RESEARCH ARTICLE

Does Parent of Origin Matter? Methylation Studies

Should be Performed on Patients with Multiple
Copies of the Prader–Willi/Angelman
Syndrome Critical Region
Umut Aypar,1 Pamela R. Brodersen,1 Patrick A. Lundquist,2 D. Brian Dawson,2 Erik C. Thorland,1
and Nicole Hoppman1*
1
Cytogenetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
2
Molecular Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota

Manuscript Received: 31 July 2013; Manuscript Accepted: 22 May 2014

Deletion of 15q11.2-q13 results in either Prader–Willi syndrome

(PWS) or Angelman syndrome (AS) depending on the parent of How to Cite this Article:
origin. Duplication of the PWS/AS critical region (PWASCR) has Aypar U, Brodersen PR, Lundquist PA,
also been reported in association with developmental delay and Dawson DB, Thorland EC, Hoppman N.
autism, and it has been shown that they also show a parent-of- 2014. Does parent of origin matter?
origin effect. It is generally accepted that maternal duplications Methylation studies should be performed
are pathogenic. However, there is conflicting evidence as to the on patients with multiple copies of the
pathogenicity of paternal duplications. We have identified 35 Prader–Willi/Angelman syndrome critical
patients with gain of the PWASCR using array comparative region.
genomic hybridization. Methylation testing was performed to
Am J Med Genet Part A 9999:1–7.
determine parent of origin of the extra copies. Of the 35 cases, 22
had a supernumerary marker chromosome 15 (SMC15), 12 had a
tandem duplication, and 1 had a tandem triplication. Only one
patient had a paternal duplication; this patient does not have Prader–Willi/Angelman syndrome critical region (PWASCR). Ap-
features typical of patients with maternal duplication of the proximately 99% of PWS and 80% of AS cases are caused by large
PWASCR. Three of the mothers had a tandem duplication (two deletions, uniparental disomy for chromosome 15, or imprinting
were paternal and one was maternal origin). While one of the two center defects, respectively, while approximately 10% of AS is likely
mothers with paternal duplication was noted not to have autism, due to point mutations in UBE3A [Clayton-Smith and Laan, 2003;
the other was noted to have learning disability and depression. Goldstone, 2004; Kishino et al., 1997]. Duplications of the critical
Based on our data, we conclude that SMC15 are almost exclu- region, both intrachromosomal and due to supernumerary marker
sively maternal in origin and result in an abnormal phenotype. chromosomes derived from chromosome 15 (SMC15), have also
Tandem duplications/triplications are generally of maternal been described and are associated with intellectual disability,
origin when ascertained on the basis of abnormal phenotype; developmental delay, seizures, and autism [Battaglia et al.,
however, tandem duplications of paternal origin have also been 1997]. It has been suggested that like deletions, these duplications
identified. Therefore, we suggest that methylation testing be may also show a parent-of-origin effect. It is generally accepted that
performed for cases of tandem duplications/triplications since duplications of maternal origin are associated with this abnormal
the pathogenicity of paternal gains is uncertain. phenotype. However, there is conflicting evidence as to the patho-
Ó 2014 Wiley Periodicals, Inc. genicity of duplications of paternal origin. It is also known that the

Key words: 15q11.2-q13; PWASCR; autism; array CGH; MS- Conflict of interest: none.


MLPA Correspondence to:
Nicole Hoppman, Cytogenetics Laboratory, Department of Laboratory
Medicine and Pathology, Mayo Clinic, 971 Hilton, 200 1st Street SW,
INTRODUCTION Rochester, MN 55905.
E-mail: hoppman.nicole@mayo.edu
Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are Article first published online in Wiley Online Library
distinct disorders caused by lack of paternal (PWS) or maternal (wileyonlinelibrary.com): 00 Month 2014
(AS) expression of imprinted genes at 15q11-15q13, termed the DOI 10.1002/ajmg.a.36663

Ó 2014 Wiley Periodicals, Inc. 1

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

mothers of patients with interstitial duplications who also carry a Thermotron and baked for 10 min at 100˚C. Bacteria artificial
duplication often have it on their paternal chromosome and are chromosomes (BACs) located within the critical region, including
phenotypically normal [Cook et al., 1997]. RP11-16E12, D15Z1, SNRPN, D15S10, RP11-10K20, 289D12,
In order to further elucidate this question, we identified patients CTD-3061G5, CTD-2317M11, and RP11-160D9 (Invitrogen),
with additional copies of the PWASCR by array comparative were used for FISH confirmation. Briefly, each BAC was extracted
genomic hybridization (array CGH) and performed methyla- from E. coli using the Qiagen Plasmid Maxi kit according to the
tion-sensitive multiplex ligation-dependent probe amplification manufacturer’s instructions and was then labeled with Spectrum
(MS-MLPA) in order to determine the parent of origin of the extra Orange (Abbott Molecular) using the Nick Translation Kit (Abbott
copies. To our knowledge, this is one of the largest series of patients Molecular) according to the manufacturer’s instructions. A FISH
with multiple copies of PWASCR. The aim of this study is to help probe working solution was made by adding 3 ml of labeled BAC to
understand the potential parent of origin effect and develop a 6 ml of control probe (CEP15/D15Z1) and 7 ml of LSI/WCP1
testing/reporting algorithm when such duplications are identified. hybridization buffer (Abbott Molecular).
Slides were pre-treated according to standard cytogenetic pro-
tocols followed by application of 3 ml of probe working solution to
MATERIALS AND METHODS the hybridization site and a coverslip. Slides were denatured at 75˚C
Patients for 5 min and hybridized at 37˚C for 70 hr using a ThermoBriteTM.
A total of 35 cases with multiple copies of the PWASCR as detected The coverslip was removed and slides were washed with 0.4 SSC at
by array CGH were ascertained through the results database of the 72˚C for 2 min and rinsed in 0.1% NP-40/2 SSC for 1 min in the
Cytogenetics Laboratory at the Mayo Clinic. All cases were initially dark at room temperature. Ten microliters of counterstain (10%
detected by array CGH except for cases 4, 6, 10, 15, 22, and 23, which DAPI) was added to the hybridization area and a coverslip was then
were initially detected by conventional karyotyping and the array applied.
CGH was performed subsequently. The phenotypic information
was limited and only available from the reason for testing and G-Banding Confirmation
minimal phone conversation with the ordering physician at the
G-banding was performed according to standard cytogenetic meth-
time of testing. The results are presented on Table I and the absence
ods using trypsin and Leishman stain.
of a phenotypic trait could either be due to the patient not having
those symptoms or due to data not being available. Parental testing
was performed on only a subset of these cases through either or both MS-MLPA
FISH and array CGH testing. The mothers of cases 24, 28, and 30
MS-MLPA was performed using Salsa MS-MLPA Kit ME028
were found to be carriers of the tandem duplication. Limited
(MRC-Holland). Briefly, 200 ng of genomic DNA was denatured
phenotypic information was available for the mothers.
at 98˚C for 10 min. Probes were then hybridized at 60˚C for 16–
24 hr. Upon completion, each reaction was divided into two tubes
DNA Extraction and Array CGH of equal volume; the ligation reaction alone was performed in one
Genomic DNA extraction was performed using the PureGene tube while ligation and simultaneous digestion with HhaI was
method (Gentra) on total blood leukocytes. Array CGH was per- performed in the second, both at 49˚C for 30 min. Each reaction
then underwent PCR amplification using universal primers accord-
formed on three array platforms; the Signature Genomics BAC array
ing to the manufacturer’s protocol. Upon completion, 8.5 ml Hi-Di
as published in Bejjani et al. [2005] and the Agilent 44 k and 180 k
Formamide and 0.5 ml GS-500HD Rox size standard were added to
array. To perform the Agilent array CGH, 1 mg of patient genomic
1 ml of each reaction. This mixture was denatured at 95˚C for 5 min
DNA was labeled with Cy5 and an equal amount of gender-matched
control DNA was labeled with Cy3 using the Genomic DNA and placed on an ice-water slurry for an additional 5 min. Samples
Labeling Kit (Agilent) according to the manufacturer’s protocol. were then subjected to capillary electrophoresis on an ABI 3100
Each sample was purified using a Multiscreen PCR 96-well plate. (Applied Biosystems). MS-MLPA results were analyzed using
Equal volumes (19.5 ml) of both patient and control DNA were GeneMarker version 1.8 (SoftGenetics, LLC) in order to determine
copy number and methylation status of the PWASCR. The fluo-
mixed along with Cot-1 DNA and 2X hybe buffer (Agilent). The
rescent signals from the copy number probes are compared to
mixtures were denatured at 95˚C for 3 min and then incubated at
normal controls, resulting in ratios of 1.5 for duplications and 2.0
37˚C for 30 min. Each mixture was then applied to either an Agilent
for triplications. The methylation probes are maternally imprinted
4  44 K or 4  180 K oligonucleotide array CGH slide and incu-
bated while rotating at 65˚C for 24 hr. Slides were washed and (maternal allele methylated). Therefore, when compared to normal
scanned on a G2565CA Microarray Scanner System (Agilent). Data controls, the ratio of methylated probes will increase accordingly in
was analyzed using DNA Analytics version 4.0 (Agilent). the presence of additional copies of the maternal allele but not due
to the presence of additional paternal alleles.

FISH Confirmation
Peripheral blood lymphocytes were cultured in a T-25 flask for 72 hr RESULTS
in PB-Max plus excess thymidine and were harvested according to To date we have identified 35 patients with multiple copies of the
standard cytogenetic protocols. Metaphases were dropped in a PWASCR tested at the Mayo Clinic Cytogenetics laboratory.
AYPAR ET AL. 3

TABLE I. List of Cases With Additional Copies of the PWASCR Region Including the Nature of the Abnormality, Parent of Origin,
Inheritance, and Phenotype
Patient Age Gender Parent of origin Inheritance Array results Mechanism
1 39y F Maternal Unknown arrcgh 15q11.2q13.2(20,249,886-28,606,801)x idic(15)
4,15q13.2q13.3(28,742,018-30,686,850)x
3 [hg17]
2 15m F Maternal Unknown arrcgh 15q11.2q13.1(20,249,886-26,829,558)x idic(15)
4 [hg17]
3 3y M Maternal Unknown arrcgh 15q11.2q13.1(RP11-125E1->RP11-300A12)x4 idic(15)
4 10y M Maternal Unknown arr 15q11.2q13.1(20,249,886-26,829,558)x4 [hg18] idic(15)
5 24y M Maternal Unknown arr 15q11.2q13.2(20,316,992-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-30,494,175)x3 [hg18]
6 9y M Maternal Unknown arr 15q11.2q13.1(20,316,992-26,232,997)x4 [hg18] idic(15)
7 19m M Maternal Unknown arr 15q11.2q13.1(20,249,886-26,739,139)x4 [hg18] idic(15)
8 2y F Maternal Unknown arr 15q11.2q13.2(20,316,992-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-30,302,272)x3 [hg18]
9 23y F Maternal Unknown arr 15q11.2q13.2(20,249,886-28,441,228)x4 [hg18] idic(15)
10 19m M Maternal Unknown arr 15q11.2q13.2(20,249,886-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-30,686,850)x3 [hg18]
11 8m F Maternal Unknown arr 15q11.2q13.2(20,249,886-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-29,651,982)x3 [hg18]
12 3y M Maternal de novo arr 15q11.2q13.2(20,316,992-28,606,801)x idic(15)
4,15q13.2q13.3(28,709,209-30,648,918)x3 [hg18]
13 7y F Maternal Unknown arr 5p15.33p15.31(2,939,277-7,161,408)x idic(15)
1,15q11.2q13.2(20,316,992-28,441,228)x
4,15q13.2q13.3(28,517,805-30,648,918)x3 [hg18]
14 6y M Maternal Unknown arr 15q11.2q13.2(20,316,992-28,606,801)x idic(15)
4,15q13.2q13.3(28,709,209-30,648,918)x3 [hg18]
15 21m F Maternal Unknown arr 15q11.2q13.2(20,316,992-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-30,648,918)x3 [hg18]
16 6y M Maternal Unknown arrcgh 15q11.2q13.2(20,249,886-28,153,416)x idic(15)
4,15q13.2q13.3(28,441,169-30,226,235)x3
17 12y M Maternal Unknown arr 15q11.2q13.1(20,316,992-26,232,997)x3 [hg18] idic(15)
18 3m M Maternal Unknown arr 15q11.2q13.1(20,316,992-26,805,893)x4 [hg18] idic(15)
19 19m M Maternal Unknown arr 15q11.2q13.1(20,316,992-26,711,098)x idic(15)
4,15q13.1q13.2(26,739,080-28,865,125)x3 [hg18]
20 4y M Maternal Unknown arr 1p13.1p12(117,096,152-117,653,987)x idic(15)
3,15q11.2q13.2(20,316,992-28,606,801)x
4,15q13.2q13.3(28,709,209-30,648,918)x3 [hg18]
21 7y M Maternal Unknown arr 15q11.2q12(20,731,330-23,776,787)x ring(15)
3,15q11.2(22,084,310-22,583,016)x4 [hg18]
22 1d M Maternal Unknown arrcgh 15q11.2q12(20,249,886-24,878,968)x3 ring(15)
23 12y M Maternal Unknown arr 15q11.2q13.1(20,249,886-26,829,558)x4 [hg18] Tandem triplication
24 13y M Maternal Maternal arr 15q11.2q13.1(21,258,545-26,739,139)x Tandem duplication
3,16p11.2(29,581,455-30,098,069)x3 [hg18]
25 3y M Maternal Unknown arrcgh 15q11.2q13.1(21,208,377-26,193,908)x3 Tandem duplication
26 4y F Maternal de novo arrcgh 15q11.2q13.1(20,851,479-26,194,049)x3 Tandem duplication
27 16y F Maternal Unknown arr 15q11.2q13.1(20,316,992-26,884,937)x3 [hg18] Tandem duplication
28 20m M Maternal Maternal arr 15q11.2q13.1(20,249,886-26,884,937)x3 [hg18] Tandem duplication
29 10y M Maternal Unknown arr 15q11.2q13.1(20,851,679-26,193,908)x3 [hg18] Tandem duplication
30 3y M Maternal Maternal arr 2q33.1(199,889,249-200,158,689)x Tandem duplication
3 dn,15q11.2q13.1(21,258,545-26,193,908)x3 mat [hg18]
31 2y M Maternal de novo arr 1q21.1(144,500,220-147,076,487)x Likely tandem duplication
3 mat,15q11.2q13.1(20,316,992-26,199,055)x
3 dn,15q13.2q13.3(28,709,209-30,405,675)x2 [hg18]
32 18m M Maternal de novo arr 15q11.2q13.1(21,208,377-26,193,908)x3 [hg18] Tandem duplication
33 2y M Maternal Unknown arr 8q23.1(107,865,666-108,462,184)x Tandem duplication
3,15q11.2q13.1(20,316,992-26,805,893)x3 [hg18]
34 2y M Maternal Unknown arr 15q11.2q13.1(21,219,452-26,199,055)x Tandem duplication
3,18p11.31(5,829,720-6,151,830)x1 [hg18]
35 10y F Paternal Unknown arrcgh 15q11.2q13.1(20,249,686-26,739,280)x3 Tandem duplication
Mother of 24 33y F Paternal Unknown Only FISH was performed Tandem duplication
Mother of 28 20y F Paternal Unknown Only FISH was performed Tandem duplication
Mother of 30 25y F Maternal Unknown arr 2q33.1(199,889,249-200,158,689)x Tandem duplication
2,15q11.2q13.1(21,258,545-26,193,908)x3 [hg18]

Figure 1A,E show representative duplications detected by array also be reliably detected by array CGH (Fig. 2A,F); however, FISH
CGH; these are interstitial duplications since only two signals were and chromosomes are necessary to distinguish between interstitial
observed by metaphase FISH (Fig. 1B,F, left) and three were triplications (Fig. 2B,C) and SMC15 (Fig. 2G,H). Because of the size
observed by interphase FISH (Fig. 1B,F, right). Triplications can and the proximity of the duplications and the triplications, only
4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

FIG. 1. Array CGH, metaphase FISH, interphase FISH, and MS-MLPA results of cases with (A–D) maternal interstitial duplication (Patient 29)
and (E–H) paternal interstitial duplication (Patient 35). Array CGH results are plotted on log 2 scale on which the duplication plots to þ0.58.
Peak ratios are determined by comparing patients with normal control (duplication 3 copies/2 copies ¼ 1.5 (C and G)). Methylation probes are
maternally imprinted, therefore, 2 maternal copies (in a patient with maternal duplication)/1 maternal copy in a normal control ¼ 2 (D); 1
maternal copy (in a patient with paternal duplication)/1 maternal copy in a normal control ¼ 1 (H).
AYPAR ET AL. 5

interphase FISH can detect these interstitial duplications and stitial duplications present with autism spectrum disorder, hypo-
triplications. tonia, developmental delay, intellectual disability, seizures, and
MS-MLPA was subsequently performed to confirm the number infrequently dysmorphic features [reviewed in Hogart
of copies detected by array CGH as well as determine the methyla- et al., 2010]. In this study, 11 out of 35 patients had tandem
tion pattern of the multiple copies, which indicates parent of origin. duplications of maternal origin. These patients in general had
It is important to determine parent of origin of the additional copies similar phenotypes as those reported in the literature including
rather than inheritance since majority of these cases are de novo and developmental delay, intellectual disability, dysmorphic features,
an inherited additional copy from one parent could potentially have and autism.
a methylation pattern consistent with inheritance from the other Supernumerary isodicentric chromosome 15 and tandem trip-
parent. The duplication and triplication of PWASCR observed on lications of maternal origin have been reported in patients with
Figure 1A,E and Figure 2A,F on array CGH were confirmed by MS- similar clinical features as those with maternal interstitial dupli-
MLPA (Figs. 1C,G and 2D,I, respectively). One of the duplications cations, but at a greater degree of severity, which include hypoto-
had an additional copy of the PWASCR that was not methylated, as nia, dysmorphic features, intellectual disability, developmental
demonstrated by a ratio of 1, suggesting a paternal origin (Fig. 1H), delay, seizures and autism spectrum disorder [reviewed in Hogart
while the triplications with two additional copies of the PWASCR et al., 2010]. In our patient population, 23 out of 35 had tripli-
had a methylation ratio approaching 3, indicating maternal origin cations of this region, either due to supernumerary markers or a
of the interstitial triplication and the SMC15 (Fig. 2E,J). tandem triplication, and they tended to have a more severe
Of the 35 cases, 22 had a supernumerary marker chromosome phenotype compared to tandem duplications. These cases make
(20 cases with isodicentric markers and two cases with rings) and 13 up the majority of the cases with developmental delay, intellectual
had an interstitial duplication or triplication (12 cases with dupli- disability, seizures/infantile spasms, dysmorphic features, autism,
cations, one case with a triplication). Confirmation FISH and/or and hypotonia.
chromosome studies were not possible for one patient due to lack of Only one of the 35 patients and two of the three mothers had a
specimen; however since this patient has only one extra copy this tandem duplication with a paternal methylation pattern (Fig. 1H).
patient most likely has a tandem duplication (Patient 31; Table I). Paternally derived duplications have been reported in patients who
MS-MLPA demonstrated that all of the supernumerary marker are unaffected as well as patients who are mildly affected with a
chromosomes and all but one of the interstitial gains were of range of features including autism spectrum disorders, develop-
maternal origin maternally. Patients with maternally derived inter- mental delay, moderate intellectual disability, and behavioral prob-
stitial duplications had phenotypic features such as developmental lems [reviewed in Hogart et al., 2010]. Our patient had a reason for
delay, intellectual disability, dysmorphic features, and autism referral of attention deficit disorder and short stature, with no
(Table I). Furthermore, 23 of the 35 had triplication of this region mention of the typical features of patients with maternal duplica-
due to supernumerary markers and a tandem triplication, which tion of the PWASCR. Even though extensive phenotypic informa-
showed a more severe phenotype compared to tandem duplica- tion was not available for the parents, it was noted that one of the
tions. These cases had features such as developmental delay, mothers did not have autism, however, the other had learning
intellectual disability, seizures/infantile spasms, dysmorphic fea- disability and depression. While the phenotype of our patient and at
tures, autism, hypotonia, and other features such as visual im- least one of the mothers supports the hypothesis that the duplica-
pairment, dyskinesia, growth delay, finger deformity, and lack of tions of paternal origin result in a milder phenotype, the discrep-
coordination (Table I). ancies in the phenotypes reported and the small number of these
Only one patient with tandem duplication had a paternal patients both in the literature and in this study makes it difficult to
methylation pattern. This patient had a reason for referral of make a strong conclusion. However, the small number of cases
attention deficit disorder and short stature, with no mention of reported may suggest an ascertainment bias as the unaffected and
the typical features of patients with maternal duplication of the even mildly affected patients with paternal copies of PWASCR are
PWASCR. less likely to be seen in the clinic. Finally, it is possible that in some
Of the three mothers, two had a duplication of paternal origin. patients, the extra paternal copies of the PWASCR are not causing
While one of the two mothers was noted not to have autism, the their phenotypic abnormalities and are just incidental findings.
other was noted to have learning disability and depression. Inter- Supernumerary marker chromosomes containing the PWASCR
estingly, one of the mothers had a duplication of maternal origin; are almost always of maternal origin due to the meiotic errors
however, no phenotype information was available. occurring in the maternal germline with an increased rate due to
advanced maternal age [Van Dyke et al., 1987]. In our study, the
SMC 15s were exclusively maternal in origin and resulted in an
DISCUSSION abnormal phenotype. There have been only three cases reported in
To our knowledge, this is one of the largest retrospective studies to the literature with a paternally derived supernumerary marker
date of patients with multiple copies of the PWASCR. The dupli- chromosome 15, however, these patients had maternal UPD of
cations and triplications observed in these 35 patients and 3 chromosome 15 resulting in PWS [Baumer et al., 2001; Werner
mothers represent both intrachromosomal gains and SMC15s. et al., 2004; Saitoh et al., 2007]. Therefore, in these cases the
The phenotypes associated with multiple copies of the PWASCR expression of the imprinted genes from the paternally derived
of maternal origin in our patient cohort are in accordance with marker chromosome likely resulted in a partial rescue of the
those previously reported. Patients with maternally derived inter- PWS phenotype.
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

FIG. 2. Array CGH, metaphase FISH, interphase FISH, chromosome, and MS-MLPA results of cases with (A–E) interstitial triplication (Patient
23) and (F–J) supernumerary isodicentric chromosome 15 (Patient 4). Array CGH results are plotted on log 2 scale on which the triplication
plots to þ1 (A and F). Peak ratios are determined by comparing patients with normal control (triplication 4 copies/2 copies ¼ 2 (D and I)).
Methylation probes are maternally imprinted, therefore, 3 maternal copies in a patient with maternal triplication/1 maternal copy in a normal
control ¼ 3 (E and J).
AYPAR ET AL. 7

are present in normal individuals and, if so, will help determine the
pathogenicity of these rearrangements.

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