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E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.

Aberrant expression of imprinted genes, such as those coding for the insulin-like growth factor 2 (IGF2) and neuronatin (NNAT), is a characteristic of a variety of embryonic neoplasms, including Wilms tumor (WT). In case of IGF2, it is generally accepted that loss of imprinting in a differentially methylated region of the IGF2/H19 locus results in biallelic expression and, thus, upregulation of the gene. In this study we examined methylation pattern at potential regulatory elements of the paternally expressed NNAT gene in a cohort of WT patients in order to further characterize the molecular mechanism causing overexpression of this regulatory gene. We demonstrate that transcriptional upregulation of NNAT in WT is grossly independent of the bladder cancer-associated protein (BLCAP) gene, an imprinted gene within the imprinted domain of the NNAT locus. However, expression of the BLCAP transcript isoform v2a formerly known to be selectively expressed from the paternal allele in brain was associated with high expression of NNAT. This contrasts the situation we found at the IGF2/H19 locus, which shows high overexpression of IGF2 and inversely correlated expression of the H19 gene in WT. An analysis of DNA methylation in two potential regulatory regions of the NNAT locus by pyrosequencing revealed significant hypomethylation of the tumors compared to normal kidney tissue. Interestingly, the difference in DNA methylation was highest at CpGs that were observed within three putative binding sites of the CCCTC-binding factor CTCF. Most importantly, hypomethylation of both NNAT regulatory regions is significantly associated with the upregulation of NNAT expression and the BLCAP_v2a transcript. Our data indicate that the methylation status of a not-yet-described regulatory element within the NNAT locus that contains four potential CTCF binding sites determines the expression level of NNAT and the nearby located BLCAP_v2a transcript, thereby suggesting a functional role in the aberrant upregulation of NNAT in WT.

Background Hepatoblastoma (HB) is an embryonal liver neoplasm of early childhood with a poor prognosis for patients with distant metastases and vascular invasion. We and others have previously shown that the overexpression of insulin-like growth factor 2 ( IGF2 ), loss of imprinting at the IGF2 / H19 locus, and amplification of pleomorphic adenoma gene 1 ( PLAG1 ) are common features in HB, suggesting a critical role of the IGF axis in hepatoblastomagenesis. In this study, we investigated the role of the insulin-like growth factor binding protein 3 (IGFBP3), a known competitor of the IGF axis, in pediatric liver cancers. Results The IGFBP3 gene was highly expressed in normal pediatric livers but was heavily downregulated in four HB cell lines and the majority of HB primary tumors (26/36). Detailed methylation analysis of CpG sites in the IGFBP3 promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of IGFBP3 in HB cell lines. Consequently, the treatment of HB cell lines with 5-aza-2'-deoxycytidine resulted in DNA demethylation and reactivation of the epigenetically silenced IGFBP3 expression. Interestingly, IGFBP3 promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring IGFBP3 expression in HB cells resulted in reduced colony formation, migration, and invasion. Conclusion This study provides the first direct evidence that the reactivation of IGFBP3 decreases aggressive properties of pediatric liver cancer cells and that IGFBP3 promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a hereditary microangiopathic condition causing stroke in young adults. The responsible gene has recently been identified as the Notch3 gene. Notch3 encodes a large transmembrane receptor with 34 extracellularly localised epidermal growth factor-like (EGF) repeat domains. We screened 71 unrelated CADASIL families for mutations in two exons coding for the first five EGF-like repeats and found mutations in 70% of the families (n = 50). Two types of mutations were identified: 48 families (96%) had missense mutations and two families (4%) had small in-frame deletions. Seven mutations occurred multiple times. All of them are C to T transitions that affect CpG dinucleotides, suggesting that their multiple occurrence is due to the hypermutability of this sequence. All mutations, including the two deletions, result in the gain or loss of a cysteine residue, thus substantiating the pivotal role of an uneven number of cysteine residues within EGF-like repeat domains of Notch3 in the pathogenesis of CADASIL. To study the potential effects of these mutations 3D homology models of the first six EGF domains were generated on the basis of NMR data from human fibrillin-1. These models predict domain misfolding for a subset of mutations.

OBJECTIVE To evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients. METHODS We screened for deletions and sequenced mitochondrial transfer RNA genes in skeletal muscle DNA from 225 index patients with clinical symptoms suggestive of a mitochondrial disorder and with biochemically demonstrable RC deficiency in skeletal muscle. RESULTS We found pathogenic mitochondrial DNA mutations in 29% of the patients. The detection rate was significantly higher in adults (48%) than in the paediatric group (18%). Only one pathogenic mutation was detected in the neonatal group. In addition, we describe seven novel transfer RNA sequence variations with unknown pathogenic relevance (six homoplasmic and one heteroplasmic) and 13 homoplasmic polymorphisms. One heteroplasmic transfer RNALeu(UUR) A>G mutation at position 3274 is associated with a distinct neurological syndrome. CONCLUSIONS We provide an estimation of the frequency of mitochondrial transfer RNA mutations and deletions in paediatric and adult patients with respiratory chain deficiencies.

Two sons of a consanguineous marriage developed biventricular cardiomyopathy. One boy died of severe heart failure at the age of 6 years, the other was transplanted because of severe heart failure at the age of 10 years. In addition, focal palmoplantar keratoderma and woolly hair were apparent in both boys. As similar phenotypes have been described in Naxos disease and Carvajal syndrome, respectively, the genes for plakoglobin (JUP) and desmoplakin (DSP) were screened for mutations using direct genomic sequencing. A novel homozygous 2 bp deletion was identified in an alternatively spliced region of DSP. The deletion 5208_5209delAG led to a frameshift downstream of amino acid 1,736 with a premature truncation of the predominant cardiac isoform DSP-1. This novel homozygous truncating mutation in the isoform-1 specific region of the DSP C-terminus caused Carvajal syndrome comprising severe early-onset heart failure with features of non-compaction cardiomyopathy, woolly hair and an acantholytic form of palmoplantar keratoderma in our patient. Congenital hair abnormality and manifestation of the cutaneous phenotype in toddler age can help to identify children at risk for cardiac death.

Background/Purpose of the study Despite tremendous progress in therapy, about 30 % of patients with hepatoblastoma still succumb to the disease. Thus, the development of improved therapies as well as the identification of prognostic factors are urgently needed. Methods In the present study, expression and promoter methylation of the N-myc downstream-regulated gene ( NDRG2 ), a tumor suppressor gene contributing to the regulation of the Wnt signalling pathway, was analysed in 38 hepatoblastoma samples by real-time reverse transcription-PCR and pyrosequencing, respectively. Results The NDRG2 gene was highly expressed in normal pediatric liver tissue, but was significantly downregulated in heptoblastoma primary tumors. Detailed methylation analysis of CpG sites in the NDRG2 promoter region revealed a general high degree of DNA methylation in hepatoblastoma, which correlated with the suppression of NDRG2 . By analyzing clinicopathological features we could demonstrate a strong association between low NDRG2 expression and tumor metastasis. Importantly, the overall survival analysis by Kaplan–Meier revealed that high NDRG2 expression was correlated with a higher survival rate in hepatoblastoma patients. Conclusion Our data show that downregulation of NDRG2 may play an important role in advanced hepatoblastomas.

COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.

Mutations in the human dysferlin gene ( DYSF) cause autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal muscles: limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Recently, an interaction between caveolin-3 and dysferlin in normal and dystrophic muscle (primary caveolin-3 deficiency; LGMD1C) was shown. In this study, clinical,morphological and genetic analysis was carried out in four independent LGMD2B/MM patients. All patients presented with an adult-onset, slowly progressive muscular dystrophy with variable involvement of proximal and distal muscles. We found complete lack of dysferlin in the four LGMD2B/MM patients. Secondary reduction of caveolin-3 was detected in three out of the four patients. Regular caveolae were detected along the basal lamina in two patients by electron microscopy. We provide further evidence that dysferlin and caveolin-3 interact in human skeletal muscle. It remains to be elucidated whether the loss of this interaction contributes to pathogenic events in muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy and usually follows an autosomal dominant trait. Clinically, FSHD affects facial muscles and proximal upper limb and girdle muscles, but may present with variable clinical phenotypes even within the same family. Most genetically confirmed FSHD patients exhibit unspecific morphological signs of a degenerative myopathy. We report on five unrelated patients who carried the pathogenic FSHD mutation on chromosome 4q35. Muscle biopsies revealed numerous rimmed vacuoles and filamentous cytoplasmic inclusions in all cases. Clinically, the patients suffered from weakness and atrophy predominantly of the lower limb muscles. In conclusion, we suggest considering FSHD in the differential diagnosis of adult-onset distal myopathies with rimmed vacuoles.