Dietary cholesterol supplementation and inhibitory factor 1 serum levels in two dizygotic Smith-Lemli-Opitz syndrome twins: a case report.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is a rare genetic neurodevelopmental disorder caused by the defect in the 7-dehydrocholesterol reductase. This defect leads to the deficiency of cholesterol biosynthesis with accumulation of 7-dehydrocholesterol. Inhibitory factor 1 (IF1) is a well-known mitochondrial protein. Recently, it has been discovered in the human serum where it is reported to be involved in the HDL-cholesterol intake. Here we report the IF1 presence in the serum of two paediatric SLOS dizygotic twins treated with dietary cholesterol supplementation. CASE PRESENTATION: The patients showed a typical phenotype. They started dietary supplementation with cholesterol when 2 months old. The cholesterol intake was periodically titrated on the basis of weight increase and the twin 1 required a larger supplementation than the twin 2 during the follow-up. When 6.4-year-old, they underwent IF1 assay that was 7-fold increased in twin 2 compared to twin 1 (93.0 pg/ml vs 13.0 pg/ml, respectively). CONCLUSIONS: We report, for the first time, the presence of circulating IF1 in the serum of SLOS patients, showing different levels among them. Our findings confirm that IF1 could be a novel research target in cholesterol-related disorders and also in SLOS, and could contribute to the general debate on IF1 as a new modulator of cholesterol levels.

Smith-Lemli-Opitz Mutations in Unexplained Stillbirths.

OBJECTIVE: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive syndrome caused by a defect in cholesterol biosynthesis with mutations in 7-dehydrocholesterol reductase (DHCR7). A total of 3% of Caucasians carry DHCR7 mutations, theoretically resulting in a homozygote frequency of 1/4000. However, SLOS occurs in only 1/20,000 to 60,000 live births. Our objective was to assess DHCR7 mutations in unexplained stillbirths. STUDY DESIGN: Prospective, multicenter, population-based case-control study of all stillbirths and a representative sample of live births enrolled in five geographic areas. Cases with stillbirth due to obstetric complications, infection, or aneuploidy, and those with poor quality deoxyribonucleic acid (DNA) were excluded. DNA was extracted from placental tissue stored at -80°C, and exons 3 to 9 of the DCHR7 gene were amplified, purified, and subjected to bidirectional sequencing to identify mutations. RESULTS: One-hundred forty four stillbirths were unexplained and had adequate DNA for analysis. Nine stillbirths of 139 (6.5%) had a single mutation in one allele in coding exons 3 to 9 of DHCR7 (Table 1). One case (0.7%) was a compound heterozygote for mutations in exons 3 to 9 of DHCR7; this fetus had no clinical or histologic features of SLOS. CONCLUSION: We detected SLOS mutations in only 0.7% of stillbirths. This does not support a strong association between unrecognized DHCR7 mutations and stillbirth.

Vulnerability of DHCR7+/- mutation carriers to aripiprazole and trazodone exposure.

Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from DHCR7+/- human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in DHCR7-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of 13C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that DHCR7 carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.

Investigation of 7-dehydrocholesterol reductase pathway to elucidate off-target prenatal effects of pharmaceuticals: a systematic review.

Mendelian diseases contain important biological information regarding developmental effects of gene mutations that can guide drug discovery and toxicity efforts. In this review, we focus on Smith-Lemli-Opitz syndrome (SLOS), a rare Mendelian disease characterized by compound heterozygous mutations in 7-dehydrocholesterol reductase (DHCR7) resulting in severe fetal deformities. We present a compilation of SLOS-inducing DHCR7 mutations and the geographic distribution of those mutations in healthy and diseased populations. We observed that several mutations thought to be disease causing occur in healthy populations, indicating an incomplete understanding of the condition and highlighting new research opportunities. We describe the functional environment around DHCR7, including pharmacological DHCR7 inhibitors and cholesterol and vitamin D synthesis. Using PubMed, we investigated the fetal outcomes following prenatal exposure to DHCR7 modulators. First-trimester exposure to DHCR7 inhibitors resulted in outcomes similar to those of known teratogens (50 vs 48% born-healthy). DHCR7 activity should be considered during drug development and prenatal toxicity assessment.

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis.

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

Sterol metabolism disorders and neurodevelopment-an update.

Cholesterol has numerous quintessential functions in normal cell physiology, as well as in embryonic and postnatal development. It is a major component of cell membranes and myelin, and is a precursor of steroid hormones and bile acids. The development of the blood brain barrier likely around 12-18 weeks of human gestation makes the developing embryonic/fetal brain dependent on endogenous cholesterol synthesis. Known enzyme defects along the cholesterol biosynthetic pathway result in a host of neurodevelopmental and behavioral findings along with CNS structural anomalies. In this article, we review sterol synthesis disorders in the pre- and post-squalene pathway highlighting neurodevelopmental aspects that underlie the clinical presentations and course of Smith-Lemli-Opitz Syndrome (SLOS), mevalonic aciduria (MVA) or the milder version hyper-immunoglobulinemia D and periodic fever syndrome (HIDS), Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis (ABS1), congenital hemidysplasia with icthyosiform nevus and limb defects (CHILD) syndrome, CK syndrome, sterol C4 methyl oxidase (SC4MOL) deficiency, X-linked dominant chondrodysplasia punctata 2(CDPX2)/ Conradi Hunermann syndrome, lathosterolosis and desmosterolosis, We also discuss current controversies and share thoughts on future directions in the field.

Increasing cholesterol synthesis in 7-dehydrosterol reductase (DHCR7) deficient mouse models through gene transfer.

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis: the conversion of 7-dehydrocholesterol (7DHC) to cholesterol (C), catalyzed by 7-dehydrocholesterol reductase (DHCR7). This disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. There are few proven treatment options. That most commonly used is a high cholesterol diet that seems to enhance the quality of life and improve behavioral characteristics of patients, although these positive effects are controversial. The goal of our study was to investigate the possibility of restoring DHCR7 activity by gene transfer. We constructed an adeno-associated virus (AAV) vector containing the DHCR7 gene. After we infused this vector into affected mice, the introduced DHCR7 gene could be identified in liver, mRNA was expressed and a functional enzyme was produced. Evidence of functionality came from the ability to partially normalize the serum ratio of 7DHC/C in treated animals, apparently by increasing cholesterol production with concomitant decrease in 7DHC precursor. By 5 weeks after treatment the mean ratio (for 7 animals) had fallen to 0.05 while the ratio for untreated littermate controls had risen to 0.14. This provides proof of principle that gene transfer can ameliorate the genetic defect causing SLOS and provides a new experimental tool for studying the pathogenesis of this disease. If effective in humans, it might also offer a possible alternative to exogenous cholesterol therapy. However, it would not offer a complete cure for the disorder as many of the negative implications of defective synthesis are already established during prenatal development.

Differences between predicted and established diagnoses of Smith-Lemli-Opitz syndrome in the Polish population: underdiagnosis or loss of affected fetuses?

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic disorder in which an error in cholesterol biosynthesis results in congenital anomalies/mental deficits. The results of our previous newborn screening, based on the carrier frequency of the two most common SLOS-causing mutations in Poland (p.W151X and p.V326L), would make SLOS one of the most frequent recessive disorders in our country (with an incidence of 1:2,300 - 1:3,937). This prompted us to carry out a 3-year (2006-2008) national surveillance program in which about 2,000 physicians were asked to identify potential SLOS patients pre- and postnatally based on clinical identification forms. The incidence of SLOS in Poland was estimated to be from 1:60,941 to 1:105,395 (1: 83,168 ± 22,227) live births, and its 3-year prevalence 1:866,273 ± 16,242. The mean carrier frequency was calculated to be from 1:123 to 1:165. The notable discrepancy between our previous carrier newborn screening and these prospective data may result from reduced fertility in SLOS carriers, intrauterine death of affected fetuses, or underdiagnosis in postnatal life. Since we did not notice significant data supporting the first two aspects, our study may support the suggestion that screening for the most frequent DHCR7 alleles does not reflect the true disease rates in the Polish population. Hence, further studies in which maternal urinary steroids (7-dehydroestriol/estriol and 8-dehydropregnanetriol/pregnanetriol ratios) would serve as screening markers in early pregnancies may be justified.

Steroid production and excretion by the pregnant mouse, particularly in relation to pregnancies with fetuses deficient in Delta7-sterol reductase (Dhcr7), the enzyme associated with Smith-Lemli-Opitz syndrome.

This study has shown that the mouse has a great increase in steroid production during pregnancy in similar fashion to the human. Many steroids were provisionally identified in maternal urine of the wild-type mouse. The major progesterone metabolites appear to be hydroxylated pregnanolones, particularly with hydroxyl groups in the 16alpha position. Rather than estriol being the major end-product of feto-placental steroid synthesis as in the human, the pregnant mouse produces and excretes large amounts of androgen metabolites, ranging in polarity from androstanetriols to androstanepentols. These steroids have 15alpha- or 18-hydroxyl groups with additional hydroxylation at uncharacterized positions. From metabolite data the peak of pregnancy progesterone production appears to be between 7.5 and 14.5 gestational days, while for C(19) metabolites peak excretion is later. The starting-point of the studies was to study pregnancy steroid production by a mouse model for Smith-Lemli-Opitz syndrome, 7-dehydrosterol reductase (DHCR7) deficiency. In human pregnancies with DHCR7 deficient fetuses large amounts of 7- and 8-dehydrosteroids are excreted, products secondary to high fetal 7- and 8-dehydrocholesterol (DHC) accumulation. This agrees with existing evidence that human feto-placental steroid synthesis utilizes little maternal cholesterol as precursor. In contrast, this study has shown that pregnant mice carrying dhcr7 deficient fetuses with relatively high DHC production had essentially undetectable maternal excretions of steroids with Delta(7)- and Delta(8)-unsaturation. As mutant mouse mothers have essentially normal cholesterol production (little or no DHC build-up), this suggests maternal cholesterol is primarily utilized for pregnancy steroid synthesis in the mouse.

Pregnancy and life after preimplantation genetic diagnosis of Smith-Lemli-Opitz syndrome.

OBJECTIVE: To use preimplantation genetic diagnosis to achieve Smith-Lemli-Opitz syndrome -free pregnancies in two couples at high risk of producing an affected child. DESIGN: Case report. SETTING: A private IVF unit. PATIENT(S): Two couples carrying the W151X mutation in the DHCR7 gene. INTERVENTION(S): Removal and testing for the W151X mutation in blastomeres from embryos after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of blastomeres indicating whether corresponding embryos were mutation-free, for the purpose of transferring only unaffected embryos. RESULT(S): Delivery of healthy children without the W151X mutation in the DHCR7 gene. CONCLUSION(S): This is the first report of preimplantation genetic diagnosis for Smith-Lemli-Opitz syndrome, allowing transfer of mutation-free embryos and successful pregnancies.

Mild Smith-Lemli-Opitz syndrome: further delineation of 5 Polish cases and review of the literature.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder caused by reduced activity of 7-dehydrocholesterol reductase, resulting in an increased concentrations of 7-dehydrocholesterol and 8-dehydrocholesterol in body fluids and tissues. Phenotypically it is characterized by wide range of abnormalities, from mild to lethal forms what causes difficulties in its clinical diagnostics. To further delineate the physical spectrum of the mild form of Smith-Lemli-Opitz syndrome, especially with regard to genotype-phenotype correlation, we describe 5 Polish patients with mild phenotype (one with novel mutation in DHCR7 gene and four published before) and analyze 18 other cases from the literature. As the conclusion we give recommendation for tests toward SLOS in cases with "idiopathic" intellectual impairment and/or behavioral anomalies, as well as in biochemically doubtful but clinically fitting cases with overall gestalt and history of this syndrome.

Age and origin of major Smith-Lemli-Opitz syndrome (SLOS) mutations in European populations.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) (MIM 270 400) is an autosomal recessive multiple congenital anomalies/mental retardation syndrome caused by mutations in the Delta7-sterol reductase (DHCR7, E.C.1.3.1.21) gene. The prevalence of SLOS has been estimated to range between 1:15000 and 1:60000 in populations of European origin. METHODS AND RESULTS: We have analysed the frequency, origin, and age of DHCR7 mutations in European populations. In 263 SLOS patients 10 common alleles (c.964-1G>C, p.Trp151X, p.Thr93Met, p.Val326Leu, p.Arg352Trp, p.Arg404Cys, p.Phe302Leu, p.Leu157Pro, p.Gly410Ser, p.Arg445Gln) were found to constitute approximately 80% of disease-causing mutations. As reported before, the mutational spectra differed significantly between populations, and frequency peaks of common mutations were observed in North-West (c.964-1G>C), North-East (p.Trp151X, p.Val326Leu) and Southern Europe (p.Thr93Met). SLOS was virtually absent from Finland. The analysis of nearly 8000 alleles from 10 different European populations confirmed a geographical distribution of DHCR7 mutations as reported in previous studies. The common Null mutations in Northern Europe (combined ca. 1:70) occurred at a much higher frequency than expected from the reported prevalence of SLOS. In contrast the most common mutation in Mediterranean SLOS patients (p.Thr93Met) had a low population frequency. Haplotypes were constructed for SLOS chromosomes, and for wild-type chromosomes of African and European origins using eight cSNPs in the DHCR7 gene. The DHCR7 orthologue was sequenced in eight chimpanzees (Pan troglodytes) and three microsatellites were analysed in 50 of the SLOS families in order to estimate the age of the three major SLOS-causing mutations. CONCLUSIONS: The results indicate a time of first appearance of c.964-1G>C and p.Trp151X some 3000 years ago in North-West and North-East Europe, respectively. The p.Thr93Met mutations on the J haplotype has probably first arisen approximately 6000 years ago in the Eastern Mediterranean. Together, it appears that a combination of founder effects, recurrent mutations, and drift have shaped the present frequency distribution of DHCR7 mutations in Europe.

Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome).

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis, which is catalyzed by 7-dehydrocholesterol reductase (DHCR7). The disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. Pathogenesis of SLOS is complex due to multiple roles of cholesterol and may be further complicated by unknown effects of aberrant metabolites that arise when 7-dehydrocholesterol (7-DHC), the substrate for DHCR7, accumulates. A viable mouse model for SLOS has recently been developed, and here we characterize cholesterol metabolism in this model with emphasis on changes during the first few weeks of postnatal development. Cholesterol and 7-DHC were measured in "SLOS" mice and compared with measurements in normal mice. SLOS mice had measurable levels of 7-DHC at all ages tested (up to 1 year), while 7-DHC was below the threshold for detection in normal mice. In perinatal to weaning age SLOS mice, cholesterol and 7-DHC levels changed dramatically. Changes in brain and liver were independent; in brain cholesterol increased several fold while 7-DHC remained relatively constant, but in liver cholesterol first increased then decreased again while 7-DHC first decreased then increased. In older SLOS animals the ratio of 7-DHC/cholesterol, which is an index of biochemical severity, tended to approach, but not reach, normal. While these mice provide the best available genetic animal model for the study of SLOS pathogenesis and treatment, they probably will be most useful at early ages when the metabolic effects of the mutations are most dramatic. To correlate any experimental treatment with improved sterol metabolism will require age-matched controls. Finally, determining the mechanism by which these "SLOS" mice tend to normalize may provide insight into the future development of therapy.

DHCR7 mutation carrier rates and prevalence of the RSH/Smith-Lemli-Opitz syndrome: where are the patients?

RSH/Smith-Lemli-Opitz (SLOS) is an inborn error of metabolism with protean manifestations. Its exact incidence and prevalence are not known; however, the carrier rate for the most frequently occurring mutation, the null mutation IVS8-1G > C, is approximately 1 in 100 for the Caucasian population in North America (1%) and possibly as high as 1 in 50 to 1 in 30 in Central European populations (2-3.3%). Based on the allele frequencies and the proportion of this mutation observed in various patient populations, the expected incidence of RSH/SLOS in those populations was calculated and reported to be between 1 in 1,590 and 1 in 17,000. However, around the world the observed prevalence and incidence are much lower than those calculated from the individual mutation carrier rates observed in any given population. The discrepancy between the expected incidence and prevalence can be explained only in part by the neonatal and infancy deaths of the most severely affected children with RSH/SLOS and the under ascertainment of mild and atypical cases at the mild end of the spectrum. RSH/SLOS may be responsible for a high number of miscarriages. Recent observations estimate the prevalence of SLOS at 16 weeks of gestation as similar to that observed at birth (approximately 1 in 60,000) suggesting that either reduced fertility of carrier couples or losses of affected embryos or fetuses in the first trimester play a significant role in reducing the second trimester prevalence of RSH/SLOS. It is possible that the estimates of carrier rates based on population screening for the most commonly occurring mutations may not reflect the true carrier rates in the population. In order to reconcile the above-mentioned paradoxes, we propose a model based on a higher than observed carrier frequency of the most common mutation and on very high fetal loss of homozygotes for that mutation.

Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase.

Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.

Development and characterization of a hypomorphic Smith-Lemli-Opitz syndrome mouse model and efficacy of simvastatin therapy.

Smith-Lemli-Opitz syndrome (SLOS) is a genetic syndrome caused by mutations in the 3beta-hydroxysterol Delta(7)-reductase gene (DHCR7). SLOS patients have decreased cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. Dietary cholesterol supplementation improves systemic biochemical abnormalities; however, because of the blood-brain barrier, the central nervous system (CNS) is not treated. Simvastatin therapy has been proposed as a means to treat the CNS. Mice homozygous for a null disruption of Dhcr7, Dhcr7(Delta3-5/Delta3-5), die soon after birth, thus they cannot be used to study postnatal development or therapy. To circumvent this problem, we produced a hypomorphic SLOS mouse model by introducing a mutation corresponding to DHCR7(T93M). Both Dhcr7(T93M/T93M) and Dhcr7(Delta3-5/T93M) mice are viable. Phenotypic findings in Dhcr7(T93M/Delta3-5) mice include CNS ventricular dilatation and two to three syndactyly. Biochemically, both Dhcr7(T93M/T93M) and Dhcr7(T93M/Delta3-5) mice have elevated tissue 7-DHC levels; however, the biochemical defect improved with age. This has not been observed in human patients, and is due to elevated Dhcr7 expression in mouse tissues. Dietary cholesterol therapy improved sterol profiles in peripheral, but not CNS tissues. However, treatment of Dhcr7(T93M/Delta3-5) mice with simvastatin decreased 7-DHC levels in both peripheral and brain tissues. Expression of Dhcr7 increased in Dhcr7(T93M/Delta3-5) tissues after simvastatin therapy, consistent with the hypothesis that simvastatin therapy improves the biochemical phenotype by increasing the expression of a Dhcr7 allele with residual enzymatic activity. We conclude that simvastatin treatment is efficacious in improving the SLOS-associated sterol abnormality found in the brain, and thus has the potential to be an effective therapeutic intervention for behavioral and learning problems associated with SLOS.

SLOS carrier frequency in Poland as determined by screening for Trp151X and Val326Leu DHCR7 mutations.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Previous studies estimated the prevalence of SLOS between 1 in 10,000 to 1 in 70,358 based on case frequency surveys. Although panethnic, SLOS appears to be most frequent in Central European populations (Czech Republic 1 in 10,000, Slovakia 1 in 15,000 - 1 in 20,000). In Polish individuals with SLOS two DHCR7 mutations, c.452G>A (p.Trp151X) and c.976G>T (p.Val326Leu), account for 65.2% of all observed DHCR7 mutations. We analyzed 2169 samples for the p.Trp151X mutation and 2087 for the p.Val326Leu mutation. The combined carrier frequency of these two mutations of was 2.40+/-0.32%, yielding a calculated incidence of SLOS in Poland of 2.5 4x10(-4)-4.3 5x10(-4) (1 in 2,300 to 1 in 3,937) placing SLOS among the most common recessive genetic disorders in Poland.

Immunohistochemical and microarray analyses of a mouse model for the smith-lemli-opitz syndrome.

The Smith-Lemli-Opitz syndrome is a mental retardation/malformation syndrome with behavioral components of autism. It is caused by a deficiency in 3beta-hydroxysteroid-Delta7-reductase (DHCR7), the enzyme required for the terminal enzymatic step of cholesterol biosynthesis. The availability of Smith-Lemli-Opitz syndrome mouse models has made it possible to investigate the genesis of the malformations associated with this syndrome. Dhcr7 gene modification (Dhcr7-/-) results in neonatal lethality and multiple organ system malformations. Pathology includes cleft palate, pulmonary hypoplasia, cyanosis, impaired cortical response to glutamate, and hypermorphic development of hindbrain serotonergic neurons. For the current study, hindbrain regions microdissected from gestational day 14 Dhcr7-/-, Dhcr7+/- and Dhcr7+/+ fetuses were processed for expression profiling analyses using Affymetrix oligonucleotide arrays and filtered using statistical significance (S-score) of change in gene expression. Of the 12,000 genes analyzed, 91 were upregulated and 98 were downregulated in the Dhcr7-/- hindbrains when compared to wild-type animals. Fewer affected genes, representing a reduced affect on these pathways, were identified in heterozygous animals. Hierarchical clustering identified altered expression of genes associated with cholesterol homeostasis, cell cycle control and apoptosis, neurodifferentiation and embryogenesis, transcription and translation, cellular transport, neurodegeneration, and neuronal cytoskeleton. Of particular interest, Dhcr7 gene modification elicited dynamic changes in genes involved in axonal guidance. In support of the microarray findings, immunohistochemical analyses of the netrin/deleted in colorectal cancer axon guidance pathway illustrated midline commissural deficiencies and hippocampal pathfinding errors in Dhcr7-/- mice. The results of these studies aid in providing insight into the genesis of human cholesterol-related birth defects and neurodevelopmental disorders and highlight specific areas for future investigation.

Photosensitive Smith-Lemli-Opitz syndrome is not caused by a single gene mutation: analysis of the gene encoding 7-dehydrocholesterol reductase in five U.K. families.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by a disorder in cholesterol metabolism. SLOS is caused by mutations in the DHCR7 gene which encodes 7-dehydrocholesterol reductase, an enzyme that catalyses the final step in cholesterol biosynthesis. We have previously established the clinical and photobiological features of the photosensitivity that is frequently a feature of SLOS. OBJECTIVES: In this study, we have performed mutational analysis of the DHCR7 gene in individuals from five families with SLOS. In each family, one member was affected by severe photosensitivity as a manifestation of SLOS. METHODS: Fifteen samples (including family controls) were screened using polymerase chain reaction amplification and direct automated sequencing. RESULTS: Six different DHCR7 mutations were identified of which five were single point mutations that caused missense amino acid substitutions (P51H, T93M, L99P, E448K and R450L). The other was a splice site mutation (G-->C in splice acceptor site) affecting the intron 8-exon 9 splice junction (IVS8-1 G-->C). This splice site mutation and four of the five missense mutations have been previously published as causal in SLOS but the P51H is a novel mutation which has not previously been reported. CONCLUSIONS: This is the first study in which DHCR7 gene mutational analysis has been performed on SLOS subjects with severe photosensitivity and indicates that no single mutation is responsible for the photosensitivity which characterizes this disorder.

Identification of nine novel DHCR7 missense mutations in patients with Smith-Lemli-Opitz syndrome (SLOS).

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital anomaly syndrome caused by deficiency of 7-dehydrocholesterol reductase (DHCR7), which catalyzes the last step of endogenous cholesterol synthesis. Surveys of SLOS patients have identified more than one hundred point mutations of the DHCR7 gene, most of which are missense mutations. Here, we report the identification of nine novel missense mutations of the DHCR7 gene.