Expanding the mutation spectrum in ICF syndrome: Evidence for a gender bias in ICF2.

BACKGROUND: Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare, genetically heterogeneous, autosomal recessive disorder. Patients suffer from recurrent infections caused by reduced levels or absence of serum immunoglobulins. Genetically, 4 subtypes of ICF syndrome have been identified to date: ICF1 (DNMT3B mutations), ICF2 (ZBTB24 mutations), ICF3 (CDCA7 mutations), and ICF4 (HELLS mutations). AIM: To study the mutation spectrum in ICF syndrome. MATERIALS AND METHODS: Genetic studies were performed in peripheral blood lymphocyte DNA from suspected ICF patients and family members. RESULTS: We describe 7 ICF1 patients and 6 novel missense mutations in DNMT3B, affecting highly conserved residues in the catalytic domain. We also describe 5 new ICF2 patients, one of them carrying a homozygous deletion of the complete ZBTB24 locus. In a meta-analysis of all published ICF cases, we observed a gender bias in ICF2 with 79% male patients. DISCUSSION: The biallelic deletion of ZBTB24 provides strong support for the hypothesis that most ICF2 patients suffer from a ZBTB24 loss of function mechanism and confirms that complete absence of ZBTB24 is compatible with human life. This is in contrast to the observed early embryonic lethality in mice lacking functional Zbtb24. The observed gender bias seems to be restricted to ICF2 as it is not observed in the ICF1 cohort. CONCLUSION: Our study expands the mutation spectrum in ICF syndrome and supports that DNMT3B and ZBTB24 are the most common disease genes.

The expanding field of IgG4-mediated neurological autoimmune disorders.

At least 13 different disease entities affecting the central nervous system, peripheral nervous system and connective tissue of the skin or kidneys are associated with immunoglobulin G4 (IgG4) immune reactivity. IgG4 has always been considered a benign, non-inflammatory subclass of IgG, in contrast to the well-known complement-activating pro-inflammatory IgG1 subclass. A comprehensive review of these IgG4 autoimmune disorders reveals striking similarities in epitope binding and human leukocyte antigen (HLA) associations. Mechanical interference of extracellular ligand-receptor interactions by the associated IgG4 antibodies seems to be the common/converging disease mechanism in these disorders.

Pathogenic immune mechanisms at the neuromuscular synapse: the role of specific antibody-binding epitopes in myasthenia gravis.

Autoantibodies against three different postsynaptic antigens and one presynaptic antigen at the neuromuscular junction are known to cause myasthenic syndromes. The mechanisms by which these antibodies cause muscle weakness vary from antigenic modulation and complement-mediated membrane damage to inhibition of endogenous ligand binding and blocking of essential protein-protein interactions. These mechanisms are related to the autoantibody titre, specific epitopes on the target proteins and IgG autoantibody subclass. We here review the role of specific autoantibody-binding epitopes in myasthenia gravis, their possible relevance to the pathophysiology of the disease and potential implications of epitope mapping knowledge for new therapeutic strategies.

Mild muscular features in tenascin-X knockout mice, a model of Ehlers-danlos syndrome.

INTRODUCTION: Tenascin-X (TNX) is an extracellular matrix (ECM) glycoprotein, the absence of which in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), a group of inherited connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. A mouse model of TNX-deficient type EDS has been used to characterize the dermatological, orthopedic, and obstetrical features. The growing insight in the clinical overlap between myopathies and inherited connective tissue disorders asks for a study of the muscular characteristics of inherited connective tissue diseases. Therefore, this study aims to define the muscular phenotype of TNX knockout (KO) mice. MATERIALS AND METHODS: We performed a comprehensive study on the muscular phenotype of these TNX KO mice, consisting of standardized clinical assessment, muscle histology, and gene expression profiling of muscle tissue. Furthermore, peripheral nerve composition was studied by histology and electron microscopy. RESULTS: The main findings are the presence of mild muscle weakness, mild myopathic features on histology, and functional upregulation of genes encoding proteins involved in ECM degradation and synthesis. Additionally, sciatic nerve samples showed mildly reduced collagen fibril density of endoneurium. DISCUSSION: The muscular phenotype of TNX KO mice consists of mild muscle weakness with histological signs of myopathy and of increased turnover of the ECM in muscle. Furthermore, mildly reduced diameter of myelinated fibers and reduction of collagen fibril density of endoneurium may correspond with polyneuropathy in TNX-deficient EDS patients. This comprehensive assessment can serve as a starting point for further investigations on neuromuscular function in TNX KO mice.

[Facioscapulohumeral muscular dystrophy]. / Facioscapulohumerale spierdystrofie.

Facioscapulohumeral muscular dystrophy is clinically mainly characterized by progressive weakness of the facial, shoulder and upper arm muscles. It is an autosomal dominant heriditary disease, caused by a contraction of a repetitive DNA element at the end of the long arm of chromosome 4. This contraction causes the local relaxation of the chromatin structure and likely dysregulation of one or more genes. Oral health care providers can play a significant role in the early recognition, as the often asymmetric facial weakness is frequently the first symptom. Adequate oral health care is needed because of the facial weakness.

Evaluation of blood gene expression levels in facioscapulohumeral muscular dystrophy patients.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the expression of DUX4 in skeletal muscles. A number of therapeutic approaches are being developed to antagonize the events preceding and following DUX4 expression that leads to muscular dystrophy. Currently, the possibility to evaluate treatment response in clinical trials is hampered by the lack of objective molecular biomarkers connecting the disease cause to clinical performance. In this study we employed RNA-seq to examine gene expression in PAXgene tubes obtained from two independent cohorts of FSHD patients. Analysis of gene expression profiles did not lead to the identification of genes or pathways differentially expressed in FSHD patients, or associated with disease severity. In particular, we did not find evidence that the DUX4 and PAX7 signatures were differentially expressed. On the other hand, we were able to improve patient classification by including single genes or groups of genes in classification models. The best classifier was ROPN1L, a gene known to be expressed in testis, coincidentally the typical location of DUX4 expression. These improvements in patient classification hold the potential to enrich the FSHD clinical trial toolbox.

Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4.

Deafness with fixation of the stapes (DFN3) is the most frequent X-linked form of hearing impairment. The underlying gene has been localized to a 500-kilobase segment of the Xq21 band. Here, it is reported that a candidate gene for this disorder, Brain 4 (POU3F4), which encodes a transcription factor with a POU domain, maps to the same interval. In five unrelated patients with DFN3 but not in 50 normal controls, small mutations were found that result in truncation of the predicted protein or in nonconservative amino acid substitutions. These findings indicate that POU3F4 mutations are a molecular cause of DFN3.

Clinical spectrum of immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome).

BACKGROUND: Immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome) is a rare autosomal recessive disease characterised by facial dysmorphism, immunoglobulin deficiency and branching of chromosomes 1, 9 and 16 after PHA stimulation of lymphocytes. Hypomethylation of DNA of a small fraction of the genome is an unusual feature of ICF patients which is explained by mutations in the DNA methyltransferase gene DNMT3B in some, but not all, ICF patients. OBJECTIVE: To obtain a comprehensive description of the clinical features of this syndrome as well as genotype-phenotype correlations in ICF patients. METHODS: Data on ICF patients were obtained by literature search and additional information by means of questionnaires to corresponding authors. RESULTS AND CONCLUSIONS: 45 patients all with proven centromeric instability were included in this study. Facial dysmorphism was found to be a common characteristic (n = 41/42), especially epicanthic folds, hypertelorism, flat nasal bridge and low set ears. Hypo- or agammaglobulinaemia was demonstrated in nearly all patients (n = 39/44). Opportunistic infections were seen in several patients, pointing to a T cell dysfunction. Haematological malignancy was documented in two patients. Life expectancy of ICF patients is poor, especially those with severe infections in infancy or chronic gastrointestinal problems and failure to thrive. Early diagnosis of ICF is important since early introduction of immunoglobulin supplementation can improve the course of the disease. Allogeneic stem cell transplantation should be considered as a therapeutic option in patients with severe infections or failure to thrive. Only 19 of 34 patients showed mutations in DNMT3B, suggesting genetic heterogeneity. No genotype-phenotype correlation was found between patients with and without DNMT3B mutations.

Scapular dyskinesis in myotonic dystrophy type 1: clinical characteristics and genetic investigations.

OBJECTIVE: To study scapular winging or other forms of scapular dyskinesis (condition of alteration of the normal position and motion of the scapula) in myotonic dystrophy type 1 (DM1), which is generally considered to be a distal myopathy, we performed an observational cohort study. METHODS: We performed a prospective cohort study on the clinical features and progression over time of 33 patients with DM1 and pronounced, mostly asymmetric scapular winging or other forms of scapular dyskinesis. We also explored if scapular dyskinesis in DM1 has the same genetic background as in facioscapulohumeral muscular dystrophy type 1 (FSHD1). RESULTS: The cohort included patients with congenital (n = 3), infantile (n = 6) and adult-onset DM1 (n = 24). Scapular girdle examination showed moderate shoulder girdle weakness (mean MRC 3) and atrophy of trapezius, infraspinatus, and rhomboid major, seemingly similar as in FSHD1. Shoulder abduction and forward flexion were limited (50-70°). In five patients, scapular dyskinesis was the initial disease symptom; in the others it appeared 1-24 years after disease onset. Follow-up data were available in 29 patients (mean 8 years) and showed mild to severe increase of scapular dyskinesis over time. In only three patients, DM1 coexisted with a FSHD mutation. In all other patients, FSHD was genetically excluded. DM2 was genetically excluded in nine patients. The clinical features of the patients with both DM1 and FSHD1 mutations were similar to those with DM1 only. CONCLUSION: Scapular dyskinesis can be considered to be part of DM1 in a small proportion of patients. In spite of the clinical overlap, FSHD can explain scapular dyskinesis only in a small minority. This study is expected to improve the recognition of shoulder girdle involvement in DM1, which will contribute to the management of these patients.

Epigenetic mechanisms in health and disease.

Epigenetic modifications of our genome involve DNA methylation, covalent modifications of the histone tails, nucleosome occupancy and turnover and higher-order chromatin folding. These mitotically heritable epigenetic modifications can affect transcription regulation and are increasingly recognised to be causally involved in a broad spectrum of human conditions, ranging from monogenic to multifactorial disorders. While our understanding of these epigenetic disease mechanisms steadily increases, the challenge will be to develop new drugs that specifically deal with the epigenetic lesion.

Respiratory function in facioscapulohumeral muscular dystrophy 1.

To test the hypothesis that wheelchair dependency and (kypho-)scoliosis are risk factors for developing respiratory insufficiency in facioscapulohumeral muscular dystrophy, we examined 81 patients with facioscapulohumeral muscular dystrophy 1 of varying degrees of severity ranging from ambulatory patients to wheelchair-bound patients. We examined the patients neurologically and by conducting pulmonary function tests: Forced Vital Capacity, Forced Expiratory Volume in 1 second, and static maximal inspiratory and expiratory mouth pressures. We did not find pulmonary function test abnormalities in ambulant facioscapulohumeral muscular dystrophy patients. Even though none of the patients complained of respiratory dysfunction, mild to severe respiratory insufficiency was found in more than one third of the wheelchair-dependent patients. Maximal inspiratory pressures and maximal expiratory pressures were decreased in most patients, with a trend that maximal expiratory pressures were more affected than maximal inspiratory pressures. Wheelchair-dependent patients with (kypho-)scoliosis showed the most restricted lung function. Wheelchair-dependent patients with (kypho-)scoliosis are at risk for developing respiratory function impairment. We advise examining this group of facioscapulohumeral muscular dystrophy patients periodically, even in the absence of symptoms of respiratory insufficiency, given its frequency and impact on daily life and the therapeutic consequences.

No effect of folic acid and methionine supplementation on D4Z4 methylation in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with a contraction of the D4Z4 allele on chromosome 4qter. There is also marked DNA hypomethylation of the D4Z4 allele. The DNA hypomethylation may have a central role in the pathogenesis of FSHD. Supplemental folic acid can boost DNA methylation. We evaluated the effect of oral folic acid and methionine supplementation on the methylation level of 4qter D4Z4 alleles in peripheral-blood lymphocytes of nine patients affected with FSHD and six healthy controls. Methylation levels did not change, while recommended serum-folate concentrations were reached.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

[First facioscapulohumeral muscular dystrophy prenatal diagnosis in a Bulgarian family].

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common myopathy. It is characterized by progressive descendent involvement of facial, shoulder girdle, truncal and lower extremities muscles. FSHD locus was mapped on the terminal part of the long arm of chromosome 4 (4q35). The disease is caused by a deletion of an integral number of tandem D4Z4 repeats and dimension of the pathological fragments < or = 38kb. Prenatal diagnosis of FSHD is possible but it is potentially difficult because of the big amount and high quality of DNA required. Hereby we describe the first prenatal tests performed for a Bulgarian family.

Yeast artificial chromosome cloning of the Xq13.3-q21.31 region and fine mapping of a deletion associated with choroideremia and nonspecific mental retardation.

Microscopically detectable deletions and X;autosome translocations have previously facilitated the construction of a high-resolution interval map of the Xq21 region. Here, we have generated three yeast artificial chromosome contigs spanning approximately 7 megabases of the Xq13.3-q21.31 region. In addition, a novel deletion associated with choroideremia and mental retardation was identified and mapped in detail. The proximal deletion endpoint was positioned between the loci DXS995 and DXS232, which enabled us to confirm the critical region for a locus involved in mental retardation. The distal deletion endpoint is situated in the Xq21.33 band, which allowed us to refine the order of several markers in this region.

FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates.

The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.

Active genes in junk DNA? Characterization of DUX genes embedded within 3.3 kb repeated elements.

The human genome contains hundreds of repeats of the 3.3 kb family in regions associated with heterochromatin. We have previously isolated a 3.3 kb-like cDNA encoding a double homeodomain protein (DUX1). Demonstration that the protein was expressed in human rhabdomyosarcoma TE671 cells, and characterization of a homologous promoter suggested that functional DUX genes might be present in 3.3 kb elements. In the present study, we describe two nearly identical 3.3 kb/DUX genes derived from PAC 137F16 (DUX3), and TE671 genomic DNA (DUX5), both mapping to all the acrocentric chromosomes. Their promoters harbor a GC and a TATAA box, and the open reading frame of the intronless structural part encodes two DUX proteins differing by alternative translation initiation. The shorter protein of the DUX5 gene is identical to DUX1. Using a protein truncation test, we could show that these two proteins are encoded by total RNA, but not by poly (A)(+) RNA, from different human tissues and cell lines. Our results indicate that active genes of unusual structure are present in chromosome regions characterized by large amounts of heterochromatic repetitive DNA.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.