D-Bifunctional Protein Deficiency Diagnosis-A Challenge in Low Resource Settings: Case Report and Review of the Literature.

D-bifunctional protein deficiency (D-BPD) is a rare, autosomal recessive peroxisomal disorder that affects the breakdown of long-chain fatty acids. Patients with D-BPD typically present during the neonatal period with hypotonia, seizures, and facial dysmorphism, followed by severe developmental delay and early mortality. While some patients have survived past two years of age, the detectable enzyme activity in these rare cases was likely a contributing factor. We report a D-BPD case and comment on challenges faced in diagnosis based on a narrative literature review. An overview of Romania's first patient diagnosed with D-BPD is provided, including clinical presentation, imaging, biochemical, molecular data, and clinical course. Establishing a diagnosis can be challenging, as the clinical picture is often incomplete or similar to many other conditions. Our patient was diagnosed with type I D-BPD based on whole-exome sequencing (WES) results revealing a pathogenic frameshift variant of the HSD17B4 gene, c788del, p(Pro263GInfs*2), previously identified in another D-BPD patient. WES also identified a variant of the SUOX gene with unclear significance. We advocate for using molecular diagnosis in critically ill newborns and infants to improve care, reduce healthcare costs, and allow for familial counseling.

Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.

Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid ß-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.

First Case of Peroxisomal D-bifunctional Protein Deficiency with Novel HSD17B4 Mutations and Progressive Neuropathy in Korea.

Peroxisomal D-bifunctional protein (DBP), encoded by the HSD17B4 gene, catalyzes ß-oxidation of very long chain fatty acids (VLCFAs). The deficiency of this peroxisomal enzyme leads to the accumulation of VLCFAs, causing multisystemic manifestations including the brain, retina, adrenal gland, hearing, and skeletal system. Herein, we report the first Korean neonatal case of peroxisomal DBP deficiency and the clinical prognosis over 2 years. This patient showed craniofacial dysmorphism, club foot, and seizures with cyanosis one day after birth. Elevated VLCFAs levels were indicative of a peroxisomal disorder. Targeted exome sequencing was performed and two missense mutations p.Asp117Val and p.Phe279Ser in the HSD17B4 gene were identified. The patient had type III DBP deficiency; therefore, docosahexaenoic acid and non-soluble vitamins were administered. However, progressive nystagmus, optic nerve atrophy, and bilateral hearing defects were observed and follow-up brain imaging revealed leukodystrophy and brain atrophy. Multiple anti-epileptic drugs were required to control the seizures. Over two years, the patient achieved normal growth with home ventilation and tube feeding. Hereby, the subject's parents had support during the second pregnancy from the proven molecular information. Moreover, targeted exome sequencing is an effective diagnostic approach, considering genetic heterogeneity of Zellweger spectrum disorders.

Microglia lacking a peroxisomal ß-oxidation enzyme chronically alter their inflammatory profile without evoking neuronal and behavioral deficits.

BACKGROUND: Microglia play a central role in most neurological disorders, but the impact of microgliosis on brain environment and clinical functions is not fully understood. Mice lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal ß-oxidation, develop a fatal disorder characterized by motor problems similar to the milder form of MFP2 deficiency in humans. The hallmark of disease in mice is the chronic proliferation of microglia in the brain, but molecular pathomechanisms that drive rapid clinical deterioration in human and mice remain unknown. In the present study, we identified the effects of specific deletion of MFP2 from microglia in the brain on immune responses, neuronal functioning, and behavior. METHODS: We created a novel Cx3cr1-Mfp2-/- mouse model and studied the impact of MFP2 deficiency on microglial behavior at different ages using immunohistochemistry and real-time PCR. Pro- and anti-inflammatory responses of Mfp2-/- microglia were assessed in vitro and in vivo after stimulation with IL-1ß/INFγ and IL-4 (in vitro) and LPS and IL-4 (in vivo). Facial nerve axotomy was unilaterally performed in Cx3cr1-Mfp2-/- and control mice, and microglial functioning in response to neuronal injury was subsequently analyzed by histology and real-time PCR. Finally, neuronal function, motor function, behavior, and cognition were assessed using brainstem auditory evoked potentials, grip strength and inverted grid test, open field exploration, and passive avoidance learning, respectively. RESULTS: We found that Mfp2-/- microglia in a genetically intact brain environment adopt an inflammatory activated and proliferative state. In addition, we found that acute inflammatory and neuronal injury provoked normal responses of Mfp2-/- microglia in Cx3cr1-Mfp2-/- mice during the post-injury period. Despite chronic pro-inflammatory microglial reactivity, Cx3cr1-Mfp2-/- mice exhibited normal neuronal transmission, clinical performance, and cognition. CONCLUSION: Our data demonstrate that MFP2 deficiency in microglia causes intrinsic dysregulation of their inflammatory profile, which is not harmful to neuronal function, motor function, and cognition in mice during their first year of life.

Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal ß-oxidation-deficient mice.

An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein-2 (MFP2), the key enzyme in peroxisomal ß-oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti-inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2-/- mice were treated with the selective colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of >95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2-/- mice. Despite microglial diminution in Mfp2-/- brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2-/- mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2-/- phenotype.

Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency.

The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal ß-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal ß-oxidation in neural cells.

Identification of a chronic non-neurodegenerative microglia activation state in a mouse model of peroxisomal ß-oxidation deficiency.

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal ß-oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2(-/-) microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2(-/-) microglia were compared with those from SOD1(G93A) mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1(G93A) spinal cord, neurons were intact in Mfp2(-/-) brain and Mfp2(-/-) microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2(-/-) microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2(-/-) microglia were immunologically activated but not polarized to a pro- or anti-inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2(-/-) brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity.

Newborn screening for X-linked adrenoleukodystrophy in New York State: diagnostic protocol, surveillance protocol and treatment guidelines.

PURPOSE: To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State. METHODS: A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up. RESULTS: A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed. CONCLUSION: Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.

Next generation sequencing with copy number variant detection expands the phenotypic spectrum of HSD17B4-deficiency.

BACKGROUND: D-bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe, infantile-onset disorder of peroxisomal fatty acid oxidation. Few affected patients survive past two years of age. Compound heterozygous mutations in HSD17B4 have also been reported in two sisters diagnosed with Perrault syndrome (MIM # 233400), who presented in adolescence with ovarian dysgenesis, hearing loss, and ataxia. CASE PRESENTATION: An adult male presented with cerebellar ataxia, peripheral neuropathy, hearing loss, and azoospermia. The clinical presentation, in combination with biochemical findings in serum, urine, and muscle biopsy, suggested a mitochondrial disorder. Commercial genetic testing of 18 ataxia and mitochondrial disease genes was negative. Targeted exome sequencing followed by analysis of single nucleotide variants and small insertions/deletions failed to reveal a genetic basis of disease. Application of a computational algorithm to infer copy number variants (CNVs) from exome data revealed a heterozygous 12 kb deletion of exons 10-13 of HSD17B4 that was compounded with a rare missense variant (p.A196V) at a highly conserved residue. Retrospective review of patient records revealed mildly elevated ratios of pristanic:phytanic acid and arachidonic:docosahexaenoic acid, consistent with dysfunctional peroxisomal fatty acid oxidation. CONCLUSION: Our case expands the phenotypic spectrum of HSD17B4-deficiency, representing the first male case reported with infertility. Furthermore, it points to crosstalk between mitochondria and peroxisomes in HSD17B4-deficiency and Perrault syndrome.

Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.

Although peroxisome biogenesis and ß-oxidation disorders are well known for their neurodevelopmental defects, patients with these disorders are increasingly diagnosed with neurodegenerative pathologies. In order to investigate the cellular mechanisms of neurodegeneration in these patients, we developed a mouse model lacking multifunctional protein 2 (MFP2, also called D-bifunctional protein), a central enzyme of peroxisomal ß-oxidation, in all neural cells (Nestin-Mfp2(-/-)) or in oligodendrocytes (Cnp-Mfp2(-/-)) and compared these models with an already established general Mfp2 knockout. Nestin-Mfp2 but not Cnp-Mfp2 knockout mice develop motor disabilities and ataxia, similar to the general mutant. Deterioration of motor performance correlates with the demise of Purkinje cell axons in the cerebellum, which precedes loss of Purkinje cells and cerebellar atrophy. This closely mimics spinocerebellar ataxias of patients affected with mild peroxisome ß-oxidation disorders. However, general knockouts have a much shorter life span than Nestin-Mfp2 knockouts which is paralleled by a disparity in activation of the innate immune system. Whereas in general mutants a strong and chronic proinflammatory reaction proceeds throughout the brain, elimination of MFP2 from neural cells results in minor neuroinflammation. Neither the extent of the inflammatory reaction nor the cerebellar degeneration could be correlated with levels of very long chain fatty acids, substrates of peroxisomal ß-oxidation. In conclusion, MFP2 has multiple tasks in the adult brain, including the maintenance of Purkinje cells and the prevention of neuroinflammation but this is not mediated by its activity in oligodendrocytes nor by its role in very long chain fatty acid degradation.

Rapid whole-genome sequencing identifies a homozygous novel variant, His540Arg, in HSD17B4 resulting in D-bifunctional protein deficiency disorder diagnosis.

Rapid whole-genome sequencing (rWGS) allows for a diagnosis to be made quickly and impact medical management, particularly in critically ill children. Variants identified by this approach are often not identified using other testing methodologies, such as carrier screening or gene sequencing panels, targeted panels, or chromosomal microarrays. However, rWGS can identify variants of uncertain significance (VUSs), which challenges clinicians in the rapid return of information to families. Here we present a case of the metabolic condition D-bifunctional protein deficiency in a neonate with epilepsy and hypotonia born to consanguineous parents. Sequencing revealed a homozygous VUS in HSD17B4, c.1619A > G (p.His540Arg). Preliminary results were delivered within 3 d of sample receipt. Previous parental carrier screening included the HSD17B4 gene but was reported as negative. The molecular finding directed the clinical team to assess phenotypic overlap and investigate next steps in terms of confirmation of the findings and potential medical management of the patient. Clinical metabolic testing of fatty acids confirmed the diagnosis. Computational analysis of HSD17B4 His540Arg showed the change to likely impact dimerization based on structural insights, with the histidine conserved and selected throughout all 223 species assessed for this amino acid. This variant clusters around several pathogenic and likely pathogenic variants in HSD17B4 This case demonstrates the utility of rWGS, the potential for receiving uncertain results, and the downstream implications for confirmation or rejection of a molecular diagnosis by the clinical team.

Increased Expression of Translocator Protein (TSPO) Marks Pro-inflammatory Microglia but Does Not Predict Neurodegeneration.

PURPOSE: Activation of the innate immune system plays a significant role in pathologies of the central nervous system (CNS). In order to follow disease progression and evaluate effectiveness of potential treatments involved in neuroinflammation, it is important to track neuroinflammatory markers in vivo longitudinally. The translocator protein (TSPO) is used as a target to image neuroinflammation as its expression is upregulated in reactive glial cells during CNS pathologies. However, it remains unclear in which microglial phenotypes TSPO levels are upregulated, as microglia can display a plethora of activation states that can be protective or detrimental to the CNS. PROCEDURES: We assessed the levels of TSPO transcripts in cultured microglia that were polarized into pro- and anti-inflammatory states in vitro and in the brain of mice in which an anti-inflammatory environment was induced in vivo. In addition, we used a mouse model of peroxisomal multifunctional protein-2 (MFP2) deficiency that exhibits widespread neuroinflammation despite no neuronal loss and monitored TSPO expression by immunohistochemistry and by imaging using the TSPO radiotracer [18F]DPA-714. RESULTS: TSPO expression was selectively increased in so-called classically activated or M1 microglia but not in alternatively activated or M2 microglia in vitro. In agreement, TSPO transcript levels were not induced in an anti-inflammatory brain environment. We found that both transcript and protein levels of TSPO are significantly increased in the brain of Mfp2 -/- compared to those of the control mice and TSPO immunoreactivity colocalized predominantly with microglia in Mfp2 -/- brain. In vitro and ex vivo autoradiography in Mfp2 -/- mice using the TSPO radiotracer [18F]DPA-714 confirmed increased expression of TSPO. These data demonstrate that TSPO imaging reveals microgliosis in non-neurodegenerative brain pathologies. CONCLUSIONS: We show that induced TSPO expression marks a pro-inflammatory brain environment that is not necessarily accompanied by neuronal loss.

Autonomous Purkinje cell axonal dystrophy causes ataxia in peroxisomal multifunctional protein-2 deficiency.

Peroxisomes play a crucial role in normal neurodevelopment and in the maintenance of the adult brain. This depends largely on intact peroxisomal ß-oxidation given the similarities in pathologies between peroxisome biogenesis disorders and deficiency of multifunctional protein-2 (MFP2), the central enzyme of this pathway. Recently, adult patients diagnosed with cerebellar ataxia were shown to have mild mutations in the MFP2 gene, hydroxy-steroid dehydrogenase (17 beta) type 4 (HSD17B4). Cerebellar atrophy also develops in MFP2 deficient mice but the cellular origin of the degeneration is unexplored. In order to investigate whether peroxisomal ß-oxidation is essential within Purkinje cells, the sole output neurons of the cerebellum, we generated and characterized a mouse model with Purkinje cell selective deletion of the MFP2 gene. We show that selective loss of MFP2 from mature cerebellar Purkinje neurons causes a late-onset motor phenotype and progressive Purkinje cell degeneration, thereby mimicking ataxia and cerebellar deterioration in patients with mild HSD17B4 mutations. We demonstrate that swellings on Purkinje cell axons coincide with ataxic behavior and precede neurodegeneration. Loss of Purkinje cells occurs in a characteristic banded pattern, proceeds in an anterior to posterior fashion and is accompanied by progressive astro- and microgliosis. These data prove that the peroxisomal ß-oxidation pathway is required within Purkinje neurons to maintain their axonal integrity, independent of glial dysfunction.

Slowly progressive d-bifunctional protein deficiency with survival to adulthood diagnosed by whole-exome sequencing.

d-Bifunctional protein (DBP) deficiency is an autosomal recessive disorder of peroxisomal fatty acid oxidation caused by mutations in HSD17B4. It is typically fatal by the age of two years with symptom onset during the neonatal period, and survival until late childhood is rare. We herein report the case of a patient with DBP deficiency surviving until adulthood, who showed severe sensorineural deafness, disturbances in language acquisition, slowly progressive cerebellar ataxia, and peripheral neuropathy. This patient, in whom findings of prior investigations were nondiagnostic, had been followed up as having an early-onset spinocerebellar degeneration of unknown etiology. Whole-exome sequencing analysis at the age of 36 showed two heterozygous variants in the gene HSD17B4, which encodes DBP in this patient. A panel of peroxisomal investigations showed normal levels of very long chain fatty acids (VLCFAs) in plasma and elevated serum phytanic acid levels. Recently, an increasing number of patients with DBP deficiency surviving until adolescence/adulthood have been reported, in whom abnormalities in the levels of VLCFAs and other peroxisomal metabolites are marginal or nonexistent. Genetic analysis of HSD17B4 should be considered in adult patients with cerebellar ataxia, peripheral neuropathy, and pyramidal signs in addition to sensorineural auditory disturbance since childhood.

Clinical and Laboratory Diagnosis of Peroxisomal Disorders.

The peroxisomal disorders (PDs) are a heterogeneous group of genetic diseases in man caused by an impairment in peroxisome biogenesis or one of the metabolic functions of peroxisomes. Thanks to the revolutionary technical developments in gene sequencing methods and their increased use in patient diagnosis, the field of genetic diseases in general and peroxisomal disorders in particular has dramatically changed in the last few years. Indeed, several novel peroxisomal disorders have been identified recently and in addition it has been realized that the phenotypic spectrum of patients affected by a PD keeps widening, which makes clinical recognition of peroxisomal patients increasingly difficult. Here, we describe these new developments and provide guidelines for the clinical and laboratory diagnosis of peroxisomal patients.

Peroxisomal D-bifunctional protein deficiency: First case reports from Slovakia.

D-bifunctional protein deficiency (#OMIM 261515) is a rare autosomal recessive hereditary metabolic disorder causing severe clinical and biochemical abnormalities that are usually fatal in the course of the first years of life. This disease is classified as single enzyme peroxisomal disorder affecting the ß-oxidation pathway in this compartment. In this paper we present a full overview of the clinical presentation, magnetic resonance imaging, biochemical and molecular data of two Slovak D-bifunctional protein deficient patients. In the clinical presentation of both patients severe generalized hypotonia, depression of neonatal reflexes, craniofacial dysmorphism and seizures dominated starting from the second day of life. In both patients, who died up to two years of life, we found elevated plasma levels of very long chain fatty acids and we identified the presence of causative mutations in the HSD17B4 gene. In the first case, we found the homozygous mutation c.46G>A, which is responsible for a defect in the dehydrogenase domain. In the second patient, the heterozygous mutations c.1369A>G and c.1516C>T were present and functionally they are related to the hydratase domain of the protein. This combination of mutations in the second patient is very rare and has not been reported until now. The presence of mutations was examined in all family members, and the resulting data were successfully utilized for prenatal diagnosis.

D-bifunctional protein deficiency: a cause of neonatal onset seizures and hypotonia.

BACKGROUND: Peroxisomal disorders are classified in two major groups: (1) peroxisome biogenesis disorders and (2) single peroxisomal enzyme/transporter deficiencies. D-bifunctional protein deficiency (OMIM #261515) is included in this last group of rare diseases and leads to an impaired peroxisomal beta-oxidation. D-bifunctional protein deficiencies are divided into four types based on the degree of activity of the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase protein units. PATIENT DESCRIPTION: We present the first Portuguese reported type II D-bifunctional protein deficiency patient, whose neonatal clinical picture is indistinguishable from a Zellweger spectrum disease. The clinical features and the neuroimaging findings of polymicrogyria raised suspicion of the diagnosis. After biochemical analysis, D-bifunctional protein deficiency was confirmed with the identification of a homozygous p.Asn457Tyr (N457Y) mutation of the HSD17B4 gene. The patient's parents were carriers of the mutated allele, confirming the patient homozygosity status and allowing prenatal diagnosis in future pregnancies. CONCLUSIONS: D-bifunctional protein deficiency is a rare, severe disease and the final diagnosis can only be accomplished after HSD17B4 gene sequencing. Treatment is supportive, aimed at improving nutrition and growth, controlling the central nervous system symptoms, and limiting the eventual progression of liver disease.

Central nervous system pathology in MFP2 deficiency: insights from general and conditional knockout mouse models.

Multifunctional protein-2 (MFP2), also known as D-bifunctional protein, is a central enzyme of the peroxisomal ß-oxidation pathway. Defects in this enzyme are associated with a spectrum of neurological disorders encompassing developmental and degenerative pathologies. In order to investigate the cellular and molecular mechanisms of these neuropathologies, mouse models with general and cell type selective loss of MFP2 were generated. In this review the distinct anomalies in the CNS of adult Mfp2 knockout mice are discussed, in particular the cerebellar degeneration and neuroinflammation. The potential underlying mechanisms are considered with regard to the cellular origin and biochemical causes. Finally, the similarities and differences between the CNS phenotypes of mice lacking MFP2 and mice with peroxisome biogenesis disorders are assessed.

Peroxisomal D-bifunctional protein deficiency: three adults diagnosed by whole-exome sequencing.

OBJECTIVE: To determine the causative genetic lesion in 3 adult siblings with a slowly progressive, juvenile-onset phenotype comprising cerebellar atrophy and ataxia, intellectual decline, hearing loss, hypogonadism, hyperreflexia, a demyelinating sensorimotor neuropathy, and (in 2 of 3 probands) supratentorial white matter changes, in whom numerous prior investigations were nondiagnostic. METHODS: The patients' initial clinical assessment included history and physical examination, cranial MRI, and nerve conduction studies. We performed whole-exome sequencing of all 3 probands, followed by variant annotation and selection of rare, shared, recessive coding changes to identify the gene responsible. We next performed a panel of peroxisomal investigations in blood and cultured fibroblasts, including assessment of D-bifunctional protein (DBP) stability and activity by immunoblot and enzymologic methods, respectively. RESULTS: Exome sequencing identified compound heterozygous mutations in HSD17B4, encoding peroxisomal DBP, in all 3 probands. Both identified mutations alter a conserved residue within the active site of DBP's enoyl-CoA hydratase domain. Routine peroxisomal screening tests, including very long-chain fatty acids and phytanic acid, were normal. DBP enzymatic activity was markedly reduced. CONCLUSION: Exome sequencing provides a powerful and elegant tool in the specific diagnosis of "mild" or "atypical" neurometabolic disorders. Given the broad differential diagnosis and the absence of detectable biochemical abnormalities in blood, molecular testing of HSD17B4 should be considered as a first-line investigation in patients with compatible features.