Localization of the gene for Cowden disease to chromosome 10q22-23.

Cowden disease (CD) (MIM 158350), or multiple hamartoma syndrome, is a rare autosomal dominant familial cancer syndrome with a high risk of breast cancer. Its clinical features include a wide array of abnormalities but the main characteristics are hamartomas of the skin, breast, thyroid, oral mucosa and intestinal epithelium. The pathognomonic hamartomatous features of CD include multiple smooth facial papules, acral keratosis and multiple oral papillomas. The pathological hallmark of the facial papules are multiple trichilemmomas. Expression of the disease is variable and penetrance of the dermatological lesions is assumed to be virtually complete by the age of twenty. Central nervous system manifestations of CD were emphasized only recently and include megalencephaly, epilepsy and dysplastic gangliocytomas of the cerebellum (Lhermitte-Duclos disease, LDD). Early diagnosis is important since female patients with CD are at risk of developing breast cancer. Other lesions include benign and malignant disease of the thyroid, intestinal polyps and genitourinary abnormalities. To localize the gene for CD, an autosomal genome scan was performed. A total of 12 families were examined, resulting in a maximum lod score of 8.92 at theta = 0.02 with the marker D10S573 located on chromosome 10q22-23.

Facioscapulohumeral Disease as a myodevelopmental disease: Applying Ockham's razor to its various features.

 Facioscapulohumeral muscular dystrophy (FSHD) is an exclusively human neuromuscular disease. In the last decades the cause of FSHD was identified: the loss of epigenetic repression of the D4Z4 repeat on chromosome 4q35 resulting in inappropriate transcription of DUX4. This is a consequence of a reduction of the array below 11 units (FSHD1) or of a mutation in methylating enzymes (FSHD2). Both require the presence of a 4qA allele and a specific centromeric SSLP haplotype. Muscles become involved in a rostro-caudally order with an extremely variable progression rate. Mild disease and non-penetrance in families with affected individuals is common. Furthermore, 2% of the Caucasian population carries the pathological haplotype without clinical features of FSHD.In order to explain the various features of FSHD we applied Ockham's Razor to all possible scenarios and removed unnecessary complexities. We postulate that early in embryogenesis a few cells escape epigenetic silencing of the D4Z4 repeat. Their number is assumed to be roughly inversely related to the residual D4Z4 repeat size. By asymmetric cell division, they produce a rostro-caudal and medio-lateral decreasing gradient of weakly D4Z4-repressed mesenchymal stem cells. The gradient tapers towards an end as each cell-division allows renewed epigenetic silencing. Over time, this spatial gradient translates into a temporal gradient based on a decreasing number of weakly silenced stem cells. These cells contribute to a mildly abnormal myofibrillar structure of the fetal muscles. They also form a downward tapering gradient of epigenetically weakly repressed satellite cells. When activated by mechanical trauma, these satellite cells de-differentiate and express DUX4. When fused to myofibrils they contribute to muscle cell death in various ways. Over time and dependent on how far the gradient reaches the FSHD phenotype becomes progressively manifest. We thus hypothesize FSHD to be a myodevelopmental disease with a lifelong attempt to restore DUX4 repression.

Characterizing the face in facioscapulohumeral muscular dystrophy.

OBJECTIVE: To evaluate facial weakness in patients with FSHD to better define clinical signs, and pilot a facial weakness severity score. METHODS: 87 FSHD patients and 55 controls were video recorded while performing seven facial tasks. The videos were assessed by three independent examiners to compile an overview of signs of facial weakness. Next, videos were semi-quantitatively assessed using a newly developed 4-point facial weakness score (FWS). This score was evaluated and correlated to other FSHD disease characteristics. RESULTS: Patients had lower scores on the total FWS than controls (mean score 43 ± 28, range 4-118, vs 14 ± 9, range 0-35, p < 0.001) and on all seven individual facial tasks (all p < 0.001). 54% of patients had FWS scores outside the range of controls. Patients had more asymmetry between the left and right side of the face than controls. About 10% of the patients had very mild facial weakness. These were mostly males (89%) with longer D4Z4 repeat sizes of 7-9 units. More severe facial weakness correlated to more severe overall disease severity and shorter D4Z4 repeat size, but not to disease duration. Interobserver agreement for the FWS between three raters was low with a Fleiss Kappa of 0.437. CONCLUSION: This study provides an overview of the clinical spectrum of facial weakness and its relation to other disease characteristics. The 4-point scale we introduced to grade the severity of facial weakness enables correlation of facial weakness to disease characteristics, but is not suited as clinical outcome measure for longitudinal studies.

Only fat infiltrated muscles in resting lower leg of FSHD patients show disturbed energy metabolism.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by asymmetric dysfunctioning of individual muscles. Currently, it is unknown why specific muscles are affected before others and more particularly what pathophysiology is causing this differential progression. The aim of our study was to use a combination of (31)P magnetic resonance spectroscopic imaging (MRSI) and T1-weighted MRI to uncover metabolic differences in fat infiltrated and not fat infiltrated muscles in patients with FSHD. T1-weighted images and 3D (31)P MRSI were obtained from the calf muscles of nine patients with diagnosed FSHD and nine healthy age and sex matched volunteers. Muscles of patients were classified as fat infiltrated (PFM) and non fat-infiltrated (PNM) based on visual assessment of the MR images. Ratios of phosphocreatine (PCr), phosphodiesters (PDE) and inorganic phosphate (Pi) over ATP and tissue pH were compared between PFM and PNM and the same muscles in healthy volunteers. Of all patients, seven showed moderate to severe fatty infiltration in one or more muscles. In these muscles, decreases in PCr/ATP and increases in tissue pH were observed compared to the same muscles in healthy volunteers. Interestingly, these differences were absent in the PNM group. Our data show that differences in metabolite ratios and tissue pH in skeletal muscle between healthy volunteers and patients with FSHD appear to be specific for fat infiltrated muscles. Normal appearing muscles on T1 weighted images of patients showed normal phosphoryl metabolism, which suggests that in FSHD disease progression is truly muscle specific.

Clinical features, lectin staining, and a novel GNE frameshift mutation in hereditary inclusion body myopathy.

We present a comprehensive report of two siblings with hereditary inclusion body myopathy (HIBM). The clinical features and histological characteristics of the muscle biopsies showed the typical pattern of predominantly distal vacuolar myopathy with quadriceps sparing. This was confirmed by muscle MRI. PNA lectin staining showed an increased signal at the sarcolemma in patient muscle sections compared to control muscle, indicating reduced sialylation of glycoconjugates. Mutation analysis revealed compound heterozygous mutations in the GNE gene, encoding the key enzyme in sialic acid synthesis UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase: a missense mutation (c.2086G > A; p.V696M) previously described in HIBM patients of Indian origin, and a novel frame shift mutation (c.1295delA; p.K432RfsX17) leading to a premature stopcodon. These findings confirmed the diagnosis of HIBM on the histological, molecular and biochemical level.

[Facioscapulohumeral muscular dystrophy]. / Facioscapulohumerale spierdystrofie.

Facioscapulohumeral muscular dystrophy is clinically mainly characterized by progressive weakness of the facial, shoulder and upper arm muscles. It is an autosomal dominant heriditary disease, caused by a contraction of a repetitive DNA element at the end of the long arm of chromosome 4. This contraction causes the local relaxation of the chromatin structure and likely dysregulation of one or more genes. Oral health care providers can play a significant role in the early recognition, as the often asymmetric facial weakness is frequently the first symptom. Adequate oral health care is needed because of the facial weakness.

Epidemiology and pathophysiology of falls in facioscapulohumeral disease.

BACKGROUND AND AIMS: Muscle weakness is a potentially important, yet poorly studied, risk factor for falls. Detailed studies of patients with specific myopathies may shed new light on the relation between muscle weakness and falls. Here falls in patients with facioscapulohumeral disease (FSHD) who suffered from lower limb muscle weakness were examined. This study provides insights into the prevalence, relevance and pathophysiology of falls in FSHD. METHODS: A validated questionnaire was used as well as a prospective 3 month follow-up to examine the prevalence, circumstances and consequences of falls in 73 patients with FSHD and 49 matched healthy controls. In a subgroup of 28 subjects, muscle strength was also examined and balance was assessed electrophysiologically using body worn gyroscopes. RESULTS: In the questionnaire, 30% of the patients reported falling at least once a month whereas none of the controls did. Injuries occurred in almost 70% of the patients. The prospective study showed that patients fell mostly at home, mainly due to intrinsic (patient related) causes, and usually in a forward direction. Fallers were unstable while climbing stairs, rising from a chair and standing with eyes closed whereas non-fallers had normal balance control. Frequent fallers had greater muscle weakness than infrequent fallers. CONCLUSION: These findings demonstrate the high prevalence and clinical relevance of falls in FSHD. The relation between muscle weakness and instability among fallers is also highlighted. Because patients fell mainly at home, fall prevention strategies should focus on home adaptations. As mainly intrinsic causes underlie falls, the impact of adopting balance strategies or balance training should be explored in this patient group.

Scapular dyskinesis in myotonic dystrophy type 1: clinical characteristics and genetic investigations.

OBJECTIVE: To study scapular winging or other forms of scapular dyskinesis (condition of alteration of the normal position and motion of the scapula) in myotonic dystrophy type 1 (DM1), which is generally considered to be a distal myopathy, we performed an observational cohort study. METHODS: We performed a prospective cohort study on the clinical features and progression over time of 33 patients with DM1 and pronounced, mostly asymmetric scapular winging or other forms of scapular dyskinesis. We also explored if scapular dyskinesis in DM1 has the same genetic background as in facioscapulohumeral muscular dystrophy type 1 (FSHD1). RESULTS: The cohort included patients with congenital (n = 3), infantile (n = 6) and adult-onset DM1 (n = 24). Scapular girdle examination showed moderate shoulder girdle weakness (mean MRC 3) and atrophy of trapezius, infraspinatus, and rhomboid major, seemingly similar as in FSHD1. Shoulder abduction and forward flexion were limited (50-70°). In five patients, scapular dyskinesis was the initial disease symptom; in the others it appeared 1-24 years after disease onset. Follow-up data were available in 29 patients (mean 8 years) and showed mild to severe increase of scapular dyskinesis over time. In only three patients, DM1 coexisted with a FSHD mutation. In all other patients, FSHD was genetically excluded. DM2 was genetically excluded in nine patients. The clinical features of the patients with both DM1 and FSHD1 mutations were similar to those with DM1 only. CONCLUSION: Scapular dyskinesis can be considered to be part of DM1 in a small proportion of patients. In spite of the clinical overlap, FSHD can explain scapular dyskinesis only in a small minority. This study is expected to improve the recognition of shoulder girdle involvement in DM1, which will contribute to the management of these patients.

Effects of training and albuterol on pain and fatigue in facioscapulohumeral muscular dystrophy.

BACKGROUND: We recently reported a randomised controlled trial on the efficacy of strength training and the beta2-adrenergic agonist albuterol in patients with facioscapulohumeral muscular dystrophy (FSHD). Strength training and albuterol appeared safe interventions with limited positive effect on muscle strength and volume. We concurrently explored the prevalence and the characteristics of pain and fatigue in the participating FSHD patients, because these are probably underreported but clinically relevant symptoms in this disorder. Next, we studied the effects of albuterol and strength training on pain, experienced fatigue, health-related functional status and psychological distress. METHODS: Sixty-five patients were randomised to strength training of elbow flexors and ankle dorsiflexors or non-training. After 26 weeks, albuterol (sustained-release, 8 mg bid) was added in a randomised, double-blind, placebo-controlled design. Outcomes comprised self-reported pain, experienced fatigue, functional status and psychological distress obtained with validated questionnaires at 52 weeks. RESULTS: Eighty percent of patients reported chronic persistent or periodic, multifocal pains. Thirty-four percent of the participants were severely fatigued. Strength training and albuterol failed to have a significant effect on all outcomes. CONCLUSIONS: Pain and fatigue are important features in FSHD. Strength training and albuterol do not have a positive or negative effect on pain, experienced fatigue, functional status and psychological distress.

Respiratory function in facioscapulohumeral muscular dystrophy 1.

To test the hypothesis that wheelchair dependency and (kypho-)scoliosis are risk factors for developing respiratory insufficiency in facioscapulohumeral muscular dystrophy, we examined 81 patients with facioscapulohumeral muscular dystrophy 1 of varying degrees of severity ranging from ambulatory patients to wheelchair-bound patients. We examined the patients neurologically and by conducting pulmonary function tests: Forced Vital Capacity, Forced Expiratory Volume in 1 second, and static maximal inspiratory and expiratory mouth pressures. We did not find pulmonary function test abnormalities in ambulant facioscapulohumeral muscular dystrophy patients. Even though none of the patients complained of respiratory dysfunction, mild to severe respiratory insufficiency was found in more than one third of the wheelchair-dependent patients. Maximal inspiratory pressures and maximal expiratory pressures were decreased in most patients, with a trend that maximal expiratory pressures were more affected than maximal inspiratory pressures. Wheelchair-dependent patients with (kypho-)scoliosis showed the most restricted lung function. Wheelchair-dependent patients with (kypho-)scoliosis are at risk for developing respiratory function impairment. We advise examining this group of facioscapulohumeral muscular dystrophy patients periodically, even in the absence of symptoms of respiratory insufficiency, given its frequency and impact on daily life and the therapeutic consequences.

No effect of folic acid and methionine supplementation on D4Z4 methylation in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is associated with a contraction of the D4Z4 allele on chromosome 4qter. There is also marked DNA hypomethylation of the D4Z4 allele. The DNA hypomethylation may have a central role in the pathogenesis of FSHD. Supplemental folic acid can boost DNA methylation. We evaluated the effect of oral folic acid and methionine supplementation on the methylation level of 4qter D4Z4 alleles in peripheral-blood lymphocytes of nine patients affected with FSHD and six healthy controls. Methylation levels did not change, while recommended serum-folate concentrations were reached.

Differential loss of heterozygosity in the region of the Cowden locus within 10q22-23 in follicular thyroid adenomas and carcinomas.

The susceptibility gene for Cowden disease (CD), an autosomal dominant inherited cancer syndrome, has recently been mapped to an approximately 6-cM interval on chromosome subband 10q22-23 between the markers D10S541 and D10S564. CD is characterized by hamartomas of many organ systems, including the thyroid, breast, skin, and gastrointestinal tract, as well as carcinoma of the thyroid and breast. Follicular thyroid adenomas and carcinomas are significant component tumors in CD; thus, we sought to examine their sporadic counterpart tumors for loss of heterozygosity (LOH) of microsatellite markers in the 20-cM region within and flanking the Cowden critical interval. In all, 38 sporadic thyroid tumors were analyzed. LOH within the CD interval was observed in 5 of 19 (26%) follicular thyroid adenomas and 1 of 9 (11%) Hürthle cell adenomas. Furthermore, of these adenomas with LOH, 3 of 4 (75%) were atypical follicular adenomas, whereas 2 of 15 (13%) were typical follicular adenomas. Surprisingly, no LOH was detected in this region in 10 follicular carcinomas. The shortest region of overlap includes the markers D10S1735 and D10S1739. If the LOH observed in these sporadic tumors is related to the CD gene, then the Cowden critical interval can be revised to lie within the interval defined by D10S579 and D10S564. LOH in this narrow interval implicates the CD gene, or another gene in that interval, in follicular thyroid tumorigenesis. However, this does not explain the lack of LOH in follicular carcinomas. Taken together, it may instead be evidence against a stepwise progression from atypical adenomas to carcinomas. Alternatively, sporadic thyroid adenoma formation may be independent of that locus, but loss of this region could prevent carcinoma formation, thus implying that the CD gene may be an oncogene or growth promoter.

Cognitive evaluation in adult patients with Möbius syndrome.

OBJECTIVE: To establish the occurrence of mental retardation in a group of patients with Möbius syndrome and subsequently, if mental retardation is absent, to screen major aspects of memory and attention, in order to assess possible pervasive dysfunction in these cognitive domains which might be responsible for the current view that mental retardation occurs frequently in Möbius syndrome. METHODS: In a group of 12 Dutch Möbius patients, intellectual performance, memory function and attention were assessed using a number of standardized neuropsychological tests. RESULTS: The mean general intellectual performance did not differ significantly from that of the Dutch population. Screening of selective attention and memory did not provide indications of pervasive dysfunctions in these domains. CONCLUSION: The rate of occurrence of mental retardation in our group of Möbius patients did not differ from that in the normal Dutch population. Furthermore, there was no evidence of attention and memory dysfunction in our group of Möbius patients.

High specificity of myositis specific autoantibodies for myositis compared with other neuromuscular disorders.

Myositis specific autoantibodies (MSAs) are proven to be specific for myositis compared with other inflammatory connective tissue diseases. Their specificity compared, however, with other neuromuscular disorders, which are included in the differential diagnosis of patients in whom the diagnosis myositis is under consideration, is unknown. We prospectively screened sera from 107 patients with various neuromuscular disorders for the most common MSAs and compared the results with the findings in a group of 97 myositis patients, published previously. Special attention was paid to patients with facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant muscle disease with marked inflammation in skeletal muscle tissue. Only one patient in the neuromuscular disorders group tested positive for an MSA, compared with 41 in the myositis group, resulting in a specificity of 99%. None of the FSHD patients tested positive. We conclude that the tested MSAs are highly specific for myositis and that they are not merely associated with muscle inflammation.

Genomewide scan identifies susceptibility locus for dyslexia on Xq27 in an extended Dutch family.

CONTEXT: Dyslexia is a common disorder with a strong genetic component, but despite significant research effort, the aetiology is still largely unknown. OBJECTIVE: To identify loci contributing to dyslexia risk. METHODS: This was a genomewide linkage analysis in a single large family. Dutch families with at least two first degree relatives suffering from dyslexia participated in the study. Participants were recruited through an advertisement campaign in papers and magazines. The main outcome measure was linkage between genetic markers and dyslexia phenotype. RESULTS: Using parametric linkage analysis, we found strong evidence for a locus influencing dyslexia on Xq27.3 (multipoint lod = 3.68). Recombinations in two family members flanked an 8 cM region, comprising 11 currently confirmed genes. All four males carrying the risk haplotype had very low scores on the reading tests. The presentation in females was more variable, but 8/9 females carrying the risk haplotype were diagnosed dyslexic by our composite score, so we considered the putative risk allele to be dominant with reduced penetrance. Linkage was not found in an additional collection of affected sibling pairs. CONCLUSIONS: A locus influencing dyslexia risk is probably located between markers DXS1227 and DXS8091 on the X chromosome, closely situated to a locus indicated by a published genome scan of English sibling pairs. Although the locus may not be a common cause for dyslexia, the relatively small and gene poor region offers hope to identify the responsible gene.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

Localisation of the gene for a dominant congenital spinal muscular atrophy predominantly affecting the lower limbs to chromosome 12q23-q24.

Spinal muscular atrophies are a heterogeneous group of disorders. They differ in time of onset, clinical presentation, progression, severity and mode of inheritance. In 1985 a Dutch family was described with a dominant, non-progressive spinal muscular atrophy presenting at birth with arthrogryposis (MIM 600175). Linkage analysis was performed in this family. After having excluded the loci for Werdnig-Hoffmann's disease and for dominant distal spinal muscular atrophy with upper limb predominance, we were able to localise the gene to a 10 cM interval between the markers D12S78 and D12S1646 on chromosome 12q23-q24. Recently, dominant scapuloperoneal spinal muscular atrophy has been localised to an overlapping interval. However, the clinical appearances of scapuloperoneal spinal muscular atrophy and the present disorder make allelism unlikely. In 1994, a second Dutch family with a disorder similar to the present one was described. We excluded linkage to markers of the 12q23-q24 region in this family and thereby proved genetic heterogeneity of this type of dominant, congenital and nonprogressive spinal muscular atrophy.

Novel PTEN mutations in patients with Cowden disease: absence of clear genotype-phenotype correlations.

Cowden disease (CD) is characterised by multiple hamartomas in a variety of tissues. The pathological hallmark is the presence of a number of trichilemmomas. Several neurological symptoms are also part of CD with megalencephaly and Lhermitte-Duclos disease (LDD) as the most important features. Early recognition of CD patients is important because of the increased risk of developing malignancies. Breast cancer is the most frequent malignancy, but also urogenital, digestive tract, and thyroid cancers are found with higher frequencies. CD was localised to chromosome 10q23 and the PTEN gene (also known as MMAC1 or TEP1) was shown to be involved. Germline mutations were identified in both familial and sporadic CD patients. We identified eight PTEN mutations, of which seven were novel, in 13 CD patients. Combined with previous data we have identified 17 independent CD mutations. Gross DNA alterations in CD patients were not detected. Genotype-phenotype relations are discussed. The only correlation suggested to exist is that missense mutations are not detected in LDD patients. However, larger numbers are needed to confirm this. Association of PTEN mutations and the occurrence of malignant breast disease found in an earlier study cannot be confirmed. Clinical features of five CD patients without a PTEN mutation in the coding sequence do not differ from CD patients with a PTEN mutation. Furthermore, it is likely that we have identified the majority of CD patients in the Netherlands. From this we estimate that CD has a prevalence of about 1 in 250,000 in the Dutch population with a low mutation frequency.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.