Spinal muscular atrophy (SMA) is caused by homozygous deletion of the SMN1 gene in approximately 96% of cases. Four percent of SMA patients have a combination of the deletion or conversion on one allele and an intragenic mutation on the second one. We performed analysis of point mutations in a set of our patients with suspicion of SMA and without homozygous deletion of the SMN1 gene. A quantitative test determining SMN1 copy number (using real-time PCR and/or MLPA analysis) was performed in 301 patients and only 1 SMN1 copy was detected in 14 of them. When these 14 patients were screened for the presence of point mutations we identified 6 mutations, p.Y272C (in three patients) and p.T274I, p.I33IfsX6, and p.A188S (each in one case). The mutations p.I33IfsX6 and p.A188S were found in two SMAI patients and were not detected previously. Further, evaluation of the relationship between mutation type, copy number of the SMN2 gene and clinical findings was performed. Among our SMA patients with a SMN1 homozygous deletion, we found a family with two patients: the son with SMAII possesses 3 SMN2 copies and the nearly asymptomatic father has a homozygous deletion of SMN1 exon 7 and carries 4 SMN2 copies. Generally, our results illustrate that an increased SMN2 gene copy number is associated with a milder SMA phenotype.
- MeSH
- Point Mutation genetics MeSH
- Gene Deletion MeSH
- DNA genetics MeSH
- Adult MeSH
- Exons genetics MeSH
- Phenotype MeSH
- Financing, Organized MeSH
- Gene Dosage MeSH
- Homozygote MeSH
- Humans MeSH
- Adolescent MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Child, Preschool MeSH
- Survival of Motor Neuron 1 Protein MeSH
- Survival of Motor Neuron 2 Protein MeSH
- Cyclic AMP Response Element-Binding Protein genetics MeSH
- SMN Complex Proteins MeSH
- Nerve Tissue Proteins genetics MeSH
- RNA-Binding Proteins genetics MeSH
- Muscular Atrophy, Spinal genetics MeSH
- Nucleic Acid Amplification Techniques MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Adolescent MeSH
- Child, Preschool MeSH
Background and Aims: The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. Methods: Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. Key Results: Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. Conclusions: Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development.
BACKGROUND: Familial hemiplegic migraine (FHM) is a rare autosomal dominant subtype of migraine with aura. Missense mutations in the chromosome 19 CACNA1A calcium channel gene have been found in approximately half of the families. The T666M mutation, replacing a threonine by a methionine at residue number 666, is the most frequent mutation, reported in 14 independent FHM families; other mutations have so far been described in only 1 or 2 families each. The clinical features of T666M families have been reported, but the course is unknown. OBJECTIVE: To present a detailed description of the clinical features of new FHM families in which we identified the T666M mutation in our CACNA1A screening program. METHODS: As part of our ongoing genetic screening, mutation analysis of the CACNA1A gene was performed by single-strand conformational polymorphism analysis in 33 probands of families with FHM. RESULTS: We identified the T666M mutation in 5 unrelated FHM families. In 3 of the families, patients displayed cerebellar ataxia. In 1 family, some affected members with the mutation had attacks with confusion but without hemiparesis. In 1 family, patients had progressive cognitive dysfunction. CONCLUSIONS: The T666M mutation is the most frequent CACNA1A mutation in FHM; it was found in 5 of 33 FHM families at our laboratory, and in 19 of 39 families with a known mutation reported in the literature (including the present study). Screening for the T666M mutation should therefore be the first step when screening families with FHM. There is a remarkable clinical heterogeneity among families with the T666M mutation.
- MeSH
- Point Mutation * MeSH
- Adult MeSH
- Phenotype MeSH
- Haplotypes MeSH
- Hemiplegia etiology genetics MeSH
- Humans MeSH
- Migraine Disorders complications genetics MeSH
- Polymorphism, Single-Stranded Conformational MeSH
- Pedigree MeSH
- Calcium Channels genetics MeSH
- Family Health MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
- Germany MeSH
- United Kingdom MeSH
- United States MeSH
- MeSH
- Point Mutation MeSH
- Dyneins genetics MeSH
- Mutagenesis, Insertional MeSH
- Humans MeSH
- Mutation, Missense MeSH
- RNA Splice Sites genetics MeSH
- Molecular Sequence Data MeSH
- Mutation * MeSH
- DNA Mutational Analysis MeSH
- Myosin VIIa MeSH
- Myosins genetics MeSH
- Codon, Nonsense MeSH
- Polymorphism, Single-Stranded Conformational MeSH
- Pedigree MeSH
- Amino Acid Sequence MeSH
- Sequence Deletion MeSH
- Sequence Homology, Amino Acid MeSH
- Sequence Alignment MeSH
- Tandem Repeat Sequences MeSH
- Usher Syndromes ethnology genetics MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Geographicals
- Czech Republic MeSH
- Italy MeSH
- Morocco MeSH
- Spain MeSH
- Turkey MeSH
OBJECTIVE: Mutations in NKX2.1, NKX2.5, FOXE1 and PAX8 genes, encoding for transcription factors involved in the development of the thyroid gland, have been identified in a minority of patients with syndromic and non-syndromic congenital hypothyroidism (CH). DESIGN: In a phenotype-selected cohort of 170 Czech paediatric and adolescent patients with non-goitre CH, including thyroid dysgenesis, or non-goitre early-onset hypothyroidism, PAX8, NKX2.1, NKX2.5, FOXE1 and HHEX genes were analysed for mutations. Methods: NKX2.1, NKX2.5, FOXE1 and HHEX genes were directly sequenced in patients with syndromic CH. PAX8 mutational screening was performed in all 170 patients by single-stranded conformation polymorphism, followed by direct sequencing of samples with abnormal findings. The R52P PAX8 mutation was functionally characterized by DNA binding studies. RESULTS: We identified a novel PAX8 mutation R52P, dominantly inherited in a three-generation pedigree and leading to non-congenital, early-onset, non-goitre, non-autoimmune hypothyroidism with gradual postnatal regression of the thyroid size and function. The R52P PAX8 mutation results in the substitution of a highly conserved residue of the DNA-binding domain with a loss-of-function effect. CONCLUSIONS: The very low frequency of genetic defects in a population-based cohort of children affected by non-goitre congenital and early-onset hypothyroidism, even in a phenotype-focussed screening study, suggests the pathogenetic role of either non-classic genetic mechanisms or the involvement of genes unknown so far. Identification of a novel PAX8 mutation in a particular variant of non-congenital early-onset hypothyroidism indicates a key function of PAX8 in the postnatal growth and functional maintenance of the thyroid gland.
- MeSH
- Point Mutation MeSH
- Child MeSH
- DNA genetics chemistry MeSH
- Thyroid Dysgenesis genetics ultrasonography MeSH
- Financing, Organized MeSH
- Cloning, Molecular MeSH
- Congenital Hypothyroidism genetics ultrasonography MeSH
- Humans MeSH
- Adolescent MeSH
- Molecular Sequence Data MeSH
- Polymerase Chain Reaction MeSH
- Polymorphism, Single-Stranded Conformational MeSH
- Electrophoretic Mobility Shift Assay MeSH
- Pedigree MeSH
- Amino Acid Sequence MeSH
- Paired Box Transcription Factors genetics MeSH
- Transcription Factors genetics MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Female MeSH
- Geographicals
- Czech Republic MeSH
STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.
- MeSH
- Point Mutation * MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Membrane Proteins chemistry genetics metabolism MeSH
- Molecular Structure MeSH
- Nucleotides, Cyclic chemistry metabolism MeSH
- Protein Domains MeSH
- Binding Sites MeSH
- Hydrogen Bonding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Familial medullary thyroid carcinoma (FMTC) is an autosomal dominant inherited disease, characterized by germ-line mutations in the RET proto-oncogene, mainly in exons 10 and 11, but also in exons 13, 14 and 15. Recently, mutations in exons 8 and 16 associated with FMTC were also described. In the herein presented study, single strand conformation polymorphism (SSCP) method for rapid screening of mutations in the RET proto-oncogene and fluorescent sequencing method were used. In one Czech family with FMTC, we have identified a novel missense point mutation of the RET proto-oncogene in exon 5, that results in substitution of arginine by glycine at codon 321 in the cadherin-like domain of ret protein. It seems that this mutation causes FMTC as no other mutation was found in the classical risk exons (10, 11, 13, 14, 15 and 16) of the RET proto-oncogene. The mutation cosegregates with medullary thyroid carcinoma (MTC) or C cell hyperplasia (CCH) in two patients; two other family members are mutation carriers without clinical signs of MTC so far. To improve the diagnosis of FMTC, analysis of exon 5 of the RET proto-oncogene should be considered in families with no identified classical RET mutations.
- MeSH
- Arginine analysis MeSH
- Point Mutation MeSH
- DNA, Neoplasm analysis genetics MeSH
- Adult MeSH
- Exons MeSH
- Financing, Organized MeSH
- Genetic Testing MeSH
- Glycine analysis MeSH
- Codon genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Carcinoma, Medullary diagnosis genetics surgery MeSH
- Mutation, Missense MeSH
- Thyroid Neoplasms diagnosis genetics surgery MeSH
- Proto-Oncogene Proteins c-ret genetics MeSH
- Proto-Oncogenes MeSH
- Pedigree MeSH
- Aged MeSH
- Thyroidectomy MeSH
- Germ-Line Mutation MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
