Necroptosis in Niemann-Pick disease, type C1: a potential therapeutic target.

Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder due to mutation of the NPC1 gene. The NPC1 phenotype is characterized by progressive neuronal dysfunction, including cerebellar ataxia and dementia. There is histological evidence of neuroinflammation and progressive neuronal loss, with cerebellar Purkinje cells particularly vulnerable to loss of NPC1 function. Necroptosis was evaluated as a mechanism of neuronal loss. Receptor-interacting protein kinase 1 (RIP1) and RIP3 are key components of the necrosomal complex that regulates necroptotic cell death. We report increased expression of RIP1 and RIP3 in NPC1 fibroblasts, NPC1 iPS cell-derived neuronal precursors, and in cerebellar tissue from both NPC1 mice and patients. Our data suggest a positive correlation between NPC1 neurological disease severity and assembly of the necrosome complex. Furthermore, we demonstrate that pharmacological inhibition of RIP1 decreases cell death both in vitro and in vivo. Treatment of Npc1-mutant mice with necrostatin-1, an allosteric inhibitor of RIP1, significantly delayed cerebellar Purkinje cell loss, progression of neurological symptoms, and death. Collectively, our data identified necroptosis as a key component of the molecular network that contributes to neuronal loss in NPC1 and establish that inhibition of necroptosis is a potential therapeutic intervention.

Determination of the allelic frequency in Smith-Lemli-Opitz syndrome by analysis of massively parallel sequencing data sets.

Data from massively parallel sequencing or 'Next Generation Sequencing' of the human exome has reached a critical mass in both public and private databases, in that these collections now allow researchers to critically evaluate population genetics in a manner that was not feasible a decade ago. The ability to determine pathogenic allele frequencies by evaluation of the full coding sequences and not merely a single nucleotide polymorphism (SNP) or series of SNPs will lead to more accurate estimations of incidence. For demonstrative purposes, we analyzed the causative gene for the disorder Smith-Lemli-Opitz Syndrome (SLOS), the 7-dehydrocholesterol reductase (DHCR7) gene and determined both the carrier frequency for DHCR7 mutations, and predicted an expected incidence of the disorder. Estimations of the incidence of SLOS have ranged widely from 1:10,000 to 1:70,000 while the carrier frequency has been reported as high as 1 in 30. Using four exome data sets with a total of 17,836 chromosomes, we ascertained a carrier frequency of pathogenic DHRC7 mutations of 1.01%, and predict a SLOS disease incidence of 1/39,215 conceptions. This approach highlights yet another valuable aspect of the exome sequencing databases, to inform clinical and health policy decisions related to genetic counseling, prenatal testing and newborn screening.

Decreased cerebral spinal fluid neurotransmitter levels in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital anomaly syndrome with cognitive impairment and a distinct behavioral phenotype that includes autistic features. SLOS is caused by a defect in 3ß-hydroxysterol Δ(7)-reductase which leads to decreased cholesterol levels and elevated cholesterol precursors, specifically 7- and 8-dehydrocholesterol. However, the pathological processes contributing to the neurological abnormalities in SLOS have not been defined. In view of prior data suggesting defects in SLOS in vesicular release and given the association of altered serotonin metabolism with autism, we were interested in measuring neurotransmitter metabolite levels in SLOS to assess their potential to be used as biomarkers in therapeutic trials. We measured cerebral spinal fluid levels of serotonin and dopamine metabolites, 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) respectively, in 21 SLOS subjects. Results were correlated with the SLOS anatomical severity score, Aberrant Behavior Checklist scores and concurrent sterol biochemistry. Cerebral spinal fluid (CSF) levels of both 5HIAA and HVA were significantly reduced in SLOS subjects. In individual patients, the levels of both 5HIAA and HVA were reduced to a similar degree. CSF neurotransmitter metabolite levels did not correlate with either CSF sterols or behavioral measures. This is the first study demonstrating decreased levels of CSF neurotransmitter metabolites in SLOS. We propose that decreased levels of neurotransmitters in SLOS are caused by a sterol-related defect in synaptic vesicle formation and that CSF 5HIAA and HVA will be useful biomarkers in development of future therapeutic trials.

Nanostructure-initiator mass spectrometry (NIMS) imaging of brain cholesterol metabolites in Smith-Lemli-Opitz syndrome.

Cholesterol is an essential component of cellular membranes that is required for normal lipid organization and cell signaling. While the mechanisms associated with maintaining cholesterol homeostasis in the plasma and peripheral tissues have been well studied, the role and regulation of cholesterol biosynthesis in normal brain function and development have proven much more challenging to investigate. Smith-Lemli-Opitz syndrome (SLOS) is a disorder of cholesterol synthesis characterized by mutations of 7-dehydrocholesterol reductase (DHCR7) that impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol and lead to neurocognitive deficits, including cerebellar hypoplasia and austism behaviors. Here we have used a novel mass spectrometry-based imaging technique called cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for the in situ detection of intact cholesterol molecules from biological tissues. We provide the first images of brain sterol localization in a mouse model for SLOS (Dhcr7(-/-)). In SLOS mice, there is a striking localization of both 7DHC and residual cholesterol in the abnormally developing cerebellum and brainstem. In contrast, the distribution of cholesterol in 1-day old healthy pups was diffuse throughout the cerebrum and comparable to that of adult mice. This study represents the first application of NIMS to localize perturbations in metabolism within pathological tissues and demonstrates that abnormal cholesterol biosynthesis may be particularly important for the development of these brain regions.

Genetic disorders of cholesterol biosynthesis in mice and humans.

Over the past few years, the number of identified inborn errors of cholesterol biosynthesis has increased significantly. The first inborn error of cholesterol biosynthesis to be characterized, in the mid 1980s, was mevalonic aciduria. In 1993, Irons et al. ( 1 ) (M. Irons, E. R. Elias, G. Salen, G. S. Tint, and A. K. Batta, Lancet 341:1414, 1993) reported that Smith-Lemli-Opitz syndrome, a classic autosomal recessive malformation syndrome, was due to an inborn error of cholesterol biosynthesis. This was the first inborn error of postsqualene cholesterol biosynthesis to be identified, and subsequently additional inborn errors of postsqualene cholesterol biosynthesis have been characterized to various extent. To date, eight inborn errors of cholesterol metabolism have been described in human patients or in mutant mice. The enzymatic steps impaired in these inborn errors of metabolism include mevolonate kinase (mevalonic aciduria as well as hyperimmunoglobulinemia D and periodic fever syndrome), squalene synthase (Ss-/- mouse), 3beta-hydroxysteroid Delta14-reductase (hydrops-ectopic calcification-moth-eaten skeletal dysplasia), 3beta-hydroxysteroid dehydrogenase (CHILD syndrome, bare patches mouse, and striated mouse), 3beta-hydroxysteroid Delta8,Delta7-isomerase (X-linked dominant chondrodysplasia punctata type 2, CHILD syndrome, and tattered mouse), 3beta-hydroxysteroid Delta24-reductase (desmosterolosis) and 3beta-hydroxysteroid Delta7-reductase (RSH/Smith-Lemli-Opitz syndrome and Dhcr7-/- mouse). Identification of the genetic and biochemical defects which give rise to these syndromes has provided the first step in understanding the pathophysiological processes which underlie these malformation syndromes.

Biochemical, phenotypic and neurophysiological characterization of a genetic mouse model of RSH/Smith--Lemli--Opitz syndrome.

The RSH/Smith--Lemli--Opitz syndrome (RSH/SLOS) is a human autosomal recessive syndrome characterized by multiple malformations, a distinct behavioral phenotype with autistic features and mental retardation. RSH/SLOS is due to an inborn error of cholesterol biosynthesis caused by mutation of the 3 beta-hydroxysterol Delta(7)-reductase gene. To further our understanding of the developmental and neurological processes that underlie the pathophysiology of this disorder, we have developed a mouse model of RSH/SLOS by disruption of the 3 beta-hydroxysterol Delta(7)-reductase gene. Here we provide the biochemical, phenotypic and neurophysiological characterization of this genetic mouse model. As in human patients, the RSH/SLOS mouse has a marked reduction of serum and tissue cholesterol levels and a marked increase of serum and tissue 7-dehydrocholesterol levels. Phenotypic similarities between this mouse model and the human syndrome include intra-uterine growth retardation, variable craniofacial anomalies including cleft palate, poor feeding with an uncoordinated suck, hypotonia and decreased movement. Neurophysiological studies showed that although the response of frontal cortex neurons to the neurotransmitter gamma-amino-n-butyric acid was normal, the response of these same neurons to glutamate was significantly impaired. This finding provides insight into potential mechanisms underlying the neurological dysfunction seen in this human mental retardation syndrome and suggests that this mouse model will allow the testing of potential therapeutic interventions.

Mutation analysis and description of sixteen RSH/Smith-Lemli-Opitz syndrome patients: polymerase chain reaction-based assays to simplify genotyping.

We report the clinical and molecular data of 16 patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) with varying phenotypic severity, for which we have identified mutations in both alleles. RSH/SLOS is an autosomal recessive malformation syndrome caused by mutations in the gene encoding the sterol Delta(7)-reductase. This protein catalyzes the reduction of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis via the Kandutsch-Russell pathway. In addition to previously reported mutations (T93M, L109P, G147D, W151X, T154M, R242C, A247V, T289I, IVS8-1G-->C, Y408H, and E448K), we have identified six previously undescribed mutations (321G-->C, W177R, R242H, Y318N, L341P, and C444Y). We also report rapid polymerase chain reaction (PCR)-based assays developed to detect four of the recurring mutations (T93M, W151X, V326L, and R404C) and six other RSH/SLOS mutations (321G-->C, L109P, T154M, T289I, Y318N, and L341P). The purpose of this article is to correlate detailed clinical information with molecular data in order to improve our understanding of the genotype-phenotype correlation of RSH/SLOS and to report the development of PCR-based assays that will allow more rapid mutation analysis.

The LIM homeobox gene Lhx9 is essential for mouse gonad formation.

During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.

A simple PCR-based assay allows detection of a common mutation, IVS8-1G-->C, in DHCR7 in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive multiple malformation disorder. A deficiency of the enzyme 7-dehydrocholesterol delta 7-reductase (DHCR7) is the primary abnormality in SLOS. The gene encoding DHCR7 has been cloned, and we have identified a mutation affecting the splice acceptor site 5' of exon 9 that occurs frequently in affected individuals. We developed a novel PCR-based assay to detect this common mutation in DHCR7. Using this assay, heterozygosity was detected for this mutation in 18 of 26 and homozygosity in 1 of 26 unrelated affected individuals. The high frequency of this mutation is suggestive of either a founder effect in our group of patients or a mutational hotspot. The simplicity and reliability of this assay will allow it to be used as a clinical test to aid in diagnosis of atypical cases, in carrier testing, in prediction of prognosis based on genotype, and in prenatal molecular genetic diagnostic testing.

Mutations in the human sterol delta7-reductase gene at 11q12-13 cause Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS; also known as "RSH syndrome" [MIM 270400]) is an autosomal recessive multiple malformation syndrome due to a defect in cholesterol biosynthesis. Children with SLOS have elevated serum 7-dehydrocholesterol (7-DHC) levels and typically have low serum cholesterol levels. On the basis of this biochemical abnormality, it has been proposed that mutations in the human sterol Delta7-reductase (7-DHC reductase; E.C.1.3.1.21) gene cause SLOS. However, one could also propose a defect in a gene that encodes a protein necessary for either the expression or normal function of sterol Delta7-reductase. We cloned cDNA encoding a human sterol Delta7-reductase (DHCR7) on the basis of its homology with the sterol Delta7-reductase from Arabidopsis thaliana, and we confirmed the enzymatic function of the human gene product by expression in SLOS fibroblasts. SLOS fibroblasts transfected with human sterol Delta7-reductase cDNA showed a significant reduction in 7-DHC levels, compared with those in SLOS fibroblasts transfected with the vector alone. Using radiation-hybrid mapping, we show that the DHCR7 gene is encoded at chromosome 11q12-13. To establish that defects in this gene cause SLOS, we sequenced cDNA clones from SLOS patients. In three unrelated patients we have identified four different mutant alleles. Our results demonstrate both that the cDNA that we have identified encodes the human sterol Delta7-reductase and that mutations in DHCR7 are responsible for at least some cases of SLOS.