Neutral lipid storage disease with myopathy: clinicopathological and genetic features of nine Iranian patients.

The rare disorder known as Neutral Lipid Storage Disease with Myopathy presents with a variety of clinical manifestations, including myopathy, cardiac dysfunction, and other organ complications. Early diagnosis is crucial due to the increased risk of cardiomyopathy. We describe the clinical, histopathological, muscle imaging, and genetic findings of nine neutral lipid storage myopathy patients. Proximal weakness and asymmetric involvement may suggest lipid storage myopathy. While skeletal muscle weakness was the main manifestation in our patients, one case presented only with hyperCKemia. Additionally, three patients had fertility issues, two suffered from diabetes mellitus, two had cardiomyopathy, and one had a history of hypothyroidism. Muscle histopathology revealed lipid depositions and rimmed vacuoles, prompting peripheral blood smears to detect Jordan Anomalies. All muscle biopsies and peripheral blood smear showed lipid droplets, rimmed vacuoles, and Jordan anomaly. Identifying PNPLA2 gene mutations is important for diagnosing neutral lipid storage myopathy; our cases showed some novel mutations. This study highlights the importance of early diagnosis and comprehensive evaluation in managing neutral lipid storage myopathy cases.

Nailfold capillaroscopy reveals early peripheral microcirculation abnormalities in children affected by heterozygous familial hypercholesterolemia.

BACKGROUND: nailfold capillaroscopy (NCF) is a non-invasive imaging technique to seek peripheral microcirculation abnormalities in children and adults. Familial hypercholesterolemia is a genetic disorder caused by mutations capable of increasing blood levels of low-density lipoproteins cholesterol (LDL-C), thus triggering early atherosclerosis. The study aims at evaluating peripheral microcirculation in children with heterozygous familial hypercholesterolemia (HeFH) by means of NFC in comparison with healthy peers and at searching for possible correlations between these abnormalities and patients' lipid panel. METHODS: thirty-six HeFH patients were enrolled (13 males and 23 females. Mean age 8 ± 3 years; age range 3-13 years). They had increased levels of total cholesterol (237.9 ± 34.2 mg/dl) and LDL-C (154.2 ± 37.6 mg/dl). Both values were ≥95th gender and age specific centile. All the subjects in the study underwent NFC. RESULTS: In 69.4 % of HeFH children nailfold capillaries were tortuous (p < 0.00001 compared to healthy controls). In 41.6 % the number of capillaries was markedly reduced (<7 capillaries/mm). The mean number of capillaries was 8.4 ± 2.6/mm in HeFH and 12.2 ± 1.4/mm in healthy controls (p < 0.00001). In 100 % of the sample size capillary blood flow was slowed down (p < 0.00001). In 50 % of the sample size a blood "sludge" phenomenon was seen (p < 0.00001). No gender differences were detected. Sludge phenomenon was seen only in those with LDL-C over 99th centile (p < 0.00001). CONCLUSION: NCF allows the identification of an early peripheral microvascular dysfunction in HeFH children which is similar to that already seen in atherosclerotic disease. Prompt identification of these capillary abnormalities may be crucial in implementing early prevention measures.

Ichthyosis, cataracts, and motor delay in an infant: A case of Chanarin-Dorfman syndrome.

Chanarin-Dorfman syndrome (CDS) is a rare, autosomal recessive disorder of impaired triacylglycerol catabolism leading to cytoplasmic deposition of triglycerides in various cell types. We describe the case of an 8-month-old boy with cataracts, strabismus, motor delays, and an ichthyosiform rash since birth. Genetic testing revealed a pathogenic variant of the ABHD5 gene, suggestive of CDS, and further workup demonstrated hepatic steatosis and myopathy. His ichthyosis improved with initiation of a diet low in very long-chain fatty acids and medium-chain fatty acid supplementation.

Clinical and genetic features of sitosterolemia in Japan.

BACKGROUND AND AIM: Clinical manifestations and genetic backgrounds of Japanese patients with sitosterolemia have been unclear. MATERIALS AND METHODS: We searched PubMed for studies using the keywords "sitosterolemia" or "phytosterolemia" and "Japan". Moreover, we added information from the members of the Committee on Primary Dyslipidemia under the Research Program on Rare and Intractable Disease of the Ministry of Health, Labour and Welfare (MHLW) of Japan. RESULTS: We identified 36 patients with sitosterolemia caused by biallelic pathogenic mutations in the ATP-binding cassette subfamily G member 5 (ABCG5) or ATP-binding cassette subfamily G member 8 (ABCG8) from 31 families in Japan. The diagnosed age ranged from 0 to 64 years (median 13 years). The median sitosterol and LDL cholesterol levels were 100 µg/ml (IQR: 50-183), and 193 mg/dl (IQR: 108-295), respectively. All the patients exhibited cutaneous and/or tendon xanthomas, up to 9 (25%) patients exhibited premature coronary artery disease, 5 (16%) patients exhibited arthritis, and 8 (22%) patients exhibited blood abnormalities. Ezetimibe was administered to all the patients, including infantile cases, while statins, colestimide, evolocumab, probucol, and LDL apheresis were also used. CONCLUSION: We are providing a demographic overview of the clinical and genetic backgrounds of Japanese patients with sitosterolemia.

Features of chinese patients with sitosterolemia.

BACKGROUND: Sitosterolemia is a lipid disorder characterized by the accumulation of phytosterols in plasma and organs, caused by mutations in the ABCG5 and/or ABCG8 genes. The disease is frequently misdiagnosed and mistreated as familial hypercholesterolemia (FH). To gain a better understanding of the disease, the current status of diagnosis and treatment of Chinese patients with sitosterolemia was reviewed and summarized. METHOD: Literature search was performed. The clinical features and molecular characteristics of Chinese patients with sitosterolemia were analysed. Four children with sitosterolemia and the treatment experience were described. RESULTS: Fifty-five patients with sitosterolemia have been reported in China. These patients were aged from 3 months to 67 years at diagnosis, and the median was 8 years of age. Several complications, such as xanthomas in 47 patients (85%), thrombocytopenia in 17 patients (31%), anemia in 14 patients (25%), and cardiovascular damage in 12 patients (22%), were observed. Thirty-nine patients (71%) exhibited mutations in the ABCG5 gene, 15 patients (27%) showed mutations in ABCG8, and variations in both genes occurred in one patient (2%). A patient with two clinically rare diseases, namely, sitosterolemia and glycogen storage disease type VI (GSD VI)), is reported here for the first time. The four reported patients were treated with low cholesterol and phytosterol-limited diet alone or combined with cholestyramine. Even though decreases were observed for total plasma cholesterol (TC) and low-density-lipoprotein cholesterol (LDL-C), and these levels were as low as normal in some patients, the levels of plant sterols remained above the normal range. However, TC, LDL-C and plant sterol levels remained at high levels in patients treated with a control diet control only. CONCLUSIONS: The analysis reveals that different from Caucasians carrying mainly variations in ABCG8, most Chinese patients have mutations in the ABCG5 gene, and Arg446Ter, Gln251Ter, anArg389His might be hot-spot mutations in Chinese patients. The current survey provides clinical data to enable the development of a standardized protocol for the diagnosis and treatment of sitosterolemia in China.

ABHD5 frameshift deletion in Golden Retrievers with ichthyosis.

Ichthyoses are hereditary skin disorders characterized by the formation of scales and defects in the outermost layer of the epidermis. In dogs, at least six different breed-specific ichthyoses including a relatively common PNPLA1-related autosomal recessive ichthyosis in Golden Retrievers are known. In this study, we investigated 14 Golden Retrievers with scales that were not homozygous for the mutant PNPLA1 allele suggesting a genetically distinct new form of ichthyosis. Histopathological examinations showed lamellar, orthokeratotic hyperkeratosis, and mildly hyperplastic epidermis that led to the diagnosis of a nonepidermolytic ichthyosis. Combined linkage and homozygosity mapping in 14 cases and 30 nonaffected family members delimited a critical interval of ∼12.7 Mb on chromosome 23. Whole-genome sequencing of an affected dog revealed a single protein-changing variant within this region that was not present in 795 control genomes. The identified variant is a 14 bp deletion in the ABHD5 gene (c.1006_1019del), leading to a frameshift and altering the last 14 codons p.(Asp336Serfs*6). The genotypes at this variant showed perfect cosegregation with the ichthyosis phenotype in a large family comprising 14 cases and 72 controls. ABHD5 encodes an acyltransferase required for lipid metabolism. In humans, variants in ABHD5 cause Chanarin-Dorfman syndrome, a neutral lipid storage disease with ichthyosis. Our data in dogs together with the knowledge on the effects of ABHD5 variants in humans strongly suggest ABHD5:c.1006_1019del as candidate causative genetic variant for a new canine form of ichthyosis, which we propose to designate as Golden Retriever ichthyosis type 2 (ICH2).

Successful plasmapheresis and immunoglobulin treatment for severe lipid storage myopathy: Doing the right thing for the wrong reason.

Three consecutive skeletal muscle biopsies during a several months time-frame, showing different degrees of neutral lipid storage. This is highlighted by Oil-red-O stains (D, E, F) and electron microscopy (G, H, I). Note the impact on mitochondrial morphology with so called 'parking lots (K, L). Zooming 'in and out' into the ultrastructure, using the nanotomy platform provides interesting detailled information (http://nanotomy.org). ​.

Lipidomic and Proteomic Alterations Induced by Even and Odd Medium-Chain Fatty Acids on Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis.

Peroxisomes play an essential role in the ß-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA ß-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA ß-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA ß-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA ß-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA ß-oxidation is a regulator of hepatic cholesterol biosynthesis.

Altered Metabolic Flexibility in Inherited Metabolic Diseases of Mitochondrial Fatty Acid Metabolism.

In general, metabolic flexibility refers to an organism's capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial fatty metabolism: ß-oxidation and fatty acid biosynthesis. We focus specifically on two diseases: very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and malonyl-CoA synthetase deficiency (acyl-CoA synthetase family member 3 (ACSF3)) deficiency, which are both characterized by alterations in metabolic flexibility. On the one hand, in a mouse model of VLCAD-deficient (VLCAD-/-) mice, the white skeletal muscle undergoes metabolic and morphologic transdifferentiation towards glycolytic muscle fiber types via the up-regulation of mitochondrial fatty acid biosynthesis (mtFAS). On the other hand, in ACSF3-deficient patients, fibroblasts show impaired mitochondrial respiration, reduced lipoylation, and reduced glycolytic flux, which are compensated for by an increased ß-oxidation rate and the use of anaplerotic amino acids to address the energy needs. Here, we discuss a possible co-regulation by mtFAS and ß-oxidation in the maintenance of energy homeostasis.

Sitosterolemia: Twenty Years of Discovery of the Function of ABCG5ABCG8.

Sitosterolemia is a lipid disorder characterized by the accumulation of dietary xenosterols in plasma and tissues caused by mutations in either ABCG5 or ABCG8. ABCG5 ABCG8 encodes a pair of ABC half transporters that form a heterodimer (G5G8), which then traffics to the surface of hepatocytes and enterocytes and promotes the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. We review the literature from the initial description of the disease, the discovery of its genetic basis, current therapy, and what has been learned from animal, cellular, and molecular investigations of the transporter in the twenty years since its discovery. The genomic era has revealed that there are far more carriers of loss of function mutations and likely pathogenic variants of ABCG5 ABCG8 than previously thought. The impact of these variants on G5G8 structure and activity are largely unknown. We propose a classification system for ABCG5 ABCG8 mutants based on previously published systems for diseases caused by defects in ABC transporters. This system establishes a framework for the comprehensive analysis of disease-associated variants and their impact on G5G8 structure-function.

MTP deficiency caused by HADHB mutations: Pathophysiology and clinical manifestations.

Mutations in the HADHB gene lead to Mitochondrial Trifunctional Protein (MTP) deficiency. MTP deficiency is a rare autosomal recessive disorder affecting long-chain fatty acid oxidation. Patients affected by MTP deficiency are unable to metabolize long-chain fatty-acids and suffer a variety of symptoms exacerbated during fasting. The three phenotypes associated with complete MTP deficiency are an early-onset cardiomyopathy and early death, an intermediate form with recurrent hypoketotic hypoglycemia and a sensorimotor neuropathy with episodic rhabdomyolysis with small amount of residual enzyme activities. This review aims to discuss the pathophysiological mechanisms and clinical manifestations of each phenotype, which appears different and linked to HADHB expression levels. Notably, the pathophysiology of the sensorimotor neuropathy is relatively unknown and we provide a hypothesis on the qualitative aspect of the role of acylcarnitine buildup in Schwann cells in MTP deficiency patients. We propose that acylcarnitine may exit the Schwann cell and alter membrane properties of nearby axons leading to axonal degeneration based on recent findings in different metabolic disorders.

De Novo Development of mtDNA Deletion Due to Decreased POLG and SSBP1 Expression in Humans.

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.

The spectrum of peripheral neuropathy in disorders of the mitochondrial trifunctional protein.

Peripheral neuropathy is a known irreversible long-term complication of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MTPD), two inherited disorders of mitochondrial long-chain fatty acid oxidation. The underlying pathophysiology of neuropathy is still not fully understood. We report electrophysiological studies and neurological findings in a series of 8 LCHAD-deficient and 11 MTP-deficient patients. The median age at time of the study was 8.0 years (0.5-25 years). The overall prevalence of neuropathy was 58% with neuropathic symptoms being slightly more common in MTPD compared to LCHADD (70% vs 50%, respectively). Onset of neuropathy was significantly earlier in MTPD patients compared to LCHADD patients (median age at onset 4.7 vs 15.3 years, respectively, P = .047). In four patients, isolated peripheral neuropathy was the first and only presenting symptom, and in all four the diagnosis was missed by newborn screening. About half of the patients (45.5%) had a sensorimotor neuropathy, while 27.3% showed a pure motor form and another 27.3% an isolated sensory form. Despite early diagnosis by newborn screening and early initiation of therapy, peripheral neuropathy cannot be prevented in all patients with LCHADD/MTPD and has severe impact on the life of affected patients. Electrophysiology classifies LCHADD/MTPD neuropathy as axonal with secondary demyelination. A novel observation is that in patients with acute, fulminant onset of neuropathy, symptoms can be partly reversible. Further studies are needed to elucidate the underlying pathophysiology of axonal damage and possible therapeutic targets.

Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear. The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial. We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.

Retained visual function in a subset of patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD).

Introduction: LCHADD causes retinopathy associated with low vision, visual field defects, nyctalopia and myopia. We report a retrospective long-term single-center study of 6 LCHADD patients trying to clarify if early diagnosis has an impact on the course and outcome of chorioretinal degeneration. Methods: Long-term follow-up of visual acuity and staging of chorioretinal degeneration by fundus photography, optical coherence tomography (OCT) and autofluorescence (AF) in all six patients. Three patients (2 m/1 f; age 8-14.8 years) were diagnosed by newborn screening, a single patient early within the first year of life and treated promptly while the other two (1 m/1 f; age 23-24 years) were diagnosed later after developing symptoms. All carried HADHA variants; five were homozygous for the common p.E510Q variant, in one from the symptomatically diagnosed group p.[E510Q]; [R291*] was detected. Results: All patients showed retinal alterations, but early diagnosis was associated with a milder phenotype and a longer preservation of visual function. Among symptomatic patients, only one showed mild retinal involvement at the time of diagnosis. Conclusion: Despite the small number our study suggests that early diagnosis does not prevent retinopathy but might contribute to a milder phenotype with retained good visual acuity over time. OCT and AF are reliable non-invasive diagnostic tools to estimate the progression of early-stage retinal changes in LCHADD patients.

Early onset neutral lipid storage disease with myopathy presenting as congenital hypotonia and hepatomegaly.

Neutral lipid storage disease with myopathy is an ultra-rare, inherited autosomal recessive neuromuscular metabolic disorder caused by pathogenic variants in PNPLA2. It typically presents in adults as a progressive myopathy and is associated with myocardiopathy, hepatic involvement, and high creatine kinase levels. Only three children and adolescents with neutral lipid storage disease with myopathy have been reported. We report a female infant with congenital hypotonia born to consanguineous parents, whose mother presented with polyhydramnios during pregnancy. She demonstrated delayed acquisition of motor milestones, hepatomegaly, and elevated creatine kinase levels. Homozygous pathogenic variants in PNPLA2 were identified. Lipid accumulation was observed within the muscle fibers and Jordans' anomaly was observed in a blood smear. This is the first report to describe an infant with mildly symptomatic neutral lipid storage disease with myopathy and demonstrate hepatic involvement in a pediatric patient. Despite her mild symptoms, her ancillary test results were markedly abnormal.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD): An evidence- and consensus-based approach.

The nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD) is the fourth in a series of web-based guidelines focusing on the diet treatment for inherited metabolic disorders and follows previous publication of guidelines for maple syrup urine disease (2014), phenylketonuria (2016) and propionic acidemia (2019). The purpose of this guideline is to establish harmonization in the treatment and monitoring of individuals with VLCAD of all ages in order to improve clinical outcomes. Six research questions were identified to support guideline development on: nutrition recommendations for the healthy individual, illness management, supplementation, monitoring, physical activity and management during pregnancy. This report describes the methodology used in its development including review, critical appraisal and abstraction of peer-reviewed studies and unpublished practice literature; expert input through two Delphi surveys and a nominal group process; and external review from metabolic physicians and dietitians. It includes the summary statements of the nutrition management recommendations for each research question, followed by a standardized rating based on the strength of the evidence. Online, open access of the full published guideline allows utilization by health care providers, researchers and collaborators who advise, advocate and care for individuals with VLCAD and their families and can be accessed from the Genetic Metabolic Dietitians International (https://GMDI.org) and Southeast Regional Genetics Network (https://southeastgenetics.org/ngp) websites.