Adrenoleukodystrophy Protein Research Articles

X-linked adrenoleukodystrophy and primary adrenal insufficiency.

X-linked adrenoleukodystrophy (X-ALD; OMIM:300100) is a progressive neurodegenerative disorder caused by a congenital defect in the ATP-binding cassette transporters sub-family D member 1 gene (ABCD1) producing adrenoleukodystrophy protein (ALDP). According to population studies, X-ALD has an estimated birth prevalence of 1 in 17.000 subjects (considering both hemizygous males and heterozygous females), and there is no evidence that this prevalence varies among regions or ethnic groups. ALDP deficiency results in a defective peroxisomal β-oxidation of very long chain fatty acids (VLCFA). As a consequence of this metabolic abnormality, VLCFAs accumulate in nervous system (brain white matter and spinal cord), testis and adrenal cortex. All X-ALD affected patients carry a mutation on the ABCD1 gene. Nevertheless, patients with a defect on the ABCD1 gene can have a dramatic difference in the clinical presentation of the disease. In fact, X-ALD can vary from the most severe cerebral paediatric form (CerALD), to adult adrenomyeloneuropathy (AMN), Addison-only and asymptomatic forms. Primary adrenal insufficiency (PAI) is one of the main features of X-ALD, with a prevalence of 70% in ALD/AMN patients and 5% in female carriers. The pathogenesis of X-ALD related PAI is still unclear, even if a few published data suggests a defective adrenal response to ACTH, related to VLCFA accumulation with progressive disruption of adrenal cell membrane function and ACTH receptor activity. The reason why PAI develops only in a proportion of ALD/AMN patients remains incompletely understood. A growing consensus supports VLCFA assessment in all male children presenting with PAI, as early diagnosis and start of therapy may be essential for X-ALD patients. Children and adults with PAI require individualized glucocorticoid replacement therapy, while mineralocorticoid therapy is needed only in a few cases after consideration of hormonal and electrolytes status. Novel approaches, such as prolonged release glucocorticoids, offer potential benefit in optimizing hormonal replacement for X-ALD-related PAI. Although the association between PAI and X-ALD has been observed in clinical practice, the underlying mechanisms remain poorly understood. This paper aims to explore the multifaceted relationship between PAI and X-ALD, shedding light on shared pathophysiology, clinical manifestations, and potential therapeutic interventions.

Diagnosing X-Linked Adrenoleukodystrophy after Implementation of Newborn Screening: A Reference Laboratory Perspective.

Adrenoleukodystrophy (ALD) is caused by pathogenic variants in the ABCD1 gene, encoding for the adrenoleukodystrophy protein (ALDP), leading to defective peroxisomal β-oxidation of very long-chain and branched-chain fatty acids (VLCFA). ALD manifests in both sexes with a spectrum of phenotypes, but approximately 35% of affected males develop childhood cerebral adrenoleukodystrophy (CCALD), which is lethal without hematopoietic stem cell transplant performed before symptoms start. Hence, ALD was added to the Recommended Uniform Screening Panel after the successful implementation in New York State (2013-2016). To date, thirty-five states have implemented newborn screening (NBS) for ALD, and a few programs have reported on the successes and challenges experienced. However, the overall impact of NBS on early detection of ALD has yet to be fully determined. Here, we conducted a retrospective analysis of VLCFA testing performed by our reference laboratory (ARUP Laboratories, Salt Lake City, UT, USA) over 10 years. Rate of detection, age at diagnosis, and male-to-female ratio were evaluated in patients with abnormal results before and after NBS implementation. After NBS inclusion, a significant increase in abnormal results was observed (471/6930, 6.8% vs. 384/11,670, 3.3%; p < 0.0001). Patients with ALDP deficiency identified via NBS were significantly younger (median age: 30 days vs. 21 years; p < 0.0001), and males and females were equally represented. ALD inclusion in NBS programs has increased pre-symptomatic detection of this disease, which is critical in preventing adrenal crisis as well as the severe cerebral form.

Two Single Nucleotide Deletions in the ABCD1 Gene Causing Distinct Phenotypes of X-Linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a rare inborn error of the peroxisomal metabolism caused by pathologic variants in the ATP-binding cassette transporter type D, member 1 (ABCD1) gene located on the X-chromosome. ABCD1 protein, also known as adrenoleukodystrophy protein, is responsible for transport of the very long chain fatty acids (VLCFA) from cytoplasm into the peroxisomes. Therefore, altered function or lack of the ABCD1 protein leads to accumulation of VLCFA in various tissues and blood plasma leading to either rapidly progressive leukodystrophy (cerebral ALD), progressive adrenomyeloneuropathy (AMN), or isolated primary adrenal insufficiency (Addison's disease). We report two distinct single nucleotide deletions in the ABCD1 gene, c.253delC [p.Arg85Glyfs*18] in exon 1, leading to both cerebral ALD and to AMN phenotype in one family, and c.1275delA [p.Phe426Leufs*15] in exon 4, leading to AMN and primary adrenal insufficiency in a second family. For the latter variant, we demonstrate reduced mRNA expression and a complete absence of the ABCD1 protein in PBMC. Distinct mRNA and protein expression in the index patient and heterozygous carriers does not associate with VLCFA concentration in plasma, which is in line with the absence of genotype-phenotype correlation in X-ALD.

Leriglitazone: frustration and hope in adrenoleukodystrophy

X-linked adrenoleukodystrophy is a rare disease caused by loss of function mutations in the ABCD1 gene. The clinical spectrum in males ranges from adrenocortical insufficiency (Addison's disease) to myeloneuropathy, with prominent spastic gait and bladder disturbance, and to cerebral leukodystrophy, often resulting in rapidly progressive dementia and disability.1 ABCD1 encodes the peroxisomal membrane adrenoleukodystrophy protein, which is involved in the transmembrane transport and beta-oxidation of very long-chain fatty acids (VLCFAs).

Adrenoleukodystrophy Protein (ALDP) Antibody Formation and Graft Loss in Gene Therapy for Adrenoleukodystrophy

A nine-year boy with adrenoleukodystrophy (ALD) due to an ABCD1 whole gene deletion was diagnosed with the active cerebral form of disease characterized by demyelination and gadolinium enhancement on brain MRI. Allogeneic hematopoietic stem cell transplant (HCT) or lentiviral correction of autologous CD34+cell-based gene therapy (GT) are the only known mechanisms to arrest the progression of cerebral disease. He was treated with GCSF/plerixafor to mobilize autologous CD34+ cells which were then transduced with the ABCD1 expressing lentiviral vector. He received myeloablative conditioning with busulfan and fludarabine followed by lentivirus-based GT. Initial viral copy number (VCN) was 0.8592 in whole blood at 30 days post HCT and MRI's showed disease stabilization with significant decrease in gadolinium enhancement at 60 and 100 days after GT. Unfortunately, VCN then declined and subsequent MRI's showed re-emergence of gadolinium enhancement and disease progression at 6 months after GT. Antibodies to the ABCD1 gene product (ALDP) were detected in the patient's plasma by immunoblot at 9 months after GT. Antisera also showed specific reactivity to human peroxisomes in immunofluorescence assays, suggesting that generation of an immune response had occurred following GT. Evaluation of pre-GT plasma did not find any anti-ALDP. Evaluation of post HCT plasma from other patients undergoing either GT or allo-HCT did not show anti-ALPD reactivity suggesting this was a unique phenomenon to this patient. Peptide mapping indicated the antisera bound mostly to the C-terminal portion of ALDP in five distinct peptide regions. Subtyping of the antibody indicated it was IgG and not IgM at 9 months post GT. Specific anti-ALDP IgG Subtypes detected were IgG1 and IgG3. Immune adjuvant therapy using rituximab and daratumumab failed to significantly reduce plasma reactivity to ALDP. Due to disease advancement on MRI, an allogeneic HCT was performed approximately 12 months after initial GT with an 5/6 HLA matched umbilical cord blood unit. Donor neutrophil engraft occurred on day +17, donor platelet engraftment occurred on day +35. Chimerism studies revealed 100% engraftment in CD33+cells and 90% engraftment in CD3+ cells by 60 days post HCT. The MRI's at 30-, 60-, and 100-days post allo-HCT showed stabilizing cerebral disease and fading gadolinium enhancement. Importantly, plasma collected at 30-, 60-days, and 100-days post allo-HCT showed no reactivity to ALPD. Conclusion. This is the first case of anti-ALDP antibodies occurring after gene therapy for cerebral ALD. Antibody generation may have been related to the unique ABCD1 whole gene deletion condition within this child which is very rare as the majority of ALD patients have missense or non-sense mutations in ABCD1. Whether anti-ALDP presence was a causal or an unrelated phenomenon to the loss of the autologous vector-containing cells is not currently known at the time of submission. These findings highlight to need to scrutinize patients prior to before and after GT for the presence of antibody to the protein product being introduced. This may be especially important in the case where the patient's immune system has never seen the protein (like in this case of a whole gene deletion) and therefore would have never been "educated".

Structural and functional insights of the human peroxisomal ABC transporter ALDP.

Adrenoleukodystrophy protein (ALDP) is responsible for the transport of very-long-chain fatty acids (VLCFAs) and corresponding CoA-esters across the peroxisomal membrane. Dysfunction of ALDP leads to peroxisomal metabolic disorder exemplified by X-linked adrenoleukodystrophy (ALD). Hundreds of ALD-causing mutations have been identified on ALDP. However, the pathogenic mechanisms of these mutations are restricted to clinical description due to limited structural and biochemical characterization. Here we report the cryo-electron microscopy structure of human ALDP with nominal resolution at 3.4 Å. ALDP exhibits a cytosolic-facing conformation. Compared to other lipid ATP-binding cassette transporters, ALDP has two substrate binding cavities formed by the transmembrane domains. Such structural organization may be suitable for the coordination of VLCFAs. Based on the structure, we performed integrative analysis of the cellular trafficking, protein thermostability, ATP hydrolysis, and the transport activity of representative mutations. These results provide a framework for understanding the working mechanism of ALDP and pathogenic roles of disease-associated mutations.

ODP310 Early Onset Adrenal Dysfunction in Infants with Adrenoleukodystrophy

Abstract Introduction X-linked adrenoleukodystrophy (ALD) is caused by mutations in the ABCD1 gene encoding the adrenoleukodystrophy protein that transports very-long-chain fatty acids (VLCFAs) to peroxisomes for degradation. An early manifestation is primary adrenal insufficiency (AI). Our clinic's protocol to screen for AI involves obtaining a morning cortisol and ACTH every 6 months, followed by cosyntropin (ACTH) stimulation testing if screening ACTH &amp;gt;100pg/mL or cortisol &amp;lt;5μg/dL. The peak incidence of AI is 3-10 years old, however there have been reports of infants as young as 4.5 months. 1 We present 2 asymptomatic boys with ALD demonstrating AI already at 6-7 months. Cases Patient 1 is a 6 month old full-term male with ALD detected on New York state (NYS) newborn screen and a hemizygous pathogenic variant, c.1201C&amp;gt;T, p. R401W in the ABCD1 gene. At 6 weeks, ACTH and cortisol levels were acceptable (table 1). After screening ACTH was elevated(175pg/mL) at 5 months, ACTH stimulation testing was performed at 6 months. Baseline cortisol was 11ug/dL and stimulated cortisol was insufficient at 13ug/dL. There was no hyperpigmentation, but were neurological concerns such as inability to roll over, left arm preference, and waning moro response. A brain MRI was recommended. Patient 2 is a 7 month old full-term male with ALD detected on NYS newborn screen and a hemizygous pathogenic variant, c.1661G&amp;gt;A (p. R554H) in the ABCD1 gene. At 6 weeks, ACTH and cortisol levels were acceptable (table 2). At 7 months, ACTH was elevated (120.7pg/mL). ACTH stimulation testing was performed with baseline cortisol of 6.9ug/dL and insufficiently stimulated cortisol of 13.5ug/dL. Neurological evaluation was normal. Because of inadequate rises in cortisol to ACTH stimulation, stress dosing hydrocortisone was recommended for both patients. Discussion These infants with early onset adrenal dysfunction raise the question of whether the peak incidence of AI is earlier than previously thought. This would not be surprising as VLCFAs were found to accumulate in fetal adrenal glands. Our findings further support the need for widespread newborn screening for ALD, followed by routine adrenal screening consistent with published guidelines to avoid adrenal crisis. In a case series of 2 patients with ALD and early onset AI, the reported mutations were p. R660W and p. R401Q. 1 In patient 1, a mutation at position 401 of the ALD protein was also reported, which stresses the importance of registries to correlate genotypes and phenotypes. Early onset adrenal disease has not yet been correlated with early neurologic disease. However, the neurological changes noted in patient 1 raise this concern and highlight the importance of multidisciplinary clinics with endocrinology and neurology. Sources: 1. Eng, L., &amp; Regelmann, M. O. (2019). Early Onset Primary Adrenal Insufficiency in Males with Adrenoleukodystrophy: Case Series and Literature Review. The Journal of pediatrics,211, 211–214. https://doi.org/10.1016/j. jpeds.2019. 04. 021 Presentation: No date and time listed

Long-Term Disease Prevention with a Gene Therapy Targeting Oligodendrocytes in a Mouse Model of Adrenomyeloneuropathy.

Adrenomyeloneuropathy (AMN) is a late-onset axonopathy of spinal cord tracts caused by mutations of the ABCD1 gene that encodes adrenoleukodystrophy protein (ALDP), a peroxisomal transporter of very long-chain fatty acids (VLCFA). Disturbed metabolic interaction between oligodendrocytes (OL) and axons is suspected to play a major role in AMN axonopathy. To develop a vector targeting OL, the human ABCD1 gene driven by a short 0.3 kb part of the human myelin-associated glycoprotein (MAG) promoter was packaged into an adeno-associated viral serotype 9 (rAAV9). An intravenous injection of this vector on postnatal day 10 in Abcd1-/- mice, a model of AMN, allowed a near normal motor performance to persist for 24 months, while age-matched untreated mice developed major defects of balance and motricity. Three weeks postvector, 50-54% of spinal cord white matter OL was expressing human ALDP (hALDP) at the cervical level, and only 6-7% after 24 months. In addition, 29-32% of cervical spinal cord astrocytes at 3 weeks and 16-19% at 24 months also expressed ALDP. C26:0-lysoPC, a sensitive VLCFA marker of AMN, was lower by 41% and 50%, respectively, in the spinal cord and brain of vector-treated compared with untreated mice. In a nonhuman primate, the intrathecal injection of the rAAV9-MAG vector induced abundant ALDP expression at 3 weeks in spinal cord OL (43%, 29%, and 26% at cervical, thoracic, and lumbar levels) and cerebellum OL (35%). In addition, 33-41% of spinal cord astrocytes expressed hALDP, and 27% of cerebellar astrocytes. To our knowledge, OL targeting had not been obtained before in primates with other vectors or promoters. The current results thus provide a robust proof-of-concept not only for the gene therapy of AMN but also for other central nervous system diseases, where the targeting of OL with the rAAV9-MAG vector may be of interest.

Newborn Screen for X-Linked Adrenoleukodystrophy Using Flow Injection Tandem Mass Spectrometry in Negative Ion Mode.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder caused by pathogenic variants in the ATP-binding cassette subfamily D member 1 gene (ABCD1) that encodes the adrenoleukodystrophy protein (ALDP). Defects in ALDP result in elevated cerotic acid, and lead to C26:0-lysophosphatidylcholine (C26:0-LPC) accumulation, which is the primary biomarker used in newborn screening (NBS) for X-ALD. C26:0-LPC levels were measured in dried blood spot (DBS) NBS specimens using a flow injection analysis (FIA) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) performed in negative ion mode. The method was validated by assessing and confirming linearity, accuracy, and precision. We have also established C26:0-LPC cutoff values that identify newborns at risk for X-ALD. The mean concentration of C26:0-LPC in 5881 de-identified residual routine NBS specimens was 0.07 ± 0.02 µM (mean + 1 standard deviation (SD)). All tested true X-ALD positive and negative samples were correctly identified based on C26:0-LPC cutoff concentrations for borderline between 0.15 µM and 0.22 µM (mean + 4 SD) and presumptive screening positive at ≥0.23 µM (mean + 8 SD). The presented FIA method shortens analysis run-time to 1.7 min, while maintaining the previously established advantage of utilizing negative mode MS to eliminate isobaric interferences that could lead to screening false positives.

Identification of Two Novel Mutations of ABCD1 Gene in Pedigrees with X-Linked Adrenoleukodystrophy and Review of the Literature.

Background X-linked adrenoleukodystrophy (ALD) is an inherited peroxisomal metabolism disorder, resulting from the loss-of-function mutation of ATP-binding cassette protein subfamily D1 (ABCD1) gene. The dysfunction of ALD protein, a peroxisomal ATP-binding cassette transporter, results in the excessive saturated very long-chain fatty acids (VLCFAs) accumulation in organs including the brain, spine, and adrenal cortex. X-ALD is characterized as the childhood, adolescent, adult cerebral ALD, adrenomyeloneuropathy (AMN), adrenal insufficiency, and asymptomatic phenotypes, exhibiting a high variety of clinical neurological manifestations with or without adrenocortical insufficiency. Results In this study, we reported two cases of X-ALD, which were first diagnosed as adrenal insufficiency (Addison's disease) and treated with adrenocortical supplement. However, both of the cases progressed as neurological symptoms and signs after decades. Elevated VLCFAs level, brain MRI scan, and genetic analysis confirmed final diagnosis. In addition, we identified two novel mutations of ABCD1 gene, NM_000033.3 (ABCD1): c.874_876delGAG (p.Glu292del) and NM_000033.3 (ABCD1): c.96_97delCT (p.Tyr33Profs∗161), in exon 1 of ABCD1 gene. Sanger sequencing confirmed that the proband's mother of the first case was heterozygous carrying the same variant. Adrenal insufficiency-only type is very rare; however, it may be the starting performance of X-ALD. In addition, we summarized reported mutation sites and clinical manifestations to investigate the correlationship of phenotype-genotype of X-ALD. Conclusions The early warning manifestations should be noticed, and the probability of X-ALD should be considered. This report could be beneficial for the early diagnosis and genetic counseling for patients with X-ALD.

Biochemical Studies in Fibroblasts to Interpret Variants of Unknown Significance in the ABCD1 Gene.

Due to newborn screening for X-linked adrenoleukodystrophy (ALD), and the use of exome sequencing in clinical practice, the detection of variants of unknown significance (VUS) in the ABCD1 gene is increasing. In these cases, functional tests in fibroblasts may help to classify a variant as (likely) benign or pathogenic. We sought to establish reference ranges for these tests in ALD patients and control subjects with the aim of helping to determine the pathogenicity of VUS in ABCD1. Fibroblasts from 36 male patients with confirmed ALD, 26 healthy control subjects and 17 individuals without a family history of ALD, all with an uncertain clinical diagnosis and a VUS identified in ABCD1, were included. We performed a combination of tests: (i) a test for very-long-chain fatty acids (VLCFA) levels, (ii) a D3-C22:0 loading test to study the VLCFA metabolism and (iii) immunoblotting for ALD protein. All ALD patient fibroblasts had elevated VLCFA levels and a reduced peroxisomal ß-oxidation capacity (as measured by the D3-C16:0/D3-C22:0 ratio in the D3-C22:0 loading test) compared to the control subjects. Of the VUS cases, the VLCFA metabolism was not significantly impaired (most test results were within the reference range) in 6/17, the VLCFA metabolism was significantly impaired (most test results were within/near the ALD range) in 9/17 and a definite conclusion could not be drawn in 2/17 of the cases. Biochemical studies in fibroblasts provided clearly defined reference and disease ranges for the VLCFA metabolism. In 15/17 (88%) VUS we were able to classify the variant as being likely benign or pathogenic. This is of great clinical importance as new variants will be detected.

Glycosphingolipids with Very Long-Chain Fatty Acids Accumulate in Fibroblasts from Adrenoleukodystrophy Patients.

Adrenoleukodystrophy (X-ALD) is an X-linked genetic disorder caused by mutation of the ATP-binding cassette subfamily D member 1 gene, which encodes the peroxisomal membrane protein, adrenoleukodystrophy protein (ALDP). ALDP is associated with the transport of very-long-chain fatty acids (VLCFAs; carbon chain length ≥ 24) into peroxisomes. Defective ALDP leads to the accumulation of saturated VLCFAs in plasma and tissues, which results in damage to myelin and the adrenal glands. Here, we profiled the glycosphingolipid (GSL) species in fibroblasts from X-ALD patients. Quantitative analysis was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry with a chiral column in multiple reaction monitoring (MRM) mode. MRM transitions were designed to scan for precursor ions of long-chain bases to detect GSLs, neutral loss of hexose to detect hexosylceramide (HexCer), and precursor ions of phosphorylcholine to detect sphingomyelin (SM). Our results reveal that levels of C25 and C26-containing HexCer, Hex2Cer, NeuAc-Hex2Cer, NeuAc-HexNAc-Hex2Cer, Hex3Cer, HexNAc-Hex3Cer, and SM were elevated in fibroblasts from X-ALD patients. In conclusion, we precisely quantified SM and various GSLs in fibroblasts from X-ALD patients and determined structural information of the elevated VLCFA-containing GSLs.

Long-Term Follow-Up of Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy.

In 2009, cerebral adrenoleukodystrophy (c-ALD) became the first brain disease to be treated with lentiviral (LV)-based hematopoietic stem cell gene therapy with the ABCD1 gene in four boys (P1-P4) who had demyelinating lesions expected to be lethal in the short term and no bone marrow donor. We report the clinical and magnetic resonance imaging (MRI) follow-up over a mean of 8.8 years posttransplant. In parallel, vector genome copies, expression of transgenic ALD protein (ALDP), and viral integration sites were determined in peripheral blood cells. Prior to transplant, the four patients had a normal or near normal neurocognitive status but gadolinium-enhanced demyelination in various brain regions. Gadolinium diffusion disappeared during the first year posttransplant. P3 kept a near normal status until 8.3 years of follow-up, but P1, P2, and P4 showed major cognitive degradation around 9, 28, and 60 months posttransplant. Neurological status and demyelination stabilized until last evaluation in P2, but deteriorated in both P1 at 10 years and P4 at 3 years posttransplant. The proportion of myeloid and lymphoid cells expressing transgenic ALDP decreased by half within 5 years then stabilized around 5% to 10%. Integration site analysis revealed a durable polyclonal distribution of genetically corrected hematopoietic cells. No adverse effects were observed. The long-term arrest of demyelination at MRI and persistence of transduced hematopoietic progenitors support that LV gene therapy may be a safe and durable treatment of c-ALD. However, the neurological degradation observed in three out of four patients mitigates the benefit of this therapy, calling for an earlier intervention, more potent vectors, and additional therapeutic strategies.

The experience of allogeneic hematopoietic stem cell transplantation in a patient with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD), a progressive neurometabolic disorder that is caused by a defect in the gene ABCD1 (ATP-binding cassette, subfamily D, member 1), which encodes the peroxisomal ABC half-transporter ALD protein. Recently, allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only therapy known to prevent disease progression. In this study, we would like to present our experience of alloHSCT for X-ALD from a HLA matched related sibling by the use of reduced intensity conditioning regimen composed of fludarabine, busulfan and ATG which allows us to reduce procedure-related toxicity and prevent mortality while achieving a curative effect.

Updated Safety and Efficacy Outcomes from an Ongoing Phase 2/3 Trial of Lenti-D Autologous Hematopoietic Stem Cell Gene Therapy for the Treatment of Cerebral Adrenoleukodystrophy

Objective Adrenoleukodystrophy (ALD) is an X-linked disease caused by dysfunction of ALD protein which results in the accumulation of very-long chain fatty acids in adrenal and nervous system tissues. Cerebral ALD (CALD), affecting 35-40% of boys with ALD, is characterized by inflammatory demyelination leading to progressive loss of neurologic function and death. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has a stabilizing impact on indices of cerebral disease progression, if performed early, but can be associated with significant risks. Methods Lenti-D Drug Product (DP) is an investigational gene therapy for the treatment of CALD. Boys with CALD (≤17 years) enrolled in an open-label phase 2/3 study (Starbeam) of the safety and efficacy of Lenti-D DP underwent full myeloablation and lymphodepletion followed by infusion of autologous CD34+ cells transduced with Lenti-D lentiviral vector (elivaldogene tavalentivec). The primary efficacy endpoint is the proportion of patients who are alive and free of major functional disabilities (MFD) at Month 24. The success criterion for the primary efficacy endpoint, defined from an observational study of the natural history of CALD and supported by outcomes of allo-HSCT, will be met for the first 17 patients enrolled in the study with a point estimate of ≥76.5% (95% CI, 50.1%-93.2%) MFD-free survival at Month 24. Results As of April 2018, 29 patients received Lenti-D DP (median follow-up 34 months, min-max, 0.4-54.0). The median DP cell dose was 11 (min-max, 5.0-19.4) x106 CD34 cells/kg; median DP vector copy number was 1.2 (min-max, 0.5-2.5) copies/diploid genome. Median day of neutrophil and platelet engraftment was 13.0 (min-max, 11.0-39.0) and 30.5 (min-max, 16.0-55.0), respectively. Of the 17 patients with evaluable data at Month 24, 15 (88%, 95% CI, 63.6%-98.5%) remain alive and MFD-free with evidence of disease stabilization. The remaining 12 patients have not yet reached 24 months of follow-up; median follow-up time for this group was 4.2 months (min-max, 0.4-11.7). As of April 2018, there was no evidence of MFDs in these patients. Overall, no graft failure, GVHD, or transplant-related mortality have been reported, and there is no evidence of replication competent lentivirus or insertional oncogenesis through 54 months of follow-up. Most adverse events were consistent with myeloablative conditioning. Conclusion Lenti-D DP met the threshold for primary efficacy success in the first 17 patients to complete 24 months of follow-up. The data presented here suggest that Lenti-D DP stabilizes neurologic disease progression and may offer an alternative to allo-HSCT in patients with early cerebral disease. The study has met its enrollment target and additional follow-up is ongoing to assess durability of effects and long-term safety.

MITOCHONDRIAL DYSFUNCTION AND HYPOGAMMAGLOBULINEMIA RESULTING IN LACTIC ACIDOSIS AND RECURRENT BACTERIAL INFECTIONS

Introduction Defective mitochondrial function has been reported to cause T cell suppression and disruption of B cell isotype class switching and differentiation. We describe a patient with a mitochondrial DNA mutation and hypogammaglobulinemia. Case Description A 24-year-old Caucasian male with a history of acute disseminated encephalomyelitis in childhood presented with progressive myeloneuropathy, episodic rhabdomyolysis and lactic acidosis, and recurrent bacterial infections, including bacteremia. His IgG was 584mg/dl (700-1600 mg/dl) with post-vaccination protective titers to 6 of 23 Streptococcus pneumonia serotypes. His presentation was concerning for X-linked adrenoleukodystrophy (X-ALD), a genetic defect in the transport of very long-chain fatty acids (VLCFAs) across the peroxisomal membrane. Whole exome sequencing revealed a missense mutation (c.274G>A, amino acid change p.Gly92Arg) in the ALD protein. VLCFAs profile is abnormal in 99.9% of males with X-ALD, and his was normal. Mitochondrial gene sequencing was due to recurrent lactic acidosis, and showed a missense mutation (m.8552 T>C) at ATP6/ATP8 gene overlapping region. A muscle biopsy showed increased immature type IIC muscle fibers, suggestive for mitochondrial disorder. He was started on intravenous immunoglobulin, with subsequent resolution of fevers and bacteremia. However, he continues to have episodes of lactic acidosis. Discussion This clinical presentation suggests a possible link between mitochondrial function and humoral immunity. This patient has mutations in ATP synthase and ALD protein, which functions as a transporter to bind and hydrolyze ATP. These novel mutations are not well categorized. Functional studies may be helpful in understanding the consequence of these mutations and the effect of mitochondrial dysfunction on the immune system.

Survival and Functional Outcomes in Boys with Cerebral Adrenoleukodystrophy with and without Hematopoietic Stem Cell Transplantation

Cerebral adrenoleukodystrophy (CALD) is a rapidly progressing, often fatal neurodegenerative disease caused by mutations in the ABCD1 gene, resulting in deficiency of ALD protein. Clinical benefit has been reported following allogeneic hematopoietic stem cell transplantation (HSCT). We conducted a large multicenter retrospective chart review to characterize the natural history of CALD, to describe outcomes after HSCT, and to identify predictors of treatment outcomes. Major functional disabilities (MFDs) were identified as having the most significant impact on patients' abilities to function independently and were used to assess HSCT outcome. Neurologic function score (NFS) and Loes magnetic resonance imaging score were assessed. Data were collected on 72 patients with CALD who did not undergo HSCT (untreated cohort) and on 65 patients who underwent transplantation (HSCT cohort) at 5 clinical sites. Kaplan-Meier (KM) estimates of 5-year overall survival (OS) from the time of CALD diagnosis were 55% (95% confidence interval [CI], 42.2% to 65.7%) for the untreated cohort and 78% (95% CI, 64% to 86.6%) for the HSCT cohort overall (P = .01). KM estimates of 2-year MFD-free survival for patients with gadolinium-enhanced lesions (GdE+) were 29% (95% CI, 11.7% to 48.2%) for untreated patients (n = 21). For patients who underwent HSCT with GdE+ at baseline, with an NFS ≤1 and Loes score of 0.5 to ≤9 (n = 27), the 2-year MFD-free survival was 84% (95% CI, 62.3% to 93.6%). Mortality rates post-HSCT were 8% (5 of 65) at 100days and 18% (12 of 65) at 1 year, with disease progression (44%; 7 of 16) and infection (31%; 5 of 16) listed as the most common causes of death. Adverse events post-HSCT included infection (29%; 19 of 65), acute grade II-IV graft-versus-host disease (GVHD) (31%; 18 of 58), and chronic GVHD (7%; 4 of 58). Eighteen percent of the patients (12 of 65) experienced engraftment failure after their first HSCT. Positive predictors of OS in the HSCT cohort may include donor-recipient HLA matching and lack of GVHD, and early disease treatment was predictive of MFD-free survival. GdE+ status is a strong predictor of disease progression in untreated patients.‎ This study confirms HSCT as an effective treatment for CALD when performed early. We propose survival without MFDs as a relevant treatment goal, rather than solely assessing OS as an indicator of treatment success.

ABC transportome inventory of human pathogenic yeast Candida glabrata: Phylogenetic and expression analysis.

ATP-binding cassette (ABC) is one of the two major superfamilies of transporters present across the evolutionary scale. ABC superfamily members came to prominence due to their ability to extrude broad spectrum of substrates and to confer multi drug resistance (MDR). Overexpression of some ABC transporters in clinical isolates of Candida species was attributed to the development of MDR phenotypes. Among Candida species, Candida glabrata is an emerging drug resistant species in human fungal infections. A comprehensive analysis of such proteins in C. glabrata is required to untangle their role not only in MDR but also in other biological processes. Bioinformatic analysis of proteins encoded by genome of human pathogenic yeast C. glabrata identified 25 putative ABC protein coding genes. On the basis of phylogenetic analysis, domain organization and nomenclature adopted by the Human Genome Organization (HUGO) scheme, these proteins were categorized into six subfamilies such as Pleiotropic Drug Resistance (PDR)/ABCG, Multi Drug Resistance (MDR)/ABCB, Multi Drug Resistance associated Protein (MRP)/ABCC, Adrenoleukodystrophy protein (ALDp)/ABCD, RNase L Inhibitor (RLI)/ABCE and Elongation Factor 3 (EF3)/ABCF. Among these, only 18 ABC proteins contained transmembrane domains (TMDs) and were grouped as membrane proteins, predominantly belonging to PDR, MDR, MRP, and ALDp subfamilies. A comparative phylogenetic analysis of these ABC proteins with other yeast species revealed their orthologous relationship and pointed towards their conserved functions. Quantitative real time PCR (qRT-PCR) analysis of putative membrane localized ABC protein encoding genes of C. glabrata confirmed their basal expression and showed variable transcriptional response towards antimycotic drugs. This study presents first comprehensive overview of ABC superfamily proteins of a human fungal pathogen C. glabrata, which is expected to provide an important platform for in depth analysis of their physiological relevance in cellular processes and drug resistance.

Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy

BackgroundIn X-linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem-cell transplantation.MethodsWe enrolled boys with cerebral adrenoleukodystrophy in a single-group, open-label, phase 2–3 safety and efficacy study. Patients were required to have early-stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft-versus-host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion.ResultsA total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range, 21.6 to 42.0). All the patients had gene-marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment-related death or graft-versus-host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem-cell transplantation and later died from transplantation-related complications.ConclusionsEarly results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Additional follow-up is needed to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102; ClinicalTrialsRegister.eu number, 2011-001953-10.)

S149R, a novel mutation in the ABCD1 gene causing X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. It is a heterogeneous disorder caused by mutations in the ATP-binding cassette protein subfamily D1 (ABCD1) gene, encoding the peroxisomal membrane protein ALDP, which is involved in the transmembrane transport of very long-chain fatty acids. For the first time, we report a case of olivopontocerebellar X-ALD on the Chinese mainland. In this study, a novel mutation (c.447T>A; p.S149R) in ABCD1 was detected in a patient diagnosed with X-ALD. The mutant amino acid is well conserved among species. Bioinformatics analysis predicted the substitution to be deleterious and to cause structural changes in the adrenoleukodystrophy protein. Immunofluorescence showed an altered subcellular localization of the S149R mutant protein, which may lead to defects in the degradation of very long chain fatty acids in peroxisomes. We therefore suggest that the novel mutation, which alters ALDP structure, subcellular distribution and function, is responsible for X-ALD.