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Progressive brain calcifications and signs in a family with the L9R mutation in the

PDGFB gene

1. Clinical synopsis:

Very little is known about the natural history of PDGFB-associated PFBC. So far, nine

PDGFB mutations have been described (1–6). Four patients from the F13 family (a father and

three siblings) harboring the c.26T>G (L9R) mutation in exon 1 of the PDGFB gene (1) were

evaluated clinically and went through radiological and biochemical studies. The pedigree is

displayed in figure e-1. Mean time for clinical follow up was 5.5 years and between first and

last brain CT scans was 4.8 years. The study was approved by the local ethics committee and

the radiation protection organization at the Karolinska University Hospital

(Etikprövningsnämnden dnr 2013/924-31).

At the time of PFBC diagnosis all the affected patients (four in total) met the criteria for

migraine with aura, mean age at onset of migraine was 13.8 (range 12-16) (1). Mean age at

last exam was 35.5 years (range 23-59). The following motor scales were used in our

assessment: Tremor rating scale (TRS), Unified Parkinson’s disease rating scale (UPDRS)

part III, Unified Huntington’s disease rating scale (UHDRS), Scale for the Assessment and

Rating of Ataxia (SARA) and Inventory of non-ataxia Symptoms (INAS). Mean age at onset

for a movement disorders was 28.25 (range 17-52). All the participants reported varying

degrees of action tremor as the first movement disorder. The motor scores and radiological

findings are summarized in table e-1. Ages at onset are summarized in table e-2. The degree

of calcification on base line exams was determined using the total calcification score (TCS)

(3). The rate of calcification progression was evaluated using both the TCS and the co-

registration method described in this paper. We also attempted to measure the density of

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hydroxyapatite (HA) in calcified brain areas. Details of the radiological methods and results

are summarized in tables e-5 to e-9. Biochemical analyses are summarized in table e-10.

The patients were also screened with the Hospital Anxiety and Depression Scale (HAD-A and

HAD-D). More than 10 points in each subscale indicates possible depression or anxiety.

Neuropsychological testing of the index case III:1 (2009 and 2015) and III:3 (2015/2016)

were performed using the following batteries: 1. Brief cognitive status: Montreal Cognitive

Assessment (MoCA) and Mini Mental State Examination (MMSE); 2. General intellectual

ability (IQ): Ravens progressive matrices; 3. Evaluation of verbal episodic memory: Rey

Auditory Verbal Learning Test RAVLT (RAVLT); 4. Visuospatial episodic memory: Rey

Osterrieth Complex Figure Test (ROCFT); 5. Working memory: digit span of the Wechsler

Adult Intelligence Scale (WAIS-III); 6. Spatial/visual construction: ROCFT, Copy and Block

Design/WAIS; 7. Verbal concept formation: Similarities in WAIS-III; 8. Word fluency:

Controlled Oral Word Association Test (FAS/COWAT); 9. Picture Naming: Boston Naming

Test (BNT); 10. Information processing speed: Symbol Digit Modalities Test (SDMT); 11.

Executive function: Trail Making Test, B (TMT); 12. Motor speed: Finger-tapping test (FT),

dominant and non-dominant hand. Z scores which were computed on the basis of reference

values from test manuals and handbooks were used to compare results from various tests

(7,8). A z score ≤ -1.5 SD is compatible with a significant cognitive deficit. Patients II:3 and

III:2 did not undergo a planned cognitive evaluation. The cognitive and language features are

summarized in tables e-3 and e-4. In this study we demonstrate progressive brain

calcifications and symptoms. To illustrate the motor progression we provide video recordings

from two different occasions, for subject II:3 we have 3 video recordings (supplementary

material). Due to progression the patients were offered treatment with clodronic acid based on

the effect of bisphosphonates described in a previous report (9). The long term effect of this

treatment will be reported later.

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1.1 Case II:3

This is a 59 year old male, born in Finland, with roots in the Karelia region. He attended

school for 9 years, came to Sweden in 1976 and has worked as a truck driver for many years.

During the last two years he started to work night shifts. He is a smoker; his past medical

history (PMH) consists of migraine with aura with onset at the age of 13. He underwent 3

surgical procedures in the left shoulder due to rupture of the supraspinatus tendon and

impingement in the left shoulder. The procedures were performed in 2009 and 2011,

nevertheless he has recurrent pain in this region. He also has a history of anxiety and recurrent

depressions requiring periodical pharmacological treatment. During some of these past

episodes he likely met the criteria for alcohol use disorder but denies current abuse. He

described insidious action tremor starting at the age of 52 for which he was intermittingly

used propranolol. He also complained of impaired short term memory, a recent screening

with Montreal Cognitive Assessment (MoCA) yielded 25 points. Briefly, deficits were found

in delayed recall, attention, visuospatial abilities and word fluency. His HAD-A was 14 and

HAD-D was 9. The patient did not comply with a planned cognitive evaluation and was

reluctant to start antidepressant treatment. A widespread chronic skin infection affecting his

back prevented a lumbar puncture. The subject has described numbness in both arms, the age

at onset of this symptom is unclear, he did not comply with a planned electroneurography

(ENeG) and electromyography (EMG). Recently, vitamin B12 deficiency was diagnosed and

replenishment recommended but again he declined to follow this recommendation.

Gynecomastia was found on exam.

His motor features are subtle (video 1). Since the first physical exam in 2010 we found

reduced dexterity on the left side when the patient performed alternating hand movements. In

the first exam fine rapid postural tremor, mild dystonic posturing of the right hand, mild

chorea in the legs and bilateral mild dysmetria on finger chase were also evident. His ability

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to walk tandem and his left leg agility have become mildly impaired over time. We also found

jerky smooth pursuit, single nystagmus beats on secondary position and suggestion of

hypermetric centripetal saccades. The remaining of the neurological exam was unremarkable.

His latest motor scores were as follows: TRS 11, UPDRS part III 8, UHDRS 5 and SARA

4.5.

Imaging: Widespread calcifications were found in the lentiform nucleus. Mild calcifications

in the white matter of frontal regions were also identified. His TCS of 15 remained

unchanged. This is the only subject in this kindred without calcifications in the thalamus,

cerebellar or caudate nuclei.

Comment on II:3: A mild motor progression has occurred. Without a deeper cognitive

assessment we have to interpret the suspicion of possible cognitive impairment with caution.

The negative impact of shift work on cognitive performance has been characterized before

(10). Other factors influencing his outcome are smoking, recurrent depressions and cobalamin

deficiency.

1.2 Case III:1

This 32 year old female is the index case of this family. Onset of migraine with aura was at

age 16. She was referred to our center at age 27 due to progressive language difficulties

starting two years before. At the time the patient worked in a printing company making signs.

Angry costumers complained about signs with typographical errors made by the patient. At

the age of 26 she started to notice tremor and impaired dexterity. These symptoms are

notoriously exacerbated by stress and by migraine. Most migraine episodes were preceded by

recurrent numbness in the right hand and aura. After ruling out common causes of secondary

brain calcifications (1) she underwent a muscle biopsy and a lumbar puncture. Both exams

yielded normal findings. She suffered from recurrent loss of consciousness, an investigation

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with long-term ECG monitoring and echocardiography were unremarkable. An EEG under

standard conditions and during sleep deprivation was also normal. Syncope occurred during a

tilt test in which her systolic blood pressure fell to 40 mmHg and her pulse to 80 beats/minute.

These findings were interpreted as compatible with a vasovagal reaction.

Her motor features were also subtle (video 2). Briefly, posturing was evident at age 30, two

years later chorea was evident in her feet, finger and head. Dystonic extension of the big toes

and posturing of the left third digit were also evident. The patient is not aware of these

hyperkinesias. A bilateral mild clumsiness is evident when the subject performs foot

stamping. Mild hypometric vertical saccades are also observed. Her latest motor scores were

as follows: TRS 9, UPDRS part III 3, UHDRS 8 and SARA 0.

A speech evaluation found preserved grammar and fluency. Her language comprehension and

inference capacity were also normal when assessed with the subtle language disorders test (a

test for Swedish speakers) (11); with this test significant and persistent difficulties in

repetition tasks and reading were found. In addition, clear deficits compatible with anomia

were identified with the BNT. During a follow up evaluation at age 32 her BNT score

improved but remained significantly low. During this follow up verbal and phonemic

paraphasias were identified as a new feature of her language impairment (table e-4). The

patient’s strategy to avoid typographical errors at work is to frequently check what she writes

and to have supervision.

She has a total education of 13 years. At the neuropsychological testing 2009 as well as at

follow-up 2015, this patient performed clearly above average in comparison with her age

group, when tested with Ravens progressive matrices measuring general intellectual ability

(IQ 125). A brief cognitive screening with MMT yielded 29/30 points at baseline and 28/30 at

follow-up six years later. Her MoCA score was 29/30 in 2015, her HAD-A was 0 and HAD-D

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was 3. At baseline in 2009, clear deficits were found in tests measuring working memory

(Digit span) and Picture Naming (BNT). Tests measuring the following functions were within

in the normal range: verbal episodic memory, (RAVLT learning), visuospatial episodic

memory (ROCFT), verbal concept formation (Similarities), word fluency (FAS/COWAT),

information processing speed (SDMT), executive function (TMT B) and motor speed on

dominant and non-dominant hand (FT). Furthermore in 2009 a result clearly above average

was seen in verbal episodic memory, (RAVLT retention) and in tests measuring spatial/visual

construction a result ranging from average (ROCFT, copy) to above average (Block design)

could be noted. When re-tested in 2015, a score clearly below average remained in digit span.

An improvement from within normal range to clearly above average could be seen at follow-

up in verbal episodic memory, (RAVLT learning), word fluency (FAS/COWAT) and motor

speed (FT) on non- dominant hand. In the following tests the performance was still in the

normal range at follow-up: visuo-spatial episodic memory (ROCFT), verbal concept

formation (Similarities), information processing speed (SDMT), executive function (TMT B)

and motor speed (FT) on dominant hand. A result clearly above average remained in verbal

episodic memory (RAVLT retention) at follow-up (table e-3).

Imaging: CT scans of the brain displays progressive symmetric calcifications in the lentiform

and caudate nuclei, dentate nuclei, thalamus and frontal white matter. We also found faint

cortical calcifications in the right parietal lobes of III:1, an MRI revealed a small

developmental venous anomaly (DVA) in the left frontal region. This finding was considered

to lack clinical relevance. Her initial TCS was 34 which increased to 39 after 5 years of

follow up.

Comment: This is one of the two cases in this kindred where the TCS detected progression of

brain calcifications. The subject displays a complex phenotype with features of mild

language impairment (anomia, emergence of paraphasias and persistent impairment in

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repetition ability), reduced working memory and progressive movement disorders (chorea,

tremor and posturing). Her BNT score improved over time indicating variability in the

cognitive testing.

1.3 Case III:2

This 28 year old male has a PMH of combined alcohol and cannabis abuse. Onset of migraine

with aura was at age 14. In 2006 he was involved in a car accident that caused transient neck

pain but a CT-scan of this region was normal. Similar to his father, he went through surgery

for impingement in the left supraspinatus muscle in 2013. He interrupted his school

attendance at the age of 12 and has not completed high school education. He has a total

education of 9 years. A neuropsychiatric condition has been suspected but not proven yet.

He complained of action induced tremor starting at the age of 18. An exam at age 23 revealed

mild right arm chorea at rest, fine and fast postural tremor with occasional negative

myoclonus, as well as reduced dexterity when performing alternating hand movements with

the left. During the last evaluation in 2015 the patient described persistent action tremor, the

appearance of leg cramps, involuntary movements and gait difficulties. The description was

suggestive for dystonia which was evident on the right foot on walking. Two years later new

features were evident: impaired bilateral foot stamping, mild chorea in both feet and tremor

on finger-to-nose testing which is more prominent at the endpoint. An increase in muscle

tonus in the right arm is noticed only after contralateral activation. Mild abnormal eye

movements were also evident: gaze-evoked nystagmus beats, centrifugal hypometric saccades

and centripetal hypermetric saccades (video 3). His latest motor scores were as follows: TRS

12, UPDRS part III 6, UHDRS 7 and SARA 1.5.

His MoCA score was 26 points. In this test attention deficits were evident and to a lesser

degree impaired delayed recall and visuospatial skills. At this point his HAD-D was 5 and

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HAD-A was 11. Despite our attempts the patient did not participate in planned cognitive or

psychiatric evaluations. For periods of time the patient withdrew socially. This is the only

subject in this family with a mild elevation of albumin in the CSF (Q alb). Nevertheless,

oxysterol in plasma and CSF as well as markers of neurodegeneration in the CSF were

normal.

Imaging: Symmetric and widespread calcifications are located in the lentiform and caudate

nuclei mainly. A moderate degree of calcifications is seen in the thalamus and to a lesser

degree in the white matter of the frontal area and in the dentate nuclei. The TCS score of 34

remained unchanged.

Comment: This case is characterized by predominant behavioral features and mild

progressive motor features in the context of widespread and marked calcifications in the basal

ganglia and to a lesser degree in the thalami, frontal regions and cerebellum. This motor

progression, clear chorea, seems to occur in the absence of detectable radiological

progression. Whether there is an underlying neuropsychiatric condition and cognitive deficits

was not possible to evaluate.

1.4 Case III:3

This 23 year old female describes spontaneous migraine remission during the last year. Age at

onset of migraine with aura was 12. She developed insidious tremor starting at the age of 17.

Her main symptom over time is impaired short memory that also raised complaints at her

work. There are no records of learning disabilities during school. She has enrolled recently in

a training program in a vocational school. During her pregnancy two years ago she noticed

reduced dexterity in her right arm and hand. An EMG revealed chronic neurogenic

abnormalities corresponding to the right C5-Th1 myotomes but the ENeG was normal. A

MRI of the spinal cord was normal. Physical exams were performed in 2010 and in July 2015.

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Physical exam at age 18 revealed fine rapid postural tremor with superimposed jerks,

posturing of the wrists and hyperextension of the fingers. Five years later chorea in the lower

limbs has emerged, the subject is not aware of those movements. Also evident here are

dexterity in her right arm and hypermetric horizontal saccades (video 4). Her latest motor

scores were as follows: TRS 10, UPDRS part III 6, UHDRS 8 and SARA 2.

Biochemical analyses revealed a mild elevation of neurofilament light chain (NfL) in her CSF

but the other parameters were normal.

She has a total education of 13.5 years. She went through the neuropsychological examination

in 2015, at this point her HAD-D score was 1 and HAD-A 9. When tested with Ravens

progressive matrices, this patient reached an average level of general intellectual ability in

relation to her age group (IQ 96). A short cognitive screening yielded a MMT and MOCA

score of 27/30. A performance clearly below average was seen in tests measuring the

following functions: visuospatial episodic memory (ROCFT immediate and delayed recall),

working memory (Digit span), spatial/visual-construction (ROCFT copy and Block Design),

picture naming (BNT), information processing speed (SDMT) and executive function (TMT

B). She also had a mild impairment when repeating foreign polysyllabic words. A

performance in the normal range was seen in tests measuring verbal episodic memory

(RAVLT), verbal concept formation (Similarities), word fluency (FAS/COWAT) and motor

speed (FT) on dominant and non-dominant hand (table e-3).

Imaging: Symmetric and widespread calcifications are found mainly in the thalamus,

lentiform and caudate nucleus. Mild calcifications were found in the dentate nucleus,

periventricular regions and in the frontal white matter. We also found faint cortical

calcifications located in the parietal lobes more predominant on the left side. Her initial TCS

was 37 and in the last assessment 39.

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Comment on III:3: This is the second case in this family with radiological progression

evident on TCS. This patient is another clear example of clinical (motor symptoms), and

radiological progression in PDGFB-associated PFBC. She has a more severe impairment in

working memory and anomia than III:1. Different to subject III:1 this subject has significant

visuospatial deficits and reduced information processing speed. It is unlikely that the reduced

dexterity in the right arm is caused by the chronic abnormalities found by EMG.

2. Hounsfield unit (HU) density measurements in the F13 family

2.1 Introduction:

The modality of choice to study brain calcifications is the computerized tomography (CT)

scan (12). Calcifications on brain MRI yield different intensities even when comparable

sequences are used. These discrepancies are due to different parameter settings used in

different protocols, manufacturers and different magnetic field strength. Thus, calcifications

appear as hypo-, iso-, or hyperintense areas on MRI (13,14). The neuropathology of PDGFB

mutations in humans has not been characterized yet. The calcification in the hypomorphic

PDGFB ret/ret mouse contain mainly calcium phosphate (1). Assuming this is also the case in

patients with PDGFB mutations with brain calcifications we attempted to quantify the content

of hydroxyapatite (HA) in the calcified brain areas using the DECT technique.

Hydroxyapatite is a crystalline form of calcium phosphate.

2.2 Methods:

2.2.1 Image acquisition and analysis

Radiological studies included computed tomography (CT) scans and magnetic resonance

imaging (MRI) exams of the brain. The first CT scans were performed with axial 4 or 5 mm

slices in three cases (II:3, III:1 and III:2) and a 0.625 mm in one case (III:3). All follow up

CT scans were performed with 0.625 mm slices. MRI of the brain were performed to assess

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the presence of white matter abnormalities (WMA) and to evaluate the integrity of

infratentorial regions (table e-5). The first CT scan was done with single CT (SECT) using

120 kilo volt peak (kVp). The follow-up exams in all cases were performed using fast kVp

switching dual energy CT (DECT) (15,16). A dual energy protocol (called GSI-30) was used

(with CTDIvol(16 cm) 59 mGy) and monochromatic source images at 67 keV were created from

the raw data set. The TCS (range 0-80) was applied (1).

Co-registration with fusion software was used to optimize comparisons. 3D region of interest

(ROI) on the reference exam propagates to the follow-up scan in a semiautomatic fashion.

The changes in Hounsfield units (HU) were measured as relative and absolute values

(Integrated Registration, GE AW server 2). The average of several ROIs was used to

minimize errors. The numbers of ROIs were different between the anatomical locations and

varied from 1 to 4 depending on the size of the calcifications. Data files from the

measurement with comparison of relative change in percentage and relative change in HU in

the 3D-ROIs were used.

2.2.2 Hydroxyapatite concentrations

Scatterplots of concentrations of hydroxyapatite (HA) were done using material

decomposition of HA/water using the manufacturer´s specific software (GSI Viewer, GE AW

server 2.0).

Two neuroradiologists (HA and EJ) with more than 15 years of experience made the

assessment with TCS according to a previous description (3); the co-registration and HA

concentration evaluation was performed by one neuroradiologist (HA).

2.3 Results:

2.3.1 Validation process of measurements with DECT

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DECT can create different monochromatic energy levels, so called keV levels. In all CT

scanners calibration with water phantom from the manufacturer are used worldwide. In DECT

calibration of keV levels 60-140 are also performed with water phantom. There is not

available data on accuracy of corresponding keV-level to a corresponding kVp. Based on the

local experience the images were reconstructed at 67 keV level which seems to match the

grayscale appearance in native CT brain scans. We assessed the impact of clinical relevant

keV levels (60-65-70-75 keV) on measurements of HU variations in one patient (III:3), using

the baseline CT with 120 kVp as reference. Measurements in 5 different ROIs are displayed

in table e-6. 2D ROIs in normal brain tissue and in areas with calcifications were analyzed.

Our conclusion is that the HU variations in the normal brain due to different keV levels are

very low, in fact less than 5 HU. As expected, the HU of the calcifications increased when

lower keV levels were used.

2.3.2 TCS and co-registration

Mean time between first and last axial brain CT scan was 4.8 years. Patient II:3 has

calcifications in the basal ganglia (lentiform nucleus) and in the frontal white matter. The 3

patients in generation III have more widespread calcifications than patient II:3. All the

participants in generation III have calcifications in the basal ganglia (lentiform and caudate

nuclei) and varying degrees of calcifications in the thalamus and dentate nucleus. (Figures 1

and e-2). Faint cortical calcifications were found in the right parietal lobes of III:1.Subject

III:3 had bilateral calcifications in the parietal cortex which was more dense on the left side

(Figure e-3). We did not find evidence of calcifications in the midbrain, pons or medulla. Two

patients, III:1 and III:3, displayed progression when the TCS was applied (table e-1). Mean

TCS at base line was 30 (range 15-37) and at follow-up 31.75 (range 15-39). Calcifications in

the white matter was very variable: periventricular in patients II:3, III:2 (punctate

appearance) and III:3. In patient III:1 these calcifications were found in the frontal white

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matter (table e-7). Other than calcifications, we did not find evidence of white matter

abnormalities (WMA) or leukoencephalopathy. The TCS system has a maximum score for

each anatomical location which precludes accurate assessments of radiological progression.

Given this ceiling effect, we explored the utility of the co-registration technique. With this

method, in which HU density levels were compared, we found progression in three patients

(II:3, III:1 and III:3) (table e-8).

2.3.3 Hydroxyapatite concentrations

ROIs deployed within the white area of the calcifications, with ”brain window”

(WW70:WL40), contains of voxels with different HA concentrations with a linear relation to

HU in the range of 25-1000 mg/cm3 (Table e-9 and figure e-4). The most dense areas were

found in the lentiform nucleus in patient III:3.

Discussion:

All the 4 patients display varying degrees of motor progression but progression of

calcifications was demonstrated in 3 of them. However, the motor features described here are

subtle. All the participants in this follow-up met the criteria for migraine with aura at the time

of inclusion in our study (3). A similar pattern of cognitive deficits (reduced working memory

and anomia were found in 2 patients (III:1 and III:3). Subject III:3 has also significant

visuospatial deficits and reduced information processing speed. Cognitive assessments could

not be evaluated in the other 2 members of this kindred

We found the emergence of chorea in all the 2 patients in generation III (III:1 and III:3), in

another patient (III:2) chorea progressed without radiological progression. The father also

displayed mild chorea but only during his first exam. Females in this kindred tend to have

more widespread calcifications and highest TCS (39 points in both cases) than men (TCS 15

and 34). In addition, only the females have cortical calcifications. The index case (III:1)

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displays a mild language impairment characterized by anomia emergence of paraphasias and

persistent impairment of repetition ability. She has also progressive subtle motor

abnormalities (chorea, posturing and tremor) and mild cognitive deficits (reduced working

memory). Patient III:3 in the other hand displays also progressive subtle motor features

(chorea, posturing and tremor) but a more pronounced cognitive impairment defined by

anomia, reduced working memory and information processing speed and visuospatial deficits.

The cognitive profiles of patients III:1 and III:3 are compatible with a subcortical dysfunction

(17). We also found mild eye movement abnormalities in all the patients

Using the TCS we detected calcification progression in 2 of 4 cases. Using the co-registration

method we identified progressive calcifications in 3 participants. There is a ceiling effect that

may limit the utility of TCS. Only in one previous PDGFB mutation a slight progression of

brain calcifications has been reported (5). However, the article did not provide TCS or

information on similar assessment. The fact that calcifications are more widespread in patients

from generation III in this family and in family F (1) illustrates intrafamilial variability but

may also suggest anticipation. Studies on larger cohorts are needed in order to assess whether

anticipation occurs and to validate the utility of co-registration as a complement to the TCS.

Elevated levels of neurofilament light chain (NfL) in the CSF are considered as potential

biomarkers of several neurodegenerative conditions (18). The fact that NfL was elevated in

one case (III:3) and the progression features support the notion that PDGFB-associated PFBC

is a neurodegenerative condition. This is to our understanding the first time oxysterols and

markers of neurodegeneration are studied in PFBC. Elevated levels of homocarnosine in the

CSF from 2 subjects of a PFBC family is the only available abnormality published (19), the

genotype of this family consisted of a SLC20A2 and THAP1 deletion (20). The discrepancy in

oxysterol levels seen our patient samples when compared with the hypomorphic PDGFBret/ret

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mouse model illustrates the challenges and difficulties when working with animal models of

neurodegeneration.

There are some limitations with this study. First of all the small sample size precludes

statistical assessment of clinical progression or assessment of anticipation. In addition, there

are other limitations with the radiological assessment. The density measurements made by

comparison of CT slices are sensitive to movement of just one or two voxels; repeated

measurements were used to minimize errors. Ideally the comparison should be done using

submillimeter slices but many hospitals in Sweden do not store the complete image series and

save only the MPR images in three planes which uses thicknesses between 4-5 mm.

Furthermore, comparisons using the same CT technique are probably to prefer. However,

since there was obvious progression regardless of different keV levels we believe that the

general trend in our results would not change even if the follow-up exam had been performed

with SECT at 120 kVp. Another factor to take into consideration is the use of different CT

scanners. It is highly unlikely that a patient will be examined on the same CT scanner or with

the same technique, especially if the follow-up time we propose is 5 years. New CT scanners

or upgraded software, acquired in average every 5-10 years at our hospital, may yield

different density measurements in the areas with calcifications. Thus, different CT scanners

constitute a potential limitation of co-registration but in order to minimize the risk of error we

set a cut-off of 10 HU as a radiological relevant level. Another limitation in our

characterization is the assessment of faint or small calcifications which are possible to score

using the TCS but difficult to measure using the co-registration technique. In general,

measurements with larger ROIs are more reproducible than smaller ROIs. This situation can

be illustrated with the finding of faint cortical calcifications in 1 of our patients (III:1).

Another difficulty we ran into was to differentiate whether calcifications are confluent or not

on axial sections only. For instance, axial and sagittal sections in subject III:1 give different

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visual impressions on the degree of calcification in the frontal white matter (Figure 1 in main

text). According to the TCS method using the axial image could score the white matter

changes of subject III:1 as ”moderate” (score 3) but appear in sagittal image to be ”severe”

(score 4). Since the radiological progression is rather slow in the L9R mutation we propose

follow-up CT-scans every 5-10 years. This proposal has to be weighted by the fact that PFBC

disorders are so far incurable.

It is difficult to prove that the concentrations estimated with the DECT technique only

represent HA. Material decomposition of the source image to a HA image (and a paired water

image) should be seen as an estimation in vivo and not an absolute value. No other available

radiological technique can provide information on these concentration values. The linear

relationship we found between HA concentrations and HU supports the notion that a SECT

exam with the same kVp in follow up exams would be comparable with DECT. Follow up

with SECT will reduce the radiation dose in comparison to DECT with fast kVp switching

technique. In this way we could also reduce the amount of radiation exposure for the patients.

Other than calcifications, we did not find evidence of white matter abnormalities (WMA) or

leukoencephalopathy as described in other PDGFB mutations (4,6) (table e-11). WMA and

calcifications occur otherwise in Nasu-Hakola disease, in some mitochondrial disorders and in

Alexander disease (21–24). Additional genetic differential diagnosis include PFBC caused by

mutations in the SCL20A2, PDGFRB and XPR1 genes (1–3,25,26). Ataxia with calcifications

in the dentate nucleus are the main features of spinocerebellar ataxia type 20 (SCA20) (27).

Autosomal recessive diseases with brain calcifications include the severe early-onset Aicardi-

Goutières syndrome and Cockayne syndrome among others (28).

The radiological penetrance of PDGFB mutations is very high but the clinical penetrance is

lower. In general the clinical penetrance of PFBC diseases is around 60 % (25,29). Of all

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PDGFB gene mutation carriers, only one subject with the c.3657C>T mutation did not

present with calcifications or any clinical abnormalities on exam (6). Reduced clinical

penetrance (meaning absence of symptoms despite the presence of brain calcifications) has

been reported in 3 other PDGFB mutations (Table e-11). However, the true penetrance may

be underestimated since clinical information on PFBC cases is available in 60% of cases only.

Furthermore, there is a discrepancy about the most common features in subjects with PDGFB

mutations. For instance G. Nicolas et al found that psychiatric features were the most

common symptoms in a cohort of patients with SLC20A2 and PDGFB gene mutations (29). In

his cohort, early onset was associated with psychiatric or cognitive symtpoms while later

onset was associated with movement disorders (29). V. Tadic et al in the other hand reported

that movement disorders were more common than psychiatric and cognitive symptoms (25).

TCS was found to correlate with symptomatic status in PFBC (29). Parkinsonism was far

more common than hyperkinetic features (1,25,29). It is possible that bias ascertainment has

occurred in other PDGFB families since the motor features we describe here are subtle and

easy to miss when the assessment is not performed using validated motor rating scales and

cognitive batteries.

The PDGFB gene has 7 exons in total (12); the missense mutation c.26T>G (L9R) is located

in exon 1 which encodes the signal peptide. The substitution was predicted to lead to

disruption of the signal peptide (1). Of all the nine PDGFB mutations reported so far 4 are

located in exon 4 and one deletion includes exons 3, 4 and 5 (1,4–6,30). The latter mutation

was not necessarily associated with a more severe phenotype (4). The predicted loss of

function for the L9R and 5 other PDGFB mutations (1) was recently demonstrated in vitro

(31). Briefly, neither the protein nor its dimer could be detected by Western blots despite

expression of PDGFB. Activation of PDGFRB, the receptor of PDGFB, also failed to occur

(31). The scarcity of mutations, limited clinical descriptions and intrafamilial heterogeneity in

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PFBC preclude at the moment genotype–phenotype associations for PDGFB mutations. How

gene modifiers and compensatory mechanisms impact the wide phenotype heterogeneity of

PFBC remain also to be characterized.

It has also been difficult to establish clear clinico-anatomical associations in PFBC

syndromes. For instance ataxia has been observed in PFBC subjects without cerebellar

calcifications (32) and the presence of cerebellar calcifications is not necessarily associated

with ataxia (3). In fact ataxia was more common in those with calcifications in the BG (25).

We can propose a pattern of clinico-anatomical correlations to some degree in the F13 family.

For instance chorea which is associated with striatal lesions is seen clearly in all the

participants with calcifications in the caudate nucleus. Subject II:3 in the other hand lacks

calcifications in this nucleus as well as persistent chorea. His chorea was documented only

once (video 1). Varying degrees of postural tremor is seen in all the 4 patients but thalamic

and cerebellar calcifications are found in 3 of them. A similar correlation is suggested for the

mild eye movement abnormalities seen here compatible with cerebellar impairment.

Functional imaging will contribute to our understanding of neuroanatomical correlates in

PFBC. So far very few functional imaging studies in subjects with brain calcifications are

available (2,19,33–36). Of them only 3 describe PFBC cases (2,19,36), the other are on

sporadic cases (described as “Fahr’s disease). I. Le Ber et al found early metabolism

reduction in the striatum and in cortical areas in a PFBC family (2). Those cortical areas

(precuneus, posterior cingulate and superior temporal gyri) are involved in episodic memory

which was impaired in 3 patients. Z.K. Wszolek et al found pre- and postsynaptic

abnormalities in the nigrostriatal pathway in a PFBC family with predominant dystonia (36).

Taken together these findings in PFBC are compatible with impaired connectivity in loops

important for language, cognition and motor function.

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Our findings are in line with the progressive brain calcifications seen in the hypomorphic

PDGFBret/ret mouse model (1). Life expectancy is not reduced in these animals despite

increased permeability of BBB as well as retinal and glomerular defects (37), knockout mice

in the other hand die at perinatal age (38). Flux of the oxysterol 24S-hydroxycholesterol from

the brain into the circulation is also increased in the PDGFBret/ret mouse (39). However, recent

work by M. Vanlandewijck et al suggests a far more complex pattern of BBB disruption than

initially thought (1,40). In contrast to the initial report (1,40) the BBB is in fact more intact in

brain areas prone to calcifications than areas without calcifications (31). The pattern of

calcifications in PFBC is not specific even though some observations have been made. For

instance, calcifications are more widespread in SLC20A2 mutations than in PDGFB and

PDGFRB mutations (3,25). Cortical calcifications are very common in SLC20A2 mutations

(77% of cases) but have not been documented in PDGFRB mutations (3). We found faint

cortical calcifications in the parietal lobes of two of our cases (III:1 and III:3). These faint

calcifications could be score with TCS but not with the co-registration method. Attempts to

delineate patterns of brain calcifications for neurological diseases other than PFBC have been

proposed as well (41). G. Nicolas found that the degree of calcifications severity in subjects

with PDGFB mutations was intermediate between what is seen among subjects with

SLC20A2 (more severe) and PDGFRB (less severe) mutations (29). These authors also stated

that the TCS could be predicted using the combined effects of mutated gene, age and sex (29).

In conclusion we have demonstrated a slow rate of clinical and radiological progression in the

L9R PDGFB mutation in the course of 5 years. Whether these results can be extrapolated to

other PDGFB mutations or other forms of PFBC remains to be studied. We have also

demonstrated the utility of the co-registration method to overcome the ceiling effect of the

TCS.

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