The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Introducing the "hip call" to facilitate early surgical treatment of hip fractures: A feasibility study.

INTRODUCTION: Surgical treatment of hip fractures within 24-48 hours decreases morbidity and mortality, but goals for early surgery have not been widely achieved so far. The primary aim of this study was to investigate the feasibility of implementation of a hip call, and the secondary aim was to investigate the effect of the hip call on time for pre-operative preparation and surgery compared to a historical control cohort. MATERIALS AND METHODS: From March 4, 2019 until June 30, 2019, admission of patients at Copenhagen University Hospital, Bispebjerg, Denmark, with a suspected hip fracture triggered an acute hip call. Key personnel are summoned to secure rapid pre-operative preparation and surgery. The implementation was defined feasible, if ≥ 75% of the patients were ready for surgery within 4 hours and had surgery initiated within 24 hours of hospital arrival. The historical control cohort was patients with hip fractures in the same period in 2018. RESULTS: A total of 128 patients were included in 2019, and 99 in 2018. After implementation of hip call, 83% of patients were ready for surgery within 4 hours. After vs before hip call, 88% vs 51% were operated within 24 hours and 96% vs 79% within 36 hours. Time from admission to surgery (hh:mm) was reduced by mean 10:33 (CI 07:46-13:20), P < .001. CONCLUSION: The implementation of a hip call was feasible with 83% of patients being ready for surgery within 4 hours, and 88% being operated within 24 hours. Future large-scale studies should clarify potential benefits on clinical outcome.

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein.

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) "conventional" tail-vein injections, (2) "primed" injections, (3) "hydrodynamic" injections, or (4) direct "intrahepatic" injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse.

Comparison of Glycomacropeptide with Phenylalanine Free-Synthetic Amino Acids in Test Meals to PKU Patients: No Significant Differences in Biomarkers, Including Plasma Phe Levels.

INTRODUCTION: Management of phenylketonuria (PKU) is achieved through low-phenylalanine (Phe) diet, supplemented with low-protein food and mixture of free-synthetic (FS) amino acid (AA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese-making and does not contain Phe. Lacprodan® CGMP-20 used in this study contained a small amount of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The purpose of this study was to compare absorption of CGMP-20 to FSAA with the aim of evaluating short-term effects on plasma AAs as well as biomarkers related to food intake. METHODS: This study included 8 patients, who had four visits and tested four drink mixtures (DM1-4), consisting of CGMP, FSAA, or a combination. Plasma blood samples were collected at baseline, 15, 30, 60, 120, and 240 minutes (min) after the meal. AA profiles and ghrelin were determined 6 times, while surrogate biomarkers were determined at baseline and 240 min. A visual analogue scale (VAS) was used for evaluation of taste and satiety. RESULTS: The surrogate biomarker concentrations and VAS scores for satiety and taste were nonsignificant between the four DMs, and there were only few significant results for AA profiles (not Phe). CONCLUSION: CGMP and FSAA had the overall same nonsignificant short-term effect on biomarkers, including Phe. This combination of FSAA and CGMP is a suitable supplement for PKU patients.

Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures.

Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

Partial correction of the dwarf phenotype by non-viral transfer of the growth hormone gene in mice: Treatment age is critical.

Non-viral transfer of the growth hormone gene to different muscles of immunodeficient dwarf (lit/scid) mice is under study with the objective of improving phenotypic correction via this particular gene therapy approach. Plasmid DNA was administered into the exposed quadriceps or non-exposed tibialis cranialis muscle of lit/scid mice followed by electroporation, monitoring several growth parameters. In a 6-month bioassay, 50µg DNA were injected three times into the quadriceps muscle of 80-day old mice. A 50% weight increase, with a catch-up growth of 21%, together with a 16% increase for nose-to-tail and tail lengths (catch-up=19-21%) and a 24-28% increase for femur length (catch-up=53-60%), were obtained. mIGF1 serum levels were ~7-fold higher than the basal levels for untreated mice, but still ~2-fold lower than in non-dwarf scid mice. Since treatment age was found to be particularly important in a second bioassay utilizing 40-day old mice, these pubertal mice were compared in a third bioassay with adult (80-day old) mice, all treated twice with 50µg DNA injected into each tibialis cranialis muscle, via a less invasive approach. mIGF1 concentrations at the same level as co-aged scid mice were obtained 15days after administration in pubertal mice. Catch-up growth, based on femur length (77%), nose-to-tail (36%) and tail length (39%) increases was 40 to 95% higher than those obtained upon treating adult mice. These data pave the way for the development of more effective pre-clinical assays in pubertal dwarf mice for the treatment of GH deficiency via plasmid-DNA muscular administration.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

In situ microfluidic dialysis for biological small-angle X-ray scattering.

Owing to the demand for low sample consumption and automated sample changing capabilities at synchrotron small-angle X-ray (solution) scattering (SAXS) beamlines, X-ray microfluidics is receiving continuously increasing attention. Here, a remote-controlled microfluidic device is presented for simultaneous SAXS and ultraviolet absorption measurements during protein dialysis, integrated directly on a SAXS beamline. Microfluidic dialysis can be used for monitoring structural changes in response to buffer exchange or, as demonstrated, protein concentration. By collecting X-ray data during the concentration procedure, the risk of inducing protein aggregation due to excessive concentration and storage is eliminated, resulting in reduced sample consumption and improved data quality. The proof of concept demonstrates the effect of halted or continuous flow in the microfluidic device. No sample aggregation was induced by the concentration process at the levels achieved in these experiments. Simulations of fluid dynamics and transport properties within the device strongly suggest that aggregates, and possibly even higher-order oligomers, are preferentially retained by the device, resulting in incidental sample purification. Hence, this versatile microfluidic device enables investigation of experimentally induced structural changes under dynamically controllable sample conditions.

DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1.

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to 'sponge' splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG-RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.

A novel homologous model for gene therapy of dwarfism by non-viral transfer of the mouse growth hormone gene into immunocompetent dwarf mice.

The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/10(6) cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (P<0.001). In mGH-treated mice, mIGF-I determined on days 15, 45 and 94 were 1.5- to 3-fold higher than the control and 1.2- to 1.6-fold higher than hGH-treated mice. The described homologous model represents an important progress forming the basis for preclinical testing of non-viral gene therapy for GH deficiency.

Surface functionalized thiol-ene waveguides for fluorescence biosensing in microfluidic devices.

Thiol-ene polymers possess physical, optical, and chemical characteristics that make them ideal substrates for the fabrication of optofluidic devices. In this work, thiol-ene polymers are used to simultaneously create microfluidic channels and optical waveguides in one simple moulding step. The reactive functional groups present at the surface of the thiol-ene polymer are subsequently used for the rapid, one step, site-specific functionalization of the waveguide with biological recognition molecules. It was found that while the bulk properties and chemical surface properties of thiol-ene materials vary considerably with variations in stoichiometric composition, their optical properties remain mostly unchanged with an average refractive index value of 1.566 ± 0.008 for thiol-ene substrates encompassing a range from 150% excess ene to 90% excess thiol. Microfluidic chips featuring thiol-ene waveguides were fabricated from 40% excess thiol thiol-ene to ensure the presence of thiol functional groups at the surface of the waveguide. Biotin alkyne was photografted at specific locations using a photomask, directly at the interface between the microfluidic channel and the thiol-ene waveguide prior to conjugation with fluorescently labeled streptavidin. Fluorescence excitation was achieved by launching light through the thiol-ene waveguide, revealing bright fluorescent patterns along the channel/waveguide interface.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds.

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices.

The suitable optical properties of thiol-ene polymers combined with the ease of modifying their surface for the attachment of recognition molecules make them ideal candidates in many biochip applications. This paper reports the rapid one-step photochemical surface patterning of biomolecules in microfluidic thiol-ene chips. This work focuses on thiol-ene substrates featuring an excess of thiol groups at their surface. The thiol-ene stoichiometric composition can be varied to precisely control the number of surface thiol groups available for surface modification up to an average surface density of 136 ± 17 SH nm(-2). Biotin alkyne was patterned directly inside thiol-ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent wave-induced fluorescence (EWIF), demonstrating the success of the grafting procedure and its potential for biochip applications.

Spatial distribution of transgenic protein after gene electrotransfer to porcine muscle.

Gene electrotransfer is an effective nonviral technique for delivery of plasmid DNA into tissues. From a clinical perspective, muscle is an attractive target tissue as long-term, high-level transgenic expression can be achieved. Spatial distribution of the transgenic protein following gene electrotransfer to muscle in a large animal model has not yet been investigated. In this study, 17 different doses of plasmid DNA (1-1500 µg firefly luciferase pCMV-Luc) were delivered in vivo to porcine gluteal muscle using electroporation. Forty-eight hours post treatment several biopsies were obtained from each transfection site in order to examine the spatial distribution of the transgenic product. We found a significantly higher luciferase activity in biopsies from the center of the transfection site compared to biopsies taken adjacent to the center, 1 and 2 cm along muscle fiber orientation (p<0.05 and p<0.0001, respectively). On average, 43% of the total luciferase activity was localized in the center biopsy. In conclusion, we found that gene electrotransfer to muscle in a large animal model led to localized gene expression corresponding to the area delineated by the electrodes. High doses of plasmid DNA did not lead to a larger area of the muscle expressing the transgenic protein.

Growth responses following a single intra-muscular hGH plasmid administration compared to daily injections of hGH in dwarf mice.

In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5 µg/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P < 0.001) and 23.1% (P < 0.01) for protein and DNA administration, respectively, a difference possibly related to the electroporation methodology. The nose-to-tail linear growth increases were 15% and 9.6% for the protein and DNA treatments, respectively, but mouse insulin-like growth factor I (mIGF-I) showed a greater increase over the control with DNA (5- to 7-fold) than with protein (3- to 4-fold) administration. The weight increases of several organs and tissues (kidneys, spleen, liver, heart, quadriceps and gastrocnemius muscles) were 1.3- to 4.6-fold greater for protein than for DNA administration, which gave a generally more proportional growth. Glucose levels were apparently unaffected, suggesting the absence of effects on glucose tolerance. A gene transfer strategy based on a single hGH-DNA administration thus appears to be comparable to repeated hormone injections for promoting growth and may represent a feasible alternative for the treatment of growth hormone deficiency.

Gold nanoparticle-based optical microfluidic sensors for analysis of environmental pollutants.

Conventional methods of environmental analysis can be significantly improved by the development of portable microscale technologies for direct in-field sensing at remote locations. This report demonstrates the vast potential of gold nanoparticle-based microfluidic sensors for the rapid, in-field, detection of two important classes of environmental contaminants - heavy metals and pesticides. Using gold nanoparticle-based microfluidic sensors linked to a simple digital camera as the detector, detection limits as low as 0.6 µg L(-1) and 16 µg L(-1) could be obtained for the heavy metal mercury and the dithiocarbamate pesticide ziram, respectively. These results demonstrate that the attractive optical properties of gold nanoparticle probes combine synergistically with the inherent qualities of microfluidic platforms to offer simple, portable and sensitive sensors for environmental contaminants.

Detection of unlabeled particles in the low micrometer size range using light scattering and hydrodynamic 3D focusing in a microfluidic system.

In this paper, we describe a microfluidic device composed of integrated microoptical elements and a two-layer microchannel structure for highly sensitive light scattering detection of micro/submicrometer-sized particles. In the two-layer microfluidic system, a sample flow stream is first constrained in the out-of-plane direction into a narrow sheet, and then focused in-plane into a small core region, obtaining on-chip three-dimensional (3D) hydrodynamic focusing. All the microoptical elements, including waveguides, microlens, and fiber-to-waveguide couplers, and the in-plane focusing channels are fabricated in one SU-8 layer by standard photolithography. The channels for out-of-plane focusing are made in a polydimethylsiloxane (PDMS) layer by a single cast using a SU-8 master. Numerical and experimental results indicate that the device can realize 3D hydrodynamic focusing reliably over a wide range of Reynolds numbers (0.5 < Re < 20). Polystyrene particles of three sizes (2, 1, and 0.5 µm) were measured in the microfluidic device with integrated optics, demonstrating the feasibility of this approach to detect particles in the low micrometer size range by light scattering detection.

Genetically modified pigs for biomedical research.

During the last two decades, pigs have been used to develop some of the most important large animal models for biomedical research. Advances in pig genome research, genetic modification (GM) of primary pig cells and pig cloning by nuclear transfer, have facilitated the generation of GM pigs for xenotransplantation and various human diseases. This review summarizes the key technologies used for generating GM pigs, including pronuclear microinjection, sperm-mediated gene transfer, somatic cell nuclear transfer by traditional cloning, and somatic cell nuclear transfer by handmade cloning. Broadly used genetic engineering tools for porcine cells are also discussed. We also summarize the GM pig models that have been generated for xenotransplantation and human disease processes, including neurodegenerative diseases, cardiovascular diseases, eye diseases, bone diseases, cancers and epidermal skin diseases, diabetes mellitus, cystic fibrosis, and inherited metabolic diseases. Thus, this review provides an overview of the progress in GM pig research over the last two decades and perspectives for future development.

Clinical expression of Menkes disease in females with normal karyotype.

BACKGROUND: Menkes Disease (MD) is a rare X-linked recessive fatal neurodegenerative disorder caused by mutations in the ATP7A gene, and most patients are males. Female carriers are mosaics of wild-type and mutant cells due to the random X inactivation, and they are rarely affected. In the largest cohort of MD patients reported so far which consists of 517 families we identified 9 neurologically affected carriers with normal karyotypes. METHODS: We investigated at-risk females for mutations in the ATP7A gene by sequencing or by multiplex ligation-dependent probe amplification (MLPA). We analyzed the X-inactivation pattern in affected female carriers, unaffected female carriers and non-carrier females as controls, using the human androgen-receptor gene methylation assay (HUMAR). RESULTS: The clinical symptoms of affected females are generally milder than those of affected boys with the same mutations. While a skewed inactivation of the X-chromosome which harbours the mutation was observed in 94% of 49 investigated unaffected carriers, a more varied pattern was observed in the affected carriers. Of 9 investigated affected females, preferential silencing of the normal X-chromosome was observed in 4, preferential X-inactivation of the mutant X chromosome in 2, an even X-inactivation pattern in 1, and an inconclusive pattern in 2. The X-inactivation pattern correlates with the degree of mental retardation in the affected females. Eighty-one percent of 32 investigated females in the control group had moderately skewed or an even X-inactivation pattern. CONCLUSION: The X- inactivation pattern alone cannot be used to predict the phenotypic outcome in female carriers, as even those with skewed X-inactivation of the X-chromosome harbouring the mutation might have neurological symptoms.

Myoblasts generated by lentiviral mediated MyoD transduction of myotonic dystrophy type 1 (DM1) fibroblasts can be used for assays of therapeutic molecules.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is the most common muscle dystrophy in adults. The disease is caused by a triplet expansion in the 3'end of the myotonic dystrophy protein kinase (DMPK) gene. In order to develop a human cell model for investigation of possible effects of antisense and RNAi effector molecules we have used lentiviral mediated myoD-forced myogenesis of DM1 patient fibroblasts. FINDINGS: Transduced fibroblasts show a multinuclear phenotype and express the differentiation marker myogenin. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed a statistical significant increase in the amount of nuclear foci in DM1 patient fibroblasts after myogenesis. Finally, no nuclear foci were found after treatment with oligonucleotides targeting the repeat expansions. CONCLUSIONS: The abundance of nuclear foci in DM1 patient fibroblasts increase following myogenesis, as visualized by FISH analysis. Foci were eradicated after treatment with antisense oligonucleotides. Thus, we propose that the current cell model is suitable for testing of novel treatment modalities.