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dysmorphic
syndromes and birth defects
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Forbes D. Porter, MD, PhD, Head, Unit on Molecular Dysmorphology Diana Cozma, MD, Postdoctoral
Fellow Fernanda Scalco, PhD, Postdoctoral Fellow Halima Goodwin, CPNP, Nurse Practitioner Alison Sterner, BA, Predoctoral Fellow Elaine Tierney, MD, Special Volunteer Chris Wassif, MSc, Technical Specialist |
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We study the molecular, biochemical, and
cellular processes that underlie dysmorphic syndromes and birth defects, focusing
on the inborn errors of cholesterol synthesis, including the
Smith-Lemli-Opitz syndrome. Smith-Lemli-Opitz syndrome Correa-Cerro,
Goodwin, Scalco, Sterner, Tierney, Wassif, Porter; in collaboration with
Kelley, Javitt, 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 (Porter FD, J Clin Inves
2002;110:715-724). The SLOS phenotype is extremely variable. At the severe
end of the phenotypic spectrum, infants often die as a result of multiple
major malformations, while 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 different mutations
of DHCR7 have been identified. In support of our clinical protocol
entitled 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, IVS8-1GgC, is a single-nucleotide G-to-C change at the
minus one 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 IVS8-1GgC allele is approximately 1 percent in
Caucasians, predicting a minimum disease incidence for SLOS of at least
1/40,000 in Caucasians. Although African American patients with SLOS are
rare, the carrier frequency for the IVS8-1GgC
mutation in this 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 is not increased. In addition to basic research aimed at
understanding the pathophysiological processes underling SLOS, we have
initiated a clinical protocol to study genotype/phenotype correlations and
endocrinological, neurological, dental, speech, and behavioral aspects of
SLOS. Notably, the SLOS behavioral phenotype includes autistic features.
Therapy for SLOS includes dietary cholesterol supplementation. We have thus
far enrolled over 50 SLOS patients and have characterized abnormal
neuroactive steroids in their urine. Recruitment of patients for a protocol
studying the safety and efficacy of simvastatin therapy in SLOS is in
progress. Given that no laboratory has yet conducted a blinded protocol
studying the efficacy of dietary cholesterol therapy to ameliorate behavioral
problems associated with SLOS, we are designing a new protocol to study the
short-term efficacy of dietary cholesterol therapy to improve behavioral
problems in SLOS children. 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. Marcos J, Guo L-W, Wilson W, Porter FD,
Shackleton C. The implications of 7-dehydrosterol-7-reductase deficiency
(Smith-Lemli-Opitz syndrome) to neurosteroid production. Steroids
2004;69:51-60. Porter FD. Human malformation syndromes due to
inborn errors of cholesterol synthesis. Curr Opin Pediatr 2003;15:607-613. Wright BS, Nwokoro NA, Waye JS, Wassif CA, Eng
B, Nowaczyk NJM, Porter FD. Carrier frequency of the RSH/Smith-Lemli-Opitz
IVS8-1GgC mutation in African Americans. Am J
Med Genet 2003;120A:139-141. Mouse models of SLOS Correa-Cerro,
Wassif, Porter; in collaboration with Kelley, Loh, Lu, Rivera Using gene targeting in murine embryonic stem cells,
we have produced three SLOS mouse models ( 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. A comparison of
mutant with control neurons showed no significant difference in the response
to GABA. However, in contrast, the glutamate response of mutant neurons was
significantly decreased compared with that 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 the 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, degranulation of mast cells is undergoing
characterization. A collaboration with Peng Loh’s laboratory focuses on
understanding abnormalities of vesicle function in pancreatic tissue. The Dhcr7 -/- pups die during the first
day of life; thus, we are not able to study postnatal brain development,
myelination, and behavior or to test therapeutic interventions. For this
reason, the laboratory has developed two missense alleles (Dhcr7T93M
and Dhcr7L99P) and a conditional Dhcr7 mutant allele
(Dhcr7 loxPΔ3-5loxP).
The T93M mutation is the second most common mutation found in human 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). Characterization of these
hypomorphic SLOS mice is in progress and includes phenotypic, histological,
biochemical, and behavioral analysis. We are using 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. 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 Cozma,
Scalco, Wassif, Porter Lathosterol 5-desaturase catalyzes the
conversion of lathosterol to 7-dehydrocholesterol and is the enzymatic step immediately
preceding the defect in SLOS. Thus, to further our understanding of 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 and exhibit micrognathia and cleft palates as well as limb
patterning defects. Many of the malformations in the mutant mice resemble
those found in SLOS and are consistent with impaired hedgehog signaling
during development. Biochemically, the pups 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 so as to identify a corresponding human
malformation syndrome; the human malformation syndrome had not been
previously described. We have now identified an infant human patient with
lathosterolosis. Biochemically, fibroblasts from the patient show decreased
cholesterol and increased lathosterol levels. Mutation analysis showed that
the infant is homozygous for a single AgC
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. A unique feature of
lathosterolosis is the clinical finding of mucolipidosis in the affected
infant. The clinical presentation is not reported in SLOS and may separate
the two disorders clinically. The 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 3-beta-hydroxysterol delta(24)-reductase gene (DHCR24). DHCR24
catalyzes the reduction of desmosterol to cholesterol. We disrupted the mouse
Dhcr24 by using targeted homologous recombination in embryonic stem
cells. Surprisingly, although most Dhcr24 mutant mice die at birth,
the pups are phenotypically normal. Further evaluation of the new mouse model
is in progress. 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 result of inefficient
definitive erythropoiesis. Lhx2 also functions to pattern the dorsal
telencephalon. Lhx9 has an overlapping but distinct expression pattern
compared with Lhx2. Lhx9 mutant mice are agonadal; thus, Lhx9
is essential for gonad development. 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 Richard Kelley, MD, PhD, The
Norman Javitt, MD, PhD, Peng Loh, PhD, Laboratory
of Developmental Neurobiology, NICHD, Bai Lu, PhD, Laboratory of
Cellular and Synaptic Neurophysiology, NICHD, Juan Rivera, PhD, Molecular
Immunology and Inflammation Branch, NIAMS, Cedric Shackleton, PhD, Children’s
Hospital Oakland Research Institute, Heiner Westphal, MD, Laboratory
of Mammalian Genes and Development, NICHD, Yingzi Yang, PhD, Genetic
Disease Research Branch, NHGRI, For
further information, contact fdporter@helix.nih.gov |
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