Louise S. Merkens

The Near Universal Presence of Autism Spectrum Disorders in Children With Smith-Lemli-Opitz Syndrome

Smith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive condition caused by a defect in cholesterol synthesis. Affected children often have malformations and mental retardation. Autistic behaviors also are evident. The purpose of the present study was to determine the prevalence of autism spectrum disorders (ASDs) in children with SLOS. Fourteen children, 3–16 years old, were evaluated using three different methods to document autistic symptoms: (a) parent interview, (b) direct observation, and (c) a behavior checklist. Blood sterols were also measured at regular intervals. Each subject was determined to have Autistic Disorder, Pervasive Developmental Disorder, not otherwise specified (PDD NOS), or no diagnosis on the autism spectrum, based on DSM‐IV criteria. Correlations among variables were calculated, and blood sterol levels were compared between diagnostic groups. Approximately three‐fourths of the children with SLOS (71–86% depending on the evaluation method) had an ASD, about 50% diagnosed with Autistic Disorder and the rest with PDD NOS. The childrens baseline cholesterol, 7‐dehydrocholesterol (7‐DHC), and 8‐dehydrocholesterol (8‐DHC) levels, and cholesterol levels following supplementation did not correlate with the presence or severity of autistic symptoms. These results suggest that most children with SLOS have some variant of autism. SLOS appears to have the most consistent relationship with autism of any single gene disorder. Therefore, a link between cholesterol metabolism and autism is suggested. With further study, these findings, together with knowledge of the genetic and biochemical defects in SLOS, will likely provide valuable insights into the causes of autism in general.

Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital malformation and intellectual disability syndrome, with clinical characteristics that encompass a wide spectrum and great variability. Elucidation of the biochemical and genetic basis for SLOS, specifically understanding SLOS as a cholesterol deficiency syndrome caused by mutation in DHCR7, opened up enormous possibilities for therapeutic intervention. When cholesterol was discovered to be the activator of sonic hedgehog, cholesterol deficiency with inactivation of this developmental patterning gene was thought to be the cause of SLOS malformations, yet this explanation is overly simplistic. Despite these important research breakthroughs, there is no proven treatment for SLOS. Better animal models are needed to allow potential treatment testing and the study of disease pathophysiology, which is incompletely understood. Creation of human cellular models, especially models of brain cells, would be useful, and in vivo human studies are also essential. Biomarker development will be crucial in facilitating clinical trials in this rare condition, because the clinical phenotype can change over many years. Additional research in these and other areas is critical if we are to make headway towards ameliorating the effects of this devastating condition.

A human gene coding for a membrane-associated nucleic acid-binding protein.

Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of ∼130 kDa (GenBank accession numbers AF255303 andAF255304). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K D ∼4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass ∼150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.

Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol

Concentrations of circulating 24S-hydroxycholesterol (24SOHChol) are of interest as a practical measure of cholesterol efflux from the human brain. The current method of choice for 24SOHChol quantification is with gas chromatography-mass spectrometry (GC-MS). Liquid chromatography-mass spectrometry (LC-MS) methods to detect 24SOHChol have been described, but they lack rigorous high-performance liquid chromatography (HPLC) separation of a closely eluting isomeric oxysterol, 25-hydroxycholesterol. This is important because 25-hydroxycholesterol can be present in significant amounts and tandem mass spectrometry (MS/MS) cannot completely differentiate 24SOHChol. We describe an LC-MS method with rapid chromatographic separation of the oxysterols to permit accurate determination of plasma 24SOHChol. The availability of an LC-MS method offers advantages such as simplified sample work-up and analysis.

Effects of dietary cholesterol and simvastatin on cholesterol synthesis in Smith-Lemli-Opitz syndrome.

Deficient cholesterol and/or excessive 7-dehydrocholesterol (7-DHC) may be responsible for the pathology of Smith-Lemli-Opitz syndrome (SLOS). Both high-cholesterol diets given to ameliorate cholesterol deficiency while decreasing 7-DHC and cholesterol-enriched diets plus simvastatin to further decrease sterol synthesis have been used as potential therapies. However, the effect of dietary cholesterol and simvastatin on cholesterol synthesis in SLOS has not been reported. Twelve subjects with SLOS enrolled in the study: Nine had received a high cholesterol diet (HI) for 3 y and three were studied after 4 wk on a low cholesterol diet (LO). Cholesterol fractional synthesis rate (FSR) was measured after oral administration of deuterium oxide, using gas chromatography isotope ratio mass spectrometry. FSR was lower in HI compared with LO (HI: 1.46 ± 0.62%/d; LO: 4.77 ± 0.95%/d; p < 0.001). Three HI subjects were retested after 0.8 y taking simvastatin (HI + ST). Simvastatin tended to reduce FSR and significantly decreased (p < 0.01) plasma 7-DHC compared with cholesterol supplementation alone. The study demonstrates the utility of the deuterium incorporation method to understand the effect of therapeutic interventions in SLOS. The data suggest that dietary cholesterol supplementation reduces cholesterol synthesis in SLOS and further support the rationale for the combined treatment of SLOS with a cholesterol-enriched diet and simvastatin.

Hypomorphic Temperature-Sensitive Alleles of NSDHL Cause CK Syndrome

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development.

Profiling sterols in cerebrotendinous xanthomatosis: Utility of Girard derivatization and high resolution exact mass LC―ESI-MSn analysis

In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girards P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girards reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 μl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) ([M](+)→[M-79](+)→) spectra that provided additional structurally informative fragment ions.

ESI-MS/MS quantification of 7α-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis

BACKGROUND The genetic disorder cerebrotendinous xanthomatosis (CTX) frequently remains undiagnosed for many years. Left untreated CTX is associated with the development of cataracts, xanthomas and severe neurological dysfunction. The method routinely used to screen for CTX is GC-based measurement of elevated 5alpha-cholestanol from hydrolyzed plasma. A plasma test for CTX utilizing ESI-MS/MS methodology would be beneficial. METHODS Development of rapid, simple LC-ESI-MS/MS methodology to test plasma for CTX is described. Two hour Girard derivatization allowed for 7alpha-hydroxy-4-cholesten-3-one quantification by isotope dilution LC-ESI-MS/MS within 12 min from un-hydrolyzed affected patient plasma (5 microl). RESULTS Adequate sensitivity and reproducibility were achieved for quantification of 7alpha-hydroxy-4-cholesten-3-one, which demonstrated improved utility as a diagnostic marker of disease and to monitor treatment compared to 5alpha-cholestanol. The mean plasma concentration of 7alpha-hydroxy-4-cholesten-3-one in untreated CTX-affected patients (n=6) was 107-fold that in unaffected subjects (n=9), with the lowest concentration in affected patients >10-fold the highest concentration in unaffected subjects. CONCLUSION Quantification of the bile acid precursor 7alpha-hydroxy-4-cholesten-3-one with LC-ESI-MS/MS is a novel approach to improved diagnostic testing of plasma for CTX, amenable to high-throughput analysis and automated sample handling. Development of ESI-MS/MS methodology should make CTX testing more widely available and facilitate easier diagnosis of CTX.

Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3β-hydroxysterol-Δ7-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d7-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.

Apolipoprotein E-low density lipoprotein receptor interaction affects spatial memory retention and brain ApoE levels in an isoform-dependent manner

Human apolipoprotein E (apoE) exists in three isoforms: apoE2, apoE3 and apoE4. APOE ε4 is a major genetic risk factor for cardiovascular disease (CVD) and Alzheimers disease (AD). ApoE mediates cholesterol metabolism by binding various receptors. The low-density lipoprotein receptor (LDLR) has a high affinity for apoE, and is the only member of its receptor family to demonstrate an apoE isoform specific binding affinity (E4>E3>>E2). Evidence suggests that a functional interaction between apoE and LDLR influences the risk of CVD and AD. We hypothesize that the differential cognitive effects of the apoE isoforms are a direct result of their varying interactions with LDLR. To test this hypothesis, we have employed transgenic mice that express human apoE2, apoE3, or apoE4, and either human LDLR (hLDLR) or no LDLR (LDLR(-/-)). Our results show that plasma and brain apoE levels, cortical cholesterol, and spatial memory are all regulated by isoform-dependent interactions between apoE and LDLR. Conversely, both anxiety-like behavior and cued associative memory are strongly influenced by APOE genotype, but these processes appear to occur via an LDLR-independent mechanism. Both the lack of LDLR and the interaction between E4 and the LDLR were associated with significant impairments in the retention of long term spatial memory. Finally, levels of hippocampal apoE correlate with long term spatial memory retention in mice with human LDLR. In summary, we demonstrate that the apoE-LDLR interaction affects regional brain apoE levels, brain cholesterol, and cognitive function in an apoE isoform-dependent manner.