Identification of new mutations in Israeli patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder characterized by impaired peroxisomal betaoxidation of very-long-chain fatty acids (VLCFAs). This is probably due to reduced activation of the VLCFAs and results in demyelination of the nervous system and adrenocortical insufficiency. The ALD gene is localized on Xq28, has 10 exons and encodes a protein of 745 amino acids with significant homology to the membrane peroxisomal protein PMP70. Characterizing the disease causing mutations is of importance in prenatal diagnosis because 12-20% of women who are obligatory carriers show false-negative results when tested for VLCFA in plasma. We have analyzed DNA from blood samples of 7 Jewish (5 Sephardi and 2 Ashkenazi) and 3 Arab Israeli families suffering from ALD. Five missense-type mutations were identified: R104H, Y174C, L229P, R401Q, and G512C. A single mutation, R464X, was nonsense, and two, Y171 frameshift and E471 frameshift, were frameshift. Interestingly, a single mutation was identified in three families of Moroccan Jewish descent, probably due to a founder effect.     

X-linked adrenoleukodystrophy phenotype is independent of ABCD2 genotype

Strikingly variable clinical phenotypes can be found in X-linked adrenoleukodystrophy (X-ALD) even with the same ABCD1 mutation. ABCD2 is the closest homolog to ABCD1. Since ABCD2 overexpression complements the loss of ABCD1 in vivo and in vitro, we have investigated the possible role of the ABCD2 gene locus as determinant of X-ALD phenotypes. Sequence and segregation analysis of the ABCD2 gene, in a large X-ALD family with different phenotypes disclosed that the identical ABCD2 alleles were inherited in brothers affected by mild (noncerebral) versus severe (childhood cerebral) X-ALD phenotypes. Moreover, two independent association studies of ABCD2 polymorphisms and clinical phenotypes showed an even allele distribution in different X-ALD phenotypes and controls. Based on these findings ABCD2 can be excluded as a major modifier locus for clinical diversity in X-ALD. These findings are of particular importance for the attempt of pharmacological induction of ABCD2 as a possible therapeutic approach in X-ALD.     

Evidence that oxidative stress is a risk factor for the development of squamous cell carcinoma in renal transplant patients

Renal transplant patients are at a greatly increased risk of skin malignancy, particularly squamous cell carcinoma (SCC), a tumor closely associated with UV exposure. There is also significant interindividual skin cancer risk among transplant patients, with evidence suggesting that this derives from variation in response to oxidative stress. Our aim was to assess urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), by liquid chromatography-tandem mass spectrometry, in renal transplant patients with and without SCC. The relationships between SCC and urinary 8-oxodG were analyzed by conditional logistic regression and those between 8-oxodG and other candidate variables by linear regression, correcting for the effect of SCC. In SCC patients, urinary 8-oxodG was significantly elevated (p=0.03), both pre- and post-tumor development, compared to non-SCC transplant patients. Secondary analyses indicated that 8-oxodG was related to current heavy smoking (p=0.02) and darker skin type (p=0.02), but not measures of previous chronic sun exposure or current age and gender. Although subject numbers were limited, immunosuppression with azathioprine was positively associated with 8-oxodG in all patients combined (p=0.02). These results demonstrate, for the first time, that a subpopulation of renal transplant patients is under greater oxidative burden, and it is this population that is particularly predisposed to skin cancer.     

Diagnostic patterns of very-long-chain fatty acids in plasma of patients with X-linked adrenoleukodystrophy

Pattern recognition analysis on the levels of the very-long-chain fatty acids (VLCFAs) in plasma is described for the visual discrimination of X-linked adrenoleukodystrophy (X-ALD) patients from normal healthy group. Plasma VLCFA compositions of 58 normal subjects and 16 X-ALD patients were examined by gas chromatography as their methyl esters to determine the area percentages of behenic acid (C22:0), lignoceric acid (C24:0) and hexacosanoic acid (C26:0) in the total fatty acids, and the concentration (μg/ml) of C26:0. When star symbol plotting was applied to the VLCFA values of C22:0 (%), C24:0 (%), C26:0 (%), C24:0/C22:0, C26:0/C22:0 and C26:0 (μg/ml) after normalization to the corresponding median values in normal group, the resulting deformed hexagonal star pattern was characteristic of each patient. Therefore, simple visual comparison with the equilateral hexagon of normal group average as the control pattern enabled one readily to discriminate X-ALD patients from the normal group. Additionally, canonical discriminant analysis performed on the six unnormalized VLCFA values correctly classified 74 plasma specimens into two separate clusters according to normal subject or X-ALD patient in the canonical plot.     

Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics.

Methods

X- ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied.

Results

We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p.Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1–12.7) and 24.7 (19.8–29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation.

Conclusions

This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.     

Mutational and protein analysis of patients and heterozygous women with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder associated with impaired beta-oxidation of very-long-chain fatty acids (VLCFA), is due to mutations in a gene encoding a peroxisomal ATP-binding cassette (ABC) transporter (ALD protein [ALDP]). We analyzed the open reading frame of the ALD gene in 44 French ALD kindred by using SSCP or denaturing gradient-gel electrophoresis and studied the effect of mutations on ALDP by immunocytofluorescence and western blotting of fibroblasts and/or white blood cells. Mutations were detected in 37 of 44 kindreds and were distributed over the whole protein-coding region, with the exception of the C terminus encoded in exon 10. Except for two mutations (delAG1801 and P560L) observed four times each, nearly every ALD family has a different mutation. Twenty-four of 37 mutations were missense mutations leading to amino acid changes located in or close to putative transmembrane segments (TMS 2, 3, 4, and 5), in the EAA-like motif and in the nucleotide fold of the ATP-binding domain of ALDP. Of 38 ALD patients tested, 27 (71%) lacked ALDP immunoreactivity in their fibroblasts and/or white blood cells. More than half of missense mutations studied (11 of 21) resulted in a complete lack of ALDP immunoreactivity, and six missense mutations resulted in decreased ALDP expression. The fibroblasts and/or white blood cells of 15 of 15 heterozygous carrier from ALD kindred with no ALDP showed a mixture of positive- and negative-ALDP immunoreactivity due to X-inactivation. Since 5%-15% of heterozygous women have normal VLCFA levels, the immunodetection of ALDP in white blood cells can be applicable in a majority of ALD kindred, to identify heterozygous women, particularly when the ALD gene mutation has not yet been identified.     

Angiotensin II-induced reduction in exercise capacity is associated with increased oxidative stress in skeletal muscle

Angiotensin II (ANG II)-induced oxidative stress has been known to be involved in the pathogenesis of cardiovascular diseases. We have reported that the oxidative stress in skeletal muscle can limit exercise capacity in mice (16). We thus hypothesized that ANG II could impair the skeletal muscle energy metabolism and limit exercise capacity via enhancing oxidative stress. ANG II (50 ng·kg(-1)·min(-1)) or vehicle was infused into male C57BL/6J mice for 7 days via subcutaneously implanted osmotic minipumps. ANG II did not alter body weight, skeletal muscle weight, blood pressure, cardiac structure, or function. Mice were treadmill tested, and expired gases were analyzed. The work to exhaustion (vertical distance × body weight) and peak oxygen uptake were significantly decreased in ANG II compared with vehicle. In mitochondria isolated from skeletal muscle, ADP-dependent respiration was comparable between ANG II and vehicle, but ADP-independent respiration was significantly increased in ANG II. Furthermore, complex I and III activities were decreased in ANG II. NAD(P)H oxidase activity and superoxide production by lucigenin chemiluminescence were significantly increased in skeletal muscle from ANG II mice. Treatment of ANG II mice with apocynin (10 mmol/l in drinking water), an inhibitor of NAD(P)H oxidase activation, completely inhibited NAD(P)H oxidase activity and improved exercise capacity, mitochondrial respiration, and complex activities in skeletal muscle. ANG II-induced oxidative stress can impair mitochondrial respiration in skeletal muscle and limit exercise capacity.     

Increased oxidative stress is correlated with mitochondrial dysfunction in chagasic patients

Previously, we have shown in an experimental model of Trypanosoma cruzi infection that increased oxidative stress and antioxidant insufficiency are associated with myocardial (cellular and mitochondrial) oxidative damage and mitochondrial functional decline and might be of pathological significance in Chagas disease. In the present study, we investigated whether enhanced oxidative stress and mitochondrial functional decline are found in human chagasic patients. Our data show substantially higher plasma (two-four-fold) and mitochondrial (67%) malonylaldehyde (MDA) levels in chagasic (n = 80, group 2) compared to healthy (n = 50, group 1) subjects. Moreover, antioxidant defense was compromised in chagasic patients. Hence, we noted a 50% decline in glutathione content and losses of 31, 60, and 68% in glutathione peroxidase, superoxide dismutase (SOD), and MnSOD activities, respectively, relative to the findings in healthy controls. Further, chagasic subjects exhibited decreased mitochondrial respiratory complex (CI: 72%; CIII: 71%) activities. Nonchagasic cardiomyopathy subjects (n = 20, group 3) exhibited marginally higher plasma MDA levels compared to gp1 subjects and were not compromised in plasma antioxidant defense capacity. These data suggest that human chagasic patients sustain an antioxidant/oxidant imbalance and a mitochondrial decline of respiratory complex activities in the circulatory system. A positive correlation between increased MDA levels, MnSOD decline, and inhibition of respiratory complexes suggests that oxidative stress may contribute to mitochondrial dysfunction in chagasic patients.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

ABCD1 mutations and phenotype distribution in Chinese patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder resulting from mutations within the ABCD1 gene. Adrenomyeloneuropathy (AMN) and childhood cerebral ALD (CCALD) are most common phenotypes in the Western ALD patients. Here we performed mutation analysis of ABCD1 in 10 Chinese ALD families and identified 8 mutations, including one novel deletion (c.1477_1488 + 11del23) and 7 known mutations. Mutations c.1772G>A and c.1816T>C were first reported in the Chinese patients. Mutations c.1661G>A and c.1679C>T were demonstrated to be de novo mutations. The dinucleotide deletion 1415_16delAG, described as a mutational hotspot in different ethnic groups, was identified in two families. In addition, we performed a retrospective nation-wide mutation study of X-linked ALD in China based on a literature review. The retrospective study further confirmed the hypothesis that exon 6 is a potential mutation cluster region in the Asian populations. Furthermore, it suggested that CCALD is the most common phenotype in China.     

Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.     

Mouse very long-chain acyl-CoA synthetase in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by accumulation of very long-chain fatty acids (VLCFA). This accumulation has been attributed to decreased VLCFA beta-oxidation and peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity. The X-ALD gene, ABCD1, encodes a peroxisomal membrane ATP binding cassette transporter, ALDP, that is hypothesized to affect VLCS activity in peroxisomes by direct interaction with the VLCS enzyme. Recently, a VLCS gene that encodes a protein with significant sequence identity to known rat and human peroxisomal VLCS protein has been identified in mice. We find that the mouse VLCS gene (Vlcs) encodes an enzyme (Vlcs) with VLCS activity that localizes to peroxisomes and is expressed in X-ALD target tissues. We show that the expression of Vlcs in the peroxisomes of X-ALD mouse fibroblasts improves VLCFA beta-oxidation in these cells, implying a role for this enzyme in the biochemical abnormality of X-ALD. X-ALD mice, which accumulate VLCFA in tissues, show no change in the expression of Vlcs, the subcellular localization of Vlcs, or general peroxisomal VLCS activity. These observations imply that ALDP is not necessary for the proper expression or localization of Vlcs protein, and the control of VLCFA levels does not depend on the direct interaction of Vlcs and ALDP.     

Altered expression of ALDP in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder with variable phenotypic expression that is characterized by elevated plasma and tissue levels of very long-chain fatty acids. However, the product of the gene defective in ALD (ALDP) is a membrane transporter of the ATP-binding cassette family of proteins and is not related to enzymes known to activate or oxidize fatty acids. We generated an antibody that specifically recognizes the C-terminal 18 amino acids of ALDP and can detect ALDP by indirect immunofluorescence. To better understand the mechanism by which mutations in ALDP lead to disease, we used this antibody to examine the subcellular distribution and relative abundance of ALDP in skin fibroblasts from normal individuals and ALD patients. Punctate immunoreactive material typical of fibroblast peroxisomes was observed in cells from seven normal controls and eight non-ALD patients. Of 35 ALD patients tested, 17 had the childhood-onset cerebral form of the disease, 13 had the milder adult phenotype adrenomyeloneuropathy, 3 had adrenal insufficiency only, and 2 were affected fetuses. More than two-thirds (69%) of all patients studied showed no punctate immunoreactive material. There was no correlation between the immunofluorescence pattern and clinical phenotype. We determined the mutation in the ALD gene in 15 of these patients. Patients with either a deletion or frameshift mutation lacked ALDP immunoreactivity, as expected. Four of 11 patients with missense mutations were also immunonegative, indicating that these mutations affected the stability or localization of ALDP. In the seven immunopositive patients with missense mutations, correlation of the location and nature of the amino acid substitution may provide new insights into the function of this peroxisomal membrane protein. Furthermore, the study of female relatives of immunonegative ALD probands may aid in the assessment of heterozygote status.     

X-linked adrenoleukodystrophy mice demonstrate abnormalities in cholesterol metabolism

The neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is caused by ABCD1 mutations and characterized by very long-chain fatty acid (VLCFA) accumulation. Cholesterol-lowering normalized VLCFA in fibroblasts and plasma of X-ALD patients. We show that in cultured cells, cholesterol-loading induces ABCD1. In X-ALD mice, plasma cholesterol is elevated and not further increasable by cholesterol-feeding, whereas hepatic HMG-CoA reductase and Abcd2 are downregulated. Upon cholesterol modulation, brain VLCFA increased in X-ALD mice, but decreased in controls. In murine X-ALD fibroblasts, cholesterol-lowering did not normalize VLCFA. Thus, ALDP-deficiency and VLCFA are linked to cholesterol but species differences complicate evaluating cholesterol-lowering drugs in X-ALD mice.     

Patients with dystrophinopathy show evidence of increased oxidative stress

Duchenne muscular dystrophy (DMD) is associated with an increase in oxidative stress. We measured 24 h 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion in 24 patients with MD (DMD + Becker's MD), 23 with myotonic dystrophy, and 34 healthy controls. The 8-OHdG/creatinine ratio was higher in patients with dystrophinopathy ( upward arrow 48%, p <.01) but not myotonic dystrophy, as compared to healthy controls. These results indicate that 8-OHdG excretion can be used as a marker of oxidative stress in clinical trials with dystrophinopathy.