Human Molecular Genetics, 2000, Vol. 9, No. 9 1385-1391
© 2000 Oxford University Press
Spectrum of 
7-dehydrocholesterol reductase mutations in patients with the Smith–Lemli–Opitz (RSH) syndrome
Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, STR 541, 114 Doughty Street, Charleston, SC 29403, USA, 1Sachs’ Children’s Hospital, Department of Pediatrics, Karolinska Institute, Stockholm, Sweden, 2Coordinated Care Service, Department of Pediatrics and 3Division of Genetics and Metabolism, Department of Medicine, Children’s Hospital, Boston, MA, USA and 4Department of Veterans Affairs Medical Center, New Jersey Healthcare System, East Orange, NJ and University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA
Received 24 January 2000; Revised and Accepted 15 March 2000.
DDBJ/EMBL/GenBank accession number AF067127.
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ABSTRACT |
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The Smith–Lemli–Opitz syndrome (SLOS; also known as the RSH syndrome) is an autosomal recessive genetic disorder, leading to characteristic multi-organ developmental abnormalities, dysmorphic facies, limb malformations and mental retardation. Mutations in the gene for
7-dehydrocholesterol reductase (7-reductase), which catalyzes the last step in cholesterol biosynthesis, cause the disease. We screened 32 patients with SLOS, 28 from the USA and four from Sweden. Twenty-two different nucleotide changes, predicted to be disease-causing mutations, were identified; 20 missense mutations, one nonsense mutation and one splice-site mutation involving the exon 9 acceptor site (IVS8 –1G
C) were detected. All probands were heterozygous for mutations. Twelve of these mutations have not been reported previously, including missense mutations L148R, F168I, D175H, P179L, P243R, F284L, N287K, F302L, R404S, Y462H, R469P and one nonsense mutation E37X. Coupled with previously reported mutations, these findings bring the total of different 7-reductase mutations to 36. These are distributed throughout the coding sequence of the 7-reductase gene except exons 3 and 5, with a clustering in exon 9. Three mutations account for 54% of those observed in our cohort, the splice acceptor site mutation IVS8 –1GC (22/64 alleles, 34%), T93M (8/64, 12.5%) and V326L (5/64, 7.8%). Severity of SLOS was negatively correlated with both plasma cholesterol and relative plasma cholesterol, but not with 7-dehydrocholesterol, the immediate precursor, confirming previous observations. However, no correlation was observed between mutations and phenotype, suggesting that the degree of severity may be affected by other factors. We estimate that between 33 and 42% of the variation in the SLOS severity score is accounted for by variation in plasma cholesterol. Thus, factors other than plasma cholesterol are additionally involved in determining severity. |
INTRODUCTION |
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The Smith–Lemli–Opitz syndrome (SLOS; also know as the RSH syndrome, MIM 270400) is an autosomal recessive disorder of sterol metabolism that leads to a large spectrum of characteristic developmental malformations and dysmorphic features (1–3). On the basis of the severity of the abnormalities, patients with SLOS have been clinically subdivided into a mild form, SLOS I, and a more severe form, SLOS II, though a gradation between SLOS I and II is now increasingly recognized. The estimated incidence of SLOS is 1 in 20 000–40 000 births, with a probable carrier frequency of 1.4% in the Caucasian population (2,4–6). SLOS is thought to be less prevalent in Asian and African populations. The underlying pathogenetic basis for SLOS has been shown to be a loss of
7-dehydrocholesterol reductase (7-reductase), which catalyzes the reduction of the C7–C8 double bond of 7-dehydrocholesterol to produce cholesterol, the last step in cholesterol biosynthesis (7–9). Reduced enzyme activity leads to a deficit of cholesterol and accumulation of precursor sterols, such as 7-dehydrocholesterol (7-DHC), 8-dehydrocholesterol (8-DHC) or cholest-5,8-dien-3 (-ol). To date, 19 different mutations of the 7-reductase gene have been reported in SLOS patients from the USA and Europe (10–14).
Although the genetic defect has now been identified, the pathophysiology of SLOS is poorly understood. A better characterization of the structure–function analyses for 7-reductase may shed further light on the pathophysiology. We report here a mutational analysis of the 7-reductase gene in 32 SLOS patients identified in the USA and Norway. Clinical and biochemical data are available in almost our entire cohort, permitting genotype–phenotype correlations.
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RESULTS |
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Mutation detection
A diagnosis of SLOS was made in 32 probands based upon characteristic clinical findings and plasma 7-DHC concentration exceeding 0.1 mg/dl. In two probands, who died soon after birth, although plasma was not available for diagnosis, tissue was available and showed elevated levels of 7-DHC. Additionally, the reporting physicians suspected SLOS as a diagnosis based upon morphological features of the two newborns. DNA was available, as fibroblast cultures were established at the time of death. Twenty-two nucleotide changes predicted to be disease-causing mutations (Table 1) and six silent polymorphisms within the
7-reductase gene (Table 2) were identified in 32 SLOS patients. All probands were compound heterozygotes for mutations. Four probands were siblings from two kindreds. In two cases, only one mutation was discovered.
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Of the 62 total mutations in our cohort (Table 1), one is a newly described nonsense mutation resulting from a G
A transition at amino acid position 37, 39 are missense mutations, 11 of which are newly described mutations and 22 mutations at a specific splice site, resulting from a GC transversion at –1 of the exon 9 acceptor splicing site (IVS8 –1GC) as reported previously (10–12,15,16). These mutations were distributed in exon 4 (n = 12), exon 6 (n = 5), exon 7 (n = 2), exon 8 (n = 5), intron 8 (n = 22), exon 9 (n = 16), but none in exons 3 and 5 (Table 1). The splice-site IVS8 –1GC mutation was detected in 22/32 SLOS patients. Two other previously described missense mutations, T93M in exon 4 and V326L in exon 9, were found in six and five of the 32 patients, respectively (10–14). These three mutations constitute almost half of all the mutations in our patient group (Table 2). Using RNA isolated from fibroblast lines from probands carrying the splice-site mutation, RT–PCR analyses showed that this mutation leads to activation of a cryptic splice site, resulting in the inclusion of 134 bases of intronic sequences and frame-shift (data not shown), confirming previous characterization of the effects of this mutation on abnormal splicing (11,12,15). A summary of all known mutations, including those from the present study is shown in Figure 1. The proximity of the mutations to the predicted transmembrane domains is as indicated (Fig. 1). Note that seven missense mutations are located away from the predicted transmembrane domains (open circles, Fig. 1), three of which are located in the C-terminal region of the protein that is conserved between human, mouse, Arabidopsis thaliana and fungal sterol reductases (17).
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In two patients, we could detect only one mutation, despite extensive analyses. These individuals were heterozygous for the identified mutations; thus the second mutation remains to be identified. Our analysis was restricted to the coding exons and their immediate flanking sequences, thus more subtle mutations, or microdeletions elsewhere may have been missed.
Genotype–phenotype correlations
An accurate genotype–phenotype correlation in our study cohorts is difficult, since all of the affected individuals are compound heterozygotes for mutated 7-reductase alleles. Although many share the common splice-site mutation, the second mutation is highly variable. Table 3 shows the plasma sterol values, as well as the ratio of plasma cholesterol to total sterols, as a measure of the endogenous 7-reductase enzyme activity. It is generally assumed that the lower the 7-reductase enzyme activity, the lower will be the ratio between plasma cholesterol and total plasma sterols (cholesterol plus cholesterol precursors). That is, the more severe a block in cholesterol biosynthesis, the lower the cholesterol:precursor ratio. The severity score, on the other hand, is a clinical score, based upon phenotypic manifestations. The greater the number of observed structural and functional abnormalities, the higher the score and the greater the severity of disease.
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There were 19 females and 13 males (Table 3) ranging in age from 1 day to 31 years (6.2 ± 7.5 years). For two individuals, only incomplete data were available. Five severely affected subjects, 3, 5, 1100-3, H1 and R1, had symptom scores of 58–79 and died perinatally. Mean plasma cholesterol levels (52 ± 36 mg/dl) and total sterols (90 ± 40 mg/dl) were significantly reduced compared with age-matched controls and varied between 6 and 132 mg/dl, respectively (18). The relative cholesterol concentration, calculated as a percentage of total sterols, averaged 56 ± 22% (range 17–94%). Severity scores (mean 39 ± 19, n = 32) correlated inversely and linearly with both absolute plasma cholesterol values (mg/dl, r2 = 0.33, P < 0.001) and relative plasma cholesterol concentrations (percentage of total plasma sterols, r2 = 0.43, P < 0.001, Fig. 2). Surprisingly, correlations of severity scores with either plasma 7-DHC or 8-DHC levels, though positive, did not reach statistical significance (r2 = 0.04, P = 0.28 and r2 = 0.03, P = 0.33, respectively). Multiple regression analyses improved significantly upon the use of relative cholesterol alone compared with the inclusion of the other variables, as a predictor of severity (43 versus 46%, respectively)
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While clinical severity is inversely related to plasma cholesterol, genotype alone does not accurately predict plasma cholesterol or severity. The best example of this is the observation that, although the T93M;IVS8 –1G
C genotype was noted in five of the patients (filled squares, Fig. 2), severity scores, relative cholesterol concentrations and absolute cholesterol concentrations varied from 15 (moderate) to 74 (very severe), 61 to 38% and 78 to 29 mg/dl, respectively. Additionally, assuming that the IVS8 –1GC genotype codes for a null enzyme (14), then two individuals (52 and 70, Table 3) have the V326L;IVS8 –1GC mutations and have severity scores of 45 and 40, respectively. Additionally, individuals Nr1 and Nr2 share the N287K;IVS8 –1GC mutations and their severity scores are 42 and 35, respectively. However, caution must be expressed as these sample sizes are very small. We had two pairs of affected siblings, individuals 66 and 67 and individuals 71 and 88 (Table 3), who showed both a similar severity score and similar plasma cholesterol levels (absolute and relative). |
DISCUSSION |
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Mutational analyses of DHCR7 shows that a wide variety of missense mutations can cause SLOS, though three mutations are more prevalent than others (IVS8 –1G
C, T93M and V326L). Of these, the splice-site mutation appears to be the most common. This mutation leads to activation of an intronic cryptic splice site and frame-shift (10–12,14,15), resulting in a protein that is missing the highly conserved C-terminal domain (17). A number of mutations have been characterized by tissue culture expression studies (10,14,19). With the exception of one (R450L), almost of all the missense mutations, when expressed in either yeast or mammalian cells, may affect enzyme stability and involve putative transmembrane domains (10,14). A number of mutations, including those from the present study, are located away from the transmembrane domains. The C-terminus of 7-reductase is highly conserved across a range of species including human, plant and fungi. Thus, mutations located in this region may result in an alteration in the active site. We have identified three other sites, away from the conserved sites and predicted transmembrane domains. Thus, the effects on the specific activity of these missense mutations need to be established. This is particularly important, since the severity of the disease correlates well with the relative or absolute level of plasma cholesterol, but there is great variation in severity in individuals that have the same type of mutations. This result is consistent with our earlier observations that survival in SLOS depends very strongly on the plasma cholesterol level (20) and is in agreement with the recent observations of Cunniff et al. (21), who used an older symptom index (22), and with Kratz and Kelley (23) who examined the relationship between severity and amniotic fluid sterols. Thus, there seems little doubt that one of the major determinants of severity in SLOS is the availability of cholesterol to the developing fetus. As a first approximation, about 40% of the severity is accounted for by the variation in plasma cholesterol. Understanding the pathophysiology of the SLOS (RSH syndrome) is important because it is likely to increase our knowledge of many biological pathways, from the role of cholesterol in normal embryonic development, feto-maternal transfer of nutrients and sterols during intra-uterine life, to the supply of cholesterol to the brain for postnatal development.
Inhibition of cholesterol biosynthesis in animal models, either by gene knockout (24), or by using inhibitors leads to disruption of normal embryonic development (25–31). In SLOS, the last enzymic step in cholesterol biosynthesis is disrupted, leading to a spectrum of specific developmental abnormalities, but with considerable variance in severity, ranging from embryonic lethality to survival into adulthood.
However, the lack of any observable relationship between genotype and phenotype suggests that factors in addition to the loss of 7-DHC 7-reductase activity must play an important role in determining how much of a restriction there is in the cholesterol supply. Severity could be reduced if the fetus were able to obtain more cholesterol from the mother via the placenta, or by reduction of the double bond at C7–C8 of the sterol molecule, by another enzyme, resulting in cholesterol synthesis via an alternative pathway. At present, there is no evidence that the latter occurs. Levels of cholesterol and 7-DHC have been reported from tissues from two SLOS fetuses, and by Tint et al. (8). Another important factor may be the dysregulation of intracellular cholesterol levels. Thus, the degree of inhibition or activation of the sterol biosynthesis pathway by the loss of 7-reductase activity may have important consequences for pathophysiology. There is some evidence that this regulation may be disrupted in SLOS. Measurements in liver samples from two affected individuals showed that activity of HMG CoA reductase, the rate-limiting step for cholesterol biosynthesis, was not elevated, despite the apparent lack of cholesterol (32), and Steiner et al. (33) have reported that total sterol synthesis is actually reduced in SLOS patients. Additionally, cultured fibroblasts from more severely affected patients synthesized cholesterol at a slower rate, when compared with fibroblasts obtained from less severely affected SLOS patients (34,35). Thus, another variable in severity may be caused by the degree to which HMG CoA reductase activity is reduced in vivo, though these preliminary observations require further confirmation.
The very existence of this defect provides considerable support for a role for cholesterol in both normal development of the brain, face and the limbs (and other organs) and its importance in postnatal life for mental development (36). Inhibition of cholesterol biosynthesis leads to disruption of normal embryonic development (25,29,30). One proposed link between embryonic development and sterol biosynthesis is through the normal function of sonic hedgehog (SHH), the mammalian homolog of Drosophila hedgehog (HH). Hedgehog is a signaling molecule important in cell fate, pattern formation and differentiation, acting through at least two identified cognate receptors, Patched 1 and 2 (37–40). Mouse knockouts for all three of these genes show embryonic lethality with holoprosencephaly and mid-line deformities, abnormalities seen infrequently in SLOS, as well as patients that have gene defects in SHH. Beachy and colleagues (31,38,41–43) have shown that SHH autocleaves itself soon after translation and in the process attains a covalently attached cholesterol moiety at its carboxyl-end of the N-terminal fragment. All the biological activity of SHH appears to reside in this covalently modified peptide. However, 7-DHC, which is elevated in SLOS, as well as a number of other sterols, can appear to substitute for cholesterol in vitro (27). Thus, it is not clear whether the abnormalities of this pathway are entirely responsible for the pathophysiology observed in SLOS.
In summary, we have identified mutations in a large cohort of SLOS propositi. Three mutations appear to be more prevalent (IVS8 –1GC, T93M and V326L) and account for 50% of the spectrum of mutations. Correlations of plasma sterols show that variations in plasma cholesterol (either total or relative) account for ~40% of the variation in severity, but that the precursor sterols individually were not significantly correlated with severity. These data would suggest that increasing the availability of cholesterol to the developing affected fetus may alleviate some of the severity of SLOS, but perhaps not all of it.
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MATERIALS AND METHODS |
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Patients
Patients were evaluated by their own physicians, all qualified clinical geneticists, who filled in a checklist of the typical clinical signs for SLOS. Using these data, the severity of the syndrome was scored, as reported previously (23), as follows: categories of cerebral, ocular, oral, skeletal and genital defects were identified from the questionnaire and each was given a score of 0, 1 or 2 depending on whether 0, 1 or 2 or more abnormalities were noted, respectively. When major abnormalities of heart, liver, kidney and GI tract as well as death were listed, each of these categories was given a score of 2. The scores for all of the 10 categories were summed and the scale normalized by dividing by the maximum score of 20 then multiplying by 100. The clinical severity score was computed by the Tint laboratory and the mutational analyses was performed by the Patel laboratory, before performing phenotype–genotype correlations. Linear regression analyses between severity and plasma sterol values was performed using GraphPad Prizm (1998) and all data are expressed ± SD.
Plasma sterols were measured by gas chromatography as described previously (7). A diagnosis of SLOS was confirmed when there was a report of typical clinical signs and a plasma 7-DHC concentration that exceeded 0.1 mg/dl. In addition to 7-DHC, we always detected 8-DHC at concentrations similar to those of 7-DHC (7). Two of the severely affected patients (H1 and R1) had died before plasma was obtained and only cultured fibroblasts were available. Diagnosis of SLOS in these individuals was established from their fibroblasts, which readily converted radiolabeled lathosterol (5 
-cholest-7-en-3 ß-ol) to 7-DHC but did not transform the resultant [3H]7-DHC to cholesterol (34).
Informed consent was obtained from all participants, in accordance with local Institutional Review Board guidelines.
Exon amplification and sequencing
Genomic DNA was prepared from cultured fibroblasts or peripheral lymphocytes, in accordance with standard methods. Genomic DNA from 32 patients and from three unrelated normal Caucasians was used as the templates for PCR. Exons 3–9 and their intron–exon boundaries were amplified with the primers as reported previously (10–12), and by additional primers as shown in Table 4. PCR products were analyzed by 1% agarose gel electrophoresis, the amplified products excised, purified over columns (Qiagen, Valencia, CA) and sequenced by the Biomolecular Core Lab, using Taq FS Dye Terminator Cycle sequencing kit (Perkin Elmer, Applied Biosystems, Foster City, CA) and an ABI377 sequencer. As a routine both strands were sequenced, to confirm any nucleotide changes. Where possible, both parents were also examined by PCR and direct sequencing, to confirm segregation of any mutations discovered.
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PCR–RFLP analysis
The IVS8 –1G
C is a GC transversion at the exon 9 splice acceptor site. This results in the elimination of an AlwNI restriction recognition site and was used to detect this mutation. This assay was performed as described previously (16). |
ELECTRONIC DATABASE INFORMATION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSELECTRONIC DATABASE INFORMATIONREFERENCES |
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Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim (for SLOS, MIM 270400). GenBank, http://www.ncbi.nlm.nih.gov/genbank/ (for GenBank accession number AF067127, human DHCR7 sequence)
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ACKNOWLEDGEMENTS |
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We are grateful to all Smith–Lemli–Opitz families for their continued involvement in our research, to Drs Erwati V. Bawle, Carolyn Bay, Laura Keppen, Marilyn J. Bull, Carol Greene, Atsuko Fujimoto and their staff for sending us patient data and samples and to Dr Woody Benson for critical reading of the manuscript. Funding from the Medical University of South Carolina (S.B.P.) and a grant from the Department of Veterans Affairs Research Service (G.S.T.) is gratefully acknowledged.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 843 953 0870; Fax: +1 843 953 6480; Email: patelsb@musc.edu
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REFERENCES |
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