François Bieuzen
email: francois.bieuzen@insep.fr

Christophe Hausswirth

Jeanick Brisswalter

Julien Louis

Dr Bieuzen François

Hausswirth Christophe

Louis Julien

Brisswalter Jeanick

AGE-RELATED CHANGES IN NEUROMUSCULAR FUNCTION AND PERFORMANCE FOLLOWING A HIGH-INTENSITY INTERMITTENT TASK IN ENDURANCE TRAINED MEN

Keywords:

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

 With aging, a great number of structural and functional transformations occur leading to a decline in "optimal" physical capacity. Many of the changes in physiological functional capacity related to aging have been found to be the result of long standing sedentary lifestyle [1,[START_REF] Wright | Age-related rates of decline in performance among elite senior athletes[END_REF]. This reduction in physical capacity with aging is related to many factors including a reduction in physical activity as well as changes in body composition [START_REF] Bouchard | Fat mass but not fat-free mass is related to physical capacity in well-functioning older individuals: nutrition as a determinant of successful aging (NuAge)--the Quebec Longitudinal Study[END_REF] and/or the development of clinical diseases. Because of the increasing number of older men and women in our society, the possibility of reducing or even preventing these age-related troubles has received increased interest. Within this framework, greater attention has been focused on the need to design strategies to increase functional capacity in older persons [START_REF] Henwood | Short-term resistance training and the older adult: the effect of varied programmes for the enhancement of muscle strength and functional performance[END_REF][START_REF] Yardley | Older people's views of falls-prevention interventions in six European countries[END_REF][START_REF] Zieglemann | Use of selection, optimization and compensation strategies in health self regulation[END_REF] but also to enhance the performance of master athletes. Indeed, studies have shown that master endurance athletes are able to maintain their performance despite structural changes in the muscle classically described with aging [START_REF] Tanaka | Endurance exercise performance in Masters athletes: age-associated changes and underlying physiological mechanisms[END_REF]8]. These observations have raised several questions on the benefit of regular endurance training on preserving functional capacity with age. During long distance events, performance depends on classical endurance qualities such as maximal aerobic capacity, efficiency of locomotion and neuromuscular performance [START_REF] Hausswirth | Physiological demands of running during long distance runs and triathlons[END_REF][START_REF] Millet | Alterations of neuromuscular function after prolonged running, cycling and skiing exercises[END_REF]. However, the majority of studies have focused on the effects of resistance training but few available studies have investigated endurance trained populations [START_REF] Deley | Effects of a one-year exercise training program in adults over 70 years old: a study with a control group[END_REF][START_REF] Dreyer | Chronic exercise and skeletal muscle power in older men[END_REF][START_REF] Tarpenning | Endurance exercise and leg strength in older women[END_REF][START_REF] Tarpenning | Endurance training delays age of decline in leg strength and muscle morphology[END_REF][START_REF] Wiswell | Relationship between physiological loss, performance decrement, and age in master athletes[END_REF]. The positive effect of regular physical activity on performance and well being in older adults is well known. However, the effect on neuromuscular performance appears controversial [START_REF] Allman | Incomplete recovery of voluntary isometric force after fatigue is not affected by old age[END_REF][START_REF] Bemben | Isometric intermittent endurance of four muscle groups in men aged 20-74 yr[END_REF][START_REF] Cupido | Neuromuscular fatigue during repetitive stimulation in elderly and young adults[END_REF][START_REF] Davies | Contractile properties of elderly human triceps surae[END_REF][START_REF] Ditor | The effect of age and gender on the relative fatigability of the human adductor pollicis muscle[END_REF][START_REF] Kent-Braun | Human skeletal muscle responses vary with age and gender during fatigue due to incremental isometric exercise[END_REF][START_REF] Laforest | Effects of age and regular exercise on muscle strength and endurance[END_REF][START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF][START_REF] Larsson | Isometric and dynamic endurance as a function of age and skeletal muscle characteristics[END_REF][START_REF] Lindstrom | Skeletal muscle fatigue and endurance in young and old men and women[END_REF][START_REF] Stackhouse | Maximum voluntary activation in nonfatigued and fatigued muscle of young and elderly individuals[END_REF][START_REF] Stevens | Characterization of the human quadriceps muscle in active elders[END_REF]. These conflicting results could be mainly attributed to different methodologies as well the nature and the characteristics of training rather than the fitness level of subjects. Classically in the scientific literature, neuromuscular function is studied using a fatiguing task and assessed by the decrease in maximal force-generating capacity. This is particularly the case during long distance events where significant decrease in muscular performance is systematically described with exercise duration (e.g. [START_REF] Lepers | Neuromuscular fatigue during a long-duration cycling exercise[END_REF]). More recently, many studies have investigated neuromuscular function with fatigue using electromyography and electrical stimulation techniques [START_REF] Kent-Braun | Human skeletal muscle responses vary with age and gender during fatigue due to incremental isometric exercise[END_REF][START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF][START_REF] Ferri | Neuromuscular recovery after a strength training session in elderly people[END_REF][START_REF] Simoneau | Muscular performances at the ankle joint in young and elderly men[END_REF]. These non-invasive techniques use the measurements of M-wave and twitch amplitude and duration to better characterise the origin of neuromuscular fatigue (i.e. central and/or peripheral fatigue) [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF]. Within this context, the purpose of this study was to compare the maximal isometric contraction, EMG activity, voluntary activation, M-wave and twitch properties of the knee extensors muscles in two groups of endurance trained young and older men. Specifically, we investigated 1) age-associated changes in neuromuscular performance in endurance trained men; and 2) age-associated changes in neuromuscular performance following a high-intensity intermittent fatigue task in endurance trained men.

MATERIALS AND METHODS

 SUBJECTS AND OVERALL DESIGN -The investigation was conducted on 16 healthy older men (Age, 66.1 ± 5.8 yrs [59-79 yrs]; height, 1.75 ± 0.06 m; body mass, 76.4 ± 7.1 kg) and on 10 young men (Age, 25.4 ± 4.6 yrs [20-34 yrs]; height, 1.80 ± 0.07 m; body mass, 75.0 ± 8.6 kg). All subjects were free from present or past neuromuscular conditions that could affect motor function. Subjects were fully informed about the protocol, and informed consent was obtained prior to all testing. This study was approved by a local research ethics committee (Saint Germain-en-Laye, France). Particular care was taken in recruiting young and older individuals with the same training level. The individuals selected for this investigation (both young and older adults) were trained in endurance activities (cycling) for at least 6 years (Cycling hours per week, Young: 11.7 ± 4.0 h; Older: 10.9 ± 2.2 h) and were not reticent to perform the fatiguing task.

PROCEDURES -One week after a full familiarization session, subjects performed an exercise protocol composed of 10 sets of 10 repetitions on a horizontal leg press (Technogym, Gambettola, Italy) at an intensity of 70% of the one repetition maximum (1 RM). 1RM values were obtained during the familiarisation session the week before using standard procedures [START_REF] Ferri | Neuromuscular recovery after a strength training session in elderly people[END_REF]. The rest between sets was 90-s. The exercise consisted of a 3-s concentric contraction followed by a 3-s eccentric contraction. Each contraction started from a position of 90° of knee flexion to reach to a knee flexion of 170°. The subjects were verbally encouraged to carry out all the sets. All subjects were able to complete the 10 sets of 10 repetitions. Immediately before (Pre) and after (Post) the exercise, neuromuscular assessments were performed. The average time between the end of the fatiguing exercise and the post-fatigue neuromuscular assessments was 25 sec.

NEUROMUSCULAR ASSESSMENTS

Torque measurements -A force transducer was used to measure maximal isometric torque of the right knee extensors (KE) muscles generated voluntary and with electrical stimulation. Subjects were seated in an experimental chair, (Schnell Trainingsgesäte GmbH, Peutenhausen, Germany) with a 110° hip angle and a 100° knee angle. Arms were folded and placed on their chest avoiding any pulling from the side-handfuls of the chair. The knee axis was aligned with the ergometer axis and the ankle was attached to the ergometer leg arm extending from the transducer. In the testing session the subjects were asked to perform maximal isometric contractions (0 rad.s -1 ) of the KE muscles of 2-3-s duration. The best performance of the three trials was defined as MVC. The subjects were strongly encouraged and the three trials were executed with a 1-min rest period between each trial.

Electrically Evoked Contractions. -Electrical stimulation was applied to the right femoral nerve with a monopolar cathode ball electrode (0.5-cm diameter) pressed into the femoral triangle by the experimentalist. The anode was a 50 cm 2 (10 x 5 cm) rectangular electrode (Medicompex, Ecublems, Switzerland) located in the gluteal fold opposite the cathode. The subject positioning was exactly the same as for the torque measurements. A high-voltage stimulator (model DS7, Digitimer, Hertfordshire, United Kingdom) was used to deliver a square-wave pulse of 1 ms duration, 400 V maximal voltage at an intensity ranging from 50 to 110 mA. The optimal intensity of stimulation was set by progressively increasing the stimulus intensity until the maximal isometric twitch torque was achieved. The same intensity was used for pre and post tests. After the optimal intensity of stimulation was found, the site of stimulation was marked and three single twitches separated by two seconds were applied at rest. The same stimulus location and intensity was used for pre and post testing. The threshold for twitch torque onset was 3SD of the mean torque calculated at rest during the 100 ms period prior to the delivery of the stimulus. The following parameters were obtained from the mechanical response of the evoked twitch contractions: (1) peak twitch (Pt), i.e. the highest value of twitch torque production; (2) contraction time (Ct), i.e. the time from the origin of the mechanical response to Pt;

(3) half-relaxation time (HRt), i.e. the time to obtain half of the decline in twitch maximal torque.

During each MVC, a superimposed stimulus was applied manually once the plateau of the torque was achieved to estimate an average superimposed signal. The stimulation parameters were the same as previously described. Voluntary activation level was estimated by expressing every increment in torque electrically evoked during the maximal isometric contractions (twitch size when superimposed) as a fraction of the amplitude of the response evoked by the same stimulus in the relaxed muscle (twitch size evoked at rest). This ratio was used to estimate the extent of inactivation during a voluntary contraction. Because the potentiated evoked stimulation represents full muscle activation, the superimposed torque using the same intensity of stimulation would activate those fibres left inactivated by the voluntary contraction [START_REF] Behm | Muscle inactivation: assessment of interpolated twitch technique[END_REF]. The activation level (AL) was then estimated according to the formula [START_REF] Gandevia | Voluntary activation of human elbow flexor muscles during maximal concentric contractions[END_REF]: AL (%) = [1 -(twitch size when superimposed / twitch size evoked at rest)] x 100 Electromyographic recordings. -The electromyographic activity of the vastus lateralis (VL) muscle of the right leg was monitored with surface EMG during the single twitch and MVC tasks. The subjects were prepared for placement of EMG electrodes by shaving the skin of each electrode site, cleaning it carefully with alcohol wipe and lightly abrading it to maintain a low inter-electrode resistance of <1000 Ω. Pairs of Ag/AgCl pre-gelled surface electrodes (Medicotest, type Blue Sensor, Q-00-S, Denmark) of 40 mm diameter with a center to center distance of 25 mm were placed on the VL muscle for EMG data acquisition. According to SENIAM recommendations [START_REF] Hermens | Development of recommendations for SEMG sensors and sensor placement procedures[END_REF], the location of the electrode was place at the distal 2/3 point on the line from the anterior spina iliaca superior to the lateral side of the patella. A ground electrode was placed on a bony site over the right anterior superior spine of the iliac crest. The electrodes were secured with chirurgical tape and cloth and kept on during the fatiguing task. EMG signals were pre-amplified (Mazet Electronique Model, Electronique du Mazet, Mazet Saint-Voy, France) close to the detection site (Common Mode Rejection Ratio, CMRR = 100 dB; Z input = 10 GΩ; gain = 600, bandwidth frequency = from 6 Hz to 1600 Hz). Prior to acquisition, a third order, zero lag Butterworth antialiasing filter at 500 Hz was applied. EMG data were collected from VL muscle, digitized through an acquisition board (DT 9800-series, Data Translation, Marlboro, USA) and stored on a computer to be analyzed using custom-written add-on software (Origin 6.1 ® , OriginLab, Northampton, USA). EMG data were sampled at 10 000 Hz and quantified by using the root mean square (RMS). Maximal RMS EMG of VL muscle was set as the maximal 500 ms RMS value found over the 3 s MVC (i.e. 500 ms window width, 1ms overlap) with Origin 6.1 ® software. Peak-to-peak amplitude (PPA), peak-to-peak duration (PPD) and RMS of the M-wave (RMS M ) were determined for the VL muscle during the control twitches performed before the MVC. Amplitude was defined as the sum of absolute values for maximum and minimum points of the biphasic (one positive and one negative deflection) M wave. Duration was defined as the time from maximum to minimum points of the biphasic M wave. The RMS M was calculated over a period from maximum to minimum points of the biphasic M wave.

The maximal RMS EMG of VL muscle was normalized to the RMS M using the ratio RMS/RMS M [START_REF] Lepers | Neuromuscular fatigue during a long-duration cycling exercise[END_REF]. A reduction in the RMS without a reduction in RMS M may be interpreted as a central activation failure [START_REF] Duchateau | Reflex regulation during sustained and intermittent submaximal contractions in humans[END_REF][START_REF] Millet | Alterations of neuromuscular function after prolonged running, cycling and skiing exercises[END_REF].

STATISTICAL ANALYSIS -All data were expressed as mean ± standard deviation (SD). A two-way analysis of variance (group x time) for repeated measures was performed to analyze the effect of groups and exercise using mechanical and EMG values as dependent variables. Tukey post-hoc test was used to determine any differences among the Pre and Post fatiguing exercise and groups. A p value of ≤0.05 was accepted as the level of statistical significance for all analyses. Intrasubject variation between the three MVC trials in Pre and Post fatiguing exercise were calculated from the coefficient of variation (CV, i.e. standard deviation/mean, in %).

RESULTS

 MVC TORQUE -Isometric MVC torque demonstrated a high reliability in Pre and Post fatigue exercise (CV, Pre: 2.08 %; Post: 3.07 %). A significant effect of aging was observed on MVC before exercise, with lower MVC values for old men compared with the young men (-29.4 %, p<0.05, Figure 1a). A significant decrease in MVC was observed after exercise (p<0.05) without any difference between groups: -9.7 % for older (from 257 ± 30 N to 232 N ± 31) and -14.3 % for young (from 364 ± 68 N to 312 ± 62 N) men.

CENTRAL ACTIVATION -RMS/RMS M ratio and activation level of the VL muscle did not differ between groups before exercise (Figure 1b and1c). Furthermore, significantly lower values of central activation were recorded after the fatiguing exercise without any difference between age groups (RMS/RMS M : Old: 0.58 ± 0.23 to 0.27 ± 0.05; Young: 0.64 ± 0.40 to 0.33 ± 0.18 -AL (%): Old: 93.1 ± 4.4 to 83.0 ± 3.8; Young: 91.5 ± 3.1 to 83.1 ± 11.7).

MUSCULAR TWITCH PROPERTIES -No significant effect of aging was observed on peak twitch torque before exercise but a slower Ct was observed for the old men compared to the young men (p<0.05). The fatiguing exercise reduced Pt by 25.8 % (p<0.05) in old men without any change in Ct or HRt when compared with preexercise values. In contrast, no significant alteration of Pt, Ct or HRt was observed in the young men after the fatiguing exercise (Table 1). M-WAVE PROPERTIES -No significant effect of age was observed on peak-to-peak amplitude (PPA) or duration (PPD) of the M-wave for the VL muscle (Table 1). The fatiguing exercise did not change the M-wave properties (PPA and PPD) for endurance trained old or young men.

DISCUSSION



The objective of the present study was to examine the age-related changes in neuromuscular performance of the knee extensors muscles in endurance trained men before and after a high-intensity intermittent fatigue task. The main results of our study are; i) a significant difference in strength level between groups but no significant differences in the decrease in the MVC torque between the groups with fatigue; ii) no difference in the level of activation between the two groups before or after fatigue and a significant decrease in the level of activation after exercise for the two groups; iii) a significant difference in contraction time between older and young adults before exercise with a significant alteration in peak twitch torque observed only in older subjects with fatigue.

MVC TORQUE -MVC for the old men was ~ 29 % lower compared to the young men before exercise. Despite the fact that our subjects were endurance trained, this result is in accordance with the literature when muscle performance of young and older trained subjects is compared. Classically, a reduction in isometric muscle strength (~15 % to ~35 %) is generally observed after the age of 60 years for the quadriceps muscles. For non endurance trained adults, this reduction is generally attributed to a loss of muscle fibres, change in the proportions of muscle fibre types [START_REF] Lexell | Human aging, muscle mass, and fiber type composition[END_REF][START_REF] Porter | Aging of human muscle: structure, function and adaptability[END_REF], and a reduction in muscle volume and cross-sectional area [START_REF] Hakkinen | Muscle cross-sectional area and voluntary force production characteristics in elite strength-and endurance-trained athletes and sprinters[END_REF]. However, based on results of Coggan et al. [START_REF] Coggan | Histochemical and enzymatic characteristics of skeletal muscle in master athletes[END_REF] confirmed by Tarpenning et al. [START_REF] Tarpenning | Endurance training delays age of decline in leg strength and muscle morphology[END_REF], decreases of strength capacities for masters athletes similar to our population could be explained by other factors. Indeed, these authors have shown that fibre area and fibre distribution are maintained with aging for master runners. They suggested that the decline in muscle performance may also the result of neural factors, such as muscle recruitment and/or specific tension. Therefore, in our population, the neural factors were not affected with aging suggesting that only the specific tension [START_REF] Larsson | Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells[END_REF] may explain our results. However, we haven't tested the fibre area and fibre distribution of our population, so we cannot conclude on it. Whereas old endurance trained men have lower isometric strength capacities than trained young men, we have observed a similar reduction of the relative muscle torque for old and young men after the fatiguing exercise with a decrease in strength of -9.7 % for young men and -14.3 % for old men. This could indicate that muscular function is similarly altered with aging in our trained subjects. In the literature, the comparison of the effect of fatigue on lower limb muscles between different age untrained groups provides contradictory results. Some studies suggest that old adults fatigue less than young adults [START_REF] Ditor | The effect of age and gender on the relative fatigability of the human adductor pollicis muscle[END_REF][START_REF] Kent-Braun | Human skeletal muscle responses vary with age and gender during fatigue due to incremental isometric exercise[END_REF][START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF] whereas other investigators have observed that older men and women fatigue more than young [START_REF] Davies | Contractile properties of elderly human triceps surae[END_REF][START_REF] Stackhouse | Maximum voluntary activation in nonfatigued and fatigued muscle of young and elderly individuals[END_REF] and some other studies have demonstrated similar fatigability in young and older adults [16-18, 22, 24, 25, 27]. For one part methodological differences especially the characteristic of the fatiguing task (e.g. high vs. low intensity, dynamic vs. static exercise, exercise duration, etc.) but also the training status of the subjects could explain this apparent inconsistency in experimental results. For example, in Lanza et al.'s study [START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF], young and older men were sedentary and practiced exercise less than 20 min/day twice a week whereas in our study all subjects were regularly trained. Therefore, our results could be explained from results on endurance trained men described in the previous section [START_REF] Tarpenning | Endurance training delays age of decline in leg strength and muscle morphology[END_REF][START_REF] Coggan | Histochemical and enzymatic characteristics of skeletal muscle in master athletes[END_REF] associated with a potentially neural adaptation on our population. Based on these observation and results on central activation discussed below, we can hypothesized that endurance exercise could reduce the fatiguing effect on the neuromuscular performance with aging with a lower decrease of neural factors.

CENTRAL ACTIVATION -Before the fatiguing exercise, the young and older trained men presented no difference in RMS/RMS M ratio and activation level values. This original result could be compared with the study of Si-moneau et al. [START_REF] Simoneau | Adaptations to long-term strength training of ankle joint muscles in old age[END_REF] showing that regardless of the ankle joint position, the RMS/RMS M ratio of the triceps surae muscle was not different between old and young adults after a 1-year strength-training program. However, to the best of our knowledge, these parameters have never been investigated on endurance trained old men while neural factors were regularly suggested to explain the greater loss of strength capacities on endurance master athlete when compared to young trained men [START_REF] Tarpenning | Endurance training delays age of decline in leg strength and muscle morphology[END_REF][START_REF] Coggan | Histochemical and enzymatic characteristics of skeletal muscle in master athletes[END_REF]. Also, in our study, no difference in central activation was observed while old men produce lower maximal strength torque than young men. This suggesting that neural factors could not be the reason of the decrease of muscular performance with aging in our specific population. Secondly, we hypothesized that chronic endurance training contributed to maintain neural factors to high level although age increases. Similarly, we observed no difference between age groups in this ratio after the fatiguing exercise. However, both groups showed an alteration of the level of activation with fatigue. This decrease associated with decrease of muscular performance after an heavy exercise is classically described in the literature [START_REF] Lepers | Neuromuscular fatigue during a long-duration cycling exercise[END_REF]. In this experiment, the two methods used for estimating central activation both attest that a central component of muscle fatigue contributes substantially to the reduction of maximal voluntary force. The reduced neural drive the motoneurons may be due to spinal and supraspinal mechanisms (for review, see Ref. 31). Fatigue can occur at levels upstream of corticospinal neurons, presumably due to neurotransmitter depletion, which could result in an impaired efficiency in generating the central command. In addition, fatigue at the spinal level might result from peripheral reflex inhibition of the α-motoneurons by muscle-spindle afferents [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF].

MUSCULAR TWITCH AND M-WAVE -Our results on the twitch properties are in agreement with previous studies since we have observed that contraction time (Ct) and half-relaxation time (HRt) of the older compared with young subjects were greater in the unfatigued (Ct: +34 %; HRt: +43 %) condition. One of the main explanations of this increase has been proposed by Jakobsson et al [START_REF] Jakobsson | Use of motor units in relation to muscle fiber type and size in man[END_REF] and Lexell [START_REF] Lexell | Human aging, muscle mass, and fiber type composition[END_REF] indicating an age-related shift toward a higher percentage of type I fibres. However, this explanation was in conflict with results of Tarpenning et al. showing that Type I and Type II fibre area and distribution do not differ between age groups on endurance trained men [START_REF] Tarpenning | Endurance training delays age of decline in leg strength and muscle morphology[END_REF]. It this case, the hypothesis of modification of the specific tension of type I and type II fibre with aging [START_REF] Larsson | Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells[END_REF] could mainly explain our results; however, this hypothesis remains highly speculative and we have no data to support this. One other factor could also affect twitch contractile characteristics and could explain one part of our results. Indeed, a recent study has observed that musculotendinous stiffness decreases following strength training in elderly individuals and could be due to peripheral but also neural adaptations [START_REF] Ochala | Effect of strength training on musculotendinous stiffness in elderly individuals[END_REF]. If we associated this original result to the fact that more compliant series elastic components in the muscles tend to decrease twitch force [START_REF] O'hagan | Elbow flexor evoked twitch contractile properties in untrained men and women and bodybuilders[END_REF] and can also partly cause a slowing of the twitch contraction time [START_REF] Rice | Strength training alters contractile properties of the triceps brachii in men aged 65±78 years[END_REF], we can hypothesized that one part of our results in older men could be attributed to a reduction of the musculotendinous stiffness properties. However, we haven't tested this hypothesis, so we cannot conclude on it. Our results showed different modifications of the twitch properties after the fatiguing exercise between groups. Indeed, older men showed a decrease in Pt after exercise suggesting an alteration of the contractile properties. This reduction is generally attributed to an alteration of the excitation-contraction coupling process that could be due to several mechanisms such as reduced Ca 2+ release from the sarcoplasmic reticulum [START_REF] Westerblad | Cellular mechanisms of fatigue in skeletal muscle[END_REF], change in metabolites (H + , inorganic phosphate), and reduced capacity of cross bridges to strongly bind [START_REF] Metzger | Moss RL: pH modulation of the kinetics of a Ca2(+)-sensitive cross-bridge state transition in mammalian single skeletal muscle fibres[END_REF]. In contrast, Pt in the young men remained unchanged after the exercise. As recently shown at similar knee flexion angle [START_REF] Place | Twitch potentiation is greater after a fatiguing submaximal isometric contraction performed at short vs. long quadriceps muscle length[END_REF], this lack of decrease could suggest that fatigue was counterbalanced by potentiation for young adults, whereas twitch potentiation of older adults was too low to counterbalance the fatigue [START_REF] Baudry | Postactivation potentiation influences differently the nonlinear summation of contractions in young and elderly adults[END_REF][START_REF] Shima | Mechanomyographic and electromyographic responses to stimulated and voluntary contractions in the dorsiflexors of young and old men[END_REF].

Before the fatiguing exercise no difference was observed between young and older men on the M-wave characteristics (i.e. PPA and PPD). This result is different from those obtained on sedentary subjects, showing an age-related reduction of 20-40% in PPA [START_REF] Allman | Incomplete recovery of voluntary isometric force after fatigue is not affected by old age[END_REF][START_REF] Cupido | Neuromuscular fatigue during repetitive stimulation in elderly and young adults[END_REF][START_REF] Ditor | The effect of age and gender on the relative fatigability of the human adductor pollicis muscle[END_REF]. This results could be related to finding from the recent study by Deley et al. [START_REF] Deley | Effects of a one-year exercise training program in adults over 70 years old: a study with a control group[END_REF] indicating that one year of combined exercise training (endurance and resistance training) improves aerobic capacity, performance on field tests, and neuromuscular factors in healthy subjects over 70 years old. Within this framework, our results indicate that for regularly endurance trained subjects, there is no age-related alteration in neuromuscular propagation or the permeability of the sarcolemma to chloride, potassium, or sodium. Moreover, a second interesting result is that there is no potentiation of the amplitude and duration of the M-wave after the fatiguing exercise. Thus, we could suggest the benefits of regular endurance training are to preserve the excitability properties of the muscle.

In conclusion, the main result of our study showed a similar reduction of the maximal strength capacities of knee extensors muscles for endurance older and young men after a high intensity intermittent endurance exercise. Furthermore, central fatigue appeared similar in both groups but the alteration of contractile properties was greater in older adults. These results seem to indicate that in endurance trained men muscle functional capacities in men aged 59-76 years are maintained despite age-related reduction in strength and changes in contractile function. α , indicate significant difference between pre and post tests (p<0.05).

β , indicate significant difference between groups (p<0.05) α , indicate significant difference with before trial (p<0.05).

β , indicate significant difference between groups (p<0.05).

Figure 1

 1 Figure 1 Mean values of the MVC of the knee extensors muscles (a), the RMS/RMS M ratio (b) and the activation level (c) of the Vastus Lateralis muscle before and after the fatiguing exercise for older and young adults groups.

Table 1

 1 Muscular twitch and vastus lateralis muscle M-wave parameters (peak twitch, i.e. Pt; contraction time, i.e. Ct; half-relaxation time, i.e. HRt; peak to peak amplitude, i.e. PPA; peak to peak duration, i.e. PPD) before and after the fatiguing exercise.

		Twitch			M-wave
		Pt	Ct	HRt	PPA	PPD
		(N)	(ms)	(ms)	(mV)	(ms)
	Young	53.8 ± 17.3	57 ± 18	68 ± 14	12.9 ± 5.7	5.8 ± 1.8
	Before
	Older	54.6 ± 11.3	76 ± 18 β	85 ± 28 β	18.8 ± 8.8	6.4 ± 1.5
	Young	54.2 ± 20.4	47 ± 21	52 ± 18	12.8 ± 6.1	4.7 ± 2.6
	After
	Older	34.4 ± 16.3 α,β 68 ± 8 β	79 ± 36 β	17.8 ± 8.1	6.2 ± 1.7


Sahar Elouej

Ana Beleza-Meireles

Richard Caswell

Kevin Colclough

Sian Ellard

Jean-Pierre Pierre Desvignes

Christophe Béroud

Nicolas Lévy

Shehla Mohammed

Annachiara De Sandre-Giovannoli

Exome sequencing

Introduction

Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL, OMIM#615381), is a rare systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This disorder was clinically described for the first time by Shastry et al. in 2010 who reported seven subjects presenting with overlapping features with mandibuloacral dysplasia syndromes (MAD-A, due to recessive mutations in the LMNA gene, OMIM#248370, and MAD-B, due to recessive ZMPSTE24 mutations, OMIM#608612) such as mandibular hypoplasia, prominent eyes, stiff joints, beaked nose, and lipodystrophy, but with specific additional clinical features [1]. These included sensorineural hearing loss, hypogonadism and absent clavicular hypoplasia/acroosteolyses. MAD and MDPL belong to the group of diseases characterized by premature aging, which can be caused by inheritable nuclear envelope and/or DNA repair defects. Some of the major disorders included in this group are: Hutchinson-Gilford progeria syndrome (HGPS, OMIM# 176670) caused by dominant mutations in the LMNA gene [2,3]; Nestor-Guillermo progeria syndrome (NGPS, OMIM#614008), due to recessive mutations in the BANF1 gene [START_REF] Puente | Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome[END_REF]; Werner syndrome (WRN, OMIM#277700), due to recessive mutations in the WRN gene [START_REF] Yu | Positional cloning of the Werner's syndrome gene[END_REF]; Restrictive dermopathy (RD, OMIM#275210), mainly due to recessive null mutations in the ZMPSTE24 gene [START_REF] Navarro | Lamin a and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy[END_REF][START_REF] Navarro | Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin a precursors[END_REF]; Cockayne syndrome type A and B (CSA, OMIM#216400; CSB, OMIM#133540), due to mutations in ERCC8 and ERCC6 respectively [START_REF] Henning | The Cockayne syndrome group a gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH[END_REF][START_REF] Mallery | Molecular analysis of mutations in the CSB (ERCC6) gene in patients with Cockayne syndrome[END_REF] and Marfanprogeroid syndrome (MFLS, OMIM# 616914) due to dominant mutations in FBN1 gene. Due to the clinical similarities among these syndromes and possible atypical phenotypes, clinical diagnosis can be quite a challenge, which can be solved in most cases through molecular exploration.

MDPL syndrome is an autosomal dominant disorder caused by mutations in the POLD1 gene encoding human polymerase delta 1 (POLD1), also known as the p125 subunit, which provides the essential catalytic and proofreading activities of DNA polymerase δ (Pol δ) [START_REF] Palles | Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas[END_REF][START_REF] Weedon | An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy[END_REF]. DNA polymerase δ is one of the three main enzymes responsible for DNA replication [START_REF] Mossi | DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair[END_REF][START_REF] Waga | The DNA replication fork in eukaryotic cells[END_REF] but it is also involved in multiple DNA repair mechanisms, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair [START_REF] Nicolas | POLD1: central mediator of DNA replication and repair, and implication in cancer and other pathologies[END_REF]. The catalytic subunit encoded by POLD1 is responsible for the synthesis of DNA lagging-strand by the combined action of two activities: 5′-to 3′-polymerase activity and 3′-to 5′-exonuclease or proofreading activity [START_REF] Prindle | DNA polymerase delta in DNA replication and genome maintenance[END_REF]. POLD1 is composed of 1107 amino-acids and includes several distinct functional domains: a putative nuclear localization signal (amino-acid 4 to amino-acid [START_REF] Pelosini | Identification of a novel mutation in the polymerase delta 1 (POLD1) gene in a lipodystrophic patient affected by mandibular hypoplasia, deafness, progeroid features (MDPL) syndrome[END_REF], an exonuclease domain (amino-acid 306 to amino-acid 519), a polymerase active site (amino-acid 581 to amino-acid 910) and a ZnF domain (aminoacid 1012 to amino-acid 1083) which contains two conserved cysteine-rich metal-binding motifs (CysA and CysB) [START_REF] Prindle | DNA polymerase delta in DNA replication and genome maintenance[END_REF][START_REF] Garcia | The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit[END_REF].

To date, 19 subjects with MDPL have been reported in the literature, whose phenotypes are similar and rather mild, without shortened lifespans. To the best of our knowledge, only two mutations in the POLD1 gene have been identified to date. The first is a recurrent de novo in frame deletion of a single codon (c.1812_1814delCTC, p.Ser605del) identified in 12 unrelated patients [START_REF] Weedon | An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy[END_REF][START_REF] Reinier | Mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome in the context of inherited lipodystrophies[END_REF][START_REF] Lessel | POLD1 germline mutations in patients initially diagnosed with Werner syndrome[END_REF] with confirmed pathogenicity, related to functional studies. The second is a de novo missense mutation (c.1572C > T, p.Arg507Cys) identified in two patients and first reported in a 48 years old woman [START_REF] Lessel | POLD1 germline mutations in patients initially diagnosed with Werner syndrome[END_REF][START_REF] Pelosini | Identification of a novel mutation in the polymerase delta 1 (POLD1) gene in a lipodystrophic patient affected by mandibular hypoplasia, deafness, progeroid features (MDPL) syndrome[END_REF]. These genetic changes respectively affect the polymerase domain and the C-terminal part of the proofreading (exonuclease) domain of the polymerase. Additionally, a very recent paper [START_REF] Ajluni | Spectrum of disease associated with partial lipodystrophy (PL): lessons from a trial cohort[END_REF] reports a novel inherited POLD1 missense mutation in two related individuals (mother and daughter): c.3199 G> A; p.E1067K which was classified as likely pathogenic. The phenotype of these patients was partly different and milder compared to previously reported patients (please see the discussion section on phenotypic comparisons of POLD1 mutated patients). Interestingly, this is the first identified variant lying within the ZNF2 domain of POLD1. Finally, germline mutations in the exonuclease domains of POLD1 have been recently associated with familial polyposis and colorectal cancer predisposition [START_REF] Palles | Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas[END_REF][START_REF] Bellido | POLE and POLD1 mutations in 529 kindred with familial colorectal cancer and/or polyposis: review of reported cases and recommendations for genetic testing and surveillance[END_REF].

In this study, we report the clinical and genetic investigation of two additional patients with variably severe MDPL syndromes, using either direct search of the POLD1 recurrent mutation by Sanger sequencing or whole exome sequencing (WES).

Methods

Patients

Two patients with MDPL were included in this study. They were admitted to the Guy's and St. Thomas' NHS Foundation Trust Hospital in London. The parents of each patient gave informed and written consents for the studies performed as well as for picture publication, including uncovered faces. The explorations were performed first in a diagnostic setting, then in a research setting, complying with the ethical guidelines of the institutions involved and the declaration of Helsinki. After written informed consent was obtained from the family members, peripheral blood samples from all of the participants were drawn, and genomic DNA was extracted following standard procedures. No cell lines could be established for functional studies in these two patients.

Whole-Exome Sequencing

Whole-exome sequencing was performed on patient 1 and her unaffected parents. Briefly, exome was captured from genomic DNA using Agilent SureSelect Clinical Research Exome kit and then sequenced on an Illumina HiSeq 4000 sequencer using 70-bp paired-end reads. We used BWA [START_REF] Li | Fast and accurate short read alignment with burrows-wheeler transform[END_REF] to align sequence reads to the hg19 reference genome and GATK [START_REF] Mckenna | The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data[END_REF] to call SNVs and indels. Control quality showed that 88% of targeted bases were covered at > 20×.

Whole-Exome Data Analysis

The results were analyzed using the in-house VarAft software version 1.6, which is freely available online (http://varaft.eu/ index.php). Through exome analysis, both recessive and dominant models of inheritance have been performed (

Sanger Sequencing Confirmation

The Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/) was used to design the specific primers used for PCR amplification and direct sequencing of the surrounding region of each variant. Primers used to amplify the mutant sequence were POLD1-Ex15-F (5′-GCTCCCAGCCAATGAATG-3′), POLD1-Ex15-R (5′-CTACTATCTCTGGGCCTGTGC-3′), POLD1-Ex26-F (5′-TCCCCAGGTATCCCATCTGA-3′), and POLD1-Ex26-R (5′-TTGGAGTCAGAGGTTGGGTC-3′). PCR products were examined by agarose gel electrophoresis and subjected to Sanger sequencing. Exome results and the mutation segregation pattern were confirmed by Sanger sequencing.

Results

Patients' Clinical Features

Both patients were sporadic, isolated cases with no known consanguinity among parents. Both showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, small mouth, growth retardation, limb muscle and skin atrophy (Table 1), but those features were much more marked in patient 1 (female patient, Fig. 1) compared to patient 2 (male patient, Fig. 2). Patient 1 was one of three children born to unrelated parents. At age 18 months, as seen in the picture provided (Fig. 1a), the patient did not show any apparent clinical signs. At the age of 7-8 years, she presented with growth retardation, mid face hypoplasia with dental crowding, and low set ears. The clinical diagnosis was then subsequently revised to Hutchinson-Gilford Progeria but no testing was available at the time. She was first seen by us at the age of 23 years (Fig. 1b) having been under the care of the cardiac and respiratory teams with musculoskeletal problems and recurrent respiratory chest infections. She had been given a clinical diagnosis of Acrogeria/ Ehlers-Danlos syndrome type 4 originally but collagen studies were inconclusive. Routine investigations for failure to thrive, including thyroid and pituitary functions were negative and there was no effect from a trial of recombinant growth hormone. She was then seen at age of 24 years where she presented with extreme generalized amyotrophy and subcutaneous lipoatrophy (Fig. 1c,d). At age 24 years her height and weight were 1.20 m (<P1) and 12.6 kg (<P3), respectively and the body mass index was 8.75 kg/m 2 . She had quite prominent eyes with a small jaw (Fig. 1e,f). She required extensive dental surgery because of crowded teeth (Fig. 1b,d). She also required treatment for exposure keratopathy. She had no secondary sex characteristics development and had primary amenorrhoea. Moreover, her skin was quite thin and sclerodermatous with prominent veins and lack of subcutaneous fat. (Fig. 1f,g,h); swelling of interphalangeal joints could be observed in the hands. She had stiffness and limited movements in elbows, knees and hips. She suffered from a major scoliosis, leading to several surgical interventions (Fig. 1i) as well as from severe and recurrent respiratory tract infections. Her clinical picture evolved towards a restrictive dermopathy phenotype and bulbar dysphagia necessitating a percutaneous endoscopic gastrostomy, until death occurred at 25 years of age, following a severe pulmonary infection. Of note, this patient did not present with hearing loss and showed entirely normal psychomotor development. The oral glucose intolerance test for patient 1 indicates an impaired glucose tolerance or a prediabetes status (Table 1). Unfortunately, it was not possible to assess whether she was affected with diabetes or to get further biochemical measurements related to dyslipidemia since she passed away before her disease could be diagnosed.

The second patient was a 6-year-old boy, born to healthy non consanguineous parents, following an uneventful pregnancy. He was born in good condition and with normal birth weight. From the age of 2 years short stature was observed and he started to have poor weight gain by the age of 5 years. He was diagnosed at age four with sensory neural low frequency hearing loss for which he wears bilateral hearing aids. He was referred for genetics assessment at age 7 years because of failure to thrive and extremely mild acanthosis nigricans on the sides of his neck and also in the axillas. At physical examination he presented with mild fat loss in his lower limbs, prominent eyes, pinched nose, small chin and thin lips (Fig. 2). He had proportionate short stature and low weight for age, height 1.114 m, weight 16.2 kg and the body mass index was 13.09 kg/m 2 (< P2 for height and weight, OFC on the 25th-50th centile). He also showed joint restriction, particularly in shoulder extension (Fig. 2d). Further, genitalia examination and further explorations showed phimosis and right cryptorchidism. He had a normal psychomotor development, normal speech and language development and unremarkable skeletal survey. A recent change in hair texture was noted together with the appearance of white hair. Abdominal ultrasound did not show any pathological features and no increased intra-abdominal adipose tissue. X-rays of the skeleton showed a delayed bone age. Routine biochemical test revealed a normal oral glucose tolerance test and did not highlight any lipid profile dysregulation (Table 1).

Table 1 summarizes the clinical information concerning the two patients we report and all the patients previously reported in the literature. In particular, in order to better compare the linear growth of all the patients carrying POLD1 mutations leading to syndromic phenotypes, we computed their growth Z-scores on the basis of available information.

No chromosomal imbalance was detected by CGH array for both patients.

Molecular Genetics Explorations

Since patient 1 was first diagnosed as having a progeroid syndrome similar to HGPS or NGPS, due to strikingly overlapping clinical features, the coding exons and intronic boundaries of the LMNA, ZMPSTE24 and BANF1 genes were first screened by direct Sanger sequencing. No causative mutation was found in these genes. Therefore, samples from patient 1 and her healthy parents were subjected to whole exome sequencing in a research context, in order to search for the genetic defect underlying patient 1's disease. A summary of the bio-informatic workflow of this study is presented in Table 2. During our analysis, we have focused on missense, nonsense, splice-site, and frameshift variants upon all modes of inheritance. After sequential filtering as shown in Table 2, and keeping variants predicted as pathogenic, no homozygous variant nor compound heterozygous variants were identified in our patient, while only one heterozygous de novo variant was observed in the POLD1 gene. Moreover, important progeria and progeria-linked genes (LMNA, ZMPSTE24, BANF1, WRN, ERCC8, ERCC6, FBN1 and B4GALT7) were well covered and did not show any potentially pathogenic variant. Likewise, no mutations in genes associated to inherited lipodystrophies (i.e., AGPAT2, PPARG, BSCL2, LMNB2, CAV1, AKT2, FBN1, PIK3R1 etc.) were observed. We thus identified an unreported heterozygous c.3209 T>A (p.Ile1070Asn) missense mutation in the 26th coding exon of the POLD1 gene (NM_002691.3), which was absent from parental DNA on exome sequencing results (Fig. 3a). Bidirectional Sanger sequencing confirmed the de novo appearance of the c.3209 T > A mutation in the patient (Fig. 3b). The c.3209 T > A variant was absent from dbSNP138, the 1000 Genomes Project, ExAC databases and the Exome Variant Server. The ExAC database reports the constraint metric for missense variants in POLD1 as 2.79 (i.e. the number of observed variants is 2.79 standard deviations below the mean value expected for the null hypothesis; [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]). Missense variants have been reported at 293/1107 residues of POLD1 (26.5%), at an average allele frequency of 6.44 × 10-4 (Fig. 4). However, within the CysB region (residues 1058-1076), although missense variants occur at a similar proportion of residues (5/19) the average variant allele frequency of 5.32 × 10-5 is ~12-fold lower than that over the entire coding region. This suggests that while there has been selective pressure against coding variation in POLD1 as a whole, this is greater within the CysB region. Specifically, no ExAC variants have been reported at Ile1070, the preceding residue Val1069, or any of the conserved cysteine residues (1058, 1061, 1071, and 1076), indicating that these residues are likely to be particularly important for normal function of this region. Furthermore, this mutation was predicted as deleterious according to prediction databases such as SIFT, UMD predictor and Mutation Taster. Moreover, the CADD C-score of the variant was 25.7, clearly predicting that the p.Ile1070Asn as deleterious (deleterious variants show scores > 15). This variant lies outside the known enzymatic domains (C306-C910) but within a region termed ZnF2 domain, also called CysB motif [START_REF] Garcia | The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit[END_REF][START_REF] Netz | Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes[END_REF], located near the C-terminus (Fig. 5b). Although previously thought to be a zinc-binding motif, this region has recently been reported to form an iron-sulfur cluster of the 4Fe-4S type and to be crucial for the assembly of the active DNA polymerase δ complex [START_REF] Netz | Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes[END_REF]. To discuss the significance of these sequence alterations, we compared the local amino-acid sequences among several organisms (Fig. 5c). Inspection of the genomic context around the variant shows that residues within the CysB motif (Cys1058-Cys1076) are strongly conserved, although isoleucine 1070 itself is only present in mammals whereas leucine is observed at this position in other vertebrates (Fig. 5c).

The crystal structure for the extreme C-terminus of POLD1, including the CysB region, has not yet been solved. Furthermore, interrogation of protein structure databases did not yield any suitable templates for comparative modeling of the 4Fe-4S cluster within CysB. Therefore to evaluate the effect of the Ile1070Asn substitution, full-length sequences of wildtype and variant POLD1 were submitted to the PredictProtein server [START_REF] Yachdav | PredictProtein-an open resource for online prediction of protein structural and functional features[END_REF] for prediction of protein secondary structure and amino acid solvent accessibility. The analysis of results over the CysB region showed that the p.Ile1070Asn substitution was predicted to have a significant effect on both the local secondary structure and the solvent accessibility of amino acid side chains within this region, and in particular around residues Cys1071 and Cys1076 which form part of the ironsulfur cluster (Fig. 6).

On the basis of family history and clinical presentation, patient 2 was clinically diagnosed as having MDPL syndrome and the recurrent mutation located in exon 15 of the POLD1 gene was directly searched for. Sanger sequencing confirmed the presence of the heterozygous in-frame deletion mutation in the POLD1 gene (c.1812_1814del, p.S605del; data not shown), confirming the clinical diagnosis. Parental testing confirmed the de novo appearance of the deletion in the patient.

Discussion

Monogenic disorders characterized by premature aging are clinically heterogeneous with overlapping phenotypic presentations, making the clinical diagnosis quite a challenge, especially in cases with variable severity. Molecular genetics analysis is a key tool to confirm the clinical diagnosis and search for the causal genes in atypical forms. Through exome sequencing we could successfully characterize at the molecular level a patient (patient 1) who first had a working diagnosis of Hutchinson Gilford Progeria Syndrome, given the particularly severe clinical presentation. Whole-exome sequencing of patient 1 and her unaffected parents revealed a heterozygous de novo missense mutation c.3209 T > A (p.Ile1070Asn) in the POLD1 gene. Several elements made us retain this variant as the pathogenic variant causing the patient's clinical picture: no missense variants have been reported in the ExAC database at this position or at neighboring residues, suggesting that these nucleotides are under strong selective pressure. The p.Ile1070Asn is located within the CysB region, a highly-conserved motif across eukaryotic DNA polymerases [START_REF] Garcia | The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit[END_REF][START_REF] Netz | Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes[END_REF]: isoleucine 1070 is present in mammals whereas leucine, a chemically similar amino-acid, is observed at this position in other vertebrates. Apparently, during the divergence of mammals from other vertebrates, leucine became substituted by isoleucine, but since the side chains of these amino acids have very similar physicochemical properties, this substitution was tolerated and has been retained in all subsequent speciation. A corollary of this observation is that substitution of isoleucine for asparagine at this position, as observed in patient 1, is unlikely to be tolerated. Indeed, in silico structural analysis using the PredictProtein server shows that the p.Ile1070Asn substitution is predicted to alter the local secondary structure and solvent accessibility within the CysB region, with the likely effect that the Fe-S cluster is destabilized. Both secondary structure and amino acid accessibility probably play a key role in the correct folding of this region into the Fe-S cluster, and specifically the cysteine side chains should remain buried and in the correct orientation to bind iron. In this respect it is notable that not only is residue 1070 always the hydrophobic leucine or isoleucine, but that the preceding residue is also a highly conserved non-polar residue. The substitution of Ile1070 by asparagine, a polar residue, is likely to cause displacement of the protein backbone to favor solvation of the novel side chain, with consequences for both the local structure and positioning of the C1071 side chain. Taken together, it is very likely that the Ile1070Asn substitution will cause disruption of the Fe-S cluster, and, since this has been shown to be crucial for interaction with other subunits in the assembly of the active polymerase complex, impairment of polymerase function [START_REF] Netz | Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes[END_REF]. An earlier study also demonstrated that removal of the tandem zinc finger modules located at the C-terminus of Pol3 in yeast by targeted proteolysis, renders the Pol3 protein non-functional in vivo, and that the Cterminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit of Pol3 in vivo and in vitro [START_REF] Garcia | The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit[END_REF]. Taken together, it is highly likely that the p.Ile1070Asn variant leads to loss of polymerase δ activity, and a resulting severe phenotype sharing features with MDPL syndrome. Interestingly, a very recent study reported a mother and a daughter carrying a novel heterozygous variant in POLD1 exon 26 (c.3199G >A; p.E1067K), which is also highly conserved and which was predicted by the authors as likely pathogenic [START_REF] Ajluni | Spectrum of disease associated with partial lipodystrophy (PL): lessons from a trial cohort[END_REF]. Glutamine 1067 lies within the ZNF2 domain, just as our mutant, thus supporting its pathogenic effect. For the first time, the authors used immunofluorescence staining to analyze the nuclear morphologies in liver sections of patients carrying a POLD1 mutation: interestingly, this analysis showed a high degree of nuclear atypia and disorganization, although at a lower extent than that observed in lipodystrophic patients carrying LMNA mutations. This observation interestingly supports possibly shared pathogenic mechanisms among POLD1 and LMNA-related lipodystrophic syndromes.

Unfortunately, it was not possible to conduct more in-depth cellular studies in patient 1 since she passed away before the identification of the mutation p.Ile1070Asn and no cell line had been established until then. But it will be certainly interesting to perform similar studies, on fibroblast cell lines or, if indicated, in liver biopsies, on other patients carrying POLD1 mutations as our patient 2 or previously published patients, both affected with MDPL or with mutations predisposing to colorectal cancer, to check whether abnormal nuclear morphology is a common feature or if it seems specific to patients carrying mutations in the protein's ZNF2 domain.

In this study we also reported a novel MDPL patient carrying the recurrent in frame deletion p.S605del. In fact, this syndrome was associated in 2013 for the first time to the POLD1 gene by some of us, where the in frame deletion (c.1812_1814del, p.S605del) was reported in four unrelated MDPL patients [START_REF] Weedon | An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy[END_REF]. This study was followed by two recent reports where a total of nine patients with the same deletion were described [START_REF] Reinier | Mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome in the context of inherited lipodystrophies[END_REF][START_REF] Lessel | POLD1 germline mutations in patients initially diagnosed with Werner syndrome[END_REF]. This is likely a mutation hotspot leading to MDPL syndrome.

As previously described, the p.Ser605del mutation is located in the polymerase active domain of POLD1, while the p.Arg507Cys missense mutation is located in the C-terminal part of the exonuclease domain. Functional studies have shown that the p.Ser605del results in an enzyme which is inactive as a polymerase but retains exonuclease activity [START_REF] Weedon | An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy[END_REF], while the p.Arg507Cys is adjacent to a variant that has been described in a cell line of human colorectal cancer, which slightly reduces the fidelity of DNA replication [START_REF] Yoshida | Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer[END_REF]. Unlike these mutations, based on previous in vivo and in vitro studies as well as on novel in silico predictions described in this work, it is expected that all catalytic functions of POLD1 are affected by the p.Ile1070Asn missense mutation, and this may well result in novel and more severe clinical features. Therefore, we have re-evaluated the clinical description of patient 1 after the identification of the causative mutation. It is obvious that various clinical features of our patient overlap with those of the other MDPL patients. As shown in Table 1, patient 1 developed lipodystrophy, progeroid facial features, muscle wasting and dental crowding which were among the most prominent clinical characteristics of the previously reported patients. Normal hearing was also reported in 3 patients with MDPL syndrome by Lessel et al., indicating that this clinical feature may not always be associated with the syndrome. Our patient displayed severe postnatal growth failure, leading to very severely reduced linear growth with a Z-score of -6.6 at 24 years of age. Of note, Lessel et al. also reported 7 patients with short stature, but these showed milder growth retardation (range -0.9 to -3.7 SDS) ( 1 glucose intolerance test indicated a prediabetic status in our patient 1. Unfortunately, she passed away before further biochemical measurements could be performed to confirm her diabetic status. As well, dyslipidemia, which is an important trait related to this syndrome, could not be searched for. Moreover, some other noteworthy differences have been noted. Indeed, it is important to note that patient 1 showed a much more severe phenotype than all MDPL patients described previously carrying the p.Ser605del or p.Arg507Cys mutation, namely featuring more striking progeroid features and premature death. Furthermore, this patient showed a high susceptibility to severe respiratory tract infections, as well as arachnodactyly and bulbar dysphagia which were never described in MDPL patients before. As well, the two related patients reported by Ajluni et al. carrying a new mutation in the ZNF2 domain of POLD1 did not show the same severe clinical features observed in patient 1, and showed only some of the previously reported signs of MDPL patients (Table 1). Indeed, the patients reported by Ajluni et al. did not present with generalized lipodystrophy, progeroid features or short stature as our patient, nor severe insulin resistance or deafness as MDPL patients, nonetheless, they shared some of our patient's and previous MDPL signs as small mandibles, paucity of fat in the extremities and joint stiffness [START_REF] Ajluni | Spectrum of disease associated with partial lipodystrophy (PL): lessons from a trial cohort[END_REF]. Overall, phenotypic comparison between patients carrying POLD1 mutations shows a clear phenotypic variability. This observation is also true for instance for patients carrying LMNA gene mutations, in whom not only different mutations cans cause totally different syndromes, but different missense changes of the same aminoacid can cause different phenotypic expressivity or different disorders [START_REF] Rodriguez | Low and high expressing alleles of the LMNA gene: implications for laminopathy disease development[END_REF]. Indeed, the aminoacid sequence is known to affect the secondary structure of the proteins, thus possibly changing the interaction capabilities of the mutated protein with tissue-specific partners. Furthermore, differential allelic expression has been suggested as a potential mechanism for the phenotypic variability in laminopathies [START_REF] Rodriguez | Low and high expressing alleles of the LMNA gene: implications for laminopathy disease development[END_REF] and it could be interesting to test the existence of physiologically low and high expressing alleles in the POLD1 locus (upon SNP patterns), and the consequences of the different mutations observed in MDPL patients on POLD1 expression levels. Although functional and molecular investigations would help to confirm the involvement of the novel mutation reported in the patient's clinical phenotype, the variant p.Ile1070Asn was predicted as pathogenic through the different in silico prediction databases and deep bioinformatics analyses did not reveal another pathogenic variant that could modify the severity of the clinical phenotype, considering that 88% of the coding regions of the genome were covered at 20X. However, the presence of a modifier variant in the non-covered regions cannot be ruled out.

Last, but not least, mutations of POLD1 affecting the exonuclease domain have been described in colorectal cancer and/or polyposis predisposition [START_REF] Bellido | POLE and POLD1 mutations in 529 kindred with familial colorectal cancer and/or polyposis: review of reported cases and recommendations for genetic testing and surveillance[END_REF]. It has been suggested that mutations in the exonuclease domain may contribute to the high mutation rate observed in tumors while mutations in the polymerase domain would be responsible for the appearance of other clinical features including lipodystrophy [START_REF] Nicolas | POLD1: central mediator of DNA replication and repair, and implication in cancer and other pathologies[END_REF]. The recent study from Lessel and colleagues also highlighted the differences in the localization of "progeroid" and "malignant" types of substitutions in the POLD1 protein structure using molecular modeling data; these provide evidence of likely different mechanisms related to progeroid and cancerassociated POLD1 mutations [START_REF] Lessel | POLD1 germline mutations in patients initially diagnosed with Werner syndrome[END_REF]. Further functional studies will be needed to clarify the genotype-phenotype relationships in patients with rare deleterious germline mutations in the POLD1 gene, namely depending on the impacted domain of the protein and its corresponding function.

In conclusion, our study shows that patients carrying POLD1 mutations add to the wide phenotypic spectrum of premature aging syndromes, notably with particularly severe phenotypes. We thus report two novel cases affected with the rare MDPL syndrome and have identified a novel likely pathogenic variant in the ZNF2 domain of POLD1, which adds further evidence for the role of this gene in causing MDPL syndrome. Adding to previous reports, this work underlines the need to include this gene among the candidate genes tested in patients affected with atypical and/or severe progeroid syndromes. At this stage, since different POLD1 mutations can lead to major phenotypic variability, further functional work is needed to unravel the pathophysiological mechanisms underlying MDPL syndrome. Namely, it will be interesting to see whether mutations in different POLD1 domains can lead to nuclear disorganization, which may represent a major pathophysiological link with LMNA mutation-associated lipodystrophies. These explorations will be also essential to establish new targets for the discovery of appropriate therapeutic strategies.

R E F E R E N C E S

[1] Shastry S, Simha V, Godbole K, Sbraccia P, Melancon S, Yajnik

Fig. 1 -

 1 Fig. 1 -Clinical characteristics of patient 1 affected with MDPL syndrome. (a): Patient 1 at age 18 months with normal appearance; (b-h): pictures of patient 1 at different ages from 13 (b) to 23 years (f,g), showing prominent eyes, beaked nose, a small jaw with crowded teeth, major lipodystrophy with thin skin and prominent superficial vessels; in panels e and f dental crowding had been surgically corrected (i-l): radiographs showing the scoliosis of the mid thoracic spine and other skeletal findings, including arachnodactyly, severe osteoporosis with thin cortices and very slender diaphysis.

Fig. 2 -

 2 Fig. 2 -Clinical features of patient 2 affected with MDPL syndrome at 6 years of age. (a-c): mild lipodystrophy, beaked nose, prominent eyes, mandibular hypoplasia and hearing aids can be observed, together with reduced shoulder extension (d). (e-f): No dermatological or skeletal abnormality was observed in patient 2.

Fig. 3 -

 3 Fig. 3 -Whole exome results for patient 1.a: IGV browser visualization of whole exome sequencing results showing the heterozygous c.3209 T > A substitution in the POLD1 gene in patient 1 (upper panel), which is absent in her unaffected parents (mid and lower panels). b: The POLD1 c.3209 T > A p.Ile1070Asn was validated by Sanger sequence analysis. Sanger sequencing of patient 1 and her parents confirm the de novo appearance of the of the p.Ile1070Asn mutation in the affected child.

Fig. 4 -

 4 Fig. 4 -Variations and conservation in POLD1. Upper figure: ExAC missense variants in POLD1 (residues 1-1107) are plotted by position (x-axis) against allele frequency (y-axis); missense variants occur at 293/1107 residues (26.5%), at an average frequency of 6.44 × 10-4; data are not plotted for positions without an ExAC variant. Upper middle figure: functional domains in POLD1; orange: nuclear localization signal; blue: exonuclease ('proof-reading') domain; purple: polymerase domain; green: CysA motif; red: CysB motif; triangles below CysA/B motifs show positions of conserved cysteine residues. Lower middle figure: mutations in POLD1; red rhomboids show positions of HGMD mutations reported in colorectal adenoma, carcinoma and adenomatous polyposis; orange rhomboids show positions of HGMD mutations reported in MDPL (mandibular hypoplasia, deafness, progeroid features and lipodystrophy) syndrome; the green rhomboid shows the position of the novel variant p.I1070N. Lower figure: ConSurf grades for POLD1 1-1107, scored from 1 (minimum) to 9 (maximum).

Fig. 5 -

 5 Fig. 5 -Genetic characterization of the POLD1 gene and its protein product. a: POLD1 is located on chromosome 19 at q13.3-q13.4 and covers an approximately 3.4 kb long chromosomal region, containing 27 exons. Previous mutations in MDPL syndrome patients are indicated in blue and the new identified mutation in red. Germinal mutations reported in patients affected with colorectal adenoma are indicated in green. b: Schematic of the functional domains of POLD1 protein showing the relative locations of: nuclear localization signal in green; the exonuclease domain in red; the polymerase active site in blue and the ZnF modules (labeled I: CysA and II: CysB) in yellow. The exons encoding each domain are indicated in the corresponding color. c: Phylogenetic conservation of POLD1-CysB amino-acid sequence among different species. The position of the aminoacid substitution found in patient 1 is indicated by the dark arrow. The four invariant cysteine residues are indicated by the green arrows.

Fig. 6 -

 6 Fig. 6 -Analysis of POLD1 secondary structure using the PredictProtein server. a: Functional domains of POLD1, as shown in Fig. 5b. b: Secondary structure and solvent accessibility predictions from PredictProtein for wild-type and variant POLD1 (data shown for residues 1026-1107 only). The upper line shows a numbered scale; CysA (partial) and CysB regions are indicated by yellow boxes, and the position of residue 1070 is indicated by a red arrow; gold triangles represent positions of the four invariant cysteine residues in CysB (Cys1058/1061/1071/1076). For each sequence, secondary structure predictions (2°) are shown for the REPROFsec (upper line) and PROFsec (lower line) algorithms of the PredictProtein server (red bars, α-helix; blue bars, β-strand); solvent accessibility (acc) is indicated by yellow (buried) or blue (exposed) bars; regions of significant difference between wild-type and variant sequences are boxed by broken red lines in the Ile1070Asn panel.

Table 2

 2

	). We

Table 1 -

 1 Comparison of the phenotypic characteristics of MDPL patients with pathogenic mutations in the POLD1 gene (literature and this study).

		Present study	Ajluni	Lessel et al.,	Reinier	Pelosini	Weedon
		Patient 1	Patient 2	et al., 2017	2015 (ranges of reported values)	et al., 2015	et al., 2014	et al., 2013
				(ranges of				(ranges of
				reported				reported
				values,				values)
				POLD1
				mutated)
	Age (y) at referral	24	6	14-34	10-62	41	48	19-37
	Sex	F	M	2F	3 M/5 F	F	F	3 M/1F
	Age at onset (y)	7	5	NR	10-20 (1 NR)	3	5	2.5-4 (2 NR)
	Age of death (y)	25	NA	NR	34, ovarian cancer (1/8) NR	NR	NR
		(respiratory
		failure)
	Ethnicity	British	British	White-	3 French; 2 US, mixed	Italian	Italian	2 British; 1
				Hispanic	European; 1 Swedish; 1			Italian; 1
					Hungarian, 1 Colombian			Indian
	Birth weight (kg)	2.7	3.18	NR	2.4-3.30 (4 NR)	NA	3.6	3-4.2
	Height (m) (Z-score)	1.2 (-6.6)	1.114 (-0.9)	163-171	123-162 (-0.9;-3.7)	1.45 (-2.8)	1.62 (-0.27) 1.60-1.91
				(1.19;0.38)				(-0.50;1.99)
	Weight (kg)	12.6	16.24	50.4-53.0	21-66	33	47.7	41.3-69.7
	BMI (kg/m 2 )	8.8	13.09	18.1-19.0	13.8-26.8	15.7	18.2	14.6-19.1
	Clinical characteristics
	Lipodystrophy	+	+	+	8/8 Yes	+	+	4 Yes
	Mandibular hypoplasia	+	+	-	7/8 Yes	+	+	4 Yes
	pinched facies	+	+	-	8/8	+	+	4 yes
	Beaked nose	+	+	-	8/8	+	+	4 yes
	High-pitched voice	-	-	-	5/8 Yes	+	NR	4/4 Yes
	Graying or loss	-	+	-	3/8 Yes	-	-	0/4
	of hair
	Joint contractures	+	+	+	1/8 Yes	-	+	2/4 Yes
	Telangiectasia	+	-	-	5/8 Yes	+	-	3/4 Yes
	Acanthosis	-	+	-	0/8	-	-	0/4
	Crowded teeth	+	+	-	5/8 Yes	+	+	4/4 Yes
	Deafness (age of	-	+ (4)	-	5/8 Yes (11-14)	10	25	4/4 Yes
	onset, y)							(7-33)
	Lack of breast	+	N A	-	NR	+	+	1/4 Yes
	development							(3 NA)
	Short stature	+	+	-	7/8 Yes	+	-	0/4
	Corneal keratosis	+	-	-	0/8	-	-	4/4 Normal
	Kyphosis/scoliosis	+	-	-	NR	-	+	3/4 Yes
	Osteoporosis	+	-		2/8 Yes (1 NR)	-	-	3/4 Yes
	Hypogonadism	+	Right	+	1/8 Yes	+	-	3/4 Yes
			chryptorchidism	Polycystic	(Cryptorchidism)
			and phymosis	ovary
			under investigation	syndrome
	Cognitive function	Normal	Normal	-	6/8 Normal;	-	-	4/4 Normal
					1 Depression, anxiety;
					1 NR
	Respiratory tract	+	-	-	NR	NR	NR	NR
	infections
	Metabolic profile
	Hepatic steatosis	-	-	+	1/8 (6 NR)	(22)	+	2/4 Yes
	(years)							(1 NR)
	Hepatomegaly	-	-	-	3/8 Normal (5 NR)	+	+	1/4 Yes
								(1 NR)
	Hypertriglyceridemia	-	-	+	5/8 (2 NR)	+	25	2/4 Yes
	Diabetes (age at	?	-	+	2/8 Yes (28-43)	30	-	3/4 Yes
	diagnosis)							(14-37)
	Triglycerides	NA	NA	5.87-7.92	2.15-2.62 (2NR)	3.47	NR	1.48-2.6
	(mmol/l)
	Total Cholesterol	NA	2.9	NR	2.99-5.87 (2NR)	5.53	NR	4.2-5.4
	(mmol/l)
	HDL-C (mg/dl)	NA	49	NR	25-47 (4 NR)	47	NR	16-35
								(1 NA)

Table 1

 1 (continued)

		Present study	Ajluni	Lessel et al.,	Reinier	Pelosini	Weedon
		Patient 1	Patient 2	et al., 2017	2015 (ranges of reported values)	et al., 2015	et al., 2014	et al., 2013
				(ranges of				(ranges of
				reported				reported
				values,				values)
				POLD1
				mutated)
	Leptin (ng/ml)	NA	NA	9.0-11.5	NR	NR	5.4	4.5-8.22
								(1 NA)
	Fasting insulin	NA	357	NR	NR	190	243	169-235
	(pmol/l)							(1 NR)
	Insulin Like Growth Factor	11. 3 (13-50) 25.7	NR	NR	NR	NR	NR
	(nmol/l) (Normal ranges)
	OGTT fasting glucose	8.4	<5.6	NR	4.22-5.21 (1 NR)	NR	5.28	4.2-7.7
	(mmol/l)
	OGTT 2 h glucose	NA	NA	NR	NR	NR	10.2	8.9-16.4
	(mmol/l)
	HbA1c (mmol/mol)	NA	31.1	90.2-91.3	27.9-37.7 (4 NR)	NR	44	36-49
								(1 NR)
	Abnormal LFTs ever	-	NA	NR	NR	NR	+	3/4 Yes
	ALT (u/L) (Normal	11 (4-45)	NA	51-97	25-44 (4 NR)	53	NR	32-172
	ranges)
	ALP (U/L) (Normal	96 (35-129)	NA	NR	NR	47	NR	66-103
	ranges)							(1 NR)
	BIL (μmol/L) (Normal	6 (0-21)	NA	NR	5.1-13.6 (4 NR)	3.4	NR	5.6-12
	ranges)
	ALB (g/L)	96	NA	NR	NR	NR	NR	43-53.7
	FSH (UI/l)	8	NA	NR	NR	4.07	NR	NR
	LH (UI/l)	4.9	NA	NR	NR	2.30	NR	NR
	Hypothyroidism	-	-	NR	1/8 (14)	-	-	-
	(age at diagnosis)
	Prolactin (ng/ml)	70 (102-496) NA	NR	NR	43.85	NR	NR
	(Normal ranges)
	Fat distribution (DEXA)
	Whole-body fat	NA	NA	NR	NR	NR	24.5	14.0-22.6
	(% body mass)
	Truncal fat (%	NA	NA	40.4-58.3	NR	NR	29.9	19.1-22.6
	regional mass)							(1 NR)
	Arm fat (% regional	NA	NA	NR	NR	NR	NR	4.0-20.4
	mass)							(1 NR)
	Leg fat (% regional	NA	NA	11.9-23.2	NR	NR	NR	4.8-13.9
	mass)							(1 NR)
	Truncal fat (%)/leg	NA	NA	1.43-2.01	NR	NR	2	1.71-22.1
	fat (%)							(1 NR)
	POLD1 mutation	p.Ile1070Asn p.Ser605del	p.E1067K	7/8 p.Ser605del; 1/8	p.Ser605del p.Arg507Cys 4/4
					p.Arg507Cys			p.Ser605del

NR: not reported; NA: not available; +: Yes; -: No; LFTs = abnormal liver function tests;?: not confirmed.

Table 2 -

 2 Variant filtering results of whole exome sequencing for patient 1 (The number of variations decreases from top to bottom by applying each described filter).

		Autosomal recessive		Autosomal
	Filtering conditions/inheritance models	Homozygous in patient 1 and heterozygous in parents	Heterozygous composite	dominant de novo
	Total number of variants	3427	10,974	2150
	Missense, nonsense, splice-site or frameshift variants	458	1553	244
	MAF <1% in the 1000Genome, ExAC, ESP	0	143	32
	CADD score >10	0	111	17
	Prediction in silico (UMD, SIFT, MutationTaster etc.)	0	36	8
	Remove dbSNP138 registered variants	0	6	2
	IGV visualization	0	6	1
	Selected mutation	0	0	POLD1:p.Ile1070Asn

Table 1

 1 ) while the stature of the other patients carrying POLD1 mutations reported by Pelosini et al. and Reinier et al. were closer or into normal Z-score ranges (please see Table 1). On the other hand, our patient showed hypogonadism, which has been reported in several male patients carrying POLD1 mutations but also in a female patient by Reinier et al. and in one of the patients carrying the p.E1067K mutation in the ZNF2 domain by Ajluni et al. As shown in Table

  CS, et al. A novel syndrome of mandibular hypoplasia, deafness, and progeroid features associated with lipodystrophy, undescended testes, and male hypogonadism. J Clin Endocrinol Metab 2010;95:E192-7.[2] De Sandre-Giovannoli A, Bernard R, Cau P, Navarro C, Amiel J,

Boccaccio I, et al. Lamin a truncation in Hutchinson-Gilford progeria. Science 2003;300:2055.