TMEM165 deficiency causes a congenital disorder of glycosylation

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.     

COG5-CDG: expanding the clinical spectrum

Background

The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350–6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia.

Methods

CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis.

Results and conclusion

We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.

     

Genetic impoverishment and cross-incompatibility in remnant genotypes of Ziziphus celata (Rhamnaceae), a rare shrub endemic to the Lake Wales Ridge, Florida

The loss of genetic diversity in fragmented populations ofself-incompatible plant species may result in sexual reproductive failure andlocal extinctions. Florida ziziphus (Ziziphus celata) is aself-incompatible clonal shrub known only from five genetically depauperatepopulations on the Lake Wales Ridge, Florida, USA. Recovery of this speciesrequires identification of cross-compatible genotypes that can be used to createviable (i.e., sexually reproducing) populations. To further development of arecovery program for this highly imperiled species, we investigated its geneticstructure and sexual reproductive viability. We used random amplifiedpolymorphic DNAs (RAPDs) to investigate genetic variability within remnantpopulations and we conducted experimental compatibility trials to determine thecross-compatibility of remnant genotypes. One hundred and ninety-nine uniquestem samples collected from one ex situ and fivein situ populations were assayed for the presence orabsence of a band for 32 RAPD markers. Based on unweighted pair-group meancluster analysis (UPGMA), only 11 multi-locus genotypes (MLGs) were identified.Eight of these MLGs correspond to MLGs identified in an earlier allozyme study.In addition, we identified three new RAPD-based MLGs. Three of the five naturalpopulations consisted of only one MLG, while the largest and most geneticallydiverse population comprised only four MLGs. Coefficients of similarity rangedfrom 96.6% for the most closely related MLGs to 20.7% for the most distantlyrelated. The compatibility trials demonstrated that most MLGs arecross-incompatible. With 69% of all possible one-way crosses tested (38/55), wehave identified only eight compatible crosses via germination trials. Based onthe results of the compatibility trials, we assigned MLGs toself-incompatibility (SI) mating types. On present evidence, the currentbreeding population of Florida ziziphus may comprise as few as two SI matingtypes. These SI mating types will be used to guide future breeding efforts andan experimental introduction.     

Astrocyte-specific expression of hamster prion protein (PrP) renders PrP knockout mice susceptible to hamster scrapie.

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 +/- 5 days and died 7 +/- 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.     

Crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion infection

In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection.