7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

Frequency gradients of DHCR7 mutations in patients with Smith-Lemli-Opitz syndrome in Europe: evidence for different origins of common mutations.

Smith-Lemli-Opitz syndrome/RSH (SLOS) is a multiple congenital anomaly syndrome caused by mutations in the gene for Delta7-sterol reductase (DHCR7) which catalyses the last step in the biosynthesis of cholesterol. SLOS is among the common recessive disorders in Europeans but almost absent in most other populations. More than 40 mutations in the DHCR7 gene some of which are frequent have been described in SLOS patients of various origins. Here we report mutation analysis of the DHCR7 gene in SLOS patients from Poland (n = 15), Germany/Austria (n = 22) and Great Britain (n = 22). Altogether 35 different mutations were identified and the two null mutations IVS8-1G > C and W151X were the most frequent in the total sample. In all three populations three mutations accounted for >0.5 of SLOS chromosomes. The mutational spectra were, however, significantly different across these populations with each of the common mutations showing an east-west gradient (W151X, V326L) or vice versa (IVS8-1G > C). W151X is the most frequent (0.33) mutation in Polish SLOS patients. It has an intermediate frequency in German/Austrian patients (0.18) and is rare among British patients (0.02). V326L shows the same distribution pattern (Poland 0.23, Germany/Austria 0.18, Britain 0.02). In contrast IVS8-1G > C is most frequent in Britain (0.34) intermediate in Germany/Austria (0.20) and rare in Poland (0.03). All analysed IVS8-1G > C and V326L alleles shared the same DHCR7 haplotype, whereas the W151X mutation occurred on different haplotypes. There is evidence for both recurrent mutations and founder effects. Together this suggests that the common SLOS mutations in Europe have different geographic and historic origins and spread across the continent in opposite directions.

Mutational spectrum in the Delta7-sterol reductase gene and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive malformation syndrome, ranges in clinical severity from mild dysmorphism and moderate mental retardation to severe congenital malformation and intrauterine lethality. Mutations in the gene for Delta7-sterol reductase (DHCR7), which catalyzes the final step in cholesterol biosynthesis in the endoplasmic reticulum (ER), cause SLOS. We have determined, in 84 patients with clinically and biochemically characterized SLOS (detection rate 96%), the mutational spectrum in the DHCR7 gene. Forty different SLOS mutations, some frequent, were identified. On the basis of mutation type and expression studies in the HEK293-derived cell line tsA-201, we grouped mutations into four classes: nonsense and splice-site mutations resulting in putative null alleles, missense mutations in the transmembrane domains (TM), mutations in the 4th cytoplasmic loop (4L), and mutations in the C-terminal ER domain (CT). All but one of the tested missense mutations reduced protein stability. Concentrations of the cholesterol precursor 7-dehydrocholesterol and clinical severity scores correlated with mutation classes. The mildest clinical phenotypes were associated with TM and CT mutations, and the most severe types were associated with 0 and 4L mutations. Most homozygotes for null alleles had severe SLOS; one patient had a moderate phenotype. Homozygosity for 0 mutations in DHCR7 appears compatible with life, suggesting that cholesterol may be synthesized in the absence of this enzyme or that exogenous sources of cholesterol can be used.

Genetic defects in postsqualene cholesterol biosynthesis.

In humans and mice, four different genetic defects in the nine biosynthetic steps from lanosterol to cholesterol have been identified. They impair the activity of a putative C3-sterol dehydrogenase (Nshdl, X-linked dominant bare patches/striated mutation in mice), the sterol delta 8-delta 7 isomerase/EBP (Ebp, X-linked dominant tattered mutation in mice; chondrodysplasia punctata (CDPX2) in humans), the delta 24-sterol reductase (autosomal recessive desmosterolosis) and the delta 7-sterol reductase (DHCR7 gene, autosomal recessive Smith-Lemli-Opitz syndrome in humans). These inborn errors in postsqualene cholesterol metabolism result in dysmorphogenetic syndromes of variable severity. The X-linked dominant mutations result in mosaicism in females, as a result of X-inactivation, and midgestational lethality in males. The mechanisms by which the depletion of cholesterol or the accumulation of intermediates impair morphogenetic programs are unclear. So far, no cellular processes that require an intact cholesterol biosynthetic pathway have been identified, although the morphogenetic hedgehog-patched signaling cascade is a candidate.

Molecular genetics of the Smith-Lemli-Opitz syndrome and postsqualene sterol metabolism.

The Smith-Lemli-Opitz syndrome is a disorder of morphogenesis resulting from an enzymatic defect in the last step of cholesterol metabolism (reduction of 7-dehydrocholesterol). Analysis of the defective gene and identification of mutations therein have paved the way for the study of the molecular genetics of the disorder which is caused by numerous different mutations. Future efforts should identify a postulated intracellular signalling activity of sterol intermediates, isolate proteins that govern the sterol traffic between intracellular compartments, structurally characterize the enzyme delta 7-sterol reductase defective in the Smith-Lemli-Opitz syndrome and investigate the pathomechanism of sterol depletion-induced dysmorphogenesis.

Cholesterol biosynthesis from lanosterol. Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein.

The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an inborn disorder of sterol metabolism with characteristic congenital malformations and dysmorphias. All patients suffer from mental retardation. Here we identify the SLOS gene as a Delta7-sterol reductase (DHCR7, EC 1.3.1. 21) required for the de novo biosynthesis of cholesterol. The human and murine genes were characterized and assigned to syntenic regions on chromosomes 11q13 and 7F5 by fluorescense in situ hybridization. Among the mutations found in patients with the SLOS, are missense (P51S, T93M, L99P, L157P, A247V, V326L, R352W, C380S, R404C, and G410S), nonsense (W151X), and splice site (IVS8-1G>C) mutations as well as an out of frame deletion (720-735 del). The missense mutations L99P, V326L, R352W, R404C, and G410S reduced heterologous protein expression by >90%. Our results strongly suggest that defects in the DHCR7 gene cause the SLOS.

Molecular cloning and expression of the human delta7-sterol reductase.

Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM). Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome.