Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies.

BACKGROUND: Limb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis. METHODS: We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes. RESULTS: Analysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound - c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene - c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present. CONCLUSIONS: Genetic diagnosis was possible in 12 of 60 patients (20%). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.

Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells.

We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.

Complete mtDNA sequencing reveals mutations m.9185T>C and m.13513G>A in three patients with Leigh syndrome.

The most common mitochondrial disorder in children is Leigh syndrome, which is a progressive and genetically heterogeneous neurodegenerative disorder caused by mutations in nuclear genes or mitochondrial DNA (mtDNA). In the present study, a novel and robust method of complete mtDNA sequencing, which allows amplification of the whole mitochondrial genome, was tested. Complete mtDNA sequencing was performed in a cohort of patients with suspected mitochondrial mutations. Patients from Latvia and Lithuania (n = 92 and n = 57, respectively) referred by clinical geneticists were included. The de novo point mutations m.9185T>C and m.13513G>A, respectively, were detected in two patients with lactic acidosis and neurodegenerative lesions. In one patient with neurodegenerative lesions, the mutation m.9185T>C was identified. These mutations are associated with Leigh syndrome. The present data suggest that full-length mtDNA sequencing is recommended as a supplement to nuclear gene testing and enzymatic assays to enhance mitochondrial disease diagnostics.

Effects of food quality on trade-offs among growth, immunity and survival in the greater wax moth Galleria mellonella.

The resources available to an individual in any given environment are finite, and variation in life history traits reflect differential allocation of these resources to competing life functions. Nutritional quality of food is of particular importance in these life history decisions. In this study, we tested trade-offs among growth, immunity and survival in 3 groups of greater wax moth (Galleria mellonella) larvae fed on diets of high and average nutritional quality. We found rapid growth and weak immunity (as measured by encapsulation response) in the larvae of the high-energy food group. It took longer to develop on food of average nutritional quality. However, encapsulation response was stronger in this group. The larvae grew longer in the low-energy food group, and had the strongest encapsulation response. We observed the highest survival rates in larvae of the low-energy food group, while the highest mortality rates were observed in the high-energy food group. A significant negative correlation between body mass and the strength of encapsulation response was found only in the high-energy food group revealing significant competition between growth and immunity only at the highest rates of growth. The results of this study help to establish relationships between types of food, its nutritional value and life history traits of G. mellonella larvae.

NFAT transcription factors control HIV-1 expression through a binding site downstream of TAR region.

NFAT factors control HIV-1 transcription. We show here that, in addition to binding to two NF-kappaB/NFAT sites within the U3 HIV LTR, NFATc1 and NFATc2 bind to an NFAT site within the LTR's U5 region. Mutations in this site which abolish NFAT binding reduce the ability of NFATs to transactivate LTR-mediated transcription. Mutations in all three NFAT sites strongly interfered with LTR induction, but affected moderately the stimulatory effect of Tat.

Dupuytren's Contracture Cosegregation with Limb-Girdle Muscle Dystrophy.

Limb-girdle muscular dystrophies (LGMDs) is a heterogeneous group of muscular dystrophies that mostly affect the pelvic and shoulder girdle muscle groups. We report here a case of neuromuscular disease associated with Dupuytren's contracture, which has never been described before as cosegregating with an autosomal dominant type of inheritance. Dupuytren's contracture is a common disease, especially in Northern Europe. Comorbid conditions associated with Dupuytren's contracture are repetitive trauma to the hands, diabetes, and seizures, but it has never before been associated with neuromuscular disease. We hypothesize that patients may harbor mutations in genes with functions related to neuromuscular disease and Dupuytren's contracture development.

DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells.

Recent studies have highlighted an apparently paradoxical link between self-renewal and senescence triggered by DNA damage in certain cell types. In addition, the finding that TP53 can suppress senescence has caused a re-evaluation of its functional role in regulating these outcomes. To investigate these phenomena and their relationship to pluripotency and senescence, we examined the response of the TP53-competent embryonal carcinoma (EC) cell line PA-1 to etoposide-induced DNA damage. Nuclear POU5F1/OCT4A and P21CIP1 were upregulated in the same cells following etoposide-induced G 2M arrest. However, while accumulating in the karyosol, the amount of OCT4A was reduced in the chromatin fraction. Phosphorylated CHK2 and RAD51/γH2AX-positive nuclear foci, overexpression of AURORA B kinase and moderate macroautophagy were evident. Upon release from G 2M arrest, cells with repaired DNA entered mitoses, while the cells with persisting DNA damage remained at this checkpoint or underwent mitotic slippage and gradually senesced. Reduction of TP53 using sh- or si-RNA prevented the upregulation of OCT4A and P21CIP1 and increased DNA damage. Subsequently, mitoses, micronucleation and senescence were all enhanced after TP53 reduction with senescence confirmed by upregulation of CDKN2A/P16INK4A and increased sa-ß-galactosidase positivity. Those mitoses enhanced by TP53 silencing were shown to be multicentrosomal and multi-polar, containing fragmented and highly deranged chromosomes, indicating a loss of genome integrity. Together, these data suggest that TP53-dependent coupling of self-renewal and senescence pathways through the DNA damage checkpoint provides a mechanism for how embryonal stem cell-like EC cells safeguard DNA integrity, genome stability and ultimately the fidelity of self-renewal.

The 4154delA mutation carriers in the BRCA1 gene share a common ancestry.

Uncertainty exists whether the 4154delA mutation of the BRCA1 gene detected in unrelated individuals from Latvia, Poland and Russia is a founder mutation with a common ancestral origin. To trace back this problem we analysed the mutation-associated haplotype of the BRCA1 intragenic SNPs as well as intragenic and nearby STR markers in mutation carriers from the aforementioned populations. The mutation-associated SNP alleles were found to be "T-A-A-A-A-G" for six intragenic SNPs of the BRCA1 gene (IVS8-58delT, 3232A/G, 3667A/G, IVS16-68A/G, IVS16-92A/G, IVS18+66G/A, respectively). The alleles 195, 154, 210 and 181 were found to be associated with the 4154delA mutation for STR markers D17S1325, D17S855, D17S1328 and D17S1320, correspondingly. Further analysis of markers in the 4154delA mutation carriers from all three populations allows us to assert that all analysed mutation carriers share a common ancestry.

Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility.

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels.

Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.

Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.