Copy number variations in cryptogenic cerebral palsy
Reeval Segel, MD* Hilla Ben-Pazi, MD* Sharon Zeligson Aviva Fatal-Valevski, MD Adi Aran, MD Varda Gross-Tsur, MD Nira Schneebaum-Sender, MD Dorit Shmueli, MD Dorit Lev, MD Shira Perlberg Luba Blumkin, MD Lisa Deutsch, PhD Ephrat Levy-Lahad, MD
Correspondence to Dr. Segel: [email protected]
ABSTRACT
Objective: To determine the prevalence and characteristics of copy number variations (CNVs) in children with cerebral palsy (CP) of unknown etiology, comprising approximately 20% of the CP population.
Methods: Fifty-two participants (age 10.5 6 7.8 years; Gross Motor Function Classification System scale 2.8 6 1.3) with nonprogressive pyramidal and/or extrapyramidal signs since infancy and no identified etiology were enrolled. Individuals with evidence of acquired causes were excluded. Participants underwent neurologic and clinical genetic examinations before the genomic testing. Chromosomal microarray analysis to detect CNVs was performed using the Affymetrix platform. CNVs identified were classified as pathogenic, likely pathogenic, likely benign, or benign. Only pathogenic and likely pathogenic CNVs were defined as clinically significant. Results: Thirty-nine CNVs were found in 25 of 52 participants (48%). Sixteen participants (31%) had clinically significant CNVs: 10 pathogenic and 6 likely pathogenic, of which 7 were not previously associated with motor disability. Nine participants had likely benign CNVs. Clinically significant CNVs were more frequently de novo (12/16; p , 0.001) including in 5 of 8 individuals who had a first- or second-degree relative with a major neurologic disorder. Dysmorphic features and nonmotor comorbidities were more prevalent in individuals with clinically significant CNVs (p , 0.05 for both).
Conclusion: CNVs, most frequently de novo, are common in individuals with cryptogenic CP. We recommend CNV testing in individuals with CP of unknown etiology. Neurology® 2015;84:1660–1668 GLOSSARY CNV 5 copy number variation; CP 5 cerebral palsy; OMIM 5 Online Mendelian Inheritance in Man; SZMC 5 Shaare Zedek Medical Center; TSC 5 tuberous sclerosis complex.
Supplemental data at Neurology.org
Cerebral palsy (CP) is the most common cause of motor disability in childhood (approximately 2:1,000 live births), and is defined as an “umbrella term” of a permanent movement and/or postural disorder caused by nonprogressive abnormalities of the developing brain.1 In most affected children, CP is associated with self-evident perinatal insults, especially prematurity, asphyxia, intrauterine infection, hemorrhage, and brain infracts.2,3 However, in approximately 20%3 of individuals with CP, motor deficits have no apparent etiology,2 and these children are considered to have cryptogenic CP. Although CP is not regarded as a genetic disease, congenital anomalies are more common among individuals with CP, and familial cases have been reported, suggesting underlying genetic susceptibility.2,4 Chromosomal microdeletions and microduplications, also known as copy number variations (CNVs), have emerged as a cause for various neurodevelopmental disorders. CNVs have been reported in 7% to 21% of individuals with intellectual disability,5 autism,6 and epilepsies,7 and in 28% of individuals with movement disorders.8 In most of these, CNVs are de novo, rather than inherited events, explaining paucity of familial occurrence. *These authors contributed equally to this work. From the Medical Genetics Institute (R.S., S.Z., S.P., E.L.-L.) and Neuropediatric Unit (H.B.-P., A.A., V.G.-T.), Shaare Zedek Medical Center, Jerusalem; Pediatric Neurology Unit (A.F.-V., N.S.-S.), Dana Children’s Hospital, Tel Aviv; Jerusalem Child Development Center (D.S.), Clalit, Jerusalem; Metabolic-Neurogenetic Clinic (D.L., L.B.), Wolfson Medical Center, Holon; and Biostatistical Consulting (L.D.), BioStats, Israel. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
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We hypothesized that CNVs may have a role in cryptogenic CP. To examine this, we tested individuals with cryptogenic CP for CNVs using chromosomal microarray (CMA), and compared clinical characteristics of participants harboring clinically significant CNVs with those without such findings. METHODS Participants. Individuals with cryptogenic CP were identified by chart review in 4 CP clinics in Israel. Patients were categorized as having cryptogenic CP when etiology of the neurologic disorder remained undetermined. Inclusion criteria were as follows: individuals with disabling nonprogressive pyramidal and/or extra pyramidal signs beginning before 3 years of age were considered eligible. Patients with any known acquired etiology were excluded a priori (table 1). Such etiologies included periventricular leukomalacia in premature infants, hypoxic ischemic encephalopathy, brain infarcts, encephalitis, head trauma, and spinal cord lesions. Recruitment methods were different based on availability of electronic medical records. In Shaare Zedek Medical Center (SZMC), records of the CP cohort were systematically reviewed for recruitment using study criteria. In other centers, enrollment was based on pediatric neurologists’ referrals of children who fulfilled the study criteria. All participants were examined by a pediatric neurologist with expertise in CP (H.B.-P., A.F.-V., A.A., V.G.-T., D.S., or L.B.) and by a medical geneticist (R.S. or D.L.). Clinical characteristics were documented: perinatal history, type and anatomical distribution of movement disorder, Gross Motor Function Classification System,9,10 and Manual Ability Classification System.11 Imaging findings and comorbidities (i.e., intellectual disability, epilepsy, psychiatric disorders) were graded as follows:
Table 1
Inclusion and exclusion criteria
Inclusion criteria (all the below) Nonprogressive Pyramidal and/or extrapyramidal signs Evident before 3 y of age Exclusion criteria (any known etiology below) PVL Born between 24 and 34 gestational age and evidence of PVL on imaging HIE History of birth complication requiring resuscitation or Apgar score ,7 in 5 min or resuscitation at infancy Hemispheric lesion Hemiplegia and acquired contralateral nonstructural lesion on imaging studiesa Encephalitis Encephalitis at birth (i.e., documented CMV) or post/perinatal meningitis/encephalitis Head injury Prenatal or postnatal Spinal cord lesion Spastic paraplegia and abnormal spinal MRI scan
Abbreviations: CMV 5 cytomegalovirus; HIE 5 hypoxic-ischemic encephalopathy; PVL 5 periventricular leukomalacia. a Including stroke, tumor; excluding brain dysplasia disorders.
0 5 normal; 1 5 mild abnormality that could be found in typically developing children as well; and 2 5 definite pathologic abnormality. Medical genetic evaluation included consanguinity, family history, and dysmorphic features. When possible, parents of participants with significant CNVs were tested.
Standard protocol approvals, registrations, and patient consents. This multicenter study was approved by SZMC and Dana Children’s Hospital Internal Review Boards and the National Review Board for Genetic Studies. Written informed consent was obtained from all participants, probands, and parents.
Controls. To determine whether CNVs in our cohort are present in healthy individuals, we compared our findings with those found in healthy individuals in the Database of Genomic Variants (http://dgv. tcag.ca/dgv/app/), CAGdb (Cytogenomics Array Group Database) (http://www.cagdb.org/), ISCA (International Standards for Cytogenomic Arrays) (http://dbsearch.clinicalgenome.org/), and our in-house variant database.
Genetic testing. DNA was extracted from peripheral blood samples, using standard methods. Affymetrix Cytoscan HD arrays were used according to manufacturer’s protocol. The CytoScan HD Array provides whole-genome coverage, with more than 2.6 million markers including 750,000 single nucleotide polymorphisms. Data analysis was done using the Chromosome Analysis Suite (Affymetrix, Santa Clara, CA) and Partek Genomic Suite (Partek Inc., St. Louis, MO) for defining CNVs, and University of California Santa Cruz (http://genome.ucsc.edu/), GeneCards (http://www.genecards.org/), OMIM (Online Mendelian Inheritance in Man) (http://www.omim.org/), CAGdb (http://www.cagdb.org/), Decipher (http://www. sanger.ac.uk/PostGenomics/decipher/), and ISCA (https://www. iscaconsortium.org/) for assessment of CNV significance. Data were analyzed for deletions and gains according to the American College of Medical Genetics and Genomics guidelines.12 CNVs were compared with parents’ CNV profiles, when available. Classification of CMA results (main outcome measure). CNVs detected were divided to 4 subgroups, according to American College of Medical Genetics and Genomics guidelines12: 1. Pathogenic CNVs: CNVs documented as clinically significant in multiple peer-reviewed publications, CNVs not described in the medical literature at sizes observed in patients but that overlap a smaller interval with established clinical significance, and CNVs containing genes known to be disease-causing based on dosage effect. 2. Likely pathogenic variants of unknown significance: CNVs described in a single case report with neurologic disease, CNVs containing a gene(s) with function(s) relevant to CP, CNVs that include genes reported in OMIM as neurologic disease–causing but not reported to have a dosage effect, or CNVs that are reported in individuals with neurologic disease in online databases, but not in a peer-reviewed publication. 3. Likely benign variants of unknown significance: CNVs that are large enough to be reported (deletions .200 kilobases [kb], duplications .500 kb), but not described as diseasecausing in any of the databases, CNVs described in a small number of cases in databases of variation in the general population, but do not represent a common polymorphism, CNVs containing disease-causing genes that are not known to be dosage-sensitive, or CNVs associated with contradictory information in the medical literature and/or online databases. 4. Benign CNVs: CNVs below a critical threshold (deletions ,200 kb and duplications ,500 kb) not containing OMIM genes associated with disease, CNVs that were reported in Neurology 84
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multiple peer-reviewed publications or curated databases as a benign variant, or CNVs that are common polymorphisms. Benign CNVs were not reported. Pathogenic and likely pathogenic CNVs were considered clinically significant, and other CNVs were considered to be lacking clinical significance. Participants were categorized as (1) harboring clinically significant CNVs, or (2) lacking clinically significant CNVs. Individuals with multiple CNVs were categorized based on their most pathogenic CNV.
Statistical analysis. Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). Continuous variables are summarized by a mean and SD and compared with a 2-sample t test or analysis of variance. Categorical data are summarized by a count and percentage and compared with Fisher exact test. Logistic regression was used to model risk of CNVs as a function of various parameters. A p value of #0.05 was considered statistically significant. Nominal p values presented.
We recruited 52 participants (age 10.5 6 7.8 years; 38 males, 14 females) from Israeli CP clinics: 33 participants were recruited from SZMC cohort, 10 participants were referred from Dana Children’s Hospital, and the rest were referred from other neuropediatric clinics. Participants were clinically categorized based on motor deficits and brain imaging findings (table 2). Gross Motor Function Classification System9,10 and Manual Ability Classification System11 were 2.8 6 1.3 and 2.3 6 1.4, respectively. Parental consanguinity was reported in 7 individuals, and in 8 individuals, there were first- or second-degree relatives with significant neurologic disorders. RESULTS Clinical characteristics.
Genomic findings. We found 39 CNVs in 25 of 52 participants (48%) (figure). Sixteen participants (31%) had clinically significant CNVs, either pathogenic CNVs (n 5 10) or likely pathogenic (n 5 6; table 2). Nine participants (17%) had CNVs that were likely benign variants of unknown significance. Twentyseven participants (52%) had only benign CNVs. Of the 16 participants with clinically significant CNVs, 11 had a single CNV, and 5 had more than 1 (1.4 6 0.8, range 1–4). Of the 9 participants with likely benign CNVs, 4 had multiple CNVs (1.7 6 1.0 CNVs, range 1–4; p 5 0.68). Percentage of clinically significant CNVs was not affected by recruitment method (systematic chart review vs neurologist referral; p 5 0.34), so analyses were performed as one group. CNV inheritance. Of the 10 individuals with pathogenic CNVs, 7 had de novo deletions and 3 had CNVs inherited from a parent: a TSC1 deletion inherited from a mildly affected mother (participant 1), a paternal deletion of KANK1 (participant 7) previously reported to cause disease when inherited from a healthy father,13 and an X chromosome duplication in a male (participant 9) inherited from a healthy mother. Of the 6 individuals with likely 1662
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pathogenic CNVs, 2 had de novo deletions and 4 had inherited CNVs. Among the 9 patients with 15 likely benign CNVs, inheritance was determined in 4 participants with 8 CNVs for whom parents’ samples were available; 4 CNVs were de novo and 4 CNVs were inherited. CNV characterization. Of the 39 CNVs, 24 (62%) were deletions and 15 (38%) were duplications. Deletions were more common in participants with clinically significant CNV (18 deletions vs 6 duplications) than in participants with likely benign CNV (6 deletions vs 9 duplications; p 5 0.029). Pathogenic CNV sizes ranged from 147 kb to 11.1 megabases (Mb; median 3 Mb), likely pathogenic CNVs ranged from 152 kb to 862 kb (median 416 kb), and the size of likely benign CNVs ranged from 21 kb to 1.08 Mb (median 36 kb). As expected, clinically significant (pathogenic and likely pathogenic) CNVs were larger than likely benign CNVs (1,767 6 1,592 kb vs 164 6 314 kb; p 5 0.0011).
Genotype–phenotype correlations. Clinical presentation of participants with clinically significant CNVs. Typical presen-
In 6 of the 16 participants with clinically significant CNVs, genomic findings could explain the phenotypes: participant 3 had spastic diplegia due to a large deletion including the SPAT gene that would not have been detected using Sanger sequencing (table 3, and table e-1 on the Neurology® Web site at Neurology.org).13 Participant 4 presented with a neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. He was found to have a deletion encompassing the MEF2C gene, which has been recently described in conjunction with this phenotype.14 Participant 5 had white matter changes and spasticity. He has a deletion containing the WDR45 gene, recently described as causing a similar phenotype in young children.15 Participant 7 had spastic quadriplegia with intellectual disability, and was found to have a paternally inherited deletion including the KANK1 gene. This deletion was described in children with a similar phenotype, and is pathogenic only with paternal inheritance.16 Participant 9, a male who presented with hypotonia spasticity and severe learning disability, has an Xq28 triplication, known to be associated with this phenotype.17 Participant 10, who has choreoathetoid CP, has an 11-Mb deletion encompassing many genes, including the NKX2-1 gene, which is known to cause benign hereditary chorea.18 tation of CNVs known to cause movement disorders.
Atypical clinical presentation of CNVs known to cause move-
Three of 16 participants had an atypical presentation of a known genomic disorder: participant 1 presented with a ganglion cell tumor and was found to have a deletion containing TSC1.19
ment disorders.
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Table 2
Clinical characteristics of individuals with clinically significant (likely pathologic and pathogenic) and nonsignificant (likely benign and benign findings) CNVs
Clinical features
Total (n 5 52)
Nonsignificant CNVs (n 5 36)
Significant CNVs (n 5 16)
p Value
Age, y
10.5 6 7.8
9.6 6 8.1
12.6 6 6.8
0.1935
Sex, M/F
38/14
26/10
12/4
1.000
Birth weight, kg
3.1 6 0.6
3.0 6 0.6
3.1 6 0.5
0.7167
Gestational age, wk
39.1 6 2.4
38.9 6 2.8
39.5 6 1.1
0.4904
Apgar score at 5 min
9.4 6 0.7
9.4 6 0.8
9.3 6 0.5
0.7333
Movement disorder
0.3311
Spasticity
39/52 (75)
29/36 (80)
10/16 (63)
Dystonia
9/52 (17)
5/36 (14)
4/16 (25)
Chorea
3/52 (6)
1/36 (3)
2/16 (12)
Spasticity and dystonia
1/52 (2)
1/36 (3)
12/50 (24)
8/35 (23)
Positive Babinski sign
4/15 (27)
Clinical group
1.0000 0.5365
Generalized dystonia
12/52 (23)
6/36 (17)
6/16 (38)
Brain dysplasiaa
7/52 (13)
5/36 (14)
2/16 (12)
Spastic diplegia
17/52 (33)
14/36 (39)
3/16 (19)
Spastic hemiplegia
1/52 (2)
1/36 (3)
0/16 (0)
Spastic quadriplegia
11/52 (21)
7/36 (19)
4/16 (25)
Term PVL
4/52 (8)
3/36 (8)
1/16 (6)
Diplegia
16/52 (31)
12/36 (33)
4/16 (25)
Hemiplegia
8/52 (15)
6/36 (17)
2/16 (13)
Quadriplegia
26/52 (50)
16/36 (44)
10/16 (62)
Triplegia
2/52 (4)
2/36 (6)
0/16 (0)
GMFCS
2.8 6 1.3
2.9 6 1.3
2.4 6 1.4
MACS
2.3 6 1.4
2.4 6 1.4
2.1 6 1.4
0.5825
Speech,b %
64 6 45
66 6 45
62 6 45
0.7543
Distribution
0.6754
Functional level 0.2087
0.0295c
Cognitive function Mainstream school
16/52 (31)
15/36 (42)
1/16 (6)
Learning disabilities
5/52 (9)
3/36 (8)
2/16 (13)
Mental retardation
31/52 (60)
18/36 (50)
13/16 (81)
0.98 6 1.45
0.61 6 1.02
1.81 6 1.91
0.0283c
Epilepsy
12/51 (24)
6/36 (17)
6/15 (40)
0.1440
Psychiatric diagnosis
3/51 (6)
1/35 (3)
2/16 (13)
0.2043
Comorbidities, sum
Visual problems
13/49 (27)
6/34 (18)
7/15 (47)
0.0782
Other systemic diagnosisd
8/50 (16)
3/35 (9)
5/15 (33)
0.0464c 0.0135c
Dysmorphic features None
24/52 (46)
21/36 (58)
3/16 (19)
Minor
10/52 (19)
4/36 (11)
6/16 (37)
Major
18/52 (35)
11/36 (31)
7/16 (44)
None
44/51 (86)
29/35 (82)
15/16 (94)
1st-degree cousins
1/51 (2)
0/35 (0)
1/16 (2)
1st/2nd-degree cousins
1/51 (2)
1/35 (3)
0/16 (0)
Consanguinity
0.4111
Continued Neurology 84
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Table 2
Continued Total (n 5 52)
Clinical features
Nonsignificant CNVs (n 5 36)
Significant CNVs (n 5 16)
2nd-degree cousins
3/51 (6)
3/35 (9)
0/16 (0)
3rd-degree cousins
2/51 (4)
2/35 (6)
0/16 (0)
p Value
,0.0001c
Relative with neurologic disorder 1st-degree
5/51 (10)
1/36 (1)
9/16 (56)
2nd-degree
3/51 (6)
0/36 (0)
5/16 (31)
None
44/51 (84)
35/36 (97)
2/16 (13)
Brain imaging
1.0000
Normal
19/51 (37)
13/35 (37)
6/16 (37)
Mild abnormalities
7/51 (14)
5/35 (14)
2/16 (13)
Abnormal
25/51 (49)
17/35 (49)
8/16 (50)
Abbreviations: CNV 5 copy number variation; GMFCS 5 Gross Motor Function Classification System; MACS 5 Manual Ability Classification System; PVL 5 periventricular leukomalacia. Data are mean 6 SD or n/N (%). a Brain dysplasia: septooptic dysplasia (n 5 2), schizencephaly (n 5 2), ganglion cell tumor (n 5 1), opercular syndrome (n 5 1), vermis atrophy (n 5 1). b Speech 5 % of speech understood by parents. c p , 0.05. d Other systemic diagnosis: deafness (n 5 1), sickle cell trait (n 5 1), hypospadias (n 5 1) and cryptorchidism (n 5 1), severe vesicoureteral reflux (n 5 1), gastrointestinal obstruction (n 5 1), lung disease (n 5 1), growth hormone deficiency (n 5 2).
Participant 6, a child with generalized dystonia and increased startle, was found to have a large 22q11 deletion. Both participants (1 and 6) had symptoms that do not fulfill the classic phenotype of tuberous sclerosis complex (TSC) and 22q11 deletion syndrome, respectively.20,21 Participant 11, a child with generalized dystonia, had a duplication of GNAL gene, in which point mutations are known to cause DYT25 focal dystonia in adults.22 Figure
Distribution of participants with CNVs in cryptogenic cerebral palsy
CNVs not previously described with movement disorders. Seven participants with clinically significant CNVs had genomic findings not previously reported in association with motor symptoms. Some of these genes were associated with other neurologic phenotypes. For example, deletion of the DYNC1H1 gene in participant 8 could explain the axonal polyneuropathy but not his chorea.23 Correlation of clinically significant CNVs with family history.
Among participants with clinically significant CNVs, family history of a neurologic disease was more common (7/16) than among the rest of the participants (1/36; p , 0.0001); 5 of them had de novo CNVs. As can be expected, family members of participants with de novo CNVs had a different neurologic disease. In the single family in which 2 siblings were recruited because of CP (participants 2 and 3), both the phenotype and genotypes were different: spastic diplegia and dystonia, with a different de novo deletion in each participant. Family history included 2 siblings with 2 different manifestations of CP (participants 2 and 3), participant 5’s brother with autistic spectrum disorder, participant 6’s paternal cousin, who died after having multiple birth anomalies and epilepsy, participant 16’s brother with severe learning disability, and participant 25’s maternal cousin with mental retardation. Participant 5’s brother was available for testing, and had normal array results. Clinical characteristics associated with pathogenic CNVs:
Of 52 participants 27 (52%) had only benign CNVs, 10 participants (19%) had pathogenic CNVs, 6 (12%) had likely pathogenic VOUS, and 9 participants (17%) had CNVs that were likely benign VOUS. CNV 5 copy number variation; VOUS 5 variants of unknown significance. 1664
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Major or minor dysmorphic features were found in 81% of individuals with clinically significant CNVs compared with 42% of the individuals without clinically
Dysmorphic features and nonmotor comorbidities.
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Table 3
Pathogenic, likely pathogenic and likely benign CNVs found in individuals with cryptogenic CP (position and genes in table e-1)
Participant
Mov dis
Plegia
GMFCS
Dysmor
Imaging
CNV type
Cytoband
Size, kb
Candidate genes
Classification
dn/Inh (M/P)
TSC1
path
Inh (M)
path
dn
Pathogenic CNVs 1
S
H
1
2/1
2
del
9q34.13q34.2
147
2
Dy
D
2
1
0
del
19q13.12
1,960
3
S
D
2
2/1
0
del
2p23.1p22.2
5,214
SPAST
path
dn
4
Dy
Q
2
1
0
del
5q14.3
3,463
MEF2C
path
dn
5
S
Q
3
1
2
6
Dy
Q
4
1
0
7
S
Q
5
2/1
2
del
Xp11.23p11.22
4,287
WDR45
path
dn
dup
Xq25
32
SPG34
bVOUS
dn
del
22q11.21
2,821
path
dn
path
Inh (P)
bVOUS
dn
DYNC1H1
path
dn
del
9p24.3
226
del
4q34.1
366
14q32.31q32.33
3,230
KANK1
8
C
Q
1
1
0
del
9
S
D
2
2
2
trip
Xq28
298
FLNA
path
Inh (M)
10
C
Q
2
1
1
del
14q12q21.2
11,150
NKX2-1
path
dn
del
3p26.3
521
bVOUS
dn
pVOUS
Inh (P)
Likely pathogenic variants of unknown significance (pVOUS) 11
Dy
Q
5
1
1
dup
18p11.21
445
12
S
Q
2
2/1
2
dup
2q13
862
pVOUS
Inh (M)
13
S
Q
1
2
2
del
7q31.1
152
pVOUS
dn
14
S
D
1
2
0
dup
20p12.3p12.2
679
pVOUS
Inh (P)
15
S
Q
4
2/1
2
del
1p21.3
154
pVOUS
Inh (P)
del
5p12p11
450
bVOUS
Inh (P)
del
11q11
289
bVOUS
Inh (P)
del
Yq11.223
273
bVOUS
Inh (P)
16
S
H
2
2/1
2
GNAL
dup
17p11.2
387
pVOUS
dn
del
Yq11.223
273
bVOUS
dn
del
18q22.2
45
bVOUS
dn
Likely benign variants of unknown significance (bVOUS) 17
S
D
2
2
0
dup
2q31.2
36
bVOUS
NT
18
S
Q
5
1
0
del
2q34
24
bVOUS
NT
del
Yq11.223
273
bVOUS
NT
19
S, Dy
Q
3
2/1
1
dup
16p13.11p12.3
549
bVOUS
NT
dup
7q36.1
272
bVOUS
NT
20
C
Q
4
2
0
del
18q22.3
330
bVOUS
Inh (M)
21
S
D
1
2/1
0
del
Xp21.1
1,083
bVOUS
NT
22
S
T
1
2
0
dup
9p13.2
594
bVOUS
NT
23
S
Q
5
2
2
del
4q28.3
947
bVOUS
Inh (M)
24
Dy
Q
3
2
1
dup
Xp11.22
53
bVOUS
Inh (P)
dup
Xq26.3
29
bVOUS
dn
dup
Xq27.3
22
bVOUS
dn
dup
Xq27.1
21
bVOUS
dn
dup
8q13.3
344
bVOUS
dn
del
10q26.3
143
bVOUS
Inh (M)
25
S
Q
3.5
2/1
2
Abbreviations: bVOUS 5 benign variants of unknown significance; C 5 chorea; CNV 5 copy number variation; CP 5 cerebral palsy; D 5 diplegia; Dy 5 dystonia; del 5 deletion; dn 5 de novo; dup 5 duplication; Dysmor 5 dysmorphic; GMFCS 5 Gross Motor Function Classification System; H 5 hemiplegia; Inh 5 inherited; M 5 maternal; Mov dis 5 movement disorder; NT 5 not tested; P 5 paternal; path 5 pathogenic; pVOUS 5 pathogenic variants of unknown significance; Q 5 quadriplegia; S 5 spasticity; T 5 triplegia.
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significant CNVs (p 5 0.01). Presence of a nonmotor morbidity (seizures, intellectual disability, psychiatric manifestations, visual impairment, or other systemic illness) was more common in individuals with clinically significant CNVs (1.8 6 1.9), compared with those without such CNVs (0.6 6 1.0; p 5 0.03). Intellectual disability and learning disability were more frequent in individuals with clinically significant CNVs: 81% and 13%, respectively, vs 50% and 8%, respectively, in individuals without such CNVs (p 5 0.03). There was no correlation between presence of clinically significant CNVs and other clinical features (table 2). DISCUSSION We found that one-third (31%) of children with cryptogenic CP have pathogenic or likely pathogenic CNVs similar to the rate in other movement disorders (28%).8 Frequency of CNVs is higher than that previously reported in CP (20%)24 and may be explained by our stringent definition of cryptogenic CP, which excluded identifiable acquired causes (e.g., infection). We realize that some of the participants who were excluded, mainly on the basis of prematurity and/or stroke, may also have an underlying genomic predisposing disorder. However, it is the current understanding that neurologic outcome of patients with these etiologies is multifactorial and relies only in part on their genetic predisposition.25 There were more deletions than duplications in the clinically significant group of CNVs, as expected, based on the current knowledge of pathogenic CNVs in other disorders26 such as attention-deficit/ hyperactivity disorder27 and epilepsy.28 CNV size was largest in the pathogenic group and can be attributable, in part, to the CNV analysis criteria. The majority of pathogenic CNVs were de novo and only 3 were inherited. Two inherited cases are explained by gender-specific effects: an X chromosome duplication in a male inherited from a healthy carrier mother, and a KANK1 deletion known to cause spastic quadriplegia only when paternally inherited. The third inherited case, a TSC1 deletion, was identified in a child that did not fulfill diagnostic criteria for TSC, whose mother was considered healthy but had seizures as a child. Of the likely pathogenic variants of unknown significance, only 2 were de novo and the rest were inherited. However, these inherited CNVs contained neurologic disease–related genes, and suspected pathogenicity was based on partial penetrance. We did not test all parents of participants with benign or likely benign CNVs because association between the CNVs and CP was unclear. Dysmorphic features and nonmotor comorbidities, especially intellectual disability, were common in individuals with clinically significant CNVs. We 1666
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found at least twice as many CNVs than reported in studies of specific nonmotor comorbidities.5–7 The rate of pathogenic CNVs was similar in all centers with different systems of referrals. Most participants with a family history of neurologic disorders had significant findings, but the clinical presentations and CNVs were different. For instance, 2 brothers with phenotypically distinctive CP (participants 2 and 3) had 2 different interstitial CNVs, which could not be explained by a balanced translocation in their parents, and indeed, the parents’ karyotypes were normal. Six pathogenic CNVs contained genes associated with the clinical CP phenotype: SPAST (hereditary spastic paraparesis),13 MEF2C (dystonia),14 WDR45 (spasticity),15 KANK1 (spastic quadriparesis),16 NKX2-1 (chorea),18 and tripXq28 (spasticity).17 Three aberrations were associated with atypical presentations of a known disorder (TSC1, 22q11 deletion, GNAL).19–22 Seven CNVs have not been previously reported to cause movement disorders in children, although some contained genes previously reported in other neurodisabilities (e.g., DYNC1H1).23 CNVs not previously implicated in movement disorders or CP may have a larger role in brain development than previously thought. Since genomic rearrangements include many genes, and the same participant may have had more than one CNV, it is possible that the phenotype is determined by the combination of more than one gene/region aberration. Further investigation into these genes is warranted to better characterize the function of these genes and regions in motor development. The limitation of the study is its small sample size; in addition, as genetic information is exponentially expanding, the significance of unknown variants may clarify with time with growing research. Based on the proportion of CNV findings in our study, CMA should be considered in all patients with cryptogenic CP as a first line of workup at diagnosis, saving many unnecessary tests. We were able to provide genetic counseling to all the families who were found to have genomic rearrangements. After testing the parents, if the CNVs were de novo, we assumed that recurrence rate in their next pregnancies would be low. When the CNVs were inherited, we offered prenatal diagnosis or preimplantation genetic diagnosis. We conclude that CNVs are common in individuals with cryptogenic CP. While they are particularly common in patients with dysmorphism or nonmotor comorbidities, they are also found in patients with cryptogenic CP without these features. We recommend CMA in individuals with CP of unknown cause to accelerate diagnosis, saving resources of both
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families and medical systems. Moreover, novel CNVs associated with CP may indicate new genes involved in neuropathology. AUTHOR CONTRIBUTIONS Reeval Segel conceptualized the study, received grants/funding, was in charge of collection of all DNA samples and clinical data, interpreted genetic results, wrote the first draft together with H.B.-P., and approved writing and revision of the manuscript. Hilla Ben-Pazi assisted in conceptualizing the study, collected the majority of DNA samples and clinical data, wrote the first draft with R.S., and approved final manuscript version. Sharon Zeligson and Shira Perlberg preformed the genetic testing and analysis. Varda Gross-Tsur assisted in conceptualizing the study, provided insight about the hypothesis, and revised the manuscript. Aviva Fatal-Valevski, Adi Aran, Nira Schneebaum-Sender, Dorit Shmueli, Dorit Lev, and Luba Blumkin recruited eligible candidates, collected DNA samples, examined the participants, recorded clinical data, and approved the manuscript. Lisa Deutsch analyzed the data, reviewed and edited manuscript drafts, approved final manuscript version. Ephrat LevyLahad initiated the study, assisted in conceptualizing the study and obtaining funding, directed the analysis, provided insight about the hypothesis, and assisted in the writing and revision of the manuscript.
ACKNOWLEDGMENT
6.
7.
8.
9.
10.
11.
The authors thank Mr. Michael Goldenshluger, Yael Grinberg, and Nava Badichi for their assistance in blood drawing. The authors thank the families for their cooperation and participation.
STUDY FUNDING
12.
Supported by grants: MOF Joint Israel Ministry of Health grant (3-6185), and the joint research fund of The Hebrew University and Shaare Zedek Medical Center.
DISCLOSURE R. Segel reports grant funding from the Chief Scientist, Israel Ministry of Health (3-6185), and the joint research fund of The Hebrew University and Shaare Zedek. H. Ben-Pazi, S. Zeligson, A. Fatal-Valevski, A. Aran, V. Gross-Tsur, N. Schneebaum-Sender, D. Shmueli, D. Lev, S. Perlberg, L. Blumkin, and L. Deutsch report no disclosures relevant to the manuscript. E. Levy-Lahad reports no funding for this particular project. Grants for other projects include funding from the Breast Cancer Research Foundation (NY), Israel Science Fund, and USAID–MERC (Middle East Regional Cooperation). Ethics committee approval: The study was approved by Shaare Zedek Medical Center’s internal review board and the National IRB (MOH 033/2008). Go to Neurology.org for full disclosures.
Received September 3, 2014. Accepted in final form January 6, 2015. REFERENCES 1. Smithers-Sheedy H, Badawi N, Blair E, et al. What constitutes cerebral palsy in the twenty-first century? Dev Med Child Neurol 2014;56:323–328. 2. Hemminki K, Li X, Sundquist K, Sundquist J. High familial risks for cerebral palsy implicate partial heritable aetiology. Paediatr Perinat Epidemiol 2007;21:235–241. 3. Shevell MI, Majnemer A, Morin I. Etiologic yield of cerebral palsy: a contemporary case series. Pediatr Neurol 2003;28:352–359. 4. Richer LP, Dower NA, Leonard N, Chan AK, Robertson CM. Familial recurrence of cerebral palsy with multiple risk factors. Case Rep Pediatr 2011;2011: 307857. 5. Vissers LE, de Vries BB, Veltman JA. Genomic microarrays in mental retardation: from copy number variation to gene, from research to diagnosis. J Med Genet 2010;47: 289–297.
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Copy number variations in cryptogenic cerebral palsy Reeval Segel, Hilla Ben-Pazi, Sharon Zeligson, et al. Neurology 2015;84;1660-1668 Published Online before print March 27, 2015 DOI 10.1212/WNL.0000000000001494 This information is current as of March 27, 2015 Updated Information & Services
including high resolution figures, can be found at: http://www.neurology.org/content/84/16/1660.full.html
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References
This article cites 28 articles, 6 of which you can access for free at: http://www.neurology.org/content/84/16/1660.full.html##ref-list-1
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Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2015 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.
Reeval Segel, MD* Hilla Ben-Pazi, MD* Sharon Zeligson Aviva Fatal-Valevski, MD Adi Aran, MD Varda Gross-Tsur, MD Nira Schneebaum-Sender, MD Dorit Shmueli, MD Dorit Lev, MD Shira Perlberg Luba Blumkin, MD Lisa Deutsch, PhD Ephrat Levy-Lahad, MD
Correspondence to Dr. Segel: [email protected]
ABSTRACT
Objective: To determine the prevalence and characteristics of copy number variations (CNVs) in children with cerebral palsy (CP) of unknown etiology, comprising approximately 20% of the CP population.
Methods: Fifty-two participants (age 10.5 6 7.8 years; Gross Motor Function Classification System scale 2.8 6 1.3) with nonprogressive pyramidal and/or extrapyramidal signs since infancy and no identified etiology were enrolled. Individuals with evidence of acquired causes were excluded. Participants underwent neurologic and clinical genetic examinations before the genomic testing. Chromosomal microarray analysis to detect CNVs was performed using the Affymetrix platform. CNVs identified were classified as pathogenic, likely pathogenic, likely benign, or benign. Only pathogenic and likely pathogenic CNVs were defined as clinically significant. Results: Thirty-nine CNVs were found in 25 of 52 participants (48%). Sixteen participants (31%) had clinically significant CNVs: 10 pathogenic and 6 likely pathogenic, of which 7 were not previously associated with motor disability. Nine participants had likely benign CNVs. Clinically significant CNVs were more frequently de novo (12/16; p , 0.001) including in 5 of 8 individuals who had a first- or second-degree relative with a major neurologic disorder. Dysmorphic features and nonmotor comorbidities were more prevalent in individuals with clinically significant CNVs (p , 0.05 for both).
Conclusion: CNVs, most frequently de novo, are common in individuals with cryptogenic CP. We recommend CNV testing in individuals with CP of unknown etiology. Neurology® 2015;84:1660–1668 GLOSSARY CNV 5 copy number variation; CP 5 cerebral palsy; OMIM 5 Online Mendelian Inheritance in Man; SZMC 5 Shaare Zedek Medical Center; TSC 5 tuberous sclerosis complex.
Supplemental data at Neurology.org
Cerebral palsy (CP) is the most common cause of motor disability in childhood (approximately 2:1,000 live births), and is defined as an “umbrella term” of a permanent movement and/or postural disorder caused by nonprogressive abnormalities of the developing brain.1 In most affected children, CP is associated with self-evident perinatal insults, especially prematurity, asphyxia, intrauterine infection, hemorrhage, and brain infracts.2,3 However, in approximately 20%3 of individuals with CP, motor deficits have no apparent etiology,2 and these children are considered to have cryptogenic CP. Although CP is not regarded as a genetic disease, congenital anomalies are more common among individuals with CP, and familial cases have been reported, suggesting underlying genetic susceptibility.2,4 Chromosomal microdeletions and microduplications, also known as copy number variations (CNVs), have emerged as a cause for various neurodevelopmental disorders. CNVs have been reported in 7% to 21% of individuals with intellectual disability,5 autism,6 and epilepsies,7 and in 28% of individuals with movement disorders.8 In most of these, CNVs are de novo, rather than inherited events, explaining paucity of familial occurrence. *These authors contributed equally to this work. From the Medical Genetics Institute (R.S., S.Z., S.P., E.L.-L.) and Neuropediatric Unit (H.B.-P., A.A., V.G.-T.), Shaare Zedek Medical Center, Jerusalem; Pediatric Neurology Unit (A.F.-V., N.S.-S.), Dana Children’s Hospital, Tel Aviv; Jerusalem Child Development Center (D.S.), Clalit, Jerusalem; Metabolic-Neurogenetic Clinic (D.L., L.B.), Wolfson Medical Center, Holon; and Biostatistical Consulting (L.D.), BioStats, Israel. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
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We hypothesized that CNVs may have a role in cryptogenic CP. To examine this, we tested individuals with cryptogenic CP for CNVs using chromosomal microarray (CMA), and compared clinical characteristics of participants harboring clinically significant CNVs with those without such findings. METHODS Participants. Individuals with cryptogenic CP were identified by chart review in 4 CP clinics in Israel. Patients were categorized as having cryptogenic CP when etiology of the neurologic disorder remained undetermined. Inclusion criteria were as follows: individuals with disabling nonprogressive pyramidal and/or extra pyramidal signs beginning before 3 years of age were considered eligible. Patients with any known acquired etiology were excluded a priori (table 1). Such etiologies included periventricular leukomalacia in premature infants, hypoxic ischemic encephalopathy, brain infarcts, encephalitis, head trauma, and spinal cord lesions. Recruitment methods were different based on availability of electronic medical records. In Shaare Zedek Medical Center (SZMC), records of the CP cohort were systematically reviewed for recruitment using study criteria. In other centers, enrollment was based on pediatric neurologists’ referrals of children who fulfilled the study criteria. All participants were examined by a pediatric neurologist with expertise in CP (H.B.-P., A.F.-V., A.A., V.G.-T., D.S., or L.B.) and by a medical geneticist (R.S. or D.L.). Clinical characteristics were documented: perinatal history, type and anatomical distribution of movement disorder, Gross Motor Function Classification System,9,10 and Manual Ability Classification System.11 Imaging findings and comorbidities (i.e., intellectual disability, epilepsy, psychiatric disorders) were graded as follows:
Table 1
Inclusion and exclusion criteria
Inclusion criteria (all the below) Nonprogressive Pyramidal and/or extrapyramidal signs Evident before 3 y of age Exclusion criteria (any known etiology below) PVL Born between 24 and 34 gestational age and evidence of PVL on imaging HIE History of birth complication requiring resuscitation or Apgar score ,7 in 5 min or resuscitation at infancy Hemispheric lesion Hemiplegia and acquired contralateral nonstructural lesion on imaging studiesa Encephalitis Encephalitis at birth (i.e., documented CMV) or post/perinatal meningitis/encephalitis Head injury Prenatal or postnatal Spinal cord lesion Spastic paraplegia and abnormal spinal MRI scan
Abbreviations: CMV 5 cytomegalovirus; HIE 5 hypoxic-ischemic encephalopathy; PVL 5 periventricular leukomalacia. a Including stroke, tumor; excluding brain dysplasia disorders.
0 5 normal; 1 5 mild abnormality that could be found in typically developing children as well; and 2 5 definite pathologic abnormality. Medical genetic evaluation included consanguinity, family history, and dysmorphic features. When possible, parents of participants with significant CNVs were tested.
Standard protocol approvals, registrations, and patient consents. This multicenter study was approved by SZMC and Dana Children’s Hospital Internal Review Boards and the National Review Board for Genetic Studies. Written informed consent was obtained from all participants, probands, and parents.
Controls. To determine whether CNVs in our cohort are present in healthy individuals, we compared our findings with those found in healthy individuals in the Database of Genomic Variants (http://dgv. tcag.ca/dgv/app/), CAGdb (Cytogenomics Array Group Database) (http://www.cagdb.org/), ISCA (International Standards for Cytogenomic Arrays) (http://dbsearch.clinicalgenome.org/), and our in-house variant database.
Genetic testing. DNA was extracted from peripheral blood samples, using standard methods. Affymetrix Cytoscan HD arrays were used according to manufacturer’s protocol. The CytoScan HD Array provides whole-genome coverage, with more than 2.6 million markers including 750,000 single nucleotide polymorphisms. Data analysis was done using the Chromosome Analysis Suite (Affymetrix, Santa Clara, CA) and Partek Genomic Suite (Partek Inc., St. Louis, MO) for defining CNVs, and University of California Santa Cruz (http://genome.ucsc.edu/), GeneCards (http://www.genecards.org/), OMIM (Online Mendelian Inheritance in Man) (http://www.omim.org/), CAGdb (http://www.cagdb.org/), Decipher (http://www. sanger.ac.uk/PostGenomics/decipher/), and ISCA (https://www. iscaconsortium.org/) for assessment of CNV significance. Data were analyzed for deletions and gains according to the American College of Medical Genetics and Genomics guidelines.12 CNVs were compared with parents’ CNV profiles, when available. Classification of CMA results (main outcome measure). CNVs detected were divided to 4 subgroups, according to American College of Medical Genetics and Genomics guidelines12: 1. Pathogenic CNVs: CNVs documented as clinically significant in multiple peer-reviewed publications, CNVs not described in the medical literature at sizes observed in patients but that overlap a smaller interval with established clinical significance, and CNVs containing genes known to be disease-causing based on dosage effect. 2. Likely pathogenic variants of unknown significance: CNVs described in a single case report with neurologic disease, CNVs containing a gene(s) with function(s) relevant to CP, CNVs that include genes reported in OMIM as neurologic disease–causing but not reported to have a dosage effect, or CNVs that are reported in individuals with neurologic disease in online databases, but not in a peer-reviewed publication. 3. Likely benign variants of unknown significance: CNVs that are large enough to be reported (deletions .200 kilobases [kb], duplications .500 kb), but not described as diseasecausing in any of the databases, CNVs described in a small number of cases in databases of variation in the general population, but do not represent a common polymorphism, CNVs containing disease-causing genes that are not known to be dosage-sensitive, or CNVs associated with contradictory information in the medical literature and/or online databases. 4. Benign CNVs: CNVs below a critical threshold (deletions ,200 kb and duplications ,500 kb) not containing OMIM genes associated with disease, CNVs that were reported in Neurology 84
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multiple peer-reviewed publications or curated databases as a benign variant, or CNVs that are common polymorphisms. Benign CNVs were not reported. Pathogenic and likely pathogenic CNVs were considered clinically significant, and other CNVs were considered to be lacking clinical significance. Participants were categorized as (1) harboring clinically significant CNVs, or (2) lacking clinically significant CNVs. Individuals with multiple CNVs were categorized based on their most pathogenic CNV.
Statistical analysis. Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). Continuous variables are summarized by a mean and SD and compared with a 2-sample t test or analysis of variance. Categorical data are summarized by a count and percentage and compared with Fisher exact test. Logistic regression was used to model risk of CNVs as a function of various parameters. A p value of #0.05 was considered statistically significant. Nominal p values presented.
We recruited 52 participants (age 10.5 6 7.8 years; 38 males, 14 females) from Israeli CP clinics: 33 participants were recruited from SZMC cohort, 10 participants were referred from Dana Children’s Hospital, and the rest were referred from other neuropediatric clinics. Participants were clinically categorized based on motor deficits and brain imaging findings (table 2). Gross Motor Function Classification System9,10 and Manual Ability Classification System11 were 2.8 6 1.3 and 2.3 6 1.4, respectively. Parental consanguinity was reported in 7 individuals, and in 8 individuals, there were first- or second-degree relatives with significant neurologic disorders. RESULTS Clinical characteristics.
Genomic findings. We found 39 CNVs in 25 of 52 participants (48%) (figure). Sixteen participants (31%) had clinically significant CNVs, either pathogenic CNVs (n 5 10) or likely pathogenic (n 5 6; table 2). Nine participants (17%) had CNVs that were likely benign variants of unknown significance. Twentyseven participants (52%) had only benign CNVs. Of the 16 participants with clinically significant CNVs, 11 had a single CNV, and 5 had more than 1 (1.4 6 0.8, range 1–4). Of the 9 participants with likely benign CNVs, 4 had multiple CNVs (1.7 6 1.0 CNVs, range 1–4; p 5 0.68). Percentage of clinically significant CNVs was not affected by recruitment method (systematic chart review vs neurologist referral; p 5 0.34), so analyses were performed as one group. CNV inheritance. Of the 10 individuals with pathogenic CNVs, 7 had de novo deletions and 3 had CNVs inherited from a parent: a TSC1 deletion inherited from a mildly affected mother (participant 1), a paternal deletion of KANK1 (participant 7) previously reported to cause disease when inherited from a healthy father,13 and an X chromosome duplication in a male (participant 9) inherited from a healthy mother. Of the 6 individuals with likely 1662
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pathogenic CNVs, 2 had de novo deletions and 4 had inherited CNVs. Among the 9 patients with 15 likely benign CNVs, inheritance was determined in 4 participants with 8 CNVs for whom parents’ samples were available; 4 CNVs were de novo and 4 CNVs were inherited. CNV characterization. Of the 39 CNVs, 24 (62%) were deletions and 15 (38%) were duplications. Deletions were more common in participants with clinically significant CNV (18 deletions vs 6 duplications) than in participants with likely benign CNV (6 deletions vs 9 duplications; p 5 0.029). Pathogenic CNV sizes ranged from 147 kb to 11.1 megabases (Mb; median 3 Mb), likely pathogenic CNVs ranged from 152 kb to 862 kb (median 416 kb), and the size of likely benign CNVs ranged from 21 kb to 1.08 Mb (median 36 kb). As expected, clinically significant (pathogenic and likely pathogenic) CNVs were larger than likely benign CNVs (1,767 6 1,592 kb vs 164 6 314 kb; p 5 0.0011).
Genotype–phenotype correlations. Clinical presentation of participants with clinically significant CNVs. Typical presen-
In 6 of the 16 participants with clinically significant CNVs, genomic findings could explain the phenotypes: participant 3 had spastic diplegia due to a large deletion including the SPAT gene that would not have been detected using Sanger sequencing (table 3, and table e-1 on the Neurology® Web site at Neurology.org).13 Participant 4 presented with a neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. He was found to have a deletion encompassing the MEF2C gene, which has been recently described in conjunction with this phenotype.14 Participant 5 had white matter changes and spasticity. He has a deletion containing the WDR45 gene, recently described as causing a similar phenotype in young children.15 Participant 7 had spastic quadriplegia with intellectual disability, and was found to have a paternally inherited deletion including the KANK1 gene. This deletion was described in children with a similar phenotype, and is pathogenic only with paternal inheritance.16 Participant 9, a male who presented with hypotonia spasticity and severe learning disability, has an Xq28 triplication, known to be associated with this phenotype.17 Participant 10, who has choreoathetoid CP, has an 11-Mb deletion encompassing many genes, including the NKX2-1 gene, which is known to cause benign hereditary chorea.18 tation of CNVs known to cause movement disorders.
Atypical clinical presentation of CNVs known to cause move-
Three of 16 participants had an atypical presentation of a known genomic disorder: participant 1 presented with a ganglion cell tumor and was found to have a deletion containing TSC1.19
ment disorders.
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Table 2
Clinical characteristics of individuals with clinically significant (likely pathologic and pathogenic) and nonsignificant (likely benign and benign findings) CNVs
Clinical features
Total (n 5 52)
Nonsignificant CNVs (n 5 36)
Significant CNVs (n 5 16)
p Value
Age, y
10.5 6 7.8
9.6 6 8.1
12.6 6 6.8
0.1935
Sex, M/F
38/14
26/10
12/4
1.000
Birth weight, kg
3.1 6 0.6
3.0 6 0.6
3.1 6 0.5
0.7167
Gestational age, wk
39.1 6 2.4
38.9 6 2.8
39.5 6 1.1
0.4904
Apgar score at 5 min
9.4 6 0.7
9.4 6 0.8
9.3 6 0.5
0.7333
Movement disorder
0.3311
Spasticity
39/52 (75)
29/36 (80)
10/16 (63)
Dystonia
9/52 (17)
5/36 (14)
4/16 (25)
Chorea
3/52 (6)
1/36 (3)
2/16 (12)
Spasticity and dystonia
1/52 (2)
1/36 (3)
12/50 (24)
8/35 (23)
Positive Babinski sign
4/15 (27)
Clinical group
1.0000 0.5365
Generalized dystonia
12/52 (23)
6/36 (17)
6/16 (38)
Brain dysplasiaa
7/52 (13)
5/36 (14)
2/16 (12)
Spastic diplegia
17/52 (33)
14/36 (39)
3/16 (19)
Spastic hemiplegia
1/52 (2)
1/36 (3)
0/16 (0)
Spastic quadriplegia
11/52 (21)
7/36 (19)
4/16 (25)
Term PVL
4/52 (8)
3/36 (8)
1/16 (6)
Diplegia
16/52 (31)
12/36 (33)
4/16 (25)
Hemiplegia
8/52 (15)
6/36 (17)
2/16 (13)
Quadriplegia
26/52 (50)
16/36 (44)
10/16 (62)
Triplegia
2/52 (4)
2/36 (6)
0/16 (0)
GMFCS
2.8 6 1.3
2.9 6 1.3
2.4 6 1.4
MACS
2.3 6 1.4
2.4 6 1.4
2.1 6 1.4
0.5825
Speech,b %
64 6 45
66 6 45
62 6 45
0.7543
Distribution
0.6754
Functional level 0.2087
0.0295c
Cognitive function Mainstream school
16/52 (31)
15/36 (42)
1/16 (6)
Learning disabilities
5/52 (9)
3/36 (8)
2/16 (13)
Mental retardation
31/52 (60)
18/36 (50)
13/16 (81)
0.98 6 1.45
0.61 6 1.02
1.81 6 1.91
0.0283c
Epilepsy
12/51 (24)
6/36 (17)
6/15 (40)
0.1440
Psychiatric diagnosis
3/51 (6)
1/35 (3)
2/16 (13)
0.2043
Comorbidities, sum
Visual problems
13/49 (27)
6/34 (18)
7/15 (47)
0.0782
Other systemic diagnosisd
8/50 (16)
3/35 (9)
5/15 (33)
0.0464c 0.0135c
Dysmorphic features None
24/52 (46)
21/36 (58)
3/16 (19)
Minor
10/52 (19)
4/36 (11)
6/16 (37)
Major
18/52 (35)
11/36 (31)
7/16 (44)
None
44/51 (86)
29/35 (82)
15/16 (94)
1st-degree cousins
1/51 (2)
0/35 (0)
1/16 (2)
1st/2nd-degree cousins
1/51 (2)
1/35 (3)
0/16 (0)
Consanguinity
0.4111
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Table 2
Continued Total (n 5 52)
Clinical features
Nonsignificant CNVs (n 5 36)
Significant CNVs (n 5 16)
2nd-degree cousins
3/51 (6)
3/35 (9)
0/16 (0)
3rd-degree cousins
2/51 (4)
2/35 (6)
0/16 (0)
p Value
,0.0001c
Relative with neurologic disorder 1st-degree
5/51 (10)
1/36 (1)
9/16 (56)
2nd-degree
3/51 (6)
0/36 (0)
5/16 (31)
None
44/51 (84)
35/36 (97)
2/16 (13)
Brain imaging
1.0000
Normal
19/51 (37)
13/35 (37)
6/16 (37)
Mild abnormalities
7/51 (14)
5/35 (14)
2/16 (13)
Abnormal
25/51 (49)
17/35 (49)
8/16 (50)
Abbreviations: CNV 5 copy number variation; GMFCS 5 Gross Motor Function Classification System; MACS 5 Manual Ability Classification System; PVL 5 periventricular leukomalacia. Data are mean 6 SD or n/N (%). a Brain dysplasia: septooptic dysplasia (n 5 2), schizencephaly (n 5 2), ganglion cell tumor (n 5 1), opercular syndrome (n 5 1), vermis atrophy (n 5 1). b Speech 5 % of speech understood by parents. c p , 0.05. d Other systemic diagnosis: deafness (n 5 1), sickle cell trait (n 5 1), hypospadias (n 5 1) and cryptorchidism (n 5 1), severe vesicoureteral reflux (n 5 1), gastrointestinal obstruction (n 5 1), lung disease (n 5 1), growth hormone deficiency (n 5 2).
Participant 6, a child with generalized dystonia and increased startle, was found to have a large 22q11 deletion. Both participants (1 and 6) had symptoms that do not fulfill the classic phenotype of tuberous sclerosis complex (TSC) and 22q11 deletion syndrome, respectively.20,21 Participant 11, a child with generalized dystonia, had a duplication of GNAL gene, in which point mutations are known to cause DYT25 focal dystonia in adults.22 Figure
Distribution of participants with CNVs in cryptogenic cerebral palsy
CNVs not previously described with movement disorders. Seven participants with clinically significant CNVs had genomic findings not previously reported in association with motor symptoms. Some of these genes were associated with other neurologic phenotypes. For example, deletion of the DYNC1H1 gene in participant 8 could explain the axonal polyneuropathy but not his chorea.23 Correlation of clinically significant CNVs with family history.
Among participants with clinically significant CNVs, family history of a neurologic disease was more common (7/16) than among the rest of the participants (1/36; p , 0.0001); 5 of them had de novo CNVs. As can be expected, family members of participants with de novo CNVs had a different neurologic disease. In the single family in which 2 siblings were recruited because of CP (participants 2 and 3), both the phenotype and genotypes were different: spastic diplegia and dystonia, with a different de novo deletion in each participant. Family history included 2 siblings with 2 different manifestations of CP (participants 2 and 3), participant 5’s brother with autistic spectrum disorder, participant 6’s paternal cousin, who died after having multiple birth anomalies and epilepsy, participant 16’s brother with severe learning disability, and participant 25’s maternal cousin with mental retardation. Participant 5’s brother was available for testing, and had normal array results. Clinical characteristics associated with pathogenic CNVs:
Of 52 participants 27 (52%) had only benign CNVs, 10 participants (19%) had pathogenic CNVs, 6 (12%) had likely pathogenic VOUS, and 9 participants (17%) had CNVs that were likely benign VOUS. CNV 5 copy number variation; VOUS 5 variants of unknown significance. 1664
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Major or minor dysmorphic features were found in 81% of individuals with clinically significant CNVs compared with 42% of the individuals without clinically
Dysmorphic features and nonmotor comorbidities.
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Table 3
Pathogenic, likely pathogenic and likely benign CNVs found in individuals with cryptogenic CP (position and genes in table e-1)
Participant
Mov dis
Plegia
GMFCS
Dysmor
Imaging
CNV type
Cytoband
Size, kb
Candidate genes
Classification
dn/Inh (M/P)
TSC1
path
Inh (M)
path
dn
Pathogenic CNVs 1
S
H
1
2/1
2
del
9q34.13q34.2
147
2
Dy
D
2
1
0
del
19q13.12
1,960
3
S
D
2
2/1
0
del
2p23.1p22.2
5,214
SPAST
path
dn
4
Dy
Q
2
1
0
del
5q14.3
3,463
MEF2C
path
dn
5
S
Q
3
1
2
6
Dy
Q
4
1
0
7
S
Q
5
2/1
2
del
Xp11.23p11.22
4,287
WDR45
path
dn
dup
Xq25
32
SPG34
bVOUS
dn
del
22q11.21
2,821
path
dn
path
Inh (P)
bVOUS
dn
DYNC1H1
path
dn
del
9p24.3
226
del
4q34.1
366
14q32.31q32.33
3,230
KANK1
8
C
Q
1
1
0
del
9
S
D
2
2
2
trip
Xq28
298
FLNA
path
Inh (M)
10
C
Q
2
1
1
del
14q12q21.2
11,150
NKX2-1
path
dn
del
3p26.3
521
bVOUS
dn
pVOUS
Inh (P)
Likely pathogenic variants of unknown significance (pVOUS) 11
Dy
Q
5
1
1
dup
18p11.21
445
12
S
Q
2
2/1
2
dup
2q13
862
pVOUS
Inh (M)
13
S
Q
1
2
2
del
7q31.1
152
pVOUS
dn
14
S
D
1
2
0
dup
20p12.3p12.2
679
pVOUS
Inh (P)
15
S
Q
4
2/1
2
del
1p21.3
154
pVOUS
Inh (P)
del
5p12p11
450
bVOUS
Inh (P)
del
11q11
289
bVOUS
Inh (P)
del
Yq11.223
273
bVOUS
Inh (P)
16
S
H
2
2/1
2
GNAL
dup
17p11.2
387
pVOUS
dn
del
Yq11.223
273
bVOUS
dn
del
18q22.2
45
bVOUS
dn
Likely benign variants of unknown significance (bVOUS) 17
S
D
2
2
0
dup
2q31.2
36
bVOUS
NT
18
S
Q
5
1
0
del
2q34
24
bVOUS
NT
del
Yq11.223
273
bVOUS
NT
19
S, Dy
Q
3
2/1
1
dup
16p13.11p12.3
549
bVOUS
NT
dup
7q36.1
272
bVOUS
NT
20
C
Q
4
2
0
del
18q22.3
330
bVOUS
Inh (M)
21
S
D
1
2/1
0
del
Xp21.1
1,083
bVOUS
NT
22
S
T
1
2
0
dup
9p13.2
594
bVOUS
NT
23
S
Q
5
2
2
del
4q28.3
947
bVOUS
Inh (M)
24
Dy
Q
3
2
1
dup
Xp11.22
53
bVOUS
Inh (P)
dup
Xq26.3
29
bVOUS
dn
dup
Xq27.3
22
bVOUS
dn
dup
Xq27.1
21
bVOUS
dn
dup
8q13.3
344
bVOUS
dn
del
10q26.3
143
bVOUS
Inh (M)
25
S
Q
3.5
2/1
2
Abbreviations: bVOUS 5 benign variants of unknown significance; C 5 chorea; CNV 5 copy number variation; CP 5 cerebral palsy; D 5 diplegia; Dy 5 dystonia; del 5 deletion; dn 5 de novo; dup 5 duplication; Dysmor 5 dysmorphic; GMFCS 5 Gross Motor Function Classification System; H 5 hemiplegia; Inh 5 inherited; M 5 maternal; Mov dis 5 movement disorder; NT 5 not tested; P 5 paternal; path 5 pathogenic; pVOUS 5 pathogenic variants of unknown significance; Q 5 quadriplegia; S 5 spasticity; T 5 triplegia.
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significant CNVs (p 5 0.01). Presence of a nonmotor morbidity (seizures, intellectual disability, psychiatric manifestations, visual impairment, or other systemic illness) was more common in individuals with clinically significant CNVs (1.8 6 1.9), compared with those without such CNVs (0.6 6 1.0; p 5 0.03). Intellectual disability and learning disability were more frequent in individuals with clinically significant CNVs: 81% and 13%, respectively, vs 50% and 8%, respectively, in individuals without such CNVs (p 5 0.03). There was no correlation between presence of clinically significant CNVs and other clinical features (table 2). DISCUSSION We found that one-third (31%) of children with cryptogenic CP have pathogenic or likely pathogenic CNVs similar to the rate in other movement disorders (28%).8 Frequency of CNVs is higher than that previously reported in CP (20%)24 and may be explained by our stringent definition of cryptogenic CP, which excluded identifiable acquired causes (e.g., infection). We realize that some of the participants who were excluded, mainly on the basis of prematurity and/or stroke, may also have an underlying genomic predisposing disorder. However, it is the current understanding that neurologic outcome of patients with these etiologies is multifactorial and relies only in part on their genetic predisposition.25 There were more deletions than duplications in the clinically significant group of CNVs, as expected, based on the current knowledge of pathogenic CNVs in other disorders26 such as attention-deficit/ hyperactivity disorder27 and epilepsy.28 CNV size was largest in the pathogenic group and can be attributable, in part, to the CNV analysis criteria. The majority of pathogenic CNVs were de novo and only 3 were inherited. Two inherited cases are explained by gender-specific effects: an X chromosome duplication in a male inherited from a healthy carrier mother, and a KANK1 deletion known to cause spastic quadriplegia only when paternally inherited. The third inherited case, a TSC1 deletion, was identified in a child that did not fulfill diagnostic criteria for TSC, whose mother was considered healthy but had seizures as a child. Of the likely pathogenic variants of unknown significance, only 2 were de novo and the rest were inherited. However, these inherited CNVs contained neurologic disease–related genes, and suspected pathogenicity was based on partial penetrance. We did not test all parents of participants with benign or likely benign CNVs because association between the CNVs and CP was unclear. Dysmorphic features and nonmotor comorbidities, especially intellectual disability, were common in individuals with clinically significant CNVs. We 1666
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found at least twice as many CNVs than reported in studies of specific nonmotor comorbidities.5–7 The rate of pathogenic CNVs was similar in all centers with different systems of referrals. Most participants with a family history of neurologic disorders had significant findings, but the clinical presentations and CNVs were different. For instance, 2 brothers with phenotypically distinctive CP (participants 2 and 3) had 2 different interstitial CNVs, which could not be explained by a balanced translocation in their parents, and indeed, the parents’ karyotypes were normal. Six pathogenic CNVs contained genes associated with the clinical CP phenotype: SPAST (hereditary spastic paraparesis),13 MEF2C (dystonia),14 WDR45 (spasticity),15 KANK1 (spastic quadriparesis),16 NKX2-1 (chorea),18 and tripXq28 (spasticity).17 Three aberrations were associated with atypical presentations of a known disorder (TSC1, 22q11 deletion, GNAL).19–22 Seven CNVs have not been previously reported to cause movement disorders in children, although some contained genes previously reported in other neurodisabilities (e.g., DYNC1H1).23 CNVs not previously implicated in movement disorders or CP may have a larger role in brain development than previously thought. Since genomic rearrangements include many genes, and the same participant may have had more than one CNV, it is possible that the phenotype is determined by the combination of more than one gene/region aberration. Further investigation into these genes is warranted to better characterize the function of these genes and regions in motor development. The limitation of the study is its small sample size; in addition, as genetic information is exponentially expanding, the significance of unknown variants may clarify with time with growing research. Based on the proportion of CNV findings in our study, CMA should be considered in all patients with cryptogenic CP as a first line of workup at diagnosis, saving many unnecessary tests. We were able to provide genetic counseling to all the families who were found to have genomic rearrangements. After testing the parents, if the CNVs were de novo, we assumed that recurrence rate in their next pregnancies would be low. When the CNVs were inherited, we offered prenatal diagnosis or preimplantation genetic diagnosis. We conclude that CNVs are common in individuals with cryptogenic CP. While they are particularly common in patients with dysmorphism or nonmotor comorbidities, they are also found in patients with cryptogenic CP without these features. We recommend CMA in individuals with CP of unknown cause to accelerate diagnosis, saving resources of both
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families and medical systems. Moreover, novel CNVs associated with CP may indicate new genes involved in neuropathology. AUTHOR CONTRIBUTIONS Reeval Segel conceptualized the study, received grants/funding, was in charge of collection of all DNA samples and clinical data, interpreted genetic results, wrote the first draft together with H.B.-P., and approved writing and revision of the manuscript. Hilla Ben-Pazi assisted in conceptualizing the study, collected the majority of DNA samples and clinical data, wrote the first draft with R.S., and approved final manuscript version. Sharon Zeligson and Shira Perlberg preformed the genetic testing and analysis. Varda Gross-Tsur assisted in conceptualizing the study, provided insight about the hypothesis, and revised the manuscript. Aviva Fatal-Valevski, Adi Aran, Nira Schneebaum-Sender, Dorit Shmueli, Dorit Lev, and Luba Blumkin recruited eligible candidates, collected DNA samples, examined the participants, recorded clinical data, and approved the manuscript. Lisa Deutsch analyzed the data, reviewed and edited manuscript drafts, approved final manuscript version. Ephrat LevyLahad initiated the study, assisted in conceptualizing the study and obtaining funding, directed the analysis, provided insight about the hypothesis, and assisted in the writing and revision of the manuscript.
ACKNOWLEDGMENT
6.
7.
8.
9.
10.
11.
The authors thank Mr. Michael Goldenshluger, Yael Grinberg, and Nava Badichi for their assistance in blood drawing. The authors thank the families for their cooperation and participation.
STUDY FUNDING
12.
Supported by grants: MOF Joint Israel Ministry of Health grant (3-6185), and the joint research fund of The Hebrew University and Shaare Zedek Medical Center.
DISCLOSURE R. Segel reports grant funding from the Chief Scientist, Israel Ministry of Health (3-6185), and the joint research fund of The Hebrew University and Shaare Zedek. H. Ben-Pazi, S. Zeligson, A. Fatal-Valevski, A. Aran, V. Gross-Tsur, N. Schneebaum-Sender, D. Shmueli, D. Lev, S. Perlberg, L. Blumkin, and L. Deutsch report no disclosures relevant to the manuscript. E. Levy-Lahad reports no funding for this particular project. Grants for other projects include funding from the Breast Cancer Research Foundation (NY), Israel Science Fund, and USAID–MERC (Middle East Regional Cooperation). Ethics committee approval: The study was approved by Shaare Zedek Medical Center’s internal review board and the National IRB (MOH 033/2008). Go to Neurology.org for full disclosures.
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Copy number variations in cryptogenic cerebral palsy Reeval Segel, Hilla Ben-Pazi, Sharon Zeligson, et al. Neurology 2015;84;1660-1668 Published Online before print March 27, 2015 DOI 10.1212/WNL.0000000000001494 This information is current as of March 27, 2015 Updated Information & Services
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