Title: Voxel based analysis in neuroferritinopathy determines clinical correlates of

disease severity. MJ Keogh et al.

Online Resources

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Figure 1. Total cerebellar volumes as determined by serial ROI assessment of 3D-T1w axial slices and

normalised to total intra-cranial volume. The plot shows individual subject data points and mean cerebellar

volumes + standard deviation of the mean for patients and controls. There was a significant difference between

the two groups (p<0.001, unpaired t-test). Intra-rater reliability had a coefficient of variation of 3.6% as

determined from repeated measurements made on one dataset on three separate occasions each spaced 7 days

apart.

Figure 2. Image intensities and probability data in the dentate region in (a) qualitative T1w raw images, (b)

unsmoothed T1w GM segmented images, (c) smoothed T1w GM segmented images, (d) quantitative T2 maps,

(e) smoothed 8 voxel T2 VBR images.

Figure 3: Delineation of ROIs of regions detected to correlate with UDRS score on T1 images in a single patient

(a) head of caudate, (b) lateral GPi

Figure 4. Image intensities and probability data in the globus pallidus internus (a) qualitative T1w raw images,

(b) unsmoothed T1w GM segmented images, (c) smoothed T1w GM segemented images, (d) quantitative T2

maps, (e) smoothed 8 voxel T2 VBR images.

Figure 5: T1w slice through the cerebellum in a control subject and patient with neuroferritinopathy (top row).

Outlined in red are corresponding lateral aspects of the right dentate nuclei, with iron deposition and cavitation

seen in the patient. Bottom row: smoothed grey matter segmented slices through the same region showing that

the dentate nucleus in the patient is in a region with increased signal intensity.

Figure 6: T2w slice through the cerebellum in a control subject and patient with neuroferritinopathy (top row).

Outlined in red are corresponding lateral aspects of the right dentate nuclei, with iron deposition and cavitation

seen in the patient. Bottom row: quantitative T2 maps through the same region following smoothing which show

that the dentate nucleus in the patient is in a region with similar intensity to the control.

Figure 7. T1w image intensities and probability data of the caudate nucleus and lateral globus pallidus. Left –

caudate: (a) raw T1w image intensity, (b) unsmoothed grey matter segmented probability, and (c) smoothed

grey matter probability against patient UDRS score for 9 patients. Right – Globus pallidus internus: (a) raw

image intensity, (b) unsmoothed grey matter probability, and (c) smoothed grey matter probability against

patient UDRS score for all 10 patients. *p<0.05, ** p<0.005.

Cluster-level Voxel-level Coordinates AreaP(corr) Size

(voxels)p(uncor) T-value x y z side

<0.001 244 <0.0001 23.57 -16 -52 -35 L Posterior cerebellar lobe, dentate nucleus<0.001 221 <0.0001 22.89 14 -53 -33 R Posterior cerebellar lobe, dentate nucleus<0.001 1099 <0.0001 21.70 -8 -14 -8 L Left mid brain/substantia nigra

<0.0001 19.43 8 -14 -8 R Right midbrain/substantia nigra<0.0001 14.70 10 -7 -3 R Right sub lobar extranuclear white matter

<0.001 124 <0.0001 9.30 15 -25 11 R Right thalamus/pulvinar<0.001 73 <0.0001 8.79 -18 -26 2 L Left thalamus

Table 1. Grey matter segmented VBM analysis of patients compared to controls (Pt > controls) (n=10). Voxel size 2x2x2 mm3. Only voxel level significance considered. A threshold of T=7.088, P<0.05 (FWE) was used between groups.

Cluster-level Voxel-level Coordinates AreaP(corr) Size

(pixels)p(corr) T-value x y z side

<0.001 3474 <0.0001 19.31 -14 -1 -5 L Lentiform nucleas/medial globus pallidus<0.0001 18.46 10 -1 -5 R Sub lobar, extra nuclear white matter<0.0001 12.79 -16 -30 3 L Thalamus

<0.001 6373 <0.0001 15.42 -38 -60 -31 L Posterior cerebellar lobe (Tuber)14.86 14 -76 -30 R Posterior cerebellar lobe (pyramis)

<0.0001 14.24 -29 -68 -28 L Posterior cerebellar lobe (Pyramis)<0.001 188 <0.0001 11.72 16 -30 4 R Thalamus<0.001 21 <0.0001 10.53 -5 -91 -4 L Occipital lobe, lingual gyrus<0.001 23 <0.0001 9.94 17 -34 64 R Pre central gyrus

Table 2. White matter segmented VBM analysis of patients compared to controls (controls > patients) (n=10). Voxel size 2x2x2 mm3. Only voxel level significance considered. A threshold of T=7.17, P<0.05 (FWE) was used between groups.

Group Dentate nucleus Globus pallidus internusT1w T2w T1w T2w

Iron deposition Cavitation Iron deposition Cavitation Iron deposition Cavitation Iron deposition CavitationControls (n=10) 1 (10%) 0 (0%) N/A N/A 4 (40%) 0 (0%) N/A N/APatients (n=10) 10 (100%) 1 (10%) 10 (100%) 2 (20%) 10 (100%) 9 (90%) 10 (100%) 9 (90%)

Table 3. Clinical neuroradiology assessment of the dentate nucleus and globus pallidus internus of raw T1w and T2w images of patients and controls. Clinical interpretation of relative T1w hyperintensity and hypointensity consistent with iron deposition and cavitation respectively, were recorded for both regions in patients and controls. T2w hypointensity and hyperintensity consistent with iron deposition and cavitation respectively were recorded in patients only due to a lack of T2w images for controls.

Cluster-level Voxel-level Coordinates AreaP(uncorr) Size

(pixels)p(uncor) T-value x y z side

Negative correlation with UDRS score

0.011 85 <0.0001 7.66 10 8 14 R Right caudate body0.011 85 <0.0001 7.13 -10 9 10 L Left caudate body0.007 96 <0.0001 6.83 16 -2 4 R Right lentiform and lateral GP0.004 113 <0.0001 6.34 -20 -6 5 L Left lentiform and lateral GP

Table 4:Grey matter weighted VBM correlation analysis with UDRS score (n=10). Voxel size 2x2x2 mm3. Only voxel level significance considered. An threshold of T=4.8, p<0.001 uncorrected was used.

Study Imaging modality

Number of

patients

Brain region

Globus Pallidus

Putamen Caudate Thalamus Dentate Substantia Nigra

Cortex Cerebellum

McNeil et al 2008[2]

T2* 21 38% hypointensity

28% hypointensity

14% hypointensity

19% hypointensity

95% hypointensity

81% hypointensity

Motor cortex only affected (71%)

Keogh et al 2012[1]

T2* 9 Iron deposition in infancy

- Iron deposition in adulthood

Iron deposition in teens

- Iron deposition in infancy

Iron deposition in adulthood (motor cortex only)

McNeil et al 2011[3]

T2 10 50% cavitation, 10% hypointense, 20% EOTT

60% cavitation 10% cavitation 20% hypointense

90% hypointense

50% hypointense, 40% hyperintense,10% cavitation

R2* 10 No correlation assessed

Rim R2* negative correlation UDRS

No R2* correlation with disease

Rim R2* positive correlation with UDRS

- -

This study

T1 10 Increasing cavitation correlates with UDRS

T1 hyperintensity in patients

Increasing T1 intensity correlates with UDRS

T1 hyperintensity in patients

T1 hyperintensity in patients

T1 hyperintensity in patients

Motor cortex and lingual gyrus shows evidence of iron deposition

Cerebellar atrophy present

Table 5. A table outlining the results of previous neuroimaging studies in neuroferritinopathy together with the new findings from this study. All data relate to abnormal

findings in patients.

References

1. Keogh MJ, Jonas P, Coulthard A, Chinnery PF, Burn J (2012) Neuroferritinopathy: a new inborn error

of iron metabolism. Neurogenetics 13:93-96

2. McNeill A, Birchall D, Hayflick SJ, Gregory A, Schenk JF, Zimmerman EA, Shang H, Miyajima H,

Chinnery PF (2008) T2* and FSE MRI distinguishes four subtypes of neurodegeneration with brain

iron accumulation. Neurology 70:1614-1619

3. McNeill A, Gorman G, Khan A, Horvath R, Blamire AM, Chinnery PF (2012) Progressive Brain Iron

Accumulation in Neuroferritinopathy Measured by the Thalamic T2* Relaxation Rate. AJNR Am J

Neuroradiol