CLINICAL REPORT
De Novo SHANK3 Mutation Causes Rett Syndrome-Like Phenotype in a Female Patient Munetsugu Hara,1 Chihiro Ohba,2 Yushiro Yamashita,3 Hirotomo Saitsu,2 Naomichi Matsumoto,2 and Toyojiro Matsuishi3* 1
Department of Neonatology, Medical Center for Maternal and Child Health, St. Mary’s Hospital, Kurume, Fukuoka, Japan
2
Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama, Japan Departments of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka, Japan
3
Manuscript Received: 12 December 2013; Manuscript Accepted: 13 August 2014
Rett syndrome (RTT) is a neurodevelopmental disorder predominantly affecting females. Females with the MECP2 mutations exhibit a broad spectrum of clinical manifestations ranging from classical Rett syndrome to asymptomatic carriers. Mutations of genes encoding cyclin-dependent kinase-like 5 (CDKL5) and forkhead box G1 (FOXG1) are also found in early onset RTT variants. Here, we present the first report of a female patient with RTT-like phenotype caused by SHANK3 (SH3 and multiple ankylin repeat domain 3) mutation, indicating that the clinical spectrum of SHANK3 mutations may extend to RTT-like phenotype in addition to (severe) developmental delay, absence of expressive speech, autistic behaviors and intellectual disability.
How to Cite this Article: Hara M, Ohba C, Yamashita Y, Saitsu Y, Matsumoto N, Matsuishi T. 2015. De novo SHANK3 mutation causes Rett syndromelike phenotype in a female patient. Am J Med Genet Part A 167A:1593–1596.
deletion; whole-exome sequencing
aptic membrane) of glutamatergic synapses, making these molecules vulnerable to Shank3 mutation [Herbert, 2011]. Mutations in SHANK3 and NLG3/4 encoding these molecules affecting neurite development were identified in autism spectrum disorders (ASD) or ID [Durand et al., 2007]. Here, we present a de novo mutation of SHANK3 in a female patient with RTT-like phenotype.
INTRODUCTION
PATIENT AND METHODS Clinical Report
Ó 2015 Wiley Periodicals, Inc.
Key words: Rett syndrome; SHANK3; de novo; frameshift;
Rett syndrome [RTT (OMIM #312750)] is a neurodevelopmental disorder affecting primarily females, with a frequency of 1 in 10,000 live female births. Most cases of RTT are caused by de novo mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). Approximately 90–95% of typical RTT cases harbor lossof-function mutations in X-linked MECP2 [Amir et al., 1999]. RTT females seem to develop normally until 6–18 months and regress thereafter. Clinical manifestations include microcephaly, loss of achieved psychomotor abilities, intellectual disability (ID), and autistic behaviors [Hagberg et al., 1983]. In RTT variants, mutations of CDKL5 and FOXG1 have been identified [Neul and Zoghbi, 2004]. Other similar disorders include Pitt–Hopkins syndrome due to TCF4 mutations and Pitt–Hopkins-like features with CNTNAP2 or NRXN1 abnormality including severe ID, autism, and breathing abnormalities [Smeets et al., 2011]. SHANK3 (SH3 and multiple ankylin repeat domain 3) mutations were found in developmental delay, severe delay or absence of expressive speech, autistic behaviors, schizophrenia, and ID. SHANK3 provides scaffolding for signaling molecules in the postsynaptic density (a multi-protein complex attached to the postsyn-
Ó 2015 Wiley Periodicals, Inc.
A female patient aged 25 years and 4 months, the first child of healthy non-consanguineous parents, was born at 39 weeks of gestation after an uneventful pregnancy. Prenatal and neonatal Conflict of interest: Nothing to declare. Grant sponsor: Japan Society for the Promotion of Science; Grant numbers: 30389283, 13313587, 21591338; Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant numbers: 11105137, 12024421; Grant sponsor: Applying Health Technology; Grant number: 12944231; Grant sponsor: Comprehensive Research on Disability Health and Welfare; Grant number: 13802019; Grant sponsor: Research on Intractable Diseases; Grant numbers: 21-110, 22-133. Correspondence to: Toyojiro Matsuishi, M.D., Ph.D., Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 30 April 2015 DOI 10.1002/ajmg.a.36775
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1594 periods were normal. Her birth weight was 2,860 g, length 50.0 cm, and her head circumference 33.5 cm (all within the normal range). Apgar score was 10 at 5 min. Family history is negative for ID and other neurological/psychiatric disorders. Her early motor milestones were normal; she controlled her head at 4 months, started crawling at 7 months, sat unassisted at 9 months; however, her motor milestones were delayed; she walked alone at 24 months and ran at 30 months of age. Both her speech development and nonverbal communication were also delayed. She spoke her first meaningful word at 2 years old and a few meaningful words at 3 years old. However, she was able to brush her hair, brush her teeth and to run to a swing with well-coordinated movements between 2 years and 3 years old. She began to regress in verbal communication, gross and fine motor skills after 4 years and 6 months old, and became unable to walk after 5 years old. Her purposeful hand movement was impaired at 4 years and 6 months old. She began to occasionally hold her breath after screaming “yeow” loudly at 4 years old. She also exhibited autistic behavior, such as impaired reciprocal social interaction and communication, keeping a ritualistic manner of holding the artificial plastic grass, and repetitive stereotyped hand movements. The onset of stereotyped hand movements, which are the hallmark of Rett syndrome, bimanual wringing, mouthing, and squeezing appeared at 4 years 10 months old (Fig. 1). They changed with emotional states and tended to appear when she was standing or walking. Elbow splints were used to correct abnormal movement. She was referred to our clinic at 10 years and 4 months old. Neurological examination revealed RTT-like phenotype. She developed her first seizures with tonic movement of the upper limbs at 3 years 8 months old. Her brain magnetic resonance imaging (MRI) did not show any abnormalities at 3 years and 8 months old. Her electroencephalography (EEG) exhibited right frontal spike discharge at 5 years old. G-banded chromosomal analysis showed a normal karyotype (46, XX). She has been treated with clonazepam, but she has had a few seizure attacks each year since the age of 9. She suffered from sleep disturbance from the age of 10. Her menarche was delayed until 18 years old. At 25 years old, her height was 145.0 cm ( 2.5 SD), her weight was 26.0 kg ( 3.4 SD), and her head circumference remained within the normal range at 50.0 cm ( 0.1 SD). Taken together, she was tentatively diagnosed with RTT-like phenotype. Whole-exome sequencing was performed after written informed consent was obtained from her parents. The research protocol was approved by the Ethics Committees of the Kurume University School of Medicine and Yokohama City University School of Medicine.
Sanger Sequencing and Multiplex LigationDependent Probe Amplification (MLPA) MECP2, CDKL5, FOXG1 mutations were screened by Sanger sequencing, and parental samples were also sequenced with respect to identified variants. We also performed MLPA to detect exon deletion or duplication of MECP2 and CDKL5.
Whole Exome Sequencing (WES) Genomic DNA was isolated from peripheral blood leukocytes, captured using the SureSelect Human All Exon v4 Kit (51 Mb;
FIG. 1. Repetitive stereotyped hand movements, such as wringing, are observed.
Agilent Technologies, Santa Clara, CA) and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA) with 101 bp paired-end reads. Data processing, variant calling, and variant annotation were performed as previously described [Saitsu et al., 2013].
RESULTS No nucleotide changes in MECP2, CDKL5, and FOXG1, and no copy number variations by MLPA of MECP2 and CDKL5 were found in the patient. WES was performed in the patient and her parents and the exome sequencing performance is shown in Table SI (see supporting information online. WES detected 239 rare protein-altering and splice-site variants in the patient (Table SI). We filtered out common single nucleotide polymorphisms (SNPs) that met the following two criteria: variants showing minor allele frequencies 1% in dbSNP 135 and variants found in more than three of our in-house 573 control exomes. All genes were surveyed for de novo, compound heterozygous or homozygous mutations and we identified three de novo mutations; CRK (GenBank accession number NM_016823.3),
HARA ET AL. SHANK3 (NM_033517.1), CAPN6 (NM_014289.3) and one compound heterozygous mutation; DNAH11 (NM_001277115.1). No homozygous mutation was detected. These five mutations were confirmed by Sanger sequencing. The mutations were absent in the 6,500 exomes sequenced by the National Heart, Lung, and Blood Institute exome project and our in-house 573 control exomes. Five mutations are rare, but a de novo mutation of CRK is predicted to be a polymorphism by web-prediction tools (Table SII—in supporting information online). Recessive mutations of DNAH11 have been reported to cause primary ciliary defects [Pan et al., 1998], but the clinical phenotypes of this patient were incompatible. De novo mutation in CAPN6, encoding calpain 6, a member of the calcium-dependent cysteine proteases, is predicted to be pathogenic, but CAPN6 abnormality in association with human phenotypes has never been described. As SHANK3 mutations have been known to be the cause of autism spectrum disorders [Herbert, 2011; Uchino and Waga, 2013] and the SHANK3 mutation [c.3100del (p. Ala1034fs 44)] is a frameshift mutation, we concluded that the de novo SHANK3 mutation is the cause for the clinical phenotype in this patient.
DISCUSSION This is the first report of a female patient with RTT-like phenotype caused by a de novo SHANK3 mutation. SHANK3 mutations have been repeatedly reported in ASD, schizophrenia, intellectual disability and poor language. Revised diagnostic criteria have been reported for the diagnosis of typical (classical) and atypical RTT [Neul et al., 2010]. The clinical criteria for typical RTT include a period of regression followed by recovery or stabilization; the patient must meet all main criteria and all exclusion criteria; supportive criteria are not required, although often present. This patient gained purposeful hand movement such as brushing her teeth at 2–3 years and later loss of purposeful hand movements at approximately 4 years old. She spoke a few meaningful words after 3 years but partially regressed in verbal communication until 4 years and 6 months, thereafter she spoke no meaningful words. She was able to walk alone at 24 months but unable to walk after 5 years. The onset of stereotyped hand movements, bimanual wringing, mouthing, and squeezing appeared at 4 years 10 months old. She fulfilled four main criteria, with typical RTT [Neul et al., 2010]. However, her mental and motor deterioration appeared later (4 years and 6 months) than those of typical RTT (12–18 months) and she did not show any decelerated head growth. Furthermore, of 11 supportive criteria, she showed nine symptoms: breathing disturbance, impaired sleep pattern, abnormal muscle tone, scoliosis, growth retardation, small cold hands and feet, inappropriate laughing/ screaming spells, diminished response to pain, and intense eye communication. She also met the all exclusion criteria of brain injury secondary to trauma peri- or post-natally, neurometabolic disease, or severe infection that causes neurological problems. She also met the sensitive recent revised diagnostic criteria of typical or classical RTT including required three conditions, four main criteria, two exclusion criteria, and nine of eleven supportive criteria [Neul et al., 2010, Percy et al., 2010]. These criteria exclude acquired microcephaly, because 18% of typical RTT patients do not show microcephaly [Percy et al., 2010]. Therefore, she fulfilled all
1595 the revised criteria of typical RTT. However, we prefer to use the term RTT-like phenotype for this patient as her mental and motor deterioration appeared at an unusually later age. SHANK3 abnormalities (mutations and copy number changes) were found in patients with neurodevelopmental disorders including ASD and ID [Uchino and Waga, 2013]. SHANK3/Shank3 is highly expressed in the striata of human and mice [Herbert, 2011]. Shank3B / mice displayed an anxiety-like behavior and excessive, self-injurious grooming, deficits in social interaction and discriminating social novelty, and infrequent seizures during handling in routine husbandry procedures. Shank3B / mice showed low signal intensity of neuronal signaling in the corticostriatal synaptic circuitry, and on stimulation, lower amplitude and frequency of miniature excitatory postsynaptic currents only in striatum. However these mice did not display any gross anatomical or histological brain abnormality. Medium spiny neurons were slightly larger and caudate volume was enlarged in Shank3B / mice [Pec¸a et al., 2011]. SHANK3 plays crucial roles as master organizers of the postsynaptic density (PSD), allowing multi-meric complexes to form with postsynaptic receptors, signaling molecules and cytoskeletal proteins in dendritic spines and PSDs, and exerts neuroligin–neurexin interaction at glutamate synapses [Durand et al., 2007]. Both neuroligin and neurexin play critical roles in the formation and functioning of synapses, particularly in the alignment and activation of glutamate and GABA synapses. Chao et al. reported dysfunction in GABA signaling mediated autism-like stereotypies and RTT features in a mouse RTT model [Chao et al., 2010]. GABA may play an important role in RTT phenotype. Studies on ASD revealed the impairments in neuroligin-neurexin pathway responsible for the proper localization and assembly of the PSD due to the mutations of SHANK3, NLG3/4 and contactin associated proteinlike 2 [Boccuto et al., 2013]. The mutations of these genes encoding the assembly proteins of the PSD induce neurodevelopmental disorders resembling RTT [Smeets et al., 2011]. We identified a frameshift single nucleotide deletion (c.3100del, p.Ala1034fs 44) in SHANK3. Recent studies reported, in two siblings heterozygous for a guanine insertion in exon 21 of SHANK3, that the balanced translocation of 12 and 22 chromosomes disrupted exon 21 of SHANK3, and led to the deletion of the homer-binding site enabling homer proteins to bind to SHANK [Bonaglia et al., 2001; Durand et al., 2007]. Although the pathologic mechanism of the lesion is uncertain, later deterioration of mental and motor skills might be important in RTT-like phenotype with SHANK3 mutation. SHANK3 is one of the most GC-rich genes in the genome with the CpG-islands in exon 21 [Durand et al., 2007; Betancur and Buxbaum, 2013]. GC-rich regions are conserved between human and mouse [Durand et al., 2007]. In mouse brain, synaptogenesis occurs during the first 2 weeks after birth, according to the elevation of the methylation rate of CpG-islands [Uchino and Waga, 2013]. Waga and co-workers revealed by ChIP assay that Mecp2 bound to methylated CpG-islands of Shank3 and that the expression levels of the variable Shank3 transcripts in Mecp2 null mice differed from those of wild-type mice in neocortical tissue, especially from P14 to P28 [Waga et al., 2014]. Taken together, synaptogenesis of the patients with RTT at the early developmental stage may be vulnerable to aberrant SHANK3 transcripts affected by the MeCP2
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dysfunction. The precise mechanisms of SHANK3 abnormality leading to RTT-like phenotype still remain elusive. We identify a de novo SHANK3 mutation in a patient with RTTlike phenotype. Our data suggest that disturbance of SHANK3Homer interaction may be one of the critical steps associated with RTT-like phenotype.
Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsa¨ter H, Sponheim E, Goubran-Botros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E, Roge´ B, He´ron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T. 2007. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet 39:25–27.
ACKNOWLEDGMENTS
Hagberg B, Aicardi J, Dias K, Ramos O. 1983. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: Report of 35 cases. Ann Neurol 14:471–479.
This work was partly supported by Grants-in-Aid for Scientific Research (no. 30389283 to M.H., no. 13313587 to N.M., and no. 21591338 to T.M.) from the Japan Society for the Promotion of Science; the Strategic Research Program for Brain Sciences (no. 11105137 to N.M.); a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) (12024421 to N.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a research grant from the Ministry of Health, Labor and Welfare of Japan (Intramural Research Grant [21B-5] for Neurological and Psychiatric Disorders of NCNP; Research on Applying Health Technology (12944231 to N.M.); Comprehensive Research on Disability Health and Welfare (13802019 to N.M.); and Research on Intractable Diseases (21-110 and 22-133 to T.M.).
REFERENCES Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23: 185–188. Betancur C, Buxbaum JD. 2013. SHANK3 haploinsufficiency: A “common” but underdiagnosed highly penetrant monogenic cause of autism spectrum disorders. Mol Autism 4:17. Boccuto L, Lauri M, Sarasua SM, Skinner CD, Buccella D, Dwivedi A, Orteschi D, Collins JS, Zollino M, Visconti P, Dupont B, Tiziano D, Schroer RJ, Neri G, Stevenson RE, Gurrieri F, Schwartz CE. 2013. Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders. Eur J Hum Genet 21:310–316. Bonaglia MC, Giorda R, Borgatti R, Felisari G, Gagliardi C, Selicorni A, Zuffardi O. 2001. Disruption of the ProSAP2 gene in a t(12;22)(q24.1; q13.3) is associated with the 22q13.3 deletion syndrome. Am J Hum Genet 69:261–268. Chao HT, Chen H, Samaco RC, Xue M, Chahrour M, Yoo J, Neul JL, Gong S, Lu HC, Heinz N, Ekker M, Rubenstein JL, Noebels JL, Rosenmund C, Zoghbi HY. 2010. Dysfunction in GABA signaling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468:263–269.
Herbert MR. 2011. SHANK3, the synapse, and autism. N Engl J Med 365:173–175. Neul JL, Kaufmann WE, Glaze DG, Christodoulou J, Clarke AJ, BahiBuisson N, Leonard H, Bailey ME, Schanen NC, Zappella M, Renieri A, Huppke P, Percy AK, Rett Search Consortium. 2010. Rett syndrome: Revised diagnostic criteria and nomenclature. Ann Neurol 68:944–950. Neul JL, Zoghbi HY. 2004. Rett syndrome: A prototypical neurodevelopmental disorder. Neuroscientist 10:118–128. Pan Y, McCaskill CD, Thompson KH, Hicks J, Casey B, Shaffer LG, Craigen WJ. 1998. Paternal isodisomy of chromosome 7 associated with complete situs inversus and immotile cilia. Am J Hum Genet 62:1551–1555. Pec¸a J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, Lascola CD, Fu Z, Feng G. 2011. Shank3 mutant mice display autistic-like behaviors and striatal dysfunction. Nature 472:437–442. Percy AK, Neul JL, Glaze DG, Motil KJ, Skinner SA, Khwaja O, Lee HS, Lane JB, Barrish JO, Annese F, McNair L, Graham J, Bames K. 2010. Rett syndrome diagnostic criteria: Lessons from the natural history study. Ann Neurol 68:951–955. Saitsu H, Nishimura T, Muramatsu K, Kodera H, Kumada S, Sugai K, Kasai-Yoshida E, Sawaura N, Nishida H, Hoshino A, Ryujin F, Yoshioka S, Nishiyama K, Kondo Y, Tsurusaki Y, Nakashima M, Miyake N, Arakawa H, Kato M, Mizushima N, Matsumoto N. 2013. De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Nat Genet 45: 445–449. Smeets EEJ, Pelc K, Dan B. 2011. Rett syndrome. Mol Syndromol 2: 113–127. Uchino S, Waga C. 2013. SHANK3 as an autism spectrum disorderassociated gene. Brain Dev 35:106–110. Waga C, Asano H, Sanagi T, Suzuki E, Nakamura Y, Tsuchiya A, Itoh M, Goto Y, Kohsaka S, Uchino S. 2014. Identification of two novel Shank3 transcripts in the developing mouse neocortex. J Neurochem 128:280–293.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
De Novo SHANK3 Mutation Causes Rett Syndrome-Like Phenotype in a Female Patient Munetsugu Hara,1 Chihiro Ohba,2 Yushiro Yamashita,3 Hirotomo Saitsu,2 Naomichi Matsumoto,2 and Toyojiro Matsuishi3* 1
Department of Neonatology, Medical Center for Maternal and Child Health, St. Mary’s Hospital, Kurume, Fukuoka, Japan
2
Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama, Japan Departments of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka, Japan
3
Manuscript Received: 12 December 2013; Manuscript Accepted: 13 August 2014
Rett syndrome (RTT) is a neurodevelopmental disorder predominantly affecting females. Females with the MECP2 mutations exhibit a broad spectrum of clinical manifestations ranging from classical Rett syndrome to asymptomatic carriers. Mutations of genes encoding cyclin-dependent kinase-like 5 (CDKL5) and forkhead box G1 (FOXG1) are also found in early onset RTT variants. Here, we present the first report of a female patient with RTT-like phenotype caused by SHANK3 (SH3 and multiple ankylin repeat domain 3) mutation, indicating that the clinical spectrum of SHANK3 mutations may extend to RTT-like phenotype in addition to (severe) developmental delay, absence of expressive speech, autistic behaviors and intellectual disability.
How to Cite this Article: Hara M, Ohba C, Yamashita Y, Saitsu Y, Matsumoto N, Matsuishi T. 2015. De novo SHANK3 mutation causes Rett syndromelike phenotype in a female patient. Am J Med Genet Part A 167A:1593–1596.
deletion; whole-exome sequencing
aptic membrane) of glutamatergic synapses, making these molecules vulnerable to Shank3 mutation [Herbert, 2011]. Mutations in SHANK3 and NLG3/4 encoding these molecules affecting neurite development were identified in autism spectrum disorders (ASD) or ID [Durand et al., 2007]. Here, we present a de novo mutation of SHANK3 in a female patient with RTT-like phenotype.
INTRODUCTION
PATIENT AND METHODS Clinical Report
Ó 2015 Wiley Periodicals, Inc.
Key words: Rett syndrome; SHANK3; de novo; frameshift;
Rett syndrome [RTT (OMIM #312750)] is a neurodevelopmental disorder affecting primarily females, with a frequency of 1 in 10,000 live female births. Most cases of RTT are caused by de novo mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). Approximately 90–95% of typical RTT cases harbor lossof-function mutations in X-linked MECP2 [Amir et al., 1999]. RTT females seem to develop normally until 6–18 months and regress thereafter. Clinical manifestations include microcephaly, loss of achieved psychomotor abilities, intellectual disability (ID), and autistic behaviors [Hagberg et al., 1983]. In RTT variants, mutations of CDKL5 and FOXG1 have been identified [Neul and Zoghbi, 2004]. Other similar disorders include Pitt–Hopkins syndrome due to TCF4 mutations and Pitt–Hopkins-like features with CNTNAP2 or NRXN1 abnormality including severe ID, autism, and breathing abnormalities [Smeets et al., 2011]. SHANK3 (SH3 and multiple ankylin repeat domain 3) mutations were found in developmental delay, severe delay or absence of expressive speech, autistic behaviors, schizophrenia, and ID. SHANK3 provides scaffolding for signaling molecules in the postsynaptic density (a multi-protein complex attached to the postsyn-
Ó 2015 Wiley Periodicals, Inc.
A female patient aged 25 years and 4 months, the first child of healthy non-consanguineous parents, was born at 39 weeks of gestation after an uneventful pregnancy. Prenatal and neonatal Conflict of interest: Nothing to declare. Grant sponsor: Japan Society for the Promotion of Science; Grant numbers: 30389283, 13313587, 21591338; Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant numbers: 11105137, 12024421; Grant sponsor: Applying Health Technology; Grant number: 12944231; Grant sponsor: Comprehensive Research on Disability Health and Welfare; Grant number: 13802019; Grant sponsor: Research on Intractable Diseases; Grant numbers: 21-110, 22-133. Correspondence to: Toyojiro Matsuishi, M.D., Ph.D., Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 30 April 2015 DOI 10.1002/ajmg.a.36775
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1594 periods were normal. Her birth weight was 2,860 g, length 50.0 cm, and her head circumference 33.5 cm (all within the normal range). Apgar score was 10 at 5 min. Family history is negative for ID and other neurological/psychiatric disorders. Her early motor milestones were normal; she controlled her head at 4 months, started crawling at 7 months, sat unassisted at 9 months; however, her motor milestones were delayed; she walked alone at 24 months and ran at 30 months of age. Both her speech development and nonverbal communication were also delayed. She spoke her first meaningful word at 2 years old and a few meaningful words at 3 years old. However, she was able to brush her hair, brush her teeth and to run to a swing with well-coordinated movements between 2 years and 3 years old. She began to regress in verbal communication, gross and fine motor skills after 4 years and 6 months old, and became unable to walk after 5 years old. Her purposeful hand movement was impaired at 4 years and 6 months old. She began to occasionally hold her breath after screaming “yeow” loudly at 4 years old. She also exhibited autistic behavior, such as impaired reciprocal social interaction and communication, keeping a ritualistic manner of holding the artificial plastic grass, and repetitive stereotyped hand movements. The onset of stereotyped hand movements, which are the hallmark of Rett syndrome, bimanual wringing, mouthing, and squeezing appeared at 4 years 10 months old (Fig. 1). They changed with emotional states and tended to appear when she was standing or walking. Elbow splints were used to correct abnormal movement. She was referred to our clinic at 10 years and 4 months old. Neurological examination revealed RTT-like phenotype. She developed her first seizures with tonic movement of the upper limbs at 3 years 8 months old. Her brain magnetic resonance imaging (MRI) did not show any abnormalities at 3 years and 8 months old. Her electroencephalography (EEG) exhibited right frontal spike discharge at 5 years old. G-banded chromosomal analysis showed a normal karyotype (46, XX). She has been treated with clonazepam, but she has had a few seizure attacks each year since the age of 9. She suffered from sleep disturbance from the age of 10. Her menarche was delayed until 18 years old. At 25 years old, her height was 145.0 cm ( 2.5 SD), her weight was 26.0 kg ( 3.4 SD), and her head circumference remained within the normal range at 50.0 cm ( 0.1 SD). Taken together, she was tentatively diagnosed with RTT-like phenotype. Whole-exome sequencing was performed after written informed consent was obtained from her parents. The research protocol was approved by the Ethics Committees of the Kurume University School of Medicine and Yokohama City University School of Medicine.
Sanger Sequencing and Multiplex LigationDependent Probe Amplification (MLPA) MECP2, CDKL5, FOXG1 mutations were screened by Sanger sequencing, and parental samples were also sequenced with respect to identified variants. We also performed MLPA to detect exon deletion or duplication of MECP2 and CDKL5.
Whole Exome Sequencing (WES) Genomic DNA was isolated from peripheral blood leukocytes, captured using the SureSelect Human All Exon v4 Kit (51 Mb;
FIG. 1. Repetitive stereotyped hand movements, such as wringing, are observed.
Agilent Technologies, Santa Clara, CA) and sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA) with 101 bp paired-end reads. Data processing, variant calling, and variant annotation were performed as previously described [Saitsu et al., 2013].
RESULTS No nucleotide changes in MECP2, CDKL5, and FOXG1, and no copy number variations by MLPA of MECP2 and CDKL5 were found in the patient. WES was performed in the patient and her parents and the exome sequencing performance is shown in Table SI (see supporting information online. WES detected 239 rare protein-altering and splice-site variants in the patient (Table SI). We filtered out common single nucleotide polymorphisms (SNPs) that met the following two criteria: variants showing minor allele frequencies 1% in dbSNP 135 and variants found in more than three of our in-house 573 control exomes. All genes were surveyed for de novo, compound heterozygous or homozygous mutations and we identified three de novo mutations; CRK (GenBank accession number NM_016823.3),
HARA ET AL. SHANK3 (NM_033517.1), CAPN6 (NM_014289.3) and one compound heterozygous mutation; DNAH11 (NM_001277115.1). No homozygous mutation was detected. These five mutations were confirmed by Sanger sequencing. The mutations were absent in the 6,500 exomes sequenced by the National Heart, Lung, and Blood Institute exome project and our in-house 573 control exomes. Five mutations are rare, but a de novo mutation of CRK is predicted to be a polymorphism by web-prediction tools (Table SII—in supporting information online). Recessive mutations of DNAH11 have been reported to cause primary ciliary defects [Pan et al., 1998], but the clinical phenotypes of this patient were incompatible. De novo mutation in CAPN6, encoding calpain 6, a member of the calcium-dependent cysteine proteases, is predicted to be pathogenic, but CAPN6 abnormality in association with human phenotypes has never been described. As SHANK3 mutations have been known to be the cause of autism spectrum disorders [Herbert, 2011; Uchino and Waga, 2013] and the SHANK3 mutation [c.3100del (p. Ala1034fs 44)] is a frameshift mutation, we concluded that the de novo SHANK3 mutation is the cause for the clinical phenotype in this patient.
DISCUSSION This is the first report of a female patient with RTT-like phenotype caused by a de novo SHANK3 mutation. SHANK3 mutations have been repeatedly reported in ASD, schizophrenia, intellectual disability and poor language. Revised diagnostic criteria have been reported for the diagnosis of typical (classical) and atypical RTT [Neul et al., 2010]. The clinical criteria for typical RTT include a period of regression followed by recovery or stabilization; the patient must meet all main criteria and all exclusion criteria; supportive criteria are not required, although often present. This patient gained purposeful hand movement such as brushing her teeth at 2–3 years and later loss of purposeful hand movements at approximately 4 years old. She spoke a few meaningful words after 3 years but partially regressed in verbal communication until 4 years and 6 months, thereafter she spoke no meaningful words. She was able to walk alone at 24 months but unable to walk after 5 years. The onset of stereotyped hand movements, bimanual wringing, mouthing, and squeezing appeared at 4 years 10 months old. She fulfilled four main criteria, with typical RTT [Neul et al., 2010]. However, her mental and motor deterioration appeared later (4 years and 6 months) than those of typical RTT (12–18 months) and she did not show any decelerated head growth. Furthermore, of 11 supportive criteria, she showed nine symptoms: breathing disturbance, impaired sleep pattern, abnormal muscle tone, scoliosis, growth retardation, small cold hands and feet, inappropriate laughing/ screaming spells, diminished response to pain, and intense eye communication. She also met the all exclusion criteria of brain injury secondary to trauma peri- or post-natally, neurometabolic disease, or severe infection that causes neurological problems. She also met the sensitive recent revised diagnostic criteria of typical or classical RTT including required three conditions, four main criteria, two exclusion criteria, and nine of eleven supportive criteria [Neul et al., 2010, Percy et al., 2010]. These criteria exclude acquired microcephaly, because 18% of typical RTT patients do not show microcephaly [Percy et al., 2010]. Therefore, she fulfilled all
1595 the revised criteria of typical RTT. However, we prefer to use the term RTT-like phenotype for this patient as her mental and motor deterioration appeared at an unusually later age. SHANK3 abnormalities (mutations and copy number changes) were found in patients with neurodevelopmental disorders including ASD and ID [Uchino and Waga, 2013]. SHANK3/Shank3 is highly expressed in the striata of human and mice [Herbert, 2011]. Shank3B / mice displayed an anxiety-like behavior and excessive, self-injurious grooming, deficits in social interaction and discriminating social novelty, and infrequent seizures during handling in routine husbandry procedures. Shank3B / mice showed low signal intensity of neuronal signaling in the corticostriatal synaptic circuitry, and on stimulation, lower amplitude and frequency of miniature excitatory postsynaptic currents only in striatum. However these mice did not display any gross anatomical or histological brain abnormality. Medium spiny neurons were slightly larger and caudate volume was enlarged in Shank3B / mice [Pec¸a et al., 2011]. SHANK3 plays crucial roles as master organizers of the postsynaptic density (PSD), allowing multi-meric complexes to form with postsynaptic receptors, signaling molecules and cytoskeletal proteins in dendritic spines and PSDs, and exerts neuroligin–neurexin interaction at glutamate synapses [Durand et al., 2007]. Both neuroligin and neurexin play critical roles in the formation and functioning of synapses, particularly in the alignment and activation of glutamate and GABA synapses. Chao et al. reported dysfunction in GABA signaling mediated autism-like stereotypies and RTT features in a mouse RTT model [Chao et al., 2010]. GABA may play an important role in RTT phenotype. Studies on ASD revealed the impairments in neuroligin-neurexin pathway responsible for the proper localization and assembly of the PSD due to the mutations of SHANK3, NLG3/4 and contactin associated proteinlike 2 [Boccuto et al., 2013]. The mutations of these genes encoding the assembly proteins of the PSD induce neurodevelopmental disorders resembling RTT [Smeets et al., 2011]. We identified a frameshift single nucleotide deletion (c.3100del, p.Ala1034fs 44) in SHANK3. Recent studies reported, in two siblings heterozygous for a guanine insertion in exon 21 of SHANK3, that the balanced translocation of 12 and 22 chromosomes disrupted exon 21 of SHANK3, and led to the deletion of the homer-binding site enabling homer proteins to bind to SHANK [Bonaglia et al., 2001; Durand et al., 2007]. Although the pathologic mechanism of the lesion is uncertain, later deterioration of mental and motor skills might be important in RTT-like phenotype with SHANK3 mutation. SHANK3 is one of the most GC-rich genes in the genome with the CpG-islands in exon 21 [Durand et al., 2007; Betancur and Buxbaum, 2013]. GC-rich regions are conserved between human and mouse [Durand et al., 2007]. In mouse brain, synaptogenesis occurs during the first 2 weeks after birth, according to the elevation of the methylation rate of CpG-islands [Uchino and Waga, 2013]. Waga and co-workers revealed by ChIP assay that Mecp2 bound to methylated CpG-islands of Shank3 and that the expression levels of the variable Shank3 transcripts in Mecp2 null mice differed from those of wild-type mice in neocortical tissue, especially from P14 to P28 [Waga et al., 2014]. Taken together, synaptogenesis of the patients with RTT at the early developmental stage may be vulnerable to aberrant SHANK3 transcripts affected by the MeCP2
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dysfunction. The precise mechanisms of SHANK3 abnormality leading to RTT-like phenotype still remain elusive. We identify a de novo SHANK3 mutation in a patient with RTTlike phenotype. Our data suggest that disturbance of SHANK3Homer interaction may be one of the critical steps associated with RTT-like phenotype.
Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsa¨ter H, Sponheim E, Goubran-Botros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E, Roge´ B, He´ron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T. 2007. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet 39:25–27.
ACKNOWLEDGMENTS
Hagberg B, Aicardi J, Dias K, Ramos O. 1983. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: Report of 35 cases. Ann Neurol 14:471–479.
This work was partly supported by Grants-in-Aid for Scientific Research (no. 30389283 to M.H., no. 13313587 to N.M., and no. 21591338 to T.M.) from the Japan Society for the Promotion of Science; the Strategic Research Program for Brain Sciences (no. 11105137 to N.M.); a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) (12024421 to N.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a research grant from the Ministry of Health, Labor and Welfare of Japan (Intramural Research Grant [21B-5] for Neurological and Psychiatric Disorders of NCNP; Research on Applying Health Technology (12944231 to N.M.); Comprehensive Research on Disability Health and Welfare (13802019 to N.M.); and Research on Intractable Diseases (21-110 and 22-133 to T.M.).
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
