Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease.

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.

Insulin-like growth factor (IGF-I) induces myotube hypertrophy associated with an increase in anaerobic glycolysis in a clonal skeletal-muscle cell model.

Insulin-like growth factor-I (IGF-I) is an important autocrine/paracrine mediator of skeletal-muscle growth and development. To develop a definitive cultured cell model of skeletal-muscle hypertrophy, C2C12 cells were stably transfected with IGF-I and clonal lines developed and evaluated. Quantitative morphometric analysis showed that IGF-I-transfected myotubes had a larger area (2381+/-60 micrometer2 versus 1429+/-39 micrometer2; P<0.0001) and a greater maximum width (21.4+/-0.6 micrometer versus 13.9+/-0.3 micrometer; P<0.0001) than control C2C12 myotubes, independent of the number of cell nuclei per myotube. IGF-I-transfected myotubes had higher levels of protein synthesis but no difference in DNA synthesis when compared with control myotubes, indicating the development of hypertrophy rather than hyperplasia. Both lactate dehydrogenase and alanine aminotransferase activities were increased (3- and 5-fold respectively), and total lactate levels were higher (2.3-fold) in IGF-I-transfected compared with control myotubes, indicating an increase in anaerobic glycolysis in the hypertrophied myotubes. However, expression of genes involved in skeletal-muscle growth or hypertrophy in vivo, e.g. myocyte nuclear factor and myostatin, was not altered in the IGF-I myotubes. Finally, myotube hypertrophy could also be induced by treatment of C2C12 cells with recombinant IGF-I or by growing C2C12 cells in conditioned media from IGF-I-transfected cells. This quantitative model should be uniquely useful for elucidating the molecular mechanisms of skeletal-muscle hypertrophy.

Counselling issues in familial hypertrophic cardiomyopathy.

To illustrate the variable clinical presentations and rates of progression in familial hypertrophic cardiomyopathy (FHC), phenotypes and genotypes were compared in three FHC families with different genetic defects. In the first family, the FHC abnormality was a protein truncating mutation (Gln969X) in the cardiac myosin binding protein C gene. The second family had a missense change (Asn755Lys) in the same gene. A missense mutation (Arg453Cys) in the cardiac beta myosin heavy chain gene was present in the third family. Penetrance associated with the Gln969X defect was 27% in the age range 0 to 40 years. This was considerably less than the 93% penetrance (0 to 40 years) observed in the two families with missense mutations. The variable penetrance in FHC, as well as the unpredictability of sudden cardiac death, complicates clinical diagnosis and management, including genetic counselling. Although a genetic disease with a predominantly adult onset, there are counselling issues in FHC which set it aside from other adult onset disorders.

Molecular pathology of familial hypertrophic cardiomyopathy caused by mutations in the cardiac myosin binding protein C gene.

DNA studies in familial hypertrophic cardiomyopathy (FHC) have shown that it is caused by mutations in genes coding for proteins which make up the muscle sarcomere. The majority of mutations in the FHC genes result from missense changes, although one of the most recent genes to be identified (cardiac myosin binding protein C gene, MYBPC3) has predominantly DNA mutations which produce truncated proteins. Both dominant negative and haploinsufficiency models have been proposed to explain the molecular changes in FHC. This study describes two Australian families with FHC caused by different mutations in MYBPC3. The first produces a de novo Asn755Lys change in a cardiac specific domain of MYBPC3. The second is a Gln969X nonsense mutation which results in a truncated protein. Neither mutation has previously been found in the MYBPC3 gene. The consequences of DNA changes on the function of cardiac myosin binding protein C are discussed in relation to current molecular models for this disorder.

Spontaneous regression of post-percutaneous transluminal coronary angioplasty aneurysm.

We report a case of a 67-year-old male with spontaneous regression of post-percutaneous transluminal coronary angioplasty (PTCA) aneurysm. This case substantiates the benign prognosis of post-PTCA aneurysms.

The natural history of left ventricular wall thickening in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is associated with mutations of genes coding for major sarcomeric proteins, but the mechanism of hypertrophy is unknown. As hypertrophy may not develop until adolescence, an altered response to physiological growth stimuli may regulate the hypertrophy process. AIMS: This study examined the relationship between age and changes in left ventricular (LV) wall thickness in patients with HCM. METHODS: Forty-three patients who had definite electrocardiographic and echocardiographic evidence of HCM were studied with serial 2D and M-mode echocardiograms at least two years apart (mean interval 5.5 +/- 3.0 years). LV cavity dimensions, septal and posterior wall thicknesses, and LV mass indices were compared with data from an age- and gender-matched control group. RESULTS: In patients with HCM aged ten to 20 years (n = 9), there was an increase in septal wall thickness during the study period from 15.9 +/- 6.2 mm to 19.3 +/- 2.1 mm (p < 0.01). This increase (3.4 +/- 2.5 mm) greatly exceeded the change in septal thickness observed in the control group between the ages of ten and 20 years (0.8 +/- 0.3 mm, p < 0.01). There was a lesser increase in posterior wall thickness from 9.8 +/- 2.1 mm to 11.5 +/- 3.5 mm (p = 0.07). In patients with HCM aged 21-40 years (n = 11), there was also an increase in septal wall thickness during the study period from 16.0 +/- 2.2 mm to 17.8 +/- 3.0 mm (p < 0.05), but no change in septal thickness in the control group. In contrast, the patients aged > 40 years (n = 23) showed no significant change in either septal or posterior wall thickness during the study period. LV mass index increased in the ten to 20 years age group from 128 +/- 24 g/m2 to 164 +/- 20 g/m2 (p = 0.01), but this increase was not observed in the older age groups. CONCLUSIONS: LV hypertrophy is progressive, particularly in the septum, during adolescence and early adult life in patients with HCM. As progressive hypertrophy may continue after somatic growth has ceased, an abnormal myocardial response to physiological growth regulators is less likely to be the principal stimulus to hypertrophy. Gene-gene interactions, changes in haemodynamic load or environmental factors may modulate the development of hypertrophy. Serial measurements of ventricular wall thickness in the first two decades of life, and probably until the fourth decade of life are advisable in patients suspected of having HCM.

DNA testing in familial hypertrophic cardiomyopathy: clinical and laboratory implications.

Counselling and clinical assessment in familial hypertrophic cardiomyopathy (FHC) is difficult, particularly in the young, since echocardiographic and ECG changes may not be diagnostic and clinical severity can vary. From 1990, when the beta-cardiac myosin heavy chain gene was implicated in the aetiology of FHC, considerable information about the molecular genetics of this disorder has emerged. However, an important question facing health professionals is the practical significance of DNA testing in FHC. The present study describes a DNA-based approach to screening for five commonly reported mutations involving the beta-cardiac myosin heavy chain gene. Approximately 11% of randomly selected families had an abnormality detected.

Handlebar problems in bicycling.

Handlebar problems are common among serious bicyclists. Compression neuropathy, more commonly ulnar than median, is frequent but seldom produces permanent injury or deficit if promptly recognized and managed by attending to bicycle fit, energy-absorbing gloves and handlebar treatments, and cycling technique. Overuse symptoms may be seen if bicycle design and fit couple with repetitive motion to aggravate wrist tendons or connective tissues of the neck and shoulder. Mechanical and medical management are effective in relieving symptoms and preventing recurrence.

Blast criteria for open spaces and enclosures.

A review of the history of blast research is presented from before World War II to the present time. The mechanisms of blast injury and the nature of the injuries are described. Casualty criteria applicable to man's exposure to single blasts in open terrain as a function of overpressure duration and orientation are given. Damage-risk criteria for man exposed to repeated blasts of low and high intensity are described. The hazards from blast waves entering open structures are described with criteria for personnel located in a standard two-man open foxhole. Methods of establishing the air blast dose in a variety of exposure conditions are illustrated. The present state-of-the-art on personnel protection afforded by rigid and soft protective garments is given. Information on animal response to blast waves generated inside enclosures from the firing of recoilless weapons and the detonation of high-explosive charges is discussed along with the problem of defining the extent of performance decrement in relation to the air blast dose.

Nonauditory injury threshold for repeated intense freefield impulse noise.

Exposure to impulse noise is an important occupational health concern. The risk of injury to auditory structures is well recognized and provides the cornerstone for present safety standards. For freefield impulse noise, nonauditory injury is dependent on peak pressure, positive phase duration (or impulse), and number of exposures. Trivial laryngeal petechiae are shown to precede nonauditory injury to more critical organs (ie, pulmonary and gastrointestinal systems). This study identifies the critical impulse noise thresholds causing trivial laryngeal petechial changes resulting from exposure to 5, 25, and 100 repetitions of specific levels of impulse noise. Because of anatomical differences, sheep should be slightly more susceptible to impulse noise laryngeal petechial changes than man; therefore, it seems reasonable to set the absolute limits for human occupational exposure levels below those causing laryngeal petechiae in sheep for persons wearing adequate hearing protection. This study does not address human auditory injury that may occur above or below these exposure limits even with proper hearing protection.

Physical correlates of eardrum rupture.

Eardrum (tympanic membrane) rupture in humans and animals in relation to various blast pressure-time patterns was reviewed. There were few systematic studies on eardrum rupture as a consequence of blast overpressure. Most reports did not describe the area of the eardrum destroyed. The peak overpressures required to produce a 50% incidence of eardrum rupture (P50) were summarized. Most of the animal data pertained to dogs. The highest P50 for dogs, 296 kPa, was associated with smooth-rising overpressure. For complex wave patterns occurring inside open shelters subjected to nuclear blasts, the P50 was 205 kPa. For fast-rising blasts in a shock tube it was 78 kPa, and 105 kPa for statically applied pressures. The duration of the overpressure was not a factor unless it was very short. The influence of the orientation of the head to the oncoming blast was demonstrated. An ear facing the blast may receive reflected overpressures several times that for one side-on to the blast. An ear on the downstream side of the head was exposed to about the same overpressure as the side-on ear. A P50 for humans of 100 kPa and a threshold of 35 kPa has been used widely in blast criteria. A recent study suggests a threshold (P1) of about 20 kPa, and gives the overpressures required to produce minor, moderate, and major eardrum ruptures. These data were presented in the form of curves showing the overpressures as a function of duration required to inflict a P1 and a P50 of eardrum rupture of the three levels of severity.

Cloth ballistic vest alters response to blast.

Ballistic wounds have been and will remain the principal cause of casualties in combat. Cloth ballistic vests (CBV) play an important role in limiting critical wounds from fragments and small-arms fire. There is an increased risk of primary blast injury on the modern battlefield. In a previous study, volunteers were exposed to short-duration blast waves of low peak pressure (18.6 +/- 0.8 kPa). Pressure measurements made in the distal esophagus as an estimate of intrathoracic pressure (ITP) were significantly higher (p less than 0.05) when the standard U.S. Army ballistic jacket was worn (8.7 +/- 1.2 kPa) than when fatigues alone were worn (7.4 +/- 0.7 kPa). In this study 58 sheep were exposed to nominal blast levels of 115, 230, 295, and 420 kPa peak pressure in groups of 12, 18, 16, and 12, respectively. Half of each group was fitted with a CBV. Lung weight index (LWI), lung weight expressed as a percentage of body weight, was used as a measure of blast injury. Use of the CBV was associated with a significant increase in LWI (p less than 0.05) which averaged 21% for the two middle exposure groups. At the 420 kPa level, two of six non-CBV animals died as opposed to five of six animals wearing the CBV. Intrathoracic pressure was generally higher in the CBV group. Likely mechanisms of injury enhancement include an increase in target surface area and an alteration of the effective loading function on the thorax. This information may be useful in the triage and treatment of casualties exposed to intense blast environments.

Late follow-up (41 to 102 months) of medically treated patients with coronary artery spasm and minor atherosclerotic coronary obstructions.

From a series of 37 patients with coronary artery spasm and less than 70% diameter narrowing treated initially with verapamil and nitrates, 33 were followed up 41 to 102 months (mean 62). One patient died from carcinoma of the lung and 3 could not be traced. Before diagnosis, 3 had nontransmural myocardial infarction and 10 had either ventricular tachycardia and fibrillation or atrioventricular block. During follow-up there were no cardiac deaths or myocardial infarctions. Asymptomatic periods of more than 3 months occurred in 23 patients during follow-up: 18 with asymptomatic periods of more than 1 year were pain free at the time of study and 5 with asymptomatic periods of 3 to 6 months had infrequent pain. Ten patients had no asymptomatic periods. Symptomatic status at last review was related to initial response to therapy: 13 of 18 patients (72%) currently asymptomatic became asymptomatic with initial therapy compared with 5 of 15 patients (33%) currently experiencing pain (p = 0.06). Twenty-six patients were currently receiving therapy: 22 verapamil, 80 to 640 mg/day (mean 280), 2 nifedipine, 1 diltiazem and amiodarone and 1 isosorbide (15 were receiving additional isosorbide). Twelve patients were not receiving therapy or were receiving very low dosage therapy, including 8 with asymptomatic periods of more than 1 year. Patients with coronary spasm and less than 70% diameter narrowing treated medically have low mortality and morbidity rates over 5-year follow-up. Many have long asymptomatic periods and some may be able to stop therapy indefinitely.