We study the molecular, biochemical, and cellular processes that underlie dysmorphic syndromes and birth defects. Our current focus is on inborn errors of cholesterol synthesis, including the Smith-Lemli-Opitz syndrome.
Smith-Lemli-Opitz syndrome
Correa-Cerro, Goodwin, Scalco, Sparks, Sterner, Tierney, Wassif, Porter; in collaboration with Javitt, Kelley, Shackleton, Watson
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. Our laboratory initially cloned the human 3beta-hydroxysterol-delta-7-reductase gene (DHCR7) and demonstrated mutations of the gene in SLOS patients. To date, over 100 mutations of DHCR7 have been identified. We have genotyped over 50 SLOS patients (see below) and have continued to identify novel mutations of the gene. To further our understanding of the mechanisms underlying SLOS’s broad phenotypic spectrum, we have used deuterium oxide labeling to measure residual DHCR7 activity in fibroblasts from patients with known genotypes and well-characterized phenotypes.
The most common SLOS mutation, IVS8-1GgC, is a single-nucleotide G-to-C change at the minus one position of the splice acceptor site 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, creating an allele with no enzymatic function; that allele 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 IVS8-1GgC allele is approximately 1 percent in Caucasians, a frequency that predicts a minimum disease incidence for SLOS of at least 1 in 40,000 Caucasians. Although African American patients with SLOS are rare, the carrier frequency for the IVS8-1GgC mutation in the African American population is approximately 0.8 percent. Recently, we demonstrated that transcripts from the nonsense alleles, W151X and Q98X, undergo nonsense-mediated decay (NMD). Although NMD can be suppressed for the common W151X allele, DHCR7 enzymatic activity does not increase.
In addition to basic work to understand the pathophysiological processes underling SLOS, we initiated a clinical protocol to study genotype/phenotype correlations and the endocrinological, neurological, dental, speech therapy, and behavioral aspects of SLOS; we have enrolled over 50 patients. Notably, the SLOS behavioral phenotype includes autistic features. Therapy for SLOS includes dietary cholesterol supplementation. In collaboration with researchers at the Children’s Hospital Oakland Research Institute, we have characterized abnormal neuroactive steroids in urine from these patients (Marcos et al., Steroids 2004;69:51). A controlled, blinded cross-over protocol studying the safety and efficacy of simvastatin therapy in SLOS is in progress (Wassif et al., Mol Genet Metab 2005;85:96). In the absence of a blinded protocol studying the efficacy of dietary cholesterol therapy to ameliorate behavioral problems associated with SLOS, we have initiated a new protocol to study the short-term efficacy of dietary cholesterol therapy to improve behavioral problems in SLOS children.
Correa-Cerro LS, Porter FD. 3beta-hydroxysterol delta7-reductase and the Smith-Lemli-Opitz Syndrome. Mol Genet Metab 2005;84:112-126.
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;42:350-357.
Lalovic A, Merkens L, Russel L, Arsenault-Lapierre G, Nowaczyk MJM, Porter FD, Steiner RD, Turecki G. Cholesterol metabolism and suicidality in Smith-Lemli-Opitz syndrome carriers. Am J Psychiatry 2004;161:2123-2126.
Scalco FB, Correa-Cerro LS, Wassif CA, Moretti-Ferreira D, Porter FD. DHCR7 mutations in Brazilian Smith-Lemli-Opitz syndrome patients. Am J Med Genet 2005;136A:278-281.
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.
Mouse models of SLOS
Brownson, Correa-Cerro, Gewandter, Lyman, Wassif, Porter; in collaboration with Fliesler, Kelley, Loh, Lu
Using gene targeting in murine embryonic stem cells, we have produced three SLOS mouse models (Wassif et al., Hum Mol Gen 2001;10:555) that include a null mutation, a hypomorphic point mutation, and a conditional mutation. Similar to human patients, homozygous null (Dhcr7 –/–) mouse pups have variable craniofacial anomalies, are growth retarded, feed poorly, appear weak, and, as a result, die during the first day of life. 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 seen in these mutant mouse pups, we measured the response of cortical neurons to the neurotransmitters GABA and glutamate. We observed no significant difference in the response to GABA between mutant and control neurons. However, the glutamate response of mutant neurons was significantly lower than that of control cortical neurons. A decreased glutamate response is consistent with the phenotypic observation of the mutant animals’ poor feeding. 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 the mouse model may yield insight into the etiology of some of the neurological dysfunction seen in SLOS.
Given that the Dhcr7 –/– pups die during the first day of life, we were not able to study postnatal brain development, myelination, or behavior or test therapeutic interventions. Accordingly, we developed a missense allele (Dhcr7T93M) and a conditional Dhcr7 mutant allele (Dhcr7 loxPΔ3-5loxP). As noted, the T93M mutation is the second most common mutation found in human patients. Dhcr7T93M/T93M and Dhcr7T93M/Δ3-5 are viable. Biochemically, the mutant pups 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 researchers at the Children’s Hospital Oakland Research Institute and using our hypomorphic mouse model, we are investigating adeno-associated viral gene therapy for SLOS. We continue to use the three SLOS mouse models to understand the pathophysiological processes that underlie the birth defects and clinical problems found in SLOS. Specifically, we are involved in various biochemical, molecular, and proteomic approaches to investigating these issues. Accordingly, 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 are characterizing degranulation of mast cells; and our collaboration with Peng Loh’s laboratory focuses on understanding abnormalities of vesicle function in pancreatic tissue.
Cooper MK, Wassif CA, Krakowiak PA, Taipale J, Gong R, Kelley RI, Porter FD, Beachy PA. A defective response to hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 2003;33:508-513.
Lathosterolosis and desmosterolosis
Scalco, Wassif, Porter
Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholesterol, the enzymatic step immediately preceding the defect in SLOS. Thus, to further our understanding of the relative roles of cholesterol deficiency and 7-dehydrocholesterol excess in SLOS, we disrupted the mouse lathosterol 5-desaturase gene (Sc5d) by using targeted homologous recombination in embryonic stem cells. The Sc5d –/– pups were stillborn with micrognathia, cleft palates, and limb patterning defects. Many of the malformations in the pups resembled malformations found in SLOS and are consistent with impaired hedgehog signalling during development. The mice exhibited markedly elevated serum and tissue lathosterol levels and decreased cholesterol levels.
One goal of producing a lathosterolosis mouse model was to identify a corresponding human malformation syndrome; we have now found a human patient with lathosterolosis, a malformation syndrome not previously described. Fibroblasts from the patient show decreased cholesterol and increased lathosterol levels. Mutation analysis revealed 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 were heterozygous for the same mutation. The infant’s phenotype resembled that of 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 mucolipidosis in the affected child, a clinical presentation not found in SLOS that may serve to separate the two disorders clinically. 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 is attributable to 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 were phenotypically normal.
Krakowiak PA, Wassif CA, Kratz L, Cozma D, Kovářová M, Harris G, Grinberg A, Yang Y, Hunter AGW, Tsokos M, Kelley RI, Porter FD. Lathosterolosis: an inborn error of human and murine cholesterol synthesis due to lathosterol 5-desaturase deficiency. Hum Mol Genet 2003;12:1631-1641.
Characterization of LIM homeobox genes Lhx2 and Lhx9
Wassif, Porter; in collaboration with Westphal, Yang
Lhx2 and Lhx9 are two closely related LIM homeobox genes that are essential for the development of several organ systems. Lhx2 mutant mice are anophthalmic, exhibit forebrain malformations, and die in utero as a consequence of inefficient definitive erythropoiesis. Lhx2 also functions in the patterning functions to pattern the dorsal telencephalon (Monuki et al., Neuron 2001;32:591). Lhx9 has an overlapping but distinct expression pattern compared with Lhx2. Lhx9 mutant mice are agonadal; thus, Lhx9 is essential for gonad development (Birk et al., Nature 2000;403:909). Both Lhx2 and Lhx9 are expressed in the developing limb; however, neither Lhx2 nor Lhx9 mutant mice have limb defects. In collaboration with both the Westphal and Yang laboratories, we are analyzing Lhx2/Lhx9 compound mutants to determine the combined functions of the two genes. Double Lhx2 and Lhx9 mutant embryos have limb truncation defects. Thus, these two LIM homeobox genes are functionally redundant with respect to limb development. Characterization of the role of Lhx2/Lhx9 in limb development is in progress.
Collaborators
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
Juan Rivera, PhD, Molecular Immunology and Inflammation Branch, NIAMS, Bethesda, MD
Cedric Shackleton, PhD, Children’s Hospital Oakland Research Institute, Oakland, CA
Gordon Watson, PhD, Children’s Hospital Oakland Research Institute, Oakland, CA
Heiner Westphal, MD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD
Yingzi Yang, PhD, Genetic Disease Research Branch, NHGRI, Bethesda, MD
For further information, contact fdporter@helix.nih.gov.