Portal de Periódicos da CAPES

RSH/Smith-Lemli-Opitz Syndrome: Mutations and Metabolic Morphogenesis

1998; Elsevier BV; Volume: 63; Issue: 2 Linguagem: Inglês

10.1086/301987

1537-6605

Richard I. Kelley,

MicroRNA in disease regulation

For almost 30 years following its description in 1964, the RSH syndrome—now more often called the “Smith-Lemli-Opitz syndrome” (SLOS)—was just one of many autosomal recessive, multiple anomaly syndromes that filled the pages of dysmorphology atlases (MIM 270400; Smith et al., 1964Smith DW Lemli L Opitz JM A newly recognized syndrome of multiple congenital anomalies.J Pediatr. 1964; 64: 210-217Abstract Full Text PDF PubMed Scopus (497) Google Scholar). Its specific combination of craniofacial anomalies, polydactyly, cleft palate, and genital malformations in males was readily diagnosed by dysmorphologists but otherwise caused little interest. In 1993, however, SLOS was abruptly lifted from relative obscurity by the discovery that affected patients have as much as 2,000-fold–increased blood levels of 7–dehydrocholesterol (7DHC), the immediate precursor of cholesterol in the Kandutsch-Russell pathway of cholesterol biosynthesis (Irons et al., 1993Irons M Elias ER Salen G Tint GS Batta AK Defective cholesterol biosynthesis in Smith-Lemli-Opitz syndrome.Lancet. 1993; 341: 1414Abstract PubMed Scopus (303) Google Scholar; Tint et al., 1994Tint GS Irons M Elias ER Batta AK Frieden R Chen TS Salen G Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome.N Engl J Med. 1994; 330: 107-113Crossref PubMed Scopus (642) Google Scholar). The now apparent high specificity of increased 7DHC levels for diagnosis of SLOS (Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar) has made possible not only definitive diagnosis of SLOS but also prenatal diagnosis by measurement of 7DHC in amniotic fluid or chorionic villus (Abuelo et al., 1995Abuelo DN Tint GS Kelley R Batta AK Shefer S Salen G Prenatal detection of the cholesterol biosynthetic defect in the Smith-Lemli-Opitz syndrome by the analysis of amniotic fluid sterols.Am J Med Genet. 1995; 56: 281-285Crossref PubMed Scopus (55) Google Scholar; Rossiter et al., 1995Rossiter JP Hofman KJ Kelley RI Smith-Lemli-Opitz syndrome: prenatal diagnosis by quantification of cholesterol precursors in amniotic fluid.Am J Med Genet. 1995; 56: 272-275Crossref PubMed Scopus (45) Google Scholar; Mills et al., 1996Mills K Mandel H Montemagno R Soothill P Gershoni-Baruch R Clayton PT First trimester prenatal diagnosis of Smith-Lemli-Opitz syndrome (7-dehydrocholesterol reductase deficiency).Pediatr Res. 1996; 39: 816-819Crossref PubMed Scopus (40) Google Scholar). Not unexpectedly, the availability of a laboratory test for SLOS also has led to an expansion of its clinical phenotype to include near-normal children with no discrete malformations, as well as severely malformed fetuses that die in utero. Studies of blood sterol levels of SLOS patients show that clinical severity correlates best not with the absolute level of 7DHC but inversely either with the level of cholesterol or with the level of cholesterol as a fraction of total sterols (Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar). The most severely affected, “type II” SLOS patients (Curry et al., 1987Curry CJ Carey JC Holland JS Chopra D Fineman R Golabi M Sherman S et al.Smith-Lemli-Opitz syndrome-type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality.Am J Med Genet. 1987; 26: 45-57Crossref PubMed Scopus (149) Google Scholar) typically have blood cholesterol levels of 1–10 mg/dl (0.03–0.3 mmol/liter) and die in the newborn period because of multiple internal anomalies (Tint et al., 1995Tint GS Salen G Batta AK Shefer S Irons M Elias ER Abuelo DN et al.Correlation of severity and outcome with plasma sterol levels in variants of the Smith-Lemli-Opitz syndrome.J Pediatr. 1995; 127: 82-87Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). At the other extreme, ∼10% of SLOS patients have minimal disease and normal or low-normal cholesterol levels at diagnosis (Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar). Although, historically, a clinical distinction often was made between classic (“type I”) SLOS and the more severe, type II patients, there is, in reality, a clinical and biochemical continuum from mild to severe SLOS (Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar). The biosynthesis of cholesterol and its related family of “isoprenoid” compounds, including coenzyme Q and dolichols, begins with the conversion of 3-hydroxy-3-methylglutaric acid to mevalonic acid and progresses to the synthesis of polyisoprenoids, such as geraniol-PP and farnesol-PP. After the formation of the first sterol, lanosterol, from squalene, there follows a succession of demethylations and double-bond rearrangements, the last of which is the reduction of the 7-8 double bond of 7DHC to form cholesterol, mediated by the microsomal enzyme, 3β-hydroxysterol-Δ7-reductase (DHCR7; E.C.1.3.1.21). The finding of marked elevations of 7DHC in SLOS immediately implicated DHCR7 as the site of the genetic defect (Tint et al., 1994Tint GS Irons M Elias ER Batta AK Frieden R Chen TS Salen G Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome.N Engl J Med. 1994; 330: 107-113Crossref PubMed Scopus (642) Google Scholar). Although the chromosomal location of DHCR7 was unknown in 1993, attention soon turned to 7q32.1 because of two unrelated SLOS patients, one of whom was biochemically confirmed, who were found to have translocations at that position (Alley et al., 1995Alley TL Gray BA Lee SH Scherer SW Tsui LC Tint GS Williams CA et al.Identification of a yeast artificial chromosome clone spanning a translocation breakpoint at 7q32.1 in a Smith-Lemli-Opitz syndrome patient.Am J Hum Genet. 1995; 56: 1411-1416PubMed Google Scholar). However, earlier this year, Fabian Moebius and colleagues reported the cloning of a human microsomal DHCR7 gene and mapped it to chromosomal position 11q12-13 (Moebius et al., 1998Moebius FF Fitzky BU Lee JN Paik YK Glossmann H Molecular cloning and expression of the human delta 7-sterol reductase.Proc Natl Acad Sci USA. 1998; 95: 1899-1902Crossref PubMed Scopus (182) Google Scholar). The same laboratory has now reported that all 13 patients with classical SLOS whom they studied had mutations in DHCR7 (Fitzky et al., 1998Fitzky BU Witsch-Baumgartner M Erdel M Lee JN Paik Y-K Glossmann H Utermann G et al.Mutations in the delta-7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome.Proc Natl Acad Sci USA. 1998; 95: 8181-8186Crossref PubMed Scopus (316) Google Scholar). In this and the July issue of the Journal, two other groups working independently, one at the National Institutes of Health and headed by Forbes Porter and another that is a consortium of Dutch centers, report mutations of the same DHCR7 gene in six additional patients with SLOS (Wassiff et al., 1998Wassiff CA Maslen C Kachilele–Linjewile S Lin D Linck LM Conner WE Steiner RD et al.Mutations in the human sterol ‡7-reductase gene at 11q12-13 cause Smith-Lemli-Opitz syndrome.Am J Hum Genet. 1998; 63: 55-62Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar; Waterham et al., 1998Waterham HR Wijburg FA Hennekam RCM Vreken P Poll-The BT Dorland L Duran M et al.The Smith-Lemli-Opitz syndrome is caused by mutations in the 7–dehydrocholesterol reductase gene.Am J Hum Genet. 1998; 63 (in this issue): 329-338Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), confirming that, for most patients with a clinical diagnosis of SLOS and increased 7DHC levels, the gene for DHCR7 harbors the responsible mutations. However, because one pair of siblings with phenotypic SLOS and moderately increased levels of 7DHC has been found to have an apparent defect of sterol transport (Anderson et al., 1998Anderson AJ, Stephan MJ, Walker WO, Newman RJ, Kelley RI (1998) Variant Smith-Lemli-Opitz syndrome with atypical sterol metabolism. Am J Med Genet (in press)Google Scholar), mutations in genes other than DHCR7 are possible. The three articles describing DHCR7 mutations reveal interesting aspects of the biochemistry and population genetics of SLOS. The historically relatively high incidence of SLOS—∼1/20,000–30,000 births among those of northern- and central-European background (Opitz, 1994Opitz JM RSH/SLO (“Smith-Lemli-Opitz”) syndrome: historical, genetic, and developmental considerations.Am J Med Genet. 1994; 50: 344-346Crossref PubMed Scopus (71) Google Scholar; Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar)—suggests either heterozygote advantage or one or more founder mutations. Possibly, both mechanisms are involved. Of 19 different DHCR7 mutations found in, coincidentally, 19 patients, there were 13 missense mutations, 1 nonsense mutation, and 5 frameshift mutations. Of these mutations, three occurred in four or more patients, the most interesting being a 134-bp insertion found in 7 of 38 alleles. This particular mutation is predicted to abolish all DHCR7 activity and was homozygous in 2 of the 19 patients studied, both of whom had a diagnosis of type II SLOS. Because the most severely affected patients have measurable cholesterol levels at birth, typically 5–10 mg/dl (Tint et al., 1995Tint GS Salen G Batta AK Shefer S Irons M Elias ER Abuelo DN et al.Correlation of severity and outcome with plasma sterol levels in variants of the Smith-Lemli-Opitz syndrome.J Pediatr. 1995; 127: 82-87Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Cunniff et al., 1997Cunniff C Kratz LE Moser A Natowicz MR Kelley RI Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am J Med Genet. 1997; 68: 263-269Crossref PubMed Scopus (174) Google Scholar), there may be another genetic source of DHCR7 activity. Alternatively, there could be either a pathway of cholesterol synthesis not requiring DHCR7 or more maternal-to-fetal transfer of cholesterol than currently is thought. The finding of many different DHCR7 mutations in this relatively common malformation syndrome suggests a heterozygote advantage, such as increased production of vitamin D from the mildly increased plasma levels of 7DHC in SLOS parents (Opitz and de la Cruz, 1994Opitz JM de la Cruz F Cholesterol metabolism in the RSH/Smith-Lemli-Opitz syndrome: summary of an NICHD conference.Am J Med Genet. 1994; 50: 326-338Crossref PubMed Scopus (52) Google Scholar; Kelley, 1995Kelley RI Diagnosis of Smith-Lemli-Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts.Clin Chim Acta. 1995; 236: 45-58Crossref PubMed Scopus (200) Google Scholar). The diversity of DHCR7 mutations also means that measurement of plasma and tissue sterol levels will remain the primary method for diagnosis of SLOS. However, mutational analysis may be useful in the evaluation of suspected SLOS patients with normal or equivocally increased 7DHC levels. As work progressed on the molecular and biochemical characterization of SLOS, major advances also were being made in the study of HPE and the “hedgehog” class of embryonic signaling proteins, two areas of genetics at first seemingly unrelated to SLOS. However, these three areas of research now have converged in a remarkable way that provides new insights into human morphogenesis. This part of the SLOS story actually begins before the first clinical description of SLOS when, in the early 1960s, researchers discovered that inhibitors of enzymes of the distal cholesterol biosynthetic pathway, including DHCR7, caused HPE, pituitary agenesis, and, less frequently, limb and genital anomalies in the pups of pregnant rats or mice (Roux and Aubry, 1966Roux C Aubry MM Action tératogène chez le rat d'un inhibiteur de la synthèse du cholesterol, le AY 9944.C R Seances Soc Biol Fil. 1966; 160: 1353-1357PubMed Google Scholar; Roux et al., 1980Roux C Dupuis R Horvath C Talbot JN Teratogenic effect of an inhibitor of cholesterol synthesis (AY 9944) in rats: correlation with maternal cholesterolemia.J Nutr. 1980; 110: 2310-2312Crossref PubMed Scopus (51) Google Scholar). An even more convincing SLOS phenotype recently has been produced by treatment of pregnant, cholesterol-deficient mice with an inhibitor of DHCR7 (Lanoue et al., 1997Lanoue L Dehart DB Hinsdale ME Maeda N Tint GS Sulik KK Limb, genital, CNS, and facial malformations result from gene/environment-induced cholesterol deficiency: further evidence for a link to sonic hedgehog.Am J Med Genet. 1997; 73: 24-31Crossref PubMed Scopus (100) Google Scholar). HPE, a failure of normal bilobar development of the forebrain and a prominent characteristic of all animal models of fetal exposure to inhibitors of DHCR7, was reported in only one SLOS patient prior to 1993 (McKeever and Young, 1990McKeever PA Young ID Smith-Lemli-Opitz syndrome II: a disorder of the fetal adrenals?.J Med Genet. 1990; 27: 465-466Crossref PubMed Scopus (42) Google Scholar). However, by use of an increased 7DHC level as a diagnostic biochemical marker, SLOS now has been identified in at least seven patients with HPE (Kelley et al., 1996Kelley RL Roessler E Hennekam RC Feldman GL Kosaki K Jones MC Palumbos JC et al.Holoprosencephaly in RSH/Smith-Lemli-Opitz syndrome: does abnormal cholesterol metabolism affect the function of Sonic Hedgehog?.Am J Med Genet. 1996; 66: 478-484Crossref PubMed Scopus (182) Google Scholar; author's unpublished data). Although all of these HPE patients had other physical characteristics of SLOS, in most of them the diagnosis of SLOS could not have been made without measurement of plasma sterols, because the facial anomalies associated with HPE, such as midline cleft lip and hypotelorism, obscured the diagnostically important facial dysmorphism of SLOS. At the same time that dysmorphologists were dusting off their biochemistry books to study cholesterol metabolism, Jeffery Porter, Phil Beachy, and their colleagues at Johns Hopkins made the startling discovery that covalent addition of cholesterol to Sonic hedgehog (Shh), an embryonic signaling protein, was an essential part of Shh's “autoprocessing” reaction (Porter et al., 1995Porter JA von Kessler DP Ekker SC Young KE J. LJ Moses K Beachy PA The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.Nature. 1995; 374: 363-366Crossref PubMed Scopus (425) Google Scholar, Porter et al., 1996Porter JA Young KE Beachy PA Cholesterol modification of Hedgehog signaling proteins in animal development.Science. 1996; 274: 255-259Crossref PubMed Scopus (1028) Google Scholar). In this reaction, precursor Shh protein in the presence of cholesterol cleaves itself to form a nonsignaling COOH-terminal half and a mature, cholesterol-substituted, N-terminal half, “Shh-N.” Shh-N appears to possess all Shh signaling activity, which in vertebrates includes patterning of development in the ventral forebrain and limb buds, among other structures. Although the covalently attached cholesterol moiety is not essential for intrinsic signaling activity of Shh–N, it appears to have a role in the attachment and localization of Shh-N to cell membranes (Porter et al., 1996Porter JA Ekker SC Park WJ von Kessler DP Young KE Chen CH Ma Y et al.Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain.Cell. 1996; 86: 21-34Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). The first link between Shh and HPE was made when Chiang et al., 1996Chiang C Litingtung Y Lee E Young KE Corden JL Westphal H Beachy PA Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.Nature. 1996; 383: 407-413Crossref PubMed Scopus (2456) Google Scholar reported that homozygous Shh knockout mice developed HPE. This discovery soon was followed by the report that mutations in SHH, the 7q36-linked gene encoding SHH in humans, caused autosomal dominant HPE (Roessler et al., 1996Roessler E Belloni E Gaudenz K Jay P Berta P Scherer SW Tsui L et al.Mutations in the human Sonic Hedgehog gene cause holoprosencephaly.Nat Genet. 1996; 14: 357-360Crossref PubMed Scopus (905) Google Scholar). The possibility that cholesterol might play an important role in embryonic forebrain development had been suggested somewhat earlier by the discovery that transgenic mice deficient in the synthesis of megalin (Willnow et al., 1996Willnow TE Hilpert J Armstrong SA Rohlmann A Hammer RE Burns DK Herz J Defective forebrain development in mice lacking gp330/megalin.Proc Natl Acad Sci USA. 1996; 93: 8460-8464Crossref PubMed Scopus (394) Google Scholar), an important component of a system for delivery of maternal LDL cholesterol to the embryonic neuroepithelium, also develop HPE. Possibly related CNS malformations also were found in transgenic mice lacking apolipoprotein B, another component of the embryonic cholesterol-delivery system (Farese et al., 1995Farese Jr, RV Ruland SL Flynn LM Stokowski RP Young SG Knockout of the mouse apolipoprotein B gene results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes.Proc Natl Acad Sci USA. 1995; 92: 1774-1778Crossref PubMed Scopus (200) Google Scholar). This unexpected convergence of HPE, SLOS, Shh, and cholesterol metabolism immediately focused attention on the possibility that the covalent attachment of cholesterol to Shh-N and related hedgehog proteins is the link between the abnormal cholesterol metabolism of SLOS and abnormal morphogenesis of SLOS. Like many other wonderful unitary theories that preceded it, the proposal that abnormalities of cholesterol-mediated Hedgehog autoprocessing causes HPE and other malformations is probably wrong. The story is indeed much more complex. Addressing the relationship between Shh function and abnormal cholesterol metabolism, Michael Cooper, Phil Beachy, and coworkers (Cooper et al., 1998Cooper MK Porter JA Young KA Beachy PA Plant-derived and synthetic teratogens inhibit the ability of target tissues to respond to Sonic hedgehog signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (739) Google Scholar) have shown that autoprocessing of Hedgehog is not impaired when cholesterol in the reaction medium is replaced by 7DHC or any of many other 27-carbon sterols tested. Although the possibility that 7DHC-modified Shh-N might not have normal signaling function was not assessed directly, additional in vitro studies with AY-9944 (which produces SLOS-like sterol abnormalities) and teratogens that cause SLOS-like malformations in rats strongly suggested that the defect in Shh signaling resided in the target tissue and not in the Shh-N signal itself. For example, treatment of target tissue (chick neural-plate ectoderm) for 48 h with AY-9944, Triparanol, or jervine at concentrations that produce HPE in whole chick embryos completely blocked signaling by recombinant Shh-N (Cooper et al., 1998Cooper MK Porter JA Young KA Beachy PA Plant-derived and synthetic teratogens inhibit the ability of target tissues to respond to Sonic hedgehog signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (739) Google Scholar). Moreover, studies in my laboratory suggest that it is not just impaired cholesterol biosynthesis in the target tissue that limits Shh signaling. For example, whereas both AY-9944 and the plant alkaloid tomatidine cause severe impairment of cholesterol biosynthesis and marked increases in 7DHC in cultured cells and chick embryos, only AY-9944 is teratogenic and blocks Shh signaling (author's unpublished observations). However, as shown by Cooper et al., 1998Cooper MK Porter JA Young KA Beachy PA Plant-derived and synthetic teratogens inhibit the ability of target tissues to respond to Sonic hedgehog signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (739) Google Scholar, all of the known synthetic and plant teratogens that cause HPE also impair intracellular cholesterol transport, as measured by uptake and esterification of extracellular cholesterol. One possible target of teratogen-induced abnormal intracellular cholesterol metabolism is “Patched,” a putative hedgehog receptor (Ingham et al., 1991Ingham PW Taylor AM Nakano Y Role of the Drosophila patched gene in positional signalling.Nature. 1991; 353: 184-187Crossref PubMed Scopus (342) Google Scholar; Stone et al., 1996Stone DM Hynes M Armanini M Swanson TA Gu Q Johnson RL Scott MP et al.The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog.Nature. 1996; 384: 129-134Crossref PubMed Scopus (928) Google Scholar; Goodrich et al., 1997Goodrich LV Milenkovic L Higgins KM Scott MP Altered neural cell fates and medulloblastoma in mouse patched mutants.Science. 1997; 277: 1109-1113Crossref PubMed Scopus (1366) Google Scholar) that contains a sterol-sensing domain (Stone et al., 1996Stone DM Hynes M Armanini M Swanson TA Gu Q Johnson RL Scott MP et al.The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog.Nature. 1996; 384: 129-134Crossref PubMed Scopus (928) Google Scholar). Shh is just one of several similarly functioning hedgehog proteins, all of which may interact with Patched in a sterol-sensitive manner. Thus, it is possible that impaired signaling activities of other hedgehog proteins, such as Desert hedgehog (Bitgood et al., 1996Bitgood MJ Shen L McMahan AP Sertoli cell signaling by Desert hedgehog regulates the male germline.Curr Biol. 1996; 6: 298-304Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) and Indian hedgehog (Iwasaki et al., 1997Iwasaki M Le AX Helms JA Expression of Indian hedgehog, bone morphogenetic protein 6 and gli during skeletal morphogenesis.Mech Dev. 1997; 69: 197-202Crossref PubMed Scopus (94) Google Scholar), also play a role in the abnormal morphogenesis of SLOS tissues that are not targets of Shh signaling. For example, although steroid abnormalities often are presumed to be the cause of the hypogenitalism of SLOS, the persistence of Müllerian remnants in some severely affected SLOS males (Bialer et al., 1987Bialer MG Penchaszadeh VB Kahn E Libes R Krigsman G Lesser ML Female external genitalia and mullerian duct derivatives in a 46,XY infant with the Smith-Lemli-Opitz syndrome.Am J Med Genet. 1987; 28: 723-731Crossref PubMed Scopus (62) Google Scholar) could be explained by inadequate signaling of Desert Hedgehog, which is prominently expressed in Sertoli cells, the source of Müllerian inhibitory factor (Bitgood et al., 1996Bitgood MJ Shen L McMahan AP Sertoli cell signaling by Desert hedgehog regulates the male germline.Curr Biol. 1996; 6: 298-304Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Despite the attractiveness of and experimental support for the role of impaired Hedgehog signaling in SLOS morphogenesis, other, less-specific disturbances also could be involved. In several 10–20-wk SLOS fetuses tested in my laboratory, cholesterol is severely deficient in all tissues, with 7DHC and related cholesterol precursors typically constituting ⩾75% of fetal sterols. Such a severe disturbance in membrane sterol composition is likely to affect many critical developmental processes that involve cell-cell interactions, as suggested by Sulik and colleagues in their recent studies with the SLOS-mimicking teratogen, BM 15,776 (Dehart et al., 1997Dehart DB Lanoue L Tint GS Sulik KK Pathogenesis of malformations in a rodent model for Smith-Lemli-Opitz syndrome.Am J Med Genet. 1997; 68: 328-337Crossref PubMed Scopus (59) Google Scholar). Even in tissue culture, SLOS fibroblasts interact abnormally with themselves and with the growth surface (author's unpublished observations). Where do all these remarkable findings leave our SLOS patients and their families? What clinical benefit can be derived from these discoveries? What other syndromes are likely to involve abnormalities of cholesterol metabolism or Hedgehog proteins? Although most geneticists interested in SLOS are learning more about cholesterol metabolism and Sonic hedgehog than they ever imagined, the most important changes are in the homes of the SLOS children. A reading of any issue of the newsletter of the RSH/Smith-Lemli-Opitz Advocacy and Exchange, the family support group for SLOS, will show how profoundly some SLOS children and adults are affected when their cholesterol-deficiency syndrome finally is treated. Growth improves, older children learn to walk, and adults speak for the first time in years. Equally important is how much better the children feel. Sometimes after just days or weeks of cholesterol treatment, head banging stops, agitation passes to calm, and older children and adults verbalize how much better they feel. As we become more involved in the treatment of SLOS patients, it is also important to note that DHCR7 first was cloned not as the SLOS gene but as the gene for a promiscuous drug-binding protein (Moebius et al., 1997Moebius FF Striessnig J Glossmann H The mysteries of sigma receptors: new family members reveal a role in cholesterol synthesis.Trends Pharmacol Sci. 1997; 18: 67-70Abstract Full Text PDF PubMed Scopus (104) Google Scholar, Moebius et al., 1998Moebius FF Fitzky BU Lee JN Paik YK Glossmann H Molecular cloning and expression of the human delta 7-sterol reductase.Proc Natl Acad Sci USA. 1998; 95: 1899-1902Crossref PubMed Scopus (182) Google Scholar). We should remember this fact not only in evaluating the behavioral pharmacology of SLOS but also in considering DHCR7 and cholesterol biosynthesis as potentially significant ancillary sites of action of various drugs in any patient. The discovery of mutations of DHCR7 as the cause of SLOS will lead to the inevitable DHCR7 knockout mouse and, we hope, to animal models of different therapy modalities. Mutational analysis will allow more-accurate carrier testing as well as preimplantation testing. In the meantime, work on various aspects of sterol metabolism in SLOS continues. An earlier observation now under active reinvestigation is that mothers carrying SLOS fetuses have much lower than normal levels of serum unconjugated estriol (McKeever and Young, 1990McKeever PA Young ID Smith-Lemli-Opitz syndrome II: a disorder of the fetal adrenals?.J Med Genet. 1990; 27: 465-466Crossref PubMed Scopus (42) Google Scholar). If confirmed as a consistent marker for SLOS, the finding of a low maternal serum estriol level may allow detection of this relatively common disorder in families without a known risk for SLOS. Another important clinical question to be answered is whether differences in human maternal cholesterol levels influence the delivery of cholesterol to the developing embryo, as some studies of AY-9944 in rats have suggested (Barbu et al., 1984Barbu V Roux C Dupuis R Gardette J Maziere JC Teratogenic effect of AY 9944 in rats: importance of the day of administration and maternal plasma cholesterol level.Proc Soc Exp Biol Med. 1984; 176: 54-59Crossref PubMed Scopus (28) Google Scholar). Finally, the new molecular embryology of SLOS and its connection with HPE and, possibly, with Shh already has spawned a small industry of sterol teratology. Dysmorphologists naturally will look at other biochemically undiagnosed malformation syndromes and wonder whether therein lurk new disorders of cholesterol biosynthesis. Indeed, an apparent defect in desmosterol (24-dehydrocholesterol) metabolism already has been described in a single stillborn infant with some characteristics of SLOS (FitzPatrick et al., 1998FitzPatrick DR Keeling JW Evans MJ Kan AE Bell JE Porteous ME Mills K et al.Clinical phenotype of desmosterolosis.Am J Med Genet. 1998; 75: 145-152Crossref PubMed Scopus (141) Google Scholar). However, SLOS as a “metabolic malformation” syndrome may be the exception rather than the rule. In this era of weekly discoveries of new genes for old syndromes, most mutations that cause multiple congenital malformation syndromes—that is, disturbances of the body plan—have not been disorders of intermediary metabolism but, instead, mutations of homeobox genes and other transcriptional regulators and signaling systems. Even the widespread and severe biochemical disturbances of a classic prenatal metabolic disorder such as Zellweger syndrome have few effects on the body plan. Thus, SLOS and possibly a few other sterol defects may be exceptional, because their abnormal sterol biochemistry, which cannot be corrected by maternal sources, appears to disrupt the action of Hedgehog proteins, important embryonic proteins with diverse effects on the activity of transcriptional regulators. Nevertheless, because clinical screening for abnormal cholesterol biosynthesis was unknown before the discovery of increased 7DHC levels in SLOS, we should remind ourselves as we see our patients with unexplained syndromes that there are many other categories of obligate fetal metabolite biosynthesis for which no systematic metabolic screening exists and for which no genetic defects are known.