Online Supplementary file
Mutations in CYP2U1, DDHD2 and GBA2 genes are rare causes of complicated spastic paraparesis
with thin corpus callosum and intellectual disability
*1
Andrea Citterio, *1Alessia Arnoldi, 1 Elena Panzeri, 2 Maria Grazia D’Angelo, 3Massimiliano Filosto,
Robertino Dilena, 5Filippo Arrigoni, 1Marianna Castelli, 6Cristina Maghini, 6Chiara Germiniasi,
7
Francesca Menni, 8Andrea Martinuzzi, 1,9 Nereo Bresolin,1Maria Teresa Bassi.
4
Scientific Institute IRCCS E. Medea, 1Laboratory of Molecular Biology, 2 Neuromuscular Disorders
Unit, 23842 Bosisio Parini, Lecco, Italy; 3Clinical Neurology, Section for Neuromuscular Diseases and
Neuropathies, University Hospital “Spedali Civili”, Brescia, Italy ; 4Unit of Clinical Neurophysiology,
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Scientific Institute IRCCS
E.Medea, 5 Neuroimaging Unit, 6 Neurorehabilitation Unit, 23842 Bosisio Parini, Lecco, Italy;
7
Pediatric Clinic 1, Department of Physiopathology and Transplantation, Università degli Studi di
Milano, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 8Scientific
Institute IRCCS E. Medea, Conegliano Research Center, Conegliano, Italy; 9Dino Ferrari Centre,
Neurology Unit, IRCCS Ca’ Granda, Ospedale Maggiore Policlinico Foundation, Dept. of
Physiopathology and Transplantation, University of Milan, Italy.
Key words: amplicon–based targeted resequencing, spastic paraparesis, CYP2U1, DDHD2, GBA2.
Corresponding author: Maria Teresa Bassi PhD, E. Medea Scientific Institute, Laboratory of Molecular
Biology, Via D. L. Monza 20, 23842 Bosisio Parini, Lecco, Italy; phone 0039031877111, fax
0039031877499; email mariateresa.bassi@bp.lnf.it, mariateresa.bassi@lanostrafamiglia.it.
Online resource Patient and Methods
Clinical report of patient P574 with CYP2U1 mutation
The proband is the second-born child (aged 4 years and 8 months) of an Egyptian consanguineous
family. His elder sister is 6 year-old and is healthy. The mother had previously two miscarriages in the
first 3 months of pregnancy. The proband was born at term by caesarean section for fetal distress, but
after birth he did not present any sign of asphyxia. His weight at birth was 3540 g. At age 8 months he
was admitted to the hospital because of a urinary tract infection: a vesico-ureteral reflux and a right
kidney hypoplasia were diagnosed. However after this infection, he did not suffer of any other urinary
problems. His developmental milestones were delayed; social smiling appeared at age 3 months while
he was able to sit kneeling on his legs at age 1 year. He started cruising although with clumsiness and
frequent falls at age 18 months. Babbling started at the age of 8 months.
At the first neurological examination (at our clinic) at age 22 months, he showed severe spasticity at the
lower limbs, polikinetic deep tendon reflexes, ankle clonus and Babinski sign. At the upper limbs there
was only a slight clumsiness of the distal fine movements with slightly brisk deep tendon reflexes. He
showed mild drooling, without dysphagia, with normal feeding. Expressive language was limited to
two words, although comprehension of simple commands was good. Sitting normally, crawling or
walking alone was not possible, although he was able to sit keeping a crouching position and to cruise
for few steps with spastic gait using his hands for support.
Brain and spinal cord MRI were normal. Brainstem evoked potentials with auditory threshold, visual
evoked potentials, somatosensory evoked potentials and electroencephagram were normal. Nerve
conduction studies were normal, while needle electromyography showed only slight chronic
neurogenic signs. Routine blood test and metabolic screening was normal.
At the last neurological examination, the patient (aged 4 years and 8 months) walked only with aid
(bilateral support), showing a pareto-spastic gait and a severe spasticity at the lower limb. He had an
impaired expressive language development (phonological, morphological and syntactic disorder), with
good language comprehension and good global psychological development.
The neurophysiology tests were unchanged.
Clinical report of affected sibs of the P1242 family with DDHD2 mutation
Both sibs of family P1242 (born to not consanguineous parents) showed psychomotor delay that
developed into intellectual disability and very early-onset progressive spasticity. In both sibs, onset of
the first symptoms of spasticity was before age 1 year.
At the time of the first examination in our clinic, the older brother, aged 16 years, was presenting a
severe spasticity of the lower and upper limbs with brisk reflexes and moderate weakness associated
with mild amyotrophy of the distal portion of the limbs, mild dysarthria, mild drooling, mental
retardation and mild cerebellar ataxia. He never acquired ambulation and sphinteric control.
At the last neurological examination the patient (age 23) showed a worsening of the clinical condition
with severe spastic tetraplegia, trunk ataxia with severe dysarthria and drooling; cognitive decline was
evident but he was still able to answer to simple questions. An isolated episode of epileptic seizure had
occurred few months before this evaluation.
Brain MRI images show severe thinning of the corpus callosum, diffuse reduction of white matter
volume and an abnormal hyper-intense signal on T2-weighted and FLAIR images of the residual white
matter in fronto-parietal regions. MR spectroscopy was not performed.
His younger sister underwent to the first neurological evaluation in our clinic at age 10 years. She
presented with a spastic paraparesis with brisk reflexes at both upper and lower limbs, mild distal
amyotrophy of distal portion of the lower limbs with mild ataxia and dysarthria. Her walking showed
severe unsteadiness and inconstant need of support.
Mental retardation was documented through a Wechsler scale (WISC-R) with a verbal intelligent
Quotient of 61. Mild behavioral disturbances were reported by the family and were also consistently
observed by the neurologist.
At the last neurological examination, at age 16 years, the patient showed a more severe spastic
paraplegia (able to walk for short routes only with aid), moderate-severe cerebellar signs and more
evident behavioral and attention disturbances. Sphinteric control, acquired at the proper time, was
maintained. Brain MRI overlaps the picture displayed by the older brother.
Strabismus, mild hypomimia, pes cavus and mild scoliosis were observed in both patients.
In the two siblings electroneuromyography was normal. Only a mild reduction of motor nerve velocity
was observed in the older brother. Motor and sensory evoked potential were presenting increased
latency in both sibs.
In both sibs metabolic (organic acid and amminoacid, very long chain fatty acid, lysosomial enzymes)
and sialotrasnferrin dosage were within the normal range; mutations in mitochondrial DNA were
excluded.
Clinical report of affected sibs of the P922 family with GBA2 mutation
The proband (II-1) is a 24-year-old man who started to complain of difficulty in walking at the age of
10. The disorder has gradually worsened over time and the patient presented progressive stiffness of the
lower limbs and frequent falls.
The parents are consanguineous and healthy.
At the neurological examination lower limb marked spasticity, mild weakness and wasting, brisk deep
tendon reflexes, ankle clonus and right Babinski sign were evident. At the upper limbs there were
moderate spasticity and brisk deep tendon reflexes with normal strength and no muscle atrophy. He
also presented with bilateral eyelid partial ptosis, mild dysmetria, mildly reduced visual acuity and
bilateral flatfoot. Although a defined cognitive impairment was not demonstrated, he was a slow
learner in school. He also complained of increasing urinary incontinence.
Brain and spine MRI was normal as well as electromyography and nerve conduction studies. Motor
evoked potentials showed increased central conduction time in all the four limbs.
His 24-year-old twin sister (II-2) did not complain of any symptom. She delivered a normal pregnancy
at age 19. Clinical examination showed lower limb moderate spasticity, brisk deep tendon reflexes and
ankle clonus. At the upper limbs brisk tendon reflexes with positive Hoffman sign were present.
Bilateral eyelid ptosis and mild dysmetria were noted.
The 22-year-old brother (II-3) also was asymptomatic but neurological examination showed lower limb
brisk deep tendon reflexes and ankle clonus. At the upper limbs brisk tendon reflexes and a positive
Hoffman sign were present. No significant spasticity was noted. Bilateral eyelid ptosis, mild dysmetria,
more evident on the right arm, and bilateral flatfoot were also reported.
Amplicon-based high-throughput pooled-sequencing methods
Target specific amplicons for the three genes CYP2U1, DDHD2 and GBA2, were prepared by using the
sets of primers indicated in supplementary table 1. The primers were designed to amplify by long-range
PCR all coding exons and large part of the flanking intronic sequences; the amplicon size ranges
between 803 and 3770 bp. Target specific amplicons were processed using a microfluidic platform, the
Fluidigm 48,48 Access Array System (Fluidigm Corporation, San Francisco, CA, USA). It allows
amplifying 48 specific amplicons from 48 samples in 2304 reaction chambers simultaneously in a
reaction volume of 35 nl each. In this pilot experiment we used 48 patients, 46 new and 2 patients
mutated one in CYP2U1 (P574) and one in DDHD2 (P1242) as positive controls.
Amplification reactions were performed according to the Long Range PCR on the 48.48 Access Array
IFC-Advanced development protocol (Fluidigm). Each amplicon library was treated with ExoSAP,
while the correct size of the amplicons was checked by using the Agilent DNA 12000 kit (Agilent
Technology, Boblingen, Germany). The total DNA obtained for each amplicon library was quantified
with Qubit dsDNA BR Assay system (Life Technology Corp., Carlsbad, CA, USA) and processed
according to Nextera XT DNA sample Preparation protocol (Illumina, San Diego, CA, USA).
Briefly, each sample was tagmented (tagged and fragmented) by the Nextera XT transposome and then
PCR amplified by adding index primers required for clusters formation. PCR products were then
purified by using the AMPure XP beads (Beckman Coulter, Milano Italy); this provides a size selection
step that removes very short library fragments from the population. Each library was normalized and
pooled for MiSeq (Illumina) loading.
Data analysis was performed by using MiSeq Reporter and Integrative Genomics Viewer (IGV) v.
2.3.20. At least 93% of the NGS fragments had an average coverage of 399 reads and passed quality
control. The remaining 7% that did not pass the quality control were re-sequenced by traditional Sanger
sequencing. Overall, all amplicons from the three genes were produced with the exception of the
amplicons 1 of CYP2U1 and GBA2 genes (corresponding to exon 1 for both genes); due to the lack of
PCR product these amplicons did not produce any cluster. Exon 1 for both genes was sequenced by
Sanger method. Only DDHD2 mutation of patient P1242 (previously identified by Sanger sequencing)
was correctly identified also by targeted resequencing (Supplementary Figure 1). The CYP2U1
mutation located in exon 1 was not detected due to the failure of long range amplification of exon 1
(amplicon 1) of the gene. A mutation in exon1 3 of GBA2 was identified (Supplementary Figure 1) and
confirmed by Sanger sequencing as described in the result section.
Online resource Figure 1 Legend
Partial schematic view (by IGV software) of the reads alignment encompassing the mutations identified
by targeted resequencing in exon 13 of GBA2 (P922) and in exon 16 of DDHD2 (P1242) genes.
Online resource Figure 1
Online resource Table 1 Primer sets used for long range amplification
Primer Name
Sequence
CYP2U1NGS-amp1F
AAG GGA AAC CAA CAG CCA ATC AG
CYP2U1NGS-amp1R
CTT CTT CTG TGT TTT CTG GAC TT
CYP2U1NGS-amp2F
AGG GCT GCT GCC GAC AAC AG
CYP2U1NGS-amp2R
CAT CTT ACC AAC TCC CTA TGA GTT
CYP2U1NGS-amp3F
GAA GAA TAC AGG GAG GAT GGT G
CYP2U1NGS-amp3R
CAT TTA GAT GAG CTC TTC TGA CCT
CYP2U1NGS-amp4F
GTC AGC TGG TAA GAG CCA GG
CYP2U1NGS-amp4R
CAC GAC AGA GTC CAC TTC ATC
DDHD2NGS-amp1F
GTT CTC AGC ACC CAG AAG CAG
DDHD2NGS-amp1R
GGC TTT CGG CTC CAT TGT CC
DDHD2NGS-amp2F
TGT TGC AAT CTA GTT TCG AGC ATG
DDHD2NGS-amp2R
DDHD2NGS-amp4F
CTT TAC AAA CCG TCA AAC CAT TCC
GAA TAT AAC TCA GAA TTG TAT GTT
GGG
CAA GGA GTT ATT ACC AGG TAT AAA
TTT
CTA CTT GAC TAG AAA TTT ATA CCT GG
DDHD2NGS-amp4R
TTA GCC AGT TAG AAT AAG ACA CCC
DDHD2NGS-amp5F
GGG TGT CTT ATT CTA ACT GGC TAA
DDHD2NGS-amp5R
DDHD2NGS-amp7R
CAT CTG AAG ATG GAA ATG GTT ATA CA
TTA AGC TTG AAG GCT TAA AAT TGG
AAG
CCA AGA ATG ACA GAG ATA ATT AAA
AGT C
GAC TTT TAA TTA TCT CTG TCA TTC TTG
G
CTG GTT AGT CTG CTC CAT TGC T
DDHD2NGS-amp8F
AGC AAT GGA GCA GAC TAA CCA G
DDHD2NGS-amp8R
ATC CCA CAA AGC ACA AGG ACA C
DDHD2NGS-amp9F
AGC ATT ATC TTC CCA CAG TGC C
DDHD2NGS-amp3F
DDHD2NGS-amp3R
DDHD2NGS-amp6F
DDHD2NGS-amp6R
DDHD2NGS-amp7F
Amplicon
Size (bp)
Exon
Amplicon
Number
1333
EX1
Amplicon 1
1741
EX2
Amplicon 2
1860
EX3
Amplicon 3
2198
EX4-EX5
Amplicon 4
EX1
Amplicon 1
2371
EX2-3
Amplicon 2
3401
EX4-5
Amplicon 3
2658
EX6
Amplicon 4
1554
EX7
Amplicon 5
EX8-EX9
Amplicon 6
1621
EX10
Amplicon 7
1834
EX11
Amplicon 8
998
803
DDHD2NGS-amp9R
GGT TGT TGG CAT CAT TAG CCT CA
DDHD2NGS-amp10F
GGA GGG AGA CTT CGT TAC TTT CT
DDHD2NGS-amp10R
TTG AGA CGG GTG AGT CCA GG
GBA2NGS-amp1F
CAA CCT GAG CTC TGC CTC AG
GBA2NGS-amp1R
CTT ACT CCC TTA ATA TGG TAG CTG
GBA2NGS-amp2F
GGA AAC CAT AAA TGG TCC TTT CAG
GBA2NGS-amp2R
CTG AAC TTT GGT GAA GGT TCC TC
GBA2NGS-amp3F
CTC TGA AAG GGG AGG AGC TG
GBA2NGS-amp3R
CAT GGA AAC AAG GCC TAC TTA TTA C
GBA2NGS-amp4F
GTA ATA AGT AGG CCT TGT TTC CAT G
GBA2NGS-amp4R
CTA GGC TCT TCC CAG ACA CC
GBA2NGS-amp5F
AAC TTC TGT GGT CCC TCA GAT TG
GBA2NGS-amp5R
CAC TTA ATG GCT TTC TGG GTC TTT
2763
EX12-EX13-EX14-EX15-EX16
Amplicon 9
3770
EX17
Amplicon 10
2964
EX1
Amplicon 1
1187
EX2-EX3
Amplicon 2
1556
EX4-EX5-EX6
Amplicon 3
1764
EX7-EX8-EX9-EX10-EX11EX12
Amplicon 4
2405
EX13-EX14-EX15-EX16-EX17
Amplicon 5