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.

SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency.

Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.

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.

Quantitative magnetization transfer characteristics of the human cervical spinal cord in vivo: application to adrenomyeloneuropathy.

Magnetization transfer (MT) imaging has assessed myelin integrity in the brain and spinal cord; however, quantitative MT (qMT) has been confined to the brain or excised tissue. We characterized spinal cord tissue with qMT in vivo, and as a first application, qMT-derived metrics were examined in adults with the genetic disorder Adrenomyeloneuropathy (AMN). AMN is a progressive disease marked by demyelination of the white matter tracts of the cervical spinal cord, and a disease in which conventional MRI has been limited. MT data were acquired at 1.5 Tesla using 10 radiofrequency offsets at one power in the cervical cord at C2 in 6 healthy volunteers and 9 AMN patients. The data were fit to a two-pool MT model and the macromolecular fraction (M(ob)), macromolecular transverse relaxation time (T(2b)) and the rate of MT exchange (R) for lateral and dorsal column white matter and gray matter were calculated. M(ob) for healthy volunteers was: WM = 13.9 +/- 2.3%, GM = 7.9 +/- 1.5%. In AMN, dorsal column M(ob) was significantly decreased (P < 0.03). T(2b) for volunteers was: 9 +/- 2 micros and the rate of MT exchange (R) was: WM = 56 +/- 11 Hz, GM = 67 +/- 12 Hz. Neither T(2b) nor R showed significant differences between healthy and diseased cords. Comparisons are made between qMT, and conventional MT acquisitions.

Auditory function in adrenomyeloneuropathy.

Auditory brainstem responses (ABR), ipsilateral and contralateral acoustic reflexes and the masking level difference for speech (MLD) were studied in 29 patients with adrenomyeloneuropathy (AMN). Abnormalities were seen for all ABR components with Waves V and III affected to the greatest degree. For male patients with AMN, the I-III, III-V and I-V interpeak latency intervals were abnormal for a majority of patients. For female patients with AMN, the I-V and III-V interpeak latency intervals were abnormal for a majority of patients with the I-III interval less affected. Contralateral acoustic reflexes were elevated or absent for approximately 50% of ears. Ipsilateral acoustic reflexes were abnormal for 25% of ears. MLDs were significantly reduced in 72% of patients. When considered in terms of the earliest ABR wave abnormality, the earlier components of the ABR (i.e., Waves III and I) were the initial components impaired for the majority of ears. Word recognition in quiet was relatively unimpaired for all subjects. Despite the presence of marked ABR abnormalities, patients with AMN denied the presence of significant difficulty hearing.

Attitudes of families affected by adrenoleukodystrophy toward prenatal diagnosis, presymptomatic and carrier testing, and newborn screening.

Families affected by adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) were surveyed to elicit attitudes toward prenatal, presymptomatic and carrier testing, and newborn screening in order to determine the level of support that these families have for current and future genetic testing protocols. Identifying attitudes toward genetic testing, including newborn screening, is especially important because of new data regarding therapeutic options and the possible addition of ALD to newborn screening regimens. The Kennedy Krieger Institute (KKI) database identified 327 prospective participants. Families that were willing to participate in the study received an anonymous questionnaire for completion. Frequencies were generated using SPSS software for Windows. Questionnaires were returned from 128 families for a response rate of 39%. Sons who were at risk for inheriting the ALD gene would be tested by 93% of respondents, and 89.3% would ideally have this testing performed prenatally or in the newborn period. Eighty-nine percent would test an at-risk daughter and 51.2% would ideally have this testing performed prenatally or shortly after birth. ALD newborn screening for males and females was supported by 90% of respondents. If newborn screening for ALD/AMN commences, or there is a new diagnosis of ALD, genetic professionals need to be prepared to have extensive conversations with families regarding the benefits and limitations of current therapeutic and genetic testing options.

"Lorenzo's oil" therapy for X-linked adrenoleukodystrophy: rationale and current assessment of efficacy.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder that damages the nervous system and is associated with the accumulation of saturated very long chain fatty acids (SVLCFA). Oral administration of "Lorenzo's oil" (LO), a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, normalizes the SVLCFA levels in plasma, but its clinical efficacy and the clinical indications for its use have been controversial for more than 15 years. We review the biochemical effects of LO administration and the rationale for its use and present a current appraisal of its capacity to reduce the risk for the childhood cerebral phenotype when administered to asymptomatic boys and to slow progression of adrenomyeloneuropathy in patients without cerebral involvement. We also present current efforts to provide definitive evaluation of its clinical efficacy and discuss its possible role in the new therapeutic opportunities that will arise if newborn screening for X-ALD is validated and implemented.

Survival analysis of haematopoietic cell transplantation for childhood cerebral X-linked adrenoleukodystrophy: a comparison study.

BACKGROUND: Favourable outcomes have been reported for patients with childhood cerebral adrenoleukodystrophy (CCALD) who had received haematopoietic cell transplantation (HCT) at the early stage of cerebral involvement. However, comparative data for non-transplanted CCALD patients are limited. We analysed survival of CCALD patients who had not received HCT and, in a subgroup with early cerebral disease, compared survival in those who underwent HCT with those who did not. METHODS: Retrospective survival analyses were done on 283 CCALD patients identified at the Kennedy Krieger Institute who had not received HCT, focusing on a 30-member early stage cerebral subgroup whose neurological disability and MRI severity scores matched those in a 19-member transplanted subgroup previously reported. A Kaplan-Meier survival curve and log-rank test were used for survival analysis and to estimate the difference between the survival probabilities of the groups with statistical significance set at alpha=0.05. FINDINGS: Mean age at onset of symptoms in the entire 283 non-transplanted group was 7 years (SD 2 years). 131 (46%) patients died during the mean follow-up period of 5.9 years (5.3) at a mean age of 12.3 years (4.9). 5-year survival was 66%. The 5-year survival probability of 54% in the early stage group was significantly poorer (chi(2)=7.47, p=0.006) than the 5-year survival of 95% in the transplanted group with early stage cerebral disease. INTERPRETATION: HCT done in the early and progressive stages of CCALD is beneficial, and our data support the recommendation that transplantation be offered to patients in the early stages of CCALD.

Magnetic resonance imaging detection of lesion progression in adult patients with X-linked adrenoleukodystrophy.

BACKGROUND: An inherited disorder, X-linked adrenoleukodystrophy (X-ALD) is known to cause progressive inflammatory demyelination. OBJECTIVE: To analyze the adult pattern of disease progression in X-ALD. DESIGN, SETTING, AND PATIENTS: We retrospectively assessed magnetic resonance (MR) images obtained in adult patients who had developed cerebral disease between January 1, 1985, and December 31, 2005. We identified 103 adult patients with X-ALD with lesions on their MR images. Of these, 56 had serial MR examinations at least 1 year apart and were included in this study. Main Outcome Measure Progression of X-ALD lesions on MR images. RESULTS: On initial presentation, 17 patients with X-ALD had corticospinal tract lesions without splenium or genu involvement, 24 had symmetric corticospinal tract lesions with additional involvement of the splenium or genu, and 15 did not have corticospinal tract involvement but had other white matter lesions. In 18 of 21 patients with progressive lesions, corticospinal tract involvement preceded or occurred concurrently with progressive inflammatory demyelination. CONCLUSIONS: Brain MR imaging abnormalities in adults with X-ALD progress slower than those reported in childhood. The involvement of the corticospinal tracts is prominent and may at times represent a variant course of progressive inflammatory demyelination.

X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is caused by a defect in the gene ABCD1, which maps to Xq28 and codes for a peroxisomal membrane protein that is a member of the ATP-binding cassette transporter superfamily. X-ALD is panethnic and affects approximately 1:20,000 males. Phenotypes include the rapidly progressive childhood, adolescent, and adult cerebral forms; adrenomyeloneuropathy, which presents as slowly progressive paraparesis in adults; and Addison disease without neurologic manifestations. These phenotypes are frequently misdiagnosed, respectively, as attention-deficit hyperactivity disorder (ADHD), multiple sclerosis, or idiopathic Addison disease. Approximately 50% of female carriers develop a spastic paraparesis secondary to myelopathic changes similar to adrenomyeloneuropathy. Assays of very long chain fatty acids in plasma, cultured chorion villus cells and amniocytes, and mutation analysis permit presymptomatic and prenatal diagnosis, as well as carrier identification. The timely use of these assays is essential for genetic counseling and therapy. Early diagnosis and treatment can prevent overt Addison disease, and significantly reduce the frequency of the severe childhood cerebral phenotype. A promising new method for mass newborn screening has been developed, the implementation of which will have a profound effect on the diagnosis and therapy of 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.

Combined liquid chromatography-tandem mass spectrometry as an analytical method for high throughput screening for X-linked adrenoleukodystrophy and other peroxisomal disorders: preliminary findings.

Utilizing combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the analytical method, we have demonstrated a ten to sixtyfold excess of lysophosphatidyl choline containing hexacosanoic acid (26:0) in dried blood spots on a filter paper matrix from 25 male patients with X-linked adrenoleukodystrophy and nine patients with peroxisome biogenesis disorders compared to 19 controls. There was no overlap between normal subjects versus affected subjects.

Therapy of X-linked adrenoleukodystrophy.

Current therapies for X-linked adrenoleukodystrophy (X-ALD) include replacement therapy with adrenal steroids, which is mandatory for all patients with impaired adrenal function but does not alter neurological progression significantly; dietary therapy with "Lorenzo's Oil," which appears to have a preventive effect in asymptomatic boys whose brain MRI is normal; and hematopoietic stem cell transplantation in patients in the early stage of the cerebral inflammatory phenotype. Application of these interventions requires careful assessment of the patients' phenotype, which often changes over time. Family screening provides important opportunities for disease prevention.

Cognitive evaluation of neurologically asymptomatic boys with X-linked adrenoleukodystrophy.

BACKGROUND: Various studies have demonstrated abnormal neuropsychological function in boys with the childhood cerebral phenotype of X-linked adrenoleukodystrophy. Not much is known about the cognitive function of neurologically asymptomatic boys with X-linked adrenoleukodystrophy who have normal brain magnetic resonance imaging results. OBJECTIVE: To describe the cognitive profile of 52 neurologically asymptomatic boys with X-linked adrenoleukodystrophy (mean +/- SD age, 6.7 +/- 3.6 years). METHODS: Neuropsychological tests included evaluation of IQ (full-scale IQ, verbal IQ, and performance IQ), 5 major cognitive domains (language, visuospatial skills, perception, visuomotor or graphomotor skills, memory, and attention or executive function), adaptive skills, and academic achievement. Standardized z scores relative to age-appropriate published norms were generated. Association between age and cognitive performance was evaluated using nonparametric Spearman rank correlation and robust median regression adjusting for full-scale IQ and socioeconomic status. RESULTS: All but 4 patients had normal cognitive function. There was a negative correlation between age and visual perception as well as age and visuomotor skills after adjustment for full-scale IQ and socioeconomic status. CONCLUSIONS: This study provides, to our knowledge, the first evidence of overall normal cognitive function in neurologically and radiologically normal boys with X-linked adrenoleukodystrophy, indicating no evidence of neurodevelopmental abnormalities despite the inherent ABCD1 mutation. Subtle deterioration with age was observed in some functional domains. This suggests that prevention and timely institution of therapy can potentially preserve cognitive function seen in patients with the cerebral X-linked adrenoleukodystrophy phenotype. X-linked adrenoleukodystrophy should be considered a candidate disorder for neonatal screening.

Sensorimotor function and axonal integrity in adrenomyeloneuropathy.

BACKGROUND: Gait abnormalities and sensorimotor disturbances are principal defects in adrenomyeloneuropathy (AMN). However, to our knowledge, their association with overall impairment and neuroanatomical changes has not been defined. OBJECTIVES: To understand how sensorimotor impairments create mobility deficits and to analyze how these impairments are related to specific metrics of axonal integrity. DESIGN: Cross-sectional study assessing impairments, including vibration sensation, strength, spasticity, and global measures of walking and balance. Fractional anisotropy was measured to evaluate the integrity of the corresponding brainstem tracts. PARTICIPANTS: Men with AMN and healthy control subjects. RESULTS: Individuals with sensory loss only showed minimal walking deficits. Concomitant strength and sensory loss resulted in slower walking, with abnormal knee control; increased spasticity led to an exaggerated trunk motion and a knee-flexed (crouched) posture. Hip strength was an independent predictor of walking velocity in subjects with AMN. Subjects with sensory loss only had greater sway amplitudes during standing balance testing, which did not worsen with additional impairments. There were significant associations among sway amplitude, great toe vibration sense, and dorsal column fractional anisotropy. Brainstem fractional anisotropy in AMN was significantly negatively correlated with impairment, indicating that overall tract integrity is associated with sensorimotor abnormalities in AMN. CONCLUSIONS: Impairment measures capture specific abnormalities in walking and balance that can be used to direct rehabilitation therapy in AMN. Tract-specific magnetic resonance imaging metrics, such as fractional anisotropy (used herein to evaluate structure-function relationships), significantly reflect disease severity in AMN.

Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxisomal localization, and interaction with the Pex1p x Pex6p complex.

Peroxisome biogenesis disorders (PBDs) are fatal autosomal recessive diseases and are caused by impaired peroxisome biogenesis. PBDs are genetically heterogeneous and classified into 13 complementation groups (CGs). CG8 is one of the most common groups and has three clinical phenotypes, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD). We recently isolated PEX26 as the pathogenic gene for PBD of CG8. Pex26p functions in recruiting to peroxisomes the complexes of the AAA ATPase peroxins, Pex1p and Pex6p. In the present work, we identified four distinct mutations in PEX26 from five patients of CG8 PBD including 2 with ZS and 3 with IRD, in addition to 7 mutant alleles in 8 patients in the first report describing the pathogenic PEX26 gene for CG8 PBD. Phenotype-genotype analyses revealed that temperature-sensitive (ts) peroxisome assembly gave rise to a milder IRD in contrast to the non-ts phenotype of the cells from ZS patients. Furthermore, we present several lines of evidence that show that the instability, insufficient binding to Pex1p x Pex6p complexes, or mislocalization of patient-derived Pex26p mutants is most likely responsible for the CG8 PBDs.

Adreno-leukodystrophy: oxidative stress of mice and men.

X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that affects primarily nervous system myelin and axons as well as the adrenal cortex. Several divergent clinical phenotypes can occur in the same family; thus, there is no correlation between the clinical phenotype and the mutation in the ABCD1 gene in this disease. The most urgent and unresolved clinical issue is the fulminant inflammatory (immune) demyelination of the central nervous system in which a variety of cellular participants, cytokines, and chemokines are noted. A knockout mouse model exhibits mitochondrial deficits and axonal degeneration, but not inflammatory demyelination. To determine whether oxidative stress and damage might play a pathogenic role, we assessed standard biochemical and immunohistochemical markers of such activity both in our knockout mouse model and patients. We find that oxidative stress, as judged by increased immunoreactivity for the mitochondrial manganese-superoxide dismutase, is present in the knockout mouse liver, adrenal cortex, and renal cortex, tissues that normally express high levels of ABCD1 but no evidence of oxidative damage. The brain does not exhibit either oxidative stress or damage. On the other hand, both the human adrenal cortex and brain show evidence of oxidative stress (e.g. hemoxygenase-1 and manganese-superoxide dismutase) and oxidative damage, particularly from lipid peroxidation (4-hydroxynonenal and malondialdehyde). The presence of nitrotyrosylated proteins is strong circumstantial evidence for the participation of the highly toxic peroxynitrite molecule, whereas the demonstration of interferon gamma and interleukin-12 is indicative of a TH1 response in the inflammatory demyelinative lesions of the cerebral phenotype. These differences between the adreno-leukodystrophy mouse and human patients are intriguing and may provide a clue to the phenotypic divergence in this disease.