Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Long-term disease course of two patients with multiple sulfatase deficiency differs from metachromatic leukodystrophy in a broad cohort.

Multiple sulfatase deficiency (MSD) is a lysosomal storage disease caused by a deficiency of formylglycine-generating enzyme due to SUMF1 defects. MSD may be misdiagnosed as metachromatic leukodystrophy (MLD), as neurological and neuroimaging findings are similar, and arylsulfatase A (ARSA) deficiency and enhanced urinary sulfatide excretion may also occur. While ARSA deficiency seems a cause for neurological symptoms and later neurodegenerative disease course, deficiency of other sulfatases results in clinical features such as dysmorphism, dysostosis, or ichthyosis. We report on a girl and a boy of the same origin presenting with severe ARSA deficiency and neurological and neuroimaging features compatible with MLD. However, exome sequencing revealed not yet described homozygosity of the missense variant c.529G > C, p.Ala177Pro in SUMF1. We asked whether dynamics of disease course differs between MSD and MLD. Comparison to a cohort of 59 MLD patients revealed different disease course concerning onset and disease progression in both MSD patients. The MSD patients showed first gross motor symptoms earlier than most patients with juvenile MLD (<10th percentile of Gross-Motor-Function in MLD [GMFC-MLD] 1). However, subsequent motor decline was more protracted (75th and 90th percentile of GMFC-MLD 2 (loss of independent walking) and 75th percentile of GMFC-MLD 5 (loss of any locomotion)). Language decline started clearly after 50th percentile of juvenile MLD and progressed rapidly. Thus, dynamics of disease course may be a further clue for the characterization of MSD. These data may contribute to knowledge of natural course of ultra-rare MSD and be relevant for counseling and therapy.

Comparison of early pregnancy serum concentration of neopterin, neopterin/creatinine ratio, C-reactive protein, and chitotriosidase, in pregnant women with birth at term and spontaneous preterm birth.

Inflammatory mechanisms are involved in achieving a normal pregnancy and in the development of certain pregnancy complications. These changes are more intense in pregnant women that suffer of pregnancy complications, such as spontaneous preterm birth (SPB). This study compared the course of inflammatory markers (IM) [neopterin (Neo), neopterin/creatinine ratio (Neo/Cre), C-reactive protein (CRP), and chitotriosidase (Chito)] serum concentration in the early pregnancy of women with birth at term (BT) and preterm birth (PB). IM concentration was measured in 90 sera sampled from 45 pregnancies with BT and 30 sera from 15 pregnancies with PB. Two sera were sampled from each pregnant woman: one in the first trimester and another one in the second trimester. Early pregnancy IM concentration showed a direct correlation with gestational age: Neo (rho=0.262, P=0.004), Neo/Cre (rho=0.372, P<0.001), CRP (rho=0.187, P=0.041), and Chito (rho=0.039, P=0.66). The correlation was present in both categories of patients with BT and PB. Patients with PB before 34 week of pregnancy (wp) and 32 wp showed higher Neo and Neo/Cre concentration than BT patients. A significant association was found between the risk of PB before 34 wp, PB before 32 wp, and Neo concentration (PB <34 wp: odds ratio (OR) =5.13, P=0.035) (PB <32 wp: OR=8.2, P=0.020) and, respectively, Neo/Cre concentration (PB <34 wp: OR=5.29, P=0.015) (PB <32 wp: OR=9.25, P=0.006). No association between CRP or Chito and PB age was found. IM concentration correlates with the gestational age at the time of blood sampling. Increased Neo and Neo/Cre concentration are associated with PB. Further studies are needed to evaluate the usefulness of these markers in clinical practice.

Human heterologous liver cells transiently improve hyperammonemia and ureagenesis in individuals with severe urea cycle disorders.

BACKGROUND: Urea cycle disorders (UCDs) still have a poor prognosis despite several therapeutic advancements. As liver transplantation can provide a cure, liver cell therapy (LCT) might be a new therapeutic option in these patients. METHODS: Twelve patients with severe UCDs were included in this prospective clinical trial. Patients received up to six infusions of cryopreserved human heterologous liver cells via a surgically placed catheter in the portal vein. Portal vein pressure, portal vein flow, and vital signs were monitored continuously. Calcineurin inhibitors and steroids were used for immunosuppression. In four patients, ureagenesis was determined with stable isotopes. Number and severity of hyperammonemic events and side effects of immunosuppression were analyzed during an observation period of up to 2 years. RESULTS: No study-related mortality was observed. The application catheter dislocated in two children. No significant side effects of catheter application or cell infusion were noted in the other ten patients. The overall incidence of infections did not differ significantly from a historical control group, and no specific side effects of immunosuppression were found. Seven patients were treated per protocol and could be analyzed for efficacy. Severe metabolic crises could be prevented in all of these patients, moderate crises in four of seven. Ureagenesis increased after cell infusion in all patients investigated. CONCLUSIONS: We found a favorable safety profile with respect to catheter placement, intraportal liver cell infusion, and immunosuppression. More than half of the children treated per protocol experienced metabolic stabilization and could be safely bridged to liver transplantation.

Multigene panel next generation sequencing in a patient with cherry red macular spot: Identification of two novel mutations in NEU1 gene causing sialidosis type I associated with mild to unspecific biochemical and enzymatic findings.

BACKGROUND: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. METHODS: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. CASE REPORT AND RESULTS: Cherry red macular spots were first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations as well as cerebral MRI were unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to < 1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699C > A, p.S233R in exon 4 and c.803A > G; p.Y268C in Exon 5 in NEU1 transcript NM_000434.3), leading to amino acid changes predicted to impair protein function. DISCUSSION: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

A novel autosomal recessive TERT T1129P mutation in a dyskeratosis congenita family leads to cellular senescence and loss of CD34+ hematopoietic stem cells not reversible by mTOR-inhibition.

The TERT gene encodes for the reverse transcriptase activity of the telomerase complex and mutations in TERT can lead to dysfunctional telomerase activity resulting in diseases such as dyskeratosis congenita (DKC). Here, we describe a novel TERT mutation at position T1129P leading to DKC with progressive bone marrow (BM) failure in homozygous members of a consanguineous family. BM hematopoietic stem cells (HSCs) of an affected family member were 300-fold reduced associated with a significantly impaired colony forming capacity in vitro and impaired repopulation activity in mouse xenografts. Recent data in yeast suggested improved cellular checkpoint controls by mTOR inhibition preventing cells with short telomeres or DNA damage from dividing. To evaluate a potential therapeutic option for the patient, we treated her primary skin fibroblasts and BM HSCs with the mTOR inhibitor rapamycin. This led to prolonged survival and decreased levels of senescence in T1129P mutant fibroblasts. In contrast, the impaired HSC function could not be improved by mTOR inhibition, as colony forming capacity and multilineage engraftment potential in xenotransplanted mice remained severely impaired. Thus, rapamycin treatment did not rescue the compromised stem cell function of TERTT1129P mutant patient HSCs and outlines limitations of a potential DKC therapy based on rapamycin.

Clinical Research in Vulnerable Populations: Variability and Focus of Institutional Review Boards' Responses.

BACKGROUND: Children and patients with cognitive deficits may find it difficult to understand the implication of research. In the European Union (EU), clinical studies outside the EU directives concerning medicinal products or medical devices, i.e., "miscellaneous clinical studies", have no legally mandated timelines for institutional review boards' (IRB) decisions. GOAL: To evaluate the review process of IRBs for two different "miscellaneous" multicenter clinical research protocols involving vulnerable subjects (children and adult stroke patients). METHODS: Descriptive and comparative statistics. Protocol 1 is a prospective, multicenter, cross-sectional screening study of a symptomatic pediatric population at risk for Fabry disease involving genetic testing (NCT02152189). Protocol 2 is a prospective, multicenter, randomized, controlled, open-label, blinded endpoint post-market study to evaluate the effectiveness of stent retrievers (NCT02135926). After having obtained positive initial IRB votes at the main study site, both protocols were subsequently submitted to the remaining IRBs. RESULTS: Protocol 1 was submitted to 19 IRBs. No IRB objected to the study. Median time-to-final vote was 34 (IQR 10-65; range 0 to 130) days. Two IRBs accepted the coordinating center's IRB votes without re-evaluation. Changes to the informed consent documents were asked by 7/19 IRBs, amendments to the protocol by 2. Protocol 2 was submitted to 16 IRBs. Fifteen decisions were made. No IRB objected to the study. Median time-to final vote was 59 (IQR 10 to 65; range 0 to 128) days, which was not statistically significantly different compared with protocol 1 (Wilcoxon test). Two IRBs accepted a previous IRB decision and did not conduct an independent review. Eight/16 IRBs required changes to the informed consent documents; two IRBs recommended an amendment of the protocol. CONCLUSION: Both clinical research protocols involving vulnerable populations were well accepted. IRB workflows and decision times varied substantially. Some IRBs accepted a previous IRB decision without the necessity of another reevaluation process. Requested changes were focused on the informed consent documents. A more standardized approach across jurisdictions is desirable.

Language and cognition in children with metachromatic leukodystrophy: onset and natural course in a nationwide cohort.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare, genetic neurodegenerative disease. It leads to progressive demyelination resulting in regression of development and early death. With regard to experimental therapies, knowledge of the natural course of the disease is highly important. We aimed to analyse onset and character of first symptoms in MLD and to provide detailed natural course data concerning language and cognition. METHODS: Patients with MLD were recruited nationwide within the scope of the German research network LEUKONET. 59 patients' questionnaires (23 late-infantile, 36 juvenile) were analysed. RESULTS: Time from first symptoms (at a median age of 1.5 years in late-infantile and 6 years in juvenile MLD) to diagnosis took one year in late-infantile and two years in juvenile patients on average. Gait disturbances and abnormal movement patterns were first signs in all patients with late-infantile and in most with juvenile MLD. Onset in the latter was additionally characterized by problems in concentration, behaviour and fine motor function (p=0.0011, p<0.0001, and p=0.0012). Half of late-infantile patients did not learn to speak in complete sentences after an initially normal language acquisition. They showed a rapid language decline with first language difficulties at a median age of 2.5 years and complete loss of expressive language within several months (median age 32, range 22-47 months). This was followed by total loss of communication at a median age of around four years. In juvenile patients, language decline was more protracted, and problems in concentration and behaviour were followed by decline in skills for reading, writing and calculating around four years after disease onset. CONCLUSIONS: Our data reflect the natural course of decline in language and cognition in late-infantile and juvenile MLD in a large cohort over a long observation period. This is especially relevant to juvenile patients where the disease course is protracted and prospective studies are hardly feasible. Knowledge of first symptoms may lead to earlier diagnosis and subsequently to a better outcome following therapeutic intervention. Our data may serve as a reference for individual treatment decisions and for evaluation of clinical outcome after treatment intervention.

Aspartylglucosaminuria: unusual neonatal presentation in Qatari twins with a novel aspartylglucosaminidase gene mutation and 3 new cases in a Turkish family.

Aspartylglucosaminuria is a rare autosomal recessive lysosomal storage disorder leading early to a progressive intellectual disability. Monozygous Qatari twins presented with an unusual perinatal manifestation characterized by severe muscular hypotonia, scarce spontaneous movements, multiple contractures, and respiratory insufficiency. Biochemical investigations suggested aspartylglucosaminuria, and a novel homozygous mutation c.439T>C (p.S147P) was found in the aspartylglucosaminidase gene. However, it cannot be excluded that the unusual neonatal presentation is due to an additional autosomal recessive disease in this multiply consanguineous family. The classical aspartylglucosaminuria phenotype (progressive speech delay, psychomotor retardation, and behavioral abnormalities) was observed in 3 Turkish siblings. Although aspartylglucosaminuria was suspected early, the definite diagnosis was not confirmed until the age of 18 years. A novel homozygous mutation c.346C>T (p.R116W) was found. These 5 cases emphasize that aspartylglucosaminuria is panethnic and may possibly present with prenatal manifestation. Screening for aspartylglucosaminuria should be done in all patients with unexplained psychomotor retardation.

Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations.

Mucopolysaccharidosis III D (Sanfilippo disease type D, MPS IIID) is a rare autosomal recessive lysosomal storage disorder previously described in only 20 patients. MPS IIID is caused by a deficiency of N-acetylglucosamine-6-sulphate sulphatase (GNS), one of the enzymes required for the degradation of heparan sulphate. So far only seven mutations in the GNS gene have been reported. The clinical phenotype of 12 new MPS IIID patients from 10 families was studied. Mutation analysis of GNS was performed in 16 patients (14 index cases). Clinical signs and symptoms of the MPS IIID patients appeared to be similar to previously described patients with MPS III. Early development was normal with onset of behavioral problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication and subsequent deterioration of motor functions. Sequence analysis of the coding regions of the gene encoding GNS (GNS) resulted in the identification of 15 novel mutations: 3 missense mutations, 1 nonsense mutation, 4 splice site mutations, 3 frame shift mutations, 3 large deletions and 1 in-frame small deletion. They include the first missense mutations and a relatively high proportion of large rearrangements, which warrants the inclusion of quantitative techniques in routine mutation screening of the GNS gene.

GM1 gangliosidosis and Morquio B disease: expression analysis of missense mutations affecting the catalytic site of acid beta-galactosidase.

Alterations in GLB1, the gene coding for acid beta-D-galactosidase (beta-Gal), can result in GM1 gangliosidosis (GM1), a neurodegenerative disorder, or in Morquio B disease (MBD), a phenotype with dysostosis multiplex and normal central nervous system (CNS) function. While most MBD patients carry a common allele, c.817TG>CT (p.W273L), only few of the >100 mutations known in GM1 can be related to a certain phenotype. In 25 multiethnic patients with GM1 or MBD, 11 missense mutations were found as well as one novel insertion and a transversion causing aberrant gene products. Except c.602G>A (p.R201H) and two novel alleles, c.592G>T (p.D198Y) and c.1189C>G (p.P397A), all mutants resulted in significantly reduced beta-Gal activities (<10% of normal) upon expression in COS-1 cells. Although c.997T>C (p.Y333H) expressed 3% of normal activity, the mutant protein was localized in the lysosomal-endosomal compartment. A homozygous case presented with late infantile GM1, while a heterozygous, juvenile case carried p.Y333H together with p.R201H. This allele, recently found in homozygous MBD, gives rise to rough endoplasmic reticulum (RER)-located beta-Gal precursors. Thus, unlike classical MBD, the phenotype of heterozygotes carrying p.R201H may rather be determined by poorly active, properly transported products of the counter allele than by the mislocalized p.R201H precursors.

Neonatal manifestation of multiple sulfatase deficiency.

INTRODUCTION: Multiple sulfatase deficiency is biochemically characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. CASE REPORT: We report clinical, biochemical, and molecular findings in a female newborn affected with a severe form of multiple sulfatase deficiency (Mendelian Inheritance in Man (MIM) # 272200). She presented with primary microcephaly, facial anomalies including depressed nasal bridge, nasal hypoplasia, anteverted nostrils, smooth philtrum, limited mobility of hip and knee joints, mild ichthyosis, as well as muscular hypotonia. The diagnosis is based on detection of excessive mucopolysacchariduria and enzymatic assays performed in leucocytes which showed complete deficiency of all of the measured sulfatases. Sequencing of the coding region of the underlying gene, SUMF1, could not identify any mutation. However, failure to detect the corresponding mRNA by reverse transcription polymerase chain reaction proves defective SUMF1 expression. CONCLUSION: The diagnosis of neonatal MSD should be considered when dealing with the association of distinct facial anomalies, limited joint mobility, ichthyosis, and muscular hypotonia.

A novel mutation in UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTAG) in two siblings with mucolipidosis type III alters a used glycosylation site.

The N-acetylglucosaminyl-1-phosphotransferase (termed phosphotransferase) catalyzes the initial step in the formation of mannose 6-phosphate (M6P) residues required for the efficient transport of soluble lysosomal enzymes. The phosphotransferase is a multisubunit enzyme composed of three subunits (alpha2beta2gamma2) that are products of two genes. The gene encoding the gamma-subunit (GNPTAG) appears to be defective in patients with mucolipidosis type III (ML III). We have analyzed the GNPTAG gene in two siblings with ML III showing elevated activities of several lysosomal enzymes in cultured fibroblasts serum and diminished activities in cultured fibroblasts. Immunoprecipitation of metabolically labeled cathepsin D (CtsD) from fibroblasts revealed that the sorting/transport of this lysosomal protease was affected. Addition of ammonium chloride inhibiting pH-dependent processes, such as the CtsD-M6P receptor interaction, indicated that 15 to 20% of the newly synthesized CtsD is transported in ML III fibroblasts in an M6P-dependent manner. By direct sequencing a novel homozygous mutation, c.347_349delACA (p.Asn116del), was identified affecting a potential N-linked glycosylation site. Western blot analysis of extracts from control fibroblasts detect a 97 kDa glycosylated dimer whereas ML III cells contain a GNPTAG dimer of reduced molecular mass. These data suggest that the loss of the used glycosylation site in the gamma subunit may affect the intracellular localization of GNPTAG and the overall efficiency of M6P formation.

Different phenotypes in human prostate cancer: alpha6 or alpha3 integrin in cell-extracellular adhesion sites.

The distribution of alpha6/alpha3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized alpha6/alpha3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The alpha6/alpha3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA). The associations were assessed by statistical methods. Eighty percent of the tumors expressed the alpha6 or alpha3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both alpha6 and alpha3 subunits, type II exclusively expressed alpha6 integrin, and type III expressed alpha3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The alpha6/alpha3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, alpha6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the alpha6 subunit did not colocalize with the alpha subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the alpha6beta1 and/or alpha3beta1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.