Christina Hung

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism–dystonia

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism–dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome

Background Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. Methods A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. Results We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. Conclusions We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome.

Transcobalamin II Deficiency at Birth

Transcobalamin II deficiency (# MIM 275350) is a rare, recessively inherited disorder of cobalamin transport that leads to intracellular cobalamin depletion with secondary impairment of methionine synthetase and methyl-malonyl CoA mutase activities. Affected individuals may suffer from long-term neurological sequelae if therapy with intramuscular hydroxocobalamin is not initiated promptly. We report two sisters with complete absence of transcobalamin due to homozygosity for a novel mutation (c.insC110) in the TCN2 gene that leads to a premature stop codon and non-functional protein. The older sister, now 4.5 years old, presented at 6 weeks of age with pancytopenia, protein losing enteropathy and a rapidly declining clinical course. Prompt therapy with 1mg hydroxocobalamin/day led to full recovery within days. Her now 1.5 year old sister was diagnosed shortly after birth and was started on hydroxocobalamin prior to onset of clinical symptoms. Interestingly, urinary methylmalonic acid excretion was increased significantly during the first days of life suggesting that functional cobalamin deficiency is present also during fetal life, although not giving rise to clinical symptoms until well after birth.

Mutations in RIT1 cause Noonan syndrome – additional functional evidence and expanding the clinical phenotype

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS‐mitogen activated protein kinase (RAS‐MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK‐ERK signaling compared to wild‐type, underscoring gain‐of‐function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS‐MAPK/MEK‐ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Laboratory and genetic evaluation of Gaucher disease

ZusammenfassungDer Morbus Gaucher (MG) ist eine vererbte lysosomale Speicherkrankheit, dem ein Mangel des Enzyms Glukocerebrosidase zugrunde liegt. Die Diagnose eines MG kann aufgrund der entsprechenden klinischen Symptome vermutet und mittels Enzymanalytik in Leukozyten, mononukleären Zellen, Fibroblasten und Trockenblut auf Filterkarten bestätigt werden. Niedrige Enzymaktivitäten sollten unbedingt durch eine molekulargenetische Untersuchung des GBA Genes bestätigt werden. Obwohl es keine weitreichende Genotyp-Phänotyp-Korrelation gibt, führt das Vorliegen einer p.N370S Mutation auf mindestens einem Allel zu einer nicht-neuronopathischen Verlaufsform eines MG, während Homozygotie der p.L444P Mutation in den meisten Fällen zu einem neuronopathischen MG führt. Die progrediente Speicherung von Glukosylzeramid in mononukleären Zellen und Makrophagen führt zu erhöhten Konzentrationen der Chitotriosidase-Aktivität und von CCL18/PARC, welche als Biomarker für den Schweregrad des MG als auch das therapeutische Ansprechen verwendet werden können. Bei etwa 6 % der Gaucher-Patienten ist die Chitotriosidase-Aktivität aufgrund einer Nullmutation im Chitotriosidase-Gen nicht nachweisbar.SummaryGaucher disease (GD) is an inherited lysosomal storage disorder due to deficiency of glucocerebrosidase. Diagnosis of GD may be suspected based on clinical symptoms and confirmed by the analysis of glucocerebrosidase in total white cells, mononuclear cells, fibroblasts and dried blood on filter paper. Low enzyme activities should be followed by molecular analysis of the GBA gene. Although there is no obvious genotype–phenotype correlation, the presence of p.N370S protects from neurological involvement whereas homozygosity of p.L444P mostly leads to a neuronopathic form of GD. Progressive storage of glucosylceramide in mononuclear cells and macrophages results in elevated levels of chitotriosidase and CCL18/PARC which may be used as biomarker to assess disease severity and efficacy of treatment. Chitotriosidase activities cannot be analysed in at least 6% of GD patients due to a null mutation in the corresponding gene.

Analysis of lyso-globotriaosylsphingosine in dried blood spots.

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset α-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75±0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77±0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.

Lyso-globotriaosylsphingosine (lyso-Gb3) levels in neonates and adults with the Fabry disease later-onset GLA IVS4+919G>A mutation

Lyso-globotriaosylsphingosine (lyso-Gb3) is a useful biomarker in the diagnosis and monitoring of treatment for Fabry disease. However, it is unclear whether lyso-Gb3 is elevated in patients with later-onset Fabry disease. Thus, we measured lyso-Gb3 levels from dried blood spots (DBS) from male newborns with the Fabry disease later-onset phenotype, IVS4+919G>A mutation, and their family members. The lyso-Gb3 levels were below the detection limit in normal control newborns and were slightly higher in adults. In males of all ages with the IVS4+919G>A mutation, lyso-Gb3 levels were elevated and were higher than in age-matched controls. The elevation of lyso-Gb3 levels in males with the IVS4+919G>A mutation was only slightly elevated compared with patients with the classical Fabry phenotype. The measurement of lyso-Gb3 levels is useful in the diagnosis of Fabry disease, including the later-onset phenotype. The DBS lyso-Gb3 level was not elevated in IVS4+919G>A heterozygotes, and is not useful for their diagnosis. Since lyso-Gb3 levels are elevated from birth in Fabry disease males, “an elevated lyso-Gb3 level” may be of little values for deciding when to begin enzyme replacement therapy.

Birth weight in patients with mucopolysaccharidosis type II: Data from the Hunter Outcome Survey (HOS)

There is a need to identify early disease markers to facilitate diagnosis of mucopolysaccharidosis type II (MPS II; Hunter syndrome). Mean birth weight and its association with disease severity was investigated in 609 patients enrolled in the Hunter Outcome Survey (HOS). This analysis indicated that birth weight is not an early marker of MPS II and is not associated with disease severity. It remains important to investigate the utility of other factors for early/pre-symptomatic diagnosis.

The Diagnostic Path to Pompe Disease

Pompe disease (PD) is a lysosomal disease that primarily affects skeletal and smooth muscles due to a deficiency of acid alpha glucosidase. The resulting clinical phenotype reflects a continuum ranging from severe infantile to later onset forms of PD. Early diagnosis is essential to prevent long-term complications and/or disease progression through initiation of enzyme-replacement therapy. The diagnostic path to PD starts with the ascertainment of medical and family history as well as an in-depth clinical and neurological evaluation. The involvement of proximal muscle groups including hip and thigh muscles as well as the diaphragm is characteristic for later onset PD. Once PD is considered creatine kinase (CK) should be measured, realising that levels may be within normal limits in end stage disease. Ultimately, diagnostic confirmation is readily achieved by enzyme analysis in dried blood spots and/or leukocytes followed by molecular analysis. Muscle biopsy is not sensitive enough for diagnostic testing of PD but may be an important diagnostic test for the differential diagnosis of myopathies. Future diagnostic approaches include whole exome and genome sequencing, which may contribute to our understanding of genotype–phenotype correlation and phenotype prediction.

A defect in the inner kinetochore protein CENPT causes a new syndrome of severe growth failure

Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.