Resumos dos Trabalhos que serão apresentados  no  58th Annual Meeting of American Academy of Neurology - San Diego , 1-8 de abril ,2006

1) Treatment with Intravenous Immunoglobulin Improves Muscle Strength in a Patient with Dysferlin-Deficient Muscular Dystrophy

Simone Spuler, Joanna Zabojszcza, Male Friedrich-Freksa, Berlin, Germany

OBJECTIVE: To investigate whether ivig improves muscle strength in dysferlin-deficient muscular dystrophy. BACKGROUND: Recently, we reported that complement inhibitory factor CD55 is downregulated in skeletal muscle of mice and patients with dysferlin-deficient muscular dystrophy (LGMD2B). The absence of CD55 increased susceptibility of dysferlin-deficient human myotubes to complement attack in vitro (Wenzel et al. J Immunol. 2005 Nov 1;175:6219-25). One of the possible therapeutic effects of intravenous immunoglobulines (ivig) is inhibition of complement factors C4 and C5. We therefore tested whether ivig might be beneficial in LGMD2B. DESIGN/METHODS: As compassionate use a single female 32 year old patient with dysferlin-deficient muscular dystrophy was treated with ivig (Intraglobin, Biotest AG, Taunus, Germany) for six months after informed consent was obtained. After an induction cycle of 0.4 g/kg body weight for 5 days ivig was applied once a month at 0.4 g/kg body weight. Muscle strength was tested by manual muscle testing. A muscle biopsy specimen was analysed before and after treatment for deposition of sarcolemmal C5b9 membrane attack complex. RESULTS: Ivig was tolerated well. After three months the patient reported a marked improvement in her ability to walk and in her strength when holding a heavy bag. Muscle strength improved further until treatment-month six. All muscle groups tested improved during treatment, some muscles such as the tibial anterior muscle improved from grade of paresis of 2/5 to almost full strength at 4-5/5. C5b9 membrane attack complex, present on almost all muscle fibres in the pretreatment muscle specimen, were nearly absent in muscle obtained after treatment. CONCLUSIONS/RELEVANCE: The effect of ivig in LGMD2B should be tested in a controlled clinical trial.

2) The Course and Outcome of Pregnancy and Delivery in Women with Facioscapulohumeral Muscular Dystrophy

Emma Ciafaloni, Eva K. Pressman, Allison M. Loi, Alexis M. Smirnow, Debra J. Guntrum, Rabi N. Tawil, Rochester, NY

OBJECTIVE: To evaluate pregnancy history and outcome in women with facioscapulohumeral muscular dystrophy (FSHD). BACKGROUND: Very little is known about the course and outcome of pregnancy and delivery in FSHD. The ability of Neurologists and Obstetricians to counsel women with FSHD prior to and during pregnancy is limited. Moreover, the effect of gestation on disease course is unknown. DESIGN/METHODS: Women with FSHD enrolled in The National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members were offered participation. Those who returned signed consents were sent a detailed questionnaire and a release form for obtaining medical records pertaining to their pregnancies.
Pregnancy outcomes were compared to published national data from the CDC with Chi square analysis. RESULTS: Twenty-six women returned questionnaires reporting 55 gestations and 2 ectopic pregnancies. Miscarriages were reported in 13 (23.6%) of the gestations vs. 15% in national data (P = 0.073). Preterm births occurred in 3 pregnancies (7%) vs. 11% nationally (P = NS). Complications with pre-eclampsia, preterm labor, premature rupture of membranes and diabetes were uncommon (1-5 pregnancies, 2-11%) and similar to a general population. Operative vaginal deliveries and primary cesarean sections were performed in 21% and 35% of gestations, respectively. This is higher than in the general population where the reported rates are 9% and 16% respectively (P = 0.03 and 0.01). FSHD women generally coped well with their muscle disease in pregnancy and after delivery; however, 24% reported worsening symptoms with pregnancy. All but 2 women would choose to have their pregnancies again. CONCLUSIONS/RELEVANCE: Pregnancy outcomes in FSHD women are generally favorable. The patients responding to this survey reported a higher incidence of operative deliveries, similar to those reported in other muscular dystrophies. Progression of FSHD symptoms during and following pregnancy is higher than expected and warrants further investigation. Supported by: FSH Society.

3) A CINRG Pilot Trial of Oxatomide in Steroid-Naive Duchenne Muscular Dystrophy

Gunnar M. Buyse, Diana Escolar, Nathalie Goemans, Erik K. Henricson, Marleen van den Hauwe, Alejandro Jara Vallejos, Leuven, Belgium, Cheng Shao, Kantilal M. Patel, Robert McCarter, Robert Leshner, Washington, DC, Julaine Florence, Saint Louis, MO, Jill Mayhew, Richmond, VA

OBJECTIVE: With CINRG (Cooperative International Neuromuscular Research Group), we have tested the hypothesis that oxatomide specifically targets the pathophysiological cascade of DMD by blocking the mast cell mediator cascade and inhibiting dendritic cell's release of cytokines, and hence may improve muscle strength in DMD patients. BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common and devastating type of muscular dystrophy. Mast cells have been implicated in the pathophysiology of DMD and its animal models. The mast cell stabilizer oxatomide has shown promise in a pre-clinical screening platform that uses the homologous mdx.jpg mouse model of DMD. DESIGN/METHODS: We tested the efficacy and safety of oxatomide in 14 ambulant steroid-naive DMD boys aged 5 - 10 years, in a pilot open-label two-center clinical trial with a 3 months medication-free lead-in period followed by a 6 months treatment period with oral oxatomide (30 mg/day). Drug efficacy was tested by measuring muscle strength quantitatively (QMT, 10 muscle groups) and manually (MMT, 34 muscle groups), and by timed functional tests. RESULTS: Repetitive measure analysis was applied using linear mixed-effects models, comparing intercepts and slopes of lead-in and treatment periods. Within the limitations of a smaller non-controlled trial, we conclude that 6 months oxatomide treatment does not improve muscle strength or function in a group of 5 to 10 year old DMD patients. There were no serious adverse events and overall oxatomide was well tolerated. Supported by: Janssen-Cilag.

4) rAAV Gene Delivery of Follistatin for Muscle Enhancement in Limb-Girdle Muscular Dystrophy Type 2D

Lizabeth L. Rizo, Christopher J. Shilling, Amanda Haidet, Priya Umapathi, Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar, Columbus, OH

OBJECTIVE: To determine the efficacy of recombinant adeno-associated virus (rAAV) delivering follistatin (FS), a potent inhibitor of myostatin, to a mouse model for limb-girdle muscular dystrophy type 2D, i.e., alpha-sarcoglycan (aSG) deficiency. BACKGROUND: LGMD2D is a debilitating muscle disease of children and young adults. There is no proven treatment to delay the disease progression. Inhibition of myostatin, a negative growth modulator for muscle, can functionally improve normal or dystrophic muscle. This has been observed across species lines, through genetic manipulation, naturally occurring mutations or protein-specific antibodies. We have employed a novel gene therapy approach using a rAAV vector carrying a FS transgene. DESIGN/METHODS: Four-week-old a-SG knock-out mice were injected bilaterally into the quadriceps and tibialis anterior muscles with 1x10e10 viral genomes of rAAV1-FS and evaluated for muscle strength and endurance using hindlimb and forelimb grip strength as well as Rotarod. At age 120 days, gross muscle evaluation and analysis of muscle weight, fiber number, and cross-sectional area (CSA) on H&E sections was performed with the addition of a FS ELISA assay. RESULTS: We found increased serum FS levels accompanied by a local and remote increase in muscle mass in treated animals (p<0.05). Muscle hyperplasia was observed in rAAV-FS mice versus controls. The number of central nucleated fibers was increased in FS animals favoring enhanced muscle regeneration. Motor function tests showed improvements in muscle strength in FS-treated mice. CONCLUSIONS/RELEVANCE: This data supports a therapeutic effect in muscular dystrophy by myostatin inhibition. This approach represents a clinically applicable gene therapy method to enhance muscle mass and function in a model for LGMD2D with potential for treatment of other forms of muscular dystrophy. No toxicity was encountered. The FS transgene could also be used to complement gene replacement therapy. For example, adding muscle mass in patients with more advanced dystrophies could improve their candidacy for gene replacement therapy.

5)  Pilot Trial of Diltiazem in Facioscapulohumeral Muscular Dystrophy

Bakri Elsheikh, Elizabeth Bollman, Mario Peruggia, Wendy King, Worthington, OH, Gloria Galloway, New Albany, OH, John Kissel, Columbus, OH

OBJECTIVE: We conducted an open label, pilot trial of the calcium ion influx inhibitor diltiazem in patients with FSHD. BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is currently untreatable. There have been few therapeutic trials performed in this disorder, in large part because both the genetic defect and underlying pathogenic mechanisms of the disease are poorly understood. Recently, multiple anecdotal reports and patient testimonials on the Internet have suggested that calcium channel blockers are beneficial in patients with FSHD, based on the rationale that calcium influx into muscle cells may lead to myonecrosis in this disease. DESIGN/METHODS: Twenty patients with FSHD (12 male and 8 female, aged 21 - 60) were enrolled. All had the typical clinical picture and a chromosome 4q35 short fragment <35 kb. Patients were given diltiazem (Cardizem, Biovail Pharmaceuticals) at a dose of 30mg TID for six months. The primary outcome measure was change in muscle strength by the manual muscle testing (MMT). A secondary outcome was change in lean muscle mass assessed through dual energy x-ray absoptiometry (DEXA) . RESULTS: One male patient dropped out before starting diltiazem because of bradycardia that developed after his screening visit. For the remaining patients, the mean change from baseline at six months in average muscle score based on MMT was -0.0046 + 0.1355 and was not significant (p=0.8883). There was also no significant change in lean body mass as determined by DEXA P=0.2713. CONCLUSIONS/RELEVANCE: Calcium channel blockade with diltiazem had no effect on strength or muscle mass in patients with FSHD. These results do not support a role for altered calcium homeostasis in the pathogenesis of FSHD. Supported by: Muscular Dystrophy Association, General Clinical Research Center/ the Ohio State University.

6) Life Expectancy and Quality of Life in Oculopharyngeal Muscular Dystrophy Patients with the (GCG)₉ Expansion on PABPN1

Sergiu C. Blumen, Ralph L. Carasso, Rivka Inzelberg, Puiu Nisipeanu, Maya Opedisano, Hadera, Israel, Nava Blumen, Tel Hashomer, Israel

OBJECTIVE: To determine life expectancy (LE) and evaluate quality of life (QOL) among heterozygotes, (GCG)9-(GCG)6, and homozygotes, (GCG)9-(GCG)9, with oculopharyngeal muscular dystrophy (OPMD), belonging to the Bukhara Jewish cluster. BACKGROUND: The (GCG)9 expansion in the PABPN1 gene on chromosome 14q is the most common mutation producing OPMD in both French Canadians and Bukhara Jews. However data on its effect on LE and QOL is still sparse. DESIGN/METHODS: During office visits or telephone conversations with patients and close family members we evaluated severity of dysphagia, visual difficulties, ambulation problems and overall QOL among 39 heterozygotes and six, genetically proven, homozygotes with OPMD , using a five degree scale ranging from "excellent" to "very poor". We enquired about age at death in 34 patients who passed away during the last 25 years and plotted it against official LE data for Israeli men and women obtained from the National Bureau of Statistics. RESULTS: After seven or more years from OPMD onset most patients rated their overall QOL as "poor" or "very poor" and that strongly correlated with severity of dysphagia. All patients with significant ambulation problems also had severe dysphagia. The QOL was considered "very poor" by all six homozygotes and their caregivers; in addition to early onset of the classical OPMD features, they also had significant ophthalmoplegia, cognitive and behavioral disturbances leading, in most cases, to psychiatric care. Two homozygotes died before their 51th birthday; the other four are 53 years old or younger. Among 32 OPMD heterozygotes who died in the last 25 years, longevity was similar to that of the general population. CONCLUSIONS/RELEVANCE: 1. In most OPMD patients QOL is poor mainly due to dysphagia. 2. Among OPMD homozygotes QOL is very poor due to both dysphagia and cognitive-affective problems. 3. Whereas LE seems unchanged in OPMD heterozygotes, it may be significantly reduced among homozygotes.

7)  Adenovirus-Mediated Utrophin Gene Delivery as a Therapy for Duchenne Muscular Dystrophy

Jatinderpal R. Deol, Renald Gilbert, Mylene Bourget, Gawiyou Danialou, Joon-Shik Moon, Newark, NJ, Basil J. Petrof, Josephine Nalbantoglu, George Karpati, Montreal, QC, Canada

OBJECTIVE: To construct a fully deleted (gutted) adenovirus (AdV) capable of high expression levels of full-length isogenic utrophin (utr) in dystrophin (dys)-deficient muscle fibers. BACKGROUND: The introduction of gutted AdV vectors encoding full-length dys into muscles leads to significant mitigation of the dystrophic phenotype. However, dys is a neoantigen, causing deleterious immune responses. An alternative approach is to use utr, a functional homologue of dys, normally present only at the neuromuscular junction. DESIGN/METHODS: We have created a gutted AdV encoding full-length murine utr cDNA. The purified recombinant adenovirus was injected into tibialis anterior (TA) muscles of neonate and adult mdx.jpg mice for evaluation at 10, 30, 90, 180, and 360 days post-injection (pi). End points include utr expression, the restoration of the dys-associated protein complex, and the reversal of the physiological dystrophic phenotype. RESULTS: In neonates at 10 days pi, the mean number of extrasynaptic utr-positive fibers in vector vs control-injected TA was 1596 vs 114, corresponding to ~58 % transduction level. The levels in adults were 685 and 112 for vector and control injected TAs respectively, corresponding to ~23% transduction level. There was ~14 and 6 fold increase in utr expression over control levels in neonates and adults. In adults at 30 days pi, the mean number of utr positive fibers was 770 corresponding to ~35% transduction level. However, that number dropped substantially to an average of 80 at 60 days pi. At the same timepoint in neonates, the mean number was 794. CONCLUSIONS/RELEVANCE: There was a time-related decline of the initial high utr transduction of the TA muscles. We believe that this is mainly due to the fact that even the initial extrasynaptic utr level is not sufficiently high to prevent necrosis and loss of some of the transduced fibers. Further augmentation of the initial extrasynaptic utr should be helpful. Supported by: CIHR and l'AFM.

8)  Dysphagia in Facioscapulohumeral Muscular Dystrophy

Mariëlle Wohlgemuth, Nijmegen, Netherlands, Bert J. M. de Swart, Johanna G. Kalf, Frank B. M. Joosten, Arnhem, Netherlands, Antonius M. van der Vliet, George W. Padberg, Nijmegen, Netherlands

OBJECTIVE: First, demonstrating dysphagia in FSHD patients with swallowing complaints. Secondly, assessment of swallowing function to find the cause of dysphagia in FSHD and subsequently provide practical advice to improve swallowing and prevent aspiration. BACKGROUND: Dysphagia is considered no part of FSHD. However, occasionally patients report difficulties in swallowing. A questionnaire survey on pulmonary function among 87 FSHD patients, both ambulant and wheelchair dependent, revealed problems related to swallowing in 10 patients. DESIGN/METHODS: Clinical assessment of the oral muscles was followed by observation of eating and drinking. Subsequently, videofluoroscopy and measurement of tongue volume by MRI were performed. The study protocol was approved by the local medical ethics committee. RESULTS: Eight FSHD patients consented to further studies. All patients showed signs of weakness in jaws, lips and tongue. Videofluoroscopy showed signs of mild to moderate oropharyngeal dysfunction (loss of food, piecemeal deglutition, extended pharyngeal transit) in 7 patients. Six patients showed tongue atrophy on visual inspection, three of them showed small tongue volumes by MRI, confirming previous observations of occasional lingual involvement in FSHD. CONCLUSIONS/RELEVANCE: This study shows that dysphagia does occur in FSHD, but seldom leads to a life-threatening situation. Therefore, dysphagia should not be an exclusion criterion in FSHD. We presented evidence of the involvement of jaw and ligual muscles as part of the more advanced clinical condition. Supported by: Prinses Beatrix Fonds and Stichting FSHD.

9) CINRG Open Label Pilot Clinical Trial of Coenzyme Q10 in Steroid Treated Duchenne Muscular Dystrophy

Diana Escolar, Erik Henricson, Carolina Tesi-Rocha, Robert McCarter, Washigton, DC, Heather Gordish, Washington, DC

OBJECTIVE: To evaluate the effect of Coenzyme Q10 (CoQ10) on muscle strength in steroid treated DMD. BACKGROUND: CoQ10 treatment in the mdx.jpg mouse increases strength similar to corticosteroids. This pilot study assessed whether CoQ10 improved strength in steroid-treated DMD to determine whether a randomized controlled study is warranted. DESIGN/METHODS: We conducted a 9-month, prospective open label study in ambulant DMD children. Each participant served as his own control. Enrollees included 13 ambulant steroid-treated DMD boys, aged 5-11 years, naive to CoQ10. Participants were followed for 3 months before beginning 6 months of CoQ10 therapy and maintained a therapeutic level of 2.5 ug/ml or higher. Participants underwent monthly strength evaluations using quantitative muscle testing (QMT) of elbow flexors and extensors, knee flexors and extensors and grip. Manual muscle strength testing (MMT), timed functional tests functional evaluation (FE) and left ventricular ejection fraction (LVEF) were also studied. CoQ10 levels and safety labs were monitored monthly. RESULTS: Eleven of 13 participants completed the trial (withdrawn = 1 noncompliance, 1 failure to reach therapeutic levels). Comparison of slopes between non-treatment and treatment periods showed improvement in right (p=0.011) and left (p=0..013) elbow flexion and elbow flexor/extensor average scores (p= 0.038). Similar non-significant trends were seen in grip score and total QMT score. There was no significant change by MMT, TFT and LVEF. One participant developed headaches attributed to high blood levels of CoQ10. CONCLUSIONS/RELEVANCE: CoQ10 increased strength in elbow flexors and extensors after 6 months of treatment. This significant effect was not reflected in other muscle groups, but increasing strength trends were observed in all muscle groups except knee flexor/extensors. CoQ10 failed to produce an improvement in LVEF. Our data shows association between CoQ10 and increased strength sufficient to recommend the design of randomized controlled trial. Supported by: CNMC GCRC NCRR 1MO1RR020359-01; Serving Up a Cure Committee; K-23 RR16281-01 from NCRR.

10) Biological Effects of a Potent, Muscle-Targeted, Orally Bioavailable Calpain Inhibitor (C101) for Duchenne Muscular Dystrophy (DMD)

Norman W. Barton, Phoenix, MD, Alfred Stracher, Marie A. Badalamente, Stony Brook, NY, Theodore Carver, Theresa Michele, Leslie DeVos, Hunt Valley, MD, Leo Kesner, Brooklyn, NY, Amy Deng, Kevin Wang, Monica Oli, Alachua, FL

OBJECTIVE: To investigate the biological effects of C101 in muscle cell cultures in vitro and dystrophin deficient mdx.jpg mice in vivo. BACKGROUND: Calpain is a ubiquitous calcium-activated neutral protease. Dystrophin deficiency causes increased sarcolemmal permeability, calcium influx into muscle, calpain activation and initiation of a proteolytic cascade culminating in myonecrosis. C101 is a leupeptin analogue to which L-aminocarnitine is covalently bound. It is specifically designed to target muscle via the high affinity OCTN2 carnitine transporter. DESIGN/METHODS: The potency of C101 compared with leupeptin was assessed in human rhabdomyosarcoma cells that expressed high levels of OCTN2. Calpain activation was induced by addition of a calcium channel opener (maitotoxin) to cells grown to 80% confluence; 145- and 150-kD calpain-specific breakdown products of II-spectrin (SBDPs) were monitored over time by quantitative Western Blot analysis. The concentrations of C101 and leupeptin required to produce 50% reductions (IC₅₀) in the levels of SBDPs, which serve as unique biomarkers of calpain activity, were then determined under standard assay conditions. The mdx.jpg murine model (Jackson Labs, Bar Harbor, ME) was chosen to assess the inhibitory effects of C101 on myofiber degeneration associated with dystrophin deficiency. Thirty-day old mdx.jpg mice received daily intraperitoneal injections of C101 for 60 days at doses of 5 and 10 mg/kg; controls received physiologic saline. Selected muscles were harvested after 60 days for histological analysis and measurement of mean myofiber diameter. RESULTS: The IC₅₀ for C101 for prevention of 145-kD SBDP formation was 54-fold less than the value obtained for leupeptin (70 vs. 3750 M); a 130-fold reduction was observed for the 150-kD SBDP (400 vs. 51900 M). C101 preserved muscle morphology and significantly increased myofiber diameter in mdx.jpg mice compared with disease controls. CONCLUSIONS/RELEVANCE: C101 is a potent, oral, muscle-targeted calpain inhibitor that may be effective in treating DMD patients. Supported by: Ceptor Corporation, 200 International Circle, Suite 5100, Hunt Valley, MD 21030-1350.

11) Haploinsufficiency of Utrophin Gene Worsens Diaphragm and Quadriceps Muscle Dystrophy in mdx.jpg Mice

Lan Zhou, Jill A. Rafael-Fortney, Columbus, OH, Georgiana Cheng, Yuefang Zhou, Xiaohua Zhou, Bendi Gong, John D. Porter, Bethesda, MD, Henry J. Kaminski, Cleveland, OH

OBJECTIVE: To compare dystrophic changes of quadriceps and diaphragm muscles in mdx.jpg and mdx.jpg mice with utrophin haploinsufficiency (mdx.jpg/utr+/-).. BACKGROUND: Dystrophin deficient mice (mdx.jpg) display a mild muscular dystrophy (MD) phenotype as compared to Duchenne muscular dystrophy (DMD) patients. Mice lacking both dystrophin and utrophin (dko) suffer premature death from MD, mimicking human DMD. The muscle pathology of diaphragm, which manifests earlier in dko mice, is indistinguishable in mdx.jpg and dko mice around 10 weeks of age when dko mice die. It is thus thought that utrophin may fail to compensate for the effects of dystrophin loss on fibrosis in diaphragm. DESIGN/METHODS: Three pairs of mdx.jpg and mdx.jpg/utr+/- male littermates were sacrificed at 6 months of age. Muscle degeneration, inflammation, and fibrosis were evaluated by H&E staining and collagen III immunostaining. Muscle fibrosis was also quantified by measuring hydroxyproline content. RESULTS: At age 6 months, mdx.jpg quadriceps showed many small regenerated muscle fibers with increased central nuclei, and a few scattered endomysial inflammatory cells. There was no myofiber degeneration or endomysial fibrosis. In contrast, the quadriceps of mdx.jpg/utr+/- mice showed marked fiber size variation, multiple medium-sized foci of endomysial inflammatory cells, and notable increased endomysial collagen deposition. The diaphragm of both mdx.jpg and mdx.jpg/utr+/- mice showed prominent endomysial inflammation. The endomysial fibrosis, however, is much worse in mdx.jpg/utr+/- diaphragm as compared to mdx.jpg diaphragm. Consistent with these morphological findings, mean hydroxyproline content (m g/mg muscle tissue) was significantly increased in mdx.jpg/utr+/- quadriceps (1.16) and diaphragm (3.27) as compared to mdx.jpg quadriceps (0.53, p=0.02) and diaphragm (1.90, p=0.03), respectively. CONCLUSIONS/RELEVANCE: Our findings suggest that utrophin does compensate, although insufficiently for the effects of dystrophin loss in mdx.jpg diaphragm and quadriceps. With more severe muscle pathology, mdx.jpg/utr+/- mice may provide a better animal model than mdx.jpg mice for testing potential therapies for muscle fibrosis. Supported by: R24 EY 14837 and R01 EY-015306.

12) Muscle Pathology in 31 Patients with Calpain3 Gene Mutations

Aleksandra A. Nadaj, Malgorzata Dorobek, Klaudia Nestorowicz, Barbara Ryniewicz, Elzbieta Szmidt-Salkowska, Anna Fidzianska, Anna M. Kaminska, Warsaw, Poland

OBJECTIVE: The aim of the study was to find correlation between genotype and muscle pathology in 31 patients with the calpain 3 (CAPN3) gene mutations. BACKGROUND: Mutations in CAPN3 gene have been proven to be responsible for limb-girdle muscular dystrophy (LGMD) type 2A. A variety of mutations in CAPN3 gene have been found in many countries. Numerous myopathological changes typical for muscular dystrophy were described in LGMD 2A patients. The most striking pathological feature however was the presence of high proportion of lobulated fibers (LFs), considered as a halmark pathological lesion of the disease. DESIGN/METHODS: We selected 31 Polish patients with CAPN3 gene mutations. In 16 patients the homozygous occurence of 550delA was detected (group 1). In 5 patients different homozygous mutations other than 550delA were demonstrated (group 2) and 10 patients were compound heterozygotes for previously described mutations in CAPN3 gene (group 3). For the purpose of this study, all biopsies were reevaluated semiquantitavely. RESULTS: All examined patients had a limb-girdle phenotype and myopathic changes in muscle biopsy. In all biopsies typical features of muscular dystrophy were noted. LFs, defined as fibers with peculiar pattern of oxidative enzyme reaction on histochemical preparations, were observed in 16 patients from all three examined groups. The number of biopsies with LFs was highest in group 1 (almost 2/3 of cases) whereas in group 2 only 1 from 5 biopsies showed the occurence of LFs. In group 3, LFs were noted in a half of the patients. No correlation between the duration of the disease and the number of LFs was noted. CONCLUSIONS/RELEVANCE: LFs are often present in LGMD 2A patients with various mutation in CAPN3 gene. It seems that such fibers are more frequent in patients with 550delA mutation. These findings may be helpful in establishing diagnostic strategies in LGMD patients.

13) Alpha-Dystroglycan (-DG) Hypoglycosylation in Limb Girdle Muscular Dystrophies (LGMD)

Antal Szabo, Debrecen, Hajdu-Bihar, Hungary, Michael Sinnreich, Maria J. Molnar, Budapest, Hungary, George Karpati, Montreal, QC, Canada

OBJECTIVE: To establish the incidence of -DG hypoglycosylation in LGMD and test the diagnostic use of lectins. BACKGROUND: -DG is a key molecule of the surface membrane of muscle fibers connecting dystrophin to the extracellular matrix. The hyperglycosylated domain of -DG is essential for this function. Hypoglycosylation of -DG has been demonstrated in myopathies, the most common is related to a fukutin-related protein (FKRP) gene mutation. In our experience, in approximately half of the LGMD cases, a specific molecular diagnosis is presently not possible. In such cases, hypoglycosylation of -DG due to genetic FKRP deficiency or other glycosylation defects may be present. DESIGN/METHODS: We investigated 53 consecutive patients with an LGMD phenotype lacking a definitive molecular diagnosis including deficiency of calapin 3, dysferlin, caveolin, the 4 isoforms of sarcoglycans, myotilin, and -dystroglycan. Skeletal muscle biopsies were studied with immunocytochemistry and Western blot using antibodies against the hyperglycosylated domain of -DG. and different plant lectins including peanut agglutinin (PNA), concanavalin-a (CON-A), and wheat germ agglutinin (WGA) with binding affinity to specific carbohydrate residues. RESULTS: Six of the 53 cases showed marked reduction or absence of the immunoreactivity for the hyperglycosylated domain (but not of the common domain) of -DG by both immunocytochemistry and Western blot. None of these cases showed the common mutation of FKRP deficiency verified by sequencing the appropriate gene. The muscle biopsies of the 6 cases also showed reduced binding of WGA and increased staining with PNA. CONCLUSIONS/RELEVANCE: Hypoglycosylation demonstrated in approximately 10% of our idiopathic LGMD cases is probably related to a deficiency of still unidentified glycosyl-transferase enzymes. In our series, unlike another reported study, FKRP-related LGMD was not found. Appropriate studies for hypoglycosylation of -DG, including lectin binding, in idiopathic LGMD cases may lead to the discovery of hitherto unidentified causes of LGMD.

14)  Recombinant AAV and Corticosteroid Therapy in mdx.jpg Mice: In Consideration for Clinical Trials

Christopher J. Shilling, Xiao Xiao, Pittsburgh, PA, R. Jude Samulski, Jade Samulski, Angelique S. Camp, Chapel Hill, NC, Jerry R. Mendell, Columbus, OH

OBJECTIVE: To evaluate the efficacy and safety of combining recombinant adeno-associated virus (rAAV) gene transfer and corticosteroids, in an acute and chronic paradigm, to simulate potential gene therapy for Duchenne muscular dystrophy (DMD). BACKGROUND: Corticosteroids represent the only known treatment to clinically improve boys with DMD. It is estimated that approximately 50% of the DMD population in the USA is treated with corticosteroids, mainly prednisone. Many consider it to be the standard of care since it is the only known clinically effective form of treatment. Gene therapy is on the horizon as a potential treatment option for some patients with DMD. It is important to establish potential interactions between viral gene transfer and corticosteroid use to satisfy issues raised by patients, clinicians, scientists and regulatory agencies. To address this issue we have combined rAAV-mini-dystrophin gene transfer in the mdx.jpg mouse with two regimens of corticosteroid use. DESIGN/METHODS: Six-week-old mdx.jpg mice on chronic (1mg/kg/day, up to 52 days) and acute (2mg/kg given 6-hours pre-rAAV-injection and 24- & 48-hours post-rAAV-injection) regimens of prednisilone (pred) and non-pred treated controls were injected unilaterally into tibialis anterior (TA) muscles with 1x10¹¹ vg/kg of rAAV-mini-dystrophin. Multiple time-points of evaluation extended over 90 days including transgene expression and inflammatory cell infiltration. RESULTS: Pred treatment did not preclude transgene expression compared to non-pred treated mdx.jpg mice. This is true for both acute pred administration, as well as chronic pred administration (more closely simulating the patient on long-term corticosteroids). Of particular importance, there was no evidence of toxicity or cytotoxicity from gene delivery in concurrence with corticoid steroid use as compared to controls. CONCLUSIONS/RELEVANCE: This data impacts future DMD gene therapy protocols and sets the stage for clinical trials including prednisone-treated patients. This will be of importance in regulatory and IRB approvals for gene therapy studies for this disease. Supported by: Muscular Dystrophy Association

15) Absence of T-Cells and B-Cells Does Not Improve Functional Outcome in mdx.jpg Mice

Paul T. Golumbek, Richard M. Keeling, Anne M. Connolly, Saint Louis, MO

OBJECTIVE: Does the absence of T-cells and B-cells (Ig) secondary to a genetic rag-/- mutation improve strength in mdx.jpg mice. BACKGROUND: mdx.jpg mice lack dystrophin protein and serve as our best animal model of Duchenne muscular dystrophy (DMD). Prednisone produces improvement in functional outcome/strength in both boys with DMD as well as the mdx.jpg mouse model. If prednisone acts by suppressing the immune function of T-cells and/or B-cells (Ig) or if they play an important negative role in the eitiology of DMD/mdx.jpg weakness then reducing their actions should improve functional outcome measures. DESIGN/METHODS: Mice harboring a recombinase-activating gene mutation (rag-) are unable to produce T-cells and B-cells (Ig) throughout their lifetime. Groups of twenty male mdx.jpg-rag- and mdx.jpg-Rag+ mutant mice were produced and longitudinal studies (36 weeks) of strength were performed. Two independent measures of strength, grip strength testing and hanging wire testing, were observed. Consistent with previous literature, two nadirs of strength (demonstrated by both measures) were observed. RESULTS: No improvement, by either strength measure, is demonstrated by the addition of the rag- mutation. CONCLUSIONS/RELEVANCE: This implies that T-cells and/or B-cells (Ig) while demonstrable in mdx.jpg-Rag+ mouse muscle by histology are playing a negligible role in functional outcome. Also, prednisone treatment acts via non T-cell/B-cell dependent mechanism(s) in mdx.jpg mice. Supported by: Muscular Dystrophy Association.

16) Artificial Zinc Finger Proteins for the Transcriptional Regulation of Utrophin

Yifan Lu, Chai Tian, Renald Gilbert, George Karpati, Josephine Nalbantoglu, Montreal, QC, Canada

OBJECTIVE: To pursue transcriptional regulation of the utrophin (utr) gene through the targeting of artificial transcription factors to activate the utrophin A promoter in a constitutive manner (i.e. without need for the sustained presence of neural factors). BACKGROUND: Utr is a structural and functional homologue of dystrophin. In normal mature muscle fibers, utr is only present at the surface membrane of the neuromuscular and myotendinous junctions. Utr is anchored to the sarcolemma by the same molecular elements as dystrophin. It is anticipated that if a sufficient amount of utr would be accumulated and maintained in the extrasynaptic sarcolemma, dystrophin deficiency would not have deleterious effects on muscle fibers. To upregulate the utr promoter, articficial zinc finger proteins (ZFPs) were used as transcription factors. DESIGN/METHODS: Four GC-rich sequences were chosen in the UtrA promoter and the appropriate ZFPs were designed. The DNA binding domains were linked to an activation domain provided by the VP16 protein of herpes simplex virus, tagged with the Flag epitope for convenience of monitoring ZFP expression and a nuclear localization signal (NLS) to ensure nuclear trafficking. RESULTS: In reporter assays, one of the designed ZFPs (ZFP51) performed better than all the other ones. ZFP51 bound its recognition sequence with good specificity as determined by electrophoretic mobility shift assay. Furthermore, C2C12 cells stably transfected with ZFP51 expressed higher levels of utrophin mRNA as well as protein by Western blot analysis (on average 3.5-fold increase as normalized to actin). Intramuscular administration of a helper-dependent adenoviral vector expressing ZFP51 resulted in upregulation of extrasynaptic utrophin in the mdx.jpg anterior tibialis muscle. The upregulation of utrophin was accompanied by restoration of the dystrophin-associated glycoprotein complex. CONCLUSIONS/RELEVANCE: Artificial ZFPs may provide a convenient way of upregulating endogenous utr promoter and may be therapeutically useful in dystrophin deficiency. Supported by: Canadian Institutes of Health Research, Muscular Dystrophy Association (USA).

17)  Structure Function Relationship of Dysferlin Inferred from Analysis of Mutational Databases and Computerized Structural Predictions

Christian Therrien, George Karpati, Michael Sinnreich, Montreal, QC, Canada

OBJECTIVE: To better characterize the dysferlin protein through combination of database analysis and computerized secondary structure prediction tools. BACKGROUND: Dysferlin is a sarcolemmal protein which is mutated in limb girdle muscular dystrophy type 2B, Myoshi Myopathy and distal anterior compartment myopathy. Currently, no therapy is available for these disorders. Dysferlinopathies are recessively inherited diseases, suggesting that gene replacement could be an effective treatment that would not require correction of the mutant alleles. Prior to the design of gene therapy approaches, additional knowledge about structure-function relationships of the dysferlin protein is necessary. DESIGN/METHODS: We used a combination of database analysis of own and published series of dysferlin gene mutations as well as computerized secondary structure prediction tools to better characterize the dyferlin protein. RESULTS: We found that over 80 % of missense mutations affected residues that were conserved between members of the ferlin family, suggesting that those residues are important for structure and function. Dysferlin was found to be especially sensitive to the alteration of residues buried inside the core protein. Protein prediction tools identified seven putative C2 domains, one more than commonly reported. Multiple sequence alignment of dysferlin C2 domains against the mouse synaptotagmin III C2A domain indicated that only certain of the predicted C2 domains are potentially able to coordinate Ca2+. CONCLUSIONS/RELEVANCE: More detailed biochemical studies will be needed to further characterize these specialized structures and to substantiate these findings. The characterization of molecular interactions between dysferlin and membrane lipids or proteins will undoubtedly be helpful for understanding ferlin biology and for the development of future therapeutic strategies. Supported by: CIHR-MDAC-ALS Partnership program, Association Francaise contre les Myopathies, Fonds de Recherche en Sante Quebec.

18)  Neuronal Nitric Oxide Synthase (nNOS) Activity and Ultrastructural Changes in Caveolin-3 Deficient Muscle Cause Mechanical Irritability

Corrado Angelini, Luigi Fulizio, Anna C. Nascimbeni, Marina Fanin, Padova, Italy, Giovanna Cenacchi, Bologna, Padova, Italy

OBJECTIVE: To study the pathogenesis of caveolin-3-deficent limb girdle muscular dystrophy 1C in muscle and myoblast cultures. BACKGROUND: Neuronal nitric oxide synthase (nNOS) associates directly with the dystrophin associated complex and is absent in dystrophinopathies and sarcoglycanopathies, its role is not established in caveolinopathy. Caveolin-1 interacts with eNOS and inhibits its catalytic activity, whereas caveolin-3 interacts with nNOS in skeletal muscle fibers.Caveolin interaction with NOS is thought to hold the enzymes in an inactive conformation and only releases their inhibitory constraints through caveolin dissociation ,allowing the signaling to take place. DESIGN/METHODS: Six caveolin-3 deficient molecularly characterized muscle biopsies were studied by electron microscopy, nNOS immunohistochemistry and Western Blotting. Their changes were compared to nNOS immunohistochemistry and blotting in dystrophinopathies, sarcoglycanopathies and myositis and quantified as percent of control. Myoblasts were grown simultaneously from the biopsy of a caveolin-3 mutated patient with rippling muscle disease (RMD) and from control muscle and their ultrastructural features were compared. RESULTS: Immunohistochemistry showed a reduction nNOS signal in caveolin-3 deficient biopsies sarcolemma and ranged between 20 -80 percent of control, western blotting was also variably expressed. Electron microscopic study of biopsies in caveolin-3 defient patients showed abnormal T-tubules ,vacuolar dilatations of sarcoplasmic reticulum (SR) and papillary projections of sarcolemma. In caveolin-3 deficient myoblasts we observed several abnormal nuclei with increased euchromatin.Caveolin-3 deficient myotubes had more undifferentiated features and a stem-like appearance compared to control myotubes. CONCLUSIONS/RELEVANCE: The mislocalization of caveolin-3 and the associated variability of nNOS, as well the observed changes in T-tubules and SR might explain the increased irritability to the mechanically induced muscle contraction in RMD. Other clinical features such as hyperCKemia and proximal/distal weakness arise from altered sarcolemma and signal transduction might be due to other genes interplay co-determining.the phenotype. Supported by: AFM grant.

19)  Prominent Signs of Disease Progression: Data from the National Institute of Healths (NIH) Registry of Myotonic Dystrophy (DM) and Facioscapulohumeral Dystrophy (FSHD) Patients and Family Members

James E. Hilbert, Colleen M. Donlin-Smith, William B. Martens, Richard T. Moxley, III, Rochester, NY, Registry Scientific Committee

OBJECTIVE: To analyze the progression of DM-type 1 and FSHD. BACKGROUND: DM1 and FSHD are autosomal dominant diseases and the most common adult muscular dystrophies. In addition to muscle weakness, DM1 typically manifests in myotonia and complications of the brain, eyes, smooth muscles, cardiovascular, and endocrine systems. FSHD patients often have hearing loss, asymmetric muscle wasting, and retinal vascular pathology. Most studies describing the progression of these diseases are limited in size, scope, and length of longitudinal assessment. We have analyzed the National Registry that contains one of the largest populations of DM and FSHD patients tracked prospectively in the United States. DESIGN/METHODS: The Registrys database derives from ongoing medical record review and patient information forms. The database contains disease-specific and psycho-social information collected prospectively for 2 yrs. RESULTS: We have currently enrolled 399 DM1 and 404 FSHD patients (52% female). Age, duration of disease, BMI, and demographics are similar in both groups. Muscle weakness is predominant in both populations and 60% require assistive devices. Physical therapy (71% vs. 47%) and wheelchair dependency (9% vs. 3%) are greater in FSHD (p<0.001). DM1 patients have greater (p<0.001) associated medical problems including gastrointestinal reflux (37% vs. 24%), constipation (33% vs. 22%), and pneumonia (24% vs.16%). Psycho-social data indicate that negative employment effects (58%) and depression are common (30%) in both populations. These and other symptoms have been followed prospectively for 2 yrs (49% of patients). Increases in peptic ulcers (10%) occur in DM1, while hypertension (8%) increases in FSHD. CONCLUSIONS/RELEVANCE: Data indicates progression of smooth muscle problems in DM1 and vascular symptoms in FSHD. Analysis over the next 5 yrs will provide a more comprehensive evaluation of disease progression and how these manifestations impact clinical care, quality of life, and economic burden. Supported by: NIH Contract #N01-AR-5-2274.

20)  Disruptions in Cognition and Personality in Adult-Onset Myotonic Dystrophy Type 1

Claire V. Flaherty-Craig, Ally Walgren, Harrisburg, PA, Zachary Simmons, Milind Kothari, Hershey, PA

OBJECTIVE: This study examined the relationship between acquired cognitive and personality change in adult-onset Myotonic Dystrophy Type 1 (DM-1), and disease related disruptions to pituitary functioning. We hypothesized that disruptions to the pituitary-hypothalamic-cingulate cortical network would lead to disruptions in anterior cingulate gyrus regulatory processes, resulting in impairments in visuospatial processing and personality. BACKGROUND: DM-1 is known to be associated with pituitary dysfunction, cognitive impairments and changes in personality. Cognitive dysfunction has been evidenced for executive functioning (Rubinsztein et al., 1997). Recognized personality changes include apathy (Rubinsztein et al., 1998), avoidance (Delaporte, 1998), monotonous affect, hindered experience of pleasure, and poor initiative (Bungener et al., 1998). DESIGN/METHODS: A neuropsychological battery was administered to seven adult-onset DM-I subjects, diagnosed by neurological exam and genetic screening. Pituitary functioning was gauged by history of diabetes or cataract. Exclusion criteria included history of mental retardation, other medical or psychiatric diagnosis. Subjects were evaluated with COGNISTAT and RBANS sub-tests of orientation, attention, language, visuospatial processing, reasoning, the Stroop test of visual attention and the COWA fluency test. Personality was evaluated with the SCIDS-II. Spearman rank correlations were computed to evaluate the relationships between neuropsychological and clinical findings. RESULTS: Cateracts: Significant correlations were seen for the RBANS Visuospatial Index (p<.034) and SCIDS-II schizotypal personality trait (p<.01).
Diabetes: Significant correlations were seen for the Stroop Color-Word Index (p<.034) and Stroop Visual Interference Index (p<.034). CONCLUSIONS/RELEVANCE: Current findings support the contention that symptoms of cognitive impairment and personality change in adult onset DM-1 relate to disruption in the pituitary-hypothalamic-cingulate pathway, with consequent disruption to visual attentional processing and emotional temper. The interface of these changes results in an alteration in personality, characterized by diminished capacities for attentional self-monitoring, and emotional withdrawal. This has direct relevance to patient care, as management with stimulant medication could result in greater motivation to comply with treatment recommendations.

21)  Differences in Distribution and Type of Myotonic Discharges in Genetically Confirmed Myotonic Dystrophy Type 1 (DM1) Versus Type 2 (DM2)

Eric Logigian, Emma Ciafaloni, Christine Quinn, Nuran Dilek, Richard Moxley, Charles Thornton, Rochester, NY

OBJECTIVE: To compare electrical myotonia in DM1 and DM2. BACKGROUND: Electrical myotonia is distally-predominant in DM1, but its distribution in DM2 is unknown. Classical myotonic discharges wax and wane in frequency and amplitude; waning discharges are common in DM, but are less specific. The types of discharge in DM have not been systematically investigated. DESIGN/METHODS: 15 DM1 and 14 DM2 genetically-confirmed patients prospectively underwent concentric needle EMG of 8 muscles: Deltoid, Biceps, Flexor carpi radialis, First dorsal interosseous (FDI), Tibialis anterior (TA), Vastus lateralis (VL), Tensor Fascia Lata (TFL), and thoracic paraspinals (TPs). Electromyographers (EL, EC) blinded to DM type, made 8 needle insertions per muscle and recorded the presence and type of discharge evoked by each insertion. RESULTS: Limb weakness was predominantly distal in DM1 and proximal in DM2. Overall, myotonia was more often evokable in DM1 (55% of insertions) than DM2 (39%). The exceptions were TFL with myotonia in 23% of DM2 insertions (10% in DM1), and VL with myotonia in 58% of DM2 insertions (48% in DM1). In TPs, the % was similar (47% DM1, 46% DM2). The major discharge type was "waxing-waning" in DM1 (63% of discharges), vs "waning" in DM2 (68%). Three DM2 (21%), but no DM1, patients had only "waning" myotonia. In both DM1 and DM2, limb myotonia increased from proximal to distal. The % of insertions (DM1/DM2) evoking myotonia was 38%/21% in deltoid vs 94%/55% in FDI. In TFL, it was 10%/23% vs 78%/47% in TA. CONCLUSIONS/RELEVANCE: Except for some proximal leg muscles, myotonia is more easily evokable in DM1 than in DM2 muscles. In DM1, classical "waxing-waning" discharges predominate, but in DM2, less-specific "waning discharges" are the rule, making electrical diagnosis more challenging. In DM1, limb muscle weakness and myotonia are both worse distally, but in DM2, proximally-predominant weakness appears to be dissociated from distally-predominant myotonia. Supported by: RO1AR49077-01.

22)  Effects of Mexiletine on Muscle Strength and Cardiac Parameters in Myotonic Dystrophies: A Longitudinal Study

Giovanni Meola, San Donato Milanese, Milan, Italy, Valeria Sansone, Marta Panzeri, Maria Cotelli, Marco Calabria, Brescia, Italy, Luigi De Ambroggi, Milan, Italy

OBJECTIVE: To determine the effects of mexiletine on muscle strength and cardiac function in myotonic dystrophy type 1 (DM1) and type 2 (DM2) over time. BACKGROUND: Mexiletine's efficacy on myotonia is well-known. However, its effects on muscle strength and safety over time are still yet to be determined. DESIGN/METHODS: 30 patients with moderately severe adult DM1 (CTG range 600-800) and 10 patients with genetically confirmed DM2 matched for age, disease duration and severity were treated with 200mg bid mexiletine. Muscle strength using the 5-point modified MRC scale (MMRC) and cardiac parameters (12 lead EKG, 24-hour Holter EKG and 2D echocardiograms) were determined at baseline and over time (mean follow-up: DM1: 4,4 years + 3,8; DM2: 6 years + 3). Results were compared to 15 untreated patients with DM1 and 10 untreated patients with DM2 matched for age, disease severity and duration. RESULTS: In DM1, MMRC decreases significantly over time in the treated (p = 0.002) and untreated groups (p = 0.015). IN DM2, MMRC decreases significantly over time only in the untreated group (p = 0.02). QRS complex duration, heart rate, longest RR interval did not change significantly over time in the treated versus untreated groups. Only in the treated group of patients with DM1, PR interval increased significantly over time (176,1 msec + 30,3 versus 180,3 msec + 33,2, p < 0,05). None of the patients developed major AV block. Side effects resulted in 6/65 drop-outs. CONCLUSIONS/RELEVANCE: Our data suggest that mexiletine may maintain muscle strength in DM2 patients. It appears safe and well-tolerated: cardiac function does not seem to be worsened by treatment. Larger trials are necessary to: (i) confirm these preliminary data; (ii) to determine ideal timing of treatment and patient inclusion criteria for treatment; (iii) to establish correlations between its demonstrated efficacy as an antimyotonia agent and muscle strength maintainance.

23)  Health-Related Quality of Life in Myotonic Dystrophy Type 1 (DM1) and Type 2 (DM2): Correlations with Indexes of Cognitive and Behavioral Abnormalities

Valeria Sansone, Sandra Gandossini, Maria Cotelli, Marco Calabria, Brescia, Italy, Orazio Zanetti, Brescia, Italy, Giovanni Apolone, Milan, Italy, Giovanni Meola, San Donato Milanese, Milan, Italy

OBJECTIVE: To investigate quality of life perception in adult patients with myotonic dystrophy type 1 (DM1) and type 2 (DM2) and to see how this is related to disease severity, hypersomnia, cognitive and behavioral abnormalities. BACKGROUND: How patients with myotonic dystrophies perceive their quality of life in relation to their neuromuscular and cognitive disabilities is unclear. DESIGN/METHODS: 47 patients with moderately severe DM1 (CTG range 600-800) and 24 patients with genetically proven DM2 matched for age, disease severity and duration were subjected to: (i) neuromuscular assessment (5-point MRC scale, MMRC); (ii) neuropsychological tests; (iii) SCID- inventory and self-assessment axiety and depression rating scales; (iv) Epworth Sleep Scale (ESS); (v) Apathy Evaluation Scale; and (vi) SF-36 questionnaire. Results were compared to 50 healthy age-, sex- and education-matched controls. RESULTS: DM1 and DM2 patients scored worse than controls in SF-36 domains of physical functionioning, physical role, and general health resulting in a significantly different physical health index compared to controls (p < 0.0001). Both DM1 and DM2 patients had significantly higher total apathy scores compared to controls (p < 0.005). Mental score index positively correlates with greater impairment in frontal tests (Wisconsin Card Sorting Test) (r = 0,32). No correlations were found between all SF-36 measures and MMSE and MMRC scores. Higher educational levels (r = 0,39; p < 0,05) and lower ESS scores (r = -0,32; p < 0,05) correlate with better physical well-being in both DM1 and DM2. CONCLUSIONS/RELEVANCE: Impairment in physical activities and general health perceived by patients with DM1 and DM2 does not seem to affect their mental and emotional well-being. This is partially explained by the presence of abnormal scores on tests of frontal lobe function and by higher apathy scores compared to healthy controls. This may have important clinical implications in cognitive and behavioral treatment strategies.