Lisa E. Kratz

Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism

RSH/Smith-Lemli-Opitz (RSH/SLO) syndrome is an autosomal recessive malformation syndrome recently shown to be associated with a severe deficiency of cholesterol biosynthesis and markedly elevated plasma and tissue levels of 7-dehydrocholesterol (7-DHC), the immediate precursor of cholesterol in the Kandutsch-Russell biosynthetic pathway. Because these biochemical abnormalities permit a reassessment of RSH/SLO on biochemical criteria rather than less specific physical criteria, we review here the clinical and biochemical characteristics of our first 80 patients with abnormally increased levels of 7-DHC. The study population included 68 index patients and 12 additional relatives identified by quantification of 7-DHC and cholesterol in plasma, amniotic fluid, or cultured fibroblasts, lymphoblasts, or amniocytes. As demonstrated in other clinical syndromes when redefined biochemically, we have found a wider range of clinical expression of RSH/SLO than previously recognized. These newly recognized atypical RSH/SLO patients included several with no malformations other than syndactyly of the toes and, at the other extreme, patients with frank holoprosencephaly or multiple visceral anomalies who died in utero. Syndactyly of toes 2 and 3 was the most common malformation, occurring in all but one of 80 patients. The best biochemical predictor of clinical severity was the plasma cholesterol level, which decreased with increasing clinical severity. However, at least 10% of patients, including one newborn infant, had normal cholesterol levels at the time of diagnosis and would have been missed without specific quantification of 7-DHC. Not unexpectedly, several patients carrying a clinical diagnosis of RSH/SLO were found to have normal levels of all plasma sterols and apparently normal cholesterol biosynthesis in cultured cells. A comparison of the frequency of anomalies in our biochemically identified patients with similar data from previously reported clinical series suggests that up to 25% of reports of RSH/SLO in the literature may describe genetic conditions other than RSH/SLO with 7-DHC-emia.

Mutations in the gene encoding 3β-hydroxysteroid-Δ8,Δ7-isomerase cause X-linked dominant Conradi-Hunermann syndrome

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported,. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 ( ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3β-hydroxysteroid-Δ8,Δ 7-isomerase (sterol-Δ8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-Δ8-isomerase ( EBP) has been identified and mapped to Xp11.22–p11.23 (Refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-Δ8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-Δ8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hunermann syndrome

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported,. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 ( ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3β-hydroxysteroid-Δ8,Δ 7-isomerase (sterol-Δ8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-Δ8-isomerase ( EBP) has been identified and mapped to Xp11.22–p11.23 (Refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-Δ8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-Δ8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay

Defects in cholesterol synthesis result in a wide variety of symptoms, from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in individuals with Smith-Lemli-Opitz syndrome. We report here the identification of mutations in sterol-C4-methyl oxidase–like gene (SC4MOL) as the cause of an autosomal recessive syndrome in a human patient with psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl oxidase (SMO), which catalyzes demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-Methylsterols are meiosis-activating sterols (MASs). They exist at high concentrations in the testis and ovary and play roles in meiosis activation. In this study, we found that an accumulation of MASs in the patient led to cell overproliferation in both skin and blood. SMO deficiency also substantially altered immunocyte phenotype and in vitro function. MASs serve as ligands for liver X receptors α and β(LXRα and LXRβ), which are important in regulating not only lipid transport in the epidermis, but also innate and adaptive immunity. Deficiency of SMO represents a biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined.

Hepatitis C virus selectively perturbs the distal cholesterol synthesis pathway in a genotype-specific manner

Hepatitis C virus (HCV) subverts host cholesterol metabolism for key processes in its lifecycle. How this interference results in the frequently observed, genotype‐dependent clinical sequelae of hypocholesterolemia, hepatic steatosis, and insulin resistance (IR) remains incompletely understood. Hypocholesterolemia typically resolves after sustained viral response (SVR), implicating viral interference in host lipid metabolism. Using a targeted cholesterol metabolomic platform we evaluated paired HCV genotype 2 (G2) and G3 patient sera for changes in in vivo HCV sterol pathway metabolites. We compared HCV genotypic differences in baseline metabolites and following antiviral treatment to assess whether sterol perturbation resolved after HCV eradication. We linked these metabolites to IR and urine oxidative stress markers. In paired sera from HCV G2 (n = 13) and G3 (n = 20) patients, baseline sterol levels were lower in G3 than G2 for distal metabolites (7‐dehyrocholesterol (7DHC) 0.017 versus 0.023 mg/dL; Padj = 0.0524, cholesterol 140.9 versus 178.7 mg/dL; Padj = 0.0242) but not the proximal metabolite lanosterol. In HCV G3, SVR resulted in increased levels of distal metabolites (cholesterol [Δ55.2 mg/dL; Padj = 0.0015], 7DHC [Δ0.0075 mg/dL; Padj = 0.0026], lathosterol [Δ0.0430 mg/dL Padj = 0.0405]). In contrast, lanosterol was unchanged after SVR (P = 0.9515). Conclusion: HCV G3, but not G2, selectively interferes with the late cholesterol synthesis pathway, evidenced by lower distal sterol metabolites and preserved lanosterol levels. This distal interference resolves with SVR. Normal lanosterol levels provide a signal for the continued proteolysis of 3‐hydroxyl‐3‐methylglutaryl coenzyme A reductase, which may undermine other host responses to increase cholesterol synthesis. These data may provide a hypothesis to explain why hypocholesterolemia persists in chronic HCV infection, particularly in HCV G3, and is not overcome by host cholesterol compensatory mechanisms. (HEPATOLOGY 2012;56:49–56)

Clinical Variability in Rett Syndrome

The clinical variability of Rett syndrome, associated with mutations in the MECP2 gene, varies from classically symptomatic female patients to asymptomatic female patients, and male patients who have none of the diagnostic features considered pathognomonic of this disease. Multiple factors contribute to this variability. In our studies, mutations closer to the amino-terminus, prior to amino acid 255, led to severe clinical manifestations, such as inability to walk, severe dysphagia, and urinary organic acid abnormalities, compared with mutations toward the carboxyl-terminus. However, we found no correlation between severity and mutation type (missense versus nonsense). Despite the importance of mutation location to clinical severity, the widely varying severity within the same mutation suggests that in females, X-chromosome inactivation or other epigenetic phenomena also have roles in determining severity. We propose that stages 1 and 2 of the disease are a consequence of failed, time-linked, postnatal expression of MeCP2 in cerebellar neurons. This, in association with glutamate N-methyl-D-aspartate receptor—mediated neuroexcitotoxic injury to the differentiating neurons, results in the transient age-specific autistic-like behavior, motor, and cognitive dysfunction associated with these stages. (J Child Neurol 2003; 18:662-668).

3-methylglutaconic acidemia in Smith-Lemli-Opitz syndrome.

ABSTRACT: The branched-chain organic acid, 3-methylglutaconic acid, is an intermediate (as the CoA thioester) in the leucine degradative pathway as well as the mevalonate shunt, a pathway that links isoprenoid metabolism with mitochondrial acetyl-CoA metabolism. Because the majority of patients with abnormal 3-methylglutaconic aciduria or acidemia appear to have normal leucine metabolism, we have speculated that some patients with 3-methylglutaconic aciduria may have defects of polyisoprenoid or sterol biosynthesis leading to overflow of isoprenoid precursors to 3-methylglutaconate via the mevalonate shunt. We therefore measured plasma levels of 3-methylglutaconic acid in patients with a known defect of sterol biosynthesis, Smith-Lemli-Opitz syndrome, and found that the patients with the lowest cholesterol levels had abnormally increased plasma levels of 3-methylglutaconic acid, similar in magnitude to those of patients with idiopathic 3-methylglutaconic aciduria. This finding suggests that some patients with unexplained 3-methylglutaconic aciduria may have defects of isoprenoid or sterol biosynthesis underlying their abnormal organic aciduria.

Midgestational maternal urine steroid markers of fetal Smith-Lemli- Opitz (SLO) syndrome (7-dehydrocholesterol 7-reductase deficiency)

Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome associated with 7-dehydrocholesterol (7DHC) 7-reductase deficiency. Although SLOS can be detected in an affected fetus before midpregnancy by measurement of 7DHC levels in amniotic fluid or chorionic villus cells, a noninvasive, more routine method is needed. Accordingly, this study was instigated to search for specific steroids in maternal urine in an affected pregnancy that reflect the 7-reductase deficiency of the fetus, ie, steroids retaining 7,8-unsaturation. Steroids were characterized by gas chromatography/mass spectrometry after urinary extraction, conjugate separation, and derivatization. Most steroids in maternal urine from a patient carrying a SLOS fetus were identified as progesterone metabolites, and these were entirely conventional, showing no evidence of additional unsaturation. Unsaturated homologues of the cortisol metabolites were also not detected. However, unsaturated homologues of pregnane-3,16,20-triols and pregnane-3,17,20-triol were found. Most likely, these are 7,8-unsaturated homologues, but 8,9-unsaturation is also possible because of the known activity of delta7-delta8-isomerase on 7DHC, which results in 8DHC being a prominent sterol in SLOS. Among these novel human steroids, the following were provisionally characterized: 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol, 5beta-pregn-7(or 8)-ene-3alpha,16alpha,20alpha-triol, and 5alpha-pregn-7(or 8)-ene-3,16alpha,20alpha-triol. Confirmation of the position of unsaturation will require steroid synthesis. These novel steroids are not present in normal pregnancy urine and, therefore, are valuable for prenatal diagnosis of SLOS. In addition, separate studies have shown that 5beta-pregn-7(or 8)-ene-3alpha,17alpha,20alpha-triol is present in urine of children and adults with SLOS, and so is a useful analyte for confirmation of the disorder throughout life.

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.

Desmosterolosis—phenotypic and molecular characterization of a third case and review of the literature

Desmosterolosis, a rare disorder of cholesterol biosynthesis, is caused by mutations in DHCR24, the gene encoding the enzyme 24‐dehydrocholesterol reductase (DHCR24). To date, desmosterolosis has been described in only two patients. Here we report on a third patient with desmosterolosis who presented after delivery with relative macrocephaly, mild arthrogryposis, and dysmorphic facial features. Brain MRI revealed hydrocephalus, thickening of the tectum and massa intermedia, mildly effaced gyral pattern, underopercularization, and a thin corpus callosum. The diagnosis of desmosterolosis was established by detection of significant elevation of plasma desmosterol levels and reduced enzyme activity of DHCR24 upon expression of the patients DHCR24 cDNA in yeast. The patient was found to be a compound heterozygote for c.281G>A (p.R94H) and c.1438G>A (p.E480K) mutations. Structural and evolutionary analyses showed that residue R94 resides at the flavin adenine dinucleotide (FAD) binding site and is strictly conserved throughout evolution, while residue E480 is less conserved, but the charge shift substitution is accompanied by drastic changes in the local protein environment of that residue. We compare the phenotype of our patient with previously reported cases.