We study the molecular, biochemical, and cellular processes that underlie dysmorphic syndromes and birth defects. Our work currently focuses on the inborn errors of cholesterol synthesis, including Smith-Lemli-Opitz syndrome SLOS, 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 due to multiple major malformations. In contrast, 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 7dehydrocholesterol to cholesterol.

Smith-Lemli-Opitz Syndrome
Correa-Cerro, Cozma, Krakowiak, Wassif, Wright, Porter
Our laboratory initially cloned the human 3bhydroxysterol D7-reductase gene (DHCR7) and demonstrated mutations of the gene in SLOS patients. To date, over 70 different mutations of DHCR7 have been identified in SLOS patients. In support of our clinical protocol Clinical and Basic Investigations into Smith-Lemli-Opitz Syndrome,” we have genotyped over 50 SLOS patients and have continued to identify novel mutations of the gene. To further our understanding of the mechanisms underlying the broad phenotypic spectrum in this human malformation syndrome, 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, IVS81G –› 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. This 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 on the order of 1 percent in Caucasians. Surprisingly, although few patients of African heritage have been identified, the carrier frequency for the same allele in African Americans was found to be 0.73 percent. These carrier frequencies predict a disease incidence for SLOS of at least 1/40,000 and 1/75,000 in Caucasians and African Americans, respectively.

Murine Models for SLOS
Correa-Cerro, Wassif, Porter
Using gene targeting in murine embryonic stem cells, we have produced three SLOS mouse models, including a null mutation and two hypomorphic point mutations. The point mutations, which have been described in human patients, are T93M and L99P. Mouse pups that are homozygous for the null mutation, similar to human patients, 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 characteri-zation shows that the pups have markedly elevated serum and tissue 7dehydrocholesterol (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 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 difference in the response to GABA, whereas the glutamate response of mutant neurons decreased significantly. A decreased 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 the mouse model may yield insight into the etiology of some of the neurological dysfunction seen in SLOS. One limitation of the null SLOS mouse model is that it dies during the first day of life, rendering it unsuitable for the study of therapeutic interventions for SLOS. We produced two hypomorphic mouse mutations, T93M and L99P, to avoid this limitation. The mice are currently being characterized for phenotypic, biochemical, neuromuscular, neurological, and behavioral abnormalities.

Clinical Studies in SLOS Patients
Porter
In addition to basic research designed to understand the pathophysiological processes underling SLOS, we have initiated a clinical protocol to study genotype/phenotype correlations, endocrinological aspects, and neurological, dental, speech, and behavioral aspects of SLOS. To date, we have enrolled over 40 SLOS patients. Therapy for SLOS includes dietary cholesterol supplementation. Even though we have noted improvements in growth and behavior, the long-term effect, if any, of dietary cholesterol supplementation on the nervous system remains to be seen. 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 and thus be used to monitor therapeutic interventions. We found elevated MTI values in SLOS patients compared with controls. In addition, in contrast to normal pediatric patients in whom maximum MTI values are observed by 20 months of age, MTI values show an age-dependent increase in SLOS patients up to 10 years of age. We are continuing to obtain longitudinal serial data to determine if MTI will provide a tool for monitoring future therapeutic interventions. Endocrine investigations have shown that about half our SLOS patients have compensated adrenal insufficiency, a finding that will affect their clinical management. We have also made progress in another area by defining a specific behavioral phenotype associated with SLOS that includes autistic and obsessive-compulsive traits.

Lathosterolosis
Cozma, Krakowiak, Wassif, Porter
Lathosterol 5-desaturase catalyzes the conversion of lathosterol to 7-dehydrocholes-terol, 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 disrupted the mouse lathosterol 5desaturase gene (Sc5d) by using targeted homologous recombination in embryonic stem cells. The Sc5d-/- pups are stillborn and have micrognathia, cleft palates, and limb patterning defects. Many of the malformations in the mutant mice resemble malformations found in SLOS and are consistent with impaired hedgehog signalling during development. Biochemically, the mice have markedly elevated serum and tissue lathosterol levels and decreased cholesterol levels.

One goal of producing a lathosterolosis mouse model was to gain phenotypic insight to help identify a corresponding human malformation syndrome. We have now identified a human patient (an infant) with lathosterolosis. The human malformation syndrome has not been described previously. Biochemically, fibroblasts from the patient show decreased cholesterol and increased lathosterol levels. Mutation analysis showed that the patient is homozygous for a single A-to-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. 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. One unique aspect 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. This lysosomal storage disorder can be replicated in embryonic fibroblasts from the Sc5d mutant mouse model.

Characterization of LIM Homeobox Genes Lhx2 and Lhx9
Wassif, Porter
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, exhibit forebrain malformations, and die in utero due to inefficient definitive erythropoiesis. Recent work has shown that Lhx2 functions to pattern the dorsal telencephalon. Lhx9 has an overlapping but a distinct expression pattern compared with Lhx2. Lhx9 mutant mice are agonadal, leading to the conclusion that Lhx9 is essential for gonad development. In collaboration with LMGD, NICHD, GDRB, and NHGRI, we are analyzing Lhx2/Lhx9 compound mutants to determine the combined functions of the two genes. Our group’s characterization of double Lhx2 and Lhx9 mutant embryos has demonstrated that the two LIM homeobox genes are functionally redundant with respect to limb development.