Development of a Murine Model for SLOS
Wassif, Porter
Using gene targeting in murine embryonic stem cells to disrupt Dhcr7, we have produced a murine model of SLOS. We then used the embryonic stem cells to produce germ line-transmitting chimeric animals. Mice heterozygous for the Dhcr7 mutation, like human carriers, are normal. When the heterozygous mice are mated, however, mice homozygous for the mutant allele result. Similar to human patients, the homozygous mutant pups have variable craniofacial anomalies, are growth retarded, feed poorly, and appear weak. The pups die during the first day of life due to failure to feed. Biochemical characterization shows that the pups have markedly elevated serum and tissue 7-dehydrocholesterol (7DHC) 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 the 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 differencewhen the response to GABA was measured. However, in contrast, the glutamate response of mutant neurons was significantly decreased compared with the response observed in 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 our mouse model may yield insight into the etiology of some of the neurological dysfunction seen in SLOS. Thus, this mouse model will be helpful in elucidating the biochemical and cellular basis of the neuropathophysiology underlying the behavioral and learning disabilities seen in this multiple congenital anomaly/mental retardation syndrome and will allow us to test potential therapeutic interventions.

Impaired LDL Cholesterol Transport in SLOS Fibroblasts
Wassif, Porter
Cells obtain cholesterol either by de novo synthesis or from the extracellular environment by the binding and uptake of LDL. The primary defect in SLOS is in de novo synthesis of cholesterol. Thus, patients with SLOS are currently being treated with dietary cholesterol supplementation. However, we have now found that besides the primary defect in de novo cholesterol biosynthesis, fibroblasts from SLOS patients have a defect in LDL cholesterol metabolism. Staining of SLOS fibroblasts with filipin, a fluorescent compound that binds to unesterified cholesterol, showed an accumulation of unesterified sterols in SLOS fibroblasts cultured for four days in lipoprotein-deficient serum (LPDS). We did not observe the increased filipin staining after seven days of culture in LPDS. The results suggested that the increased filipin staining resulted from an accumulation of unesterified LDL cholesterol rather than from an accumulation of 7-DHC. Further studies of fibroblasts from nine SLOS patients have shown that LDL degradation was less in SLOS fibroblasts than in control and carrier fibroblasts. Neither LDL binding nor LDL uptake is decreased. Thus, the decline in LDL degradation is attributable to a defect in intracellular LDL cholesterol metabolism and can be induced in fibroblasts by exposing them to 7-DHC, thereby demonstrating that 7-DHC or a metabolite of 7-DHC perturbs intracellular cholesterol metabolism. In support of these findings, we have found that, under appropriate growth conditions, SLOS fibroblasts develop lysosomal inclusion bodies similar in appearance to those found in Niemann Pick type C disease. We hypothesize that 7-DHC may perturb the function of the Niemann Pick type C protein by interacting with its sterol sensing domain. Based on these studies, we conclude that children with SLOS have a primary defect in cholesterol biosynthesis due to mutations in the DHCR7 gene and a secondary defect in intracellular LDL cholesterol metabolism due to elevated levels of 7-DHC. Clinically, the secondary defect in LDL cholesterol metabolism may limit the effectiveness of the current dietary therapy.

Disruption of the Mouse D7-Sterol-C5(6)-Desaturase Gene
Krakowiak, Wassif, Porter
In the enzymatic step immediately preceding the defects in SLOS, _7-sterol-C5(6)-desaturase catalyzes the conversion of lathosterol to 7-DHC. To further our understanding of the roles of decreased cholesterol versus increased 7-DHC in SLOS, we disrupted the mouse _7-sterol-C5(6)-desaturase gene (Sc5d) by using targeted homologous recombination in embryonic stem cells. The Sc5d -/- pups are stillborn and exhibit micrognathia and cleft palates as well as limb patterning defects.

Clinical Investigations of Smith-Lemli-Opitz Syndrome
Nwokoro, Porter
We have initiated a clinical protocol to study genotype/phenotype correlations as well as the endocrinological, neurological, dental, speech, and behavioral aspects of SLOS. Currently, we have enrolled 43 SLOS patients. Therapy for SLOS includes dietary cholesterol supplementation. Although improvements in growth and behavior have been noted, it is not clear if dietary cholesterol supplementation will have any effects, long-term or otherwise, on the nervous system. We are investigating whether an MRI enhancement technique known as Magnetic Transfer Imaging (MTI), which is highly sensitive to changes in myelin, can provide a quantitative measure of abnormal myelin in SLOS patients. Thus, we set out to determine whether MTI can be used to monitor therapeutic interventions in the disease. In SLOS patients, we found elevated MTI values compared with controls. In addition, MTI values show an age-dependent increase in SLOS patients up to 10 years of age. This result stands in contrast to normal pediatric patients, in whom maximum MTI values are observed by 20 months of age. Although we are only now obtaining longitudinal serial data, the results suggest ongoing changes in the myelin structure in SLOS patients; therefore, MTI may provide a tool for monitoring therapeutic interventions. In addition to the promising applications of MTI, our endocrine investigations have shown that over half of our SLOS patients have compensated adrenal insufficiency; such information will influence the clinical management of these patients. We have also made progress in defining a specific behavioral phenotype associated with SLOS that includes autistic and obsessive-compulsive traits.

Characterization of LIM Homeobox Genes Lhx2 and Lhx9
Wassif, Porter in collaboration with H. Westphal
Lhx2 and Lhx9 are two closely related LIM homeobox genes that are essential for the development of multiple organ systems. Lhx2 mutant mice are anophthalmic, evidence forebrain malformations, and die in utero due to inefficient definitive erythropoiesis. Recent work has shown that Lhx2 functions to pattern the dorsal telencephalon while Lhx9 has an overlapping but a distinct expression pattern compared with Lhx2. While Lhx9 mutant mice are agonadal, Lhx9 is essential for gonad development. In collaboration with members of the LMGD (NICHD), we are analyzing Lhx2/Lhx9 compound mutants to determine the combined functions of the two genes. Characterization of double Lhx2 and Lhx9 mutant embryos by our group has demonstrated that these two LIM homeobox genes are functionally redundant with respect to limb development.