Jonas Denecke

A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patients. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patients MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.

A Defect in Dolichol Phosphate Biosynthesis Causes a New Inherited Disorder with Death in Early Infancy

The following study describes the discovery of a new inherited metabolic disorder, dolichol kinase (DK1) deficiency. DK1 is responsible for the final step of the de novo biosynthesis of dolichol phosphate. Dolichol phosphate is involved in several glycosylation reactions, such as N-glycosylation, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and C- and O-mannosylation. We identified four patients who were homozygous for one of two mutations (c.295T-->A [99Cys-->Ser] or c.1322A-->C [441Tyr-->Ser]) in the corresponding hDK1 gene. The residual activity of mutant DK1 was 2%-4% when compared with control cells. The mutated alleles failed to complement the temperature-sensitive phenotype of DK1-deficient yeast cells, whereas the wild-type allele restored the normal growth phenotype. Affected patients present with a very severe clinical phenotype, with death in early infancy. Two of the patients died from dilative cardiomyopathy.

Normal Clinical Outcome in Untreated Subjects with Mild Hyperphenylalaninemia

There is international consensus that patients with phenylalanine (Phe) levels <360 μM on a free diet do not need Phe-lowering dietary treatment whereas patients with levels >600 μM do. Clinical outcome of patients showing Phe levels between 360 and 600 μM in serum on a free nutrition has so far only been assessed in a small number of cases. Therefore, different recommendations exist for patients with mild hyperphenylalaninemia. We investigated in a nationwide study 31 adolescent and adult patients who persistently displayed serum Phe levels between 360 and 600 μM on a normal nutrition with a corresponding genotype. Because of limited accuracy of measurements, Phe levels should be looked on as an approximation, but not as an absolute limit in every instance. In addition to serum Phe levels, the assessment program consisted of comprehensive psychological testing, magnetic resonance imaging of the head, 1H magnetic resonance spectroscopy, and genotyping. We found a normal intellectual (intelligence quotient, 103 ± 15; range, 79–138) and educational (school performance and job career) outcome in these subjects as compared with healthy control subjects (intelligence quotient, 104 ± 11; range, 80–135). Magnetic resonance imaging revealed no changes of cerebral white matter in any patient, and 1H magnetic resonance spectroscopy revealed brain Phe levels below the limit of detection (<200 μM). In the absence of any demonstrable effect, dietary treatment is unlikely to be of value in patients with mild hyperphenylalaninemia and serum Phe levels <600 μM on a free nutrition, and should no longer be recommended. Because of a possible late-onset phenylketonuria, Phe levels of untreated patients should be monitored carefully at least during the first year of life. Nevertheless, problems of maternal phenylketonuria should still be taken into account.

p.S143F mutation in lamin A/C: A new phenotype combining myopathy and progeria

We report a young girl with a phenotype combining early‐onset myopathy and a progeria. She had myopathy and marked axial weakness during the first year of life; progeroid features, including growth failure, sclerodermatous skin changes, and osteolytic lesions, developed later. We identified the underlying cause to be a hitherto unreported de novo missense mutation in the LMNA gene (S143F) encoding the nuclear envelope proteins lamins A and C. Although LMNA mutations have been known to cause Hutchinson–Gilford progeria syndrome and Emery–Dreifuss muscular dystrophy, this is the first report of a patient combining features of these two phenotypes because of a single mutation in LMNA. Ann Neurol 2005;57:148–151

Tetrahydrobiopterin responsiveness in a large series of phenylketonuria patients

In a group of 87 consecutive patients with hyperphenylalaninaemia born since 1990, only 3 patients showed a (temporary) decrease of serum phenylalanine levels after tetrahydrobiopterin (BH4) loading in usual doses (20 mg/kg body weight).

Congenital Disorder of Glycosylation Type Id: Clinical Phenotype, Molecular Analysis, Prenatal Diagnosis, and Glycosylation of Fetal Proteins

Congenital disorder of glycosylation type Id is an inherited glycosylation disorder based on a defect of the first mannosyltransferase involved in N-glycan biosynthesis inside the endoplasmic reticulum. Only one patient with this disease has been described until now. In this article, a second patient and an affected fetus are described. The patient showed abnormal glycosylation of several plasma proteins as demonstrated by isoelectric focusing and Western blot. Lipid-linked oligosaccharides in the endoplasmic reticulum, reflecting early N-glycan assembly, revealed an accumulation of immature Man5GlcNAc2-glycans in fibroblasts of the patient. Chorion cells of the affected fetus showed the same characteristic lipid-linked oligosaccharides pattern. However, the fetus had a normal glycosylation of several plasma proteins. Some fetal glycoproteins are known to be derived from the mother, but even glycoproteins that do not cross the placenta were normally glycosylated in the affected fetus. Maternal or placental factors that partially compensate for the glycosylation defect in the fetal stage must be proposed and may be relevant for the therapy of these disorders in the future.

Lessons learned from additional research analyses of unsolved clinical exome cases

BackgroundGiven the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery.MethodsWe designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent–offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols.ResultsAnalysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3).ConclusionAn efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

Frontal lobe-dependent functions in treated phenylketonuria: blood phenylalanine concentrations and long-term deficits in adolescents and young adults.

SummaryEven early-treated phenylketonurics may suffer from phenylalanine-related deficits. Elevated phenylalanine concentrations can interfere with the development and function of the CNS. Outcome beyond childhood has not been extensively investigated. This long-term study was performed to determine whether adolescents and young adults with PKU show frontal lobe-dependent deficits when compared to diabetic patients. The comparative study covered 35 PKU patients, 13–21 years of age (mean 17.8 years), and 35 diabetic patients matched for sex, age and socioeconomic status. Patients were assessed for IQ (Culture Fair Intelligence Test), information processing (Trail Making Test), and selective and sustained attention (Stroop Task, Test d-2). Assessments were repeated within a 3-year follow-up. PKU patients showed no increase in blood phenylalanine concentrations at follow-up. They had significantly poorer test results than the diabetic patients at both assessment times. Within the tests, however, this was due to reduced performance speed but not to deficits in specific frontal lobe-dependent functions. Elevated phenylalanine concentrations seem to exert a global effect slowing performance speed. This effect is enduring in adolescence and early adulthood.

Phenylketonuria: No Specific Frontal Lobe-Dependent Neuropsychological Deficits of Early-Treated Patients in Comparison with Diabetics

Neuropsychologic studies have shown that even phenylketonuric patients treated early suffer from phenylalanine-related deficits in all age periods, from childhood to adulthood. This study was performed to determine whether phenylketonuric children show specific frontal lobe–dependent deficits when compared with diabetic patients. The comparative study included 42 phenylketonuric patients, 10 to 18 y of age [mean 14.7 (years, months), SD 2.9], and 42 diabetic patients matched for sex, age, and socioeconomic status. Patients were assessed for intelligence quotient (Culture Fair Intelligence Test), information processing (Wisconsin Card Sorting Test, Trail-Making Test), and selective (Stroop task) as well as sustained attention (Test d-2). Phenylketonuric patients had significantly poorer results than the diabetic patients. Within all tests, however, this was due to reduced performance speed, not to deficits in specific functions. Patients did not show deficits in insight and learning. The selection abilities and the sustained attention of the phenylketonuric patients were not impaired. Performance speed and blood phenylalanine levels were negatively correlated. Elevated phenylalanine levels may cause an imbalance in neurotransmitter metabolism. However, this seems to refer to a global neurotoxic effect rather than to specific effects on the dopaminergic system, which would affect specifically the activation of the frontal lobes.

Hypoglycosylation due to dolichol metabolism defects.

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.