Chromosome 10q-linked FSHD identifies DUX4 as principal disease gene.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is an inherited muscular dystrophy clinically characterised by muscle weakness starting with the facial and upper extremity muscles. A disease model has been developed that postulates that failure in somatic repression of the transcription factor DUX4 embedded in the D4Z4 repeat on chromosome 4q causes FSHD. However, due to the position of the D4Z4 repeat close to the telomere and the complex genetic and epigenetic aetiology of FSHD, there is ongoing debate about the transcriptional deregulation of closely linked genes and their involvement in FSHD. METHOD: Detailed genetic characterisation and gene expression analysis of patients with clinically confirmed FSHD and control individuals. RESULTS: Identification of two FSHD families in which the disease is caused by repeat contraction and DUX4 expression from chromosome 10 due to a de novo D4Z4 repeat exchange between chromosomes 4 and 10. We show that the genetic lesion causal to FSHD in these families is physically separated from other candidate genes on chromosome 4. We demonstrate that muscle cell cultures from affected family members exhibit the characteristic molecular features of FSHD, including DUX4 and DUX4 target gene expression, without showing evidence for transcriptional deregulation of other chromosome 4-specific candidate genes. CONCLUSION: This study shows that in rare situations, FSHD can occur on chromosome 10 due to an interchromosomal rearrangement with the FSHD locus on chromosome 4q. These findings provide further evidence that DUX4 derepression is the dominant disease pathway for FSHD. Hence, therapeutic strategies should focus on DUX4 as the primary target.

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant myopathy characterized by slowly progressive skeletal muscle weakness and wasting. While a regenerative response is often provoked in many muscular dystrophies, little is known about whether a regenerative response is regularly elicited in FSHD muscle, prompting this study. For comparison, we also examined the similarly slowly progressing myotonic dystrophy type 2 (DM2). To first investigate regeneration at the transcriptomic level, we used the 200 human gene Hallmark Myogenesis list. This myogenesis biomarker was elevated in FSHD and control healthy myotubes compared to their myoblast counterparts, so is higher in myogenic differentiation. The myogenesis biomarker was also elevated in muscle biopsies from most independent FSHD, DM2 or Duchenne muscular dystrophy (DMD) studies compared to control biopsies, and on meta-analysis for each condition. In addition, the myogenesis biomarker was a robust binary discriminator of FSHD, DM2 and DMD from controls. We also analysed muscle regeneration at the protein level by immunolabelling muscle biopsies for developmental myosin heavy chain. Such immunolabelling revealed one or more regenerating myofibres in 76% of FSHD muscle biopsies from quadriceps and 91% from tibialis anterior. The mean proportion of regenerating myofibres per quadriceps biopsy was 0.48%, significantly less than 1.72% in the tibialis anterior. All DM2 muscle biopsies contained regenerating myofibres, with a mean of 1.24% per biopsy. Muscle regeneration in FSHD was correlated with the pathological hallmarks of fibre size variation, central nucleation, fibrosis and necrosis/regeneration/inflammation. In summary, the regenerative response in FSHD muscle biopsies correlates with the severity of pathology.

Cis D4Z4 repeat duplications associated with facioscapulohumeral muscular dystrophy type 2.

Facioscapulohumeral muscular dystrophy, known in genetic forms FSHD1 and FSHD2, is associated with D4Z4 repeat array chromatin relaxation and somatic derepression of DUX4 located in D4Z4. A complete copy of DUX4 is present on 4qA chromosomes, but not on the D4Z4-like repeats of chromosomes 4qB or 10. Normally, the D4Z4 repeat varies between 8 and 100 units, while in FSHD1 it is only 1-10 units. In the rare genetic form FSHD2, a combination of a 4qA allele with a D4Z4 repeat size of 8-20 units and heterozygous pathogenic variants in the chromatin modifier SMCHD1 causes DUX4 derepression and disease. In this study, we identified 11/79 (14%) FSHD2 patients with unusually large 4qA alleles of 21-70 D4Z4 units. By a combination of Southern blotting and molecular combing, we show that 8/11 (73%) of these unusually large 4qA alleles represent duplication alleles in which the long D4Z4 repeat arrays are followed by a small FSHD-sized D4Z4 repeat array duplication. We also show that these duplication alleles are associated with DUX4 expression. This duplication allele frequency is significantly higher than in controls (2.9%), FSHD1 patients (1.4%) and in FSHD2 patients with typical 4qA alleles of 8-20 D4Z4 units (1.5%). Segregation analysis shows that, similar to typical 8-20 units FSHD2 alleles, duplication alleles only cause FSHD in combination with a pathogenic variant in SMCHD1. We conclude that cis duplications of D4Z4 repeats explain DUX4 expression and disease presentation in FSHD2 families with unusual long D4Z4 repeats on 4qA chromosomes.

Muscle pathology grade for facioscapulohumeral muscular dystrophy biopsies.

INTRODUCTION: As we move toward planning for clinical trials in facioscapulohumeral muscular dystrophy (FSHD), a better understanding of the clinical relationship with morphological changes in FSHD muscle biopsies will be important for stratifying patients and understanding post-therapeutic changes in muscle. METHODS: We performed a prospective cross-sectional study of quadriceps muscle biopsies in 74 genetically confirmed FSHD participants (64 with FSHD type 1 and 10 with FSHD type 2). We compared a 12-point muscle pathology grade to genetic mutation, disease severity score, and quantitative myometry. RESULTS: Pathology grade had moderate correlations with genetic mutation (rho = -0.45, P < 0.001), clinical severity score (rho = 0.53, P < 0.001), disease duration (rho = 0.31, P = 0.03), and quantitative myometry (rho = -0.47, P < 0.001). We found no difference in the frequency of inflammation between FSHD types 1 and 2. CONCLUSIONS: The pathology grade of quadriceps muscle may be a useful marker of disease activity in FSHD, and it may have a role in stratification for future clinical trials.

Developmental exposure to methylmercury and resultant muscle mercury accumulation and adult motor deficits in mice.

Developmental methylmercury (MeHg) exposure can have lasting consequences on neural development and motor function across the lifespan. Recent evidence for MeHg targeting of myogenic pathways has drawn attention to the possibility that developing skeletal muscle plays a role in the motor deficits stemming from early life MeHg exposure. In this study we examined a potential role for muscle in influencing MeHg developmental toxicity in offspring of female mice exposed to MeHg via drinking water. Dams had access to 0, 0.5 or 5.0 ppm MeHg chloride in drinking water from two weeks prior to mating through weaning. Blood, brain and muscle tissue was harvested from dams at weaning and pups at postnatal days (PND) 6, 21 and 60 for analysis of total Hg. Muscle tissue sections were examined with histological stains. Behavioral testing of offspring was conducted at PND 60 and included locomotor activity, inverted screen, grip strength and rotarod tests to assess motor function. Total Hg (tHg) levels in dam muscles at weaning were 1.7-3-fold higher than Hg levels in blood or brain. In PND6 male and female pups, muscle and brain tHg levels were 2 to 4-fold higher than blood tHg. Brain tHg levels decreased more rapidly than muscle tHg levels between PND 6 and 21. Premised on modeling of growth dilution, brain tissue demonstrated an elimination of tHg while muscle tissue exhibited a net uptake of tHg between PND 6 and 21. Despite overall elevated Hg levels in developing muscle, no gross morphological or cytological phenotypes were observed in muscle at PND 60. At the higher MeHg dose, grip strength was reduced in both females and males at PND 60, whereas only male specific deficits were observed in locomotor activity and inverted screen tests with marginally significant deficits on rotarod. These findings highlight a potential role for developing skeletal muscle in mediating the neuromuscular insult of early life MeHg exposure.

HIV-1 viral replication below 50 copies/ml in patients on antiretroviral therapy is not associated with CD8+ T-cell activation.

OBJECTIVES: To determine if the expression of CD38 on CD8+ T-cells could be used as a marker of viral replication <50 copies/ml in peripheral blood. METHODS: In a cross-sectional study of patients attending a single HIV clinic in London, an ultra-sensitive HIV RNA viral load assay, with a limit of detection of 3 copies/ml, was used to determine HIV-1 replication in plasma in 70 patients who had sustained viral suppression <50 copies/ml by bDNA assays. Immune activation using the expression of CD38 on CD8+ T-cells was also assessed in patients on antiretroviral therapy (ART) with sustained viral suppression, individuals with persistent low-level viraemia <400 copies/ml and subjects failing ART (viral load >400 copies/mi). RESULTS: There was no significant difference in the percentage of CD8+CD38++ T-cells between patients with <50 copies or <3 copies/ml. Immune activation was significantly increased in patients with persistent low-level viraemia and in subjects failing ART. CD4+ T-cell counts in patients on long-term successful ART are inversely associated with CD8+ T-cell activation. CONCLUSIONS: T-cell activation in patients on long-term successful ART is not due to residual low-level viral replication in the blood compartment of HIV-1. CD8+ T-cell activation in this patient group appears to be associated with poor CD4+ T-cell recovery.

The influence of 10 min of the Johrei healing method on laboratory stress.

OBJECTIVE: Johrei has been shown to decrease exam stress responses but its immediate effects have not been assessed. DESIGN: In a randomised, blinded, counter-balanced design, 33 medical students were asked to calculate mental arithmetic in the Paced Auditory Serial Addition Task (PASAT), which served as an acute stressor prior to two conditions, 10 min of Johrei or a control resting condition involving 10 min without Johrei in a cross-over trial; after each, saliva was collected and mood tested. SETTING: University EEG laboratory. INTERVENTION: Johrei, a non-touch healing method. MAIN OUTCOME MEASURES: Profile of mood states (POMS-Bi); state anxiety (STAI); salivary variables: cortisol, DHEA, IgA. RESULTS: Mood scores on 5/6 of the POMS-Bi subscales were slightly but significantly more positive in the Johrei condition. State anxiety was similarly decreased. IgA levels were unchanged but cortisol levels were found to be slightly but non-significantly lower after Johrei than after the control condition and DHEA levels slightly but non-significantly raised, with a negative correlation between cortisol and DHEA levels. CONCLUSIONS: This study gives some indication that Johrei can reduce negative mood and increase positive mood states after the acute effects of a laboratory stressor in comparison to a resting control condition.

Immunohistochemical Characterization of Facioscapulohumeral Muscular Dystrophy Muscle Biopsies.

BACKGROUND: Posited pathological mechanisms in Facioscapulohumeral Muscular Dystrophy (FSHD) include activation in somatic tissue of normally silenced genes, increased susceptibility to oxidative stress, and induction of apoptosis. OBJECTIVE: To determine the histopathological changes in FSHD muscle biopsies and compare to possible pathological mechanisms of disease. METHODS: We performed a cross-sectional study on quadriceps muscle biopsies from 32 genetically confirmed FSHD participants, compared to healthy volunteers and myotonic dystrophy type 1 as disease controls. Biopsies were divided into groups to evaluate apoptosis rates, capillary density, myonuclear and satellite cell counts. RESULTS: Apoptosis rates were increased in FSHD (n=10, 0.74%) compared to myotonic dystrophy type 1 (n=10, 0.14%, P=0.003) and healthy volunteers (n=14, 0.13%, P=0.002). Apoptosis was higher in FSHD patients with the smallest residual D4Z4 fragments. Capillary density was decreased in FSHD1 (n=10, 316 capillaries/mm2) compared to healthy volunteers (n=15, 448 capillaries/mm2, P=0.001). No differences were seen in myonuclear or satellite cell counts. CONCLUSIONS: Preliminary evidence for increased apoptosis rates and reduced capillary density may reflect histopathological correlates of disease activity in FSHD. The molecular-pathological correlates to these changes warrants further investigation.

Serotype-specific pneumococcal antibodies in breast milk of Gambian women immunized with a pneumococcal polysaccharide vaccine during pregnancy.

BACKGROUND: In breast-feeding populations, immunization during pregnancy with pneumococcal polysaccharide offers a potentially useful approach to preventing pneumococcal disease in young infants. METHODS: Breast milk samples were collected at 0, 2, 4 and 6 months after delivery from Gambian women vaccinated during pregnancy (24-32 weeks gestation) with Pneumovax II (n = 56) or Mengivax A&C (n = 57). Specimens were examined for secretory immunoglobulin A (s-IgA) concentration, subclass distribution and avidity specific to pneumococcal serotypes 4, 6B, 14, 19F and 23F and the antigen mixture in Pneumovax II by enzyme-linked immunosorbent assay. Colostral s-IgA and IgG concentrations in paired maternal sera were compared. RESULTS: Colostral s-IgA concentrations specific to all pneumococcal polysaccharide antigens investigated were significantly higher (P < 0.05) among Pneumovax II vaccinees. Titers specific to serotypes 4, 6B and 14 and the vaccine formula remained significantly higher during 6 months, and those for 19F were higher during 4 months. Significantly higher concentrations of vaccine antigen-specific s-IgA antibody were sustained for 6 months after delivery (P = 0.011). Comparison of colostral s-IgA and IgG in serum revealed a significant correlation only among Mengivax A&C vaccinees for pneumococcal polysaccharide 23F (rs= 0.68; P < or = 0.0001). Vaccination elicited trends toward increased s-IgA2, reaching significance for serotype 14 and the vaccine formula. Immunization elicited significantly higher s-IgA avidities specific to all pneumococcal polysaccharide antigens studied during 6 months. CONCLUSIONS: The public health value of immunization during pregnancy with pneumococcal polysaccharide vaccine in breast-feeding populations warrants further evaluation, particularly in populations with a high incidence of pneumococcal disease in early infancy.

The impact of self-hypnosis and Johrei on lymphocyte subpopulations at exam time: a controlled study.

In a prospective randomised controlled trial, 48 students were randomly assigned to stress reduction training before exams with self-hypnosis, Johrei or a mock neurofeedback relaxation control. Peripheral blood lymphocyte subpopulations and self-reported stress (Perceived Stress Scale) were measured before training and 1-2 months later as exams approached. Absolute number and percentages of CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocytes, CD3(-)CD56(+) Natural Killer cells (NK cells) and NK cell cytotoxic activity was measured from venous blood. Stressed participants showed small but significant declines in both CD3(-)CD56(+) NK cell percentages and NK cell cytotoxic activity levels while CD3(+)CD4(+) T cell percentages increased, changes supported by correlations with perceived stress. The effects of stress were moderated in those who learned Johrei at exam time; 11/12 showed increases in CD3(-)CD56(+) NK cell percentages with decreased percentages of CD3(+)CD4(+) T cells, effects not seen in the relaxation control group. Stress was also buffered in those who learned and practised self-hypnosis in whom CD3(-)CD56(+) NK cell and CD3(+)CD4(+) T cell levels were maintained, and whose CD3(+)CD8(+) T cell percentages, shown previously to decline with exams, increased. The results compliment beneficial effects on mood of self-hypnosis and Johrei. The results are in keeping with beneficial influences of self-hypnosis and provide the first evidence of the suggestive value of the Japanese Johrei procedure for stress reduction, which clearly warrants further investigation.

Tryptophan and quality of life in colorectal cancer.

We tested the hypothesis that reduced tryptophan availability due to immunological stimulation in colorectal cancer impairs quality of life (QoL) by measuring serum tryptophan levels, and correlating them with serum immunological markers and with QoL indices. Serum tryptophan level was significantly reduced in cancer patients compared with healthy controls. Serum tryptophan/kynurenine ratio and neopterin level were significantly increased in cancer, with a significant correlation between the two variables. Reduced serum tryptophan correlated significantly with worse QoL scores. The results support the hypothesis that in colorectal cancer, QoL impairment may be due to serum tryptophan depletion mediated by immunological activation.

A biomarker panel (Bioscore) incorporating monocytic surface and soluble TREM-1 has high discriminative value for ventilator-associated pneumonia: a prospective observational study.

INTRODUCTION: Ventilator-associated pneumonia (VAP) increases mortality in critical illness. However, clinical diagnostic uncertainty persists. We hypothesised that measuring cell-surface and soluble inflammatory markers, incorporating Triggering Receptor Expressed by Myeloid cells (TREM)-1, would improve diagnostic accuracy. METHODS: A single centre prospective observational study, set in a University Hospital medical-surgical intensive Care unit, recruited 91 patients into 3 groups: 27 patients with VAP, 33 ventilated controls without evidence of pulmonary sepsis (non-VAP), and 31 non-ventilated controls (NVC), without clinical infection, attending for bronchoscopy. Paired samples of Bronchiolo-alveolar lavage fluid (BALF) and blood from each subject were analysed for putative biomarkers of infection: Cellular (TREM-1, CD11b and CD62L) and soluble (IL-1ß, IL-6, IL-8, sTREM-1, Procalcitonin). Expression of cellular markers on monocytes and neutrophils were measured by flow cytometry. Soluble inflammatory markers were determined by ELISA. A biomarker panel ('Bioscore'), was constructed, tested and validated, using Fisher's discriminant function analysis, to assess its value in distinguishing VAP from non VAP. RESULTS: The expression of TREM-1 on monocytes (mTREM-1) and neutrophils (nTREM-1) and concentrations of IL-1ß, IL-8, and sTREM-1 in BALF were significantly higher in VAP compared with non-VAP and NVC (p<0.001). The BALF/blood mTREM-1 was significantly higher in VAP patients compared to non-VAP and NVC (0.8 v 0.4 v 0.3 p<0.001). A seven marker Bioscore (BALF/blood ratio mTREM-1 and mCD11b, BALF sTREM-1, IL-8 and IL-1ß, and serum CRP and IL-6) correctly identified 88.9% of VAP cases and 100% of non-VAP cases. CONCLUSION: A 7-marker bioscore, incorporating cellular and soluble TREM-1, accurately discriminates VAP from non-pulmonary infection.

Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease.

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.

Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by genomic expansions of CTG or CCTG repeats. When transcribed, these mutations give rise to repeat expansion RNAs that form nuclear inclusions and compromise the function of myonuclei. Here, we have used in situ hybridization and immunofluorescence to compare DM1 and DM2 and search for proteins that associate with the RNA nuclear (ribonuclear) inclusions. Although muscle disease is generally more severe in DM1, the ribonuclear inclusions were 8- to 13-fold more intense in DM2, implying greater amounts of repeat expansion RNA. Expression of repeat expansion RNA in myoblasts has been implicated in the pathogenesis of congenital DM1. However, we found that repeat expansion RNA is also expressed in myoblasts in DM2, a disorder that has not been associated with a congenital phenotype. Of 10 putative CUG binding proteins tested for colocalization with mutant RNA, only proteins in the muscleblind family were recruited into ribonuclear inclusions. Previous studies have shown activation of the protein kinase, PKR, by expanded CUG repeats in vitro. However, breeding experiments utilizing PKR knockout mice indicate that this kinase is not required for disease pathogenesis in a transgenic mouse model of DM1. We conclude that ribonuclear inclusions are a key feature of the muscle pathology in DM and that sequestration of muscleblind proteins may have a direct role in the disease process.

Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy.

We investigated the associations of baseline epidermal nerve fiber (ENF) densities and morphology (percent ENF swellings) and quantitative sensory testing (QST) with clinically defined human immunodeficiency virus (HIV)-associated distal polyneuropathy (DSP) and whether these measures are predictive of development of symptomatic DSP over time. Fifty-seven HIV-infected subjects with and without DSP and 19 controls participated. Mean ENF densities were lower at the distal leg and proximal thigh in asymptomatic or symptomatic DSP than in controls. Mean ENF densities did not differ significantly among the HIV groups. Percent ENF swellings was higher in patients with symptomatic DSP than controls at the distal leg, and was also greater at the proximal thigh in patients with asymptomatic or symptomatic DSP than in controls. The percent ENF swellings at the distal leg correlated with the thresholds for both minimal (HP 0.5) and intermediate (HP 5.0) heat pain (HP) intensity. A higher percent ENF swellings in the distal leg [hazard ratio (HR) 1.16, 95% CI 1.02-1.31] and HP 0.5 thresholds (HR 1.03, 95% CI 1.01-1.05) were the only two measures associated with a shorter time to development of symptomatic DSP. Quantitation of ENF swellings and heat pain thresholds deserve further study as predictors of symptomatic neuropathy.

The presence of interleukin-2 receptor alpha in the serum of colorectal cancer patients is unlikely to result only from T cell up-regulation.

It is unclear whether the presence of interleukin-2 soluble receptor alpha (IL-2 sRalpha) in the serum of colorectal cancer patients is solely due to T cell activation. In this study, we therefore investigated whether T cell activation, indicated by the up-regulation of the CD25 and HLA-DR markers, or cell-mediated immunity (CMI) were associated with increased serum levels of IL-2 sRalpha in patients with advanced colorectal carcinoma. The levels of serum IL-2 sRalpha and the proportion of T cells expressing HLA-DR (DR(+) T cells) were measured as markers for chronic activation. CMI was assessed by delayed-type hypersensitivity reaction (DTH) to intradermal injections of recall antigens. Eighty-seven colorectal liver metastases (CLM) patients and 23 'cancer-free' control subjects were studied. DR(+) T cells were found to be more prevalent ( P<0.0001) in CLM patients (median: 21.1%) than in controls (median: 3.4%), but DR(+) T cell up-regulation was not correlated with serum IL-2 sRalpha levels. CMI positivity was significantly reduced ( P=0.002) in CLM patients compared with controls, and this reduction was significantly associated ( P=0.05) with an increase in the number of DR(+) T cells. Although survival was significantly shorter ( P=0.0003) in patients with increased serum IL-2 sRalpha levels than in subjects with normal levels, no association was found between survival and DR(+) T cell up-regulation. These findings were consistent with the hypothesis of an additional source of serum IL-2 sRalpha other than T cell up-regulation in CLM patients -- either from other immune cells, or from tumour products.

Colostrum obtained from women vaccinated with pneumococcal vaccine during pregnancy inhibits epithelial adhesion of Streptococcus pneumoniae.

Prevention of nasopharyngeal colonization may reduce the burden of pneumococcal infection during infancy. Colostrum obtained from Gambian mothers who had been vaccinated with either Pneumovax II or Mengivax A&C (n=8 per group) during pregnancy was examined for inhibition of adherence of Streptococcus pneumoniae serotypes 6B and 14 to pharyngeal epithelial cells in vitro. Pneumococcal adherence was significantly reduced in the presence of breast milk (P< or =.0001 for S. pneumoniae serotype 14; P=.036 for serotype 6B), independent of the concentration of secretory IgA antibodies. Maternal vaccination with polyvalent pneumococcal polysaccharide vaccine boosts the capacity of colostrum to inhibit adherence of pneumococci to pharyngeal epithelial cells. In breast-feeding populations, maternal vaccination might prevent pneumococcal disease in young infants.