Cholesterol Deficiency and Genetic Syndromes

Forbes D. Porter, MD, PhD, Head, Section on Molecular Dysmorphology
Xiao-sheng Jiang, PhD, Postdoctoral Fellow
Marie Lindegaard, MD, Special Volunteer
Li Song, MD, Special Volunteer
Susan Sparks, MD, PhD, Special Volunteer
Elaine Tierney, MD, Special Volunteer
Julia Cupp, BA, Predoctoral Fellow
Meghan Lyman, BA, Predoctoral Fellow
Erin Merkel, BA, Predoctoral Fellow
Elizabeth Wasmuth, BA, Predoctoral Fellow
Chris Wassif, MSc, Technical Specialist
Nicole Yanjanin, RN, Research Nurse
Halima Goodwin, CPNP, Nurse Practitioner

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 (SLOS), lathosterolosis, desmosterolosis, X-linked dominant chondrodysplasia punctata type 2 (CDPX2), and CHILD syndrome. We conduct both basic and clinical research. Our 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 found in the various disorders. Our clinical research focuses on characterization and treatment of patients with SLOS. Our emphasis on both basic and clinical research allows us to integrate laboratory and clinical data in order to increase our understanding of the pathological mechanisms underlying SLOS and to improve the clinical care of SLOS patients.

Smith-Lemli-Opitz syndrome

Goodwin, Sparks, Tierney, Wassif, Porter; in collaboration with Bailey-Wilson, Javitt, Platt, Shackleton

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 result of multiple 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 3beta-hydroxysterol delta 7-reductase gene (DHCR7) and demonstrated mutations of the gene in SLOS patients (Correa-Cerro et al., J Med Genet 2005;42:350). To date, together with others we have identified over 100 mutations of DHCR7. 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 science work focusing on understanding 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 them in a longitudinal natural history study. Parents frequently report improved behavior in SLOS children on dietary cholesterol supplementation. However, we had yet to conduct a blinded protocol studying the efficacy of dietary cholesterol therapy to ameliorate behavioral problems associated with SLOS. 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 (Wassif et al., Mol Genet Metab 2005;85:96; Correo-Cerro et al. 2006). Accordingly, we initiated a controlled, blinded cross-over protocol to study the safety and efficacy of simvastatin therapy in SLOS. Previously, we showed that SLOS fibroblasts have a secondary defect in intracellular cholesterol transport. As part of a Bench-to-Bedside proposal, we continue, 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 exhibit autistic characteristics. We are currently working in collaboration with groups from both NHGRI and the Kennedy Krieger Institute in Baltimore to analyze this association further. We have also received funding from Autism Speaks to initiate a multicenter double-masked placebo-controlled trial of cholesterol in hypocholesterolemic autism spectrum disorder.

Bukelis I, Porter FD, Zimmerman AW, Tierney E. Smith-Lemli-Opitz syndrome and autism spectrum disorder. Am J Psych 2007;164:1655-1661.
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;15:839-851.
Goodwin H, Brooks B, Porter FD. Acute postnatal cataract formation in Smith-Lemli-Opitz Syndrome. Am J Med Genet 2008;146:208-211.
Porter FD. Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis, and management. Eur J Hum Genet 2008;16:535-541.

Mouse models of SLOS

Cupp, Jiang, Song, Lindegaard, Merkel, Wassif, Wasmuth, Porter; in collaboration with Fliesler, Javitt, Remaley, Shackleton, Watson, Yergey

Using gene targeting in murine embryonic stem cells, we produced three SLOS mouse models: a null mutation, a hypomorphic point mutation, and a conditional mutation. Mouse pups homozygous for the null mutation (Dhcr7^−/−) exhibit variable craniofacial anomalies, are growth-retarded, feed poorly, and appear weak. Dhcr7^−/− pups die during the first day of life because they fail 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 these 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 dysfunction, are major clinical problems in SLOS. The impaired glutamate response observed in our mouse model may yield insight into the etiology of some of the neurological dysfunction in SLOS.

Given that the Dhcr7^−/− pups die during the first day of life, we are not able to study postnatal brain development, myelination, and behavior or to test therapeutic interventions. For this reason, we developed a mis-sense allele (Dhcr7^T93M) and a conditional Dhcr7 mutant allele (Dhcr7^{loxPΔ3-5loxP}). The ^T93M mutation is the second most common mutation found in human patients. Dhcr7^T93M/T93M and Dhcr7^T93M/Δ3-5 mice are viable and demonstrate SLOS with a gradient of biochemical severity (Dhcr7Δ3-5/Δ3-5 > Dhcr7^T93M/Δ3-5 > Dhcr7^T93M/T93M). We used Dhcr7^T93M/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. Treatment of mice with simvastatin improved the biochemical defect in both peripheral and central nervous system tissue, suggesting that simvastatin therapy may be used to treat some of the behavioral and learning problems in children with SLOS. In collaboration with Cedric Shackleton and Gordon Watson, we are using our hypomorphic mouse model to investigate adeno-associated viral gene therapy for SLOS.

As part of our clinical studies on SLOS, we previously identified a novel oxysterol, 27-hydroxy-7-dehydrocholesterol (27-7DHC), derived from 7-DHC in SLOS patients. We thus investigated whether 27-7DHC contributes to the pathology of SLOS and found a strong negative correlation between plasma 27-7DHC and cholesterol levels in SLOS patients. In addition, previous work showed that low cholesterol levels impair hedgehog signaling. Therefore, we hypothesized that increased 27-7DHC levels would have detrimental effects during development in response to suppression of cholesterol levels. To test our hypothesis, we produced SLOS mice (Dhcr7^−/−) expressing a CYP27 transgene. CYP27Tg mice have elevated CYP27 expression and 27-hydroxycholesterol levels but normal cholesterol levels. Dhcr7^−/− mice are growth-retarded, exhibit a low incidence of cleft palate (9 percent), and die during the first day of life. Dhcr7^−/−:CYP27Tg embryos are stillborn and have multiple malformations, including growth retardation, micrognathia, cleft palate (77 percent), lingual and dental hypoplasia, ankyloglossia, umbilical hernia, cardiac defects, cloacae, curled tails, and limb defects. We observed autopod defects (polydactyly, syndactyly, and oligodactyly) in 77 percent of the mice. Consistent with our hypothesis, sterol levels were halved in the liver and were 20-fold lower in brain tissue of Dhcr7^−/−:CYP27Tg than of 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.

Recognizing its potential impact on prenatal therapy, we have been characterizing maternal-fetal cholesterol transport in Abca1, Abcg1, and Sr-b1 mutant mice. We found that disruption of Abca1 decreased maternal-fetal cholesterol transport and conversely the elevated expression of Abca1 caused an increased in maternal-fetal cholesterol transport. In our SLOS mouse model, we were able to show, as a proof of principle, that increased expression of Abca1 increased cholesterol content in SLOS mutant embryos.

We continue to use the SLOS mouse models to understand the pathophysiological processes that underlie the birth defects and clinical problems in SLOS and are using various biochemical, molecular, and proteomic approaches to investigate these issues. 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;15:839-851.
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;119:1876-1885.
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;203:1161-1171.
Lindegaard ML, Wassif CA, Boris V, Marcelo A, Wasmuth E, Shamburek R, Nielsen LB, Remaley AT, Porter FD. Characterization of placental cholesterol transport: ABCA1 is a potential target for in utero therapy of Smith-Lemli-Opitz syndrome. Hum Mol Genet 2008;17:3806-3813.
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;72:802-808.

Lathosterolosis, desmosterolosis, and HEM dysplasia

Jiang, Wassif, Porter

Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholesterol, representing the enzymatic step immediately preceding the defect in SLOS. Thus, to understand further the roles of decreased cholesterol versus increased 7-dehydrocholesterol in SLOS, we disrupted the mouse lathosterol 5-desaturase gene (Sc5d) by using targeted homologous recombination in embryonic stem cells. The Sc5d^−/− pups are stillborn, present with micrognathia and cleft palate, and exhibit limb-patterning defects. Many of the malformations in these mutant mice resemble malformations in SLOS and are consistent with impaired hedgehog signaling during development. Biochemically, the mice have markedly elevated serum and tissue lathosterol levels and reduced cholesterol levels.

One goal of producing a lathosterolosis mouse model was to gain phenotypic insight for the purpose of identifying a corresponding human malformation syndrome. We have now identified a human infant patient with lathosterolosis, a human malformation syndrome that has not yet been described. Biochemically, fibroblasts from the patient show decreased cholesterol and increased lathosterol levels. Mutation analysis showed that the patient is homozygous for a single A→C nucleotide change at position 137 in SC5D, resulting in a mutant enzyme in which the amino acid serine is substituted for tyrosine at position 46. Both parents were heterozygous for the mutation. The infant’s phenotype resembled severe SLOS. Malformations found in both the human patient and mouse model include 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 clinically distinguish SLOS from lathosterolosis. This 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. It results from a mutation of the 3beta-hydroxysterol delta 24-reductase gene (DHCR24). DHCR24 catalyzes the reduction of desmosterol to cholesterol. We disrupted the mouse Dhcr24 gene by using targeted homologous recombination in embryonic stem cells. Surprisingly, although most Dhcr24 mutant mice die at birth, the pups are phenotypically normal.

Others have shown that mutations of the lamin B receptor (LBR) cause HEM dysplasia in humans and ichthyosis in mice. LBR has both lamin B–binding and sterol Δ14-reductase domains. Although only a minor sterol abnormality has been reported, 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 the hypothesis that LBR and DHCR14 are redundant sterol Δ14-reductases, we obtained ichthyosis mice (Lbr^−/−) and disrupted Dhcr14. Dhcr14^−/− mice are 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^+/− 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) and, consistent with a demyelinating process, evidenced vacuolation and swelling of the myelin sheaths in the spinal cord upon pathological evaluation. We observed neither vacuolation nor swelling of the myelin sheaths in either Lbr^−/− or Dhcr14^−/− mice. In contrast to Lbr^−/− mice, Lbr^+/−:Dhcr14^−/− mice had normal skin and did not display the Pelger-Huët anomaly. Peripheral tissue sterols were normal in all three mutant mice, although we found significantly elevated levels (50 percent of total sterols) of cholesta-8,14-dien-3β-ol and cholesta-8,14,24-trien-3β-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 result from impaired lamin B receptor function rather than from 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 3β-hydroxysterol Δ¹⁴-reductase deficiency. Hum Mol Genet 2007;16:1176-1187.

Niemann-Pick type C

Jiang, Wassif, Yanjanin, Porter; in collaboration with Ioannou, Patterson, Pavan, Platt, Yergey

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. The project continues to receive support from the Ara Parsegian Medical Research Foundation and Dana’s Angels Research Trust. To date, we have enrolled 26 NPC patients. To complement the clinical work, we have begun to apply proteomic approaches to both mouse and human biomaterials to identify biological pathways disrupted in NPC.

Collaborators

Joan Bailey-Wilson, PhD, Inherited Diseases Research Branch, NHGRI, Bethesda, MD
Steven Fliesler, PhD, St. Louis University, St. Louis, MO
Yiannis Ioannou, PhD, Mount Sinai School of Medicine, New York, NY
Norman Javitt, MD, PhD, New York University Medical School, New York, NY
Zheng Li, PhD, Clinical Neuroscience Branch, NIMH, Bethesda, MD
Daniel Ory, MD, Washington University, St. Louis, MO
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
Alan Remaley, MD, PhD, Molecular Diseases Branch, NHLBI, Bethesda, MD
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, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact fdporter@helix.nih.gov.