Forbes D. Porter, MD, PhD, Head, Section on Molecular Dysmorphology
Lina Correa-Cerro, MD, PhD, Postdoctoral Fellow
Xiao-sheng Jiang, PhD, Postdoctoral Fellow
Kirstyn Brownson, BA, Predoctoral Fellow
Meghan Lyman, BA, Predoctoral Fellow
Alison Sterner, BA, Predoctoral Fellow
Susan Sparks, MD, PhD, Special Volunteer
Elaine Tierney, MD, Special Volunteer
Chris Wassif, MSc, Technical Specialist
Halima Goodwin, CPNP, Nurse Practitioner
Nicole Yanjanin, RN, Research Nurse

We study the molecular, biochemical, and cellular processes that underlie dysmorphic syndromes and birth defects that result from 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. To understand the biochemical, molecular, cellular, and developmental processes that underlie the birth defects and clinical problems associated with these disorders, our basic research focuses on mouse models of the various disorders while our clinical research focuses on characterization and treatment of patients with SLOS. Our emphasis on both basic and clinical research allows for the integration of laboratory and clinical data, permitting us to increase our understanding of the pathological mechanisms underlying SLOS and improve the clinical care of SLOS patients.

Smith-Lemli-Opitz syndrome

Goodwin, Sparks, Sterner, Tierney, Wassif, Porter; in collaboration with Bailey-Wilson, Chignell, Javitt, Kelley, Shackleton

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 consequence 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 (Correa-Cerro et al., Mol Genet Metab 2005;84:112). Our laboratory initially cloned the human 3β-hydroxysterol Δ⁷-reductase gene (DHCR7) and demonstrated mutations of the gene in SLOS patients. To date, over 100 different mutations of DHCR7 have been identified. In support of our clinical protocols, we have genotyped over 60 SLOS patients and continue to identify novel mutations of the gene (Waye et al., Hum Mutat 2005;26:59). To further our understanding of the mechanisms underlying the broad phenotypic spectrum in SLOS, we have 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.

The most common SLOS mutation, IVS8-1G→C, is a single nucleotide G to C change at the -1 position of the splice acceptor in the eighth intron. Aberrant splicing to a cryptic splice acceptor results in the insertion of 134 base pairs of intronic sequence into the mRNA. The mutation results in an allele with no enzymatic function and accounts for about one-third of the identified mutant alleles. The second most common SLOS mutation is T93M. Other common mutations include W151X, V326L, R404C, and R352W. SLOS may be more common than typically thought. The carrier frequency for the IVS8-1G→C allele is approximately 1 percent in Caucasians, which predicts a minimum disease incidence for SLOS of at least 1 in 40,000 in Caucasians. We demonstrated that transcripts from the nonsense alleles W151X and Q98X undergo nonsense-mediated decay (NMD). Using aminoglycoside antibiotics, we demonstrated that NMD can be suppressed for the common W151X allele. Unfortunately, DHCR7 enzymatic activity does not increase.

In addition to our basic science work focused on understanding the pathophysiological processes underling SLOS, we are actively recruiting and following SLOS patients in three clinical protocols. Given that SLOS patients suffer from cholesterol deficiency, they can 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, we had yet to conduct a blinded protocol studying the efficacy of dietary cholesterol therapy to ameliorate behavioral problems associated with SLOS. Thus, we have initiated a protocol to study the short-term efficacy of dietary cholesterol therapy to improve behavioral problems in 40 SLOS children. In vitro and mouse experiments have suggested that simvastatin may be efficacious in improving the sterol abnormality in SLOS. Thus, we have began a controlled, blinded cross-over protocol studying the safety and efficacy of simvastatin therapy in SLOS. Enrollment of patients for the study is almost complete.

One of the most interesting aspects of SLOS is its distinct behavioral phenotype. Most patients with SLOS have autistic characteristics. We are currently collaborating with groups from both NHGRI and the Kennedy Krieger Institute in Baltimore to analyze this association further.

Mouse models of SLOS

Brownson, Correa-Cerro, Jiang Lyman, Sterner, Wassif, Porter; in collaboration with Fliesler, Javitt, Kelley, Loh, Lu, Parsegian, Rivera, 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 that are homozygous for the null mutation (Dhcr7-/-) have variable craniofacial anomalies, are growth retarded, feed poorly, and appear weak. Dhcr7-/- pups 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. To characterize further the neurological abnormalities observed in these mutant mouse pups, we measured the response of cortical neurons to the neurotransmitters GABA and glutamate. Comparing mutant with control neurons, we observed no significant difference in the response to GABA. However, the glutamate response of mutant neurons was significantly lower than the response observed in control cortical neurons. A low glutamate response is consistent with the phenotypic observation of poor feeding by the mutant animals. Glutamate receptors are involved in neuronal pattern formation, long-term potentiation and depression, memory acquisition, and learning. Neurological dysfunctions, including poor feeding, hypotonia, mental retardation, and behavioral problems, 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 seen in SLOS.

In a continuing collaboration with Bai Lu's laboratory, we are characterizing synapse formation in neurons derived from SLOS mice. In collaboration with Juan Rivera's laboratory, we have characterized degranulation of mast cells. We are collaborating with Peng Loh's and Adrian Parsegian's laboratories to understand abnormalities of vesicle function due to alteration of membrane biophysical properties.

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 test therapeutic interventions. For this reason, we have developed a missense 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 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 improves the sterol composition in peripheral tissues but not in the central nervous system. Treatment of mice with simvastatin improves 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 investigating adenoassociated viral gene therapy for SLOS by using the Dhcr7T93M/Δ3-5 hypomorphic mouse model.

Lathosterolosis, desmosterolosis, and HEM dysplasia

Brownson, Jiang, Lyman, Wassif, Porter

Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholesterol, which is the enzymatic step immediately preceding the defect in SLOS (Krakowiak et al., Hum Mol Genet 2003;12:1631). Thus, to further our understanding of the roles of lowered cholesterol versus elevated 7-dehydrocholesterol in SLOS, we disrupted the mouse lathosterol 5-desaturase gene (Sc5d) with targeted homologous recombination in embryonic stem cells. The Sc5d-/- pups are stillborn and have micrognathia, cleft palate, and limb patterning defects. Many of the malformations found in these mutant mice resemble malformations found 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 in producing a lathosterolosis mouse model was to gain phenotypic insight so as to identify a corresponding human malformation syndrome. This lysosomal storage disorder can be replicated in embryonic fibroblasts from the Sc5d mutant mouse model. We have now identified a human infant patient with lathosterolosis, a human malformation syndrome not been previously described. Biochemically, fibroblasts from the patient show reduced cholesterol and elevated lathosterol levels. Mutation analysis showed that the patient is homozygous for a single A to 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 are heterozygous for the mutation. Phenotypically, the infant resembled severe SLOS. Malformations found in both the human patient and the 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 clinically distinguish lathosterolosis from SLOS.

Desmosterolosis is another inborn error of cholesterol synthesis that resembles SLOS. Desmosterolosis results from mutation of the 3β-hydroxysterol Δ²⁴-reductase gene (DHCR24). DHCR24 catalyzes the reduction of desmosterol to cholesterol. Using targeted homologous recombination in embryonic stem cells, we disrupted the mouse Dhcr24. Surprisingly, although most Dhcr24 mutant mice die at birth, the pups are phenotypically normal.

Hydrops-ectopic calcification-moth-eaten (HEM) is an autosomal recessive disorder that results in a lethal skeletal dysplasia. HEM dysplasia results from mutation of the lamin B receptor (LBR) gene. Mutation of this gene in the mouse causes the ichthyosis phenotype. LBR encodes a bifunctional protein with a lamin binding domain and a sterol Δ¹⁴-reductase domain. A second gene, DHCR14, also encodes a protein with sterol Δ¹⁴-reductase activity. We hypothesized that the two genes are redundant with respect to cholesterol synthesis. To evaluate the role of these two genes in cholesterol synthesis and embryonic development, we obtained ichthyosis mice and disrupted Dhcr14. Molecular, biochemical, and phenotypic evaluation of these mice is currently in progress.

Niemann-Pick type C

Yanjanin, Porter; in collaboration with Patterson, Pavan

Niemann-Pick type C is a neurodegenerative disorder that results from a defect in intracellular lipid and cholesterol transport. As part of a Bench-to-Bedside initiative, we have recently initiated a new clinical protocol focused on identifying and characterizing biomarkers that could be used in a subsequent therapeutic trial.

COLLABORATORS

Joan Bailey-Wilson, PhD, Inherited Disease Research Branch, NHGRI, Bethesda, MD
Colin Chignell, PhD, Laboratory of Pharmacology and Chemistry, NIEHS, Research Triangle Park, NC
Steven Fliesler, PhD, St. Louis University, St. Louis, MO
Norman Javitt, MD, PhD, New York University Medical School, New York, NY
Richard Kelley, MD, PhD, The Johns Hopkins University, Baltimore, MD
Peng Loh, PhD, Section on Cellular Neurobiology, NICHD, Bethesda, MD
Bai Lu, PhD, Laboratory of Cellular and Synaptic Neurophysiology, NICHD, Bethesda, MD
Adrian Parsegian, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
Marc Patterson, MD, Columbia University, New York, NY
William Pavan, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD
Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
Cedric Shackleton, PhD, Children's Hospital Oakland Research Institute, Oakland, CA
Matthew 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, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD

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