Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene

International audienceStraightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations

Droplet Digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

International audienceBackground - Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. Methods - To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. Results - We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. Conclusions - This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd

Asiakastyytyväisyys ja kehittämiskohteet : case Matkailukeskus Rauhalahti

Tämän opinnäytetyön tarkoituksena on selvittää Matkailukeskus Rauhalahden kesän 2015 asiakastyytyväisyyskyselyiden pohjalta Matkailukeskus Rauhalahden kehitystarpeet ja asiakasprofiili. Lisäksi työssäni vertaan tekemääni asiakasprofiilia vuoden 2012 opinnäytetyönä tehtyyn Matkailukeskus Rauhalahden asiakasprofiilitutkimukseen. Asiakaskyselylomakkeita jaettiin asiakkaille kesän aikana noin 5000 kpl. Palautuneista lomakkeista 800 oli suomenkielisiä. Suomenkielisistä lomakkeista jouduttiin valitettavasti hylkäämään 329 kappaletta, joten jäljelle jäi 471 lomaketta tutkittavaksi. Asiakastyytyväisyyslomakkeen lopussa asiakas pystyi täyttämään yhteystietonsa ja osallistumaan huvilalahjakortin arvontaan. Tarkoituksena on kartoittaa Matkailukeskus Rauhalahden kehittämiskohteet asiakkaiden näkökulmasta ja esittää niihin mahdollisia ratkaisuja. Lisäksi tavoitteena on verrata tutkimuksen pohjalta tehtyä asiakasprofiilia vuoden 2012 asiakasprofiiliin ja huomioida mahdolliset muutokset. Lisäksi analysoin asiakastyytyväisyyskyselyn tuloksia, jotta voidaan nähdä, mikä Matkailukeskus Rauhalahdessa on asiakkaiden mielestä hyvää ja mikä huonompaa. Opinnäytetyöni teoriaosuus koostuu asiakasprofiiliin ja asiakastyytyväisyyteen liittyvistä asioista, kuten esimerkiksi asiakaskokemuksesta. Työni kertoo, kuinka tärkeää esimerkiksi asiakkaiden segmentointi on yritykselle ja miten yritys voi hyötyä asiakaskokemuksen parantamisesta. Kerron myös erilaisista asiakastyytyväisyystutkimuksista ja niiden hyödyistä yrityksille. Tutkimus on kvantitatiivinen eli määrällinen. Se on tehty asiakastyytyväisyyslomakkeilla ja tutkimuksessa on huomioitu vain suomenkieliset lomakkeet. Tutkimus suoritettiin Matkailukeskus Rauhalahden kesäkauden aikana, eli toukokuun 2015 lopusta elokuun 2015 loppuun. Kyselyssä kysyttiin asiakkaiden taustatietoja, kuten ikää, sukupuolta ja yhteiskunnallista asemaa. Lisäksi kysyttiin, mistä asiakas on saanut tietonsa Matkailukeskus Rauhalahdesta ja mikä vaikutti asiakkaan päätökseen majoittua Matkailukeskus Rauhalahteen. Myös asiakkaiden majoitusmuotoa ja majoitusöiden lukumäärää tiedusteltiin lomakkeessa. Opinnäytetyöni tutkimusosio on tehty kehittämisnäkökulmasta.The purpose of this thesis was to find the needs of development and the customer profile for Matkailukeskus Rauhalahti. The research was made from customer satisfaction questionnaire that was made during summer 2015. In my thesis I compare my customer profile to the one made in 2012 by Sarianne Suominen. There were about 5000 questionnaires given to the customers in summer 2015 and there were 800 Finnish questionnaires that came back. I had to disqualify 329 of them because of incomplete questionnaires, so I had 471 questionnaires to make my research with. Matkailukeskus Rauhalahti tempted customers to fill the forms in with a lottery, where customers could win a gift card to stay in a holiday villa. The purpose was to present the developing needs from the customers’ aspect and provide solutions to them. I also compare the customer profile I made to the one made in 2012 and pay attenton to possible changes. I also analyze the results of the questionnaire, so we can see what customers think is good and what is bad in Matkailukeskus Rauhalahti. The theory part in this thesis is made of things that relate to customer profiles and customer satisfaction, such as for example customer experience. My thesis tells how important customer segmentation is for the companies and how a company can profit from making their customer experience better. I also tell about different customer satisfaction researches and how they can profit a company. The research in this thesis is quantitative. It is made with customer satisfactory questionnaires in Finnish. Research was made from May 2015 to August 2015. There were questions about customers’ age, sex and their social status. There were also questions about how the customer got his/hers information about Matkailukeskus Rauhalahti and which factors affected their decision to stay in Matkailukeskus Rauhalahti. The questionnaire also asked about how many nights the customer stayed and in what kind of accommodation. The research was made from the customers’ point of view

X-linked muscular dystrophy in a Labrador Retriever strain: phenotypic and molecular characterisation

International audienceBackground: Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the Golden Retriever muscular dystrophy (GRMD) model remains the most used in preclinical studies. Here, we report a new spontaneous dystrophinopathy in a Labrador Retriever strain, named Labrador Retriever muscular dystrophy (LRMD).Methods: A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing.Results: The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2-Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability.Conclusions: This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes

Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes

International audiencehe frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs

Non Random Distribution of DMD Deletion Breakpoints and Implication of Double Strand Breaks Repair and Replication Error Repair Mechanisms

International audienceBACKGROUND:Dystrophinopathies are mostly caused by copy number variations, especially deletions, in the dystrophin gene (DMD). Despite the large size of the gene, deletions do not occur randomly but mainly in two hot spots, the main one involving exons 45 to 55. The underlying mechanisms are complex and implicate two main mechanisms: Non-homologous end joining (NHEJ) and micro-homology mediated replication-dependent recombination (MMRDR).OBJECTIVE:Our goals were to assess the distribution of intronic breakpoints (BPs) in the genomic sequence of the main hot spot of deletions within DMD gene and to search for specific sequences at or near to BPs that might promote BP occurrence or be associated with DNA break repair.METHODS:Using comparative genomic hybridization microarray, 57 deletions within the intron 44 to 55 region were mapped. Moreover, 21 junction fragments were sequenced to search for specific sequences.RESULTS:Non-randomly distributed BPs were found in introns 44, 47, 48, 49 and 53 and 50% of BPs clustered within genomic regions of less than 700bp. Repeated elements (REs), known to promote gene rearrangement via several mechanisms, were present in the vicinity of 90% of clustered BPs and less frequently (72%) close to scattered BPs, illustrating the important role of such elements in the occurrence of DMD deletions. Palindromic and TTTAAA sequences, which also promote DNA instability, were identified at fragment junctions in 20% and 5% of cases, respectively. Micro-homologies (76%) and insertions or deletions of small sequences were frequently found at BP junctions.CONCLUSIONS:Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible

Additional file 3: Figure S5. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

BMP4-treated hiPSCs/hESCs express dystrophin protein. (a) Western blot in six pluripotent stem cell lines (hiPSCs 1, 3 and 4, DMD hiPSCs 1 and 3, hESCs 2) at day 4 either without or after BMP4 treatment. (b) Western blot in hiPSCs 1 from days 0 through 4 after a single BMP4 treatment. (Dystrophin antibody: DYS1; Muscle biopsy protein extract from a healthy individual serves as a control, α-tubulin was used as loading control)

Additional file 8: Figure S4. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

In silico translation of the novel DMD transcript. Representation of the open reading frame (ORF) resulting in translation of the novel DMD transcript. Possible translated proteins from this ORF are represented in red. The largest predicted protein is composed of 3562 amino acids (≈412 kDa) ( http://web.expasy.org/translate )

Additional file 7: Figure S3. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

The new DMD exon 1 belongs to a retrovirus-like sequence. (a) Alignment of the new exon 1 region (highlighted in light blue) among 100 vertebrate species with the conserved upstream/downstream sequence marked by red arrows ( https://genome.ucsc.edu ). (b) Schematic representation of the approximately 8 kb region in the DMD gene that is conserved among a sub-group of anthropoids. It is composed of simple repeats, Alu sequences and the whole human endogenous retrovirus-like sequence HuERS-P1 ( http://www.dfam.org/entry/DF0000214 ) flanked by two LTR8 elements

Additional file 1: Table S1. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

Human embryonic and induced pluripotent stem cell line list. Details concerning the stem cell lines used in the present study