Genetic mapping of a second myotonic dystrophy locus.

We report the mapping of a second myotonic dystrophy locus, myotonic dystrophy type 2 (DM2). Myotonic dystrophy (DM) is a multi-system disease and the most common form of muscular dystrophy in adults. In 1992, DM was shown to be caused by an expanded CTG repeat in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) on chromosome 19 (refs 2-6). Although several theories have been put forth to explain how the CTG expansion causes the broad spectrum of clinical features associated with DM, it is not understood how this mutation, which does not alter the protein-coding region of a gene, causes an affect at the cellular level. We have identified a five-generation family (MN1) with a genetically distinct form of myotonic dystrophy. Affected members exhibit remarkable clinical similarity to DM (myotonia, proximal and distal limb weakness, frontal balding, cataracts and cardiac arrhythmias) but do not have the chromosome-19 D CTG expansion. We have mapped the disease locus (DM2) of the MN1 family to a 10-cM region of chromosome 3q. Understanding the common molecular features of two different forms of the disease should shed light on the mechanisms responsible for the broad constellation of seemingly unrelated clinical features present in both diseases.

An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)

Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.

Rapid cloning of expanded trinucleotide repeat sequences from genomic DNA.

Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.

Targeting parents for the treatment of pediatric obesity in boys with Duchenne muscular dystrophy: a case series.

Obesity is a major public health concern in children. Obesity occurs frequently in boys with Duchenne muscular dystrophy (DMD), complicating treatment and impairing functioning. Parent-focused interventions to facilitate weight loss have been successful in other pediatric samples but have not been studied with this population. The current investigation examined the feasibility and potential efficacy of parent-focused treatment to improve healthy eating and physical activity of parents and eating and weight in their sons with DMD. Three families participated in this case series. Resulting changes in body weight among boys with DMD were an outcome variable. Findings indicate inconsistent changes in boys' weight, decreases in parent weight, increases in healthy foods available in the home, and increases in children's perceived quality of life. Participant ratings of treatment suitability and satisfaction were generally favorable. These preliminary findings support the use of parent-focused psychoeducation for the treatment of obesity in children with DMD.

Net energy analysis of alcohol production from sugarcane.

Energy requirements were calculated for the agricultural and the industrial phase of ethyl alcohol production from sugarcane grown in Louisiana. Agricultural energy requirements comprised 54 percent of all energy inputs, with machinery, fuel, and nitrogen fertilizer representing most of the energy subsidies. Overall net energy benefits (output:input) for alcohol production ranged from 1.8:1 to 0.9:1 depending on whether crop residues or fossil fuels were used for industrial processes.

Mississippi deltaic wetland survival: sedimentation versus coastal submergence.

Seasonal sedimentation, measured with the aid of artificial marker horizons, was markedly different in deteriorating as compared with stable marshes in the Mississippi River deltaic plain. Deteriorating marshes receive most sediment during storm events, whereas stable marshes receive substantial amounts of sediments during the spring river flood. The deteriorating marshes are accreting at a faster rate (1.5 centimeters per year at streamside, 0.9 centimeter per year at inland areas) than the stable marshes (1.3 centimeters per year at streamside, 0.6 centimeter per year at inland areas). However, relative to local apparent sea-level rise as measured by tide gauges in each area, the deteriorating marshes are not maintaining their intertidal elevation as well as the stable marshes. These results indicate the importance of considering accretion relative to submergence.

Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19q13 (DM1) or 3q21 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). Several mechanisms have been invoked to explain how this mutation, which does not alter the protein-coding portion of a gene, causes the specific constellation of clinical features characteristic of DM. We now report that DM2 is caused by a CCTG expansion (mean approximately 5000 repeats) located in intron 1 of the zinc finger protein 9 (ZNF9) gene. Parallels between these mutations indicate that microsatellite expansions in RNA can be pathogenic and cause the multisystemic features of DM1 and DM2.

Heterozygosity for a protein truncation mutation of sodium channel SCN8A in a patient with cerebellar atrophy, ataxia, and mental retardation.

BACKGROUND: The SCN8A gene on chromosome 12q13 encodes the voltage gated sodium channel Na(v)1.6, which is widely expressed in neurons of the CNS and PNS. Mutations in the mouse ortholog of SCN8A result in ataxia and other movement disorders. METHODS: We screened the 26 coding exons of SCN8A in 151 patients with inherited or sporadic ataxia. RESULTS: A 2 bp deletion in exon 24 was identified in a 9 year old boy with mental retardation, pancerebellar atrophy, and ataxia. This mutation, Pro1719ArgfsX6, introduces a translation termination codon into the pore loop of domain 4, resulting in removal of the C-terminal cytoplasmic domain and predicted loss of channel function. Three additional heterozygotes in the family exhibit milder cognitive and behavioural deficits including attention deficit hyperactivity disorder (ADHD). No additional occurrences of this mutation were observed in 625 unrelated DNA samples (1250 chromosomes). CONCLUSIONS: The phenotypes of the heterozygous individuals suggest that mutations in SCN8A may result in motor and cognitive deficits of variable expressivity, but the study was limited by lack of segregation in the small pedigree and incomplete information about family members. Identification of additional families will be required to confirm the contribution of the SCN8A mutation to the clinical features in ataxia, cognition and behaviour disorders.

Synaptonemal complexes during premeiotic DNA synthesis in oocytes of Drosophila melanogaster.

Well-synchronized populations of oocytes obtained by means of the "pupal system" (Grell, 1973a) have been examined to determine the time of appearance of the synaptonemal complex. The complex first appears in the most advanced oocytes between 132 and 138 hr of female development. Between 138 and 156 hr the complex apparently undergoes a fourfold increase in length. At 150 and 156 hr the complex system is extensive and present in virtually all oocytes. Previous studies using the pupal system have placed the period of premeiotic DNA synthesis between 132 and 162 hr. Thus, indirect evidence indicates that a significant portion of synaptonemal complex formation is coextensive with the main DNA replication in the oocyte. Direct evidence that DNA synthesis and complex formation occur simultaneously in oocytes has been obtained by electron microscope autoradiography. By definition, then, the stage of synaptonemal complex formation in Drosophila must include premeiotic interphase.

Spinocerebellar ataxia type 8: clinical features in a large family.

OBJECTIVE: To compare the clinical and genetic features of the seven-generation family (MN-A) used to define the spinocerebellar ataxia 8 (SCA8) locus. BACKGROUND: The authors recently described an untranslated CTG expansion that causes a novel form of SCA (SCA8) characterized by reduced penetrance and complex patterns of repeat instability. METHODS: Clinical and molecular features of 82 members of the MN-A family were evaluated by neurologic examination, quantitative dexterity testing, and, in some individuals, MRI and sperm analyses. RESULTS: SCA8 is a slowly progressive, predominantly cerebellar ataxia with marked cerebellar atrophy, affecting gait, swallowing, speech, and limb and eye movements. CTG tracts are longer in affected (mean = 116 CTG repeats) than in unaffected expansion carriers (mean = 90, p < 10-8). Quantitative dexterity testing did not detect even subtle signs of ataxia in unaffected expansion carriers. Surprisingly, all 21 affected MN-A family members inherited an expansion from their mothers. The maternal penetrance bias is consistent with maternal repeat expansions yielding alleles above the pathogenic threshold in the family (>107 CTG) and paternal contractions resulting in shorter alleles. Consistent with the reduced penetrance of paternal transmissions, CTG tracts in all or nearly all sperm (84 to 99) are significantly shorter than in the blood (116) of an affected man. CONCLUSIONS: The biologic relationship between repeat length and ataxia indicates that the CTG repeat is directly involved in SCA8 pathogenesis. Diagnostic testing and genetic counseling are complicated by the reduced penetrance, which often makes the inheritance appear recessive or sporadic, and by interfamilial differences in the length of a stable (CTA)n tract preceding the CTG repeat.

Molecular genetics of spinocerebellar ataxia type 8 (SCA8).

We previously reported that a transcribed but untranslated CTG expansion causes a novel form of ataxia, spinocerebellar ataxia type 8 (SCA8) (Koob et al., 1999). SCA8 was the first example of a dominant spinocerebellar ataxia that is not caused by the expansion of a CAG repeat translated into a polyglutamine tract. This slowly progressive form of ataxia is characterized by dramatic repeat instability and a high degree of reduced penetrance. The clinical and genetic features of the disease are discussed below.

Clinical and genetic characteristics of a five-generation family with a novel form of myotonic dystrophy (DM2).

We report the clinical and genetic characteristics of a five-generation family (MN1) with an unusual form of myotonic dystrophy (DM). Affected individuals have clinical features that are similar to DM including myotonia, distal weakness, frontal balding, polychromatic cataracts, infertility and cardiac arrhythmias. Genetic analyses reveal that affected individuals do not have the CTG expansion associated with DM, nor is the disease locus linked to the DM region of chromosome 19. We have also excluded the MN1 disease locus from the chromosomal regions containing the genes for the muscle sodium (alpha- and beta-subunits) and chloride channels, both of which are involved in other myotonic disorders. We have recently mapped the disease locus (DM2) in this family to a 10 cM region of chromosome 3q [Ranum LPW, Rasmussen PF, Benzow KA, Koob MD, Day JW. Nat Genet 1998;19:196-198]. The genetically distinct form of myotonic dystrophy in the MN1 kindred shares some of the clinical features of previously reported families with proximal myotonic myopathy (PROMM). The size of the MN1 family (25 affected individuals) makes it a unique resource for both clinical and genetic studies. This second form of myotonic dystrophy may help resolve the confusion that remains about how the CTG repeat expansion in the 3' untranslated portion of the myotonin protein kinase gene causes the multisystem involvement of DM.

Time course of miniature postsynaptic potentials at the Mauthner fiber giant synapse of the hatchetfish.

The hatchetfish Mauthner fiber is presynaptic to 8-14 large myelinated axons in the medulla; the large ('giant') synapses formed by these fibers appear to be nicotinic cholinergic. Miniature postsynaptic potentials (mPSPs) were recorded from single identified synapses. The mPSPs were averaged to more accurately determine their shape; the rise time was approximately 70 microseconds, and the fall usually was biphasic with time constants of decay for the two phases of 280 and 800 microseconds. In 25% of the records analyzed a third, slow tail of decay was seen which had an average decay constant of 4.2 ms. The biphasic decay of mPSPs largely accounts for the similar shape of the postsynaptic current following a presynaptic impulse, which is described in the accompanying paper.

Postsynaptic currents at the Mauthner fiber giant synapse of the hatchetfish.

Postsynaptic currents (PSCs) at the giant synapse between Mauthner and giant fibers of the hatchetfish Gasteropelecus were studied under voltage clamp. This axo-axonic synapse lies in the central nervous system beneath the floor of the 4th ventricle where electrodes can be closely positioned both pre- and postsynaptically. Transmission is nicotonic cholinergic. The PSCs produced by Mauthner fiber impulses rise rapidly to a peak and decay in two phases; an early more rapid phase is followed by a late slower phase. The slope conductance of the peak amplitude of the PSCs declines at more inside positive potentials. The late phase of decay is exponential and voltage dependent, becoming faster for PSCs evoked at more inside positive potentials. At potentials positive to about -40 mV the late phase merges with the early phase. The decay rate constant of the slowest phase is exponentially related to voltage for potentials negative to about -10 mV, but becomes less voltage dependent for more positive potentials. The peak current is independent of whether it is evoked during inward or outward active currents of the electrically excitable membrane, and two phase decays are observed in PSCs of reduced quantal content. Thus, changes in slope conductance and two phase decays are not due to series resistance or interactions between quanta. PSCs can be modeled by a 3 state reaction scheme in which closed channels open when they bind transmitter and then can pass to a second closed state with receptor still bound such that they must return through the open state before losing their transmitter and returning to the resting, closed state.

Effect of pelvic tilt on standing posture.

Low back dysfunction is associated in many cases with lumbar lordosis, and tilting the pelvis posteriorly is often recommended for therapeutic purposes. The influence of pelvic tilt on the spinal curves has not been studied. The purpose of this study was to use an objective noninvasive method to determine the effect of the pelvic tilt on the spinal curves in the sagittal plane. Thirty-two healthy subjects and 15 patients with chronic low back dysfunction (CLBD) were studied. Patients with CLBD and healthy subjects were instructed in performing active anterior and posterior pelvic tilt maneuvers, first in the supine and then in the standing position. Comparisons between the Patient Group and the Healthy Group were made for several variables representing the severity of spinal curves, pelvic orientation, hip orientation, and knee orientation. A computerized system, the Iowa Anatomical Position System, was used to obtain coordinates of external body surface landmarks from which pelvic tilt measurements were determined. The results showed that the voluntary pelvic tilt did not alter the thoracic spinal curve. For both the Healthy Group and the Patient Group, the lumbar curve was altered by the pelvic tilt: anterior tilt increased the depth of the lumbar curve and posterior tilt decreased the depth of the lumbar curve. The amount of pelvic tilt was the same whether knees were extended or flexed approximately 10 degrees. Pelvic tilt also tended to influence the orientation of the head and other parts of the body.

Comparative effectiveness of somatotropin and anabolic steroids in feedlot steers.

Crossbred steers (n = 252, BW = 379 +/- 28 kg) were allotted to 42 pens in a 2 x 3 factorial arrangement of treatments: control or steroid implant (STR; estradiol benzoate+progesterone [three lighter blocks reimplanted on d 84] and trenbolone acetate [reimplanted on d 63]), and either 0, 80, or 160 mg/wk of recombinant bovine somatotropin (bST). Steers were adapted to the finishing diet (12% roughage equivalent, 13% CP) before the start of the experiment and fed for 84 or 119 d. Blood samples were taken on d 0, 14, 28, 56, and 84 for plasma urea N (PUN), serum somatotropin (ST), plasma insulin-like growth factor I (IGF-I), and plasma amino acid assay. Few interactions were noted (P > .1). Gain was increased by both treatments: 1.30 vs 1.66 kg/d for control vs. STR (P < .001) and 1.44, 1.49, and 1.51 kg/d (linear, P = .07) for 0, 80, and 160 mg of bST/wk, respectively. Gain efficiency was also improved: 169 vs 205 g/kg (P < .001) and 177, 189, and 195 g/kg (linear, P < .001), respectively. Average PUN was decreased (P < .001) 29% by STR and decreased 17 and 29% by 80 and 160 mg of bST/wk, respectively (linear, P < .001). Somatotropin decreased mean serum ST compared with controls; STR increased ST 36% compared with controls. Average plasma IGF-I was increased (P < .001) 12% by STR and 13 and 19% (linear, P < .001) by 80 and 160 mg of bST/wk, respectively. Both STR and bST influenced (P < .05) plasma amino acid profiles. Indicators of carcass fatness were decreased linearly (P < .05) by bST; STR implant tended to decrease carcass fatness and increase longissimus muscle area, which was related to carcass weight. The anabolic effects of STR and bST were found to be additive and possibly independent in feedlot steers.

An improved method for muscle force assessment in neuromuscular disease.

We describe here the reliability and validity of methods to quantify involuntary muscle torque induced by non-invasive nerve stimulation. A rigid apparatus was used to hold the subject's limb in a predetermined position and confine movement to a specific direction (i.e. ankle dorsiflexion or thumb adduction). An incorporated strain gauge was used to measure isometric torque, and all data were recorded by a data acquisition program. The innervating nerves were stimulated by surface electrodes, using either single stimuli to generate a twitch, or short trains of stimuli to produce tetanic contraction of the individual muscle under study. The average peak tetanic torque generated by the dorsiflexor muscles in healthy control was 20.4 +/- 3.8 Nm and varied by 3.7% with repeated testing. The mean torque generated by the adductor pollicis muscle in controls was 1.5 +/- 0.4 Nm and varied by 4.6% with repeated testing. In patient populations significant changes in activated torque were readily quantified, and the effects of treatment can be easily assessed. Furthermore, several specific parameters of recorded isometric contractions were measured; e.g. time between stimulus and torque onset, peak rate of torque development, time to peak torque, half-relaxation time, and others (none of which are measurable when using voluntary contraction of muscle). Compared to current assessment methods, monitoring muscle torque generated by nerve stimulation improves objectivity, reliability, and quantitative capabilities. The presented method has significant potential both in diagnosing neuromuscular disorders and determining treatment efficacy.

Randomized, blinded trial of weekend vs daily prednisone in Duchenne muscular dystrophy.

OBJECTIVE: To perform a double-blind, randomized study comparing efficacy and safety of daily and weekend prednisone in boys with Duchenne muscular dystrophy (DMD). METHODS: A total of 64 boys with DMD who were between 4 and 10 years of age were randomized at 1 of 12 centers of the Cooperative International Neuromuscular Research Group. Efficacy and safety of 2 prednisone schedules (daily 0.75 mg/kg/day and weekend 10 mg/kg/wk) were evaluated over 12 months. RESULTS: Equivalence was met for weekend and daily dosing of prednisone for the primary outcomes of quantitative muscle testing (QMT) arm score and QMT leg score. Secondary strength scores for QMT elbow flexors also showed equivalence between the 2 treatment groups. Overall side effect profiles of height and weight, bone density, cataract formation, blood pressure, and behavior, analyzed at 12 months, did not differ between weekend and daily dosing of prednisone. CONCLUSIONS: Weekend dosing of prednisone is equally beneficial to the standard daily dosing of prednisone. Analysis of side effect profiles demonstrated overall tolerability of both dosing regimens. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that weekend prednisone dosing is as safe and effective as daily prednisone in preserving muscle strength and preventing body mass index increases in boys with DMD over a 12-month period.

The science of hypoxia in the Northern Gulf of Mexico: a review.

The Mississippi River is one of the world's 10 largest rivers, with average freshwater discharge into the northern Gulf of Mexico (GOM) of 380km(3) year(-1). In the northern GOM, anthropogenic nitrogen is primarily derived from agricultural fertilizer and delivered via the Mississippi River. The general consensus is that hypoxia in the northern Gulf of Mexico is caused primarily by algal production stimulated by excess nitrogen delivered from the Mississippi-Atchafalaya River Basin and seasonal vertical stratification of incoming stream flow and Gulf waters, which restricts replenishment of oxygen from the atmosphere. In this paper, we review the controversial aspects of the largely nutrient-centric view of the hypoxic region, and introduce the role of non-riverine organic matter inputs as other oxygen-consuming mechanisms. Similarly, we discuss non-nutrient physically-controlled impacts of freshwater stratification as an alternative mechanism for controlling in part, the seasonality of hypoxia. We then explore why hypoxia in this dynamic river-dominated margin (RiOMar) is not comparable to many of the other traditional estuarine systems (e.g., Chesapeake Bay, Baltic Sea, and Long Island Sound). The presence of mobile muds and the proximity of the Mississippi Canyon are discussed as possible reasons for the amelioration of hypoxia (e.g., healthy fisheries) in this region. The most recent prediction of hypoxia area for 2009, using the current nutrient-centric models, failed due to the limited scope of these simple models and the complexity of this system. Predictive models should not be the main driver for management decisions. We postulate that a better management plan for this region can only be reached through a more comprehensive understanding of this RiOMar system-not just more information on river fluxes (e.g., nutrients) and coastal hypoxia monitoring programs.

Clinical features of facioscapulohumeral muscular dystrophy 2.

OBJECTIVE: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss of DNA methylation and heterochromatin markers at the D4Z4 repeat that are similar to patients with D4Z4 contractions (FSHD1). This commonality suggests that a change in D4Z4 chromatin structure unifies FSHD1 and FSHD2. The aim of our study was to critically evaluate the clinical features in patients with FSHD2 in order to establish whether these patients are phenotypically identical to FSHD1 and to establish the effects of the (epi-) genotype on the phenotype. METHODS: This cross-sectional study studied 33 patients with FSHD2 from 27 families, the largest cohort described to date. All patients were clinically assessed using a standardized clinical evaluation form. Genotype analysis was performed by pulsed field gel electrophoresis and PCR; D4Z4 methylation was studied by methylation-sensitive Southern blot analysis. RESULTS: FSHD2 is identical to FSHD1 in its clinical presentation. Notable differences include a higher incidence (67%) of sporadic cases and the absence of gender differences in disease severity in FSHD2. Overall, average disease severity in FSHD2 was similar to that reported in FSHD1 and was not influenced by D4Z4 repeat size. In FSHD2, a small effect of the degree of hypomethylation on disease severity was observed. CONCLUSIONS: Clinically, patients with FSHD2 are indistinguishable from patients with FSHD1. The present data suggest that FSHD1 and FSHD2 are the result of the same pathophysiologic process.