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Blended phenotype of AP4E1 deficiency and Angelman syndrome caused by paternal isodisomy of chromosome 15
Brain & Development 42 (2020) 289–292 www.elsevier.com/locate/braindev
Case Report
Blended phenotype of AP4E1 deficiency and Angelman syndrome caused by paternal isodisomy of chromosome 15 Hiroaki Murakami a,⇑, Tomoko Uehara b, Yoshinori Tsurusaki c, Yumi Enomoto c, Yukiko Kuroda a, Noriko Aida d, Kenjiro Kosaki b, Kenji Kurosawa a,⇑ a
Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan b Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan c Clinical Research Institute, Kanagawa Children’s Medical Center, Yokohama, Japan d Department of Radiology, Kanagawa Children’s Medical Center, Yokohama, Japan
Received 20 November 2019; received in revised form 13 December 2019; accepted 27 December 2019
Abstract Atypical phenotype of an imprinting disease can develop with a recessive homozygous variant due to uniparental isodisomy. We present a girl with severe intellectual disability, developmental delay, distinctive facial features, and other neuropsychiatric features. Trio whole exome sequencing revealed a novel homozygous frameshift variant in AP4E1 [NM_007347.5:c.2412dupT:p.(Gly805T rpfs*8)] and uniparental isodisomy of chromosome 15 [iUPD(15)]. Single nucleotide polymorphism mapping analysis of exome data showed that the homozygous AP4E1 variant was derived from her heterozygous carrier father and unmasked by paternal iUPD(15). Brain magnetic resonance imaging confirmed the brain abnormalities characteristic of AP4 deficiency including the dilated ventricles and hypointensity in the globus pallidus in susceptibility-weighted imaging. This is the first case report of a combination of AP4E1 deficiency and Angelman syndrome. Our patient indicates that whole exome sequencing could uncover an atypical phenotype caused by multiple genetic factors including the uniparental isodisomy. Ó 2020 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Blended phenotype; Uniparental isodisomy; Angelman syndrome; AP4E1; The neurodegeneration with brain iron accumulation (NBIA)
1. Introduction Adaptor protein complex-4 (AP4) is one of five adaptor protein complexes that are ubiquitously expressed and function in vesicle trafficking. Biallelic pathogenic variants in genes encoding the AP4 subunits, AP4B1, AP4E1, AP4M1, and AP4S1 have been shown to cause intellectual disability (ID), developmental delay (DD), ⇑ Corresponding authors at: Division of Medical Genetics, Kanagawa Children’s Medical Center, 2-138-4 Mutsukawa, Minami-ku, Yokohama 232-8555, Japan. E-mail addresses: [email protected] (H. Murakami), [email protected] (K. Kurosawa).
microcephaly, speech delay, and spastic paraplegia [1,2]. To date, three nonsense variants [p.(Trp710*), p. (Gln1093*), and p.(Arg1105*)], two small deletions [c.830delT:p.(Leu277Profs*2) and c.1036_1037delCT:p. (Leu346Valfs*3)], and one gross deletion (192 kb deletion including the 50 side of AP4E1) of AP4E1 have been identified [1,3–6]. All of these were homozygous variants. Angelman syndrome (AS) is a well-known neurodevelopmental disorder caused by the lack of function of the maternally inherited copy of the gene encoding ubiquitin protein ligase E3A (UBE3A) at chromosome 15q11.2. AS is characterized by ID/DD, speech
https://doi.org/10.1016/j.braindev.2019.12.008 0387-7604/Ó 2020 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
290
H. Murakami et al. / Brain & Development 42 (2020) 289–292
impairment, epilepsy, and paroxysms of laughter [7]. A microdeletion of 15q11.2q13, paternal uniparental disomy of chromosome 15, a UBE3A mutation, or an imprinting defect can disrupt the maternal copy of UBE3A. Here, we describe a girl with a complex phenotype of AS associated with a homozygous pathogenic variant of AP4E1 caused by paternal isodisomy of chromosome 15 [iUPD(15)]. 2. Clinical report The proband was a 2-year-old female. She was born at term after an uneventful pregnancy with birth weight 3430 g [+0.5 standard deviation (SD)], height 51.8 cm (+1.1 SD), and occipital frontal circumference (OFC)
34 cm (+0.2 SD). Her developmental milestones were severely delayed. She began to control her head at 4 months, roll over at 11 months, and sit without support at 24 months. She was referred to our hospital at the age of 2 years and 6 months for an assessment of ID/DD. On physical examination, her weight was 13.5 kg (+1.1 SD), height 89.0 cm (+0.4 SD), and OFC 50.0 cm (+1.4 SD). She could not stand unaided and speak any meaningful words. She had distinctive facial feature including strabismus, wide ala nasi, short philtrum, wide open mouth, prognathism and pointed chin (Fig. 1A). She showed stereotypic laughter, drooling, and sleep disturbance. Brain magnetic resonance imaging (MRI) at the age of 2 years showed no specific findings, except for a mild delay of myelination. Her karyotype was normal. At the age of 3 years, she
Fig. 1. A. Photograph of the patient showing strabismus, wide ala nasi, short philtrum, wide and open mouth, prognathism and pointed chin. Written consent to publish photographs was obtained from each family. B. SNP array analysis confirmed the iUPD(15). The positions of UBE3A (15q11.2) and AP4E1 (15q21.2) are shown at the bottom. The Y-axis shows B-allele frequency (BAF). C. Sanger sequencing confirmed a novel homozygous variant of AP4E1 derived from heterozygous carrier father. D. MRI of the patient at 5 years and 9 months of age. Axial view of SWI. White asterisk indicates mild asymmetrical ventriculomegaly. Arrow indicates slight hypointensity of the globus pallidus.
H. Murakami et al. / Brain & Development 42 (2020) 289–292
developed epilepsy. Her seizures were suppressed by treatment with carbamazepine, levetiracetam, and valproate. 3. Results Written informed consent was obtained from her parents in accordance with the Kanagawa Children’s Medical Center Review Board and Ethics Committee. Whole exome sequencing (WES) data were analyzed as described previously [8]. The algorithm used for homozygosity data mapping was described previously [9]. Analysis of her WES data showed a pattern of single nucleotide polymorphisms (SNPs) consistent with paternal iUPD(15). We confirmed her diagnosis of AS by methylation-specific PCR of SNRPN (data not shown) and paternal iUPD(15) by SNP array (Fig. 1B). WES also revealed a novel homozygous frameshift mutation in AP4E1 at 15q21.2 [NM_007347:exon18:c.2412dupT: p.(Gly805Trpfs*8)]. Sanger sequencing confirmed that it was inherited from her heterozygous carrier father (Fig. 1C). We could not identify other pathogenic recessive or dominant variants that might affect the phenotype of this patient. Brain MRI at the age of 5 years and 9 months revealed mild asymmetrical ventriculomegaly and slight hypointensity in the globus pallidus in susceptibility-weighted imaging (SWI) (Fig. 1D). We could not confirm the accumulation of any substances in the globus pallidus by magnetic resonance spectroscopy (MRS).
291
4. Discussion This report presents a female patient with severe ID/ DD and other neuropsychiatric features. Trio WES analysis revealed that paternal iUPD(15) caused AS and unmasked a recessive disease resulting from a homozygous AP4E1 variant. This is the sixth report of recessive variants in AP4E1, and the first report of a blended phenotype of AS and AP4E1 deficiency. The clinical findings of isolated AP4E1 deficiency and AS overlap considerably (Table 1). Our patient’s mild spastic tetraplegia and brain abnormalities may be caused by AP4E1 deficiency, but sleep disturbance and strabismus may be caused by AS. Normal head circumference of this patient was atypical as AP4E1 deficiency or Angelman syndrome both of which shows microcephaly. We could not identify the reason for the atypical feature of the patient, but the familial macrocephaly was related in this family. These results indicate that complex phenotypes in subjects with AS caused by UPD(15) warrant further genetic analysis of unmasked recessive disorders. We cannot exclude the possibility that other recessive variants have an effect in such subjects. AP4 deficiency is being considered as a novel type of the neurodegeneration with brain iron accumulation (NBIA) spectrum of disorders [2]. The bilateral SWI hypointensity of the globus pallidus have been shown in 4 patients harboring a homozygous variant of AP4M1 and AP4E1 [2,3]. In line with these findings, functional perturbation of the AP4 may result in iron
Table.1 Clinical findings of patients with AP4E1 mutations and cases with AS in the present and past studies. Patient
Sex Genetic change Neonatal hypotonia Head circumference Spastic tetraplegia Severe ID Speech impairment Independent walking Stereotypic laughter Drooling Sleep disturbance Epilepsy Strabismus Wide mouth Brain MRI Age at examination (years) Dilated ventricles Cerebral atrophy Abnormal white matter SWI hypointensity of the globus pallidus
Moreno-De-Luca et al. (2011)
Jamra et al. (2011)
IV-4
IV-5
III-5
III-11
F 192 kb deletion at 15q21.2 + 3 SD + + + + + NA + NA NA
M
+ 3 SD + + + + + NA + NA NA
M c.542 + 1_ +4del + 3 SD + + + NA NA +
23 + + + +
22 + NA
NA NA NA NA NA
F, female; M, male; +, present; , absent; SD, standard deviation; NA, not available.
AS (Dagli et al., 2012)
Present study
F
NA NA
+ 4 SD + + + + NA + NA +
+ Microcephaly + + + + + + + + +
F p. (Gly805Trpfs*8) + +0.2 SD + + + + + + + + +
NA NA NA NA NA
NA
5 + +
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accumulation, especially in the globus pallidus. Brain imaging of our patient identified the same pattern of bilateral SWI hypointensity in the globus pallidus (Fig. 1D). However, this finding was equivocal, and we could not confirm iron accumulation in the globus pallidus by MRS, consistent with the mild spastic paraplegia in our patient. Considering the younger age on MRI examination, follow-up assessments will be needed. In conclusion, we described a rare clinical case with a blended phenotype consisting of AP4E1 deficiency and AS caused by paternal iUPD(15). Our case indicates that WES is a valuable tool for understanding an atypical phenotype caused by homozygosity of a recessive allele in a patient with an imprinting disease.
[2]
[3]
[4]
[5]
[6]
Acknowledgments We thank the patient and her family for participating in this work. This research was supported in part by the Initiative on Rare and Undiagnosed Diseases (Grant number: 17ek0109151) from the Japan Agency for Medical Research and Development.
[7] [8]
[9]
References [1] Moreno-De-Luca A, Helmers SL, Mao H, Burns TG, Melton AMA, Schmidt KR, et al. Adaptor protein complex-4 (AP-4)
deficiency causes a novel autosomal recessive cerebral palsy syndrome with microcephaly and intellectual disability. J Med Genet 2011;48:141–4. Roubertie A, Hieu N, Roux CJ, Leboucq N, Manes G, Charif M, et al. AP4 deficiency: a novel form of neurodegeneration with brain iron accumulation?. Neurol Genet 2018;4:1–6. Abou Jamra R, Philippe O, Raas-Rothschild A, Eck SH, Graf E, Buchert R, et al. Adaptor protein complex 4 deficiency causes severe autosomal-recessive intellectual disability, progressive spastic paraplegia, shy character, and short stature. Am J Hum Genet 2011;88:788–95. Kong XF, Bousfiha A, Rouissi A, Itan Y, Abhyankar A, Bryant V, et al. A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease. PLoS ONE 2013;8. Trujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, Ko¨ster J, Marais A, et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J Hum Genet 2017;25:176–82. Dillon OJ, Lunke S, Stark Z, Yeung A, Thorne N, Gaff C, et al. Exome sequencing has higher diagnostic yield compared to simulated disease-specific panels in children with suspected monogenic disorders. Eur J Hum Genet 2018;26:644–51. Dagli A, Buiting K, Williams CA. Molecular and clinical aspects of Angelman syndrome. Mol Syndromol 2012;2(3–5):100–12. Kuroda Y, Murakami H, Yokoi T, Kumaki T, Enomoto Y, Tsurusaki Y, et al. Two unrelated girls with intellectual disability associated with a truncating mutation in the PPM1D penultimate exon. Brain Dev 2019;41:538–41. Takenouchi T, Inaba M, Uehara T, Takahashi T, Kosaki K, Mizuno S. Biallelic mutations in NALCN: expanding the genotypic and phenotypic spectra of IHPRF1. Am J Med Genet Part A 2018;176:431–7.
Case Report
Blended phenotype of AP4E1 deficiency and Angelman syndrome caused by paternal isodisomy of chromosome 15 Hiroaki Murakami a,⇑, Tomoko Uehara b, Yoshinori Tsurusaki c, Yumi Enomoto c, Yukiko Kuroda a, Noriko Aida d, Kenjiro Kosaki b, Kenji Kurosawa a,⇑ a
Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan b Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan c Clinical Research Institute, Kanagawa Children’s Medical Center, Yokohama, Japan d Department of Radiology, Kanagawa Children’s Medical Center, Yokohama, Japan
Received 20 November 2019; received in revised form 13 December 2019; accepted 27 December 2019
Abstract Atypical phenotype of an imprinting disease can develop with a recessive homozygous variant due to uniparental isodisomy. We present a girl with severe intellectual disability, developmental delay, distinctive facial features, and other neuropsychiatric features. Trio whole exome sequencing revealed a novel homozygous frameshift variant in AP4E1 [NM_007347.5:c.2412dupT:p.(Gly805T rpfs*8)] and uniparental isodisomy of chromosome 15 [iUPD(15)]. Single nucleotide polymorphism mapping analysis of exome data showed that the homozygous AP4E1 variant was derived from her heterozygous carrier father and unmasked by paternal iUPD(15). Brain magnetic resonance imaging confirmed the brain abnormalities characteristic of AP4 deficiency including the dilated ventricles and hypointensity in the globus pallidus in susceptibility-weighted imaging. This is the first case report of a combination of AP4E1 deficiency and Angelman syndrome. Our patient indicates that whole exome sequencing could uncover an atypical phenotype caused by multiple genetic factors including the uniparental isodisomy. Ó 2020 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Blended phenotype; Uniparental isodisomy; Angelman syndrome; AP4E1; The neurodegeneration with brain iron accumulation (NBIA)
1. Introduction Adaptor protein complex-4 (AP4) is one of five adaptor protein complexes that are ubiquitously expressed and function in vesicle trafficking. Biallelic pathogenic variants in genes encoding the AP4 subunits, AP4B1, AP4E1, AP4M1, and AP4S1 have been shown to cause intellectual disability (ID), developmental delay (DD), ⇑ Corresponding authors at: Division of Medical Genetics, Kanagawa Children’s Medical Center, 2-138-4 Mutsukawa, Minami-ku, Yokohama 232-8555, Japan. E-mail addresses: [email protected] (H. Murakami), [email protected] (K. Kurosawa).
microcephaly, speech delay, and spastic paraplegia [1,2]. To date, three nonsense variants [p.(Trp710*), p. (Gln1093*), and p.(Arg1105*)], two small deletions [c.830delT:p.(Leu277Profs*2) and c.1036_1037delCT:p. (Leu346Valfs*3)], and one gross deletion (192 kb deletion including the 50 side of AP4E1) of AP4E1 have been identified [1,3–6]. All of these were homozygous variants. Angelman syndrome (AS) is a well-known neurodevelopmental disorder caused by the lack of function of the maternally inherited copy of the gene encoding ubiquitin protein ligase E3A (UBE3A) at chromosome 15q11.2. AS is characterized by ID/DD, speech
https://doi.org/10.1016/j.braindev.2019.12.008 0387-7604/Ó 2020 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
290
H. Murakami et al. / Brain & Development 42 (2020) 289–292
impairment, epilepsy, and paroxysms of laughter [7]. A microdeletion of 15q11.2q13, paternal uniparental disomy of chromosome 15, a UBE3A mutation, or an imprinting defect can disrupt the maternal copy of UBE3A. Here, we describe a girl with a complex phenotype of AS associated with a homozygous pathogenic variant of AP4E1 caused by paternal isodisomy of chromosome 15 [iUPD(15)]. 2. Clinical report The proband was a 2-year-old female. She was born at term after an uneventful pregnancy with birth weight 3430 g [+0.5 standard deviation (SD)], height 51.8 cm (+1.1 SD), and occipital frontal circumference (OFC)
34 cm (+0.2 SD). Her developmental milestones were severely delayed. She began to control her head at 4 months, roll over at 11 months, and sit without support at 24 months. She was referred to our hospital at the age of 2 years and 6 months for an assessment of ID/DD. On physical examination, her weight was 13.5 kg (+1.1 SD), height 89.0 cm (+0.4 SD), and OFC 50.0 cm (+1.4 SD). She could not stand unaided and speak any meaningful words. She had distinctive facial feature including strabismus, wide ala nasi, short philtrum, wide open mouth, prognathism and pointed chin (Fig. 1A). She showed stereotypic laughter, drooling, and sleep disturbance. Brain magnetic resonance imaging (MRI) at the age of 2 years showed no specific findings, except for a mild delay of myelination. Her karyotype was normal. At the age of 3 years, she
Fig. 1. A. Photograph of the patient showing strabismus, wide ala nasi, short philtrum, wide and open mouth, prognathism and pointed chin. Written consent to publish photographs was obtained from each family. B. SNP array analysis confirmed the iUPD(15). The positions of UBE3A (15q11.2) and AP4E1 (15q21.2) are shown at the bottom. The Y-axis shows B-allele frequency (BAF). C. Sanger sequencing confirmed a novel homozygous variant of AP4E1 derived from heterozygous carrier father. D. MRI of the patient at 5 years and 9 months of age. Axial view of SWI. White asterisk indicates mild asymmetrical ventriculomegaly. Arrow indicates slight hypointensity of the globus pallidus.
H. Murakami et al. / Brain & Development 42 (2020) 289–292
developed epilepsy. Her seizures were suppressed by treatment with carbamazepine, levetiracetam, and valproate. 3. Results Written informed consent was obtained from her parents in accordance with the Kanagawa Children’s Medical Center Review Board and Ethics Committee. Whole exome sequencing (WES) data were analyzed as described previously [8]. The algorithm used for homozygosity data mapping was described previously [9]. Analysis of her WES data showed a pattern of single nucleotide polymorphisms (SNPs) consistent with paternal iUPD(15). We confirmed her diagnosis of AS by methylation-specific PCR of SNRPN (data not shown) and paternal iUPD(15) by SNP array (Fig. 1B). WES also revealed a novel homozygous frameshift mutation in AP4E1 at 15q21.2 [NM_007347:exon18:c.2412dupT: p.(Gly805Trpfs*8)]. Sanger sequencing confirmed that it was inherited from her heterozygous carrier father (Fig. 1C). We could not identify other pathogenic recessive or dominant variants that might affect the phenotype of this patient. Brain MRI at the age of 5 years and 9 months revealed mild asymmetrical ventriculomegaly and slight hypointensity in the globus pallidus in susceptibility-weighted imaging (SWI) (Fig. 1D). We could not confirm the accumulation of any substances in the globus pallidus by magnetic resonance spectroscopy (MRS).
291
4. Discussion This report presents a female patient with severe ID/ DD and other neuropsychiatric features. Trio WES analysis revealed that paternal iUPD(15) caused AS and unmasked a recessive disease resulting from a homozygous AP4E1 variant. This is the sixth report of recessive variants in AP4E1, and the first report of a blended phenotype of AS and AP4E1 deficiency. The clinical findings of isolated AP4E1 deficiency and AS overlap considerably (Table 1). Our patient’s mild spastic tetraplegia and brain abnormalities may be caused by AP4E1 deficiency, but sleep disturbance and strabismus may be caused by AS. Normal head circumference of this patient was atypical as AP4E1 deficiency or Angelman syndrome both of which shows microcephaly. We could not identify the reason for the atypical feature of the patient, but the familial macrocephaly was related in this family. These results indicate that complex phenotypes in subjects with AS caused by UPD(15) warrant further genetic analysis of unmasked recessive disorders. We cannot exclude the possibility that other recessive variants have an effect in such subjects. AP4 deficiency is being considered as a novel type of the neurodegeneration with brain iron accumulation (NBIA) spectrum of disorders [2]. The bilateral SWI hypointensity of the globus pallidus have been shown in 4 patients harboring a homozygous variant of AP4M1 and AP4E1 [2,3]. In line with these findings, functional perturbation of the AP4 may result in iron
Table.1 Clinical findings of patients with AP4E1 mutations and cases with AS in the present and past studies. Patient
Sex Genetic change Neonatal hypotonia Head circumference Spastic tetraplegia Severe ID Speech impairment Independent walking Stereotypic laughter Drooling Sleep disturbance Epilepsy Strabismus Wide mouth Brain MRI Age at examination (years) Dilated ventricles Cerebral atrophy Abnormal white matter SWI hypointensity of the globus pallidus
Moreno-De-Luca et al. (2011)
Jamra et al. (2011)
IV-4
IV-5
III-5
III-11
F 192 kb deletion at 15q21.2 + 3 SD + + + + + NA + NA NA
M
+ 3 SD + + + + + NA + NA NA
M c.542 + 1_ +4del + 3 SD + + + NA NA +
23 + + + +
22 + NA
NA NA NA NA NA
F, female; M, male; +, present; , absent; SD, standard deviation; NA, not available.
AS (Dagli et al., 2012)
Present study
F
NA NA
+ 4 SD + + + + NA + NA +
+ Microcephaly + + + + + + + + +
F p. (Gly805Trpfs*8) + +0.2 SD + + + + + + + + +
NA NA NA NA NA
NA
5 + +
292
H. Murakami et al. / Brain & Development 42 (2020) 289–292
accumulation, especially in the globus pallidus. Brain imaging of our patient identified the same pattern of bilateral SWI hypointensity in the globus pallidus (Fig. 1D). However, this finding was equivocal, and we could not confirm iron accumulation in the globus pallidus by MRS, consistent with the mild spastic paraplegia in our patient. Considering the younger age on MRI examination, follow-up assessments will be needed. In conclusion, we described a rare clinical case with a blended phenotype consisting of AP4E1 deficiency and AS caused by paternal iUPD(15). Our case indicates that WES is a valuable tool for understanding an atypical phenotype caused by homozygosity of a recessive allele in a patient with an imprinting disease.
[2]
[3]
[4]
[5]
[6]
Acknowledgments We thank the patient and her family for participating in this work. This research was supported in part by the Initiative on Rare and Undiagnosed Diseases (Grant number: 17ek0109151) from the Japan Agency for Medical Research and Development.
[7] [8]
[9]
References [1] Moreno-De-Luca A, Helmers SL, Mao H, Burns TG, Melton AMA, Schmidt KR, et al. Adaptor protein complex-4 (AP-4)
deficiency causes a novel autosomal recessive cerebral palsy syndrome with microcephaly and intellectual disability. J Med Genet 2011;48:141–4. Roubertie A, Hieu N, Roux CJ, Leboucq N, Manes G, Charif M, et al. AP4 deficiency: a novel form of neurodegeneration with brain iron accumulation?. Neurol Genet 2018;4:1–6. Abou Jamra R, Philippe O, Raas-Rothschild A, Eck SH, Graf E, Buchert R, et al. Adaptor protein complex 4 deficiency causes severe autosomal-recessive intellectual disability, progressive spastic paraplegia, shy character, and short stature. Am J Hum Genet 2011;88:788–95. Kong XF, Bousfiha A, Rouissi A, Itan Y, Abhyankar A, Bryant V, et al. A novel homozygous p.R1105X mutation of the AP4E1 gene in twins with hereditary spastic paraplegia and mycobacterial disease. PLoS ONE 2013;8. Trujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, Ko¨ster J, Marais A, et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J Hum Genet 2017;25:176–82. Dillon OJ, Lunke S, Stark Z, Yeung A, Thorne N, Gaff C, et al. Exome sequencing has higher diagnostic yield compared to simulated disease-specific panels in children with suspected monogenic disorders. Eur J Hum Genet 2018;26:644–51. Dagli A, Buiting K, Williams CA. Molecular and clinical aspects of Angelman syndrome. Mol Syndromol 2012;2(3–5):100–12. Kuroda Y, Murakami H, Yokoi T, Kumaki T, Enomoto Y, Tsurusaki Y, et al. Two unrelated girls with intellectual disability associated with a truncating mutation in the PPM1D penultimate exon. Brain Dev 2019;41:538–41. Takenouchi T, Inaba M, Uehara T, Takahashi T, Kosaki K, Mizuno S. Biallelic mutations in NALCN: expanding the genotypic and phenotypic spectra of IHPRF1. Am J Med Genet Part A 2018;176:431–7.