Browsing by Author "Wegener, Eike"

Rett syndrome, one of the most common causes of mental retardation in females, is caused by mutations in the X chromosomal gene MECP2. Mice deficient for MeCP2 recapitulate some of the symptoms seen in patients with Rett syndrome. It has been shown that reactivation of silent MECP2 alleles can reverse some of the symptoms in these mice. We have generated a knockin mouse model for translational research that carries the most common nonsense mutation in Rett syndrome, R168X. In this article we describe the phenotype of this mouse model. In male MeCP2(R168X) mice life span was reduced to 12-14 weeks and bodyweight was significantly lower than in wild type littermates. First symptoms including tremor, hind limb clasping and inactivity occurred at age 27 days. At age 6 weeks nest building, rotarod, open-field and elevated plus maze experiments showed impaired motor performance, reduced activity and decreased anxiety-like behavior. Plethysmography at the same time showed apneas and irregular breathing with reduced frequency. Female MeCP2R168X mice showed no significant abnormalities except decreased performance on the rotarod at age 9 months. In conclusion we show that the male MeCP2R168X mice have a phenotype similar to that seen in MECP2 knockout mouse models and are therefore well suited for translational research. The female mice, however, have a much milder and less constant phenotype making such research with this mouse model more challenging.

Background: Mutations in proteins involved in the glycosylphosphatidylinositol anchor biosynthesis and remodeling pathway are associated with autosomal recessive forms of intellectual disability. Recently mutations in the PGAP1 gene that codes for PGAP1, a protein localized in the endoplasmic reticulum responsible for the first step of the remodeling of glycosylphosphatidylinositol was linked to a disorder characterized by psychomotor retardation and facial dysmorphism. Whole exome sequencing (WES) was performed in siblings with severely delayed myelination and psychomotor retardation. Mutations in PGAP1 were confirmed by Sanger sequencing and RNA analysis. A literature search was performed to describe the emerging phenotype of PGAP1 related disease. Case presentation: WES resulted in the detection of two novel compound heterozygous mutations in PGAP1, one base pair insertion leading to a frame shift c.334_335InsA (p.A112fs) and a splice site mutation leading to exon skipping c.G1173C (p.L391L). A symptom not described in PGAP1 related disorder before but prominent in the siblings were recurrent apnea especially during sleep that persisted at least until age 2 years. Sequential cerebral MRI at age one and two year(s) respectively revealed frontal accentuated brain atrophy and significantly delayed myelination. Conclusion: We report siblings with two novel mutations in PGAP1. Other that the common symptoms related to PGAP1 mutations including non-progressive psychomotor retardation, neonatal feeding problems, microcephaly and brain atrophy these patients displayed severely delayed myelination and recurrent apneas thereby widing the clinical spectrum associated with such mutations.

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.

Background: Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients. Methods: In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients. Results: Increased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden. Conclusions: MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Read-through of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2(R168X) mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients.

So far very few patients with sequence variants in the closely related tectonic genes TCTN1-3 have been described. By multi-gene panel next-generation sequencing (NGS) in patients with Joubert syndrome, we identified two more patients and summarize what is currently known about the phenotypes associated with sequence variants in these genes. In a boy aged 12 years with intellectual disability and the classical molar tooth sign on MRI, a homozygous splice-site sequence variant in TCTN3 leading to in-frame skipping of exon 7 was detected. A previously described non-truncating sequence variant in TCTN3 was also associated with Joubert syndrome, whereas four truncating sequence variants were detected in patients with Meckel-Gruber or Mohr-Majewski syndrome. The second patient, a boy aged 7 years with severe psychomotor retardation, was found to carry a homozygous canonic splice-site sequence variant in TCTN2. So far, only three sequence variants associated with Joubert syndrome and two with Meckel-Gruber syndrome have been described in this gene. Reviewing the clinical data on patients with sequence variants in the tectonic genes TCTN1-3 reveals that all of them have a neurological phenotype with vermis hypoplasia or occipital encephalocele associated with severe intellectual disability in the surviving patients. In contrast, other features frequently seen in patients with ciliopathies such as nephronophthisis, liver fibrosis, retinal dystrophy or coloboma have not been reported. Our patients emphasize the usefulness and efficacy of a comprehensive NGS panel approach. A concise genetic diagnosis may help to prevent unnecessary investigations and improve the clinical management of these patients.