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The Smith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive multiple congenital anomaly/mental retardation disorder caused by an inborn error of post-squalene cholesterol biosynthesis. Deficient cholesterol synthesis in SLOS is caused by inherited mutations of 3β-hydroxysterol-Δ7 reductase gene (DHCR7). DHCR7 deficiency impairs both cholesterol and desmosterol production, resulting in elevated 7DHC/8DHC levels, typically decreased cholesterol levels and, importantly, developmental dysmorphology. The discovery of SLOS has led to new questions regarding the role of the cholesterol biosynthesis pathway in human development. To date, a total of 121 different mutations have been identified in over 250 patients with SLOS who represent a continuum of clinical severity. Two genetic mouse models have been generated which recapitulate some of the developmental abnormalities of SLOS and have been useful in elucidating the pathogenesis. This mini review summarizes the recent insights into SLOS genetics, pathophysiology and potential therapeutic approaches for the treatment of SLOS.
Genetic defects in enzymes responsible for post-squalene cholesterol biosynthesis have recently emerged as important causes of congenital dysmorphology syndromes. Identification of the genetic defect in the Smith–Lemli–Opitz syndrome (SLOS, MIM270400) (1–3) led to the discovery that other similar dysmorphology syndromes were caused by inborn errors of cholesterol biosynthesis in human and mouse: desmosterolosis (4), lathosterolosis (5), CHILD syndrome (6, 7), CDPX2 (a form of chondrodysplasia punctata) and hydrops-ectopic calcification-moth-eaten skeletal (HEM, also known as Greenberg) dysplasia (8–11). All of these syndromes have been linked to deficiency of the specific enzymes required for the production of cholesterol from lanosterol, as summarized in Table 1. Patients with these disorders present with complex malformation syndromes involving different organs and systems. These disorders have led to a new appreciation for the essential role that cholesterol plays in development. Given the multiple and critical roles that sterols play, it is not surprising that mutations in genes encoding sterol biosynthetic enzymes can have a dramatic effect on eucaryotic development. Antley–Bixler syndrome (ABS) was previously classified as a sterol synthesis disorder. However, a recent large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have fibroblast growth factor receptor (FGFR) mutations, whereas those with ambiguous genitalia are caused by genetic defects of P450 oxidoreductase (POR), which is responsible for steroidogenesis (12, 13). Therefore, ABS with POR mutations belongs to a disorder related to steroid metabolism instead of sterol synthesis (14). The detailed descriptions of these malformation syndromes have been extensively reviewed (15–17). In the present review, some recent insights into SLOS genetics, pathophysiology and potential therapeutic approaches are highlighted.
SLOS is an autosomal recessive disorder; loss-of-function mutations in both copies of DHCR7 on chromosome 11q13 are necessary to have disease. Both male and females are equally affected. DHCR7 reduces the C7-C8 double bond in the sterol B ring to form cholesterol or desmosterol depending upon the precursor. Desmosterol can be converted to cholesterol by 3β-hydroxysterol-Δ24 reductase (DHCR24). Therefore, SLOS patients usually have low plasma cholesterol levels and invariably have elevated levels of cholesterol precursors 7-dehydrocholesterol (and its spontaneous isomer 8-dehydrocholesterol) and absent desmosterol. SLOS is currently recognized as one of the most common autosomal recessive inherited disorders after cystic fibrosis and phenylketonuria in the North American Caucasian population. Based on elevated 7DHC levels, the incidence of SLOS is estimated to range from 1/10,000 to 1/60,000 of live births, depending upon the region. SLOS seems to be more prevalent among peoples of Caucasian descent, but infrequently reported in peoples of Asian or African background. In the US, the incidence of SLOS has been estimated to be approximately 1/50,000 (18, 19). A recent prenatal diagnostic study in the US, using maternal serum unconjugated estriol as a predictor for SLOS, indicated an incidence of 1/60,000 births (20). Another 3-year prospective population surveillance performed in Canada reported that the minimum incidence was 1/70,358 live births with a minimum prevalence of approximately 1/950,000 (21). There is, however, a discrepancy between the incidence of SLOS and the higher prevalence of carrier status of mutant alleles of DHCR7. Population screens for mutant DHCR7 alleles suggest a 3–4% carrier frequency in Caucasian populations, giving a hypothetical birth incidence about 1/2500–1/4500, assuming no foetal loss (17, 22, 23). This twofold discrepancy suggests that the true incidence may be higher due to unrecognized miscarriages or missed diagnoses in mildly affected individuals (e.g. subtle physical stigmata, minor behaviour or mental problems). More milder cases of SLOS have now been reported, due to increased awareness and access to a diagnostic test (24–27). Severe mutations such as homozygosity for null alleles may result in intrauterine or perinatal lethality (28–30). Prenatal mortality for the most severely affected cases of SLOS may be as high as 80% (22). In cases where a woman suffers an early spontaneous abortion, but who subsequently gives birth to a normal child (who may be wild type or heterozygous for a mutant DHRC7 allele) will not be further investigated. As early spontaneous abortion is not uncommon, perhaps this pattern may have masked the true incidence of SLOS. In this context, a true estimate of the incidence of SLOS is required using a more widespread screening.
Correa-Cerro and Porter recently summarized 105 different mutant alleles in a review of 559 published DHCR7 mutations (17). Sixteen additional novel missense mutations have since been reported (31–33). The mutational spectrum in SLOS appears consistent with that of most other autosomal recessive disorders, with missense mutations accounting for the vast majority of mutations, with less frequent null mutations (e.g. frameshift, nonsense or splice-site mutations). A number of SLOS cases (seven cases in published reports and two cases from our unpublished data) have been identified with only a single mutation (1, 3, 34), screened by bidirectional sequencing of PCR-amplified coding exons and adjacent intronic sequences. To date, a total of 121 mutations (685 alleles) in DHCR7 have been identified (Fig. 1). Mutations are located throughout the coding region (exons 3–9), including 105 missense, five nonsense, three splice-site and eight nucleotide insertions or deletions. The missense mutations account for 87.6% of the total spectrum. Fifty per cent of the missense mutations are located in one of the nine predicted transmembrane domains. In descending order, the commonest DHCR7 mutations, with the relative frequencies over 4%, are IVS8-1G>C (27.3%), T93M (10.4%), W151X (5.7%), V326L (4.8%) and R404C (4.5%). These five mutations account for 50–60% of reported mutant alleles. The other less common mutations (indicated in red typescript, Fig. 1) include R352W (2.8%), E448K (2.8%), G410S (1.9%), R242C (1.6%), S169L (1.6%) and F302 (1.5%).
The classic paradigm for the pathogenesis of an inborn error of metabolism includes the accumulation of a toxic precursor and/or deficiency of an essential product as a result of an enzyme deficiency. In the case of SLOS, accumulation of sterol precursors, such as 7DHC (7DHC has two conjugated Δ5,7 double bonds in the B-ring, but cholesterol has a unique Δ5 double bond), could be potentially toxic (but has not been convincingly demonstrated) (35, 36), and cholesterol deficiency is almost certainly detrimental (37, 38), but how sterol deficiency leads to dysmorphology has yet to be elucidated. A number of potential popular mechanisms have been championed, such as disruption of sonic hedgehog signalling, yet none of these have led to any mechanistic insight.
Although the phenotype severity correlates inversely with blood cholesterol levels and with predicted or measured DHCR7 activity, there exists considerable phenotype heterogeneity in patients with identical DHCR7 mutations (34, 39–41). The SLOS phenotypic spectrum is broad and variable, from early embryonic non-viability to varying levels of severity postnatally, including distinctive facial appearance, growth and mental retardation, autistic behaviour, hypotonia, failure to feed, decreased lifespan and variable structural anomalies of the heart, the lungs, the brain, the gastrointestinal tract, the limbs, the genitalia and the kidneys. Other factors in addition to genotype and the cholesterol and/or 7DHC levels at the time of diagnosis have been considered to influence the clinical severity. These factors could involve maternal factors that may increase the supply of cholesterol to the developing foetus, the amounts of cholesterol that can accumulate in the neural tissues before the blood–brain barrier is formed, perhaps the rate at which potentially toxic precursors can accumulate or be removed from the foetal tissues, and undefined other genetic and epigenetic factors (42–44). Recently, the maternal apolipoprotein E genotype (ApoE) was implicated in phenotype heterogeneity (45). Maternal ApoE2 genotypes were associated with a severe SLOS phenotype, whereas ApoE genotypes without the E2 allele were associated with a milder phenotype (45). However, as the numbers of mothers with the ApoE2/ApoE2 genotypes were small, some precaution should be expressed.
Notably, despite the apparently limited mother–foetus cholesterol transport, severely affected SLOS patients with homozygous ‘null’ mutations have detectable levels of cholesterol and do not invariably manifest a severe phenotype (40, 46). A recent study that the DHCR7 transcriptional signal was detectable in cultured fibroblasts from a patient with the ‘null’ mutations (W151X/IVS8-1G>C) suggests that the IVS8-1G>C splice mutation may be ‘leaky’ (46, 47), a phenomenon described in other genetic disorders (48). It is theoretically possible that another enzyme may be able to reduce the double bond at C7-8 of sterol precursors, albeit at a very low level. There is considerable amino acid sequence homology between DHCR7 and other sterol synthesis enzymes or ones that share the putative reductase domain, such as sterol Δ8 isomerase, sterol C14 reductase, the lamin B receptor and TM7SR2. These reductase motifs in DHCR7 are sterol reductase motif 1, SR1, amino acids 213–228, GNFFYNYMMGIEFNPR in a loop between the fourth and fifth transmembrane regions, sterol reductase 2, SR2, amino acids 439–462, LLTHRCLRDEHRCASKYGRDWERY in the carboxy terminus (CT) and a 12 amino acid sterol reductase signature motif, SRSM, amino acids 394–405, LLXSGWWGXXRH in the fourth loop which contains the binding site for NADPH. These motifs remain to be confirmed experimentally, but many of the missense mutations map to these motifs – Q224K and R228W are located in SR1; R443H, R443C, C444Y, E448K, E448Q, R450L and Y462H in SR2; S397L, R404S and H405Y in SRSM.
Two genetically manipulated murine models that mimic the biochemical and phenotypic hallmarks of the SLOS have been generated. Targeted disruption of Dhcr7 results in early postnatal death with severe sterol metabolic alterations and a number of developmental abnormalities (49–51), confirming the need for de novo cholesterol synthesis for normal mammalian embryogenesis.
Although a number of neurophysiological abnormalities, involving glutamate and serotonin pathways, have been reported in Dhcr7 −/− mice (50, 52), it remains unclear whether underlying neurodevelopmental defects contribute significantly to neonatal lethality of Dhcr7 null mice (49, 50). To investigate the consequences of cholesterol deficiency in postnatal central nervous system (CNS) development, we have generated transgenic mice expressing human DHCR7 under the control of a CNS-specific nestin promoter. Minimally expressed transgene in Dhcr7 null background results in a stochastically partial rescue (12%) of neonatal lethality. Rescued Dhcr7 −/− mice (TgRKO) survived 3–17 days postnatally, showing severe growth retardation and impaired movement suggestive of a neurological dysfunction (53). TgRKO mice present with profound cholesterol deficiency in the brain and less severe in peripheral tissues, indicating that the dietary cholesterol was not available to the brains of rescued mice. CNS defects are observed in TgRKO mice, including a dilated ventricular system and a number of structural defects. Although the total sterol level is not reduced in postnatal brains of rescued mice (consisting of approximately 10% cholesterol and approximately 90% of different sterol precursors), a delayed postnatal myelination and defective neurogenesis are clearly evident in TgRKO mice. In this context, conditional knockout of squalene synthase (the committed step for cholesterol synthesis) in neurons or oligodendrocytes causes either early postnatal death due to abnormal neurogenesis or premature death resulting from hypomyelination, indicating that normal CNS cholesterol synthesis is critical for postnatal survival (54, 55). Our study indicates that CNS defects, involving abnormal myelination and neurogenesis, contribute significantly to the early postnatal lethality of Dhcr7 −/− mice and may be important in the severity of SLOS in humans. Another conclusion is that even small and subtle changes in brain sterol metabolism were sufficient to enable partial rescue, suggesting that mechanism(s) involved in the CNS dysfunction may involve mechanisms other than the bulk requirement for cholesterol in membranes. One such pathway may be the disruption of critical neurosteroid synthesis within the CNS, a pathway highlighted by preliminary studies by Shackleton and his colleagues (56).
Another interesting development is the creation of a hypomorph mouse, the Dhcr7T93M. Mice homozygous for Dhcr7T93M or heterozygous for compound mutations (Dhcr7T93M/null) are viable, fertile and appear to have normal growth. Phenotypically Dhcr7T93M/T93M and Dhcr7T93M/null mice have mild dilatation of the third and lateral ventricles, and T93M/null mice have 2–3 toe syndactyly (57). The viability of these mice may allow investigations of therapeutic approaches for management of SLOS.
Currently, the pathophysiological mechanisms involved in SLOS have not been fully elucidated. The most popular theory advanced has been that the key morphogen, Sonic hedgehog (and its related proteins Indian and Desert hedgehog), is affected, as this protein needs covalently attached cholesterol for regulated short and long-range signalling processes (58–61). Shh post-translational autoprocessing (51, 62) and expression in CNS and lung are not affected in the Dhcr7 −/− embryos (51), validating the previous conclusions that precursor sterols participate as efficiently as cholesterol in the Shh processing reaction (58) and can also substitute for cholesterol for structural requirements such as constituents of bilayer membranes (63). With regard to the latter, cholesterol is a known important component of membrane lipid rafts, and any subtle defects in raft formation could conceivably alter signalling pathways (64, 65). An appropriate composition of cholesterol in the cell membranes is crucial for optimal enzymatic activity, ion and metabolite transport or channelling, protein–protein and protein–lipid interactions, and signal transduction. The pathogenetic mechanisms of cholesterol deficiency caused by abnormal cholesterol biosynthesis may extend beyond disruption of morphogenic pathways. For example, the role of cholesterol in normal synaptogenesis is independent of the Shh pathway (66).
Prenatal diagnosis of SLOS, using elevated 7DHC, has now been performed in many pregnancies (67, 68). An elevated 7DHC/cholesterol ratio in foetal tissues or amniotic fluid (AF) is diagnostic of SLOS, and chorionic villus (CV) sampling at 11–12 weeks of gestation (69) has been reported. A direct correlation between the level of 7DHC in AF and the clinical severity of SLOS has been demonstrated in 20 pregnancies eventually confirmed to have SLOS (70). Interestingly, the levels of cholesterol in the AF of foetuses with SLOS are not significantly different from those of controls (71), supporting the concept that some of the pathophysiology may involve cholesterol precursor molecules and/or their metabolites.
Maternal serum or urine levels of unconjugated estriol (μE3) to screening for SLOS foetal pregnancies is a promising non-invasive new method for early diagnosis (20). Synthesis of μE3, a pregnancy-related steroid hormone, is dependent on cholesterol produced by the foetal tissues for synthesis by the placenta. Low to undetectable levels of μE3 have been observed in the urine, amniotic fluid and serum of pregnant women carrying foetuses affected with SLOS. In pregnancies affected with SLO, the levels of μE3 in maternal serum are abnormally low. Additionally, the utilization of precursor sterols in the foetal adrenals and gonads leads to the synthesis of abnormal equine-type oestrogens, which enter the maternal plasma and urine. These compounds are detectable using gas chromatography-mass spectrometry (72), although this test is not available for routine clinical use. It is also possible that this test may distinguish between affected and unaffected foetuses as early as 12 weeks of gestation, when the foetal production of steroids increases rapidly.
The diagnosis of SLOS by sonographic evaluation alone of the foetus is not reliable, except possibly in cases with severe malformations and a previous history of affected family members.
Prenatal diagnosis of SLOS by molecular analysis on DNA extracted from CV samples in families with known genotype has been reported (73, 74), although the high risk of spontaneous abortion limits its widespread use at this time.
There is no known cure for SLOS. Surgical repair of physical anomalies such as congenital heart defects, cleft palate, genital anomalies, craniofacial, gastrointestinal and limb defects involves a number of specialities in the care of survivors. Much of the treatment is supportive. The inability to synthesize cholesterol leads to predictable deficiency in adrenal steroid synthesis and may require steroid replacement. As bile acids are also synthesized from cholesterol, deficiency of this leads to inability to absorb dietary cholesterol and fat-soluble vitamin deficiencies. Dietary cholesterol supplementation restores both adrenal and bile salt deficiencies. Once sufficient cholesterol has been converted to bile salts by the liver, the absorption of dietary cholesterol should be restored. On a balanced diet, this should ensure a relatively normal cholesterol delivery to endocrine organs for sufficient steroid hormone synthesis. As the adrenals in SLOS depend upon lipoprotein-delivered means for cortisol synthesis, adrenal insufficiency should be considered when faced with a child with a prolonged illness. Intravenous plasma therapy has been reported for a case diagnosed antenatally, although it is unclear whether this is necessary or effective in reducing morbidity and mortality (75). Treatment strategies of dietary cholesterol supplementation are focused on supplying exogenous crystalline cholesterol by various vehicles (i.e. oil-based or aqueous suspension) in an attempt to increase body cholesterol levels and to secondarily decrease the levels of 7DHC/8DHC, through feedback inhibition of HMG-CoA reductase. Dietary cholesterol supplementation is now routine, although there are no controlled studies to validate their efficacy, and this issue remains unresolved. Not all children show a positive response to cholesterol supplementation (76). The response to therapy has varied between patients and protocols. Merkens et al. (77) reported that total sterols in plasma, LDL and HDL increased with dietary cholesterol supplementation. LDL and HDL cholesterol increased significantly, while the concentrations of 7DHC or 8DHC in LDL did not change. While plasma cholesterol levels and the ratio of cholesterol to total sterols increased, a significant decrease in 7DHC was not seen. A significant negative correlation was noted between cholesterol and 7DHC levels. The data indicate that those with the best response to dietary cholesterol in terms of cholesterol levels also had the lowest 7DHC levels, which would support the current practice of cholesterol supplementation as a treatment modality for SLOS in terms of improving the biochemical phenotype and providing a pool of cholesterol for transport to the tissues.
Another novel therapy proposed is the use of a ‘statin’ drug, simvastatin, an HMG-CoA reductase inhibitor, aimed at blocking the cholesterol synthesis pathway to avoid the formation of large amounts of precursor sterols, such as 7DHC/8DHC, thereby limiting exposure to potentially toxic metabolites. Simvastatin can also cross the blood–brain barrier and may provide a means to treat the biochemical defect present in the CNS of SLOS patients (78). A major effect of statin therapy is the transcriptional upregulation of genes controlled by the transcriptional factor SREBP, one of which is DHCR7. Thus, if any residual activity is present in the mutant DHCR7, its upregulation could increase intracellular cholesterol synthesis. Simvastatin use in SLOS patients resulted in a paradoxical increase in serum and cerebral spinal fluid cholesterol levels (79). Treatment of mutant and control fibroblasts with simvastatin significantly increased DHCR7 expression, whether cells were cultured in either cholesterol-containing or cholesterol-deficient media (41). To date, reports of statin therapy in SLOS have been confined to case reports, and further carefully constructed trials in larger series of cases are needed. Determination of residual DHCR7 enzymatic activity may be helpful in selecting SLOS patients being considered for a beneficial response of statins, as this treatment could theoretically be detrimental in subjects with little or no DHCR7 activity (33, 80).
Finally, we propose another therapy, that of direct cholesterol delivery to the CNS by low-pressure catheter infusion for the treatment of the one clinical manifestation in SLOS that has no direct treatment. Mental retardation, behavioural disturbance and movement disorders are major causes of morbidity. Therapy aimed specifically at this organ is likely to have a greater benefit than the reversal of non-CNS biochemical parameters. Restoration of adequate cholesterol stores by direct delivery is feasible, and as the turnover of cholesterol in the CNS is very slow, its effects may last for months, perhaps years, before re-treatment may be required. Toxicity of overdosing with cholesterol should be limited, as the normal CNS mechanisms regulating sterol exit out of the brain remain intact. Additionally, a slight overreplacement of cholesterol should lead to a suppression of the sterol synthesis pathway and allow for clearance of precursors. Finally, repletion of cholesterol in the brain may allow the brain to remodel and develop normally, especially if this can be carried out as early as possible after diagnosis.
Funding for this work has been provided by a grant from the National Institutes of Health, NHLBI, HL689660 (SBP).