CHOLESTEROL DEFICIENCY AND GENETIC SYNDROMES
, Head, Section on Molecular Dysmorphology
Halima Goodwin, CPNP, Nurse Practitioner
Xiao-sheng Jiang, PhD, Postdoctoral Fellow
Chris Wassif, MSc, Technical Specialist
Nicole Yanjanin, RN, Research Nurse
Monisha Bahri, MD, Special Volunteer
Marie Lindegaard, MD, Special Volunteer
Li Song, MD, Special Volunteer
Susan Sparks, MD, PhD, Special Volunteer
Elaine Tierney, MD, Special Volunteer
Kirstyn Brownson, BA, Predoctoral Fellow
Meghan Lyman, BA, Predoctoral Fellow
Erin Merkel, BA, Predoctoral Fellow
Elizabeth Wasmuth, BA, Predoctoral Fellow
We study the molecular, biochemical, and cellular processes that underlie dysmorphic syndromes and birth defects attributable to inborn errors of cholesterol synthesis. Human malformation/mental retardation syndromes caused by inborn errors of cholesterol synthesis include Smith-Lemli-Opitz syndrome, lathosterolosis, desmosterolosis, X-linked dominant chondrodysplasia punctata type 2 (CDPX2), and CHILD syndrome. We conduct both basic and clinical research. Basic research uses mouse models of these disorders to understand the biochemical, molecular, cellular, and developmental processes that underlie the birth defects and clinical problems associated with the disorders. Clinical research focuses on characterizing and treating patients with SLOS. Our emphasis on basic and clinical research allows for the integration of laboratory and clinical data, thereby permitting us both to increase our understanding of the pathological mechanisms underlying SLOS and to improve the clinical care of SLOS patients.
Smith-Lemli-Opitz syndrome
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation syndrome characterized by dysmorphic facial features, mental retardation, hypotonia, poor growth, and variable structural anomalies of the heart, lungs, brain, limbs, gastrointestinal tract, and genitalia. The SLOS phenotype is extremely variable. At the severe end of the phenotypic spectrum, infants often die as a result of several major malformations. At the mild end of the phenotypic spectrum, SLOS combines minor physical malformations with behavioral and learning problems. The syndrome is attributable to an inborn error of cholesterol biosynthesis that blocks the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. Our laboratory initially cloned the human 3 beta-hydroxysterol delta 7-reductase gene (DHCR7) and demonstrated mutations of the gene in SLOS patients. To date, over 100 different mutations of DHCR7 have been identified. To further our understanding of the mechanisms underlying the broad phenotypic spectrum in SLOS, we used deuterium oxide labeling to measure residual DHCR7 activity in fibroblasts from patients with known genotypes and well-characterized phenotypes. In collaboration with other laboratories, we have been identifying and characterizing the biological activity of aberrant oxysterols, steroids, and neuroactive steroids.
In addition to our basic research on the pathophysiological processes underling SLOS, we are actively recruiting and following SLOS patients in three clinical protocols. Given that SLOS patients have a cholesterol deficiency, they may be treated with dietary cholesterol supplementation. To date, we have evaluated over 60 SLOS patients and continue to follow many of these patients in a longitudinal natural history study. Parents frequently report improved behavior in SLOS children on dietary cholesterol supplementation. However, a blinded protocol studying the efficacy of dietary cholesterol therapy to ameliorate behavioral problems associated with SLOS has not been conducted. Thus, we designed and implemented a protocol to study the short-term efficacy of dietary cholesterol therapy to improve behavioral problems in 40 SLOS children. Basic laboratory experiments using patient cells and our SLOS mouse model suggested that simvastatin may be efficacious in improving the sterol abnormality in SLOS. Thus, we initiated a controlled, blinded cross-over protocol studying the safety and efficacy of simvastatin therapy in SLOS. Previously we had shown that SLOS fibroblasts exhibit a secondary defect in intracellular cholesterol transport. As part of a Bench-to-Bedside effort, we have begun, in collaboration with Fran Platt’s laboratory, to study impaired cholesterol and glycosphingolipid transport in SLOS and to investigate novel therapeutic interventions.
One of the most interesting facets of SLOS is its distinct behavioral phenotype. Most patients with SLOS have autistic characteristics. We are currently working with groups from both the NHGRI and the Kennedy Krieger Institute in Baltimore to further analyze the association with autistic characteristics.
Bukelis I, Porter FD, Zimmerman AW, Tierney E. Smith-Lemli-Opitz syndrome and autism spectrum disorder. Am J Psychiatry 2007;164:1655-61.
Correa-Cerro LS, Wassif CA, Waye JS, Krakowiak PA, Cozma D, Dobson NR, Levin SW, Anadiotis G, Steiner RD, Krajewska-Walasek M, Nowaczyk MJM, Porter FD. DHCR7 nonsense mutations and characterization of mRNA nonsense mediated decay in Smith-Lemli-Opitz syndrome. J Med Genet 2005;42:350-7.
Goodwin H, Brooks B, Porter FD. Acute postnatal cataract formation in Smith-Lemli-Opitz syndrome. Am J Med Genet, in press.
Wassif CA, Krakowiak PA, Wright BS, Correa-Cerro LS, Sterner AL, Javitt N, Yergey AL, Porter FD. Fractional cholesterol synthesis and simvastatin induction of cholesterol synthesis in Smith-Lemli-Opitz syndrome. Mol Genet Metab 2005;85:96-107.
Waye JS, Krakowiak PA, Wassif CA, Sterner AL, Nowaczyk MJM, Eng B, Nakamura LM, Porter FD. Identification of eleven novel DHCR7 missense mutations in patients with Smith-Lemli-Opitz syndrome (SLOS). Hum Mutat 2005;26:59-62.
Mouse models of SLOS
Using gene targeting in murine embryonic stem cells, we have produced three SLOS mouse models that include a null mutation, a hypomorphic point mutation, and a conditional mutation. Mouse pups homozygous for the null mutation (Dhcr7−/−) have variable craniofacial anomalies, are growth-retarded, feed poorly, and appear weak; they die during the first day of life due to failure to feed. Biochemical characterization showed that the mutant pups had markedly elevated serum and tissue 7-DHC levels as well as reduced serum and tissue cholesterol levels. Cleft palate was present in 9 percent of the Dhcr7−/− pups and is found in approximately one-third of all SLOS patients. Further investigation of the neurological abnormalities in the mice showed that cortical neurons had an impaired response to glutamate; a decreased glutamate response is consistent with the phenotypic observation of poor feeding by the mutant animals. Neurological dysfunctions, including poor feeding, hypotonia, mental retardation, and behavioral problems, are major clinical problems in SLOS. The impaired glutamate response observed in this mouse model may yield insight into the etiology of some of the neurological dysfunction seen in SLOS.
Given that Dhcr7−/− pups die during the first day of life, we are unable to study postnatal brain development, myelination, or behavior or to test therapeutic interventions. For this reason, we developed a mis-sense allele (Dhcr7T93M) and a conditional Dhcr7 mutant allele (Dhcr7loxPΔ3-5loxP). The T93M mutation is the second most common mutation found in human SLOS patients. Dhcr7T93M/T93M and Dhcr7T93M/Δ3-5 mice are viable. Biochemically, the mice have SLOS with a gradient of biochemical severity (Dhcr7Δ3-5/Δ3-5 > Dhcr7T93M/Δ3-5 > Dhcr7T93M/T93M). We used Dhcr7T93M/Δ3-5 mice to test the efficacy of therapeutic interventions on tissue sterol profiles. As expected, dietary cholesterol therapy improved the sterol composition in peripheral tissues but not in the central nervous system while treatment of mice with simvastatin improved the biochemical defect in both peripheral and central nervous system tissue, suggesting that simvastatin therapy can be used to treat some of the behavioral and learning problems encountered in children with SLOS. In collaboration with Cedric Shackleton and Gordon Watson, we are using this hypomorphic mouse model to investigate adeno-associated viral gene therapy for SLOS.
As part of our clinical studies on SLOS, we previously identified the novel oxysterol 27-hydroxy-7-dehydrocholesterol (27-7DHC), which is derived from 7-DHC in SLOS patients, and investigated whether it contributes to the pathology of SLOS; indeed, we found a strong negative correlation between plasma 27-7DHC and cholesterol levels in SLOS patients. Previous work showed that low cholesterol levels impair hedgehog signaling. We thus hypothesized that increased 27-7DHC levels would have detrimental effects during development due to suppression of cholesterol levels. To test our hypothesis, we produced SLOS mice (Dhcr7−/−) expressing a CYP27 transgene. The CYP27Tg mice exhibited elevated CYP27 expression and 27-hydroxycholesterol levels but normal cholesterol levels. Whereas Dhcr7−/− mice exhibit growth retardation, demonstrate a low incidence of cleft palate (9 percent), and die during the first day of life, Dhcr7−/−:CYP27Tg embryos are growth-retarded, stillborn, and have several malformations, micrognathia, cleft palate (77 percent), lingual and dental hypoplasia, ankyloglossia, umbilical hernia, cardiac defects, cloacae, curled tails, and limb defects. We also observed autopod defects (polydactyly, syndactyly, and oligodactyly) in 77 percent of the embryos. Consistent with our hypothesis, sterol levels were two-fold lower in liver and 20-fold lower in brain tissue in Dhcr7−/−:CYP27Tg versus Dhcr7−/− embryos. Recognition of the role of 27-7DHC in SLOS may explain some of the phenotypic variability and may lead to the development of a therapeutic intervention.
Enlisting a variety of biochemical, molecular, and proteomic approaches, we continue to use the SLOS mouse models to understand the pathophysiological processes that underlie the birth defects and clinical problems associated with SLOS. We are also using the mouse models to test various prenatal and postnatal therapeutic interventions.
Correa-Cerro LS, Wassif CA, Kratz L, Miller GF, Munasinghe JP, Grinberg A, Fliesler SJ, Porter FD. Development and characterization of a hypomorphic Smith-Lemli-Opitz syndrome mouse model and efficacy of simvastatin therapy. Hum Mol Genet 2006;15:839-51.
Gondré-Lewis MC, Petrache HI, Wassif CA, Harries D, Parsegian A, Porter FD, Loh YP. Aberrant secretory granule biogenesis linked to cholesterol deficiency in Smith-Lemli-Opitz syndrome (SLOS). J Cell Sci 2006;119:1876-85.
Kovarova M, Wassif CA, Odom S, Liao K, Porter FD, Rivera J. Cholesterol-deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness. J Exp Med 2006;203:1161-71.
Marcos J, Shackleton C, Buddhikot M, Porter FD, Watson G. Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome). Steroids 2007;72:802-8.
Lathosterolosis, desmosterolosis, and HEM dysplasia
Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholesterol, representing the enzymatic step immediately preceding the defect in SLOS. Thus, in order to further our understanding of the relative roles of decreased cholesterol and increased 7-dehydrocholesterol in SLOS, we used targeted homologous recombination in embryonic stem cells to disrupt the mouse lathosterol 5-desaturase gene (Sc5d). Sc5d−/− pups are stillborn and have micrognathia and cleft palate as well as limb patterning defects. Many of the malformations found in these mutant mice resemble the malformations found in SLOS and are consistent with impaired hedgehog signaling during development. Biochemically, the mice exhibit markedly elevated serum and tissue lathosterol levels and decreased cholesterol levels.
One goal of producing a lathosterolosis mouse model was to gain the phenotypic insight needed to facilitate the identification of a corresponding human malformation syndrome. We have now identified a human patient with lathosterolosis. This human malformation syndrome has not previously been described. Biochemically, fibroblasts from the infant patient showed decreased cholesterol and increased lathosterol levels. Mutation analysis showed that the patient was homozygous for a single A→C nucleotide change at position 137 in SC5D, which results in a mutant enzyme in which the amino acid serine is substituted for tyrosine at position 46. Both parents were heterozygous for the mutation. Phenotypically, the infant resembled severe SLOS. Malformations found in both the human patient and the mouse model included growth failure, abnormal nasal structure, abnormal palate, micrognathia, and postaxial polydactyly. A unique feature of lathosterolosis is the clinical finding of mucolipidosis in the affected infant. This clinical presentation is not reported in SLOS and may help in clinically separating the two disorders. The lysosomal storage disorder can be replicated in embryonic fibroblasts from the Sc5d mutant mouse model.
Desmosterolosis is another inborn error of cholesterol synthesis that resembles SLOS. Desmosterolosis is the result of a mutation in the 3beta-hydroxysterol delta 24-reductase gene (DHCR24). DHCR24 catalyzes the reduction of desmosterol to cholesterol. We disrupted the mouse Dhcr24 by using targeted homologous recombination in embryonic stem cells. Surprisingly, although most Dhcr24 mutant mice die at birth, the pups are phenotypically normal.
Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosis in mice. LBR has both a lamin B–binding and a sterol Δ14-reductase domain. Although only a minor sterol abnormality has been observed, it has been proposed that LBR is the primary sterolΔ14-reductase and that impaired sterol Δ14-reduction underlies HEM dysplasia; however, DHCR14 also encodes a sterol Δ14-reductase. To test our hypothesis that the sterol Δ14-reductases of LBR and DHCR14 have an overlapping redundancy, we obtained ichthyosis mice (Lbr−/−) and disrupted Dhcr14. Dhcr14−/− mice were phenotypically normal. We found no sterol abnormalities in either Lbr−/− or Dhcr14−/− tissues at 1 and 21 days of age. We then bred the mice to obtain compound mutant mice. Lbr−/−:Dhcr14−/− and Lbr−/−:Dhcr14+/− mice died in utero. Lbr+/−:Dhcr14−/− mice appeared normal at birth but, by 10 days of age, were growth-retarded and neurologically abnormal (ataxia and tremors). Pathological evaluation demonstrated vacuolation and swelling of the myelin sheaths in the spinal cord, consistent with demyelination. We did not observe such vacuolation/swelling in either Lbr−/− or Dhcr14−/− mice. In contrast to Lbr−/− mice, Lbr+/−:Dhcr14−/− mice had normal skin and did not exhibit the Pelger-Huët anomaly. Peripheral tissue sterols were normal in all three mutant mice. However, we found significantly elevated levels (50 percent of total sterols) of cholesta-8,14-dien-3b-ol and cholesta-8,14,24-trien-3b-ol in brain tissue from 10-day-old Lbr+/−:Dhcr14−/− mice. In contrast, we observed relatively small transient elevations of Δ14-sterols in Lbr−/− and Dhcr14Δ4-7/Δ4-7 brain tissue. Our data support the idea that HEM dysplasia and ichthyosis are attributable to impaired lamin B receptor function rather than to impaired sterol Δ14-reduction. Impaired sterol Δ14-reduction gives rise to a novel murine phenotype for which a corresponding human disorder has yet to be identified.
Wassif CA, Brownson KE, Sterner AL, Forlino A, Zerfas PM, Wilson WK, Starost MF, Porter FD. HEM dysplasia and ichthyosis are likely laminopathies and not due to 3b-hydroxysterol Δ14-reductase deficiency. Hum Mol Genet 2007;16:1176-87.
Niemann-Pick type C
Niemann-Pick type C (NPC) is a neurodegenerative disorder caused by a defect in intracellular lipid and cholesterol transport. As part of a Bench-to-Bedside initiative, we initiated a clinical protocol to identify and characterize biomarkers that could be used in a subsequent therapeutic trial. To date, we have enrolled 20 NPC patients.
COLLABORATOR
Joan Bailey-Wilson, PhD, Inherited Diseases Research Branch, NHGRI, Bethesda, MD
Brian Brooks, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Steven Fliesler, PhD, St. Louis University, St. Louis, MO
Norman Javitt, MD, PhD, New York University Medical School, New York, NY
Marc Patterson, MD, Columbia University, New York, NY
William Pavan, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD
Fran Platt, PhD, Oxford University, Oxford, UK
Cedric Shackleton, PhD, Children’s Hospital Oakland Research Institute, Oakland, CA
Mathew Starost, DVM, PhD, Division of Veterinary Resources, Office of the Director, NIH, Bethesda, MD
Gordon Watson, PhD, Children’s Hospital Oakland Research Institute, Oakland, CA
Alfred Yergey, PhD, Program in Physical Biology, NICHD, Bethesda, MD
For further information, contact fdporter@helix.nih.gov.
