Gene therapy for aromatic L-amino acid decarboxylase deficiency: Requirements for safe application and knowledge-generating follow-up.

The autosomal recessive defect of aromatic L-amino acid decarboxylase (AADC) leads to a severe neurological disorder with manifestation in infancy due to a pronounced, combined deficiency of dopamine, serotonin and catecholamines. The success of conventional drug treatment is very limited, especially in patients with a severe phenotype. The development of an intracerebral AAV2-based gene delivery targeting the putamen or substantia nigra started more than 10 years ago. Recently, the putaminally-delivered construct, Eladocagene exuparvovec has been approved by the European Medicines Agency and by the British Medicines and Healthcare products Regulatory Agency. This now available gene therapy provides for the first time also for AADC deficiency (AADCD) a causal therapy, leading this disorder into a new therapeutic era. By using a standardized Delphi approach members of the International Working Group on Neurotransmitter related Disorders (iNTD) developed structural requirements and recommendations for the preparation, management and follow-up of AADC deficiency patients who undergo gene therapy. This statement underlines the necessity of a framework for a quality-assured application of AADCD gene therapy including Eladocagene exuparvovec. Treatment requires prehospital, inpatient and posthospital care by a multidisciplinary team in a specialized and qualified therapy center. Due to lack of data on long-term outcomes and the comparative efficacy of alternative stereotactic procedures and brain target sites, a structured follow-up plan and systematic documentation of outcomes in a suitable, industry-independent registry study are necessary.

Novel mutations in the mitochondrial complex I assembly gene NDUFAF5 reveal heterogeneous phenotypes.

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27 months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19 years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.

Consensus recommendation for a diagnostic guideline for acid sphingomyelinase deficiency.

Disclaimer:This diagnostic guideline is intended as an educational resource and represents the opinions of the authors, and is not representative of recommendations or policy of the American College of Medical Genetics and Genomics (ACMG). The information should be considered a consensus based on expert opinion, as more comprehensive levels of evidence were not available in the literature in all cases. BACKGROUND: Acid sphingomyelinase deficiency (ASMD) is a rare, progressive, and often fatal lysosomal storage disease. The underlying metabolic defect is deficiency of the enzyme acid sphingomyelinase that results in progressive accumulation of sphingomyelin in target tissues. ASMD manifests as a spectrum of severity ranging from rapidly progressive severe neurovisceral disease that is uniformly fatal to more slowly progressive chronic neurovisceral and chronic visceral forms. Disease management is aimed at symptom control and regular assessments for multisystem involvement. PURPOSE AND METHODS: An international panel of experts in the clinical and laboratory evaluation, diagnosis, treatment/management, and genetic aspects of ASMD convened to review the evidence base and share personal experience in order to develop a guideline for diagnosis of the various ASMD phenotypes. CONCLUSIONS: Although care of ASMD patients is typically provided by metabolic disease specialists, the guideline is directed at a wide range of providers because it is important for primary care providers (e.g., pediatricians and internists) and specialists (e.g., pulmonologists, hepatologists, and hematologists) to be able to identify ASMD.Genet Med advance online publication 13 April 2017.

Late onset of symptoms in an atypical patient with the cblJ inborn error of vitamin B12 metabolism: diagnosis and novel mutation revealed by exome sequencing.

Inborn errors of vitamin B(12) (cobalamin) metabolism are characterized by decreased production of active cobalamin cofactors and subsequent deficiencies in the activities of methionine synthase and methylmalonyl-CoA mutase. With the recent discovery of the cblJ defect in two patients with phenotypes mimicking the cblF defect, there are nine genes known to be involved in cobalamin metabolism. The new defect is caused by mutations in the ABCD4 gene, encoding an ABC transporter. At the moment, there is no clear distinction between the cblJ and cblF defects either clinically or biochemically, and both defects result in blocks in the transport of cobalamin from the lysosome to the cytoplasm. A patient was diagnosed with hyperhomocysteinemia and methylmalonic aciduria at the age of 8 years. Incorporations of both [(14)C]propionate and [(14)C]methyltetrahydrofolate in cultured fibroblasts were within reference ranges and thus too high to allow for complementation analysis. We observed decreased synthesis of both adenosylcobalamin and methylcobalamin and accumulation of unmetabolized cyanocobalamin. Exome sequencing was performed to identify causative mutation(s) and Sanger re-sequencing was performed to validate segregation of mutation in the family. By this approach, a homozygous mutation, c.423C>G, in the ABCD4 gene was identified. Here, we report the successful application of exome sequencing for diagnosis of a rare inborn error of vitamin B(12) metabolism in a patient whose unusual presentation precluded diagnosis using standard biochemical and genetic approaches. The patient represents only the third known patient with the cblJ disorder.

Corrigendum to "Evaluation of 3-O-methyldopa as a biomarker for aromatic L-amino acid decarboxylase deficiency in 7 Brazilian cases" [27/100744/2021/ pages: 1-4].

[This corrects the article DOI: 10.1016/j.ymgmr.2021.100744.].

STIG study: real-world data of long-term outcomes of adults with Pompe disease under enzyme replacement therapy with alglucosidase alfa.

BACKGROUND: Pompe disease is one of the few neuromuscular diseases with an approved drug therapy, which has been available since 2006. Our study aimed to determine the real-world long-term efficacy and safety of alglucosidase alfa. METHODS: This multicenter retrospective study (NCT02824068) collected data from adult Pompe disease patients receiving ERT for at least 3 years. Demographics and baseline characteristics, muscle strength, lung function (FVC), walking capability (6MWT), and safety were assessed once a year. Evaluation was done on the group and individual levels, using quantitative linear models (t test) and general univariate linear models (ANOVA). FINDINGS: Sixty-eight adult Pompe disease patients from four countries (Spain, Taiwan, Italy, Germany (STIG)) participated. The mean follow-up was 7.03 years ± 2.98. At group level in all outcome measures, an initial improvement followed by a secondary decline was observed. After 10 years, the 6MWT%pred showed the most sustained positive effect (p = 0.304). The MRC%max remained stable with a mild decline (p = 0.131), however, FVC%pred deteriorated significantly (p < 0.001) by 14.93% over 10 years of ERT. The progression rate of FVC%pred under ERT could be explained in most of the patients (83.5%) by the disease severity at baseline. Furthermore, our study shows a decline in the FVC combined with an increase in non-invasive and invasive ventilation requirements in adult Pompe disease patients over time. CONCLUSIONS: The STIG real-world study confirms an initial efficacy of ERT in the first years with a secondary sustained decline in multiple outcome measures. Further efforts are required to establish a more valid long-term monitoring and improved therapies.

Evaluation of 3-O-methyldopa as a biomarker for aromatic L-amino acid decarboxylase deficiency in 7 Brazilian cases.

Aromatic L-amino acid decarboxylase (AADCD) deficiency is an autosomal recessive neurometabolic disorder, caused by biallelic mutations in the DDC gene, that impairs the synthesis or metabolism of neurotransmitters leading to severe motor dysfunction. The main clinical signs are oculogyric crisis, hypotonia, hypokinesia, and dystonia. The biochemical diagnosis can be performed in cerebrospinal fluid by neurotransmitter analysis, which requires an invasive lumbar puncture, and the sample needs to be shipped frozen to a reference laboratory, usually across a country border. Measurement of AADC activity in plasma is also possible, but available in a few labs globally. 3-O-methyldopa (3-OMD) is a catabolic product of L-dopa and it is elevated in patients with AADC deficiency. The quantification of 3-OMD can be performed in dried blood spots (DBS), a sample that could be shipped at room temperature. 3-OMD levels of AADCD patients and controls were quantified in DBS by liquid chromatography tandem mass spectrometry. DBS samples from 7 Brazilian patients previously diagnosed with AADCD were used to validate the 3-OMD quantification as a screening procedure for this condition. All AADCD patients had at least a four-fold increase of 3-OMD. Thus, 3-OMD seems to be a reliable marker for AADCD, with potential use also in the newborn screening of this disease.

Gene therapy in the putamen for curing AADC deficiency and Parkinson's disease.

This commentary provides an overview of the putamen as an established target site for gene therapy in treating aromatic l-amino acid decarboxylase (AADC) deficiency and Parkinson's disease, two debilitating neurological disorders that involve motor dysfunction caused by dopamine deficiencies. The neuroanatomy and the function of the putamen in motor control provide good rationales for targeting this brain structure. Additionally, the efficacy and safety of intraputaminal gene therapy demonstrate that restoration of dopamine synthesis in the putamen by using low doses of adeno-associated viral vector serotype 2 to deliver the hAADC gene is well tolerated. This restoration leads to sustained improvements in motor and nonmotor symptoms of AADC deficiency and improved uptake and conversion of exogenous l-DOPA into dopamine in Parkinson's patients.

Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

Trabecular microfracture and the influence of pyridinium and non-enzymatic glycation-mediated collagen cross-links.

The propensity of individual trabeculae to fracture (microfracture) may be important clinically since it could be indicative of bone fragility. Whether or not an overloaded trabecula fractures is determined in part by its structural ductility, a mechanical property that describes how much deformation a trabecula can sustain. The overall goal of this study was to determine the structural ductility of individual trabeculae and the degree to which it is influenced by pyridinium and non-enzymatic collagen cross-links. Vertically oriented rodlike trabeculae were taken from the thoracic vertebral bodies of 32 cadavers (16 male and 16 female, 54 - 94 years of age). A total of 221 trabeculae (4 - 9 per donor) were tested to failure in tension using a micro-tensile loading device. A subset of 76 samples was analyzed to determine the concentration of hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP) cross-links as well as pentosidine, a marker of non-enzymatic glycation. Structural ductility (defined as the ultimate strain of the whole trabecula) ranged from 1.8% to 20.2% strain (8.8 +/- 3.7%, mean +/- SD) and did not depend on age (P = 0.39), sex (P = 0.57), or thickness of the sample at the point of failure (P = 0.36). Pentosidine was the only marker of collagen cross-linking measured that was found to be correlated with structural ductility (P = 0.01) and explained about 9% of the observed variance. We conclude that the ductility of individual trabeculae varies tremendously, can be substantial, and is weakly influenced by non-enzymatic glycation.

Over-expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma.

AIM: To describe the significant over-expression of fibroblast growth factor receptor 3 (FGFR3), which is a signal transduction and cell proliferation related gene in hepatocellular carcinoma (HCC). METHODS: Following DNA microarray, Northern blot and quantitative real-time PCR were employed to confirm FGFR3 expression difference in HCC tissues and surrounding non-neoplastic liver tissue. FGFR3 expression levels were further determined by immunohistochemical study in 43 cases of HCC. RESULTS: Northern blot results showed the significant over-expression of FGFR3 in HCC tissues, which was consistent with that from DNA microarray. Quantitative real-time PCR demonstrated that the mean ratio of FGFR3 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA in HCC tissue was 0.250, whereas the ratio in non-neoplastic liver tissue was 0.014. Statistical analyses of 43 cases of HCC revealed that HCC scored higher than the matched non-neoplastic liver tissues. Examination of clinicopathological features revealed a strong correlation of over-expression of FGFR3 with poor tumor differentiation and high nuclear grade. CONCLUSION: Over-expression of FGFR3 may play an important role in liver carcinogenesis. FGFR3 may be an ideal candidate as a molecular marker in the diagnosis of HCC and a potential therapeutic target.