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• W2130130554 abstract "CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development. CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development. X-linked intellectual disability (XLID) disorders account for 16% of all intellectual disabilities in males.1Stevenson R.E. Schwartz C.E. X-linked intellectual disability: unique vulnerability of the male genome.Dev Disabil Res Rev. 2009; 15: 361-368Crossref PubMed Scopus (49) Google Scholar This high frequency arises in part because males, unlike females, have only one X chromosome. To date, 91 genes involved in XLID have been cloned with demonstrated causative mutations and another 35 XLID syndromes have been mapped.2Greenwood Genetic Center (GGC). XLMR Update - July 2010. Available at http://www.ggc.org/xlmr.htm.Google Scholar Despite this progress, ∼50% of affected families lack an identified causative mutation and thus remain undiagnosed.3Gécz J. Shoubridge C. Corbett M. The genetic landscape of intellectual disability arising from chromosome X.Trends Genet. 2009; 25: 308-316Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar CK syndrome (CKS) is a recently described XLID disorder named after the first reported patient.4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar It is characterized by mild to severe cognitive impairment, seizures beginning in infancy, microcephaly, cerebral cortical malformations, and a thin body habitus.4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar Distinctive features include downslanting palpebral fissures, a high nasal bridge, a high arched palate, micrognathia, and disproportionate short stature without distinctive radiographic findings (Figure S1, available online). Affected males also have behavior problems, including aggression, attention deficit hyperactivity disorder, and irritability.4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar Using DNA from the first described family,4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar we performed an X chromosome single-nucleotide variant (SNV) scan of family members giving consent to the protocol (H07-02142), approved by the clinical research ethics board at the University of British Columbia. We identified linkage to Xq28 (Figure 1). Two-point linkage analysis was performed with MLink from the FASTLINK software package, version 4.0P,5Schäffer A.A. Gupta S.K. Shriram K. Cottingham Jr., R.W. Avoiding recomputation in linkage analysis.Hum. Hered. 1994; 44: 225-237Crossref PubMed Scopus (621) Google Scholar and the Allegro program, version 1.1b.6Gudbjartsson D.F. Jonasson K. Frigge M.L. Kong A. Allegro, a new computer program for multipoint linkage analysis.Nat. Genet. 2000; 25: 12-13Crossref PubMed Scopus (674) Google Scholar Multipoint linkage analysis was performed with the Allegro program. The maximum two-point and multipoint LOD scores were, respectively, 1.43 (θ = 0) and 2.29 (θ = 0) with marker rs941400. Haplotype and microsatellite analysis narrowed the interval to 5 Mb, marker DXS1684 to the telomere (Figure 1). Analysis of the 36.5 NCBI build of the human genome sequence revealed 133 positional candidate genes, expressed sequence tags (ESTs), and noncoding RNAs annotated within this region. By long-range PCR we amplified 1,535,643 bp of genomic sequence containing 111,381 bp of coding sequence and used Illumina reversible terminator-based sequencing to sequence the amplicons. Sufficient sequence coverage for unambiguously identifying variants was obtained for 85.3%, 80.6%, and 87.5% of the coding sequence in individuals V-3, III-4, and IV-11, respectively (Figure S2). Analysis identified a total of 6200 SNVs and 581 indels (Figure 1). Of the SNVs, 5106 were not found in four reference genomes.7Bentley D.R. Balasubramanian S. Swerdlow H.P. Smith G.P. Milton J. Brown C.G. Hall K.P. Evers D.J. Barnes C.L. Bignell H.R. et al.Accurate whole human genome sequencing using reversible terminator chemistry.Nature. 2008; 456: 53-59Crossref PubMed Scopus (2520) Google Scholar, 8Levy S. Sutton G. Ng P.C. Feuk L. Halpern A.L. Walenz B.P. Axelrod N. Huang J. Kirkness E.F. Denisov G. et al.The diploid genome sequence of an individual human.PLoS Biol. 2007; 5: e254Crossref PubMed Scopus (1339) Google Scholar, 9Wheeler D.A. Srinivasan M. Egholm M. Shen Y. Chen L. McGuire A. He W. Chen Y.J. Makhijani V. Roth G.T. et al.The complete genome of an individual by massively parallel DNA sequencing.Nature. 2008; 452: 872-876Crossref PubMed Scopus (1405) Google Scholar, 10Wang J. Wang W. Li R. Li Y. Tian G. Goodman L. Fan W. Zhang J. Li J. Zhang J. et al.The diploid genome sequence of an Asian individual.Nature. 2008; 456: 60-65Crossref PubMed Scopus (736) Google Scholar Capillary resequencing of 44,925 bp confirmed 86% of the SNV and indel observations. Of the 1347 SNVs and one indel unique to the proband, one SNV and one indel met the following criteria: (1) is absent from dbSNP, (2) is confirmed by capillary sequencing, (3) changes an amino acid change, and (4) segregates with CKS. The SNV was a mutation in F8 (c.1064G>A [p.Arg355Gln]), which encodes the blood coagulation factor 8 associated with hemophilia A. However, this mutation was considered clinically irrelevant because these males do not have bleeding problems. The indel was in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL [MIM 300275]) (NM 015922.1:c.696_698del [p.Lys232del]) (Figure 1). The NSDHL mutation was not observed in 150 North American control chromosomes or in the 357 genomes evaluated for indels as part of the 1000 Genomes Project. We did not observe NSDHL mutations among 79 males (58 syndromic and 21 nonsyndromic) with intellectual disability (Table S1). During the course of our studies, however, Tarpey et al.11Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O'Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (468) Google Scholar reported that 1 of 208 families with X-linked intellectual disability had an NSDHL mutation (c.1098dup [p.Arg367SerfsX33, reported as p.R367fsX31 by Tarpey et al.11Tarpey P.S. Smith R. Pleasance E. Whibley A. Edkins S. Hardy C. O'Meara S. Latimer C. Dicks E. Menzies A. et al.A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.Nat. Genet. 2009; 41: 535-543Crossref PubMed Scopus (468) Google Scholar]) (Figure 2). Careful clinical evaluation of this family by F.L.R. showed that the p.Arg367SerfsX33 mutation, which extends the protein past the native stop codon and into the 3′ untranslated region (Figure 2), also causes CKS in this family (Figure 3).Figure 3Males Affected with CKSShow full caption(A) Affected males from family 1 (V-3 and IV-8) hemizygous for the c.696_698del (p.Lys232del) NSDHL mutation and from family 2 (III-1, III-4, III-7, and II-7) hemizygous for the c.1098dup (p.Arg367SerfsX33) NSDHL mutation.(B) Summary of clinical features in males with CKS from families 1 and 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Affected males from family 1 (V-3 and IV-8) hemizygous for the c.696_698del (p.Lys232del) NSDHL mutation and from family 2 (III-1, III-4, III-7, and II-7) hemizygous for the c.1098dup (p.Arg367SerfsX33) NSDHL mutation. (B) Summary of clinical features in males with CKS from families 1 and 2. The NSDHL enzyme, which localizes to the surface of the endoplasmic reticulum and lipid droplets, is a C4 demethylase involved in postsqualene cholesterol biosynthesis.12Caldas H. Herman G.E. NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets.Hum. Mol. Genet. 2003; 12: 2981-2991Crossref PubMed Scopus (68) Google Scholar, 13Gachotte D. Barbuch R. Gaylor J. Nickel E. Bard M. Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13794-13799Crossref PubMed Scopus (76) Google Scholar, 14Mo C. Valachovic M. Randall S.K. Nickels J.T. Bard M. Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis.Proc. Natl. Acad. Sci. USA. 2002; 99: 9739-9744Crossref PubMed Scopus (55) Google Scholar Because CKS males and their mothers had normal plasma cholesterol, steroid hormone levels, and lipoprotein profiles (Table 1), we cultured lymphoblastoid cells expressing p.Lys232del or p.Arg367SerfsX33 NSHDL in cholesterol-poor medium and measured sterols as described.15Kelley R.I. 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 (212) Google Scholar Although of lesser severity, the sterol aberrations were similar to those reported for the allelic disorder congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome (CHILD [MIM 308050]) (Figure 4) (R.I.K., unpublished data) and in mice with Nsdhl mutations.16Liu X.Y. Dangel A.W. Kelley R.I. Zhao W. Denny P. Botcherby M. Cattanach B. Peters J. Hunsicker P.R. Mallon A.M. et al.The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.Nat. Genet. 1999; 22: 182-187Crossref PubMed Scopus (132) Google Scholar The aberrations include accumulation of 4-methyl sterol intermediates, 4,4-dimethyl sterol intermediates, lathosterol, and desmosterol.16Liu X.Y. Dangel A.W. Kelley R.I. Zhao W. Denny P. Botcherby M. Cattanach B. Peters J. Hunsicker P.R. Mallon A.M. et al.The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.Nat. Genet. 1999; 22: 182-187Crossref PubMed Scopus (132) Google ScholarTable 1Serum Cholesterol, Lipoprotein, and Sterol Profiles for Members in Family 1 and Family 2p.Lys232del NSDHL Positivep.Arg367SerfsX33NSDHL Positivep.Lys232del NSDHL NegativeIII-2III-5III-8IV-2IV-6IV-7IV-8V-1aIndividual V-1 was 32 wks pregnant at the time of blood work. Reference ranges for total cholesterol and lipoproteins are based on those reported by Piechota and Statszewski.40 All values were within the normal range for pregnancy in the third trimester.V-2V-3II-2III-1IV-10IV-11SexFFFFMFMFFMFMFMAge (yrs)6753654311162223211941214244X inactivation ratiobX inactivation ratio for an additional p.Lys232del NSDHL-positive female (IV-9) is 58:42; for a p.Lys232del NSDHL-negative female (V-4), the ratio is 36:64.69:3189:1186:1471:29NRNRNR70:3090:10NRNRNRNRNRCholesterol (mg/dL)183.7384.4230.9181.0164.3225.1228.5266.8169.4138.8176.7182.5238.217624S (ng/ml)48.531.1NR43.6NR80.870.962.165.646.4NRNRNR40.6LipoproteinsLDL (mg/dL)107.5266.1129.995.999.0150.4163.6139.299.886.2106115147.377.7HDL (mg/dL)49.143.746.475.056.145.642.581.256.129.847.922.470.463.4SteroidsEstradiol (pg/ml)27.2NR24.2217.913.397.034.1NR249.820.4NRNR38.7NRTestosterone (ng/ml)0.7< 0.20.600.4< 0.20.86.81.60.43.8NRNR0.69NRDHEAS (ug/dl)106.829.577.4117.970.0333.6187.962.6143.7138.8NRNRNRNRCortisol (ug/dl)NR10.711.914.19.710.817.924.314.6NRNRNRNRNRProgesterone (ng/ml)NR<0.2NR21.4NR4.1NR109.11.89NRNRNRNRNRAbbreviations are as follows: 24S, 24S-hydroxycholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; DHEAS, 5-Dehydroepiandrosterone sulfate; F, female; M, male; NR, not reported or not checked.Normal value ranges are as follows:Cholesterol: adult male (110.2–220.4); adult female (162.4–201.8); pediatric male (125.7–230); pediatric female (106.3–216.6).24S: males and females ages 11–70 years (30.1–105.9).LDL: adult male (58–116); adult female (58–131.5); pediatric male and female (< 110).HDL: adult and pediatric male and female (> 34.8).Estradiol: adult male (15–45); adult female (30–450); adult postmenopausal female (< 59.9); prepubertal (< 10.9).Testosterone: adult male (2.8–8.8); adult female (0.1–0.8); prepubertal (< 0.2).DHEAS: adult female (33.2–431); prepurbertal (7.4–66.3).Cortisol: morning levels; adult male/female (5–25).Progesterone: adult postmenopausal females (< 1); adult preovulatory females (< 1); adult midcycle females (5–20); adult females in third trimester of pregnancy (48.4–425).a Individual V-1 was 32 wks pregnant at the time of blood work. Reference ranges for total cholesterol and lipoproteins are based on those reported by Piechota and Statszewski.40Piechota W. Staszewski A. Reference ranges of lipids and apolipoproteins in pregnancy.Eur. J. Obstet. Gynecol. Reprod. Biol. 1992; 45: 27-35Abstract Full Text PDF PubMed Scopus (100) Google Scholar All values were within the normal range for pregnancy in the third trimester.b X inactivation ratio for an additional p.Lys232del NSDHL-positive female (IV-9) is 58:42; for a p.Lys232del NSDHL-negative female (V-4), the ratio is 36:64. Open table in a new tab Abbreviations are as follows: 24S, 24S-hydroxycholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; DHEAS, 5-Dehydroepiandrosterone sulfate; F, female; M, male; NR, not reported or not checked. Normal value ranges are as follows: Cholesterol: adult male (110.2–220.4); adult female (162.4–201.8); pediatric male (125.7–230); pediatric female (106.3–216.6). 24S: males and females ages 11–70 years (30.1–105.9). LDL: adult male (58–116); adult female (58–131.5); pediatric male and female (< 110). HDL: adult and pediatric male and female (> 34.8). Estradiol: adult male (15–45); adult female (30–450); adult postmenopausal female (< 59.9); prepubertal (< 10.9). Testosterone: adult male (2.8–8.8); adult female (0.1–0.8); prepubertal (< 0.2). DHEAS: adult female (33.2–431); prepurbertal (7.4–66.3). Cortisol: morning levels; adult male/female (5–25). Progesterone: adult postmenopausal females (< 1); adult preovulatory females (< 1); adult midcycle females (5–20); adult females in third trimester of pregnancy (48.4–425). NSDHL mutations associated with CHILD are presumed to eliminate or greatly decrease NSDHL function because they include nonsense, frameshift, and deletion mutations.17Bornholdt D. König A. Happle R. Leveleki L. Bittar M. Danarti R. Vahlquist A. Tilgen W. Reinhold U. Poiares Baptista A. et al.Mutational spectrum of NSDHL in CHILD syndrome.J. Med. Genet. 2005; 42: e17Crossref PubMed Scopus (68) Google Scholar To test this, we assessed NSDHL expression in fibroblasts cultured from the affected skin of CHILD patients. Consistent with the nonsense mutations causing either nonsense-mediated mRNA decay or rapid degradation of a truncated protein, the cultures were a mosaic of cells with and without NSDHL expression (Figure S3). Using the Swiss-Model server18Bordoli L. Kiefer F. Arnold K. Benkert P. Battey J. Schwede T. Protein structure homology modeling using SWISS-MODEL workspace.Nat. Protoc. 2009; 4: 1-13Crossref PubMed Scopus (983) Google Scholar to predict the tertiary structure of NSDHL, we found that p.Lys232del disrupts a β-pleated sheet (Figure 4). By immunoblotting, the steady-state level of NSDHL in patient cells expressing either p.Lys232del or p.Arg367SerfsX33 NSDHL was markedly reduced despite comparable mRNA levels as measured by qRT-PCR (Figure 4). Deletion of the analogous amino acid Glu221 from mouse Nsdhl confirmed a stabilizing role for this amino acid when the protein was expressed in HEK293 cells (Figure S4). Also, immunoblotting for p.Lys232del and p.Arg367SerfsX33 NSDHL expressed in HEK293 cells detected low or undetectable steady-state levels unless the proteosome was inhibited with MG132 (Figure 4). The p.Lys232del and p.Arg367SerfsX33 NSDHL had a distribution similar to that of wild-type NSDHL and partially colocalized with the endoplasmic reticulum protein calnexin (Figure 4). To test whether the mutant protein retained enzymatic activity, we assessed complementation in S. cerevisiae deficient for the NSDHL ortholog Erg26.19Lucas M.E. Ma Q. Cunningham D. Peters J. Cattanach B. Bard M. Elmore B.K. Herman G.E. Identification of two novel mutations in the murine Nsdhl sterol dehydrogenase gene and development of a functional complementation assay in yeast.Mol. Genet. Metab. 2003; 80: 227-233Crossref PubMed Scopus (17) Google Scholar The appropriate cDNAs were cloned into the pAG-416-GPD-DEST vector and inserted as single copies into the yeast strain SGD200, which is deficient for Erg26.13Gachotte D. Barbuch R. Gaylor J. Nickel E. Bard M. Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.Proc. Natl. Acad. Sci. USA. 1998; 95: 13794-13799Crossref PubMed Scopus (76) Google Scholar Interestingly, both p.Lys232del and p.Arg367SerfsX33 NSDHL complemented at 30°C (Figure 4). Immunoblotting detected protein levels comparable to those of wild-type NSDHL at 30°C but detected little mutant protein when the yeast were grown at 37°C (Figure 4). Therefore, at a permissive temperature of 30°C, the mutant NSDHL proteins are able to correctly fold and function at a level comparable to wild-type, whereas at the restrictive temperature of 37°C, abnormal folding of the mutant proteins results in protein degradation. Given that NSDHL mutations associated with CHILD syndrome and the Nsdhl loss-of-function alleles found in Bpa mice do not show complementation at the permissive temperature,19Lucas M.E. Ma Q. Cunningham D. Peters J. Cattanach B. Bard M. Elmore B.K. Herman G.E. Identification of two novel mutations in the murine Nsdhl sterol dehydrogenase gene and development of a functional complementation assay in yeast.Mol. Genet. Metab. 2003; 80: 227-233Crossref PubMed Scopus (17) Google Scholar we conclude that the p.Lys232del and p.Arg367SerfsX33 mutations are temperature-sensitive hypomorphic alleles of NSDHL. From this, we postulate that these hypomorphic alleles retain sufficient function to allow survival of males and to mitigate the severe features of CHILD syndrome, particularly in cooler tissues such as skin. Because the developing brain synthesizes cholesterol de novo,20Dietschy J.M. Turley S.D. Thematic review series: brain Lipids. Cholesterol metabolism in the central nervous system during early development and in the mature animal.J. Lipid Res. 2004; 45: 1375-1397Crossref PubMed Scopus (770) Google Scholar we used in situ hybridization and immunohistochemistry to assess NSDHL expression and NSDHL localization, respectively, in the mouse and human brain. The mouse and human tissues were obtained in accordance with protocols approved by the University of British Columbia's ethical review board and institutional policies. In both species, cortical neurons and glia express NSDHL throughout development (Figure S5). Therefore, we hypothesized that deficiency of NSDHL could cause the cortical brain malformations observed in males with CKS (Figure 5).4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar Indeed, histopathological studies of embryonic day 10.5 (E10.5) forebrains from male mice with a Bpa8H loss-of-function allele of Nsdhl19Lucas M.E. Ma Q. Cunningham D. Peters J. Cattanach B. Bard M. Elmore B.K. Herman G.E. Identification of two novel mutations in the murine Nsdhl sterol dehydrogenase gene and development of a functional complementation assay in yeast.Mol. Genet. Metab. 2003; 80: 227-233Crossref PubMed Scopus (17) Google Scholar showed a thin and disorganized cortex and, as measured by TUNEL and BrdU incorporation, significantly increased numbers of apoptotic cells as well as increased cellular proliferation (Figure 5). This paradoxical observation can be explained by the toxic and proliferative effects of methylsterols (L.E.K. and R.I.K., unpublished data). From these observations, we hypothesized that accumulation of methylsterols, not cholesterol deficiency alone, causes CKS. Three patient observations support this: (1) as measured by isotope dilution liquid chromatography-tandem mass spectrometry,21DeBarber A.E. Lütjohann D. Merkens L. Steiner R.D. Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol.Anal. Biochem. 2008; 381: 151-153Crossref PubMed Scopus (41) Google Scholar postnatal plasma 24S-hydroxycholesterol levels, a measure of brain cholesterol turnover,22Lütjohann D. von Bergmann K. 24S-hydroxycholesterol: a marker of brain cholesterol metabolism.Pharmacopsychiatry. 2003; 36: S102-S106PubMed Google Scholar did not differ from controls for absolute 24S-hydroxycholesterol levels or 24S-hydroxycholesterol: cholesterol ratios (Table 1); (2) the cerebrospinal fluid (CSF) cholesterol level of one affected male was normal, whereas his CSF methylsterol levels were elevated (data not shown); and (3) the phenotype and neuropathology of males with CKS are distinctly different than that observed in humans or mice with deficiency of sterol delta-7-reductase,23Wassif C.A. Zhu P. Kratz L. Krakowiak P.A. Battaile K.P. Weight F.F. Grinberg A. Steiner R.D. Nwokoro N.A. Kelley R.I. et al.Biochemical, phenotypic and neurophysiological characterization of a genetic mouse model of RSH/Smith—Lemli—Opitz syndrome.Hum. Mol. Genet. 2001; 10: 555-564Crossref PubMed Scopus (138) Google Scholar, 24Kelley R.I. Hennekam R.C.M. Smith-Lemli-Opitz Syndrome.in: Valle D. Beaudet A.L. Vogelstein B. Kinzler K.W. Antonarakis S.E. Ballabio A. The Online Metabolic & Molecular Bases of Inherited Disease. McGraw-Hill, New York2001Google Scholar the last step in the synthesis of cholesterol.25Kandutsch A.A. Russell A.E. Preputial gland tumor sterols. 3. A metabolic pathway from lanosterol to cholesterol.J. Biol. Chem. 1960; 235: 2256-2261Abstract Full Text PDF PubMed Google Scholar Accumulation of substrate and consequent toxicity, with or without cholesterol deficiency, also explains the diversity of phenotypes observed with defects of cholesterol biosynthesis. These include Greenberg dysplasia (MIM 215140), mevalonic aciduria (MIM 610377), X-linked dominant chondrodysplasia punctata (CDPX2 [MIM 302960]), lathosterolosis (MIM 607330) and desmosterolosis (MIM 602398), as well as Smith-Lemli-Opitz syndrome (SLOS [MIM 270400]), CHILD syndrome, and CKS.26Korade Z. Xu L. Shelton R. Porter N.A. Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2010; 51: 3259-3269Crossref PubMed Scopus (98) Google Scholar, 27Fliesler S.J. Retinal degeneration in a rat model of smith-lemli-opitz syndrome: thinking beyond cholesterol deficiency.Adv. Exp. Med. Biol. 2010; 664: 481-489Crossref PubMed Scopus (28) Google Scholar Similarly, in Insig double-knockout mice, the accumulation of cholesterol precursors in the presence of normal cholesterol levels causes phenotypes ranging from facial clefting28Engelking L.J. Evers B.M. Richardson J.A. Goldstein J.L. Brown M.S. Liang G. Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin.J. Clin. Invest. 2006; 116: 2356-2365Crossref PubMed Scopus (71) Google Scholar to hair-growth defects,29Evers B.M. Farooqi M.S. Shelton J.M. Richardson J.A. Goldstein J.L. Brown M.S. Liang G. Hair growth defects in Insig-deficient mice caused by cholesterol precursor accumulation and reversed by simvastatin.J. Invest. Dermatol. 2010; 130: 1237-1248Crossref PubMed Scopus (41) Google Scholar and consistent with this, the pathology is ameliorated or reversed by blocking the pathway with HMG-CoA reductase inhibitors.28Engelking L.J. Evers B.M. Richardson J.A. Goldstein J.L. Brown M.S. Liang G. Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin.J. Clin. Invest. 2006; 116: 2356-2365Crossref PubMed Scopus (71) Google Scholar, 29Evers B.M. Farooqi M.S. Shelton J.M. Richardson J.A. Goldstein J.L. Brown M.S. Liang G. Hair growth defects in Insig-deficient mice caused by cholesterol precursor accumulation and reversed by simvastatin.J. Invest. Dermatol. 2010; 130: 1237-1248Crossref PubMed Scopus (41) Google Scholar Study of SLOS also implicates the accumulation of enzymatic substrates, not cholesterol deficiency alone, as the cause of disease.26Korade Z. Xu L. Shelton R. Porter N.A. Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2010; 51: 3259-3269Crossref PubMed Scopus (98) Google Scholar, 30Kelley R.I. Hennekam R.C. The Smith-Lemli-Opitz syndrome.J. Med. Genet. 2000; 37: 321-335Crossref PubMed Scopus (381) Google Scholar First, cultured fibroblasts with mutations predicted to have no DHCR7 activity can synthesize cholesterol at rates that can be as high as 50% of all sterols; this suggests that cells have alternate pathways for synthesizing cholesterol.30Kelley R.I. Hennekam R.C. The Smith-Lemli-Opitz syndrome.J. Med. Genet. 2000; 37: 321-335Crossref PubMed Scopus (381) Google Scholar Second, the oxidized derivatives of 7-dehydrocholesterol retard growth of cultured rat embryos, are toxic to cultured cells, and induce gene-expression changes similar to those observed in cells deficient for 7-dehydrocholesterol reductase activity.26Korade Z. Xu L. Shelton R. Porter N.A. Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2010; 51: 3259-3269Crossref PubMed Scopus (98) Google Scholar, 31Gaoua W. Chevy F. Roux C. Wolf C. Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1999; 40: 456-463PubMed Google Scholar Understanding the role of these substrates in human biology is thus crucial to treating these disorders and understanding the role of cholesterol in human behavior.32Golomb B.A. Stattin H. Mednick S. Low cholesterol and violent crime.J. Psychiatr. Res. 2000; 34: 301-309Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 33Lalovic A. Merkens L. Russell L. Arsenault-Lapierre G. Nowaczyk M.J. Porter F.D. Steiner R.D. Turecki G. Cholesterol metabolism and suicidality in Smith-Lemli-Opitz syndrome carriers.Am. J. Psychiatry. 2004; 161: 2123-2126Crossref PubMed Scopus (41) Google Scholar Alternatively, the anomalies of CKS might be attributable, at least in part, to deficient hedgehog signaling as has been suggested in SLOS30Kelley R.I. Hennekam R.C. The Smith-Lemli-Opitz syndrome.J. Med. Genet. 2000; 37: 321-335Crossref PubMed Scopus (381) Google Scholar and in studies of Nsdhl-deficient mouse placentas.34Jiang F. Herman G.E. Analysis of Nsdhl-deficient embryos reveals a role for Hedgehog signaling in early placental development.Hum. Mol. Genet. 2006; 15: 3293-3305Crossref PubMed Scopus (19) Google Scholar Autoprocessing of the hedgehog protein requires cholesterol as a cofactor and covalent adduct.35Chiang C. Litingtung Y. Lee E. Young K.E. Corden J.L. Westphal H. Beachy P.A. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.Nature. 1996; 383: 407-413Crossref PubMed Scopus (2558) Google Scholar Also, cells defective in cholesterol biosynthesis have a defective response to Sonic hedgehog signaling because of reduced Smoothened activity.36Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Teratogen-mediated inhibition of target tissue response to Shh signaling.Science. 1998; 280: 1603-1607Crossref PubMed Scopus (774) Google Scholar, 37Cooper M.K. Wassif C.A. Krakowiak P.A. Taipale J. Gong R. Kelley R.I. Porter F.D. Beachy P.A. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis.Nat. Genet. 2003; 33: 508-513Crossref PubMed Scopus (321) Google Scholar In contrast to SLOS or mutations of Sonic hedgehog, however, CKS individuals do not have polydactyly, syndactyly, genital anomalies, or, as judged by MRI, a rostral-caudal gradient of neuropathology, the forme fruste of holoprosencephaly. Thus, deficient hedgehog signaling does not fully explain the pathology of CKS and again suggests a pathology arising primarily from accumulation of methylsterols. Interestingly, the pathology of CKS is also distinct from that of CHILD syndrome. This disorder, which affects females, is characterized by normal intellect, unilateral ichthyosiform skin lesions typically involving only the right side of the body, alopecia, ipsilateral limb defects with epiphyseal stippling, and occasional internal malformations.38Happle R. Koch H. Lenz W. The CHILD syndrome. Congenital hemidysplasia with ichthyosiform erythroderma and limb defects.Eur. J. Pediatr. 1980; 134: 27-33Crossref PubMed Scopus (146) Google Scholar Mutations of NSDHL causing CHILD syndrome are presumed to be lethal to males on the basis of the skewing of the sex ratio and mouse models.16Liu X.Y. Dangel A.W. Kelley R.I. Zhao W. Denny P. Botcherby M. Cattanach B. Peters J. Hunsicker P.R. Mallon A.M. et al.The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.Nat. Genet. 1999; 22: 182-187Crossref PubMed Scopus (132) Google Scholar, 17Bornholdt D. König A. Happle R. Leveleki L. Bittar M. Danarti R. Vahlquist A. Tilgen W. Reinhold U. Poiares Baptista A. et al.Mutational spectrum of NSDHL in CHILD syndrome.J. Med. Genet. 2005; 42: e17Crossref PubMed Scopus (68) Google Scholar Mouse models have skewing of X inactivation as adults (Figure S6).39Cunningham D. Spychala K. McLarren K.W. Garza L.A. Boerkoel C.F. Herman G.E. Developmental expression pattern of the cholesterogenic enzyme NSDHL and negative selection of NSDHL-deficient cells in the heterozygous Bpa(1H)/+ mouse.Mol. Genet. Metab. 2009; 98: 356-366Crossref PubMed Scopus (15) Google Scholar Also, we found that fibroblast cultures from affected skin of three CHILD patients had X inactivation ratios of 77:23, 96:4, and 92:8 (Figure S6). In the mouse model, the development of skewing is progressive, suggesting that the pathology of CHILD syndrome arises from cell death.39Cunningham D. Spychala K. McLarren K.W. Garza L.A. Boerkoel C.F. Herman G.E. Developmental expression pattern of the cholesterogenic enzyme NSDHL and negative selection of NSDHL-deficient cells in the heterozygous Bpa(1H)/+ mouse.Mol. Genet. Metab. 2009; 98: 356-366Crossref PubMed Scopus (15) Google Scholar In contrast, mothers carrying an NSDHL mutation causing CKS have X inactivation ratios ranging from 90:10 to 58:42 (Table 1), a range common in the general population; this provides additional in vivo support that the NSDHL mutations observed with CKS are hypomorphic. In summary, CKS expands the phenotypes associated with NSDHL mutations. In CHILD syndrome17Bornholdt D. König A. Happle R. Leveleki L. Bittar M. Danarti R. Vahlquist A. Tilgen W. Reinhold U. Poiares Baptista A. et al.Mutational spectrum of NSDHL in CHILD syndrome.J. Med. Genet. 2005; 42: e17Crossref PubMed Scopus (68) Google Scholar and in the bare patches and striated Nsdhl mutant mice,16Liu X.Y. Dangel A.W. Kelley R.I. Zhao W. Denny P. Botcherby M. Cattanach B. Peters J. Hunsicker P.R. Mallon A.M. et al.The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.Nat. Genet. 1999; 22: 182-187Crossref PubMed Scopus (132) Google Scholar there is male lethality and tissue deficiency among carrier females. In contrast, males with CKS survive, and their mothers have no physical abnormalities.4du Souich C. Chou A. Yin J. Oh T. Nelson T.N. Hurlburt J. Arbour L. Friedlander R. McGillivray B.C. Tyshchenko N. et al.Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.Am. J. Med. Genet. A. 2009; 149A: 2469-2478Crossref PubMed Scopus (24) Google Scholar This diversity of phenotypes arising from dysfunction of NSDHL is likely the consequence of variations in flux through the cholesterol biosynthesis pathway (Figure 6). Our findings provide an entry point for further dissection of the role of cholesterol synthesis intermediates in human development. The authors thank Daniel Goldowitz, Jan M. Friedman, Ken Inoue, David Cooke, Martin Bard, and Rosemarie Rupps for critical review of this manuscript. We thank Colin Ross for genotyping support, Daniel Goldowitz for mouse tissues, and the family for their collaboration. This work was supported in part by a British Columbia Children's Foundation Telethon Award (C.D.S.), a Scottish Rite Foundation Award (C.D.S.), a Child & Family Research Institute Establishment Award (C.F.B.), the BC Clinical Genomics Network of the Michael Smith Foundation for Health Research (C.F.B.), and the Réseau de Médecine Génétique Appliquée of Québec (J.L.M. and G.A.R.). C.F.B., S.J.M.J., and M.A.M. are scholars of the Michael Smith Foundation for Health Research. Download .pdf (.87 MB) Help with pdf files Document S1. Six Figures and One Table The URLs for data presented herein are as follows:1000 Genomes Project, http://www.1000genomes.org/dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/The Greenwood Genetic Center, XLMR update, http://www.ggc.org/xlmr.htmOnline Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/The Swiss Model Server, http://swissmodel.expasy.org/SWISS-MODEL.html The dbSNP accession numbers for the sequence variants reported in this paper are ss263199175, ss263199176, and ss263199177." @default.
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• W2130130554 title "Hypomorphic Temperature-Sensitive Alleles of NSDHL Cause CK Syndrome" @default.
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