Baziel G.M. van Engelen

Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2

Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction–independent FSHD2 are unclear. Here, we show that mutations in SMCHD1 (encoding structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4 contraction–independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic diet.

Skeletal Muscle Ultrasound: Correlation Between Fibrous Tissue and Echo Intensity

In this study, we examined the correlation between muscle ultrasound and muscle structure. Echo intensity (EI) of 14 muscles of two golden retriever muscular dystrophy dogs was correlated to the percentage interstitial fibrous tissue and fat in muscle biopsy. A significant correlation between interstitial fibrous tissue and EI was found (r = 0.87; p < 0.001). The separate influence of interstitial fat on muscle EI could not be established as only little fat was present. We conclude that fibrous tissue causes increased muscle EI. The high correlation between interstitial fibrous tissue and EI makes ultrasound a reliable method to determine severity of structural muscle changes.

Autoantibodies to cytosolic 5′-nucleotidase 1A in inclusion body myositis

Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy characterized by both degenerative and autoimmune features. In contrast to other inflammatory myopathies, myositis‐specific autoantibodies had not been found in sIBM patients until recently. We used human skeletal muscle extracts as a source of antigens to detect autoantibodies in sIBM and to characterize the corresponding antigen.

Common epigenetic changes of D4Z4 in contraction‐dependent and contraction‐independent FSHD

Facioscapulohumeral muscular dystrophy (FSHD), caused by partial deletion of the D4Z4 macrosatellite repeat on chromosome 4q, has a complex genetic and epigenetic etiology. To develop FSHD, D4Z4 contraction needs to occur on a specific genetic background. Only contractions associated with the 4qA161 haplotype cause FSHD. In addition, contraction of the D4Z4 repeat in FSHD patients is associated with significant D4Z4 hypomethylation. To date, however, the methylation status of contracted repeats on nonpathogenic haplotypes has not been studied. We have performed a detailed methylation study of the D4Z4 repeat on chromosome 4q and on a highly homologous repeat on chromosome 10q. We show that patients with a D4Z4 deletion (FSHD1) have D4Z4‐restricted hypomethylation. Importantly, controls with a D4Z4 contraction on a nonpathogenic chromosome 4q haplotype or on chromosome 10q also demonstrate hypomethylation. In 15 FSHD families without D4Z4 contractions but with at least one 4qA161 haplotype (FSHD2), we observed D4Z4‐restricted hypomethylation on chromosomes 4q and 10q. This finding implies that a genetic defect resulting in D4Z4 hypomethylation underlies FSHD2. In conclusion, we describe two ways to develop FSHD: (1) contraction‐dependent or (2) contraction‐independent D4Z4 hypomethylation on the 4qA161 subtelomere. Hum Mutat 30:1–11, 2009.

A weak balance: the contribution of muscle weakness to postural instability and falls

Muscle strength is a potentially important factor contributing to postural control. In this article, we consider the influence of muscle weakness on postural instability and falling. We searched the literature for research evaluating muscle weakness as a risk factor for falls in community-dwelling elderly individuals, for evidence that strength training reduces falls, and for pathophysiological evidence from patients with neuromuscular disease that supports the link between muscle weakness and falls. In virtually all studies that included strength testing, muscle weakness was a consistent risk factor for falls in the elderly. Studies that evaluated the merits of muscle strength training often showed a reduction in fall rates, particularly when strength training was a component of a multifactorial intervention, although it was unclear whether strength training alone led to a fall reduction. Surprisingly few studies addressed the pathophysiological relationship between muscle strength and balance control. We conclude that muscle weakness is an important risk factor for falls that is potentially amenable to therapeutic intervention, and that future studies should further clarify the role of muscle weakness in balance control and the pathophysiology of falls.

Pain in Ehlers-Danlos Syndrome Is Common, Severe, and Associated with Functional Impairment

CONTEXT The Ehlers-Danlos Syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Musculoskeletal pain is mentioned in the diagnostic criteria and described as early in onset, chronic, and debilitating. However, systematic research on pain in EDS is scarce. OBJECTIVES We investigated prevalence and impact of pain and associated features in a large group of EDS patients. METHODS We performed a study among members of the Dutch EDS patient organization (n=273) and included the McGill Pain Questionnaire to investigate various aspects of pain, the Sickness Impact Profile to study functional impairment, the Symptom Checklist subscale sleep to evaluate sleep disturbances, and the Checklist Individual Strength subscale fatigue to determine fatigue severity. RESULTS The results of this study show that 1) chronic pain in EDS is highly prevalent and associated with regular use of analgesics; 2) pain is more prevalent and more severe in the hypermobility type than in the classic type; 3) pain severity is correlated with hypermobility, dislocations, and previous surgery; 4) pain is correlated with low nocturnal sleep quality; and 5) pain contributes to functional impairment in daily life, independent of the level of fatigue. CONCLUSION From this large cohort of EDS patients, we conclude that pain is common and severe in EDS. Pain is related to hypermobility, dislocations, and previous surgery and associated with moderate to severe impairment in daily functioning. Therefore, treatment of pain should be a prominent aspect of symptomatic management of EDS.

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 α (LAP2α) upon entry and exit from G0 is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2α are down-regulated in G0. Although RbS780 phosphoform and LAP2α are up-regulated upon reentry into G1 and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2α is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G1 phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2α or lamin A/C in HDFs leads to accumulation of Rb in speckles and G1 arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2α and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.

Dominant Mutations in KBTBD13, a Member of the BTB/Kelch Family, Cause Nemaline Myopathy with Cores

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecules beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

In vivo quantitative near‐infrared spectroscopy in skeletal muscle during incremental isometric handgrip exercise

The aim of this study was to investigate the performance of in vivo quantitative near‐infrared spectroscopy (NIRS) in skeletal muscle at various workloads. NIRS was used for the quantitative measurement of O2 consumption (mV˙O2) in the human flexor digitorum superficialis muscle at rest and during rhythmic isometric handgrip exercise in a broad range of work intensities (10–90% MVC=maximum voluntary contraction force). Six subjects were tested on three separate days. No significant differences were found in mV˙O2 measured over different days with the exception of the highest workload. The within‐subject variability for each workload measured over the three measurements days ranged from 15·7 to 25·6% and did not increase at the high workloads. The mV˙O2 was 0·14 ± 0·01 mlO2 min–1 100 g–1 at rest and increased roughly 19 times to 2·68 ± 0·58 mlO2 min–1 100 g–1 at 72% MVC. These results show that local muscle oxygen consumption at rest as well as during exercise at a broad range of work intensities can be measured reliably by NIRS, applied to a uniform selected subject population. This is of great importance as direct local measurement of mV˙O2 during exercise is not possible with the conventional techniques. The method is robust enough to measure over separate days and at various workloads and can therefore contribute to a better understanding of human physiology in both the normal and pathological state of the muscle.