**7.3 Reproducibility of MUNE technique**

644 Amyotrophic Lateral Sclerosis

Study Groups

Fig. 7. Baseline thenar (APB) MUNE subdivided into study groups (The lower boundary of the box is the 25th percentile, and the upper border is the 75th percentile of MUNE. The horizontal line inside the box represents the median MUNE. The whispers represent the

In population controls the mean EDB MUNE was 138 with a range of 119 - 169, in SOD1 negative family controls was 134 with a range of 107 - 180 and in asymptomatic SOD1

Once again, there was no detectable difference in the mean number of EBD motor units in the group of asymptomatic SOD1 mutation carriers compared to the group of SOD1 negative family controls (EDB p>0.95), or population controls (EDB p>0.50) (Table 2 and

**Population Controls** 13 138 (119-169)

**Controls** 30 134 (107-180) **SOD1 Mutation Carriers** 14 136 (111-187) **Sporadic MND patients** 9 70 (8-82) Table 2. EDB motor unit number estimates (MUNE number represents mean MUNE).

range of 5 - 84 and a mean EDB MUNE of 70 with a range of 8 - 82 (Tables 1 and 2).

Symptomatic sporadic MND subjects showed a definite loss of motor units with fewer motor units compared to all other groups (p<0001) with a mean thenar MUNE of 45 with a

There was no cross over between thenar and EDB MUNE results in symptomatic and

SOD1 Mut Carriers

largest and smallest observed values, i.e. the range). Data is shown in Table 1.

Population Controls

mutation carriers, 136 with a range of 111 - 187.

APB MUNE

Figure 8).

**SOD1 Negative Family** 

asymptomatic subjects.

250

200

150

100

50

0

SOD1 Neg Controls

**Extensor Digitorum Brevis Cases MUNE (Range)** 

Sporadic MND

To assess the test-retest reproducibility of the technique, 69 of the 88 SOD1 family members and population controls were followed over a 1-year period, with thenar and extensor digitorum brevis (EDB) MUNE tests repeated every 3-6 months, depending on patient availability. The difference between MUNE results from the first and second study, and if possible, first and third studies were divided by the MUNE of the first study, and expressed as a percentage change. The results were analysed using Pearson and Spearman correlation coefficients.

The test-retest correlation of thenar MUNE in asymptomatic subjects was high with a Pearson correlation coefficient of 0.93. The mean difference between MUNE results on separate occasions on the same individual was +/- 3.6%, with a range of 0-11.7% (Table 3).


Table 3. Reproducibility of mean thenar (APB) motor unit number estimates in asymptomatic subjects on separate reviews over a one-year period.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

Average Right Handgrip (lb)

150

100

50

0

thenar (APB) MUNE

during the study period. In individual cases, there was:

MND was confirmed.

corresponding with the development of symptoms.

51% loss of motor units, 4 months prior to onset of weakness in Case 1 37% loss of motor units, 10 months prior to onset of weakness in Case 2 28% loss of motor units, 6 months prior to onset of weakness in Case 3 46% loss of motor units, 6 months prior to onset of weakness in Case 4 68% loss of motor units, 8 months prior to onset of weakness in Case 5

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 647

Average APB MUNE

Fig. 10. Scatter graph showing the lack of correlation between right handgrip and right

**7.5 Detection of pre-symptomatic motor neurone loss in SOD1 mutation carriers**  The MUNE results, after validating their reproducibility, were used as a baseline to follow the number of motor units over time in individual pre-symptomatic SOD1 mutation carriers over the next 2-5 years, to determine whether pattern of motor neurone loss is either a slow attrition of motor neurones over time or whether normal numbers of motor neurones are maintained until sudden, rapid multi-focal cell death of motor neurones occurs,

During the course of the study, 5 of the SOD1 mutation carriers developed leg weakness. A significant fall in motor unit number was detected in these 5 SOD1 mutation carriers, were there was a detectable reduction of motor units, 4-10 months prior to the onset of weakness and the diagnosis of familial ALS being made. There was no detectable loss of motor units in the other 15 SOD1 mutation carriers or in the group of SOD1 mutation negative relatives,

There was further motor unit loss as weakness progressed, at which point the diagnosis of

0 50 100 150 200 250 300

For EDB MUNE, the Pearson correlation coefficient was also high, 0.88, with a mean difference between MUNE results on separate occasions on the same individual of +/- 4.6%, with a range of 0-15.7%. The test-retest correlation was high with a Pearson correlation coefficient of 0.91, when groups were broken down into the different study groups.

### **7.4 Maximal voluntary isometric contraction**

Maximal voluntary isometric contraction (MVIC), using the Jamar hand dynamometer was used to measure isometric grip strength to determine whether this correlated with the number of functional motor neurones in the thenar group of muscles as measured by MUNE. Isometric grip strength tests, thenar MUNE and MRC power were performed on 69 asymptomatic subjects twice within a 3-6 month period to assess the test-retest reproducibility of this technique. Pearson correlation coefficients between study 1 and study 2 of right hand grip strength was 0.941, left hand grip strength 0.910 and thenar MUNE results 0.937. These results indicate that the reproducibility of these techniques was high.

Right hand grip strength correlated with left hand grip strength, with Pearson correlation coefficients of 0.959 and Spearman correlation coefficients of 0.956 Two-way analyses of variance showed a no significant difference between the right and left hands (Figure 9). There was no correlation between right grip strength and right thenar motor unit number, with Pearson correlation coefficients of 0.483 and Spearman correlation coefficient of 0.34 (Figure 10).

Fig. 9. Graph showing the correlation between right and left handgrip

For EDB MUNE, the Pearson correlation coefficient was also high, 0.88, with a mean difference between MUNE results on separate occasions on the same individual of +/- 4.6%, with a range of 0-15.7%. The test-retest correlation was high with a Pearson correlation

Maximal voluntary isometric contraction (MVIC), using the Jamar hand dynamometer was used to measure isometric grip strength to determine whether this correlated with the number of functional motor neurones in the thenar group of muscles as measured by MUNE. Isometric grip strength tests, thenar MUNE and MRC power were performed on 69 asymptomatic subjects twice within a 3-6 month period to assess the test-retest reproducibility of this technique. Pearson correlation coefficients between study 1 and study 2 of right hand grip strength was 0.941, left hand grip strength 0.910 and thenar MUNE results 0.937. These results indicate that the reproducibility of these techniques was high. Right hand grip strength correlated with left hand grip strength, with Pearson correlation coefficients of 0.959 and Spearman correlation coefficients of 0.956 Two-way analyses of variance showed a no significant difference between the right and left hands (Figure 9). There was no correlation between right grip strength and right thenar motor unit number, with Pearson correlation coefficients of 0.483 and Spearman correlation coefficient of 0.34

coefficient of 0.91, when groups were broken down into the different study groups.

Average Left Handgrip (lb)

Fig. 9. Graph showing the correlation between right and left handgrip

0 50 100 150

**7.4 Maximal voluntary isometric contraction** 

(Figure 10).

150

100

50

0

Average Right Handgrip (lb)

Fig. 10. Scatter graph showing the lack of correlation between right handgrip and right thenar (APB) MUNE

### **7.5 Detection of pre-symptomatic motor neurone loss in SOD1 mutation carriers**

The MUNE results, after validating their reproducibility, were used as a baseline to follow the number of motor units over time in individual pre-symptomatic SOD1 mutation carriers over the next 2-5 years, to determine whether pattern of motor neurone loss is either a slow attrition of motor neurones over time or whether normal numbers of motor neurones are maintained until sudden, rapid multi-focal cell death of motor neurones occurs, corresponding with the development of symptoms.

During the course of the study, 5 of the SOD1 mutation carriers developed leg weakness. A significant fall in motor unit number was detected in these 5 SOD1 mutation carriers, were there was a detectable reduction of motor units, 4-10 months prior to the onset of weakness and the diagnosis of familial ALS being made. There was no detectable loss of motor units in the other 15 SOD1 mutation carriers or in the group of SOD1 mutation negative relatives, during the study period.

In individual cases, there was:

51% loss of motor units, 4 months prior to onset of weakness in Case 1

37% loss of motor units, 10 months prior to onset of weakness in Case 2

28% loss of motor units, 6 months prior to onset of weakness in Case 3

46% loss of motor units, 6 months prior to onset of weakness in Case 4

68% loss of motor units, 8 months prior to onset of weakness in Case 5

There was further motor unit loss as weakness progressed, at which point the diagnosis of MND was confirmed.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 649

Fig. 12. Progressive results of case 1 showing the change in APB and EDB motor unit estimates over time in relation to handgrip strength and power. There is a reduction of APB

The MUNE results remained stable over the first 2 years of the study. By November 2000, her left EDB MUNE had dropped to 64 (total reduction of 51%), but she only developed wasting and weakness of the anterior compartment muscles of her left leg, 4 months later, when she had MRC grade 2/5 of dorsiflexion and eversion, 3/5 plantarflexion and inversion 3/5. Proximal muscles, upper limb and right leg muscles were normal. Deep tendon reflexes were brisk in the upper and lower limbs. Over the next 6 months, her right thenar MUNE from 130 to 115 (12%) She had no detectable right arm weakness at the time and her

and EDB MUNE prior to the onset of weakness.

handgrip strength remained at around 65 pounds.

Fig. 11. Pedigree of cases 1, 2 and 3

### **7.5.1 Case study 1**

A 48-year-old lady from a family with a strong history of familial ALS. Pedigree is shown in Figure 11. This family has a point mutation in the SOD1 gene at val148gly. At the time of recruitment, October 1998, the subject was asymptomatic, with a normal neurological examination and no evidence of wasting, weakness or fasciculations. Progress MUNE results are shown in Table 4 and Figure 12.


Table 4. Case 1 progressive handgrip, dorsiflexion power and thenar and EDB MUNE results

A 48-year-old lady from a family with a strong history of familial ALS. Pedigree is shown in Figure 11. This family has a point mutation in the SOD1 gene at val148gly. At the time of recruitment, October 1998, the subject was asymptomatic, with a normal neurological examination and no evidence of wasting, weakness or fasciculations. Progress MUNE

> **Jul-00**

**R Handgrip** 60 65 68 60 60 65 65 62 60 45 Died

**-26 -24 -21 -13 -7 -4 0 +3 +5 +8 +17** 

**Nov-00** 

**Mar-01** 

**Jun -01**

**Aug-01** 

**Nov-01** 

**Aug-02** 

Fig. 11. Pedigree of cases 1, 2 and 3

results are shown in Table 4 and Figure 12.

**98** 

**Mar-99** 

**Nov-99** 

**MUNE** 130 131 126 131 115 113 115 110 83 49 **L Handgrip** 60 60 60 60 60 60 55 45 37 18

**MUNE** 122 114 110 122 83 52 40 0

**power** 5/5 5/5 5/5 5/5 5/5 5/5 5/5 3/5 0/5 0/5

**MUNE** 124 120 132 116 116 114 114 72 53 0 **L EDB power** 5/5 5/5 5/5 5/5 5/5 5/5 0/5 0/5 0/5 0/5

**MUNE** 130 125 64 0 0 0 0

Table 4. Case 1 progressive handgrip, dorsiflexion power and thenar and EDB MUNE

**Date of study Oct-98 Dec-**

**7.5.1 Case study 1** 

**Months before and after weakness** 

**R Thenar** 

**L Thenar** 

**R EDB** 

**R EDB** 

**L EDB** 

results

Fig. 12. Progressive results of case 1 showing the change in APB and EDB motor unit estimates over time in relation to handgrip strength and power. There is a reduction of APB and EDB MUNE prior to the onset of weakness.

The MUNE results remained stable over the first 2 years of the study. By November 2000, her left EDB MUNE had dropped to 64 (total reduction of 51%), but she only developed wasting and weakness of the anterior compartment muscles of her left leg, 4 months later, when she had MRC grade 2/5 of dorsiflexion and eversion, 3/5 plantarflexion and inversion 3/5. Proximal muscles, upper limb and right leg muscles were normal. Deep tendon reflexes were brisk in the upper and lower limbs. Over the next 6 months, her right thenar MUNE from 130 to 115 (12%) She had no detectable right arm weakness at the time and her handgrip strength remained at around 65 pounds.

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 651

Fig. 14. Progressive results of case 2 showing the change in APB and EDB motor unit estimates over time in relation to handgrip strength and power. There is a reduction of APB

Over the next 6 months, there was a reduction in her right thenar MUNE to 96 (20%) and her left thenar MUNE to 89 (19%), with no detectable weakness. Her right EDB MUNE also dropped from 111 to 92 (17%), but she only had detectable weakness 10 months later of MRC grade 4+/5 in right dorsiflexors, at which time her right EDB MUNE had dropped further to 71 motor units (35%). The left EDB MUNE also dropped from a baseline of 112 (2

An independent neurologist performed needle EMG examination, which showed high amplitude motor units with reduced recruitment in vastus medialis, tibialis anterior and

and EDB MUNE even though strength has remained stable.

years previously) to 89 (20%), but with no detectable weakness.

An independent neurologist performed needle EMG, which showed extensive denervation in left leg muscles with fibrillation potentials in the left tibialis posterior, gastrocnemius, vastus medialis and L5/S1 paraspinal muscles. There were no changes in the right vastus medialis, biceps brachialis, triceps, left deltoid and the tongue. It was felt that these changes were not enough to make a diagnosis of ALS. She went on to have a MRI scan of her lumbar spine showed degenerative disc disease of L4/5 and L5/S1, with no evidence of neural compression.

Over the next 6 months, her right EDB MUNE continued to drop from 114 to 72 and subsequently to 53, a 54% reduction. Her left thenar MUNE also dropped from 122 to 83 motor units, a 36% reduction. Her right foot power remained normal until August 2001, 7 months after the reduction was noted. At that time, she developed upper limb weakness, with a reduction of left handgrip strength to 45 pounds.

Repeat needle EMG examination showed severe denervation in the left tibialis anterior and gastrocnemius, but still no changes in proximal right lower limb or upper limb muscles. Once again, this was considered not to be diagnostic for ALS. Her EDB and thenar MUNE however continued to drop and she developed upper and lower limb weakness and became wheelchair bound. She subsequently died with respiratory failure in August 2002.

### **7.5.2 Case study 2**

A 43-year-old sister of case 1. She had the same strong family history of ALS, with a point mutation in SOD1 gene at val148gly. Her pedigree is shown in Figure 11. She was asymptomatic at the time of recruitment with a normal neurological examination, and no evidence of wasting, weakness or fasciculation. Her right and left thenar MUNE's remained stable for the first 2½ years of the study at around 115-120 motor units. Progress MUNE results are shown in Table 5 and Figures 14.


Table 5. Case 2 progressive handgrip, dorsiflexion power and thenar and EDB MUNE results

An independent neurologist performed needle EMG, which showed extensive denervation in left leg muscles with fibrillation potentials in the left tibialis posterior, gastrocnemius, vastus medialis and L5/S1 paraspinal muscles. There were no changes in the right vastus medialis, biceps brachialis, triceps, left deltoid and the tongue. It was felt that these changes were not enough to make a diagnosis of ALS. She went on to have a MRI scan of her lumbar spine showed degenerative disc disease of L4/5 and L5/S1, with no evidence of neural

Over the next 6 months, her right EDB MUNE continued to drop from 114 to 72 and subsequently to 53, a 54% reduction. Her left thenar MUNE also dropped from 122 to 83 motor units, a 36% reduction. Her right foot power remained normal until August 2001, 7 months after the reduction was noted. At that time, she developed upper limb weakness,

Repeat needle EMG examination showed severe denervation in the left tibialis anterior and gastrocnemius, but still no changes in proximal right lower limb or upper limb muscles. Once again, this was considered not to be diagnostic for ALS. Her EDB and thenar MUNE however continued to drop and she developed upper and lower limb weakness and became

A 43-year-old sister of case 1. She had the same strong family history of ALS, with a point mutation in SOD1 gene at val148gly. Her pedigree is shown in Figure 11. She was asymptomatic at the time of recruitment with a normal neurological examination, and no evidence of wasting, weakness or fasciculation. Her right and left thenar MUNE's remained stable for the first 2½ years of the study at around 115-120 motor units. Progress MUNE

**-42 -40 -37 -29 -20 -10 0 +11 +21 +27** 

03

Feb-04

wheelchair bound. She subsequently died with respiratory failure in August 2002.

**Date of study** Oct-98 Dec-98 Mar-99 Nov-99 Jul-00 Jan-01 Nov-01 Oct-02 Aug-

Table 5. Case 2 progressive handgrip, dorsiflexion power and thenar and EDB MUNE

**R Handgrip** 60 60 65 65 60 70 65 65 65 65 **R Thenar MUNE** 111 111 117 119 120 114 96 97 86 85 **L Handgrip** 60 55 60 65 63 65 65 60 60 60 **L Thenar MUNE** 117 119 111 89 86 79 81 **R EDB power** 5/5 5/5 5/5 5/5 5/5 5/5 4+/5 4+/5 4+/5 4+/5 **R EDB MUNE** 104 111 119 108 104 92 71 75 75 65 **L EDB power** 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 **L EDB MUNE** 112 89 80 80 81

with a reduction of left handgrip strength to 45 pounds.

results are shown in Table 5 and Figures 14.

compression.

**7.5.2 Case study 2** 

**Months pre and post weakness 1st detected** 

results

Fig. 14. Progressive results of case 2 showing the change in APB and EDB motor unit estimates over time in relation to handgrip strength and power. There is a reduction of APB and EDB MUNE even though strength has remained stable.

Over the next 6 months, there was a reduction in her right thenar MUNE to 96 (20%) and her left thenar MUNE to 89 (19%), with no detectable weakness. Her right EDB MUNE also dropped from 111 to 92 (17%), but she only had detectable weakness 10 months later of MRC grade 4+/5 in right dorsiflexors, at which time her right EDB MUNE had dropped further to 71 motor units (35%). The left EDB MUNE also dropped from a baseline of 112 (2 years previously) to 89 (20%), but with no detectable weakness.

An independent neurologist performed needle EMG examination, which showed high amplitude motor units with reduced recruitment in vastus medialis, tibialis anterior and

Protection of Motor Neurons in Pre-Symptomatic Individuals Carrying

**Months pre and post** 

**7.5.5 Case study 5** 

**Months pre and post** 

**L Thenar MUNE** 128

SOD 1 Mutations: Results of Motor Unit Number Estimation (MUNE) Electrophysiology 653

She subsequently developed left lower leg weakness with inversion and eversion of MRC grade 3/5 and dorsiflexion and plantarflexion 4/5, 6 months later. Her disease progressed rapidly and died within the 4 months of diagnosis, in March 2002 of respiratory failure.

**weakness 1st detected -33 -30 -25 -14 -6 0 +4** 

**R Thenar MUNE** 149 144 142 151 153 123 **L Handgrip** 50 60 65 60 55 58

**R EDB power** 5/5 5/5 5/5 5/5 5/5 5/5 **R EDB MUNE** 126 132 137 119 118 118 **L EDB power** 5/5 5/5 5/5 5/5 5/5 3/5 **L EDB MUNE** 123 118 64 42

wasting, weakness or fasciculations. Progress MUNE results are shown in Table 8.

**weakness 1st detected -50 -48 -44 -35 -22 -8 0** 

**Date of study** May-98 Jul-98 Nov-98 Aug-99 Sep-00 Nov-01 Jul-02 **R Handgrip** 115 110 120 115 110 115 120 **R Thenar MUNE** 127 117 107 116 138 116 121 **L Handgrip** 100 100 105 105 105 100 100 **L Thenar MUNE** 135 122 120 **R EDB power** 5/5 5/5 5/5 5/5 5/5 0/5 0/5 **R EDB MUNE** 125 114 129 106 115 0 0 **L EDB power** 5/5 5/5 5/5 5/5 5/5 5/5 4/5 **L EDB MUNE** 127 114 36 0 Table 8. Case 5 progressive handgrip, dorsiflexion power and thenar and EDB MUNE results The MUNE results remained stable over the first 3 years of the study. The right EDB MUNE dropped from 115 down to being not recordable. Thenar MUNE remained stable at about 130 motor units. He had occasional fasciculations in the right quadriceps region, weakness of the right quadriceps and MRC grade 0/5 weakness of right dorsiflexion. He was unsure

**Date of study** Apr-98 Jul-98 Dec-98 Nov-99 Apr-01 Oct-01 Mar-02 **R Handgrip** 60 66 70 80 60 70 Died

Table 7. Case 4 progressive handgrip, dorsiflexion power and thenar and EDB MUNE results

A 44-year-old man with a strong family history of ALS and a 2nd cousin once removed of case 4. His family also had a point mutation in SOD1 gene at glu100gly. At the time of recruitment, the subject was asymptomatic, with a normal neurological examination and no

extensor carpi radialis longus, bilaterally but no fibrillation potentials were seen. It was felt that these changes were not enough to make the diagnosis of ALS.

In view of her strong family history, a presumed diagnosis of familial ALS was made and she was commenced on Riluzole in February 2002.

Over the next 3 years, her EDB MUNE results have stabilised. Her weakness has not progressed significantly. In February 2004, she still had MRC grade 4+/5 power of her right dorsiflexors and no symptomatically apparent weakness in her left dorsiflexors or upper limbs.
