Chenodeoxycholic acid (CDCA) treatment during pregnancy in women with cerebrotendinous xanthomatosis (CTX): Lessons learned from 19 pregnancies.

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a rare, autosomal recessive bile acid synthesis disorder. Biallelic pathogenic variants in CYP27A1, encoding for sterol 27-hydroxylase, impair cholic acid (CA) and chenodeoxycholic acid (CDCA) synthesis and lead to accumulation of cholestanol and C27 bile alcohols. Treatment with CDCA decreases the accumulation of these harmful metabolites and slows disease progression. Currently, CDCA is contraindicated for use during pregnancy based on animal studies that showed that high-dose CDCA may cause fetal harm when administered to pregnant animals. Data regarding the safety of CDCA treatment in humans are lacking. METHODS: We present a case series of 19 pregnancies in 9 women with CTX who either received CDCA treatment throughout pregnancy or did not. RESULTS: In 11 pregnancies where mothers continued CDCA treatment, no complications were reported, and newborns were born at or near full term, with normal birth weight and Apgar scores. In 8 pregnancies where mothers did not receive CDCA, 2 newborns experienced elevated bilirubin soon after birth. One woman who stopped treatment during her pregnancy deteriorated neurologically while off treatment. CONCLUSION: The data we present support the benefit of continued CDCA treatment in pregnant women with CTX for both the affected women and their offspring.

Pregnancy and weaning regulate human maternal liver size and function.

During pregnancy, the rodent liver undergoes hepatocyte proliferation and increases in size, followed by weaning-induced involution via hepatocyte cell death and stromal remodeling, creating a prometastatic niche. These data suggest a mechanism for increased liver metastasis in breast cancer patients with recent childbirth. It is unknown whether the human liver changes in size and function during pregnancy and weaning. In this study, abdominal imaging was obtained in healthy women at early and late pregnancy and postwean. During pregnancy time points, glucose production and utilization and circulating bile acids were measured. Independently of weight gain, most women's livers increased in size with pregnancy, then returned to baseline postwean. Putative roles for bile acids in liver growth and regression were observed. Together, the data support the hypothesis that the human liver is regulated by reproductive state with growth during pregnancy and volume loss postwean. These findings have implications for sex-specific liver diseases and for breast cancer outcomes.

Synthesis of Deuterated Endochin-Like Quinolones.

Malaria continues to be a serious and debilitating disease. The emergence and spread of high-level resistance to multiple antimalarial drugs by Plasmodium falciparum has brought about an urgent need for new treatments that will be active against multidrug resistant malaria infections. One such treatment, ELQ-331 (MMV-167), an alkoxy carbonate prodrug of 4(1H)-quinolone ELQ-300, is currently in preclinical development with the Medicines for Malaria Venture. Clinical development of ELQ-331 or similar compounds will require the availability of isotopically labeled analogs. Unfortunately, a suitable method for the deuteration of these important compounds was not found in the literature. Here, we describe a facile and scalable method for the deuteration of 4(1H)-quinolone ELQ-300, its alkoxycarbonate prodrug ELQ-331, and their respective N-oxides using deuterated acetic acid.

Update on cerebrotendinous xanthomatosis.

PURPOSE OF REVIEW: Cerebrotendinous xanthomatosis (CTX) is a rare genetic lipid storage disorder with highly pleomorphic clinical phenotype. Complications of this disease can be devastating and may include severe cognitive impairment and dementia in later stages. Disease progression can be prevented or stabilized by bile acid replacement therapy, although a subset of patients with advanced disease continue to deteriorate despite therapy. RECENT FINDINGS: Delayed diagnosis of CTX continues to impede effective treatment. A clinical diagnostic algorithm for CTX was developed that can decrease the age of diagnosis of CTX. The strategy of screening children with bilateral juvenile cataracts for CTX also improved diagnosis, as this group had a 500-fold higher-rate of CTX than the general population. Improved diagnosis of CTX is critical, as patients treated early in the course of the disease have significantly better outcomes compared with those treated later. More sensitive and specific biochemical testing for CTX has been developed that is potentially more informative than blood cholestanol to assess treatment efficacy and medication compliance in CTX. SUMMARY: Because we are recognizing more severe presentations of CTX in infants and children, and delayed diagnosis and treatment worsens the prognosis, CTX is an excellent candidate disorder for newborn screening using recently reported methods for newborn dried bloodspot analysis.

Metabolic profiling in serum, cerebrospinal fluid, and brain of patients with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but <2% in patients with CTX. Metabolic changes in brain tissue corresponded well with findings in CSF. Interestingly, 7α,12α-dihydroxycholest-4-en-3-one and 5α-cholestanol did not exert toxicity in neuronal cell culture. In conclusion, we propose that increased 7α,12α-dihydroxycholest-4-en-3-one and lack of 27-hydroxycholesterol may be highly sensitive metabolic biomarkers of CTX. As CDCA cannot reliably prevent disease progression despite reduction of most accumulated metabolites, supplementation of 27-hydroxylated bile acid intermediates or replacement of CYP27A1 might be required to counter neurodegeneration in patients with progressive disease despite CDCA treatment.

Toward newborn screening of cerebrotendinous xanthomatosis: results of a biomarker research study using 32,000 newborn dried blood spots.

PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5ß-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). METHODS: Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. CONCLUSION: The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.

Newborn screening for cerebrotendinous xanthomatosis is the solution for early identification and treatment.

Cerebrotendinous xanthomatosis (CTX) is a progressive metabolic leukodystrophy. Early identification and treatment from birth onward effectively provides a functional cure, but diagnosis is often delayed. We conducted a pilot study using a two-tier test for CTX to screen archived newborn dried bloodspots (DBSs) or samples collected prospectively from a high-risk Israeli newborn population. All DBS samples were analyzed with flow injection analysis (FIA)-MS/MS, and 5% of samples were analyzed with LC-MS/MS. Consecutively collected samples were analyzed to identify CTX-causing founder genetic variants common among Druze and Moroccan Jewish populations. First-tier analysis with FIA-MS/MS provided 100% sensitivity to detect CTX-positive newborn DBSs, with a low false-positive rate (0.1-0.5%). LC-MS/MS, as a second-tier test, provided 100% sensitivity to detect CTX-positive newborn DBSs with a false-positive rate of 0% (100% specificity). In addition, 5ß-cholestane-3α,7α,12α,25-tetrol-3-O-ß-D-glucuronide was identified as the predominant bile-alcohol disease marker present in CTX-positive newborn DBSs. In newborns identifying as Druze, a 1:30 carriership frequency was determined for the c.355delC CYP27A1 gene variant, providing an estimated disease prevalence of 1:3,600 in this population. These data support the feasibility of two-tier DBS screening for CTX in newborns and set the stage for large-scale prospective pilot studies.

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios.

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.

Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development.

NSDHL is a 3ß-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhl(tm1.1Hrm)) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8-10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism.

Thyroid Hormone Status in Sitosterolemia Is Modified by Ezetimibe.

OBJECTIVES: To assess the association between biomarkers of thyroid status and 5α-stanols in patients with sitosterolemia treated with ezetimibe (EZE). STUDY DESIGN: Eight patients with sitosterolemia (16-56 years of age) were studied during 14 weeks off EZE therapy and 14 weeks on EZE (10 mg/day). Serum thyroid biomarkers (free triiodothyronine [FT3], free thyroxine [FT4], FT3/FT4 ratio, thyroid-stimulating hormone), 5α-stanols (sitostanol and cholestanol), and cholestanol precursors (total cholesterol and its synthesis marker lathosterol, and 7α-hydroxy-4-cholesten-3-one cholestenol) were measured at baseline and during the 14 weeks off EZE and on EZE. RESULTS: EZE increased FT3/FT4 (10% ± 4%; P = .02). EZE reduced plasma and red blood cells sitostanol (-38% ± 6% and -20% ± 4%; all P < .05) and cholestanol (-18% ± 6% and -13% ± 3%; all P < .05). The change in plasma cholestanol level on EZE inversely correlated with the change in FT3/FT4 (r = -0.86; P = .01). EZE lowered total cholesterol (P < .0001) and did not affect 7α-hydroxy-4-cholesten-3-one cholestanol. EZE increased (P < .0001) lathosterol initially, but the level was not sustained, resulting in similar levels at week 14 off EZE and on EZE. CONCLUSION: In patients with STSL, 5α-stanols levels might be associated with thyroid function. EZE reduces circulating 5α-stanols while increasing FT3/FT4, implying increased conversion of T4 to T3, thus possibly improving thyroid hormone status. TRIAL REGISTRATION: ClinicalTrials.govNCT01584206.

Prevalence estimation for monogenic autosomal recessive diseases using population-based genetic data.

Genetic methods can complement epidemiological surveys and clinical registries in determining prevalence of monogenic autosomal recessive diseases. Several large population-based genetic databases, such as the NHLBI GO Exome Sequencing Project, are now publically available. By assuming Hardy-Weinberg equilibrium, the frequency of individuals homozygous in the general population for a particular pathogenic allele can be directly calculated from a sample of chromosomes where some harbor the pathogenic allele. Further assuming that the penetrance of the pathogenic allele(s) is known, the prevalence of recessive phenotypes can be determined. Such work can inform public health efforts for rare recessive diseases. A Bayesian estimation procedure has yet to be applied to the problem of estimating disease prevalence from large population-based genetic data. A Bayesian framework is developed to derive the posterior probability density of monogenic, autosomal recessive phenotypes. Explicit equations are presented for the credible intervals of these disease prevalence estimates. A primary impediment to performing accurate disease prevalence calculations is the determination of truly pathogenic alleles. This issue is discussed, but in many instances remains a significant barrier to investigations solely reliant on statistical interrogation--functional studies can provide important information for solidifying evidence of variant pathogenicity. We also discuss several challenges to these efforts, including the population structure in the sample of chromosomes, the treatment of allelic heterogeneity, and reduced penetrance of pathogenic variants. To illustrate the application of these methods, we utilized recently published genetic data collected on a large sample from the Schmiedeleut Hutterites. We estimate prevalence and calculate 95% credible intervals for 13 autosomal recessive diseases using these data. In addition, the Bayesian estimation procedure is applied to data from a central European study of hereditary fructose intolerance. The methods described herein show a viable path to robustly estimating both the expected prevalence of autosomal recessive phenotypes and corresponding credible intervals using population-based genetic databases that have recently become available. As these genetic databases increase in number and size with the advent of cost-effective next-generation sequencing, we anticipate that these methods and approaches may be helpful in recessive disease prevalence calculations, potentially impacting public health management, health economic analyses, and treatment of rare diseases.

Apparent underdiagnosis of Cerebrotendinous Xanthomatosis revealed by analysis of ~60,000 human exomes.

Cerebrotendinous Xanthomatosis (CTX) is a treatable inborn error of metabolism caused by recessive variants in CYP27A1. Clinical presentation varies, but typically includes infant-onset chronic diarrhea, juvenile-onset bilateral cataracts, and later-onset tendinous xanthomas and progressive neurological dysfunction. CYP27A1 plays an essential role in side-chain oxidation of cholesterol necessary for the synthesis of the bile acid, chenodeoxycholic acid, and perturbations in this gene that reduce enzyme activity result in elevations of cholestanol. It is commonly held that CTX is exceedingly rare, but epidemiological studies are lacking. In order to provide an accurate incidence estimate of CTX, we studied the ExAC cohort of ~60,000 unrelated adults from global populations to determine the allele frequency of the 57 variants in CYP27A1 reported pathogenic for CTX. In addition, we conducted bioinformatics analyses on these CTX-causing variants and determined a bioinformatics profile to predict variants that may be pathogenic but have not yet been reported in the CTX patient literature. An additional 29 variants were identified that met bioinformatics criteria for being potentially pathogenic. Incidence was estimated based allele frequencies of pathogenic CTX variants plus those determined to be potentially pathogenic. One variant, p.P384L, previously reported in three unrelated CTX families had an allele frequency ≥ 1% in European, Latino and Asian populations. Three additional mutations had a frequency of ≥ 0.1% in Asian populations. CTX disease incidence was calculated excluding the high frequency p.P384L and separately using a genetic paradigm where this high frequency variant only causes classic CTX when paired in trans with a null variant. These calculations place CTX incidence ranging from 1:134,970 to 1:461,358 in Europeans, 1:263,222 to 1:468,624 in Africans, 1:71,677 to 1:148,914 in Americans, 1:64,267 to 1:64,712 in East Asians and 1:36,072 to 1:75,601 in South Asians. This work indicates CTX is under-diagnosed and improved patient screening is needed as early intervention prevents disease progression.

A blood test for cerebrotendinous xanthomatosis with potential for disease detection in newborns.

Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were ∼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.

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 Alzheimer's 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.

Effect of PCSK9 inhibition on plasma levels of small dense low density lipoprotein-cholesterol and 7-ketocholesterol.

BACKGROUND: Oxidized forms of cholesterol (oxysterols) are implicated in atherogenesis and can accumulate in the body via direct absorption from food or through oxidative reactions of endogenous cholesterol, inducing the formation of LDL particles loaded with oxidized cholesterol. It remains unknown whether drastic reductions in LDL-cholesterol (LDL-C) are associated with changes in circulating oxysterols and whether small dense LDL (sdLDL) are more likely to carry these oxysterols and susceptible to the effects of PCSK9 inhibition (PCSK9i). OBJECTIVE: We investigate the effect of LDL-C reduction accomplished via PCSK9i on changes in plasma levels of sdLDL-cholesterol (sdLDL-C) and a common, stable oxysterol, 7-ketocholesterol (7-KC), among 134 patients referred to our Preventive Cardiology clinic. METHODS: Plasma lipid panel, sdLDL-C, and 7-KC measurements were obtained from patients before and after initiation of PCSK9i. RESULTS: The intervention caused a significant lowering of LDL-C (-55.4 %). The changes in sdLDL-C levels (mean reduction 51.4 %) were highly correlated with the reductions in LDL-C levels (R = 0.829, p < 0.001). Interestingly, whereas changes in plasma free 7-KC levels with PCSK9i treatment were much smaller than (-6.6 %) and did not parallel those of LDL-C and sdLDL-C levels, they did significantly correlate with changes in triglycerides and very low-density lipoprotein-cholesterol (VLDL-C) levels (R = 0.219, p = 0.025). CONCLUSION: Our findings suggest a non-preferential clearance of LDL subparticles as a consequence of LDL receptor upregulation caused by PCSK9 inhibition. Moreover, the lack of significant reduction in 7-KC with PCSK9i suggests that 7-KC may be in part carried by VLDL and lost during lipoprotein processing leading to LDL formation.

Living with Cerebrotendinous Xanthomatosis: Patient, Caregiver, and Expert Perspectives.

In this article, patients with cerebrotendinous xanthomatosis (CTX) and caregivers detail their experience with lifelong symptoms, diagnosis, treatment and efficacy, and ongoing disease management. One patient and four caregivers describe the challenges associated with pursuing a correct diagnosis for years before testing confirmed a CTX diagnosis. They also detail their ongoing struggles and desire for greater access to physicians with CTX knowledge and to reliable online resources to continue their education about the disease and strategies for symptom management. The expert perspective is a direct response by three CTX researchers, including physicians who are treating patients with CTX in the United States and experts whose laboratories provide genetic and biochemical testing for CTX. They respond to many of the patient and caregiver concerns, including steps that are being taken to identify CTX earlier and provide access to confirmatory diagnostic testing sooner, and suggest the best online resources for CTX-related information and access to webinars and support groups. While the expert perspective is a direct response to the patient and caregiver authors' CTX journeys, it should be beneficial to any patient with CTX or their caregivers.

Genetically and clinically confirmed atypical cerebrotendinous xanthomatosis with normal cholestanol and marked elevations of bile acid precursors and bile alcohols.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid disorder. Affected patients often remain undiagnosed until the age of 20-30 years, when they have already developed significant neurologic disease that may not be reversible. An elevated plasma cholestanol concentration has been accepted as a diagnostic criterion for CTX for decades. OBJECTIVE: Full biochemical characterization was performed for three genetically and clinically confirmed atypical CTX cases with normal plasma cholestanol levels. METHODS: Clinical assessment and genetic/biochemical testing for patients with CTX was performed by their physician providing routine standard of care. RESULTS: We report three new atypical CTX cases with large extensor tendon xanthomas but normal plasma cholestanol levels. All three cases had marked elevations of bile acid precursors and bile alcohols in plasma and urine that decreased on treatment with chenodeoxycholic acid. We also review eight published cases of atypical CTX with normal/near normal circulating cholestanol levels. CONCLUSION: The atypical biochemical presentation of these cases provides a diagnostic challenge for CTX, a disorder for which cholestanol has been believed to be a sensitive biomarker. These cases demonstrate measurements of plasma cholestanol alone are insufficient to exclude a diagnosis of CTX. The data presented is consistent with the concept that bile acid precursors and bile alcohols are sensitive biomarkers for atypical CTX with normal cholestanol, and that such testing is indicated, along with CYP27A1 gene analyses, in patients presenting with significant tendon and/or tuberous xanthomas and/or neurologic disease in early adulthood despite normal or near normal cholesterol and cholestanol levels.

Altered liver metabolism post-wean abolishes efficacy of vitamin D for breast cancer prevention in a mouse model.

Young women have increased risk of vitamin D deficiency, which may increase breast cancer incidence. Here, we assessed the anti-cancer efficacy of vitamin D in mouse models of young-onset breast cancer. In never-pregnant mice, vitamin D supplementation increased serum 25(OH)D and hepatic 1,25(OH)2D3, reduced tumor size, and associated with anti-tumor immunity. These anti-tumor effects were not replicated in a mouse model of postpartum breast cancer, where hepatic metabolism of vitamin D was suppressed post-wean, which resulted in deficient serum 25(OH)D and reduced hepatic 1,25(OH)2D3. Treatment with active 1,25(OH)2D3 induced hypercalcemia exclusively in post-wean mice, highlighting metabolic imbalance post-wean. RNAseq revealed suppressed CYP450 expression postpartum. In sum, we provide evidence that vitamin D anti-tumor activity is mediated through immunomodulatory mechanisms and is ineffective in the post-wean window due to altered hepatic metabolism. These findings have implications for suppressed xenobiotic metabolism in postpartum women beyond vitamin D.

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.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.