Distribution of motor unit potential velocities in short static and prolonged dynamic contractions at low forces : use of the within-subject 's skewness and standard deviation variables Behaviour of motor unit potential -LRB- MUP -RRB- velocities in relation to -LRB- low -RRB- force and duration was investigated in biceps brachii muscle using a surface electrode array . Short static tests of 3.8 s -LRB- 41 subjects -RRB- and prolonged dynamic tests -LRB- prolonged tests -RRB- of 4 min -LRB- 30 subjects -RRB- were performed as position tasks , applying forces up to 20 % of maximal voluntary contraction -LRB- MVC -RRB- . Four variables , derived from the inter-peak latency technique , were used to describe changes in the surface electromyography signal : the mean muscle fibre conduction velocity -LRB- CV -RRB- , the proportion between slow and fast MUPs expressed as the within-subject skewness of MUP velocities , the within-subject standard deviation of MUP velocities -LSB- SD-peak velocity -LRB- PV -RRB- -RSB- , and the amount of MUPs per second -LRB- peak frequency = PF -RRB- . In short static tests and the initial phase of prolonged tests , larger forces induced an increase of the CV and PF , accompanied with the shift of MUP velocities towards higher values , whereas the SD-PV did not change . During the first 1.5 -- 2 min of the prolonged lower force levels tests -LRB- unloaded , and loaded 5 and 10 % MVC -RRB- the CV and SD-PV slightly decreased and the MUP velocities shifted towards lower values ; then the three variables stabilized . The PF values did not change in these tests . However , during the prolonged higher force -LRB- 20 % MVC -RRB- test , the CV decreased and MUP velocities shifted towards lower values without stabilization , while the SD-PV broadened and the PF decreased progressively . It is argued that these combined results reflect changes in both neural regulatory strategies and muscle membrane state . Introduction Diverse laboratory conditions have been used in surface electromyography -LRB- sEMG -RRB- studies in order to gain insights into the neural regulatory strategies and muscle membrane alterations . The influence of force load on sEMG can be investigated by using force tasks or position tasks . The majority of studies have been performed as force tasks , which means that the subject controls the effort by maintaining a target force while the limb position is fixed . During the position tasks , in contrast , an inertial load is applied while the subject controls a target limb position . Both force and position tasks can be performed in static or dynamic conditions . Examples of the static force tasks are the well-known isometric experiments , with higher and lower force levels . In the recent past , force tasks in dynamic conditions have been performed sporadically , such as the cycling experiments by Pozzo et al. -LRB- 2004 -RRB- and Farina et al. -LRB- 2004 -RRB- . Since Hunter et al. -LRB- 2002 -RRB- found that , with the same load torque , position tasks resulted in a shorter endurance time than force tasks , suggesting different regulatory mechanism for the both type of tasks , the position task studies gained field . Most position task experiments have been performed in static conditions , evaluating the underlying physiological phenomena during force versus position tasks -LRB- Hunter et al. 2002 , 2003 ; Hunter and Enoka 2003 ; Rudroff et al. 2005 , 2007 -RRB- . Potvin -LRB- 1997 -RRB- has described the changes in the sEMG during position tasks in dynamic conditions . Previous findings suggest that , as compared with force tasks , position tasks induce greater synaptic input into the motor neurons -LRB- Mottram et al. 2005a -RRB- and greater adaptation in the motor unit discharge -LRB- MacGillis et al. 2003 -RRB- . Changes in muscle activity during -LRB- static and dynamic -RRB- position tasks have been assessed using two of the three traditional sEMG parameters , the power spectrum and the global sEMG amplitude . However , the third parameter , the mean muscle fibre conduction velocity -LRB- CV -RRB- , has been lacking . Spectral estimates are generally accepted in fatigue experiments as equivalents of CV because they highly correlate with the CV 's changes -LRB- Bigland-Ritchie 1981 ; Eberstein and Beatie 1985 ; Arendt-Nielsen and Mills 1985 -RRB- . But this correlation holds true only for the constant forces and isometric conditions ; thus , a replacement of CV by power spectrum assessments does not always seem feasible -LRB- Farina et al. 2002 ; Broman et al. 1985 -RRB- . The favour of CV above power spectrum is , furthermore , that it renders direct and absolute values of conduction velocity , and is less sensitive to the anatomical local relationships , such as depth of the motor unit -LRB- MU -RRB- in relation to the muscle and skin surface -LRB- Farina et al. 2002 -RRB- . Yet the limitation of these global CV measurements remains the lack of sensitivity to the changes at the level of an individual motor unit potential / motor unit . To accommodate with these limitations of CV , researchers have recently been trying to disentangle the propagation velocities of individual motor unit potentials -LRB- MUPs -RRB- from sEMG . One of the methods is the inter-peak latency method -LRB- IPL -RRB- proposed by Lange et al. -LRB- 2002 -RRB- . The principle comprises calculating conduction velocities of the MUPs from the latencies between paired MUPs of two differential sEMG signals obtained parallel to the muscle fibres , and the distance between the recording electrodes . The negative peaks of two paired MUPs are then the elements determining the interpeak latency . As motor unit propagation velocity reflects the intrinsic physiological properties of a MU , such as fast-twitch or slow-twitch type -LRB- Buchthal et al. 1973 ; Andreassen and Arendt-Nielsen 1987 -RRB- , the IPL method renders many diverse MUP velocities . Lange at al. -LRB- 2002 -RRB- proposed using the standard deviation of MUP velocities as an additional measure that offers information about muscle fibre properties . Changes in these velocities during prolonged effort may indicate , for example , slowing/fatigue of the activated motor units and/or appearance of fast/newly recruited motor units . Such shifts in the activated MUs ' populations were shown by Houtman et al. -LRB- 2003 -RRB- by eliciting the MUP velocities with the IPL method and presenting their distribution in histograms . The IPL method has not been applied much . It yields insights into the diversity of MUP velocities and thereby the underlying changes in the MU activity . The method is simple and does not require expensive apparatus or software . When compared with techniques that assess the propagation patterns of MUPs by multi-channel/spatial resolution sEMG -LRB- Masuda and Sadoyama 1986 ; Rau et al. 1997 -RRB- , the IPL method is unable to distinguish and follow individual MUPs belonging to the specific MUs . In the present study , the sEMG signal was described with four parameters derived from the IPL method : -LRB- 1 -RRB- the mean muscle CV which was the average of the obtained MUP velocities ; the two statistical distribution variables , which were : -LRB- 2 -RRB- the within-subject MUP velocities ' skewness -LSB- Sk-peak velocity -LRB- PV -RRB- -RSB- and -LRB- 3 -RRB- the within-subject MUP velocities ' standard deviation -LRB- SD-PV -RRB- , and -LRB- 4 -RRB- the peak frequency -LRB- PF -RRB- , a variable expressing the amount of MUP activity -LRB- number of peaks = MUPs -RRB- per second . The aim of the present study was to investigate with the four parameters the changes in MUPs ' velocities of the biceps brachii muscle -LRB- BB -RRB- during prolonged dynamic position tasks , in dependence of -LRB- low -RRB- force and duration . It is chosen for the dynamic position tasks as study design because their physiology promised the finding of a large variety of MUP velocities -LRB- great amount of activity due to the position tasks character , and diversity because of the recruitment/derecruitment changes within the dynamic cycle -RRB- . Whole cycles of movement with their concentric and eccentric phases were analyzed together in order to evaluate a total of the sEMG activity with its evolution over time . To highlight the initial changes with the effect of force on it , the changes during the first 14.4 s of the dynamic tasks were evaluated separately . Additively , short static position tasks were performed in order to show the -LRB- early -RRB- changes on force , without any influence of movements on the signal . Methods Subjects The study involved short static and prolonged dynamic experiments . Forty-one healthy and physically active males -LRB- 24.8 ± 6.7 years , from 17 to 48 -RRB- -LRB- mean ± SD -RRB- volunteered for the first experiment and 30 randomly chosen subjects from that group -LRB- 25.4 ± 7.6 years , from 18 to 42 -RRB- participated in both experiments . Exclusion criteria were drug abuse and the practice of bodybuilding . Three from a total of 44 subjects were excluded because of the impossibility to obtain a required correlation coefficient between the sEMG signals used to estimate the parameters ' values . The experimental protocol was conducted according to the Helsinki Declaration and approved by the local ethics committee . All participants gave written informed consent . Experimental set up Maximal voluntary contraction -LRB- MVC -RRB- of elbow flexors was measured at least 5 days before the experiment with a hand-held dynamometer -LRB- Lameris Instruments , Utrecht , The Netherlands -RRB- . During the MVC measurements subjects were sitting upright . The shoulder was slightly abducted and flexed at 45 ° , the elbow was firmly sustained and flexed at 90 ° , and the forearm was supinated . The dynamometer was applied to the wrist by the break method -LRB- van der Ploeg and Oosterhuis 1991 -RRB- . The peak hold was switched off and the force was kept for at least 3 s . The mean of three maximal values was taken as a MVC . The MVC was assessed with elbow at 90 ° , although the tests were performed at the elbow angle of 135 ° -LRB- Philippou et al. 2004 -RRB- . During the experiment subjects were sitting in a chair . The upper arm was slightly abducted and comfortably supported at 45 ° of the shoulder flexion , the forearm was free . When the elbow was stretched , the line of the upper arm-forearm was at 45 ° in relation to horizontal . When the elbow was flexed to the angle of 135 ° , the forearm was horizontal . The forearm was supinated during static and dynamic tests . Static tests Subjects were asked to hold the forearm horizontally -LRB- elbow angle was then 135 ° -RRB- . A visual bar helped to maintain the correct -LRB- horizontal -RRB- position of the forearm . In the loaded tests a sack filled with lead and sand was placed in the palm . Three levels of force were applied in blocks that were 3 min apart : unloaded , loaded 10 and 20 % MVC . A block consisted of three tests -LRB- three repetitions at the same level of force -RRB- ; every test lasted for 3.8 s and was within a block separated 30 s from one another . Dynamic tests All participants of the dynamic tests underwent previously static tests , separated by 5 min . Subjects were asked to swing the forearm from the stretched -LRB- elbow angle 180 ° -RRB- to horizontal position -LRB- elbow angle 135 ° -RRB- , thus moving over an angle of 45 ° . They did it within a rate of 40 beats per minute -LRB- one up-and-down movement in one beat -RRB- , given by a metronome sound . The visual bar indicated the horizontal position to which the lower arm returned after being stretched . Four force levels were applied : unloaded , and loaded 5 , 10 and 20 % MVC . The tests lasted for 4 min and were separated by 5 min . EMG recording Measurements were performed on the short head of the BB of a dominant arm . A surface electrode array consisted of three golden-coated electrodes -LRB- Harwin , P25-3526 -RRB- , diameter 1.5 mm , insulated in synthetic material plate , with a 10 mm distance between the electrodes -LRB- Sadoyama et al. 1985 -RRB- . The skin was cleaned with 95 % ethanol . The electrode array was placed parallel to the muscle fibres -LRB- Sollie et al. 1985 -RRB- . The proximal electrode was positioned exactly on the distal one-quarter point of the upper arm , measured between the coracoid and the elbow crease . This place of the electrode was about halfway between the endplate zone and the tendon , securing a sufficient distance from the endplate -LRB- Sadoyama et al. 1985 ; Masuda and Sadoyama 1987 -RRB- . Bipolar derivation was made from the proximal to distal direction . The optimal electrode position was controlled by both the observation of the signal on the monitor and the estimation of a correlation coefficient -LRB- CC -RRB- of the two sEMG signals , which was accepted at r > 0.7 for unloaded , r > 0.85 for 5 % MVC and r > 0.9 for higher loaded tests . -LRB- During the static and dynamic tests , the maximal CC for unloaded arm was usually lower than that for loaded arm , which was consistent with Hogrel et al. 1998 , who have estimated for an unloaded arm r > 0.7 -RRB- . The ground electrode was placed on the lateral upper arm , slightly proximal from the derivation electrode . The temperature sensor was medial on the upper arm . Two obtained signals were differentially amplified -LRB- gain 2,000 -- 10,000 × -RRB- and band pass filtered at 2 -- 250 Hz by EMG apparatus -LRB- Viking IV , US -RRB- . Data processing The signals were simultaneously A/D converted -LRB- sampling 10 kHz , 12 bits acquisition -RRB- . Data were stored on a personal computer . The signal was analyzed with LabVIEW 6.1 that also facilitated a partial on-line analysis . The peak selection and the correlation coefficient assessments for both static and dynamic tests were performed on 0.2 s epochs . In the static tests , measurements were taken every second during 0.8 s -LRB- comprising 4 epochs of 0.2 s -RRB- . A static test was of 3.8 s duration and was repeated three times for each force level . The statistical analyses were performed on the data of these three repeated tests taken together . In the dynamic tests , the data were assembled every 30 s during 14.4 s -LRB- comprising 72 epochs of 0.2 s -RRB- . The test duration was 4 min . Peak selection The basic principle was that of Lange et al. -LRB- 2002 -RRB- . The software was custom designed and written in LabView . The orientation of the signals is up-negative . 1st step : finding zero line . 2nd step : finding the peak-to-peak amplitude of the largest MUP in an epoch of 0.2 s. 3d step : finding a peak-decline structure . The peak-decline is defined as a structure with a decline of ≥ 20 % compared with the largest MUP amplitude of a 0.2 epoch , over ≤ 4 ms -LRB- ≤ 40 sample points -RRB- . 3d step : finding a peak . A peak is the highest -LRB- most negative -RRB- point previous to the decline , and must be ≥ 10 μV -LRB- the threshold of the noise level -RRB- . 4th step : finding a pair peak . A pair peak is a peak in the second signal with the properties as previous , found in a time window between 1.49 and 4 ms after the peak of the first signal . This window is chosen assuming the physiological CV values of 2.5 -- 6.67 m/s . 5th step : excluding double peak . If a peak from the first signal matches two different peaks from the second signal , then the first peak from the second signal is true . If during the fatiguing tests the CV severely diminishes , then the low limit of velocity is put at 1.3 m/s , making a window of 1.49 -- 7.68 ms. As an objective pragmatic criterion to this change a lowering of the peak frequency with ≤ 30 % was assumed , as compared with the first value of the 20 % MVC test . The parameters The following calculations were performed : -LRB- 1 -RRB- basic calculation of PV , following the IPL method ; -LRB- 2 -RRB- mean CV , expressed as an average value of the PVs ; -LRB- 3 -RRB- within-subject skewness of the peak velocities -LRB- Sk-PV -RRB- , expressed as a skewness of a PVs ' population of a subject ; -LRB- 4 -RRB- within-subject standard deviation of the peak velocities -LRB- SD-PV -RRB- , expressed as a standard deviation of a PVs ' population of a subject ; and -LRB- 5 -RRB- peak frequency -LRB- PF -RRB- , expressed as a number of peaks per second . Statistics One-way analysis of variance -LRB- ANOVA -RRB- with repeated measures on force was used to compare the dependent variables for static tests -LRB- three levels -RRB- and for the initial values of the dynamic tests -LRB- four levels -RRB- . A two-way ANOVA with repeated measures on force -LRB- four levels -RRB- and time -LRB- nine levels -RRB- was used to compare variables during the dynamic tests . In the case of interactions between force and time , to describe changes over time , when appropriate , four separated ANOVAs were performed with smaller time windows of 0 -- 60 , 60 -- 120 , 120 -- 180 and 180 -- 240 s. To be assured that the dependent variables met parametric assumptions , plots of residues were produced with SPSS program , model control as suggested by Kutner et al. -LRB- 2005 , p. 1157 -RRB- . No relevant deviations of model were detected . Pearson correlation coefficients were calculated to evaluate associations between variables . A level of P < 0.05 was used to identify statistical significance . Results Subjects ' physical characteristics are presented in the Table 1 . The force of the biceps correlated positively with the upper arm circumference -LRB- r = 0.484 , P < 0.01 -RRB- . No association was found between either force or upper arm circumference and the sEMG variables . The average skin temperature increased during the dynamic tests by 1.65 °C -LRB- P < 0.001 -RRB- ; it did not change during the static tests . Table 1Characteristics of 41 participants to the static tests ; 30 of them participated in the dynamic tests as wellNMinimumMaximumMeanSDAge -LRB- years -RRB- 41164824.76.7 Height -LRB- cm -RRB- 41166.0197.0183.28.0 Weight -LRB- kg -RRB- 4160.095.074.39.2 Force -LRB- N -RRB- right38148 .5346.5246.141.9 Force -LRB- N -RRB- left -LRB- for left-handed -RRB- 3262.3267.3265.12.5 Skin thickness -LRB- mm -RRB- 411.44.22.4.7 Circumference upper arm -LRB- cm -RRB- 4124.031.027.52.0 Length of biceps -LRB- cm -RRB- a4130 .5039.5035.42.0 Length lower arm/radius -LRB- cm -RRB- b3324 .030.026.41.6 Length lower arm/palm -LRB- cm -RRB- c3330 .036.033.11.6 Initial skin temp . -LRB- ˚C -RRB- 4130.833.832.3.8 Temp . upper arm before dyn . tests -LRB- ˚C -RRB- 3030.834.232.3.9 Temp . upper arm after dyn . tests -LRB- ˚C -RRB- 3032.235.733.91.1 Room temp . -LRB- ˚C -RRB- 4120.024.022.31.0 a From the coracoid to the elbow creaseb From the lateral epicondyle to the wrist creasec From the lateral epicondyle to the middle of the palm Static tests Mean muscle fibre conduction velocity -LRB- CV -RRB- Histograms in Fig. 1 show an example of a PVs ' population in one subject during the static tests at three levels of force : unloaded , 10 and 20 % MVC . Figure 2a presents the averages of the CVs over 41 subjects , calculated from the subject 's PVs . The CV of the unloaded test was the lowest -LRB- 3.92 ± SD 0.27 m s − 1 -RRB- and it increased with augmenting force levels -LRB- effect of force P < 0.001 -RRB- . A positive correlation existed between the CVs of unloaded test with 10 % MVC , and 10 % with 20 % MVC -LRB- all r > 0.505 , P < 0.05 -RRB- . Fig. 1Distribution of peak velocities of one subject during short static tests at three force levels : unloaded , and loaded 10 and 20 % MVC . Note the shift of the velocities as a whole to the higher values with increasing level of forceFig . 2Behaviour of peak velocities -LRB- PVs -RRB- as effect of force , expressed with four parameters . a Mean conduction velocity -LRB- CV -RRB- ; b skewness of within-subject PVs ; c SD of within-subject PVs -LRB- SD -RRB- ; and d number of peaks per second -LRB- peak frequency = PF -RRB- . Averages and standard errors are given , obtained from 41 subjects in short static tests at three levels of force : unloaded , and loaded 10 and 20 % of maximal voluntary contraction . With increasing forces , the CV and amount of activity -LRB- PF -RRB- increases , accompanied with augmenting proportion of fast peaks -LRB- the skewness value diminishes -RRB- . However , the spread of peak velocities within an individual -LRB- SD -RRB- does not change Skewness of peak velocities -LRB- Sk-PV -RRB- Figure 2b presents the averages of the within-subject PVs ' skewnesses over 41 subjects -LRB- see also the histograms of PVs in Fig. 1 -RRB- . In all the tests a moderate positive skewness was found , which indicates a relative excess of lower velocities at the distribution scale . With increasing force the Sk-PV significantly diminished , indicating that the proportion of higher velocities increased -LRB- effect of force P < 0.05 -RRB- . The PVs ' population as a whole shifted towards higher values with increasing forces too , which can be seen on the histograms in Fig. 1 . Standard deviation of peak velocities -LRB- SD-PV -RRB- The averages of the within-subject 's standard deviations of PVs are shown in Fig. 2c . The SD-PV did not change when the force levels increased -LRB- effect of force n.s. -RRB- . Peak frequency -LRB- PF -RRB- Figure 2d shows the averages of PF in the static tests . The PF increased with increasing forces -LRB- effect of force P < 0.001 -RRB- . Dynamic tests Muscle fibre conduction velocity -LRB- CV -RRB- Histograms in Fig. 3 show the PVs estimated from one subject during dynamic tests at four levels of force : unloaded , loaded with 5 , 10 and 20 MVC . Figure 4a shows the averages of CV calculated from the subject 's PVs over 30 subjects . As in the static tests , the initial CV of the dynamic tests increased with level of force -LRB- effect of force P < 0.001 -RRB- . Further , a positive correlation was found between the CV of the static tests and the initial CV of the respective dynamic tests -LRB- all r > 0.409 , P < 0.05 -RRB- . The lowest initial CV was that of the unloaded test with about 4 -LRB- 3.6 -- 4.5 -RRB- m s − 1 and the highest was at 20 % MVC with approximately 4.6 -LRB- 4.0 -- 5.15 -RRB- m s − 1 , increasing from the unloaded to 20 % MVC test with 14 ± 9.6 % . During all the dynamic tests the CV significantly declined -LRB- effects of time for the unloaded test P < 0.02 ; for other tests P < 0.001 -RRB- , whereby the decline was steeper with larger forces -LRB- interaction between force and time , P < 0.001 -RRB- . At the three lowest force levels -LRB- unloaded , and loaded 5 and 10 % MVC -RRB- , the CV had two phases : a decline phase lasting for about 120 -- 150 s and a steady phase continuing to the end of a test . The steady state values approached the level of the unloaded test . During the 20 % MVC test , however , the CV continued to decline , without a stable phase . Twelve of 30 subjects -LRB- 40 % -RRB- reported fatigue during the 20 % MVC test and terminated the task prematurely between 90 and 210 s . The CV decreased for the fatigued subjects from 4.6 ± 0.3 -LRB- 4.2 -- 5.0 -RRB- to 3.6 ± 0.3 -LRB- 3.0 -- 4.1 -RRB- m s − 1 and for the continuing subjects from 4.5 ± 0.3 -LRB- 4.0 -- 5.15 -RRB- m s − 1 to 3.8 ± 0.4 -LRB- 3.0 -- 4.7 -RRB- m s − 1 . Neither the absolute initial CV nor the end CV differed significantly between the groups -LRB- P = 0.497 and P = 0.124 , respectively -RRB- . However , the relative decline of the CV tended to be larger for the fatigued subjects compared with the continuing subjects , for fatigued being about − 20 -LRB- − 7 to − 35 -RRB- % and for continuing − 15 -LRB- +7 to − 28 -RRB- % ; P = 0.074 . Fig. 3Changes in the distribution of peak velocities -LRB- PVs -RRB- over time for different levels of force . The PVs are obtained from one subject -LRB- the same as in Fig. 1 for static tests -RRB- during prolonged dynamic contractions at four levels of force : unloaded , and loaded 5 , 10 and 20 % of maximal voluntary contraction -LRB- MVC -RRB- . Every histogram represents a number of PVs within a period of 14.4 s. Initially -LRB- at time zero -RRB- , a global shift of peak velocities is visible towards higher values when forces augment . During the unloaded , and loaded 5 and 10 % MVC tests , slower peaks are moderately increasing and faster peaks are diminishing . During the 20 % MVC test , the peak velocities shift considerably as a whole towards lower regions , and the amount of peaks visibly diminishesFig . 4Effects of force and time on the behaviour of peak velocities -LRB- PVs -RRB- , expressed with four parameters . a Mean conduction velocity -LRB- CV -RRB- ; b skewness of within-subject PVs ; c SD of within-subject PVs -LRB- SD -RRB- ; and d number of peaks per second -LRB- peak frequency = PF -RRB- . Averages and standard errors are given , obtained from 30 subjects during prolonged dynamic tests at four levels of load : unloaded , and loaded 5 , 10 and 20 % of maximal voluntary contraction -LRB- MVC -RRB- . Note the difference in the decline pattern of the CV and the PF : the mean muscle conduction velocity declines over time for all levels of force , while the amount of activity remains stable for the three lower force level tests -LRB- unloaded , and loaded 5 and 10 % MVC -RRB- . In the higher force level test -LRB- 20 % MVC -RRB- , the CV starts declining immediately , while the PF declines first gradually and later on steeply . Note the stable SD values from about 90 s for the three lower force tests , while the SD of the higher force test -LRB- 20 % MVC -RRB- clearly increases Skewness of peak velocities -LRB- Sk-PV -RRB- Histograms in Fig. 3 show the distribution of PVs of one subject and Fig. 4b presents the averages of the within-subject 's skewnesses of the PVs over 30 subjects . In the initial phase , consistent with the static tests , the PVs were most positively skewed -LRB- = skewed in favour of lower velocities -RRB- in the unloaded test , and the skewness diminished with increasing forces -LRB- effect of force P < 0.001 -RRB- . That means that lower PVs dominated in the unloaded test and the proportion of higher PVs increased when forces augmented . Thus , in the 20 % MVC test , the initial PVs approached a normal distribution . In addition , with increasing forces the PVs as a whole group seem to shift towards higher values , as can be seen in the histograms Fig. 3a -- d at time zero . During the tests , the skewness increased again , except for the unloaded test , indicating growing proportion of lower PVs over time and decreasing amount of higher PVs -LRB- histograms Fig. 3a -- c -RRB- . The larger the forces the steeper increase of skewness -LRB- for all tests together : effect of time P < 0.001 ; interaction between force and time P = 0.005 ; effect of time in unloaded test P = n.s. ; for the tests 5 , 10 and 20 % MVC interaction between force and time P < 0.001 -RRB- . During the last 2 min of the 5 and 10 % MVC tests the Sk-PV stabilized . In the 20 % MVC test , however , the positivity still tended to increase up to the end of the test -LRB- over the time windows 120 -- 180 and 180 -- 240 s : effect of time for the 5 , 10 and 20 % MVC tests , n.s. ; interaction between force and time , P = 0.106 ; for the 5 and 10 % MVC tests the effect of time , n.s. ; for 20 % MVC test over 120 -- 180 s n.s. , over 180 -- 240 s , P = 0.052 -RRB- . At the end of the 20 % MVC test , the whole population of PVs appeared to shift towards the lower values too , as can be seen at the last two histograms in the Fig. 3d . Taken together , in the initial phase of activity , the proportion of fast peaks increased with increasing force . In the prolonged tests loaded up to 10 % MVC , the proportion of fast peaks declined again over the first 2 min and then stabilized at about the level of the unloaded test . During the 20 % MVC test , however , the proportion of fast peaks still tended to decline up to the end of the test , accompanied with a growing amount of slow peaks . Standard deviation of peak velocities -LRB- SD-PV -RRB- The averages of SD-PVs of 30 subjects are presented in Fig. 4c . The initial SD-PV was for all force levels similar -LRB- P = 0.65 -RRB- , which resembled the static tests . The values in the dynamic tests were significantly higher compared with those of respective static tests -LRB- paired sample t test for the unloaded , 10 and 20 % MVC tests , respectively P = 0.014 ; P = 0.027 and P = 0.001 -RRB- . During the tests the SD-PV changed significantly with time , depending on the force level -LRB- for all tests effect of time P = 0.011 , interaction between force and time P < 0.001 -RRB- . The course of the SD-PV had two phases which were different for the three lower force levels -LRB- unloaded , 5 and 10 % MVC -RRB- , compared with 20 % MVC . In the three lower force levels , the SD-PV first declined over about 90 s and then stabilized -LRB- interaction between force and time over 0 -- 240 s P = n.s. ; effect of time over the time windows 0 -- 60 s P < 0.001 , 60 -- 120 s P = 0.075 ; 120 -- 180 and 180 -- 240 s for both P > 0.343 -RRB- . However , during the 20 % MVC test , the decline , which lasted for approximately 60 s , was followed by an extreme increase -LRB- effect of time over 0 -- 240 s P < 0.001 ; effect of time over 0 -- 60 s P = 0.019 ; over 60 -- 120 s , which was in opposite direction , P = 0.019 , and 120 -- 180 and 180 -- 240 s P < 0.05 -RRB- . This pattern of results can also be seen in the histograms Fig. 3d . Peak frequency -LRB- PF -RRB- Figure 4d shows averages of PF over 30 subjects . The initial PF rose with increasing force levels -LRB- effect of force P < 0.001 -RRB- . Then , during the tests at three lowest force levels -LRB- unloaded , 5 and 10 % MVC -RRB- the PF remained stable . But during the 20 % MVC test the PF significantly diminished , at the beginning gradually and from about 120 s steeply -LRB- interaction between force and time for all the four tests , P < 0.001 ; for the three lowest force levels , P = 0.234 ; effect of time for the three lowest force levels P = 0.541 ; effect of time for 20 % MVC P < 0.001 -RRB- . There was much variability among subjects in the size of decline in 20 % MVC test . For those who were able to complete the test , the PF continued to decline up to the end , with exception of one subject in whom the PF increased instead . At 240th s the PF of the continuing subjects was reduced by − 35 -LRB- − 78 to +3 -RRB- % . Discussion Changes in the distribution of MUP velocities as an effect of -LRB- low -RRB- force and duration were described with four parameters : -LRB- 1 -RRB- the global parameter of mean CV , -LRB- 2 -RRB- the within subject skewness of a population of MUP velocities ; -LRB- 3 -RRB- the within-subject standard deviation of MUP velocities and -LRB- 4 -RRB- the amount of MUP activity , expressed as MUP frequency . First we will comment on the four parameters . Next , using these parameters , we will discuss the main findings . The four parameters The CV parameter renders a mean value of the motor unit potentials ' propagation velocities . The CV will increase or decrease , depending on the type of the activated -LRB- fast-twitch and slow-twitch -RRB- motor units . It will also change with the alterations in muscle membrane potential , which influences the depolarisation/repolarisation processes . For example , it decreases in muscular fatigue -LRB- Stalberg 1966 ; Milner-Brown and Miller 1986 -RRB- , and increases with a smaller interstimulus interval , such as that due to the rising rate coding -LRB- Gydikov and Christova 1984 ; Radicheva et al. 1986 ; Nishizono et al. 1989 -RRB- . Because of the lack of studies , it is not possible to compare the CV values with those of any other position tasks experiments . However , the estimates are consistent with those of the studies using static and dynamic force tasks , especially with those of Lange et al. -LRB- 2002 -RRB- , obtained with the inter-peak latency method . Skewness is used as an sEMG parameter in the present study for the first time . This statistical measure of deviation from a normal distribution , in this case expresses the proportion between slower and faster MUPs within an individual . It will increase with the growing proportion of slow/tonic/fatigue resistant MUs and will decrease with the augmenting proportion of fast/phasic/fatigable MUs . All the estimates were moderately positively skewed , which indicates a relative excess of lower MUP velocities . The within-subject standard deviation of MUP velocities , a variable introduced by Lange et al. -LRB- 2002 -RRB- shows the spread of MUP velocities . For a fresh and healthy muscle , it will render information about diversity of the participating MUs . In a fatigued muscle , when membrane propagation is slowing , the SD-PV will broaden as a result of the temporal dispersion of velocities . Further , the SD-PV can be expected to narrow when the same velocities repeat , such as in a higher discharge rate of a certain group of MUs . In the brief static position tasks , the standard deviations were larger than those previously estimated by Lange and colleagues -LRB- 2002 -RRB- in the staticforce tasks , with respectively 0.55 -- 0.62 and 0.3 -- 0.52 m s − 1 . This difference can be due to the different type of tasks , as data are available suggesting that different excitatory/inhibitory inputs to the motor neurons play a part in the position tasks and the force tasks -LRB- Rudroff et al. 2005 -RRB- . In the present study , the SD-PVs of the dynamic tests were larger than those of the static tests . This difference can have different explanations . With every contraction of a dynamic cycle , the muscle fibres ' diameter increases , leading to higher fibre propagation velocities -LRB- in a part of a cycle -RRB- -LRB- Arendt-Nielsen et al. 1992 -RRB- . This problem was partially restrained by using a small movement angle of 45 ° . However , the most important role in the increase of SD-PVs ' during dynamic contractions might be played by the cyclic changes in the motor units ' discharge characteristics . Several studies deliver the supporting data . For example , during dynamic contractions the amount of activity differs between the concentric and eccentric phases , suggesting different regulatory strategies for the two phases -LRB- Potvin 1997 -RRB- . Previous studies have also shown that the rate of MU discharge is related to the movement 's velocity , and the -LRB- angle -RRB- velocity alters depending on the elbow angle -LRB- Gillis 1972 ; Milner-Brown et al. 1973a ; Potvin 1997 -RRB- . Thus , the discharge rates will alter through a cycle . In addition , eccentric movements are shown to further the activation of high-threshold -LRB- fast propagating -RRB- motor units -LRB- Komi and Tesch 1979 ; Nardone et al. 1989 -RRB- . Peak frequency -LRB- MUP frequency -RRB- expresses the amount of MUPs in a time . To our knowledge , it is used as sEMG parameter in the present study for the first time . The PF is comparable with the zero crossing parameter -LRB- Lynn 1979 ; Masuda et al. 1982 ; Hagg 1981 -RRB- . It is argued that the diminishing zero crossings ' number during prolonged exercises indicates decrease in MUs ' activity , as a sign of fatigue -LRB- Inbar et al 1986 ; Hagg and Suurküla 1991 -RRB- . Lange et al. -LRB- 2002 -RRB- mentioned a number of MUPs obtained during 1.5 s measurements in static force tasks , which renders the frequency of about 4 -- 5 MUPs/s for 10 % MVC test , and about 8 MUPs/s for 20 % MVC test . These values are much lower than ours with 37 , 47 and 50 MUPs/s -LRB- for respectively unloaded , loaded 10 and 20 % MVC tests -RRB- in static position tasks . The findings are consistent with the interpretation that during position tasks more motor units are being recruited compared with force tasks , and the discharge rate of MUs is higher -LRB- Mottram et al. 2005a -RRB- . The initial changes on increasing force levels -LRB- in the static and dynamic tests -RRB- The effects of force on the behaviour of MUPs in the short static tests and the initial phase of the prolonged dynamic tests were similar . With increasing forces the CV grew higher and MUP frequency increased . In the population of MUP velocities , not only the proportion of fast MUPs increased -LRB- see the skewness in Fig. 2b -RRB- , but also the velocities as a whole shifted towards higher values -LRB- histograms in Figs. 1 , 3a -- d at time zero -RRB- . Despite of the changes in the skewness , the standard deviation of MUP velocities remained unaltered . The increases of the CV with increasing force are in accordance with the previous findings inforce tasks -LRB- Naeije and Zorn 1983 ; Broman et al. 1985 ; Sadoyama and Masuda 1987 ; Zwarts and Arendt-Nielsen 1988 ; Lange et al. 2002 -RRB- . It is generally accepted that these increases are caused by activating high threshold/fast/phasic motor units when demands of force are augmented -LRB- Henneman et al. 1965 ; Milner-Brown et al. 1973b ; Gantchev et al. 1992 ; Gazzoni et al. 2001 -RRB- . This explanation was supported by the increasing proportion of fast MUPs found in the present study . However , the global shift of MUP velocities towards higher values may be induced by either replacing slow MUs by fast ones , or by increasing the propagation velocity of the muscle membrane due to the rising rate coding -LRB- Radicheva et al. 1986 ; Nishizono et al. 1989 ; Van der Hoeven and Lange 1994 -RRB- . Little is known about changes in the within-subject standard deviation of MUP velocities . Only Lange and colleagues -LRB- 2002 -RRB- mentioned -LRB- inforce tasks -RRB- , contrary to our results , increases between 10 and 50 % MVC -LRB- and no increases between 50 and 100 % -RRB- . The experiments of Lange et al. and the present short static experiments were both isometric , and the duration of the contraction did not differ much -LRB- our 3.8 vs. their 1.5 s -RRB- . In fact , the two studies only differed in the type of task -LRB- position tasks applied by us vs. force tasks by Lange et al. -RRB- . This task difference may play a part in the discrepancy of the standard deviation , as the excitatory and inhibitory inputs for the two tasks are supposed to be different -LRB- Rudroff et al. 2005 -RRB- . Thus , for the two tasks different types of motor units -LRB- with different velocities -RRB- may be activated . In short , increases of mean CV with increasing forces in the initial phase of muscle activity may be a result of both recruitment of fast/phasic motor units and faster membrane propagation . Changes in the prolonged dynamic tests Tests loaded below 20 % MVC The main feature of the prolonged tests loaded 5 and 10 % MVC were changes in the CV , skewness and standard deviation over the first 90 -- 120 s , followed by stabilizing . Thus , the CV first declined -LRB- with steeper decline for higher forces -RRB- and then stabilized at approximately the level of the unloaded test -LRB- Fig. 4a -RRB- . The MUP velocities , which at the beginning of tests were shifted towards the higher values with increasing forces , re-shifted over the tests back to the lower values -LRB- skewness variable in Fig. 4b and histograms in Fig. 3a -- c -RRB- . Subsequently , the MUP velocities stabilized nearly at the level of the unloaded test too . The standard deviation narrowed first and later on stabilized at a new level -LRB- Fig. 4c -RRB- . The MUP frequency held steady over these tests at the primary level determined by the used force -LRB- Fig. 4d -RRB- . We suggest that this pattern of results may reflect an emerging equilibrium between phasic and tonic MU activity . No comparison is possible between the parameters used here and those in any other prolonged position tasks study . The decline of CV during contractions at low forces was in contradiction with the studies in static force tasks , which reported increases during sustained isometric contractions at forces of 10 -- 25 % MVC -LRB- Zwarts and Arendt-Nielsen 1988 ; Arendt-Nielsen et al. 1989 ; Krogh-Lund and Jorgensen 1991 ; Krogh-Lund and Jorgensen 1992 ; Krogh-Lund 1993 -RRB- . This discrepancy may be caused by either/or both the different contraction types -LRB- static vs. dynamic -RRB- or different tasks -LRB- force vs. position tasks -RRB- . Increases of the CV in prolonged isometric contractions at low force levels are supposed to be due to the recruitment of fast -LRB- anaerobic -RRB- MUs in response to the hindered blood flow -LRB- Crenshaw et al. 1997 ; Zwarts et al 1987 -RRB- . In the dynamic conditions , the blood supply is assumed to be undisturbed , so the aerobic MUs can be activated . The decline of the CV followed by stability , along with the changes in the skewness , suggest that , within the cyclically fluctuating activity , the global amount of initially recruited fast/fatigable/anaerobic MUs may successively diminish and the proportion of slow/fatigue resistant/aerobic MUs may augment . The maintaining activity of slow MUs is in accordance with the hypothesis that tonic/fatigue resistant -LRB- aerobic -RRB- MUs remain active through the whole muscular action -LRB- Grimby and Hannerz 1968 ; Hagg and Suurkula 1991 -RRB- . On the other hand , the position character of the present tasks may have contributed to the discrepancy between the decline of CV in the present experiments and increases in previous studies , as firstly , the discharge characteristics of the same motor unit differ between the force and position tasks , and secondly , motor units show greater discharge adaptation during the position tasks -LRB- Mottram et al. 2005a , b ; MacGilles et al. 2003 -RRB- . The evolution of the standard deviation parameter , with its narrowing followed by stabilizing , fits in with the idea that the rate coding may temporarily increase and consecutively adapt , resulting in a new balance . The MUP frequency variable , expressing the amount of MU activity produced as a result of recruitment and rate coding did not change during these tests . This suggests that all the changes in recruitment and rate coding do not , in principle , affect the total amount of MU activity . All subjects were able to complete the tests , and the sEMG parameters became stable in the course of time as well . Thus , one can assume that the three tests at lowest force levels were non-fatiguing . Taken together , the results of these apparently non-fatiguing dynamic position tasks suggest that following the initially increased activation of fast MUs , the proportion shifts after about 2 min in favour of slower MUs . The amount of activity seems to remain stable throughout the duration of the tests . The test at 20 % MVC The changes encountered during the prolonged test at 20 % MVC differed clearly from those at lower forces -LRB- Fig. 4 -RRB- . During the 20 % MVC test , the CV dropped below the level of the unloaded test . At the same time the proportion of low MUP velocities increased -LRB- skewness increased -RRB- , and finally the velocities ' population made a global move from higher towards lower values , while their standard deviation broadened clearly . The MUP frequency , in contrast with that of non-fatiguing tests , progressively diminished -LRB- histogram in Figs. 3d , 4d -RRB- . The CV 's decreases were in accordance with those shown by Farina et al. -LRB- 2004 -RRB- during fatiguing dynamic force tasks of vastus medialis muscle . The behaviour of the skewness and standard deviation suggests that a large majority of the MUPs became extremely slow at the end . This general slowness best matches the slowing of the muscle membrane propagation , which is generally accepted as sign of muscular fatigue -LRB- Milner-Brown et al. 1986 ; Miller et al. 1987 -RRB- . The diminishing MUP frequency can be due to the synchronisation of discharges -LRB- Datta and Stephens 1990 ; Semler and Nordstorm 1999 -RRB- and diminishing motor unit activity -LRB- Hagg 1981 ; Hagg and Suurkulla 1991 -RRB- . The non-linear decline pattern of MUP frequency , first slow and later on steep , suggests successively exhausting available MUs ' reserves , resulting in lower firing frequencies and failing recruitment . Accordingly , many subjects reported fatigue and stopped exercising . In short , during these apparently fatiguing dynamic position tasks , a global slowing of MUP velocities appears , suggesting a fatigued muscle membrane . The amount of MU activity seems to diminish progressively and finally the recruitment stops . Some aspects of the method must be explained . The load was applied to the palm , whereas the MVC was assessed from the wrist . By applying load to the palm , we intended to mimic natural circumstances , such as holding something in the hand . Assessment of the MVC from the palm was not feasible , however , due to the relative weakness of the wrist 's flexors compared with the elbow flexors , which influenced the estimates . A reasonable alternative was to measure the MCV from the wrist . Distances measured over the forearm and the palm -LRB- Table 1 -RRB- enabled calculation of the real exerted load torque , which was about 20 -- 25 % larger than the used one . The MVC was assessed at the elbow angle of 90 ° despite performing the tests at the angle of 135 ° . The reason was that , when applying the dynamometer at the wrist with high forces , the elbow angle being at 135 ° , subjects tended to overstretch the wrist and experienced pain . This was not the case at the 90 ° angle . The MVC values at 135 and 90 ° were similar , which was consistent with the findings of Philippou et al. -LRB- 2004 -RRB- , so it was chosen for assessments at 90 ° . In conclusion , we present a set of parameters derived from the interpeak latency method , which yields information about changes in MUP velocities ' distribution and amount of MUP activity . Skewness , standard deviation and peak frequency parameters appear to corroborate the results of a global muscle conduction velocity . Together they could contribute to quantifying the dynamics of motor unit activity and membrane fatigue . The interconnected results may be useful in ergonomics -LRB- for assessment of fatigue -RRB- and in sports -LRB- for eliciting specific capabilities , such as explosive or endurance capabilities -RRB- .

O O B I I O O O O O O O O O O O O O O O O O B O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

motor unit potential
surface electromyography
position task
skewness

conduction velocity distribution
dynamic contraction


Best

practice

guidelines

for

the

molecular

genetic

diagnosis

of

maturity-onset

diabetes

of

the

young

Aims/hypothesis

Mutations

in

the

GCK

and

genes

are

the

most

common

cause

of

the

monogenic

forms

of

diabetes

known

as

maturity-onset

diabetes

of

the

young

GCK

encodes

the

glucokinase

enzyme

which

acts

as

the

pancreatic

glucose

sensor

and

mutations

result

in

stable

mild

fasting

hyperglycaemia

A

progressive

insulin

secretory

defect

is

seen

in

patients

with

mutations

in

the

and

genes

encoding

the

transcription

factors

hepatocyte

nuclear

alpha

and

alpha

A

molecular

genetic

diagnosis

often

changes

management

since

patients

with

GCK

mutations

rarely

require

pharmacological

treatment

and

mutation

carriers

are

sensitive

to

sulfonylureas

These

monogenic

forms

of

diabetes

are

often

misdiagnosed

as

type

or

diabetes

Best

practice

guidelines

for

genetic

testing

were

developed

to

guide

testing

and

reporting

of

results

Introduction

Maturity-onset

diabetes

of

the

young

-LRB-

MODY

-RRB-

describes

the

dominantly

inherited

disorder

of

non-insulin-dependent

diabetes

typically

diagnosed

before

years

that

was

first

recognised

by

Tattersall

-LSB-

-RSB-

MODY

is

the

most

common

form

of

monogenic

diabetes

accounting

for

an

estimated

of

diabetes

in

Europe

-LSB-

-RSB-

but

is

often

misdiagnosed

as

type

or

type

diabetes

The

term

MODY

is

used

to

describe

a

group

of

clinically

heterogeneous

often

non-insulin-dependent

forms

of

diabetes

that

are

defined

at

the

molecular

genetics

level

by

mutations

in

different

genes

All

show

dominant

inheritance

and

are

disorders

of

beta

cell

dysfunction

but

variable

features

include

the

age

at

onset

severity

of

the

hyperglycaemia

-LRB-

and

hence

risk

of

complications

-RRB-

and

associated

clinical

features

The

most

recent

classification

of

diabetes

by

the

American

Diabetes

Association

and

the

World

Health

Organization

recognises

these

discrete

subtypes

of

MODY

-LSB-

-RSB-

Mutations

in

the

GCK

and

genes

are

the

most

frequent

cause

of

MODY

in

all

populations

studied

They

account

for

approximately

of

cases

-LRB-

see

Table

-RRB-

The

ratio

of

GCK

to

mutations

varies

between

countries

because

of

different

recruitment

strategies

for

genetic

testing

blood

glucose

screening

in

young

asymptomatic

individuals

will

identify

a

higher

proportion

of

GCK

mutations

Table

in

which

mutations

cause

MODY

Gene

symbol

-LRB-

other

symbol

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

ProteinGlucokinaseHepatocyte

nuclear

alphaHepatocyte

nuclear

alphaInsulin

promoter

differentiation

nuclear

betaChromosome

Accession

no.NM

OMIM

-LRB-

Gene

-RRB-

-LRB-

Phenotype

-RRB-

frequency

-LRB-

-RRB-

-LRB-

not

known

in

of

cases

-RRB-

Genbank

reference

sequence

refers

to

the

full

length

transcript

which

uses

exons

and

and

not

For

the

full

length

transcript

which

includes

exon

there

is

a

human

mRNA

sequence

but

no

refseq

The

convention

is

to

use

for

exons

and

with

for

exon

only

mutation

descriptions

in

the

literature

use

the

translation

start

codon

reported

by

Chartier

et

al.

in

-LSB-

-RSB-

However

an

alternative

start

codon

nine

amino

acids

upstream

was

proposed

in

-LSB-

-RSB-

and

this

alternative

start

codon

is

used

in

Hence

the

convention

is

to

report

mutations

using

the

amino

acid

methionine

at

codon

in

as

the

start

codon

with

the

A

of

this

codon

as

the

first

nucleotideaNomenclature

for

the

gene

is

complicated

because

the

gene

encodes

nine

isoforms

expressed

from

two

promoters

The

liver-specific

promoter

drives

the

expression

of

transcripts

which

include

exons

and

and

transcripts

which

include

exons

and

Transcripts

are

expressed

from

the

pancreatic

-LRB-

-RRB-

promoter

located

approximately

kb

upstream

of

the

transcription

start

site

and

exhibit

splicing

of

the

upstream

exon

to

exon

without

the

inclusion

of

sequences

from

either

exons

or

Heterozygous

loss-of-function

GCK

mutations

result

in

mild

stable

hyperglycaemia

from

birth

Microvascular

complications

are

rare

reflecting

the

fact

that

is

normally

just

above

the

upper

limit

of

the

normal

range

Treatment

with

oral

hypoglycaemic

agents

or

insulin

is

not

needed

because

it

rarely

changes

-LSB-

-RSB-

A

genetic

diagnosis

is

important

for

the

small

number

of

children

misdiagnosed

with

type

diabetes

and

treated

with

insulin

-LSB-

-RSB-

The

identification

of

GCK

mutations

in

women

with

gestational

diabetes

can

be

useful

for

obstetric

management

since

their

babies

who

do

not

inherit

the

mutation

are

at

risk

of

macrosomia

-LSB-

-RSB-

and

it

can

guide

follow-up

in

the

mothers

Transcription

factor

mutations

in

the

or

genes

cause

a

similar

progressive

diabetic

phenotype

although

the

penetrance

of

mutations

is

lower

-LRB-

S.

Ellard

and

A.

T.

Hattersley

unpublished

data

-RRB-

Sensitivity

to

sulfonylureas

means

that

some

patients

can

transfer

from

insulin

to

oral

agents

-LSB-

-RSB-

A

low

renal

threshold

for

glucose

is

a

feature

of

mutations

-LSB-

-RSB-

and

may

provide

a

useful

method

of

screening

at-risk

family

members

during

childhood

-LSB-

-RSB-

Mutations

identified

in

the

GCK

and

genes

include

missense

nonsense

splicing

small

deletions/insertions/duplications

and

splice

site

and

promoter

region

mutations

-LSB-

-RSB-

Partial

and

whole

deletions

have

recently

been

reported

in

and

GCK

-LSB-

-RSB-

The

location

of

mutations

within

the

gene

influences

the

age

at

diagnosis

the

average

age

at

diagnosis

for

patients

with

exon

mutations

that

affect

all

three

isoforms

is

younger

than

for

those

with

mutations

in

exons

that

affect

only

isoform

-LRB-

A

-RRB-

-LSB-

-RSB-

Rarer

forms

of

MODY

include

heterozygous

mutations

in

-LRB-

also

known

as

-LSB-

-RSB-

-RRB-

and

-LSB-

-RSB-

but

analysis

of

these

genes

is

not

usually

included

in

routine

molecular

genetic

testing

for

MODY

Dominantly

inherited

syndromic

forms

of

diabetes

may

also

be

described

as

MODY

subtypes

The

renal

cysts

and

diabetes

syndrome

results

from

mutations

and

other

features

include

renal

abnormalities

female

genital

malformations

hyperuricaemia

pancreatic

atrophy

and

abnormal

liver

function

tests

-LSB-

-RSB-

Mutations

in

the

CEL

variable

number

tandem

repeat

cause

a

syndrome

of

diabetes

and

pancreatic

exocrine

dysfunction

-LSB-

-RSB-

These

syndromes

and

maternally

inherited

diabetes

and

deafness

caused

by

the

mitochondrial

m.

G

mutation

are

not

included

in

these

guidelines

since

testing

is

guided

by

the

non-endocrine

pancreatic

or

extra-pancreatic

clinical

features

A

molecular

genetic

diagnosis

of

a

GCK

or

mutation

is

important

because

it

confirms

a

diagnosis

of

MODY

classifies

the

subtype

predicts

the

likely

clinical

course

and

may

change

the

patient

's

treatment

First-degree

relatives

will

be

at

risk

of

inheriting

the

mutation

and

asymptomatic

individuals

may

be

offered

predictive

genetic

testing

-LRB-

after

appropriate

genetic

counselling

-RRB-

in

order

to

provide

reassurance

-LRB-

for

those

shown

not

to

carry

the

mutation

-RRB-

or

regular

blood

glucose

monitoring

with

early

diagnosis

and

appropriate

treatment

-LRB-

for

mutation

carriers

-RRB-

Methods

A

group

of

European

clinicians

and

scientists

met

on

May

at

a

workshop

to

formulate

best

practice

guidelines

for

molecular

genetic

testing

in

MODY

Discussions

focused

on

clinical

criteria

for

selection

of

patients

for

testing

methodologies

interpretation

of

results

and

reporting

those

results

to

the

referring

clinicians

A

draft

document

was

posted

on

August

and

an

online

editing

tool

was

used

by

participants

to

produce

consensus

guidelines

Results

Clinical

criteria

for

testing

Mild

fasting

hyperglycaemia

testing

for

GCK

mutations

The

finding

of

raised

fasting

blood

glucose

in

the

range

of

mmol/l

is

unusual

in

children

and

young

adults

This

always

raises

concern

that

they

may

be

about

to

develop

type

diabetes

or

the

patient

has

type

diabetes

However

a

considerable

proportion

of

young

non-obese

patients

with

persistent

mild

fasting

hyperglycaemia

will

have

a

heterozygous

mutation

in

the

GCK

gene

In

a

cohort

of

children

with

incidental

hyperglycaemia

had

GCK

mutations

-LSB-

-RSB-

The

phenotype

associated

with

GCK

mutations

is

remarkably

similar

for

all

mutations

The

following

features

suggest

a

diagnosis

of

a

GCK

mutation

The

fasting

hyperglycaemia

is

mmol/l

-LRB-

patients

-RRB-

persistent

-LRB-

at

least

three

separate

occasions

-RRB-

and

stable

over

a

period

of

months

or

years

-LSB-

-RSB-

is

typically

just

above

the

upper

limit

of

normal

and

rarely

exceeds

In

an

OGTT

the

increment

-LSB-

-LRB-

h

glucose

-RRB-

-LRB-

fasting

glucose

-RRB-

-RSB-

is

small

-LRB-

of

patients

in

the

large

European

study

reported

by

Stride

et

al.

-LSB-

-RSB-

had

an

increment

mmol/l

-RRB-

An

increment

of

mmol/l

is

often

used

to

prioritise

testing

and

corresponds

to

the

centile

-LRB-

S.

Ellard

and

A.

T.

Hattersley

unpublished

data

-RRB-

Parents

may

have

type

diabetes

with

no

complications

or

may

not

be

diabetic

On

testing

one

parent

will

usually

have

a

mildly

raised

fasting

blood

glucose

-LRB-

range

of

mmol/l

-RRB-

unless

the

mutation

has

arisen

de

novo

Testing

of

apparently

unaffected

parents

fasting

glucose

is

important

when

considering

a

diagnosis

of

a

glucokinase

mutation

Gestational

diabetes

testing

for

GCK

mutations

Mutations

of

GCK

cause

mild

fasting

hyperglycaemia

throughout

life

and

this

is

often

diagnosed

during

pregnancy

when

routine

testing

is

performed

Since

these

patients

have

consistently

raised

fasting

blood

glucose

levels

their

babies

who

do

not

inherit

the

mutation

may

be

macrosomic

-LSB-

-RSB-

The

diagnosis

of

a

GCK

mutation

is

important

not

only

as

the

child

may

subsequently

be

picked

up

as

having

a

raised

fasting

blood

glucose

and

this

may

lead

to

concern

about

type

diabetes

but

also

because

the

guidelines

given

to

the

mother

are

different

from

the

normal

pre-type

diabetic

phenotype

as

they

will

not

deteriorate

with

time

The

following

criteria

identify

when

GCK

testing

is

appropriate

-LSB-

-RSB-

Persistently

raised

fasting

blood

glucose

in

the

range

of

mmol/l

before

during

and

after

pregnancy.An

increment

of

mmol/l

on

at

least

one

OGTT

-LRB-

either

during

or

after

pregnancy

-RRB-

A

parent

may

have

mild

type

diabetes

but

often

this

has

not

been

detected

and

so

the

absence

of

family

history

should

not

exclude

the

diagnosis

Children

and

young

adults

with

diabetes

and

a

strong

family

history

of

diabetes

testing

for

mutations

The

possibility

of

monogenic

diabetes

should

be

considered

whenever

a

parent

has

diabetes

even

if

they

are

thought

to

have

type

or

type

diabetes

The

most

common

form

of

MODY

is

caused

by

mutations

The

clinical

characteristics

of

patients

with

mutations

include

Young-onset

diabetes

-LRB-

typically

before

years

old

in

at

least

one

family

member

-RRB-

Non-insulin-dependent

outside

the

normal

honeymoon

period

-LRB-

years

-RRB-

e.g.

not

developing

ketoacidosis

in

the

absence

of

insulin

good

glycaemic

control

on

less

than

the

usual

replacement

dose

of

insulin

or

detectable

C-peptide

measured

when

on

insulin

with

glucose

mmol/l

Family

history

of

diabetes

-LRB-

at

least

two

generations

-RRB-

This

may

be

insulin

treated

and

considered

to

be

type

diabetes

or

type

diabetes

At

least

two

individuals

within

the

family

would

typically

be

diagnosed

in

their

or

There

may

also

be

an

affected

grandparent

although

often

these

are

diagnosed

after

years

OGTTs

in

early

stages

tend

to

show

a

very

large

glucose

increment

usually

mmol/l

-LSB-

-RSB-

Some

individuals

may

have

a

normal

fasting

level

but

a

value

within

the

diabetic

range

at

h.The

absence

of

pancreatic

islet

autoantibodies.Glycosuria

at

blood

glucose

levels

mmol/l

is

often

seen

as

these

patients

have

a

low

renal

threshold

-LSB-

-RSB-

Marked

sensitivity

to

sulfonylureas

resulting

in

hypoglycaemia

despite

poor

glycaemic

control

before

starting

sulfonylureas

-LSB-

-RSB-

Several

features

suggesting

monogenic

diabetes

rather

than

a

diagnosis

of

young-onset

type

diabetes

should

be

considered

no

marked

obesity

or

evidence

of

insulin

resistance

in

diabetic

family

members

absence

of

acanthosis

nigricans

and

whether

the

family

is

from

an

ethnic

background

with

a

low

prevalence

of

type

diabetes

-LRB-

e.g.

of

European

descent

-RRB-

Children

and

young

adults

with

diabetes

and

a

strong

family

history

of

diabetes

testing

for

mutations

Diabetes

caused

by

mutations

in

the

gene

is

considerably

less

common

-LRB-

Table

-RRB-

than

mutations

The

clinical

characteristics

are

similar

except

there

is

not

a

low

renal

threshold

and

the

age

of

diagnosis

may

be

later

-LSB-

-RSB-

mutations

should

be

considered

when

analysis

does

not

detect

a

mutation

but

the

clinical

features

are

strongly

suggestive

of

Patients

are

often

sensitive

to

sulfonylureas

-LSB-

-RSB-

mutations

are

associated

with

macrosomia

-LRB-

approximately

of

mutation

carriers

-RRB-

and

transient

neonatal

hypoglycaemia

-LRB-

approximately

of

mutation

carriers

-RRB-

-LSB-

-RSB-

The

possibility

of

mutations

should

be

considered

when

diabetic

family

members

have

marked

macrosomia

-LRB-

kg

at

term

-RRB-

or

if

diazoxide-responsive

neonatal

hyperinsulinism

has

been

diagnosed

in

the

context

of

familial

diabetes

Babies

with

diazoxide-responsive

neonatal

hyperinsulinaemic

hypoglycaemia

and

a

strong

family

history

of

diabetes

testing

for

mutations

Mutations

of

are

a

cause

of

neonatal

hypoglycaemia

that

remits

during

infancy

or

early

childhood

with

diabetes

developing

later

in

life

-LSB-

-RSB-

Macrosomic

babies

with

diazoxide-responsive

hyperinsulinism

and

a

strong

family

history

of

diabetes

-LRB-

see

characteristic

in

the

section

above

entitled

Children

and

young

adults

with

diabetes

and

a

strong

family

history

of

diabetes

testing

for

mutations

-RRB-

should

be

considered

for

mutation

screening

Testing

methodology

The

mutation

screening

methodology

should

be

described

in

the

report

-LSB-

e.g.

sequencing

denaturing

high-performance

liquid

chromatography

-LRB-

dHPLC

-RRB-

conformation-sensitive

capillary

electrophoresis

-LRB-

CSCE

-RRB-

-RSB-

together

with

the

sensitivity

PCR

primers

should

be

checked

for

primer

binding

site

single

nucleotide

polymorphisms

-LRB-

SNPs

a

useful

tool

is

available

at

-RRB-

Gene

dosage

analysis

may

be

useful

if

a

diagnosis

of

MODY

is

strongly

suspected

and

no

mutation

is

found

on

mutation

screening

Interpretation

of

results

The

textbox

includes

recommended

interpretations

for

the

most

common

reporting

scenarios

Reporting

Each

laboratory

has

its

own

reporting

format

and

general

guidance

on

reporting

is

available

from

the

European

Molecular

Genetics

Quality

Network

-LRB-

-RRB-

the

UK

Clinical

Molecular

Genetics

Society

-LRB-

-RRB-

and

the

Swiss

Society

of

Medical

Genetics

-LRB-

-RRB-

A

one

page

report

is

the

preferred

format

The

report

should

state

the

methodology

and

specify

the

gene

exons

and/or

mutations

tested

for

If

promoter

sequences

are

examined

then

the

report

should

specify

the

nucleotides

analysed

An

estimation

of

the

assay

sensitivity

is

particularly

useful

for

pre-screening

techniques

such

as

dHPLC

CSCE

etc.

The

use

of

mutation

nomenclature

approved

by

the

Human

Genome

Variation

Society

-LRB-

-RRB-

is

strongly

recommended

The

gene

accession

number

-LRB-

with

version

-RRB-

is

required

in

order

to

describe

mutations

unambiguously

-LRB-

see

Table

-RRB-

The

A

nucleotide

of

the

ATG

start

codon

is

numbered

Reports

describing

novel

variants

should

state

that

the

variant

is

novel

and

include

the

evidence

in

support

of

pathogenicity

This

might

include

the

absence

from

a

large

series

of

ethnically

matched

controls

or

MODY

patients

-LRB-

testing

of

normal

chromosomes

is

necessary

to

achieve

at

least

power

to

detect

a

polymorphism

present

in

of

the

population

-LSB-

-RSB-

-RRB-

Testing

of

other

affected

relatives

is

recommended

in

order

to

check

for

co-segregation

and

to

calculate

the

LOD

score

in

suitable

sized

pedigrees

-LRB-

LOD

scores

of

or

are

suggestive

or

conclusive

of

linkage

respectively

-RRB-

For

missense

variants

the

evidence

for

pathogenicity

might

include

conservation

across

species

and

a

significant

amino

acid

substitution

Several

programs

are

available

that

predict

the

pathogenicity

of

a

missense

variant

based

upon

amino

acid

conservation

-LRB-

SIFT

-RRB-

or

the

structure

and

function

of

the

protein

-LRB-

PolyPHEN

-RRB-

but

they

should

be

used

to

supplement

other

pieces

of

evidence

rather

than

in

isolation

Both

missense

and

silent

variants

can

affect

splicing

if

the

mutation

is

within

an

exon

splicing

enhancer

or

exon

splicing

silencer

Splice

predictor

software

programs

-LRB-

or

-RRB-

may

aid

interpretation

Base

substitutions

affecting

the

conserved

splice

donor

-LRB-

GT

-RRB-

site

splice

acceptor

-LRB-

AG

-RRB-

site

or

the

conserved

A

nucleotide

within

the

branch

site

are

highly

likely

to

be

pathogenic

but

splice

predictor

software

may

be

useful

in

the

interpretation

of

other

intronic

variants

Analysis

of

patient

mRNA

is

often

informative

but

lymphoblastoid

cell

lines

are

usually

required

because

of

the

low

levels

of

expression

of

the

MODY

genes

in

blood

Sequence

analysis

of

RT-PCR

products

amplified

from

lymphoblastoid

cell

mRNA

has

demonstrated

exon

skipping

retention

and

the

use

of

cryptic

splice

sites

for

a

variety

of

intronic

mutations

in

the

GCK

and

genes

-LSB-

-RSB-

Novel

promoter

variants

may

be

investigated

by

examination

of

known

transcription

binding

sites

or

by

in

vitro

transfection

experiments

-LSB-

-RSB-

They

may

also

alter

mRNA

expression

levels

which

may

be

measured

by

allele-specific

real-time

PCR

-LSB-

-RSB-

Polymorphisms

Some

laboratories

include

details

of

polymorphisms

detected

in

the

report

The

reasons

for

doing

this

include

-LRB-

-RRB-

making

all

data

available

to

the

requesting

clinician

based

on

the

rationale

that

a

polymorphism

may

later

be

reclassified

as

a

mutation

and

-LRB-

-RRB-

identifying

heterozygous

SNPs

excludes

a

gene

deletion

involving

the

exon

-LRB-

s

-RRB-

in

question

However

this

information

can

cause

confusion

or

even

misinterpretation

of

the

result

-LRB-

A.

T.

Hattersley

unpublished

data

-RRB-

and

it

certainly

adds

to

the

length

of

the

report

While

in

some

cases

there

may

be

reports

in

the

literature

of

an

association

with

type

diabetes

or

reduced

insulin

secretion

these

polymorphisms

do

not

cause

MODY

and

we

recommend

that

they

should

be

excluded

from

the

report

Treatment

Individual

treatment

recommendations

are

outside

the

jurisdiction

of

a

molecular

genetics

report

since

this

is

the

referring

clinician

's

responsibility

It

is

useful

to

include

an

appropriate

reference

if

there

is

evidence

in

the

literature

for

a

particular

treatment

-LRB-

e.g.

low-dose

sulfonylureas

in

MODY

-RRB-

associated

with

the

genetic

diagnosis

Other

issues

Genetic

counselling

should

be

provided

for

all

asymptomatic

individuals

requesting

predictive

testing

We

recommend

that

unaffected

relatives

are

offered

a

biochemical

test

first

-LRB-

fasting

blood

glucose

for

GCK

mutations

or

OGTT

for

mutations

-RRB-

If

the

biochemical

test

is

consistent

with

a

diagnosis

of

diabetes

or

hyperglycaemia

then

the

genetic

test

will

be

diagnostic

not

predictive

For

families

requesting

predictive

testing

for

children

too

young

to

provide

informed

consent

referral

to

a

specialist

clinical

genetics

unit

-LRB-

or

equivalent

-RRB-

is

strongly

recommended

Reasons

for

testing

children

include

-LRB-

-RRB-

to

remove

the

uncertainty

around

the

child

's

status

and

-LRB-

-RRB-

to

assist

with

management

as

a

negative

test

would

mean

that

monitoring

of

blood

glucose/glycosuria

would

not

be

necessary

-LSB-

-RSB-

Conclusions

Molecular

genetic

testing

is

useful

in

patients

with

MODY

because

it

confirms

a

diagnosis

of

monogenic

diabetes

predicts

likely

clinical

course

defines

risk

for

relatives

and

determines

treatment

At

the

present

time

molecular

genetic

testing

for

MODY

is

relatively

expensive

and

phenotypic

selection

prior

to

testing

is

normal

practice

With

the

development

of

new

technologies

it

is

likely

that

these

costs

will

decrease

in

time

and

that

the

analysis

of

genes

associated

with

monogenic

diabetes

may

become

routine

for

all

newly

diagnosed

patients

In

the

meantime

we

hope

that

these

guidelines

will

be

useful

in

determining

which

patients

should

be

offered

testing

and

in

the

interpretation

and

reporting

of

the

test

results

Below

is

the

link

to

the

electronic

supplementary

material

ESM

-LRB-

PDF

kb

-RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O 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O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

best practice
gck
hnf1a
hnf4a
mody
monogenic diabetes