François Boemer

Newborn Screening for Sickle Cell Disease Using Tandem Mass Spectrometry

BACKGROUND Neonatal screening programs for sickle cell disease are now widespread in North American and European countries. Most programs apply isoelectric focusing or HPLC to detect hemoglobin variants. Because tandem mass spectrometry (MS/MS) is being used for screening of inherited metabolic disorders and allows protein identification, it was worth testing for hemoglobinopathy screening. METHODS We minimized sample preparation and analysis times by avoiding prior purification, derivatization, or separation. We developed a tryptic digestion methodology to screen for the main clinically important variants (Hb S, Hb C, and Hb E) and beta-thalassemia. To ensure proper discrimination between homozygote and heterozygote variants, we selected 4 transitions with good signal intensities for each specific peptide and calculated variant/Hb A ratios for each. Method validation included intra- and interseries variability, carryover, and limit of detection. We also performed a comparative study with isoelectric focusing results on 2082 specimens. RESULTS Intraassay imprecision values (CVs) varied between 2.5% and 30.7%. Interassay CVs were between 6.3% and 23.6%. Carryover was <0.03%, and the limit of detection was fixed at 1% of Hb S. According to the MS/MS settings (detection of Hb S, Hb C, Hb E, and beta-globin production defects), the comparative study did not yield any discrepant results between the 2 techniques. CONCLUSIONS MS/MS is a reliable method for hemoglobinopathy neonatal screening.

Identification of methylenecyclopropyl acetic acid in serum of European horses with atypical myopathy

REASONS FOR PERFORMING STUDY It is hypothesised that European atypical myopathy (AM) has a similar basis as seasonal pasture myopathy in North America, which is now known to be caused by ingestion of hypoglycin A contained in seeds from the tree Acer negundo. Serum from horses with seasonal pasture myopathy contained the conjugated toxic metabolite of hypoglycin A, methylenecyclopropyl acetic acid (MCPA). STUDY DESIGN Retrospective study on archived samples. OBJECTIVES 1) To determine whether MCPA-carnitine was present in serum of European horses confirmed to have AM; 2) to determine whether Acer negundo or related Acer species were present on AM pastures in Europe. METHODS Concentrations of MCPA-carnitine were analysed in banked serum samples of 17 AM horses from Europe and 3 diseased controls (tetanus, neoplasia and exertional rhabdomyolysis) using tandem mass spectrometry. Atypical myopathy was diagnosed by characteristic serum acylcarnitine profiles. Pastures of 12 AM farms were visited by experienced botanists and plant species were documented. RESULTS Methylenecyclopropyl acetic acid-carnitine at high concentrations (20.39 ± 17.24 nmol/l; range 0.95-57.63 nmol/l; reference: <0.01 nmol/l) was identified in serum of AM but not disease controls (0.00 ± 0.00 nmol/l). Acer pseudoplatanus but not Acer negundo was present on all AM farms. CONCLUSIONS Atypical myopathy in Europe, like seasonal pasture myopathy in North America, is highly associated with the toxic metabolite of hypoglycin A, MCPA-carnitine. This finding coupled with the presence of a tree of which seeds are known to also contain hypoglycin A indicates that ingestion of Acer pseudoplatanus is the probable cause of AM. This finding has major implications for the prevention of AM.

Hepatocyte transplantation using the domino concept in a child with Tetrabiopterin non-responsive phenylketonuria.

Phenylketonuria is a metabolic disease caused by phenylalanine hydroxylase deficiency. Treatment is based on a strict natural protein-restricted diet that is associated with the risk of malnutrition and severe psychosocial burden. Oral administration of tetrahydrobiopterin can increase residual enzyme activity, but most patients with severe clinical phenotypes are nonresponders. We performed liver cell transplantation in a 6-year-old boy with severe tetrahydrobiopterin nonresponsive phenylketonuria who failed to comply with diet prescriptions. The transplanted hepatocytes were obtained in part from an explanted glycogen storage type 1b liver. Following two infusions, blood phenylalanine levels returned within the therapeutic target while the phenylalanine half-life assessed by loading tests decreased from 43 to 19 h. However, 3 months later, blood phenylalanine concentrations increased and the phenylalanine intake had to be reduced. Cell-based therapy is a promising therapeutic option in phenylketonuria, and the domino concept may solve the issue of cell sources for hepatocyte transplantation.

Mutation of the iron-sulfur cluster assembly gene IBA57 causes fatal infantile leukodystrophy

Leukodystrophies are a heterogeneous group of severe genetic neurodegenerative disorders. A multiple mitochondrial dysfunctions syndrome was found in an infant presenting with a progressive leukoencephalopathy. Homozygosity mapping, whole exome sequencing, and functional studies were used to define the underlying molecular defect. Respiratory chain studies in skeletal muscle isolated from the proband revealed a combined deficiency of complexes I and II. In addition, western blotting indicated lack of protein lipoylation. The combination of these findings was suggestive for a defect in the iron-sulfur (Fe/S) protein assembly pathway. SNP array identified loss of heterozygosity in large chromosomal regions, covering the NFU1 and BOLA3, and the IBA57 and ABCB10 candidate genes, in 2p15-p11.2 and 1q31.1-q42.13, respectively. A homozygous c.436C > T (p.Arg146Trp) variant was detected in IBA57 using whole exome sequencing. Complementation studies in a HeLa cell line depleted for IBA57 showed that the mutant protein with the semi-conservative amino acid exchange was unable to restore the biochemical phenotype indicating a loss-of-function mutation of IBA57. In conclusion, defects in the Fe/S protein assembly gene IBA57 can cause autosomal recessive neurodegeneration associated with progressive leukodystrophy and fatal outcome at young age. In the affected patient, the biochemical phenotype was characterized by a defect in the respiratory chain complexes I and II and a decrease in mitochondrial protein lipoylation, both resulting from impaired assembly of Fe/S clusters.

Neonatal Thyroid-Stimulating Hormone Concentrations in Belgium: A Useful Indicator for Detecting Mild Iodine Deficiency?

It has been proposed that neonatal thyroid-stimulating hormone (TSH) concentrations are a good indicator of iodine deficiency in the population. A frequency of neonatal TSH concentrations above 5 mU/L below 3% has been proposed as the threshold indicating iodine sufficiency. The objective of the present study was to evaluate feasibility and usefulness of nation-wide neonatal TSH concentration screening results to assess iodine status in Belgium. All newborns born in Belgium during the period 2009–2011 (n = 377713) were included in the study, except those suffering from congenital hypothyroidism and premature neonates. The frequency of neonatal TSH concentrations above 5 mU/L from 2009 to 2011 in Belgium fluctuated between 2.6 and 3.3% in the centres using the same TSH assay. There was a significant inverse association between neonatal TSH level and birth weight. The longer the duration between birth and screening, the lower the TSH level. Neonatal TSH levels were significantly lower in winter than in spring or autumn and significantly lower in spring and summer than in autumn while significantly higher in spring compared to summer. In conclusion, despite that pregnant women in Belgium are mildly iodine deficient, the frequency of neonatal TSH concentrations above 5 mU/L was very low, suggesting that the neonatal TSH threshold proposed for detecting iodine deficiency needs to be re-evaluated. Although neonatal TSH is useful to detect severe iodine deficiency, it should not be recommended presently for the evaluation of iodine status in mildly iodine deficient regions.

Folinic acid treatment for schizophrenia associated with folate receptor autoantibodies

BACKGROUND Auto-antibodies against folate receptor alpha (FRα) at the choroid plexus that block N(5)-methyltetrahydrofolate (MTHF) transfer to the brain were identified in catatonic schizophrenia. Acoustic hallucinations disappeared following folinic acid treatment. Folate transport to the CNS prevents homocysteine accumulation and delivers one-carbon units for methyl-transfer reactions and synthesis of purines. The guanosine derivative tetrahydrobiopterin acts as common co-factor for the enzymes producing dopamine, serotonin and nitric oxide. METHODS Our study selected patients with schizophrenia unresponsive to conventional treatment. Serum from these patients with normal plasma homocysteine, folate and vitamin B12 was tested for FR autoantibodies of the blocking type on serial samples each week. Spinal fluid was analyzed for MTHF and the metabolites of pterins, dopamine and serotonin. The clinical response to folinic acid treatment was evaluated. RESULTS Fifteen of 18 patients (83.3%) had positive serum FR auto-antibodies compared to only 1 in 30 controls (3.3%) (χ(2)=21.6; p<0.0001). FRα antibody titers in patients fluctuated over time varying between negative and high titers, modulating folate flux to the CNS, which explained low CSF folate values in 6 and normal values in 7 patients. The mean±SD for CSF MTHF was diminished compared to previously established controls (t-test: 3.90; p=0.0002). A positive linear correlation existed between CSF MTHF and biopterin levels. CSF dopamine and serotonin metabolites were low or in the lower normal range. Administration of folinic acid (0.3-1mg/kg/day) to 7 participating patients during at least six months resulted in clinical improvement. CONCLUSION Assessment of FR auto-antibodies in serum is recommended for schizophrenic patients. Clinical negative or positive symptoms are speculated to be influenced by the level and evolution of FRα antibody titers which determine folate flux to the brain with up- or down-regulation of brain folate intermediates linked to metabolic processes affecting homocysteine levels, synthesis of tetrahydrobiopterin and neurotransmitters. Folinic acid intervention appears to stabilize the disease process.

3-years experience review of neonatal screening for hemoglobin disorders using tandem mass spectrometry.

BACKGROUND Neonatal screening programs for sickle cell disease are common in North America and in some European countries. Isoelectric Focusing or High Performance Liquid Chromatography is the main technique used for hemoglobin variant detection. METHODS Since tandem mass spectrometry is being used for screening of inherited metabolic disorders and allows protein identification, we had developed an application to identify the most relevant hemoglobin mutations with this technology. RESULTS This approach had been previously validated and has been routinely applied in our laboratory for the last three years. We report here our experience with this new method in the field, applied to our East-Belgian population. CONCLUSIONS To conclude, mass spectrometry provides an efficient alternative approach for laboratories performing neonatal screening of hemoglobin disorders.

Samaras and seedlings of Acer pseudoplatanus are potential sources of hypoglycin A intoxication in atypical myopathy without necessarily inducing clinical signs

REASONS FOR PERFORMING STUDY Ingestion of sycamore seeds (Acer pseudoplatanus) is the likely source of hypoglycin A in atypical myopathy (AM) but ingestion of seedlings in spring might also contribute to intoxication. OBJECTIVES To test for hypoglycin A in seeds and seedlings collected on pastures where AM cases were reported and compare its concentration in serum of affected and healthy horses. STUDY DESIGN Field investigation of clinical cases. METHODS Whenever present, samaras (the winged nuts that each contain one seed) and/or seedlings were collected from pastures of 8 AM cases and 5 unaffected horses from different premises. Two AM cases were each co-grazing with an apparently healthy horse. Acylcarnitines and hypoglycin A were quantified in blood samples of all horses involved in the study. RESULTS Hypoglycin A was detected in serum of AM (5.47 ± 1.60 μmol/l) but not in healthy controls pasturing where A. pseudoplatanus trees were not present. However, hypoglycin A was detected at high concentrations (7.98 μmol/l) in serum of a clinically healthy horse grazing a pasture with seedlings and samaras and also in the 2 healthy horses co-grazing with AM cases (0.43 ± 0.59 μmol/l). Hypoglycin A was detected in all samples of seeds and spring seedlings of A. pseudoplatanus. CONCLUSIONS Atypical myopathy can be associated with the ingestion of sycamore samaras and also ingestion of seedlings. Hypoglycin A can be detected in the blood of horses with no detectable clinical signs at pasture in which there is A. pseudoplatanus. Determination of hypoglycin A concentration in blood is useful for screening for exposure in suspected cases of AM.

Neonatal screening for sickle cell disease in Central Africa: a study of 1825 newborns with a new enzyme-linked immunosorbent assay test

Objectives: To evaluate the feasibility of systematic neonatal screening for sickle cell disease in the region of Great Lakes in Central Africa using a new approach with limited costs. Methods: Between July 2004 and July 2006, 1825 newborn dried blood samples were collected onto filter papers in four maternity units from Burundi, Rwanda and the East of the Democratic Republic of Congo. We tested for the presence of haemoglobin C and S in the eluted blood by an enzyme-linked immunosorbent assay (ELISA) test using a monoclonal antibody. All ELISA-positive samples (multiple of the median (MoM)≥ 1.5) were confirmed by a simple molecular test. The statistica software version 7.1 was used to create graphics and to fix the MoM cut-off, and the χ2 of Pearson was used to compare the genotype incidences between countries. Results: Of the 1825 samples screened, 97 (5.32%) were positive. Of these, 60 (3.28%) samples were heterozygous for Hb S, and four (0.22%) for Hb C; two (0.11%) newborns were Hb SS homozygotes. Conclusions: The lower cost and the high specificity of ELISA test are appropriate for developing countries, and such systematic screening for sickle cell anaemia is therefore feasible.

Quantification of hypoglycin A in serum using aTRAQ® assay

BACKGROUND Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equines biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. METHODS Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. RESULTS Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50μmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. CONCLUSIONS The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.