Recognition of alpha-mannosidosis in paediatric and adult patients: Presentation of a diagnostic algorithm from an international working group.

Alpha-mannosidosis is an ultra-rare progressive lysosomal storage disorder caused by deficiency of alpha-mannosidase. Timely diagnosis of the disease has the potential to influence patient outcomes as preventive therapies can be initiated at an early stage. However, no internationally-recognised algorithm is currently available for the diagnosis of the disease. With the aim of developing a diagnostic algorithm for alpha-mannosidosis an international panel of experts met to reach a consensus by applying the nominal group technique. Two proposals were developed for diagnostic algorithms of alpha-mannosidosis, one for patients ≤10 years of age and one for those >10 years of age. In younger patients, hearing impairment and/or speech delay are the cardinal symptoms that should prompt the clinician to look for additional symptoms that may provide further diagnostic clues. Older patients have different clinical presentations, and the presence of mental retardation and motor impairment progression and/or psychiatric manifestations should prompt the clinician to assess for other symptoms. In both younger and older patients, either additional metabolic monitoring or referral for testing is warranted upon suspicion of disease. Oligosaccharides in urine (historically performed) or serum were considered as an initial screening procedure, while enzymatic activity may also be considered as first choice in some centres. Molecular testing should be performed as a final confirmatory step. The developed algorithms can easily be applied in a variety of settings, and may help to favour early diagnosis of alpha mannosidosis and treatment.

Impact of immunosuppressive therapy on therapy-neutralizing antibodies in transplanted patients with Fabry disease.

BACKGROUND: Inhibitory antibodies towards enzyme replacement therapy (ERT) are associated with disease progression and poor outcome in affected male patients with lysosomal disorders such as Fabry disease (FD). However, little is known about the impact of immunosuppressive therapy on ERT inhibition in these patients with FD. METHODS: In this retrospective study, we investigated the effect of long-term immunosuppression on ERT inhibition in male patients with FD (n = 26) receiving immunosuppressive therapy due to kidney (n = 24) or heart (n = 2) transplantation. RESULTS: No ERT-naïve transplanted patient (n = 8) developed antibodies within follow-up (80 ±72 months) after ERT initiation. Seven (26.9%) patients were tested ERT inhibition positive prior to transplantation. No de novo ERT inhibition was observed after transplantation (n = 18). In patients treated with high dosages of immunosuppressive medication such as prednisolone, tacrolimus and mycophenolate-mofetil/mycophenolate acid, ERT inhibition decreased after transplantation (n = 12; P = 0.0160). Tapering of immunosuppression (especially prednisolone) seemed to re-increase ERT inhibition (n = 4, median [range]: 16.6 [6.9; 36.9] %; P = 0.0972) over time. One ERT inhibition-positive patient required interventions with steroid therapy and increased doses of tacrolimus, which also lowered ERT inhibition. CONCLUSION: We conclude that the immunosuppressive maintenance therapy after transplantations seems to be sufficient to prevent de novo ERT inhibition in ERT-naïve patients. Intensified high dosages of immunosuppressive drugs are associated with decreased antibody titres and decreased ERT inhibition in affected patients, but did not result in long-term protection. Future studies are needed to establish ERT inhibition-specific immunosuppressive protocols with long-term modulating properties to warrant an improved disease course in ERT inhibition-positive males.

DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics.

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6-mutation negative individuals with non-syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.

Clinical onset and course, response to treatment and outcome in 24 patients with the cblE or cblG remethylation defect complemented by genetic and in vitro enzyme study data.

BACKGROUND: The cobalamin E (cblE) (MTRR, methionine synthase reductase) and cobalamin G (cblG) (MTR, methionine synthase) defects are rare inborn errors of cobalamin metabolism leading to impairment of the remethylation of homocysteine to methionine. METHODS: Information on clinical and laboratory data at initial full assessment and during the course of the disease, treatment, outcome and quality of life was obtained in a survey-based, retrospective study from physicians caring for patients with the CblE or CblG defect. In addition, data on enzyme studies in cultured skin fibroblasts and mutations in the MTRR and MTR gene were analysed. RESULTS: In 11 cblE and 13 cblG patients, failure to thrive, feeding problems, delayed milestones, muscular hypotonia, cognitive impairment and macrocytic anaemia were the most frequent symptoms. Delay in diagnosis depended on age at first symptom and clinical pattern at presentation and correlated significantly with impaired communication abilities at follow-up. Eighteen/22 patients presented with brain atrophy or white matter disease. Biochemical response to treatment with variable combinations of betaine, cobalamin, folate was significant. The overall course was considered improving (n = 8) or stable (n = 15) in 96% of patients, however the average number of CNS symptoms per patient increased significantly over time and 16 of 23 patients were classified as developmentally delayed or severely handicapped. In vitro enzyme analysis data showed no correlation with outcome. Predominantly private mutations were detected and no genotype- phenotype correlations evident. CONCLUSIONS: The majority of patients with the cblE and cblG defect show limited clinical response to treatment and have neurocognitive impairment.

Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Propionic acidemia: neonatal versus selective metabolic screening.

BACKGROUND: Whereas propionic acidemia (PA) is a target disease of newborn screening (NBS) in many countries, it is not in others. Data on the benefit of NBS for PA are sparse. STUDY DESIGN: Twenty PA patients diagnosed through NBS were compared to 35 patients diagnosed by selective metabolic screening (SMS) prompted by clinical findings, family history, or routine laboratory test results. Clinical and biochemical data of patients from 16 metabolic centers in Germany, Austria, and Switzerland were evaluated retrospectively. Additionally, assessment of the intelligent quotient (IQ) was performed. In a second step, the number of PA patients who have died within the past 20 years was estimated based on information provided by the participating metabolic centers. RESULTS: Patients diagnosed through NBS had neither a milder clinical course regarding the number of metabolic crises nor a better neurological outcome. Among NBS patients, 63% were already symptomatic at the time of diagnosis, and <10% of all patients remained asymptomatic. Among all PA patients, 76% were found to be at least mildly mentally retarded, with an IQ <69. IQ was negatively correlated with the number of metabolic decompensations, but not simply with the patients' age. Physical development was also impaired in the majority of patients. Mortality rates tended to be lower in NBS patients compared with patients diagnosed by SMS. CONCLUSION: Early diagnosis of PA through NBS seems to be associated with a lower mortality rate. However, no significant benefit could be shown for surviving patients with regard to their clinical course, including the number of metabolic crises, physical and neurocognitive development, and long-term complications.

Management and outcome in 75 individuals with long-chain fatty acid oxidation defects: results from a workshop.

At present, long-chain fatty acid oxidation (FAO) defects are diagnosed in a number of countries by newborn screening using tandem mass spectrometry. In the majority of cases, affected newborns are asymptomatic at time of diagnosis and acute clinical presentations can be avoided by early preventive measures. Because evidence-based studies on management of long-chain FAO defects are lacking, we carried out a retrospective analysis of 75 patients from 18 metabolic centres in Germany, Switzerland, Austria and the Netherlands with special regard to treatment and disease outcome. Dietary treatment is effective in many patients and can prevent acute metabolic derangements and prevent or reverse severe long-term complications such as cardiomyopathy. However, 38% of patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency had intermittent muscle weakness and pain despite adhering to therapy. Seventy-six per cent of patients with disorders of the mitochondrial trifunctional protein (TFP)-complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, had long-term myopathic symptoms. Of these, 21% had irreversible peripheral neuropathy and 43% had retinopathy. The main principle of treatment was a fat-reduced and fat-modified diet. Fat restriction differed among patients with different enzyme defects and was strictest in disorders of the TFP-complex. Patients with a medium-chain fat-based diet received supplementation of essential long-chain fatty acids. l-Carnitine was supplemented in about half of the patients, but in none of the patients with VLCAD deficiency identified by newborn screening. In summary, in this cohort the treatment regimen was adapted to the severity of the underlying enzyme defect and thus differed among the group of long-chain FAO defects.

Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop.

Published data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects.

Dihydropyrimidine dehydrogenase deficiency caused by a novel genomic deletion c.505_513del of DPYD.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.

[Thromboembolic events, abortions and a sick infant--unusual presentation of a vitamin deficiency]. / Thromboembolien, Aborte und ein krankes Neugeborenes. Ungewöhnliche Präsentation eines Vitaminmangels.

Homocysteine is a risk factor for the development of thromboembolic disorders and vascular diseases. Furthermore, complications during pregnancy have been ascribed to hyperhomocysteinemia. We report on a pregnant woman being substituted by high doses folic acid for hyperhomocysteinemia. Thereby, the underlying pernicious anemia was masked. After birth, the neonate was exclusively breastfed. At the age of 5 months, the infant had to be admitted to hospital due to severe vitamin B(12)-deficiency. Using parenteral vitamin B(12) substitution, homocystein levels of the mother normalized and the infant throve and prospered again.

[A rare inborn error of intracellular processing of cobalamine presenting with microcephalus and megaloblastic anemia: a report of 3 children]. / Mikrozephalus und megaloblastäre Anämie als Leitsymptome einer seltenen angeborenen Störung des intrazellulären Kobalamin-Processings--3 Kasuistiken.

INTRODUCTION: Defects of methionine synthase or methionine synthase reductase result in an impaired remethylation of homocysteine to methionine. Patients present with megaloblastic anemia, failure to thrive and various neurological manifestations including mental retardation, cerebral atrophy, muscular hypotonia or hypertonia, ataxia, seizures, nystagmus and visual disturbances. PATIENTS: We report on three children (two girls, one boy), aged 3.5-7.5 years, who presented with severe megaloblastic anemia, micro-cephalus and partly nystagmus (2/3) due to a rare inborn error of remethylation. RESULTS: Methionine synthase reductase deficiency, cblE type of homocystinuria (OMIM 236270), is a rare autosomal recessive inherited disorder described only in 14 patients worldwide. Metabolic hallmarks of the disease are hyperhomocysteinemia (median 98 micromol/l, normal range <15) without methylmalonic aciduria but often hypomethioninemia. The patients described here were diagnosed at ages of 2-18 months. The importance of an early recognition of this possibly underdiagnosed congenital disease is stressed. Treatment consisted of the application of hydroxocobalamine (1-2 mg weekly, i.m.), betaine (100-200 mg/kg daily, p.o.), folate (5-10 mg daily, p.o.) and intensive physical therapy. CONCLUSION: Defects of intracellular processing of cobalamine must be considered in all patients with neurological symptoms in combination with megaloblastic anemia. Measurements of homocysteine and methionine in plasma as well as methylmalonic acid in urine is required for confirming the diagnosis. Early treatment im-proves the outcome, although mental disability may not be prevented. Treatment has a positive impact on megaloblastic anemia but only slight effect on hyperhomocysteinemia. The long-term cardiovascular risk of hyperhomocysteinemia in cblE deficient patients is not known yet.

Benzoate treatment and the glycine index in nonketotic hyperglycinaemia.

High-dose benzoate treatment aimed at reducing plasma glycine levels to normal reduces seizures and increases wakefulness in patients with nonketotic hyperglycinaemia (NKH). Since benzoate metabolism is dependent on the available glycine pool, and since the glycine pool is variably affected by the deficiency in the glycine cleavage enzyme system, we examined the importance of interpatient variability in benzoate requirement. To correct for the dietary glycine contribution, the glycine index was introduced as the molar requirement of benzoate dose necessary to normalize plasma glycine levels and subtracting from that the dietary glycine intake, both corrected for weight. The glycine index varied between 3.62 and 4.87 mmol/kg per day in five patients with a poor neurodevelopmental outcome and between 0.92 and 1.90 mmol/kg per day in four patients with a better neurodevelopmental outcome, and was 2.54 mmol/kg per day in a single patient with an intermediate outcome. The glycine index was stable over time within each patient. Exceeding the balance by either increasing food glycine intake or decreasing the benzoate dose resulted in increased glycine levels. Exceeding the glycine tolerance by increasing benzoate resulted in elevated and toxic levels of benzoate. The glycine index is a stable, individually specific parameter in patients with NKH. It has clinical consequences for the dose of benzoate required and the role of dietary management. Through its correlation with neurodevelopmental outcome, the glycine index points to potential genetic factors that could contribute to the psychomotor retardation in NKH.

Lipid and lipoprotein profiles in children with familial hypercholesterolaemia: effects of therapy.

UNLABELLED: In 71 children with familial hypercholesterolaemia the effect of dietary and/or medical treatment was evaluated. Initial total cholesterol and low density lipoprotein (LDL)-cholesterol levels were significantly lower in children who were consecutively treated by diet (Step-One-Diet) than in those who received additional medication. By dietary treatment, the median total cholesterol level (236.5 mg/dl; range 210-510 mg/dl) was reduced by 7.4% and the median LDL-cholesterol level (162 mg/dl; range 126-423 mg/dl) by 9.9%. By dietary and medical therapy, the median total cholesterol level (330 mg/dl; range 270-424 mg/dl) was reduced by 29.7% and the median LDL-cholesterol level (263 mg/dl; 192-333 mg/dl) by 25.9%. High density lipoprotein (HDL)-cholesterol and HDL 3 remained unchanged. HDL 2 showed a significant decrease of 15.6% up to 27 mg/dl (13-42 mg/dl) on medical treatment. Apolipoprotein A I levels did not change during therapy. Initial apolipoprotein B levels were significantly higher in children who were treated by diet and medication and were reduced by 28.9% by combined therapy. In 28 patients (39.4%) an excess of lipoprotein (a) was detected. Regarding the apolipoprotein E phenotype, 32.2% of the patients carried the risk gene epsilon4 in a hetero- or homozygous form. CONCLUSION: Early dietary and/or medical treatment in hypercholesterolaemic children significantly ameliorates the lipoprotein status. The pretherapy lipoprotein status seems to prognosticate the effectiveness of therapy.

Phenylketonuria and hyperphenylalaninemia in eastern Germany: a characteristic molecular profile and 15 novel mutations.

Phenylketonuria (PKU) is an important error of amino acid metabolism which results in most patients from phenylalanine hydroxylase (PAH) deficiency. PKU displays a marked genotypic heterogeneity both within and between different populations. The aim of this study was to establish the genotypic spectrum of PKU in eastern Germany, and to compare this to the distribution of mutations in western Germany. The study population included 302 patients in 290 families who were followed at treatment centers in Berlin, Leipzig and Jena. The study showed marked genotypic variability with a total of 75 mutations, including 15 that have so far not been described (eleven missense mutations, one splicing mutation, and three small deletions). One of these novel mutations, E183Q, occurred in cis to a R408W mutation. In the non-immigrant eastern German population, the frequency of R408W accounted for 40.1% of the PKU alleles. In the immigrant Turkish population of the former West Berlin, the most prevalent mutation was IVS10-11G>A (57%). There was a marked difference of the genotypic spectrum between the population studied here and the data reported from the western part of the country.