MUTATION UPDATE OFFICIAL JOURNAL

Mutation Update for UBE3A Variants in Angelman Syndrome www.hgvs.org

Bekim Sadikovic,1 Priscilla Fernandes,2 Victor Wei Zhang,3 Patricia A. Ward,3 Irene Miloslavskaya,3 William Rhead,4 Richard Rosenbaum,5 Robert Gin,5 Benjamin Roa,2 and Ping Fang3∗ 1

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; 2 Myriad Genetics, Salt Lake City, Utah; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas; 4 Medical College of Milwaukee, Pediatrics and Pathology Milwaukee, Milwaukee, Wisconsin; 5 Littleton Pediatric Medical Center, Highlands Ranch, Colorado

3

Communicated by Nancy Spinner Received 28 February 2014; accepted revised manuscript 25 August 2014. Published online 11 September 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22687

ABSTRACT: Angelman syndrome is a neurodevelopmental disorder caused by a deficiency of the imprinted and maternally expressed UBE3A gene. Although de novo genetic and epigenetic imprinting defects of UBE3A genomic locus account for majority of Angelman diagnoses, approximately 10% of individuals affected with Angelman syndrome are a result of UBE3A loss-of-function mutations occurring on the expressed maternal chromosome. The variants described in this manuscript represent the analysis of 2,515 patients referred for UBE3A gene sequencing at our institution, along with a comprehensive review of the UBE3A mutation literature. Of these, 267 (10.62%) patients had a report issued for detection of a UBE3A gene nucleotide variant, which in many cases involved family studies resulting in reclassification of variants of unknown clinical significance (VUS). Overall, 111 (4.41%) probands had a nucleotide change classified as pathogenic or strongly favored to be pathogenic, 29 (1.15%) had a VUS, and 126 (5.0%) had a nucleotide change classified as benign or strongly favored to be benign. All variants and their clinical interpretations are submitted to NCBI ClinVar, a freely accessible human variation and phenotype database. C 2014 Wiley Periodicals, Inc. Hum Mutat 35:1407–1417, 2014. 

KEY WORDS: UBE3A; Angelman syndrome; DNA sequencing; genetic testing

Background Angelman syndrome is a unique neurodevelopmental disorder caused by the deficiency of an imprinted, maternally expressed gene called E6-AP ubiquitin-protein ligase (UBE3A; MIM #601623). This syndrome was initially described by Dr. Harry Angelman, an English pediatrician in 1965, in three children with distinct abnormalities in physical and mental development, and characteristic behavioral features [Angelman, 1965]. These features include severe developmental delay, speech impairment, gait ataxia and/or tremulousness of the limbs, hypopigmentation, dysmorphic facial features, flat Additional Supporting Information may be found in the online version of this article. ∗

Correspondence to: Ping Fang, Baylor College of Medicine, Human and Molecular

Genetics, Houston, TX. E-mail: [email protected]

occiput, microcephaly, seizures and EEG abnormalities, and a distinctive happy demeanor that includes frequent laughing, smiling, and excitability [Angelman, 1965; Clayton-Smith and Laan, 2003; Williams et al., 2006; Van Buggenhout and Fryns, 2009]. The incidence of Angelman Syndrome is estimated to be between 1/12,000 and 1/20,000, with most cases being sporadic, although familial occurrence is not uncommon [Clayton-Smith and Pembrey, 1992; Petersen et al., 1995; Steffenburg et al., 1996]. The defective chromosomal region in Angelman Syndrome maps to the proximal region of the long arm of chromosome 15, specifically 15q11.2-q13. The UBE3A gene lies within this region and is now recognized as the “Angelman syndrome” gene, based on the findings of loss of function mutations of this gene in affected patients [Kishino et al., 1997; Matsuura et al., 1997]. UBE3A is part of an imprinted region where expression of the paternally inherited allele is suppressed, whereas maternal allele is expressed in the human brain [Rougeulle et al., 1997; Vu and Hoffman, 1997] and in mouse hippocampus and cerebellum [Albrecht et al., 1997; Jiang et al., 1998]. The UBE3A gene spans 120 kb and consists of 16 exons. The open reading frame includes the last two nucleotides of exon 7 and exons 8–16 [Yamamoto et al., 1997; Kishino and Wagstaff, 1998] (exons 1–10; NM 130838). The UBE3A gene encodes a class of E3 ubiquitin-protein ligase of 865 amino acids with a C-terminal HECT (homologous to the E6-AP carboxyl terminus) domain of 350 amino acids. The C-terminal domain of the protein is essential for transferring the ubiquitin to proteins targeted for degradation by the ubiquitin-proteasome system, thus playing a role in cellular protein homeostasis [Huibregtse et al., 1995]. UBE3A is also shown to act as a transcriptional coactivator of steroid hormone receptors [Nawaz et al., 1999; Smith et al., 2002; Khan et al., 2006]. Through its interaction with a number of cellular proteins, UBE3A is involved in multiple cellular functions including cell cycle regulation [Kumar et al., 1999; Oda et al., 1999; Mishra and Jana, 2008; Louria-Hayon et al., 2009; Mishra et al., 2009], synaptic function, and synaptic plasticity [Reiter et al., 2006; Greer et al., 2010; Margolis et al., 2010; Kaphzan et al., 2011; Mabb et al., 2011; Kaphzan et al., 2012]. UBE3A is expressed from both paternal and maternal alleles of chromosome 15 in non-CNS tissues of normal individuals, whereas in CNS tissues, the paternal allele of UBE3A is silenced through genomic imprinting. This results in the expression of only the maternal UBE3A allele in neurons from normal individuals. The underlying defect in Angelman syndrome is the absence or inactivation of the maternal UBE3A allele, resulting in loss of expression of the UBE3A protein in the CNS tissues.  C

2014 WILEY PERIODICALS, INC.

There are multiple molecular mechanisms by which UBE3A can be disrupted including: (1) Deletions of the 15q11.2-q13 region, containing UBE3A, which account for 65%–70% of the cases. These deletions commonly span approximately 5–7 Mb and are commonly caused by a combination of three chromosomal breakpoints (two alternate proximal BP1 or BP2 and one distal BP3) [Knoll et al., 1990; Amos-Landgraf et al., 1999; Christian et al., 1999]. (2) Paternal uniparental disomy of chromosome 15, wherein one inherits both chromosomes 15 from the father. This mechanism accounts for 3%–7% of the cases and is most likely to be postzygotic in origin [Robinson et al., 2000]. (3) Imprinting center defects resulting from either genetic (small deletions) or epigenetic (disruption of characteristic imprinting pattern of DNA methylation) defects in the imprinting center within the 15q11.2-q13 region, which are seen in 2%–5% of patients. (4) Lastly, loss of function mutations in the maternal UBE3A allele, which account for 10% of patients [Kishino et al., 1997; Matsuura et al., 1997; Kishino and Wagstaff, 1998; Malzac et al., 1998; Lossie et al., 2001]. Approximately 10% of patients that meet the clinical criteria for Angelman syndrome have no identified molecular defect.

Variants The variants described in this manuscript represent an up to date UBE3A mutation database that summarizes published as well as variations identified from 2,515 patients referred for UBE3A gene sequencing at our institution. Of the 2,515 patients, 267 (10.62%) patients had a report issued for detection of a nucleotide variant. These variants are summarized as pathogenic, likely pathogenic, (Tables 1 and 2), variants of unknown clinical significance (VUS) (Table 2) or benign (Supp. Table S1). Overall, 111 (4.41%) probands had a variant classified as pathogenic or strongly favored to be pathogenic, 29 (1.15%) had a VUS, and 126 (5.0%) had a variant classified as benign or strongly favored to be benign. The 4.4% pathogenic/favored pathogenic rate is lower than the classically quoted 10% UBE3A pathogenic variant detection rate in Angelman patients. The main reason for this discrepancy is that this patient cohort includes patients referred for testing and that were suspected to have Angelman based on the clinical presentation, and who in fact may not have Angelman syndrome. This is normally preceded by the normal Angelman methylation test. Many of the patients may fit certain aspects of Angelman diagnosis, and would get UBE3A testing as part of a rule-out involving a more broad genetics work up. We report 73 different pathogenic variants classified as either previously published pathogenic variants [Kishino et al., 1997; Matsuura et al., 1997; Fung et al., 1998; Malzac et al., 1998; Tsai et al., 1998; Baumer et al., 1999; Fang et al., 1999; Laan et al., 1999; Russo et al., 2000; Lossie et al., 2001; Monzon et al., 2003; Hitchins et al., 2004; Molfetta et al., 2004; Rapakko et al., 2004; Espay et al., 2005; Hosoki et al., 2005; Asahina et al., 2008; Mueller and Coovadia, 2008; Sartori et al., 2008; Camprubi et al., 2009; Tzagkaraki et al., 2009; Abaied et al., 2010; Bai et al., 2011; Horsthemke et al., 2011; Al-Maawali et al., 2013; Bai et al., 2014], novel truncating mutations, or other novel splice-site and translational error variants (Table 1). Thirteen of these variants were previously published. The frameshift and nonsense variant group includes 55 previously unpublished variants, which are predicted to result in a premature stop codon. Thirty six of these variants are a result of small insertions and deletions, and 19 are single nucleotide substitutions. Novel splicesite and translation error variants which include three variants at the consensus intronic splice sites are c.302–2A>T, c.1694–2A>G, and c.2065–2A>C. These are predicted to affect the mRNA processing

1408

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

of the UBE3A gene. Two other variants in this category include a variant in the start codon and a variant in the stop codon. Both are predicted to disrupt translation and are classified as pathogenic variants. Forty four novel variants were identified which were interpreted as VUS (Table 2). Parental analysis was offered to all the cases wherein VUS were identified in the probands, and in some cases, study of additional family members was recommended. Additional family studies in 31 families allowed us to further reclassify 29 variants as either de novo, family studies support as likely pathogenic variants and maternally inherited variants. Fifteen VUS remain unclassified because of no additional information. De novo missense variants in the affected probands are determined through the analysis of both parents with the assumption that DNA samples were obtained from the biological parents and interpreted as likely pathogenic variants. We detected nine different nonsynonymous variants including c.635A>T (p.D212V), c.710T>C (p.L237P), c.788T>G (p.L263W), c.1373C>T (p.P458L), c.1633G>A (p.G545R), c.1750G>C (p.E584Q), c.1967C>T (p.T656I), c.2069T>G (p.F690C), and c.2480C>T (p.P827L), two in-frame single amino acid deletions c.2406 2408del (p.M802del) and c.1745 1747del (p.S582del), one in-frame deletion of 5 amino acids c.1365 1379del (p.M455 F459del), and one in-frame 7 amino acid duplication c.2487 2507dup (p.L835 K836insNSSKEKL). Furthermore, in silico analysis of the nonsynonymous VUS, using the variant prediction software SIFT [Kumar et al., 2009] and PolyPhen2 [Adzhubei et al., 2010] showed concordant results for all nine nonsynonymous variants as damaging (Supp. Table S2). Interpretation and reclassification of a VUS often requires that multiple family members be tested. This can result in reclassification of a variant to either benign variant or pathogenic, and sometimes can provide evidence favorable but not conclusive for either interpretation. Here we report five such variants: c.317C>A (p.T106K), c.710T>A (p.L237H), c.1304T>C (p.L435P), c.1430G>C (p.R477P), and c.1697T>A (p.M566K) (Table 2). Four out of five probands have documented classic AS phenotype in their records (Supp. Table S3). The nonsynonymous c.317C>A (p.T106K) variant was maternally inherited in the proband, proband’s sister, and in two affected maternal cousins. Furthermore, an allelic amino acid change (p.T106P) has previously been described as a pathogenic variant [Rapakko et al., 2004]. Variant prediction analysis gives discordant predictions for this variant, with SIFT predicting it to be tolerated and PolyPhen2 calling it possibly damaging (Supp. Table S2). Based on cumulative evidence, the c.317C>A is currently interpreted as a likely pathogenic variant. The c.710T>A is a nonsynonymous variant transmitted from the maternal grandfather. Both SIFT and PolyPhen2 predict this variant to be damaging (Supp. Table S2). Therefore, we currently interpret c.710T>A as a VUS but possibly pathogenic. Family studies for the variant c.1304T>C resulting in a missense substitution of leucine to proline at the 435th amino acid strongly favor it to be pathogenic. There is evidence of the maternal transmission of this variant to the three affected siblings, whereas four unaffected siblings do not carry this variant. SIFT and PolyPhen2 predict it to be damaging as well. The c.1304C>T (p.L435P) variant was not detected in the maternal grandfather’s DNA. Although we were unable to test the maternal grandmother, the fact that the variant allele segregates with the disease phenotype along with the in silico prediction data suggest that the c.1304C>T (p. L435P) variant is likely a pathogenic variant, originating de novo in the carrier mother’s paternal chromosome or inherited from the maternal grandfather’s germ line. Variant c.1430G>C (p.R477P) was detected in a patient with the classic AS phenotype. This variant was maternally inherited from the grandfather, while the proband’s unaffected maternal half

Table 1. UBE3A Pathogenic Variants Nucleotide c.2T>C c.59G>T c.59 60insA c.62G>A c.99delC c.176 177insGA c.199 202dup c.263 264del c.270delG c.275dupA c.277 280del c.302–2A>T c.311dupA c.312 315del c.316A>C c.323 327delAGAAG c.324delG c.325A>T c.362 363delAG c.389T>C c.396 397delAG c.403dup c.418 419insG c.499G>T c.547del c.575 578dupGTGA c.580dup c.688G>T c.711dupT c.717T>A c.750T>A c.875 888del14 c.914G>A c.934C>T c.936delG c.961C>T c.966delA c.972 978del c.974 980delCTTATAA c.986T>C c.1006C>T c.1045T>C c.1067dup c.1076dup c.1110 1113delTGAA c.1114G>T c.1199C>A c.1201C>T c.1249C>T c.1270G>T c.1285G>T c.1337 1344delTAGAAACA c.1347 1348delGA c.1355dup c.1361 1362del c.1371T>A c.1379delT c.1387del c.1412 1416delATTAT c.1444C>T c.1447A>T c.1451 1460del10 c.1505 1506del c.1508 1513delAAGTTA c.1516C>T c.1537dup c.1549–8A>G c.1559 1560delTC c.1571dup c.1599T>G

Amino acid p.M1? p.G20V p.C21fs p.C21Y p.C34fs p.C60fs p.G68fs p.I88fs p.M90fs p.K93fs p.K93fs p.? p.Y104∗ p.Y104∗ p.T106P p.E108fs p.K109fs p.K109∗ p.E121fs p.I130T p.R132fs p.S135fs p.L140fs p.E167∗ p.D183fs p.D193fs p.S194fs p.E230∗ p.V238fs p.Y239∗ p.Y250∗ p.K292fs p.W305∗ p.Q312∗ p.I313fs p.Q321∗ p.Q322fs p.T325fs p.T325fs p.I329T p.R336∗ p.S349P p.Y356fs p.N359fs p.N370fs p.E372∗ p.P400H p.R401∗ p.R417∗ p.E424∗ p.E429∗ p.V446fs p.N450fs p.S453fs p.F454fs p.C457∗ p.I460fs p.A463fs p.Y471∗ p.R482∗ p.R483∗ p.I484fs p.L502fs p.K503 V504del p.R506C p.A513fs p.? p.I520fs p.N524fs p.Y533∗

# Observed in this study 1

1 1 1 1 1 1 2

1 1 2

1 1 1 1 1 1

1 1

2 1 1 1 2 1 1 1

1 1 1 1 1 4

1 2 1 2 1

Classification

References

Start codon Missense Frameshift Missense Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Consensus splice site Nonsense Nonsense Missense Frameshift Frameshift Nonsense Frameshift Missense Frameshift Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Nonsense Frameshift Nonsense Nonsense Frameshift Nonsense Nonsense Frameshift Nonsense Frameshift Frameshift Frameshift Missense Nonsense Missense Frameshift Frameshift Frameshift Nonsense Missense Nonsense Nonsense Nonsense Nonsense Frameshift Frameshift Frameshift Frameshift Nonsense Frameshift Frameshift Nonsense Nonsense Nonsense Frameshift Frameshift In-frame deletion Missense Frameshift Splicing Frameshift Frameshift Nonsense

This study Mueller and Coovadia, 2008 Malzac et al., 1998 Matsuura et al., 1997 This study Baumer et al., 1999 This study This study Fang et al., 1999 This study This study This study Russo et al., 2000 This study Rapakko et al., 2004 Fang et al., 1999 Asahina et al., 2008 Camprubi et al., 2009 Laan et al., 1999 Rapakko et al., 2004 Fang et al., 1999 This study Tzagkaraki et al., 2009 Russo et al., 2000 This study Hitchins et al., 2004 This study This study Baumer et al., 1999 This study This study Malzac et al., 1998 Fang et al., 1999 Camprubi et al., 2009 Malzac et al., 1998 This study Fang et al., 1999 This study Fang et al., 1999 Camprubi et al., 2009 Camprubi et al., 2009 Malzac et al., 1998 This study This study Fang et al., 1999 This study Bai et al., 2011 This study Matsuura et al., 1997 This study This study Camprubi et al., 2009 Matsuura et al., 1997 This study This study This study Hitchins et al., 2004 This study Hitchins et al., 2004 Malzac et al., 1998 Lossie et al., 2001 Malzac et al., 1998 This study Monzon et al., 2003 Baumer et al., 1999 This study Kishino et al., 1997 Runte et al., 2004 This study Fang et al., 1999 (Continued)

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

1409

Table 1. Continued Nucleotide

Amino acid

c.1608dup c.1613 1615delGAG c.1634 1637dupGAGG c.1639 1642dup c.1665 1666insAACTA c.1688A>G c.1693+1G>A c.1694–2A>G c.1730G>A c.1790delG c.1811 1812delGT c.1814 1824del c.1893 1894delTC c.1912 1913del c.1943dupA c.1956 1963del c.1957dup c.1958 1959insA c.1972C>T c.1972del c.1985 1988delCAGA c.1985 1988dupCAGA c.2065–2A>C c.2102 2103insTATT c.2170 2174dup c.2177T>A c.2186delC c.2233C>T c.2245G>T c.2245delG c.2247 2251dup c.2270 2281del12 c.2289dup c.2295–2A>G c.2304G>A c.2337 2340dupAAGA c.2344 2345del c.2346 2348delCTT c.2370 2373del c.2409 2411dupTAT c.2411T>A c.2441dupT c.2448dupA c.2463 2521dup c.2474T>A c.2478del c.2487 2554del c.2487 2490dupCTCA c.2489C>G c.2497 2500dup c.2503 2506dup c.2503 2507dupCTTAA c.2507 2508del c.2507 2510delAAGA c.2508delA c.2521 2536dup16 c.2524 2530delGCCATCAinsAGATGTT c.2556 ∗ 11del15 c.2556 ∗ 6del10 c.2558A>T

p.E537∗ p.G538del p.V547fs p.S548fs p.V556fs p.D563G p.? p.? p.W577∗ p.G597fs p.C604fs p.I605fs p.H632fs p.S638fs p.N648fs p.M653fs p.M653fs p.M653fs p.Q658∗ p.Q658fs p.T662fs p.L664fs p.? p.K701fs p.Y725∗ p.L726∗ p.P729fs p.Q745∗ p.E749∗ p.E749fs p.T751fs p.Y757 D760del p.I764fs p.? p.W768∗ p.L781fs p.F782fs p.F782del p.D790fs p.I804dup p.I804K p.L814fs p.S817fs p.K841fs p.L825∗ p.P827fs p.S830fs p.S831fs p.S830∗ p.K834fs p.K836fs p.K836fs p.K836fs p.K836fs p.E837fs p.A846fs p.A842 T844delinsRCS p.∗ 853fs p.∗ 853fs p.X853Lext

# Observed in this study 1

1

1 1

1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1

1 3 1

1 1 1 1 1 1 1 4 2 3

3 1

Classification

References

Nonsense In-frame deletion Frameshift Frameshift Frameshift Missense Splicing Consensus splice site Nonsense Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Consensus splice site Frameshift Nonsense Nonsense Frameshift Nonsense Nonsense Frameshift Frameshift In-frame deletion Frameshift Splicing Nonsense Frameshift Frameshift In-frame deletion Frameshift In-frame duplication Missense Frameshift Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Nonsense Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift In-del Frameshift Frameshift Stop codon loss

This study Horsthemke et al., 2011 Molfetta et al., 2004 This study Espay et al., 2005 Bai et al., 2014 Sartori et al., 2008 This study This study Baumer et al., 1999 Mueller and Coovadia, 2008 This study Camprubi et al., 2009 This study Malzac et al., 1998 This study This study Russo et al., 2000 This study This study Runte et al., 2004 Fang et al., 1999 This study This study This study This study This study This study This study This study This study Hitchins et al., 2004 This study Hosoki et al., 2005 Tsai et al., 1998 Camprubi et al., 2009 This study Fang et al., 1999 This study Malzac et al., 1998 Fang et al., 1999 Laan et al., 1999 Malzac et al., 1998 This study This study This study This study Laan et al., 1999 This study This study This study Kishino et al., 1997 This study Fang et al., 1999 Lossie et al., 2001 Baumer et al., 1999 Abaied et al., 2010 Fang et al., 1999 Al-Maawali et al., 2013 This study

Nucleotide numbering for the UBE3A (NM_130838.1) variant uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. All novel pathogenic variants that were identified in this study have a number of occurrences specified in the “#” column. All previously published pathogenic variants have a citation reference indicated.

sibling was negative for this variant. Variant c.1697T>A (p.M566K) showed maternal inheritance with allelic conversion (see discussion later). Both c.1430G>C (p.R477P) and c.1697T>A (p.M566K) were predicted to be damaging (Supp. Table S2), and are interpreted as VUS but likely pathogenic.

1410

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

We detected eleven new maternally inherited variants that are likely benign or remain classified as VUS. Maternal transmission of a VUS, particularly one affecting the amino acid sequence may be clinically significant. Four such variants that were detected in the affected probands include missense changes c.349T>C (p.C117R),

Table 2. Likely Pathogenic Variants and Variants of Unknown Clinical Significance Nucleotide

Amino acid

SIFT/PolyPhen2

#

Classification info and refs

∗

De novo c.635A>T c.710T>C c.788T>G c.1373C>T c.1365 1379del c.1633G>A c.1745 1747del c.1750G>C c.1967C>T c.2069T>G c.2406 2408del c.2480C>T c.2487 2507dup

p.D212V p.L237P p.L263W p.P458L p.M455 F459del p.G545R p.S582del p.E584Q p.T656I p.F690C p.M802del p.P827L p.L835 K836ins7

Damaging Damaging Damaging Damaging NA Damaging NA Damaging Damaging Damaging NA Damaging NA See Fig. 2

1 1 1 1 1 1 1 1 1 1 1 2 1

De novo De novo De novo De novo De novo De novo De novo De novo De novo De novo De novo De novo and W.O.A.I De novo

Family studies (multiple members), likely pathogenic c.317C>A

p.T106K

Discordant

1

c.710T>A

p.L237H

Damaging

1

c.1304T>C

p.L435P

Damaging

1

c.1430G>C

p.R477P

Damaging

1

c.1697T>A

p.M566K

Damaging

1

Positive: mother, affected sister and affected maternal cousins; p.I106P has been described Positive: mother and maternal grandfather De novo p.L237P found in one patient Positive: mother and two affected siblings Negative: four unaffected siblings and maternal grandfather Positive: mother and maternal grandfather Negative: unaffected maternal half sibling Positive: mother and unaffected sibling; allelic changes in the family

NA

1

Maternally inherited, likely benign or remains as VUS c.2+20G>A c.349T>C

p.C117R

Discordant

2

c.809A>C

p.N270T

Benign

1

c.1629 1631del

p.D543del

NA

1

NA Benign NA NA NA NA NA

1 1 1 1 1 2 1

NA NA Damaging NA Benign NA

1 1 1 1 1 1

NA Discordant NA NA Discordant Discordant Damaging NA NA

2 1 1 1 1 1 1 1 1

c.2221–42T>C c.2284G>A c.2294+20A>G c.2355T>C c.2439–31T>G c.2439–37dup c.2439–40C>T Without additional information/insufficient information c.301+29T>C c.301+30G>A c.398T>G c.558A>T c.1004G>C c.1119T>C c.1209C>T c.1434G>A c.1548+34T>C c.1693+32G>A c.1762C>G c.1763A>C c.2018T>G c.2064+9T>C c.2355T>C

p.V762I p.F785F

p.V133G p.A186A p.S335T p.D373D p.D403D p.M478I

p.Q588E p.Q588P p.L673R p.F785F

Positive: mother Negative: two unaffected siblings Positive: mother Negative: one unaffected sibling second family W.O.A.I Positive: mother and one affected sibling Negative: one affected sibling and maternal grandparents Positive: mother Negative: both maternal grandparents Positive: mother Positive: maternal half sibling Positive: mother Positive: mother Positive: mother Positive: mother (one family) Positive: mother W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. Negative: mother Father not tested W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I. W.O.A.I.

Nucleotide numbering for the UBE3A (NM_130838.1) variant uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. ∗ The interpretation of de novo changes are based on the assumption that parental samples are collected from the probands’ biological parents and germline mosaicism cannot be ruled out. W.O.A.I: without additional information.

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

1411

c.809A>C (p.N270T), c.2284G>A (p.V762I), and an in-frame deletion c.1629 1631del (p.D543del) (Table 2). In silico analysis gives discordant results for c.349T>C (p.C117R), tolerated by SIFT and probably damaging by PolyPhen2 (Supp. Table S2); family study results could not facilitate the interpretation and there was not phenotype information for either index case (Supp. Table S3). The c.809A>C (p.N270T) was identified as a maternally transmitted allele in the proband and one symptomatic sibling; however, a second symptomatic sibling was negative for this variant. The c.2284G>A (p.V762I) was identified in the proband’s symptomatic maternal half sibling. In silico analyses predict both the c.809A>C (p.N270N) and the c.2284G>A (p.V762I) changes to be benign. Conclusive interpretation requires further genotype-phenotype correlation and additional supportive evidence for both variants. The c.2355T>C nucleotide change does not predict an amino acid substitution or a new splicing site; it is therefore a likely benign variant. Although maternally inherited, study for additional family members is indicated to further interpret the c.1629 1631del (p.D543del) variant. The remainder of the variants with evidence of maternal inheritance including c.2+20G>A, c.2221–42T>C, c.2294+20A>G, c.2439– 31T>G, c.2439–37dup, and c.2439–40C>T are nonconsensus splice site intronic variants and are therefore interpreted as VUS but likely benign. All of the intronic VUS in Table 2 were assessed using in silico splice site prediction software including SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, and Human Splicing Finder available in the Alamut (Interactive Boisoftware) DNA sequence analysis software package, and none were predicted to either create or abolish a predicted splicing-related DNA sequence. The final group of VUS includes fifteen previously unreported variants for which we do not have additional family studies available, or family studies are limited (Table 2). These include six nonsynonymous variants c.398T>G (p.V133G), c.1004G>C (p.S335T), c.1434G>A (p.M478I), c.1762C>G (p.Q588E), c.1763A>C (p.Q588P), and c.2018T>G (p.L673R); five intronic variants and four synonymous variants. Although intronic and synonymous variants are interpreted as VUS but likely benign, the nonsynonymous variants and the in-frame insertion are interpreted only as VUS. Variants c.398T>G (p.V133G) and c.2018T>G (p.L673R) are predicted to be damaging by both SIFT and PolyPhen2. Although clinical presentations for patient with the c.2018T>G (p.L673R) variant are consistent with AS (Supp. Table S3), parental studies to confirm maternal inheritance or de novo are indicated to confirm the pathogenicity for both VUS. Within 29 benign variants, four commonly observed variants have been previously published, including a missense c.532G>A (p.A178T), a synonymous c.2558A>G (p.X853X) variant, and two small intronic deletions, c.2221–40 -38del and c.∗ 22 35del 14 (Supp. Table S1). The most conclusive evidence allowing for classification of a benign variant is the presence of maternal transmission of that variant to a clinically unaffected individual. The following variants or linked sets of variants including: [c.2+31T>G; c.1125A>G], c.301+30G>A, c.558A>T (p.A186A) [c.532G>T (p.A178S); c.1549– 18dup], c.1693+37, and [c.1713A>G; c.2221–40 -38del] were all classified as benign due to evidence of maternal transmission to an unaffected individual. Because of the imprinting mechanism, a maternally inherited disease-causing UBE3A variant is expected to be either transmitted from the maternal grandfather or a de novo event in the mother. The identification of a maternally inherited UBE3A variant in the proband’s maternal grandmother, such as the c.1899+11T>A and the c.2102A>T (p.K701I) variants, would rule out the diagnosis of Angelman syndrome for the proband. Intronic variant c.1899+41T>C was detected in an affected individual who also concurrently tested positive for Angelman with the methylation

1412

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

Table 3. Maternal Carrier Frequency and Prenatal Study Maternal studies Number Total number of pathogenic variant positive cases (from Table 1) Mother not tested but obligate carrier Mother tested Positive Negative but obligate carrier Maternal carrier frequency

92 6 45 12 1 13/45 (28.9%)

Prenatal studies

Mother negative Mother positive Mother mosaic Total ∗

Cases

Pregnancies

Positive pregnancies

3 4 1 8

4 6 2 12

0 ∗ 2 0 ∗ 2

From the same family, one singleton and one twin pregnancies.

assay, and is therefore interpreted as likely benign. The “paternal inheritance” category of benign variants (Supp. Table S1) includes 16 different variants which were ruled out as clinically significant based on the evidence of at least one case of paternal allelic transmission. The imprinted paternal copy of the UBE3A gene is normally inactive, and therefore a variant may only be considered clinically significant if present on the maternally inherited, expressed copy of the UBE3A gene. Although evidence of paternal inheritance of a variant indicates lack of clinical significance of that variant for that specific proband, it is important to note that the same variant in the context of maternal transmission may have clinical significance.

Database All the variants in Tables 1, 2, and 3 in the standard HGVS nomenclature were submitted to the freely accessible NCBI ClinVar Database http://www.ncbi.nlm.nih.gov/clinvar/.

Biological Relevance The key biological function of the UBE3A protein is its capacity to target other proteins for degradation. As an E3 ubiquitin-protein ligase, UBE3A is essential for transferring the ubiquitin to the proteins targeted for degradation by the ubiquitin-proteasome system [Huibregtse et al., 1995] and act as a transcriptional coactivator of steroid hormone receptors [Nawaz et al., 1999; Smith et al., 2002; Khan et al., 2006]. Pathogenic variants in any of the coding exons (7–16) are thought to disrupt these functions, and when present on the maternally inherited allele, which is exclusively expressed in the neuronal tissues, can lead to pathogenesis. Figure 1 summarizes the number of all the known pathogenic and likely pathogenic variants in UBE3A, clearly demonstrating a wide distribution of these variants throughout the gene. As an E3 ubiquitin-protein ligase, UBE3A is a component of the E2-E3-ubiquitin system. Ubiquitin is covalently linked to E2 (encoded by the UbcH7 ubiquitin-conjugating enzyme) at Cys86 and transferred to the HECT domain of E3 at Cys820 to target downstream proteins for degradation. To illustrate how variants in the HECT domain (amino acid residues 497–845) may affect this essential process, we performed the analysis of the crystal structure model of the human E2-E3 complex to assess two in-frame deletions c.1745 1747del (p.S582del) and c.2406 2408del (p.M802del) (Supp. Fig. S1), based on the previously published structures of the E2–E3

Figure 1. UBE3A pathogenic and likely pathogenic variants found in this study. Established pathogenic variants include previously published pathogenic variants as well as those determined to be pathogenic through family and other studies. Likely pathogenic variants include de novo as well as familial variants determined to be most likely pathogenic. Number of frame shift and nonsense variants reflects the number of different frame shift and nonsense variants observed in the coding exons of the UBE3A gene.

complex (PDB:1C4Z and 3JWO) [Huang et al., 1999; Kamadurai et al., 2009]. In order to evaluate the process of ubiquitin transfer from E2 to E3, we constructed a structure model of the E2–E3–ubiquitin complex in absence (Supp. Figs. S1A and S1B) and presence of ubiquitin molecule (Supp. Figs. S1D and S1E) with Ser582 and M820 modeled as red spheres. Ser582 is located at middle of the N-lope of the HECT domain of E3 protein. It plays a key role in bridging two roughly globular substructures of the N-lope. These atomic interactions remain relatively stable after ubiquitin binding, highlighting the importance of Ser582 residue in maintaining the overall structure stability (Supp. Fig. S1 C). Met802 is located in the flexible N-lope of the E3, and about 5 A˚ away from the catalytic Cys820. Met802 is evolutionarily highly conserved and closely interacts with Asn822 and Ile804, which are in crucial positions to maintain the geometric configuration of the Cys820 residue (Supp. Fig. S1F). Therefore deletion of Met802 would be foreseeable to disrupt these stereochemical interactions and perturb protein stability and catalytic function.

Clinical and Diagnostic Relevance Loss of functional UBE3A gene expression is the principal and sole cause of Angelman syndrome. Although there is no cure or effective treatment that would significantly improve the clinical course of this disorder, it is very important to determine the precise molecular or cytogenetic nature of the genetic defect. The most significant clinical implication is genetic counseling. Although majority of cases of Angelman syndrome are caused either by large genomic deletions, paternal uniparental disomy, or imprinting center defects, most of these events are de novo. Therefore, in such cases there is a minimal possibility or recurrence in the family. Contrary to this, when a clinically deleterious sequence variant in UBE3A is identified in a proband, it is either a result of inheritance of this variant from an unaffected mother or a result of a de novo variant of the maternal allele in the proband. When a pathogenic variant is maternally inherited, the carrier mother has a 50% risk to pass the variant to a fetus in any subsequent pregnancy. Although there are no published estimates for incidence of de novo versus inherited UBE3A variants,

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

1413

data from our study suggest approximately 28.9% to be inherited and 71.1% to be de novo (Table 3). Specifically, from the 92 patients with definite clinically significant variants described in this report (Table 1), we performed maternal and/or extended family studies in 45 cases, resulting in 12/45 mothers testing positive; one obligate carrier negative and the remaining 32 mothers negative. The latter group suggests a de novo event in the probands, although germline mosaicism cannot be ruled out. In addition, 6/44 mothers that were not tested were obligate carriers. This to our knowledge is the largest empirical dataset providing the estimate for de novo UBE3A variants in Angelman syndrome. Therefore, at least 29% of the families where a UBE3A pathogenic variant is identified in the affected proband carry the risk of recurrence and would benefit from genetic testing for the familial variants, and could need counseling and access to family planning options. Prenatal testing for pathogenic UBE3A variants is currently an option. Our center has tested six pregnancies in four unrelated carriers resulting in two pathogenic variant positive pregnancies and three affected fetuses in one family. Also, we tested two pregnancies from one maternal mosaic case and four pregnancies from three pathogenic variant negative mothers, all resulting in healthy pregnancies (Table 3). The main clinical benefit in UBE3A genetic testing is the possibility of accurate genetic counseling and risk prediction, and as such it depends on our ability to accurately interpret the clinical significance of genetics variants in the UBE3A. In addition to the considerations, we would apply to any other functional gene when analyzing the genetic variants, the analysis is complicated by the epigenetic imprinting nature of this locus, which effectively results in only maternal allele being functionally active. Even when identifying a frameshift, nonsense, or an invariable splice site variant, which are generally classified as clinically deleterious [Richards et al., 2008], in a child with clinical features of Angelman syndrome, it is generally recommended that family studies are done to confirm either maternal inheritance or lack of paternal inheritance. The caveats to keep in mind here of course are a possibility of de novo occurrence, as well as nonpaternity. However, because of the distinct clinical presentation and the relative rarity of both Angelman syndrome and of pathogenic UBE3A variants in population, identification of such variant in a clinically symptomatic child very strongly favors it to be the underlying clinical etiology. It is our experience that in such instances, less than half of the families choose extend family testing, while it is clear that at least 29% of the nontested families will carry additional risk for a familial recurrence. Unlike with the nonsense, frameshift, or splice site variants, it is much more difficult to predict the clinical significance of variants with unknown effect on gene translation. Although it is suspected that a vast majority of synonymous variants or deep intronic variants do not affect the gene function and are generally interpreted as either benign or likely benign, the effect of nonsynonymous (amino acid changing) variants is much more difficult to predict. In silico analysis using software such as SIFT and PolyPhen2, and analysis of evolutionary conservation and protein domains can be informative, but are by no means conclusive. Functional studies using in vitro and in vivo models for gene variants can be very informative, but are beyond utility in a clinical testing environment. Therefore, studying extended family members becomes an invaluable tool for reclassification of UBE3A variants. There are three major considerations when trying to reclassify a UBE3A VUS. These include determination if a variant is de novo or inherited, evidence of maternal transmission, and/or maternal grandfather transmission through an unaffected mother and evidence of variant segregation with patients’ phenotype. Evidence of a de novo variant in a proband immediately raises a suspicion of clinically significance. Although evidence of maternal transmission

1414

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

Figure 2. Pedigree of the c.1693+37A>T variant.

also raises the suspicion for a clinically significant variant, transmission of the same allele to the unaffected sibling completely rules it out as clinically significant. This principle is illustrated in Figure 2, where a c.1693+37A>T variant that was detected in the proband was subsequently detected in the mother, as well as the maternal grandfather, which raised the suspicion for a clinical significance of this allele, only to be ruled out by its presence in an unaffected sibling. Also, note that transmission of this allele form the grandfather to the unaffected mother is an expected outcome even for a deleterious variant, as the allele would be present on the inactive paternally imprinted allele in the mother. Contrary to this pedigree scenario, evidence of transmission of the genetic variant from the mother to the affected proband, and not to an unaffected child further raises the suspicion of clinical significance. This scenario is illustrated in Figure 3, in a pedigree that shows multiple transmissions of the variant to all of the affected children, and lack of transmission to multiple unaffected children. Note that if even a single unaffected individual in this pedigree carried the c.1304C>T variant, it would be sufficient to rule it out the clinical significance. This demonstrates another important aspect of UBE3A genetic testing, which is that a maternal transmission to a single unaffected child rules out clinical significance for the variant, whereas multiple transmission to affected individuals will incrementally increase the probability for clinical significance, hence underlining the importance for extensive family studies in some cases. Another interesting aspect in interpretation of clinical significance unique to UBE3A genetic testing is the interpretation of the paternal transmission of a variant. Although evidence of a paternal transmission of a UBE3A variant to a clinically affected child conclusively rules out this variant as clinically significant, it is only the case for that particular child. In other words, this variant will be on the inactive paternally imprinted allele in the child, and it will not matter if it has an effect on the UBE3A gene. It is possible that the same variant, when maternally transmitted (on the active UBE3A allele) is going to be clinically significant. Although these general rules apply to the interpretative strategy in most pedigrees, doing extensive family studies will sometimes yield unexpected results, as illustrated in Figure 4. It depicts a pedigree where sequence analysis identified a c.1697T>A nucleotide change, predicting a methionine to lysine change at amino acid 566 (p.M566K) in the proband with the classic AS phenotype (Supp. Table S3). Maternal transmission was confirmed through the parental study. Analysis for the maternal grandparents identified an

Figure 3. Pedigree of the c.1304C>T variant.

Figure 4. Pedigree of the c.1697T>A variant.

alternate nucleotide change, c.1697T>G, in the maternal grandfather’s UBE3A gene. The c.1697T>G change predicts a Methionine to Arginine change at the same residue (p.M566R) which was subsequently transmitted to the mother. This could be explained by a germline mosaicism in the maternal grandfather or a de novo allelic conversion in the mother. These unique complexities associated with UBE3A genetic testing also illustrate that although costly and time consuming, extensive familial testing in these families is the most effective strategy for classification of UBE3A variants.

Future Prospects Approximately 80% of Angelman syndrome patients have a defect due to a large chromosomal deletion, uniparental disomy, or imprinting mutations, and another 10% carry a pathogenic UBE3A gene variant, which leaves approximately 10% of patients without a molecular diagnosis. Although there is currently no cure for this disorder, an exact molecular diagnosis is particularly important for the purpose of family counseling and recurrence risk assessment. As infants commonly present with nonspecific psychomotor delay and

seizures, which may result in a broad differential diagnosis. There is a growing list of disorders with clinical presentations similar to Angelman syndrome and these are important to rule out in a differential diagnosis. These include autosomal recessive conditions such as Mowat-Wilson (ZEB2; MIM #605802), autosomal dominant or recessive Pitt Hopkins syndrome (TCF4; MIM #602272), NRXN1 (MIM #600565), CNTNAP2 (MIM #604569), as well as X-linked disorders such as MECP2-related disorders (MECP2; MIM #300005) and Christianson syndrome (SLC9A6; MIM #300231). The advent of massively parallel sequencing technologies allows for cost and time effective genetic testing of multiple genes, gene panels, and entire genomes of individual patients. There are currently multiple clinical laboratories offering extended “tier-2” Angelman syndrome gene panels with up to 20 genes. It is unclear what proportion of the 10% of Angelman syndrome-like patients remaining after the imprinting defect and UBE3A testing would be resolved with such panels, and whether extended gene panels would be adapted as the “tier 1” genetic test in this patient population. Detailed pedigree assessment will continue to play an important role. Our data show that approximately 4% of our UBE3A sequencing reports initially result in a VUS (Tables 2 and 3, and unpublished variants), parental HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

1415

and extended family member variant analysis enabled us to reduce the VUS cases down to 1.2%. Our experiences prove that detailed variant analysis for the family members is currently the most effective way to assess the clinical significance of VUS. Whether caused by the mutation of the UBE3A gene on the transcriptionally active maternal allele, or by the imprinting defect, the underpinning disease etiology in approximately 90% of Angelman syndrome patients is the loss of functional UBE3A protein in neuronal cells. Therefore, transcriptional activation of the epigenetically silenced paternal allele in the neuronal cells presents an exciting therapeutic target. Recently, using an unbiased, high-content screen in primary cortical neurons from mice, Huang et al. (2012) have identified a number of topoisomerase I inhibitors and four topoisomerase II inhibitors that can “unsilence” the paternal Ube3a allele. Using nanomolar concentrations of Topotecan, they showed long lasting induction of the expression of the UBE3A protein, accompanied with downregulation of expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. Subsequent publications have further demonstrated that the Ube3a-ATS is an atypical RNA polymerase II transcript that represses the paternal expression of Ube3a [Meng et al., 2012], and that truncation of Ube3a-ATS unsilences paternal Ube3a and ameliorates behavioral defects in the Angelman syndrome mouse model [Meng et al., 2013]. These data are expected to provide basis for clinical trials for use of topoisomerase inhibitors in treatment of Angelman syndrome in the very near future [Beaudet, 2012].

Acknowledgments The authors wish to thank Jane R. Zhou and Anh T. Dang for their excellent technical assistance. Disclosure statement: The authors declare no conflict of interest.

References Abaied L, Trabelsi M, Chaabouni M, Kharrat M, Kraoua L, M’Rad R, Tebib N, Maazoul F, Chaabouni H. 2010. A novel UBE3A truncating mutation in large Tunisian Angelman syndrome pedigree. Am J Med Genet A 152A(1):141–146. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7(4):248–249. Al-Maawali A, Machado J, Fang P, Dupuis L, Faghfoury H, Mendoza-Londono R. 2013. Angelman syndrome due to a termination codon mutation of the UBE3A gene. J Child Neurol 28(3):392–395. Albrecht U, Sutcliffe JS, Cattanach BM, Beechey CV, Armstrong D, Eichele G, Beaudet AL. 1997. Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nat Genet 17(1):75–78. Amos-Landgraf JM, Ji Y, Gottlieb W, Depinet T, Wandstrat AE, Cassidy SB, Driscoll DJ, Rogan PK, Schwartz S, Nicholls RD. 1999. Chromosome breakage in the PraderWilli and Angelman syndromes involves recombination between large, transcribed repeats at proximal and distal breakpoints. Am J Hum Genet 65(2):370–386. Angelman H. 1965. “Puppet Children”: a report of three cases. Dev Med Child Neurol 7:681–688. Asahina N, Shiga T, Egawa K, Shiraishi H, Kohsaka S, Saitoh S. 2008. [(11)C]flumazenil positron emission tomography analyses of brain gamma-aminobutyric acid type A receptors in Angelman syndrome. J Pediatrics 152(4):546–549, 549 e1–3. Bai JL, Qu YJ, Jin YW, Wang H, Yang YL, Jiang YW, Yang XY, Zou LP, Song F. 2014. Molecular and clinical characterization of Angelman syndrome in Chinese patients. Clin Genet 85(3):273–277. Bai JL, Qu YJ, Zou LP, Yang XY, Liu LJ, Song F. 2011. A novel missense mutation of the ubiquitin protein ligase E3A gene in a patient with Angelman syndrome. Chin Med J 124(1):84–88. Baumer A, Balmer D, Schinzel A. 1999. Screening for UBE3A gene mutations in a group of Angelman syndrome patients selected according to non-stringent clinical criteria. Hum Genet 105(6):598–602. Beaudet AL. 2012. Angelman syndrome: Drugs to awaken a paternal gene. Nature 481(7380):150–152.

1416

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

Camprubi C, Guitart M, Gabau E, Coll MD, Villatoro S, Oltra S, Rosello M, Ferrer I, Monfort S, Orellana C, Martinez F. 2009. Novel UBE3A mutations causing Angelman syndrome: different parental origin for single nucleotide changes and multiple nucleotide deletions or insertions. Am J Med Genet A 149A(3):343–348. Christian SL, Fantes JA, Mewborn SK, Huang B, Ledbetter DH. 1999. Large genomic duplicons map to sites of instability in the Prader-Willi/Angelman syndrome chromosome region (15q11-q13). Hum Mol Genet 8(6):1025–1037. Clayton-Smith J, Laan L. 2003. Angelman syndrome: a review of the clinical and genetic aspects. J Med Genet 40(2):87–95. Clayton-Smith J, Pembrey ME. 1992. Angelman syndrome. J Med Genet 29(6):412–415. Espay AJ, Andrade DM, Wennberg RA, Lang AE. 2005. Atypical absences and recurrent absence status in an adult with Angelman syndrome due to the UBE3A mutation. Epileptic Disord 7(3):227–230. Fang P, Lev-Lehman E, Tsai TF, Matsuura T, Benton CS, Sutcliffe JS, Christian SL, Kubota T, Halley DJ, Meijers-Heijboer H, Langlois S, Graham JM, et al. 1999. The spectrum of mutations in UBE3A causing Angelman syndrome. Hum Mol Genet 8(1):129–135. Fung DC, Yu B, Cheong KF, Smith A, Trent RJ. 1998. UBE3A “mutations” in two unrelated and phenotypically different Angelman syndrome patients. Hum Genet 102(4):487–492. Greer PL, Hanayama R, Bloodgood BL, Mardinly AR, Lipton DM, Flavell SW, Kim TK, Griffith EC, Waldon Z, Maehr R, Ploegh HL, Chowdhury S, et al. 2010. The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc. Cell 140(5):704–716. Hitchins MP, Rickard S, Dhalla F, Fairbrother UL, de Vries BB, Winter R, Pembrey ME, Malcolm S. 2004. Investigation of UBE3A and MECP2 in Angelman syndrome (AS) and patients with features of AS. Am J Med Genet A 125A(2): 167–172. Horsthemke B, Wawrzik M, Gross S, Lich C, Sauer B, Rost I, Krasemann E, Kosyakova N, Liehr T, Weise A, Dybowske JN, Hoffmann D, et al. 2011. Parental origin and functional relevance of a de novo UBE3A variant. Eur J Med Genet 54(1):19–24. Hosoki K, Takano K, Sudo A, Tanaka S, Saitoh S. 2005. Germline mosaicism of a novel UBE3A mutation in Angelman syndrome. Am J Med Genet A 138A(2):187–189. Huang HS, Allen JA, Mabb AM, King IF, Miriyala J, Taylor-Blake B, Sciaky N, Dutton JW, Jr., Lee HM, Chen X, Jin J, Bridges AS, et al. 2012. Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature 481(7380):185–189. Huang L, Kinnucan E, Wang G, Beaudenon S, Howley PM, Huibregtse JM, Pavletich NP. 1999. Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286(5443):1321–1326. Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. 1995. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci USA 92(11):5249. Jiang YH, Armstrong D, Albrecht U, Atkins CM, Noebels JL, Eichele G, Sweatt JD, Beaudet AL. 1998. Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron 21(4):799–811. Kamadurai HB, Souphron J, Scott DC, Duda DM, Miller DJ, Stringer D, Piper RC, Schulman BA. 2009. Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex. Mol Cell 36(6):1095–1102. Kaphzan H, Buffington SA, Jung JI, Rasband MN, Klann E. 2011. Alterations in intrinsic membrane properties and the axon initial segment in a mouse model of Angelman syndrome. J Neurosci 31(48):17637–17648. Kaphzan H, Hernandez P, Jung JI, Cowansage KK, Deinhardt K, Chao MV, Abel T, Klann E. 2012. Reversal of impaired hippocampal long-term potentiation and contextual fear memory deficits in Angelman syndrome model mice by ErbB inhibitors. Biol Psychiatry 72(3):182–190. Khan OY, Fu G, Ismail A, Srinivasan S, Cao X, Tu Y, Lu S, Nawaz Z. 2006. Multifunction steroid receptor coactivator, E6-associated protein, is involved in development of the prostate gland. Mol Endocrinol 20(3):544–559. Kishino T, Lalande M, Wagstaff J. 1997. UBE3A/E6-AP mutations cause Angelman syndrome. Nat Genet 15(1):70–73. Kishino T, Wagstaff J. 1998. Genomic organization of the UBE3A/E6-AP gene and related pseudogenes. Genomics 47(1):101–107. Knoll JH, Nicholls RD, Magenis RE, Glatt K, Graham JM, Jr., Kaplan L, Lalande M. 1990. Angelman syndrome: three molecular classes identified with chromosome 15q11q13-specific DNA markers. Am J Hum Genet 47(1):149–154. Kumar P, Henikoff S, Ng PC. 2009. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4(7):1073–1081. Kumar S, Talis AL, Howley PM. 1999. Identification of HHR23A as a substrate for E6associated protein-mediated ubiquitination. J Biol Chem 274(26):18785–18792. Laan LA, v Haeringen A, Brouwer OF. 1999. Angelman syndrome: a review of clinical and genetic aspects. Clin Neurol Neurosurg 101(3):161–170. Lossie AC, Whitney MM, Amidon D, Dong HJ, Chen P, Theriaque D, Hutson A, Nicholls RD, Zori RT, Williams CA, Driscoll DJ. 2001. Distinct phenotypes distinguish the molecular classes of Angelman syndrome. J Med Genet 38(12):834–845.

Louria-Hayon I, Alsheich-Bartok O, Levav-Cohen Y, Silberman I, Berger M, Grossman T, Matentzoglu K, Jiang YH, Muller S, Scheffner M, Haupt S, Haupt Y. 2009. E6AP promotes the degradation of the PML tumor suppressor. Cell Death Differ 16(8):1156–1166. Mabb AM, Judson MC, Zylka MJ, Philpot BD. 2011. Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes. Trends Neurosci 34(6):293–303. Malzac P, Webber H, Moncla A, Graham JM, Kukolich M, Williams C, Pagon RA, Ramsdell LA, Kishino T, Wagstaff J. 1998. Mutation analysis of UBE3A in Angelman syndrome patients. Am J Hum Genet 62(6):1353–1360. Margolis SS, Salogiannis J, Lipton DM, Mandel-Brehm C, Wills ZP, Mardinly AR, Hu L, Greer PL, Bikoff JB, Ho HY, Soskis, MJ, Sahin M, et al. 2010. EphBmediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation. Cell 143(3):442–455. Matsuura T, Sutcliffe JS, Fang P, Galjaard RJ, Jiang YH, Benton CS, Rommens JM, Beaudet AL. 1997. De novo truncating mutations in E6-AP ubiquitinprotein ligase gene (UBE3A) in Angelman syndrome. Nat Genet 15(1): 74–77. Meng L, Person RE, Beaudet AL. 2012. Ube3a-ATS is an atypical RNA polymerase II transcript that represses the paternal expression of Ube3a. Hum Mol Genet 21(13):3001–3012. Meng L, Person RE, Huang W, Zhu PJ, Costa-Mattioli M, Beaudet AL. 2013. Truncation of Ube3a-ATS unsilences paternal Ube3a and ameliorates behavioral defects in the angelman syndrome mouse model. PLoS Genet 9(12):e1004039. Mishra A, Godavarthi SK, Jana NR. 2009. UBE3A/E6-AP regulates cell proliferation by promoting proteasomal degradation of p27. Neurobiol Dis 36(1):26–34. Mishra A, Jana NR. 2008. Regulation of turnover of tumor suppressor p53 and cell growth by E6-AP, a ubiquitin protein ligase mutated in Angelman mental retardation syndrome. Cell Mol Life Sci 65(4):656–666. Molfetta GA, Munoz MV, Santos AC, Silva WA, Wagstaff J, Pina-Neto JM. 2004. Discordant phenotypes in first cousins with UBE3A frameshift mutation. Am J Med Genet A 127A(3):258–262. Monzon FA, Kant J, Sanders E, Bay C. 2003. Gene symbol: UBE3A. Disease: Angelman syndrome. Hum Genet 113(4):367. Mueller OT, Coovadia A. 2008. Gene symbol: UBE3A. Disease: Angelman syndrome. Hum Genet 123(5):538. Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, O’Malley BW. 1999. The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19(2): 1182–1189. Oda H, Kumar S, Howley PM. 1999. Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. Proc Natl Acad Sci USA 96(17):9557– 9562. Petersen MB, Brondum-Nielsen K, Hansen LK, Wulff K. 1995. Clinical, cytogenetic, and molecular diagnosis of Angelman syndrome: estimated prevalence rate in a Danish county. Am J Med Genet 60(3):261–262.

Rapakko K, Kokkonen H, Leisti J. 2004. UBE3A gene mutations in Finnish Angelman syndrome patients detected by conformation sensitive gel electrophoresis. Am J Med Genet A 126A(3):248–252. Reiter LT, Seagroves TN, Bowers M, Bier E. 2006. Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase. Hum Mol Genet 15(18):2825–2835. Richards CS, Bale S, Bellissimo DB, Das S, Grody WW, Hegde MR, Lyon E, Ward BE. 2008. ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med 10(4):294–300. Robinson WP, Christian SL, Kuchinka BD, Penaherrera MS, Das S, Schuffenhauer S, Malcolm S, Schinzel AA, Hassold TJ, Ledbetter DH. 2000. Somatic segregation errors predominantly contribute to the gain or loss of a paternal chromosome leading to uniparental disomy for chromosome 15. Clin Genet 57(5):349–358. Rougeulle C, Glatt H, Lalande M. 1997. The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain. Nat Genet 17(1):14–15. Runte M, Kroisel PM, Gillessen-Kaesbach G, Varon R, Horn D, Cohen MY, Wagstaff J, Horsthemke BK. 2004. SNURF-SNRPN and UBE3A transcript levels in patients with Angelman syndrome. Hum Genet 114(6):553–561. Russo S, Cogliati F, Viri M, Cavalleri F, Selicorni A, Turolla L, Belli S, Romeo A, Larizza L. 2000. Novel mutations of ubiquitin protein ligase 3A gene in Italian patients with Angelman syndrome. Hum Mutat 15(4):387. Sartori S, Anesi L, Polli R, Toldo I, Casarin A, Drigo P, Murgia A. 2008. Angelman syndrome due to a novel splicing mutation of the UBE3A gene. J Child Neurol 23(8):912–915. Smith CL, DeVera DG, Lamb DJ, Nawaz Z, Jiang YH, Beaudet AL, O’Malley BW. 2002. Genetic ablation of the steroid receptor coactivator-ubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and defects in reproduction. Mol Cell Biol 22(2):525–535. Steffenburg S, Gillberg CL, Steffenburg U, Kyllerman M. 1996. Autism in Angelman syndrome: a population-based study. Pediatr Neurol 14(2):131–136. Tsai TF, Raas-Rothschild A, Ben-Neriah Z, Beaudet AL. 1998. Prenatal diagnosis and carrier detection for a point mutation in UBE3A causing Angelman syndrome. Am J Hum Genet 63(5):1561–1563. Tzagkaraki E, Christalena S, Helen F, Argyris D, Ariadni M, Emmanuel K. 2009. Novel human pathological mutations. Gene symbol: UBE3A. Disease: Angelman Syndrome. Human genetics 126(2):331. Van Buggenhout G, Fryns JP. 2009. Angelman syndrome (AS, MIM 105830). Eur J Hum Genet 17(11):1367–1373. Vu TH, Hoffman AR. 1997. Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain. Nat Genet 17(1):12–13. Williams CA, Beaudet AL, Clayton-Smith J, Knoll JH, Kyllerman M, Laan LA, Magenis RE, Moncla A, Schinzel AA, Summers JA, Wagstaff J. 2006. Angelman syndrome 2005: updated consensus for diagnostic criteria. Am J Med Genet A 140(5):413– 418. Yamamoto Y, Huibregtse JM, Howley PM. 1997. The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing. Genomics 41(2):263–266.

HUMAN MUTATION, Vol. 35, No. 12, 1407–1417, 2014

1417