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Decreased cholesterol synthesis as a possible aetiological factor in malformations of Trisomy 18
European Journal of Medical Genetics 49 (2006) 195–199 www.elsevier.com/locate/ejmg
Short report
Decreased cholesterol synthesis as a possible aetiological factor in malformations of Trisomy 18 Wayne W.K. Lam a,*, J. Kirk b, N. Manning c, W. Reardon d, R.I. Kelley e, D. FitzPatrick a,f a
South East of Scotland Clinical Genetics Service, MMC Building, Western General Hospital, Edinburgh EH4 2UX, UK b Department of Clinical Biochemistry, Royal Hospital for Sick Children, Edinburgh, UK c Department of Paediatric Pathology, Sheffıeld Children’s Hospital, Sheffıeld, UK d National Centre for Medical Genetics, Our Lady’s Hospital, Dublin, UK e Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA f Medical Research Council, Western General Hospital, Edinburgh, UK Available online 22 June 2005
Abstract We report a series of neonates and foetuses with trisomy 18 and abnormally low cholesterol levels and propose that down regulation of cholesterol synthesis in trisomy 18 is, in part, responsible for its phenotype. Cholesterol is a major structural lipid of cell membranes, as well as the precursor of steroid hormones and bile acids. Several human malformation syndromes have been identified biochemically as disorders of cholesterol biosynthesis. Trisomy 18, a multi-system malformation syndrome, has clinical features that overlap with those of disorders of cholesterol biosynthesis and dysregulation of this pathway may have a role in the developmental pathology. © 2005 Elsevier SAS. All rights reserved. Keywords: Trisomy 18; Edwards; Smith–Lemli–Opitz; Cholesterol; Sterol biosynthesis
1. Introduction Cholesterol is a 27-carbon mono-unsaturated sterol and is the major sterol in mammals. It serves as an important structural lipid of cell membranes, mitochondrial membranes, and myelin. Cholesterol is also the precursor for the synthesis of all known steroid hormones * Corresponding author. Tel.: +44 131 651 1012; fax: +44 131 651 1013. E-mail address: [email protected] (W.W.K. Lam). 1769-7212/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.ejmg.2005.05.011
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W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
and bile acids. While cholesterol is a constituent of most foods of animal origin, in human cells, most cholesterol derives from de novo synthesis. Most free cholesterol within the cell is located in the plasma membranes, where it is present in roughly equimolar amounts to the sum of all other lipids. In 1993, Smith–Lemli–Opitz syndrome (SLOS), a well-known autosomal recessive malformation syndrome, became the first inborn error of post-squalene cholesterol biosynthesis to be identified biochemically [1]. The elucidation of the biochemical basis of SLOS as a deficiency of 3b-hydroxysteroid D7-reductase, the last enzymatic step in the biosynthesis pathway, soon led to the finding that several other well-known disorders were also due to enzymatic defects in this pathway: Greenberg dysplasia (3b-hydroxysteroid D14reductase), CHILD syndrome (3b-hydroxysteroid dehydrogenase), X-linked dominant chondrodysplasia punctata type 2 or “Conradi-Hunermann syndrome” (3b-hydroxysteroid D8,D7-isomerase), desmosterolosis (3b-hydroxysteroid D24-reductase) and lathosterolosis (Lathosterol 5-desaturase). All of these conditions are very rare with the exception of SLOS, which has an incidence of approximately one in 40,000 births in those of European origin [2]. While each condition has a unique constellation of developmental abnormalities, as a group the disorders are characterised by growth deficiency and multi-system involvement, including, in particular, abnormalities of the cardiac (endocardial cushion defects), skeleton (polydactyly and rhizomelic shortness), and central nervous system (microcephaly and holoprosencephaly). Recently, cholesterol was found to be an essential co-factor for proper functioning of Sonic hedgehog and other members of the Hedgehog family of signalling molecules [3]. The hedgehog proteins are key determinants of embryonic patterning in many tissues, including brain, spinal cord, craniofacial structures, genital structures, cartilage, and the limbs. Trisomy 18, or Edwards’s syndrome, is a well-known malformation syndrome characterised by intrauterine growth retardation, prominent occiput, low set ears, micrognathia, short sternum, and skeletal and cardiac defects. Abnormalities of the central nervous system, including varying degrees of holoprosencephaly, have been documented, as have urogenital anomalies such as hypospadias and cryptorchidism. It is the second most common autosomal trisomy after Down syndrome (trisomy 21); however, in common with other trisomies, the mechanism by which aneuploidy of trisomy 18 causes malformations is unknown. 2. Clinical report We report a series of four neonates presenting with multiple congenital abnormalities, which were due to trisomy 18 (see Table 1). An abnormally low cholesterol level was found as part of the investigation into their congenital abnormalities (see Table 2). The cholesterol measurements were performed by two different paediatric biochemical laboratories using standard assay techniques, for gas chromatography mass spectrometry (GCMS, see Table 2). Apart from case 1, where the cholesterol measurement was taken on day 5 of life, all were measured within 24–48 h after delivery. Only in case 2 were there a second cholesterol measurement, that was performed 24 h after the initial low result, this showed that the cholesterol had increased from 1.1 to 2.0 mmol/l (control ranges 2.1–4.5 mmol/l). No increase of cholesterol precursors was seen in any of the patients.
W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
197
Table 1 Presenting clinical features of neonates with Trisomy 18 IUGR Congenital heart disease Craniofacial dysmorphisms Low set ears Excess skin over nape of neck Micrognathia Prominent occiput Limb abnormalities Arthrogryposis Radial ray defects 2/3 syndactyly Talipes Urogenital abnormalities Prominent clitoris
Case 1 + +
Case 2 + +
+
Case 3 + +
Case 4 + +
+
+ +
+
+
+ +
+
+ + + + + +
+
+
Table 2 Plasma sterol levels Cholesterol (mmol/l) 0.2 1.1 0.9 0.9 2.1–4.5
Case 1 Case 2 Case 3 Case 4 Control range
7-dehydrocholesterol (µmol/l) 0.1 Undetectable 0.05 0.1 0.7–1.96
Cholesterol measurements were carried out by the paediatric biochemical laboratories of Great Ormond Street and Sheffield Children’s Hospital.
In addition, we found the mean level of cholesterol in six mid-trimester amniotic fluids from foetuses with trisomy 18 to be significantly low compared to the level of cholesterol in control amniotic fluids from normal foetuses (see Table 3). 3. Discussion Trisomy 18 is a disorder that is well-known and the associated spectrum of abnormalities, well described [4]. Together with trisomy 21 are the commonest aneuploidies seen at Table 3 Amniotic fluid sterol levels
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Controls N = 31
Cholesterol (µmol/l) 23.8 17.5 20.4 16.9 21.2 20.6 33.9 ± 11.7 P < 0.05
7-dehydrocholesterol (nmol/l) 2.5 2.7 1.3 1.9 3.1 2.1 8.1 ± 7.7 P < 0.05
7-dehydrocholesterol/total sterol ratio (%) 0.011 0.015 0.007 0.011 0.015 0.010 0.025 ± 0.019 P < 0.05
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birth [5]. There have been attempts to delineate the underlying molecular genetic defect. Over the last decade most progress has been made with trisomy 21 and there are many reports high lighting certain regions of chromosome 21, where triplication of these region have been shown to demonstrate the majority of features seen in patients with Down syndrome [6]. Within this Down syndrome critical region several genes have been identified [7]. However, work has only recently started in trying to identify the nature of these genes [8]. In Edwards syndrome two non-contiguous regions on chromosome 18 has been identified to be important in the clinical features seen in Edwards syndrome [9]. We are not aware of any work demonstrating a possible mechanism for the pathogenesis of the clinical features seen in Edward’s syndrome. Compared with the phenotype seen in Smith–Lemli–Opitz, the most common of the cholesterol biosynthesis disorders, it is clear that there are many features which are in common with that of Edwards syndrome, also there are similarities in the distribution of congenital anomalies between the two conditions [10]. In addition some of the more characteristic features that are described in disorders of cholesterol biosynthesis, such as stippling of the epiphysis, a hallmark of chondrodysplasia punctata, or holosprosencephaly, which occurs in 5% of SLOS, have also been reported in Edward’s syndrome [11]. SLO is due to deficiency of the enzyme catalysing the penultimate step of cholesterol synthesis, resulting in deficient cholesterol in patients with SLO. Recent studies by Cooper et al. [12] and Roux et al. [13] convincingly argue that the cause of Hedgehog dysfunction in SLOS and Lathosterolosis is the cellular deficiency of cholesterol and not the effect of any precursor sterol that accumulates behind the blocks. We propose that hypocholesterolemia is a common characteristic of trisomy 18 and may have a role in causing many of the malformations it shares with SLOS and the other malformation syndromes due to defects in the cholesterol synthesis pathway [14]. The recurrent finding of abnormally low serum cholesterol level shortly after birth, together with the abnormal levels in amniotic fluids, suggest that there is dysregulation of cholesterol biosynthesis during foetal life. The low levels of 7-dehydrocholesterol as a fraction of cholesterol and the lack of increases in other precursor sterols suggest that there is down regulation of cholesterol biosynthesis prior to lanosterol, perhaps at the level of HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis. The low maternal unconjugated estriol levels seen in trisomy 18 pregnancies has long been recognised as an important biochemical marker in antenatal screening programmes for aneuploid pregnancies [15]. As in SLOS, the decreased cholesterol synthesis may be a reason for this. Thus, it seems reasonable to speculate that a genetically determined cellular deficiency of cholesterol in trisomy 18 has the same significance with regard to the low unconjugated estriol levels and malformations such as holoprosencephaly and heart defects in trisomy 18 as it does in the clinical manifestations in SLOS. However, in case 2 where we have an later cholesterol measurement there appears to be some evidence of self-correction to a low/normal level suggesting that the dysregulation of the biosynthesis pathway may be transient or that other factors may be involved in post natal life. However, given the small size of the sample, we can only speculate on the nature of these regulatory mechanisms, it is our intention to perform a more detailed study in the future with a larger cohort of patients and where we have full ethical approval to look at these issues.
W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
199
References [1] [2] [3] [4] [5] [6]
[7] [8] [9]
[10] [11] [12] [13] [14]
[15]
M. Irons, E.R. Elias, G. Salen, G.S. Tint, A.K. Batta, Defective cholesterol biosynthesis in Smith–Lemli– Opitz syndrome, Lancet 341 (1993) 1414. R.I. Kelley, G.E. Herman, Inborn errors of sterol biosynthesis, Annu. Rev. Genomics Hum. Genet. 2 (2001) 299–341. M. Muenke, M.M. Cohen Jr., Genetic approaches to understanding brain development: holoprosencephaly as a model, Ment. Retard. Dev. Disabil. Res. Rev. 6 (1) (2000) 15–21. Kenneth Lyons Jones, David W. Smith’s Recognizable Patterns of Human Malformation, fifth ed. R.J. McKinlay Gardner, Grant R. Sutherland. Chromosome Abnormalities and Genetic Counseling, third ed. (Oxford Monographs on Medical Genetics). Nadal M, Vigo CG, Melaragno MI, Andrade JA, Alonso LG, Brunoni D, Pritchard M, Estivill X. Clinical and cytogenetic characterisation of a patient with Down syndrome resulting from a 21q22.1–>qter duplication. J. Med. Genet. 38 (1) 73–6. Nelson DL, Gibbs RA. Genetics. The critical region in trisomy 21. Science 306 (5696) 619–21. Vesa J, Brown Y, Greenfield D, Korenberg JR. Molecular and cellular characterization of the Down syndrome critical region protein 2. Biochem. Biophys. Res. Commun. 328 (1) 235–42. Boghosian-Sell L, Mewar R, Harrison W, Shapiro RM, Zackai EH, Carey J, Davis-Keppen L, Hudgins L, Overhauser J. Molecular mapping of the Edwards syndrome phenotype to two noncontiguous regions on chromosome 18. Am. J. Hum. Genet. 55 (3) 476–83. Robert J. Gorlin, M. Michael Cohen, Raoul C.M. Hennekam. Syndromes of the Head and Neck, fourth ed. (Oxford Monographs on Medical Genetics). M.J. Nowaczyk, J.S. Waye, The Smith–Lemli–Opitz syndrome: a novel metabolic way of understanding developmental biology, embryogenesis, and dysmorphology, Clin. Genet. 59 (2001) 375–386. H.J.M. Kempen, J.F.C. Glatz, J.A. Gevers-Leuven, H.A. van der Voort, M.B. Katan, Serum lathosterol is an indicator of whole-body cholesterol synthesis in humans, J. Lipid Res. 29 (1988) 1149–1156. J. Gillen-Goldstein, H. Roque, B.K. Young, Steroidogenesis patterns in common trisomies, J. Perinat. Med. 30 (2002) 132–136. M.K. Cooper, C.A. Wassif, P.A. Krakowiak, J. Taipale, R. Gong, R.I. Kelley, F.D. Porter, P.A. Beachy, A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis, Nat. Genet. 33 (2003) 508–513. C. Roux, C. Wolf, N. Mulliez, W. Gaoua, V. Cormier, F. Chevy, D. Citadelle, Role of cholesterol in embryonic development, Am. J. Clin. Nutr. 71 (suppl.) (2000) 1270S–1279S.
Short report
Decreased cholesterol synthesis as a possible aetiological factor in malformations of Trisomy 18 Wayne W.K. Lam a,*, J. Kirk b, N. Manning c, W. Reardon d, R.I. Kelley e, D. FitzPatrick a,f a
South East of Scotland Clinical Genetics Service, MMC Building, Western General Hospital, Edinburgh EH4 2UX, UK b Department of Clinical Biochemistry, Royal Hospital for Sick Children, Edinburgh, UK c Department of Paediatric Pathology, Sheffıeld Children’s Hospital, Sheffıeld, UK d National Centre for Medical Genetics, Our Lady’s Hospital, Dublin, UK e Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA f Medical Research Council, Western General Hospital, Edinburgh, UK Available online 22 June 2005
Abstract We report a series of neonates and foetuses with trisomy 18 and abnormally low cholesterol levels and propose that down regulation of cholesterol synthesis in trisomy 18 is, in part, responsible for its phenotype. Cholesterol is a major structural lipid of cell membranes, as well as the precursor of steroid hormones and bile acids. Several human malformation syndromes have been identified biochemically as disorders of cholesterol biosynthesis. Trisomy 18, a multi-system malformation syndrome, has clinical features that overlap with those of disorders of cholesterol biosynthesis and dysregulation of this pathway may have a role in the developmental pathology. © 2005 Elsevier SAS. All rights reserved. Keywords: Trisomy 18; Edwards; Smith–Lemli–Opitz; Cholesterol; Sterol biosynthesis
1. Introduction Cholesterol is a 27-carbon mono-unsaturated sterol and is the major sterol in mammals. It serves as an important structural lipid of cell membranes, mitochondrial membranes, and myelin. Cholesterol is also the precursor for the synthesis of all known steroid hormones * Corresponding author. Tel.: +44 131 651 1012; fax: +44 131 651 1013. E-mail address: [email protected] (W.W.K. Lam). 1769-7212/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.ejmg.2005.05.011
196
W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
and bile acids. While cholesterol is a constituent of most foods of animal origin, in human cells, most cholesterol derives from de novo synthesis. Most free cholesterol within the cell is located in the plasma membranes, where it is present in roughly equimolar amounts to the sum of all other lipids. In 1993, Smith–Lemli–Opitz syndrome (SLOS), a well-known autosomal recessive malformation syndrome, became the first inborn error of post-squalene cholesterol biosynthesis to be identified biochemically [1]. The elucidation of the biochemical basis of SLOS as a deficiency of 3b-hydroxysteroid D7-reductase, the last enzymatic step in the biosynthesis pathway, soon led to the finding that several other well-known disorders were also due to enzymatic defects in this pathway: Greenberg dysplasia (3b-hydroxysteroid D14reductase), CHILD syndrome (3b-hydroxysteroid dehydrogenase), X-linked dominant chondrodysplasia punctata type 2 or “Conradi-Hunermann syndrome” (3b-hydroxysteroid D8,D7-isomerase), desmosterolosis (3b-hydroxysteroid D24-reductase) and lathosterolosis (Lathosterol 5-desaturase). All of these conditions are very rare with the exception of SLOS, which has an incidence of approximately one in 40,000 births in those of European origin [2]. While each condition has a unique constellation of developmental abnormalities, as a group the disorders are characterised by growth deficiency and multi-system involvement, including, in particular, abnormalities of the cardiac (endocardial cushion defects), skeleton (polydactyly and rhizomelic shortness), and central nervous system (microcephaly and holoprosencephaly). Recently, cholesterol was found to be an essential co-factor for proper functioning of Sonic hedgehog and other members of the Hedgehog family of signalling molecules [3]. The hedgehog proteins are key determinants of embryonic patterning in many tissues, including brain, spinal cord, craniofacial structures, genital structures, cartilage, and the limbs. Trisomy 18, or Edwards’s syndrome, is a well-known malformation syndrome characterised by intrauterine growth retardation, prominent occiput, low set ears, micrognathia, short sternum, and skeletal and cardiac defects. Abnormalities of the central nervous system, including varying degrees of holoprosencephaly, have been documented, as have urogenital anomalies such as hypospadias and cryptorchidism. It is the second most common autosomal trisomy after Down syndrome (trisomy 21); however, in common with other trisomies, the mechanism by which aneuploidy of trisomy 18 causes malformations is unknown. 2. Clinical report We report a series of four neonates presenting with multiple congenital abnormalities, which were due to trisomy 18 (see Table 1). An abnormally low cholesterol level was found as part of the investigation into their congenital abnormalities (see Table 2). The cholesterol measurements were performed by two different paediatric biochemical laboratories using standard assay techniques, for gas chromatography mass spectrometry (GCMS, see Table 2). Apart from case 1, where the cholesterol measurement was taken on day 5 of life, all were measured within 24–48 h after delivery. Only in case 2 were there a second cholesterol measurement, that was performed 24 h after the initial low result, this showed that the cholesterol had increased from 1.1 to 2.0 mmol/l (control ranges 2.1–4.5 mmol/l). No increase of cholesterol precursors was seen in any of the patients.
W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
197
Table 1 Presenting clinical features of neonates with Trisomy 18 IUGR Congenital heart disease Craniofacial dysmorphisms Low set ears Excess skin over nape of neck Micrognathia Prominent occiput Limb abnormalities Arthrogryposis Radial ray defects 2/3 syndactyly Talipes Urogenital abnormalities Prominent clitoris
Case 1 + +
Case 2 + +
+
Case 3 + +
Case 4 + +
+
+ +
+
+
+ +
+
+ + + + + +
+
+
Table 2 Plasma sterol levels Cholesterol (mmol/l) 0.2 1.1 0.9 0.9 2.1–4.5
Case 1 Case 2 Case 3 Case 4 Control range
7-dehydrocholesterol (µmol/l) 0.1 Undetectable 0.05 0.1 0.7–1.96
Cholesterol measurements were carried out by the paediatric biochemical laboratories of Great Ormond Street and Sheffield Children’s Hospital.
In addition, we found the mean level of cholesterol in six mid-trimester amniotic fluids from foetuses with trisomy 18 to be significantly low compared to the level of cholesterol in control amniotic fluids from normal foetuses (see Table 3). 3. Discussion Trisomy 18 is a disorder that is well-known and the associated spectrum of abnormalities, well described [4]. Together with trisomy 21 are the commonest aneuploidies seen at Table 3 Amniotic fluid sterol levels
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Controls N = 31
Cholesterol (µmol/l) 23.8 17.5 20.4 16.9 21.2 20.6 33.9 ± 11.7 P < 0.05
7-dehydrocholesterol (nmol/l) 2.5 2.7 1.3 1.9 3.1 2.1 8.1 ± 7.7 P < 0.05
7-dehydrocholesterol/total sterol ratio (%) 0.011 0.015 0.007 0.011 0.015 0.010 0.025 ± 0.019 P < 0.05
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W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
birth [5]. There have been attempts to delineate the underlying molecular genetic defect. Over the last decade most progress has been made with trisomy 21 and there are many reports high lighting certain regions of chromosome 21, where triplication of these region have been shown to demonstrate the majority of features seen in patients with Down syndrome [6]. Within this Down syndrome critical region several genes have been identified [7]. However, work has only recently started in trying to identify the nature of these genes [8]. In Edwards syndrome two non-contiguous regions on chromosome 18 has been identified to be important in the clinical features seen in Edwards syndrome [9]. We are not aware of any work demonstrating a possible mechanism for the pathogenesis of the clinical features seen in Edward’s syndrome. Compared with the phenotype seen in Smith–Lemli–Opitz, the most common of the cholesterol biosynthesis disorders, it is clear that there are many features which are in common with that of Edwards syndrome, also there are similarities in the distribution of congenital anomalies between the two conditions [10]. In addition some of the more characteristic features that are described in disorders of cholesterol biosynthesis, such as stippling of the epiphysis, a hallmark of chondrodysplasia punctata, or holosprosencephaly, which occurs in 5% of SLOS, have also been reported in Edward’s syndrome [11]. SLO is due to deficiency of the enzyme catalysing the penultimate step of cholesterol synthesis, resulting in deficient cholesterol in patients with SLO. Recent studies by Cooper et al. [12] and Roux et al. [13] convincingly argue that the cause of Hedgehog dysfunction in SLOS and Lathosterolosis is the cellular deficiency of cholesterol and not the effect of any precursor sterol that accumulates behind the blocks. We propose that hypocholesterolemia is a common characteristic of trisomy 18 and may have a role in causing many of the malformations it shares with SLOS and the other malformation syndromes due to defects in the cholesterol synthesis pathway [14]. The recurrent finding of abnormally low serum cholesterol level shortly after birth, together with the abnormal levels in amniotic fluids, suggest that there is dysregulation of cholesterol biosynthesis during foetal life. The low levels of 7-dehydrocholesterol as a fraction of cholesterol and the lack of increases in other precursor sterols suggest that there is down regulation of cholesterol biosynthesis prior to lanosterol, perhaps at the level of HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis. The low maternal unconjugated estriol levels seen in trisomy 18 pregnancies has long been recognised as an important biochemical marker in antenatal screening programmes for aneuploid pregnancies [15]. As in SLOS, the decreased cholesterol synthesis may be a reason for this. Thus, it seems reasonable to speculate that a genetically determined cellular deficiency of cholesterol in trisomy 18 has the same significance with regard to the low unconjugated estriol levels and malformations such as holoprosencephaly and heart defects in trisomy 18 as it does in the clinical manifestations in SLOS. However, in case 2 where we have an later cholesterol measurement there appears to be some evidence of self-correction to a low/normal level suggesting that the dysregulation of the biosynthesis pathway may be transient or that other factors may be involved in post natal life. However, given the small size of the sample, we can only speculate on the nature of these regulatory mechanisms, it is our intention to perform a more detailed study in the future with a larger cohort of patients and where we have full ethical approval to look at these issues.
W.W.K. Lam et al. / European Journal of Medical Genetics 49 (2006) 195–199
199
References [1] [2] [3] [4] [5] [6]
[7] [8] [9]
[10] [11] [12] [13] [14]
[15]
M. Irons, E.R. Elias, G. Salen, G.S. Tint, A.K. Batta, Defective cholesterol biosynthesis in Smith–Lemli– Opitz syndrome, Lancet 341 (1993) 1414. R.I. Kelley, G.E. Herman, Inborn errors of sterol biosynthesis, Annu. Rev. Genomics Hum. Genet. 2 (2001) 299–341. M. Muenke, M.M. Cohen Jr., Genetic approaches to understanding brain development: holoprosencephaly as a model, Ment. Retard. Dev. Disabil. Res. Rev. 6 (1) (2000) 15–21. Kenneth Lyons Jones, David W. Smith’s Recognizable Patterns of Human Malformation, fifth ed. R.J. McKinlay Gardner, Grant R. Sutherland. Chromosome Abnormalities and Genetic Counseling, third ed. (Oxford Monographs on Medical Genetics). Nadal M, Vigo CG, Melaragno MI, Andrade JA, Alonso LG, Brunoni D, Pritchard M, Estivill X. Clinical and cytogenetic characterisation of a patient with Down syndrome resulting from a 21q22.1–>qter duplication. J. Med. Genet. 38 (1) 73–6. Nelson DL, Gibbs RA. Genetics. The critical region in trisomy 21. Science 306 (5696) 619–21. Vesa J, Brown Y, Greenfield D, Korenberg JR. Molecular and cellular characterization of the Down syndrome critical region protein 2. Biochem. Biophys. Res. Commun. 328 (1) 235–42. Boghosian-Sell L, Mewar R, Harrison W, Shapiro RM, Zackai EH, Carey J, Davis-Keppen L, Hudgins L, Overhauser J. Molecular mapping of the Edwards syndrome phenotype to two noncontiguous regions on chromosome 18. Am. J. Hum. Genet. 55 (3) 476–83. Robert J. Gorlin, M. Michael Cohen, Raoul C.M. Hennekam. Syndromes of the Head and Neck, fourth ed. (Oxford Monographs on Medical Genetics). M.J. Nowaczyk, J.S. Waye, The Smith–Lemli–Opitz syndrome: a novel metabolic way of understanding developmental biology, embryogenesis, and dysmorphology, Clin. Genet. 59 (2001) 375–386. H.J.M. Kempen, J.F.C. Glatz, J.A. Gevers-Leuven, H.A. van der Voort, M.B. Katan, Serum lathosterol is an indicator of whole-body cholesterol synthesis in humans, J. Lipid Res. 29 (1988) 1149–1156. J. Gillen-Goldstein, H. Roque, B.K. Young, Steroidogenesis patterns in common trisomies, J. Perinat. Med. 30 (2002) 132–136. M.K. Cooper, C.A. Wassif, P.A. Krakowiak, J. Taipale, R. Gong, R.I. Kelley, F.D. Porter, P.A. Beachy, A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis, Nat. Genet. 33 (2003) 508–513. C. Roux, C. Wolf, N. Mulliez, W. Gaoua, V. Cormier, F. Chevy, D. Citadelle, Role of cholesterol in embryonic development, Am. J. Clin. Nutr. 71 (suppl.) (2000) 1270S–1279S.