Drug discovery for X-linked adrenoleukodystrophy: An unbiased screen for compounds that lower very long-chain fatty acids.

X-linked adrenoleukodystrophy (XALD) is a genetic neurologic disorder with multiple phenotypic presentations and limited therapeutic options. The childhood cerebral phenotype (CCALD), a fatal demyelinating disorder affecting about 35% of patients, and the adult-onset adrenomyeloneuropathy (AMN), a peripheral neuropathy affecting 40%-45% of patients, are both caused by mutations in the ABCD1 gene. Both phenotypes are characterized biochemically by elevated tissue and plasma levels of saturated very long-chain fatty acids (VLCFA), and an increase in plasma cerotic acid (C26:0), along with the clinical presentation, is diagnostic. Administration of oils containing monounsaturated fatty acids, for example, Lorenzo's oil, lowers patient VLCFA levels and reduced the frequency of development of CCALD in presymptomatic boys. However, this therapy is not currently available. Hematopoietic stem cell transplant and gene therapy remain viable therapies for boys with early progressive cerebral disease. We asked whether any existing approved drugs can lower VLCFA and thus open new therapeutic possibilities for XALD. Using SV40-transformed and telomerase-immortalized skin fibroblasts from an XALD patient, we conducted an unbiased screen of a library of approved drugs and natural products for their ability to decrease VLCFA, using measurement of C26:0 in lysophosphatidyl choline (C26-LPC) by tandem mass spectrometry as the readout. While several candidate drugs were initially identified, further testing in primary fibroblast cell lines from multiple CCALD and AMN patients narrowed the list to one drug, the anti-hypertensive drug irbesartan. In addition to lowering C26-LPC, levels of C26:0 and C28:0 in total fibroblast lipids were reduced. The effect of irbesartan was dose dependent between 2 and 10 µM. When male XALD mice received orally administered irbesartan at a dose of 10 mg/kg/day, there was no reduction in plasma C26-LPC. However, irbesartan failed to lower mouse fibroblast C26-LPC consistently. The results of these studies indicate a potential therapeutic benefit of irbesartan in XALD that should be validated by further study.

Allelic Expression Imbalance Promoting a Mutant PEX6 Allele Causes Zellweger Spectrum Disorder.

Zellweger spectrum disorders (ZSDs) are autosomal-recessive disorders that are caused by defects in peroxisome biogenesis due to bi-allelic mutations in any of 13 different PEX genes. Here, we identified seven unrelated individuals affected with an apparent dominant ZSD in whom a heterozygous mutant PEX6 allele (c.2578C>T [p.Arg860Trp]) was overrepresented due to allelic expression imbalance (AEI). We demonstrated that AEI of PEX6 is a common phenomenon and is correlated with heterozygosity for a frequent variant in the 3' untranslated region (UTR) of the mutant allele, which disrupts the most distal of two polyadenylation sites. Asymptomatic parents, who were heterozygous for PEX c.2578C>T, did not show AEI and were homozygous for the 3' UTR variant. Overexpression models confirmed that the overrepresentation of the pathogenic PEX6 c.2578T variant compared to wild-type PEX6 c.2578C results in a peroxisome biogenesis defect and thus constitutes the cause of disease in the affected individuals. AEI promoting the overrepresentation of a mutant allele might also play a role in other autosomal-recessive disorders, in which only one heterozygous pathogenic variant is identified.

Acute Illness Among Surfers After Exposure to Seawater in Dry- and Wet-Weather Conditions.

Rainstorms increase levels of fecal indicator bacteria in urban coastal waters, but it is unknown whether exposure to seawater after rainstorms increases rates of acute illness. Our objective was to provide the first estimates of rates of acute illness after seawater exposure during both dry- and wet-weather periods and to determine the relationship between levels of indicator bacteria and illness among surfers, a population with a high potential for exposure after rain. We enrolled 654 surfers in San Diego, California, and followed them longitudinally during the 2013-2014 and 2014-2015 winters (33,377 days of observation, 10,081 surf sessions). We measured daily surf activities and illness symptoms (gastrointestinal illness, sinus infections, ear infections, infected wounds). Compared with no exposure, exposure to seawater during dry weather increased incidence rates of all outcomes (e.g., for earache or infection, adjusted incidence rate ratio (IRR) = 1.86, 95% confidence interval (CI): 1.27, 2.71; for infected wounds, IRR = 3.04, 95% CI: 1.54, 5.98); exposure during wet weather further increased rates (e.g., for earache or infection, IRR = 3.28, 95% CI: 1.95, 5.51; for infected wounds, IRR = 4.96, 95% CI: 2.18, 11.29). Fecal indicator bacteria measured in seawater (Enterococcus species, fecal coliforms, total coliforms) were strongly associated with incident illness only during wet weather. Urban coastal seawater exposure increases the incidence rates of many acute illnesses among surfers, with higher incidence rates after rainstorms.

Peroxisome biogenesis disorders in the Zellweger spectrum: An overview of current diagnosis, clinical manifestations, and treatment guidelines.

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care.

The Pex1-G844D mouse: a model for mild human Zellweger spectrum disorder.

Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal ß-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.

Nonsense suppressor therapies rescue peroxisome lipid metabolism and assembly in cells from patients with specific PEX gene mutations.

Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies.

Human and great ape red blood cells differ in plasmalogen levels and composition.

BACKGROUND: Plasmalogens are ether phospholipids required for normal mammalian developmental, physiological, and cognitive functions. They have been proposed to act as membrane antioxidants and reservoirs of polyunsaturated fatty acids as well as influence intracellular signaling and membrane dynamics. Plasmalogens are particularly enriched in cells and tissues of the human nervous, immune, and cardiovascular systems. Humans with severely reduced plasmalogen levels have reduced life spans, abnormal neurological development, skeletal dysplasia, impaired respiration, and cataracts. Plasmalogen deficiency is also found in the brain tissue of individuals with Alzheimer disease. RESULTS: In a human and great ape cohort, we measured the red blood cell (RBC) levels of the most abundant types of plasmalogens. Total RBC plasmalogen levels were lower in humans than bonobos, chimpanzees, and gorillas, but higher than orangutans. There were especially pronounced cross-species differences in the levels of plasmalogens with a C16:0 moiety at the sn-1 position. Humans on Western or vegan diets had comparable total RBC plasmalogen levels, but the latter group showed moderately higher levels of plasmalogens with a C18:1 moiety at the sn-1 position. We did not find robust sex-specific differences in human or chimpanzee RBC plasmalogen levels or composition. Furthermore, human and great ape skin fibroblasts showed only modest differences in peroxisomal plasmalogen biosynthetic activity. Human and chimpanzee microarray data indicated that genes involved in plasmalogen biosynthesis show cross-species differential expression in multiple tissues. CONCLUSION: We propose that the observed differences in human and great ape RBC plasmalogens are primarily caused by their rates of biosynthesis and/or turnover. Gene expression data raise the possibility that other human and great ape cells and tissues differ in plasmalogen levels. Based on the phenotypes of humans and rodents with plasmalogen disorders, we propose that cross-species differences in tissue plasmalogen levels could influence organ functions and processes ranging from cognition to reproduction to aging.

Identification of differences in human and great ape phytanic acid metabolism that could influence gene expression profiles and physiological functions.

BACKGROUND: It has been proposed that anatomical differences in human and great ape guts arose in response to species-specific diets and energy demands. To investigate functional genomic consequences of these differences, we compared their physiological levels of phytanic acid, a branched chain fatty acid that can be derived from the microbial degradation of chlorophyll in ruminant guts. Humans who accumulate large stores of phytanic acid commonly develop cerebellar ataxia, peripheral polyneuropathy, and retinitis pigmentosa in addition to other medical conditions. Furthermore, phytanic acid is an activator of the PPAR-alpha transcription factor that influences the expression of genes relevant to lipid metabolism. RESULTS: Despite their trace dietary phytanic acid intake, all great ape species had elevated red blood cell (RBC) phytanic acid levels relative to humans on diverse diets. Unlike humans, chimpanzees showed sexual dimorphism in RBC phytanic acid levels, which were higher in males relative to females. Cultured skin fibroblasts from all species had a robust capacity to degrade phytanic acid. We provide indirect evidence that great apes, in contrast to humans, derive significant amounts of phytanic acid from the hindgut fermentation of plant materials. This would represent a novel reduction of metabolic activity in humans relative to the great apes. CONCLUSION: We identified differences in the physiological levels of phytanic acid in humans and great apes and propose this is causally related to their gut anatomies and microbiomes. Phytanic acid levels could contribute to cross-species and sex-specific differences in human and great ape transcriptomes, especially those related to lipid metabolism. Based on the medical conditions caused by phytanic acid accumulation, we suggest that differences in phytanic acid metabolism could influence the functions of human and great ape nervous, cardiovascular, and skeletal systems.

Identification of novel mutations and sequence variation in the Zellweger syndrome spectrum of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive neurodegenerative disorders that affect multiple organ systems. Approximately 80% of PBD patients are classified in the Zellweger syndrome spectrum (PBD-ZSS). Mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes are found in approximately 90% of PBD-ZSS patients. Here, we sequenced the coding regions and splice junctions of these five genes in 58 PBD-ZSS cases previously subjected to targeted sequencing of a limited number of PEX gene exons. In our cohort, 71 unique sequence variants were identified, including 18 novel mutations predicted to disrupt protein function and 2 novel silent variants. We identified 4 patients who had two deleterious mutations in one PEX gene and a third deleterious mutation in a second PEX gene. For two such patients, we conducted cell fusion complementation analyses to identify the defective gene responsible for aberrant peroxisome assembly. Overall, we provide empirical data to estimate the relative fraction of disease-causing alleles that occur in the coding and splice junction sequences of these five PEX genes and the frequency of cases where mutations occur in multiple PEX genes. This information is beneficial for efforts aimed at establishing rapid and sensitive clinical diagnostics for PBD-ZSS patients and interpreting the results from these genetic tests.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) method.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD) has until now been limited in implementation because of the lack of an accepted standard methodology. We have previously reported a technique using LC-MS/MS analysis that could provide the basis for screening of newborns for X-ALD. The target analyte diagnostic for X-ALD and other peroxisomal disorders of peroxisomal beta-oxidation is 1-hexacosanoyl-2-lyso-sn-3-glycero-phosphorylcholine (26:0-lyso-PC). We report here the validation of the analytical method using an authentic standard of the target compound. The method possesses sensitivity of <1.0fmole injected on column with a correlation coefficient (R(2)) of 0.9987. A tetradeuterated analog of 26:0-lyso-PC served as the internal standard. The sensitivity of this clinical method was confirmed using 17 newborn samples of individuals with peroxisomal disorders retrieved from state newborn screening programs. These samples were run masked with over 1000 newborn samples. All affected individuals were identified with one exception. One sample which was retrieved as an affected did not have the biochemical or genetic abnormality of X-ALD and thus is considered an error in sample identity. These studies clearly show that the method is highly sensitive and accurate in identifying individuals with a defect in peroxisomal beta-oxidation such as X-ALD.

Peroxisome biogenesis disorders.

Defects in PEX genes impair peroxisome assembly and multiple metabolic pathways confined to this organelle, thus providing the biochemical and molecular bases of the peroxisome biogenesis disorders (PBD). PBD are divided into two types--Zellweger syndrome spectrum (ZSS) and rhizomelic chondrodysplasia punctata (RCDP). Biochemical studies performed in blood and urine are used to screen for the PBD. DNA testing is possible for all of the disorders, but is more challenging for the ZSS since 12 PEX genes are known to be associated with this spectrum of PBD. In contrast, PBD-RCDP is associated with defects in the PEX7 gene alone. Studies of the cellular and molecular defects in PBD patients have contributed significantly to our understanding of the role of each PEX gene in peroxisome assembly.

Primary renal neoplasia of dogs.

BACKGROUND: Primary renal tumors are diagnosed uncommonly in dogs. HYPOTHESIS: Signs and survival will differ among different categories of primary renal tumors. ANIMALS: Data were collected from the medical records of 82 dogs with primary renal tumors diagnosed by examination of tissue obtained by ultrasound-guided biopsy, needle aspiration, surgery, or at postmortem examination. METHODS: This was a multi-institutional, retrospective study. RESULTS: Forty-nine dogs had carcinomas, 28 had sarcomas, and 5 had nephroblastomas. The dogs were geriatric (mean 8.1 years; range: 1-17) with a weight of 24.9 kg (range: 4.5-80). Tumors occurred with equal frequency in each kidney with 4% occurring bilaterally. Initial signs included one or more of hematuria, inappetance, lethargy. weight loss, or a palpable abdominal mass. Pain was reported more frequently in dogs with sarcomas (5/28). The most common hematologic abnormalities were neutrophilia (22/63), anemia (21/64), and thrombocytopenia (6/68). Polycythemia was present in 3 dogs and resolved with treatment. Hematuria (28/49), pyuria (26/49), proteinuria (24/50), and isosthenuria (20/56) were the most frequently observed abnormalities on urinalysis. Pulmonary metastases were noted on thoracic radiographs in 16% of dogs at diagnosis. Seventy-seven percent of dogs had metastatic disease at the time of death. Median survival for dogs with carcinomas was 16 months (range 0-59 months), for dogs with sarcomas 9 months (range 0-70 months), and for dogs with nephroblastomas 6 months (range 0-6 months). CONCLUSIONS AND CLINICAL IMPORTANCE: Primary renal tumors in dogs are generally highly malignant with surgery being the only treatment that improves survival.

Association of common variations on chromosome 4q25 and left atrial volume in patients with atrial fibrillation.

AIMS: Recent studies have shown that several genetic variants near the PITX2 locus on chromosome 4q25 are associated with atrial fibrillation (AF). However, the mechanism that mediates this association remains unclear. Basic murine studies suggest that reduced PITX2 expression is associated with left atrial dilatation. We sought to examine the association between single nucleotide polymorphisms (SNPs) near PITX2 and left atrial size in patients with AF. METHODS: We prospectively enrolled 96 consecutive patients (mean age 60 ± 10 years, 72% male) with drug-resistant AF (57% paroxysmal, 38% persistent, and 5% long-standing persistent) who underwent catheter ablation. Following DNA extraction from blood obtained pre-operatively, SNPs rs10033464 and rs2200733 were genotyped using the Sequenom MassARRAY. Left atrial volume (LAV) was determined using three-dimensional imaging (CT or MRI prior to first ablation) and by investigators blinded to genotype results. RESULTS: The minor allele frequencies at SNPs rs10033464 and rs2200733 were 0.14 and 0.25, respectively. Using multivariable linear regression, homozygosity for the minor allele at rs10033464 (recessive model) was independently associated with larger LAV (P = 0.002) after adjustment for age, gender, BMI, height, type, and duration of AF, left ventricular ejection fraction, history of hypertension, valve disease, and antiarrhythmic drug use. The strength of the association was reconfirmed in a bootstrap study with 1000 resamplings. In contrast, no association was found between rs2200733 variant alleles and LAV. CONCLUSION: SNP rs10033464 near the PITX2 locus on 4q25 is associated with LAV. Left atrial dilatation may mediate the association of common variants at 4q25 with AF.

Induced pluripotent stem cell models of Zellweger spectrum disorder show impaired peroxisome assembly and cell type-specific lipid abnormalities.

INTRODUCTION: Zellweger spectrum disorder (PBD-ZSD) is a disease continuum caused by mutations in a subset of PEX genes required for normal peroxisome assembly and function. They highlight the importance of peroxisomes in the development and functions of the central nervous system, liver, and other organs. To date, the underlying bases for the cell-type specificity of disease are not fully elucidated. METHODS: Primary skin fibroblasts from seven PBD-ZSD patients with biallelic PEX1, PEX10, PEX12, or PEX26 mutations and three healthy donors were transduced with retroviral vectors expressing Yamanaka reprogramming factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, copy number variation, genotyping, in vitro differentiation and teratoma formation assays. Confirmed iPSCs were differentiated into neural progenitor cells (NPCs), neurons, oligodendrocyte precursor cells (OPCs), and hepatocyte-like cell cultures with peroxisome assembly evaluated by microscopy. Saturated very long chain fatty acid (sVLCFA) and plasmalogen levels were determined in primary fibroblasts and their derivatives. RESULTS: iPSCs were derived from seven PBD-ZSD patient-derived fibroblasts with mild to severe peroxisome assembly defects. Although patient and control skin fibroblasts had similar gene expression profiles, genes related to mitochondrial functions and organelle cross-talk were differentially expressed among corresponding iPSCs. Mitochondrial DNA levels were consistent among patient and control fibroblasts, but varied among all iPSCs. Relative to matching controls, sVLCFA levels were elevated in patient-derived fibroblasts, reduced in patient-derived iPSCs, and not significantly different in patient-derived NPCs. All cell types derived from donors with biallelic null mutations in a PEX gene showed plasmalogen deficiencies. Reporter gene assays compatible with high content screening (HCS) indicated patient-derived OPC and hepatocyte-like cell cultures had impaired peroxisome assembly. CONCLUSIONS: Normal peroxisome activity levels are not required for cellular reprogramming of skin fibroblasts. Patient iPSC gene expression profiles were consistent with hypotheses highlighting the role of altered mitochondrial activities and organelle cross-talk in PBD-ZSD pathogenesis. sVLCFA abnormalities dramatically differed among patient cell types, similar to observations made in iPSC models of X-linked adrenoleukodystrophy. We propose that iPSCs could assist investigations into the cell type-specificity of peroxisomal activities, toxicology studies, and in HCS for targeted therapies for peroxisome-related disorders.

Late-onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked adrenoleukodystrophy.

BACKGROUND: Zellweger spectrum disorder is an autosomal recessively inherited multisystem disorder caused by one of the 13 different PEX gene defects resulting in defective peroxisomal assembly and multiple peroxisomal enzyme deficiencies. We report a new patient with late-onset Zellweger spectrum disorder mimicking X-linked adrenoleukodystrophy. PATIENT DESCRIPTION: This 8.5-year-old boy with normal development until 6.5 years of age presented with bilateral sensorineural hearing loss during a school hearing test. He then developed acute-onset diplopia, clumsiness, and cognitive dysfunction at age 7 years. Magnetic resonance imaging of the brain revealed symmetric leukodystrophy, although without gadolinium enhancement. Elevated plasma very long chain fatty acid levels were suggestive of X-linked adrenoleukodystrophy, but his ABCD1 gene had normal coding sequence and dosage. Additional studies of cultured skin fibroblasts were consistent with Zellweger spectrum disorder. Molecular testing identified disease-causing compound heterozygous mutations in the PEX6 gene supporting the Zellweger spectrum disorder diagnosis in this patient. CONCLUSIONS: We describe a new patient with late-onset Zellweger spectrum disorder caused by PEX6 mutations who presented with an acute neurodegenerative disease course mimicking X-linked adrenoleukodystrophy. This finding provides an additional reason that molecular confirmation is important for the genetic counseling and management of patients with a clinical and biochemical diagnosis of X-linked adrenoleukodystrophy.

The gene expression profiles of induced pluripotent stem cells from individuals with childhood cerebral adrenoleukodystrophy are consistent with proposed mechanisms of pathogenesis.

INTRODUCTION: X-linked adrenoleukodystrophy (X-ALD) is a complex disorder with variable expressivity that affects the nervous, adrenocortical and male reproductive systems. Although ABCD1 mutations are known to provide the genetic basis for X-ALD, its pathogenesis is not fully elucidated. While elevated very long chain fatty acid (VLCFA) levels in blood and reduced VLCFA catabolic activity in cultured fibroblasts are biomarkers used to identify ABCD1 mutation carriers, the roles peroxisomal lipid metabolism play in disease etiology are unknown. METHODS: Primary skin fibroblasts from two male patients with the childhood cerebral form of the disease (CCALD) caused by ABCD1 frameshift or missense mutations and three healthy donors were transduced with retroviral vectors expressing the OCT4, SOX2, KLF4 and c-MYC factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, DNA copy number variation, and genotyping analysis and tested for pluripotency through in vitro differentiation and teratoma formation. Saturated VLCFA (sVLCFA) and plasmalogen levels in primary fibroblasts and iPSCs from healthy donors as well as CCALD patients were determined through mass spectroscopy. RESULTS: Skin fibroblasts from CCALD patients and healthy donors were reprogrammed into validated iPSCs. Unlike fibroblasts, CCALD patient iPSCs show differentially expressed genes (DEGs) relevant to both peroxisome abundance and neuroinflammation. Also, in contrast to fibroblasts, iPSCs from patients showed no significant difference in sVLCFA levels relative to those from controls. In all cell types, the plasmalogen levels tested did not correlate with ABCD1 mutation status. CONCLUSION: Normal ABCD1 gene function is not required for reprogramming skin fibroblasts into iPSCs or maintaining pluripotency. Relative to DEGs found in fibroblasts, DEGs uncovered in comparisons of CCALD patient and control iPSCs are more consistent with major hypotheses regarding disease pathogenesis. These DEGs were independent of differences in sVLCFA levels, which did not vary according to ABCD1 mutation status. The highlighted genes provide new leads for pathogenic mechanisms that can be explored in animal models and human tissue specimens. We suggest that these iPSC resources will have applications that include assisting efforts to identify genetic and environmental modifiers and screening for therapeutic interventions tailored towards affected cell populations and patient genotypes.

A novel SOX9 mutation, 972delC, causes 46,XY sex-reversed campomelic dysplasia with nephrocalcinosis, urolithiasis, and dysgerminoma.

An 8-year-old phenotypic female with campomelic dysplasia (CD) and 46,XY sex-reversal presented with renal colic. Medullary nephrocalcinosis, urolithiasis, and renal malrotation were diagnosed by computed tomographic scanning. Pelvic sonogram identified an enlarged left gonad. Genetic testing revealed a novel SOX9 heterozygous deletion of a cytosine at nucleotide 972 (972delC), causing a frameshift at codon 200, introducing a stop codon 18 codons further downstream (P200fsX218). At laparoscopic gonadectomy, a left dysgerminoma was removed. This first reported case of dysgerminoma in a sex-reversed patient with CD who also had urolithiasis stresses the importance of prophylactic gonadectomy and urologic evaluations in this susceptible population.

Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules.

The assembly of class I MHC molecules and their export from the endoplasmic reticulum (ER) is governed by chaperones and accessory proteins. We present evidence that the putative cargo receptor protein Bap31 participates in the transport and the quality control of human class I molecules. Transfection of the human adenocarcinoma cell line HeLa with yellow fluorescent protein-Bap31 chimeras increased surface levels of class I in a dose-dependent manner, by as much as 3.7-fold. The increase in surface class I resulted from an increase in the rate of export of newly synthesized class I molecules to the cell surface and from an increase in the stability of the exported molecules. We propose that Bap31 performs quality control on class I molecules in two distinct phases: first, by exporting peptide-loaded class I molecules to the ER/Golgi intermediate compartment, and second, by retrieving class I molecules that have lost peptides in the acidic post-ER environment. This function of Bap31 is conditional or redundant, because we find that Bap31 deficiency does not reduce surface class I levels. Overexpression of the Bap31 homolog, Bap29, decreases surface class levels in HeLa, indicating that it does not substitute for Bap31.