Establishing the precise evolutionary history of a gene improves prediction of disease-causing missense mutations

PURPOSE: Predicting the phenotypic effects of mutations has become an important application in clinical genetic diagnostics. Computational tools evaluate the behavior of the variant over evolutionary time and assume that variations seen during the course of evolution are probably benign in humans. However, current tools do not take into account orthologous/paralogous relationships. Paralogs have dramatically different roles in Mendelian diseases. For example, whereas inactivating mutations in the NPC1 gene cause the neurodegenerative disorder Niemann-Pick C, inactivating mutations in its paralog NPC1L1 are not disease-causing and, moreover, are implicated in protection from coronary heart disease. METHODS: We identified major events in NPC1 evolution and revealed and compared orthologs and paralogs of the human NPC1 gene through phylogenetic and protein sequence analyses. We predicted whether an amino acid substitution affects protein function by reducing the organism’s fitness. RESULTS: Removing the paralogs and distant homologs improved the overall performance of categorizing disease-causing and benign amino acid substitutions. CONCLUSION: The results show that a thorough evolutionary analysis followed by identification of orthologs improves the accuracy in predicting disease-causing missense mutations. We anticipate that this approach will be used as a reference in the interpretation of variants in other genetic diseases as well. Genet Med 18 10, 1029–1036

Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

We describe the rudolph mouse, a mutant with striking defects in both central nervous system and skeletal development. Rudolph is an allele of the cholesterol biosynthetic enzyme, hydroxysteroid (17-beta) dehydrogenase 7, which is an intriguing finding given the recent implication of oxysterols in mediating intracellular Hedgehog (Hh) signaling. We see an abnormal sterol profile and decreased Hh target gene induction in the rudolph mutant, both in vivo and in vitro. Reduced Hh signaling has been proposed to contribute to the phenotypes of congenital diseases of cholesterol metabolism. Recent in vitro and pharmacological data also indicate a requirement for intracellular cholesterol synthesis for proper regulation of Hh activity via Smoothened. The data presented here are the first in vivo genetic evidence supporting both of these hypotheses, revealing a role for embryonic cholesterol metabolism in both CNS development and normal Hh signaling

Increasing cholesterol synthesis in 7-dehydrosterol reductase (DHCR7) deficient mouse models through gene transfer

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis: the conversion of 7-dehydrocholesterol (7DHC) to cholesterol (C), catalyzed by 7-dehydrocholesterol reductase (DHCR7). This disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. There are few proven treatment options. That most commonly used is a high cholesterol diet that seems to enhance the quality of life and improve behavioral characteristics of patients, although these positive effects are controversial. The goal of our study was to investigate the possibility of restoring DHCR7 activity by gene transfer. We constructed an adeno-associated virus (AAV) vector containing the DHCR7 gene. After we infused this vector into affected mice, the introduced DHCR7 gene could be identified in liver, mRNA was expressed and a functional enzyme was produced. Evidence of functionality came from the ability to partially normalize the serum ratio of 7DHC/C in treated animals, apparently by increasing cholesterol production with concomitant decrease in 7DHC precursor. By five weeks after treatment the mean ratio (for 7 animals) had fallen to 0.05 while the ratio for untreated littermate controls had risen to 0.14. This provides proof of principle that gene transfer can ameliorate the genetic defect causing SLOS and provides a new experimental tool for studying the pathogenesis of this disease. If effective in humans, it might also offer a possible alternative to exogenous cholesterol therapy. However, it would not offer a complete cure for the disorder as many of the negative implications of defective synthesis are already established during prenatal development

HEM dysplasia and ichthyosis are likely laminopathies and not due to 3beta-hydroxysterol Delta14-reductase deficiency.

Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosis in mice. LBR is a bifunctional protein with both a lamin B binding and a sterol D14-reductase domain. It previously has been proposed that LBR is the primary sterol D14-reductase and that HEM dysplasia and ichthyosis are inborn errors of cholesterol synthesis. However, DHCR14 also encodes a sterol D14-reductase and could provide enzymatic redundancy with respect to cholesterol synthesis. To test the hypothesis that LBR and DHCR14 both function as sterol D14-reductases, we obtained ichthyosis mice (Lbr2/2) and disrupted Dhcr14. Heterozygous Lbr and Dhcr14 mice were intercrossed to test for a digenic phenotype. Lbr2/2, Dhcr14D4-7/D4-7 and Lbr1/2:Dhcr14D4-7/D4-7 mutant mice have distinct physical and biochemical phenotypes. Dhcr14D4-7/D4-7 mice are essentially normal, whereas Lbr1/2:Dhcr14D4-7/D4-7 mice are growth retarded and neurologically abnormal. Neither of these mutants resembles the ichthyosis mouse and biochemically, no sterol abnormalities were detected in either liver or kidney tissue. In contrast, relatively small transient elevations of D14-sterols were observed in Lbr2/2 and Dhcr14D4-7/D4-7 brain tissue, and marked elevations were seen in Lbr1/2:Dhcr14D4-7/D4-7 brain. Pathological evaluation demonstrated vacuolation and swelling of the myelin sheaths in the spinal cord of Lbr1/2:Dhcr14D4-7/D4-7 mice consistent with a demyelinating process.This was not observed in either Lbr2/2 or Dhcr14 D4-7/D4-7 mice. Our data support the conclusions that LBR and DHCR14 provide substantial enzymatic redundancy with respect to cholesterol synthesis and that HEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis

FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2 and its active phosphorylated form (FTY720-P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and -2 in brain and liver and decreased cholesterol in an SphK2-dependent manner. FTY720 greatly increased expression of NPC1 and -2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720-P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease

FTY720/fingolimod increases NPC1 and NPC2 expression and reduces cholesterol and sphingolipid accumulation in Niemann-Pick type C mutant fibroblasts

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 or NPC2 with decreased functions leading to lysosomal accumulation of cholesterol and sphingolipids. FTY720/fingolimod, used for treatment of multiple sclerosis, is phosphorylated by nuclear sphingosine kinase 2 and its active phosphorylated form (FTY720-P) is an inhibitor of class I histone deacetylases. In this study, administration of clinically relevant doses of FTY720 to mice increased expression of NPC1 and -2 in brain and liver and decreased cholesterol in an SphK2-dependent manner. FTY720 greatly increased expression of NPC1 and -2 in human NPC1 mutant fibroblasts that correlated with formation of FTY720-P and significantly reduced the accumulation of cholesterol and glycosphingolipids. In agreement with this finding, FTY720 pretreatment of human NPC1 mutant fibroblasts restored transport of the cholera toxin B subunit, which binds ganglioside GM1, to the Golgi apparatus. Together, these findings suggest that FTY720 administration can ameliorate cholesterol and sphingolipid storage and trafficking defects in NPC1 mutant fibroblasts. Because neurodegeneration is the main clinical feature of NPC disease, and FTY720 accumulates in the CNS and has several advantages over available histone deacetylase inhibitors now in clinical trials, our work provides a potential opportunity for treatment of this incurable disease

A placebo-controlled trial of simvastatin therapy in Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/cognitive impairment syndrome characterized by the accumulation of 7-dehydrocholesterol (7DHC), a precursor sterol of cholesterol. Simvastatin, an HMG-CoA reductase inhibitor that crosses the blood-brain-barrier, has been proposed for treatment of SLOS based on in vitro and in vivo studies suggesting that simvastatin increases expression of hypomorphic DHCR7 alleles. METHODS: Safety and efficacy of simvastatin therapy in 23 mild to typical SLOS patients was evaluated in a randomized, double-blind, placebo-controlled trial. The cross-over trial consisted of two 12 month treatment phases separated by a 2 month wash-out period. RESULTS: No safety issues were identified in this study. Plasma dehydrocholesterol levels decreased significantly 8.9 ± 8.4% on placebo to 6.1 ± 5.5% on simvastatin (p<0.005) and we observed a trend toward decreased cerebral spinal fluid dehydrocholesterol levels. A significant improvement (p=0.017, paired t-test) was observed in the Aberrant Behavior Checklist-C Irritability when subjects were on simvastatin. CONCLUSIONS: This paper reports the first randomized, placebo-controlled trial designed to test the safety and efficacy of simvastatin therapy in SLOS. Simvastatin appears to be relatively safe in SLOS patients, improves the serum dehydrocholesterol/total sterol ratio, and significantly improves irritability symptoms in mild to classical SLOS patients