation of tsA-201 cells and calcium phosphate to the human cDNA, no second polyadenylation site was found transfection (6 mg of specific
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