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Two males with sick sinus syndrome in a family with 0.6 kb deletions involving major domains in MECP2
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Two males with sick sinus syndrome in a family with 0.6 kb deletions involving major domains in MECP2 Takehiko Inuia,∗,1, Kazuhiro Iwamab,c,1, Takuya Miyabayashia, Ryo Satoa, Yukimune Okuboa, Wakaba Endod, Noriko Togashia, Yosuke Kakisakad, Atsuo Kikuchid, Takeshi Mizuguchib, Shigeo Kured, Naomichi Matsumotob, Kazuhiro Haginoyaa,d a
Department of Pediatric Neurology, Miyagi Children's Hospital, 4-3-17 Ochiai, Aoba-ku, Sendai-shi, Miyagi, 989-3126, Japan Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan c Department of Pediatrics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan d Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: MECP2 Sick sinus syndrome Autonomic nervous system dysregulation Whole-exome sequencing Nord's method Large deletion
Mutations in methyl-CpG-binding protein 2 (MECP2) in males can lead to various phenotypes, ranging from neonatal encephalopathy to intellectual disability. In this study, using Nord's method of next-generation sequencing in three siblings, we identified a 0.6 kb deletion involving the transcriptional repression domain (TRD). Two males and one female had intellectual disability and apnea, but none met the criteria of Rett syndrome. Both males had sick sinus syndrome and severe tracheomalacia that resulted in early death. The mother, with skewed X-inactivation, had no symptoms. Therefore, this mutation is pathological for both males and females, resulting in sick sinus syndrome and severe tracheomalacia with strong reproducibility in males. Deletions involving major domains in MECP2 can result in a severe phenotype, and deletion of the TRD domain can cause severe autonomic nervous system dysregulation in males in these cases.
1. Introduction Rett syndrome (OMIM 312750) is an X-linked neurodevelopmental disorder presenting almost exclusively in females. Its prevalence is approximately 1 in 10000 females (Leonard et al., 2017). The clinical course of the disease consists of a normal neonatal period, followed by an arrest of development between 6 and 18 months of age. Individuals have clinical signs indicating a neurodevelopmental defect: arrested brain development, regression of acquired skills, and stereotypic hand movements (Neul et al., 2010). Individuals also show clinical signs indicating autonomic nervous system dysregulation (ANSD): gastroesophageal reflux, peripheral vasomotor disturbances, hyperventilation, apnea in an awake state, abnormal blood pressure response, and arrhythmia (Leonard et al., 2017; Axelrod et al., 2006). ANSD is suggested as the cause of sudden death (Axelrod et al., 2006). Mutations in the methyl CpG-binding protein2 (MECP2) gene were identified in 1999 in females affected by Rett syndrome (Amir et al., 1999). Subsequently, the MeCP2 protein was revealed to have three major domains: a methyl-binding domain (MBD), which binds
specifically to DNA-containing methylated ChG (Nan et al., 1993), a nuclear localization sequence (NLS) allowing trafficking of MECP2 to the nucleus (Nan et al., 1996), and a transcription-repression domain (TRD), which is responsible for MECP2-mediated repression (Nan et al., 1998). To date, 519 pathological MECP2 mutations are deposited in the International Rett database (Krishnaraj et al., 2017). In females, the nature of the variant and the position in MECP2 seem to predict the phenotype. Individuals with Arg133Cys, Arg294X, and C-terminal deletions tend to have milder phenotypes, whereas those with Arg270X, Arg255X, early truncating mutations, and large deletions tend to have more severe phenotypes (Cuddapah et al., 2014). MECP2 mutations are rare in males because de novo mutations in MECP2, especially the deamination of methylated cytosines, occur mainly in sperm, and such mutations cannot be transmitted to a male (Girard et al., 2001). In one study of more than 1200 individuals with MECP2 mutations and duplications, MECP2 mutation were seen only in 30 males (Neul et al., 2019). In this study, the clinical presentation ranged from cognitive impairment to progressive encephalopathy. Regarding Rett-causing point mutations and small deletions, some
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Corresponding author. E-mail address: [email protected] (T. Inui). 1 Takehiko Inui and Kazuhiro Iwama should be considered joint first author. https://doi.org/10.1016/j.ejmg.2019.103769 Received 16 March 2019; Received in revised form 5 September 2019; Accepted 15 September 2019 1769-7212/ © 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Takehiko Inui, et al., European Journal of Medical Genetics, https://doi.org/10.1016/j.ejmg.2019.103769
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referred to our hospital at 6 months for developmental delay. Physical examination revealed muscle weakness, apnea when awake and asleep, and microcephaly (40.3 cm, −2.3 SD). No facial dimorphism was observed. At 1 year, she was able to sit alone, and her development was stagnant. At present, she is 7 years old and can neither stand unaided nor speak meaningful words. She did not develop purposeful hand skills, spoken language, or vocal communication. She did not show regression or apparent stereotypic hand movement. II:2, A healthy 11-year-old girl.
genotype–phenotype correlation is observed (Neul et al., 2019). In this study, we describe three familial cases with 635 base pair deletions involving the TRD domain in MECP2. All individuals showed severe intellectual disability but did not fulfill the criteria for Rett syndrome. Two males showed severe autonomic dysregulation, including gastroesophageal reflux, sinus arrest, and tracheomalacia, resulting in early death. A thorough clinical description of these three individuals enables discussion of the phenotypic effect of this mutation. 2. Clinical reports (Fig. 1A)
3. Materials and methods II:1, A 1-year-old boy was born to healthy, non-consanguineous Japanese parents. He was born normally at 38 weeks of gestation, with a birth weight and head circumference of 2590 g (−0.7 SD) and 32 cm (−0.7 SD), respectively. He presented with muscle weakness and poor sucking, and tube nutrition was started in the neonatal period. At 1 month, he presented with recurrent vomiting and frequent aspiration pneumonia. He was referred to our hospital at 8 months for developmental delay. Physical examination revealed muscle weakness, central apnea when awake and asleep, and microcephaly (39 cm, −4.0 SD). No facial dysmorphism was observed. Esophageal 24 h pH monitoring showed severe gastroesophageal reflux. Brain magnetic resonance imaging at 10 months was normal. At 1 year and 1 month, he suffered from severe aspiration pneumonia requiring mechanical ventilation. Extubation failure resulted in tracheostomy placement. After tracheostomy placement, he had tracheal rhonchi, cough, and apneic spells. Bronchoscopy showed tracheomalacia (Fig. 2A). It was so severe that he required prolonged mechanical ventilation with high positiveend expiratory pressure (PEEP) and frequent sedation. At 1 year and 6 months, he developed sudden sinus arrest with decreased blood pressure and loss of consciousness, and was diagnosed with sick sinus syndrome (Fig. 2B). Atropine was effective for the bradycardia, but its effects were short-lasting. He required sedation to ameliorate tracheomalacia, but sedation aggravated sick sinus syndrome. His vital signs became unstable, his consciousness became impaired, and he suffered from repetitive pneumonia. At 1 year and 9 months, he died of pneumonia. II:4, A 3-year-old boy was born normally at 39 weeks of gestation. His birth weight and head circumference were 2740 g (−1.0 SD) and 32.5 cm (−0.5 SD), respectively. He presented with muscle weakness and poor sucking, and tube nutrition was started in the neonatal period. He was referred to our hospital at 3 months for developmental delay. Physical examination revealed muscle weakness and central apnea when awake and asleep. No facial dysmorphism was observed. At 4 months, he developed recurrent vomiting and dry cough. Esophageal 24 h pH monitoring showed severe gastroesophageal reflux. After nasojejunal tube was placed, the vomiting and dry cough disappeared, which suggests amelioration of gastroesophageal reflux. Brain computed tomography at 4 months and electroencephalogram at 6 months were normal. At 8 months, microcephaly became apparent (39 cm, −4.0 SD). At 10 months, he suffered from severe aspiration pneumonia requiring mechanical ventilation. While under intensive care, he developed sudden sinus arrest with decreased blood pressure and loss of consciousness, and was diagnosed with sick sinus syndrome (Fig. 2C). A pacemaker was placed. Extubation failure resulted in tracheostomy placement. After tracheostomy placement, he had tracheal rhonchi, cough, and apneic spells. Bronchoscopy showed tracheomalacia. He required prolonged mechanical ventilation with high PEEP and sedation. At 3 years, he died of septic shock. An abdominal x-ray showed a dilated sigmoid colon. Because the septic shock developed suddenly, and no other infection focus was detected, the bacteria were thought to have been derived from the sigmoid colon. II:3, A 7-year-old girl was born normally at 39 weeks of gestation, with a birth weight and head circumference of 2806 g (−0.5 SD) and 32.4 cm (−0.6 SD), respectively. She showed muscle weakness and frequent vomiting from early infancy but could feed well. She was
3.1. Individual information We observed three familial cases with a suspected hereditary intractable disease at Miyagi Children's Hospital. Written informed consent was obtained from the parents for diagnostic procedures, nextgeneration sequencing, and publication of this case report. The study was approved by the ethics review board of Miyagi Children's Hospital (No. 287). 3.2. Whole-exome sequencing (WES) and Sanger sequencing The genomic DNA of two affected individuals (II-3 and II-4) was isolated from saliva, captured using the SureSelect XT Human All Exon v5 Kit (50 Mb; Agilent Technologies, Santa Clara, CA, USA), and sequenced on an Illumina HiSeq2000 or 2500 (Illumina, San Diego, CA, USA) with 101 bp paired-end reads. Data processing, variant calling, annotation, and filtering of single nucleotide variants (SNVs) were performed as previously described (Iwama et al., 2018). The average read depth of the protein-coding regions ranged from 98.7× to 101×, and at least 96.3% of the target bases were sequenced by 10 or more reads. Copy number variation (CNV) was investigated using two bioinformatics tools (Tsuchida et al., 2018): eXome Hidden Markov Model (XHMM) (Fromer et al., 2012) and Nord's method, which combines the relative depth of coverage and scanning for partially mapped reads (Nord et al., 2011). The Nord program targeted more than 250 genes reported to cause neurodevelopmental disorders. The deletion was confirmed by polymerase chain reaction (PCR) and Sanger sequencing with an ABI PRISM 3500xl Autosequencer (Life Technologies, Carlsbad, CA, USA) using genomic DNA from the individuals and their family members as PCR template. 3.3. X-chromosome inactivation (XCI) analysis The X-chromosome inactivation pattern was investigated using the human androgen receptor (HUMARA) assay and fragile X methylation locus assay (FRAXA), as previously described (Kondo et al., 2012). Genomic DNA from the affected girl (II-3) and her healthy mother (I-2) was digested with two methylation-sensitive enzymes, HpaII and HhaI. PCR was performed using FAM-labeled primer sets (Supplementary Table S1). Fluorescent-labeled products were analyzed on an ABI PRISM 3130xl Genetic Analyzer using GeneMapper Software (Applied Biosystems, Foster City, CA, USA). 4. Results 4.1. Genetic testing No candidates were found by usual filtering for SNVs and XHMM for CNVs. Nord's method revealed that the affected sister (II-3) harbored a partial deletion in MECP2 (Fig. 1B). The integrative genomics viewer indicated some discordant read pairs with an abnormal insert size (Fig. 1C). Sanger sequencing revealed a 635 base pair deletion (NM_004992.3:c.559_1193del, p.Gly187Profs*6) in two affected individuals (II-3 and II-4) and their healthy mother (I-2) (Fig. 1D). 2
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Fig. 1. Copy number variations analyzed by whole-exome sequencing data. (A) Familial pedigree with phenotypes and genotypes shown. Right: gray symbols indicate a developmental delay, microcephaly and apnea, which appear regardless of gender. Left: gray symbols indicate sick sinus syndrome, tracheomalasia and gastroesophageal reflux (only males). WT and DEL indicate the wild-type allele and a 635-bp deletion, respectively. NA indicates “not analyzed” (II-1). (B) The relative coverage ratio with Nord's method revealed that the affected sister (II-3) harbored a partial deletion (red arrow) in MECP2 (highlighted in brown). (C) Integrative genomics viewer (IGV) revealed discordant read pairs with an abnormal insert size (burgundy thin line). IGV indicated that the deletion involved exon 4 of MECP2 (NM_004992.3) (D) Sanger sequencing revealed a 635-bp deletion (c.559_1193del, p.Gly187Profs*6) in the affected male (II-4, hemizygous), the affected female (II-3, heterozygous), and their healthy mother (I-2, heterozygous). The father (I-1) and a healthy sister (II-2) harbor the wild-type sequence. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
5. Discussion
HUMARA analysis revealed that the healthy mother and the affected female showed a markedly skewed (99:1) and random inactivation pattern (60:40), respectively (FRAXA analysis was not informative). This result suggests that favorable XCI skewing prevented the affected individual's mother from disease conditions.
We presented three familial cases with a 635 base pair deletion in exon 4 of MECP2. We predict that this deletion is pathological because the 635 base pair deletion causes a frameshift and loses nearly one half of coding sequence of MECP2, including two major domains, the TRD and the NLS. No individual met the diagnostic criteria of Rett syndrome (Neul et al., 2010). Both males developed apnea, gastroesophageal reflux, sick sinus syndrome, and tracheomalacia, some of which was serious. Some of these symptoms, including apnea, gastroesophageal reflux, sick sinus syndrome, and tracheomalacia, can occur in other congenital syndromes, including congenital central hypoventilation syndrome,
4.2. Deposition of genetic data The data obtained in this study were submitted to the Leiden Open Variation Database (LOVD), an online gene-centered collection and display of DNA variation (Patient ID 00238978 Variant ID 0000484081). 3
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atropine (Herrera et al., 2016). Based on these similarities, the sinus pauses in these cases were thought to result from the dysfunction of cholinergic neurons in central nervous system. Individuals with Rett syndrome have a high incidence of sudden unexpected death (26% of all deaths), which is believed to be of cardiac origin (Kerr et al., 1997). Although both animals and humans with MECP2 mutations often exhibit a long QTc (Guideri et al., 1999; McCauley et al., 2011; Herrera et al., 2015), more cases are needed to assess the significance of sinus dysfunction in the prognosis of Rett syndrome and MECP2 males. Both males had severe tracheomalacia, which required high PEEP and frequent sedation (Fraga et al., 2016). In II-1, frequent gastroesophageal reflux, vomiting and aspiration pneumonia was seen from infancy, and tracheomalacia emerged at 1 year. In II-4, although the nasojejunal tube prevented vomiting, gastroesophageal reflux, and frequent aspiration pneumonia, severe tracheomalacia emerged at 10 months. Because removal of the aggravating cause could not prevent tracheomalacia, it is plausible that a common congenital factor influenced this presentation. Several cases with MECP2 duplication developed tracheomalacia (Sanlaville et al., 2005; del Gaudio et al., 2006), which indicates that MECP2 dysfunction makes patients susceptible to tracheomalacia. In MECP2, many mutations are seen in either males or females (Neul et al., 2019), and some mutations show a range of clinical presentations (Schönewolf-Greulich et al., 2016). In our cases, two males showed a very similar clinical course; the female showed a milder presentation, and the mother, with skewed inactivation, had no symptoms. These results indicate that this mutation is pathological for both males and females, is more severe for males, and shows strong reproducibility. The MECP2 protein harbors three major domains: the MBD, NLS, and TRD domains. All three domains are essential for MECP2 function, and point mutations in these domains tend to cause typical Rett syndrome in females (Cuddapah et al., 2014). However, it is not clear whether deletions including major domains correlate with the phenotype. So far, only two other males with a large deletion in MECP2 have been reported. One case involved a deletion including the MBD, NLS, and TRD domains and showed neonatal encephalopathy. The other case involved a deletion from the from the C-terminus to the 3’ untranslated region and showed autism with muscle weakness (Table 1) (Hardwick et al., 2007; Wang et al., 2012). Given these facts, it is plausible that a deletion involving a major domain causes the severe phenotype and that the deletion of the NLS and TRD domains was correlated with severe ANSD in this case. In conclusion, this report describes the clinical aspects of individuals with a deletion involving major domains in MECP2. Autonomic dysregulation may be a unique presentation in this mutation, although further cases are required to confirm this finding.
Fig. 2. (A) Bronchoscopy finding of main carina and main bronchial branches of Ⅱ:1. The diagram represents the individual's trachea and bronchi. (1): Trachea from tracheal cannula. (2): Left main bronchi. (3) Left lower lobe bronchi. (4): Right main bronchi. Severe tracheomalacia and bronchomalacia are shown. (B) Electrocardiogram of Ⅱ:1. Sinus arrest for 6.0 s is shown. (C) Electrocardiogram of Ⅱ:4. Sinus arrest for 4.7 s is shown.
Pitt–Hopkins syndrome, and Prader–Willi syndrome as ANSD symptoms (Axelrod et al., 2006; Gallego, 2012). Our cases could be differentiated in terms of these syndromes by the following: [1] apnea that was present both awake and asleep, [2] no hyperventilation that proceeded apnea, [3] presentation with severe intellectual disability and microcephaly, and [4] mild presentation in the female sibling. No mutations were detected upon sequencing of PHOX2B, TCF4, and SNRPN. Because the syndromic sinus pauses were sustained more than 3 s, we diagnosed these males with sick sinus syndrome according to the 2018 ACC/AHA/HRS Guidelines (Kusumoto et al., 2018). Although bradycardia is often reported in both males and females with MECP2 mutations (Madan et al., 2004; Jülich et al., 2009), sick sinus syndrome is extremely rare (Shioda et al., 2018). Interestingly, however, the courses of these cases resembled that seen in Mecp2 mutant mice, with sudden sinus pauses, worsening with aging, and a transient response to
Conflicts of interest The authors declare no conflict of interest.
Table 1 Genetic testing and clinical presentation with a large deletion of MECP2 in males.
Age Deletion region Size Including exon(s) Deletion site Clinical feature
Case 1 Hardwick et al.)
Case 2 Wang et al.)
These cases
1y ChrX:153,296,172 −15,33,04,647 8.4 kbp Exons 3,4 MBD, NLS, TRD Neonatal encephalopathy
8y ChrX:153,294,275 −15,32,96,120 1.8 kbp Exon 4 C-terminus Autism
1y ChrX 153,296,086 −15,32,96,720 635 bp Exon 4 NLS,TRD Autosomal nervous system dysregulation
Deletion regions described based on GRCh37/hg19. Abbreviations: MBD, methyl-binding domain; MECP2, methyl-CpG-binding protein 2; NLS, nuclear localization sequence. TRD, transcriptional repression domain; y, year(s). 4
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Acknowledgments
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Two males with sick sinus syndrome in a family with 0.6 kb deletions involving major domains in MECP2 Takehiko Inuia,∗,1, Kazuhiro Iwamab,c,1, Takuya Miyabayashia, Ryo Satoa, Yukimune Okuboa, Wakaba Endod, Noriko Togashia, Yosuke Kakisakad, Atsuo Kikuchid, Takeshi Mizuguchib, Shigeo Kured, Naomichi Matsumotob, Kazuhiro Haginoyaa,d a
Department of Pediatric Neurology, Miyagi Children's Hospital, 4-3-17 Ochiai, Aoba-ku, Sendai-shi, Miyagi, 989-3126, Japan Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan c Department of Pediatrics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan d Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: MECP2 Sick sinus syndrome Autonomic nervous system dysregulation Whole-exome sequencing Nord's method Large deletion
Mutations in methyl-CpG-binding protein 2 (MECP2) in males can lead to various phenotypes, ranging from neonatal encephalopathy to intellectual disability. In this study, using Nord's method of next-generation sequencing in three siblings, we identified a 0.6 kb deletion involving the transcriptional repression domain (TRD). Two males and one female had intellectual disability and apnea, but none met the criteria of Rett syndrome. Both males had sick sinus syndrome and severe tracheomalacia that resulted in early death. The mother, with skewed X-inactivation, had no symptoms. Therefore, this mutation is pathological for both males and females, resulting in sick sinus syndrome and severe tracheomalacia with strong reproducibility in males. Deletions involving major domains in MECP2 can result in a severe phenotype, and deletion of the TRD domain can cause severe autonomic nervous system dysregulation in males in these cases.
1. Introduction Rett syndrome (OMIM 312750) is an X-linked neurodevelopmental disorder presenting almost exclusively in females. Its prevalence is approximately 1 in 10000 females (Leonard et al., 2017). The clinical course of the disease consists of a normal neonatal period, followed by an arrest of development between 6 and 18 months of age. Individuals have clinical signs indicating a neurodevelopmental defect: arrested brain development, regression of acquired skills, and stereotypic hand movements (Neul et al., 2010). Individuals also show clinical signs indicating autonomic nervous system dysregulation (ANSD): gastroesophageal reflux, peripheral vasomotor disturbances, hyperventilation, apnea in an awake state, abnormal blood pressure response, and arrhythmia (Leonard et al., 2017; Axelrod et al., 2006). ANSD is suggested as the cause of sudden death (Axelrod et al., 2006). Mutations in the methyl CpG-binding protein2 (MECP2) gene were identified in 1999 in females affected by Rett syndrome (Amir et al., 1999). Subsequently, the MeCP2 protein was revealed to have three major domains: a methyl-binding domain (MBD), which binds
specifically to DNA-containing methylated ChG (Nan et al., 1993), a nuclear localization sequence (NLS) allowing trafficking of MECP2 to the nucleus (Nan et al., 1996), and a transcription-repression domain (TRD), which is responsible for MECP2-mediated repression (Nan et al., 1998). To date, 519 pathological MECP2 mutations are deposited in the International Rett database (Krishnaraj et al., 2017). In females, the nature of the variant and the position in MECP2 seem to predict the phenotype. Individuals with Arg133Cys, Arg294X, and C-terminal deletions tend to have milder phenotypes, whereas those with Arg270X, Arg255X, early truncating mutations, and large deletions tend to have more severe phenotypes (Cuddapah et al., 2014). MECP2 mutations are rare in males because de novo mutations in MECP2, especially the deamination of methylated cytosines, occur mainly in sperm, and such mutations cannot be transmitted to a male (Girard et al., 2001). In one study of more than 1200 individuals with MECP2 mutations and duplications, MECP2 mutation were seen only in 30 males (Neul et al., 2019). In this study, the clinical presentation ranged from cognitive impairment to progressive encephalopathy. Regarding Rett-causing point mutations and small deletions, some
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Corresponding author. E-mail address: [email protected] (T. Inui). 1 Takehiko Inui and Kazuhiro Iwama should be considered joint first author. https://doi.org/10.1016/j.ejmg.2019.103769 Received 16 March 2019; Received in revised form 5 September 2019; Accepted 15 September 2019 1769-7212/ © 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Takehiko Inui, et al., European Journal of Medical Genetics, https://doi.org/10.1016/j.ejmg.2019.103769
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referred to our hospital at 6 months for developmental delay. Physical examination revealed muscle weakness, apnea when awake and asleep, and microcephaly (40.3 cm, −2.3 SD). No facial dimorphism was observed. At 1 year, she was able to sit alone, and her development was stagnant. At present, she is 7 years old and can neither stand unaided nor speak meaningful words. She did not develop purposeful hand skills, spoken language, or vocal communication. She did not show regression or apparent stereotypic hand movement. II:2, A healthy 11-year-old girl.
genotype–phenotype correlation is observed (Neul et al., 2019). In this study, we describe three familial cases with 635 base pair deletions involving the TRD domain in MECP2. All individuals showed severe intellectual disability but did not fulfill the criteria for Rett syndrome. Two males showed severe autonomic dysregulation, including gastroesophageal reflux, sinus arrest, and tracheomalacia, resulting in early death. A thorough clinical description of these three individuals enables discussion of the phenotypic effect of this mutation. 2. Clinical reports (Fig. 1A)
3. Materials and methods II:1, A 1-year-old boy was born to healthy, non-consanguineous Japanese parents. He was born normally at 38 weeks of gestation, with a birth weight and head circumference of 2590 g (−0.7 SD) and 32 cm (−0.7 SD), respectively. He presented with muscle weakness and poor sucking, and tube nutrition was started in the neonatal period. At 1 month, he presented with recurrent vomiting and frequent aspiration pneumonia. He was referred to our hospital at 8 months for developmental delay. Physical examination revealed muscle weakness, central apnea when awake and asleep, and microcephaly (39 cm, −4.0 SD). No facial dysmorphism was observed. Esophageal 24 h pH monitoring showed severe gastroesophageal reflux. Brain magnetic resonance imaging at 10 months was normal. At 1 year and 1 month, he suffered from severe aspiration pneumonia requiring mechanical ventilation. Extubation failure resulted in tracheostomy placement. After tracheostomy placement, he had tracheal rhonchi, cough, and apneic spells. Bronchoscopy showed tracheomalacia (Fig. 2A). It was so severe that he required prolonged mechanical ventilation with high positiveend expiratory pressure (PEEP) and frequent sedation. At 1 year and 6 months, he developed sudden sinus arrest with decreased blood pressure and loss of consciousness, and was diagnosed with sick sinus syndrome (Fig. 2B). Atropine was effective for the bradycardia, but its effects were short-lasting. He required sedation to ameliorate tracheomalacia, but sedation aggravated sick sinus syndrome. His vital signs became unstable, his consciousness became impaired, and he suffered from repetitive pneumonia. At 1 year and 9 months, he died of pneumonia. II:4, A 3-year-old boy was born normally at 39 weeks of gestation. His birth weight and head circumference were 2740 g (−1.0 SD) and 32.5 cm (−0.5 SD), respectively. He presented with muscle weakness and poor sucking, and tube nutrition was started in the neonatal period. He was referred to our hospital at 3 months for developmental delay. Physical examination revealed muscle weakness and central apnea when awake and asleep. No facial dysmorphism was observed. At 4 months, he developed recurrent vomiting and dry cough. Esophageal 24 h pH monitoring showed severe gastroesophageal reflux. After nasojejunal tube was placed, the vomiting and dry cough disappeared, which suggests amelioration of gastroesophageal reflux. Brain computed tomography at 4 months and electroencephalogram at 6 months were normal. At 8 months, microcephaly became apparent (39 cm, −4.0 SD). At 10 months, he suffered from severe aspiration pneumonia requiring mechanical ventilation. While under intensive care, he developed sudden sinus arrest with decreased blood pressure and loss of consciousness, and was diagnosed with sick sinus syndrome (Fig. 2C). A pacemaker was placed. Extubation failure resulted in tracheostomy placement. After tracheostomy placement, he had tracheal rhonchi, cough, and apneic spells. Bronchoscopy showed tracheomalacia. He required prolonged mechanical ventilation with high PEEP and sedation. At 3 years, he died of septic shock. An abdominal x-ray showed a dilated sigmoid colon. Because the septic shock developed suddenly, and no other infection focus was detected, the bacteria were thought to have been derived from the sigmoid colon. II:3, A 7-year-old girl was born normally at 39 weeks of gestation, with a birth weight and head circumference of 2806 g (−0.5 SD) and 32.4 cm (−0.6 SD), respectively. She showed muscle weakness and frequent vomiting from early infancy but could feed well. She was
3.1. Individual information We observed three familial cases with a suspected hereditary intractable disease at Miyagi Children's Hospital. Written informed consent was obtained from the parents for diagnostic procedures, nextgeneration sequencing, and publication of this case report. The study was approved by the ethics review board of Miyagi Children's Hospital (No. 287). 3.2. Whole-exome sequencing (WES) and Sanger sequencing The genomic DNA of two affected individuals (II-3 and II-4) was isolated from saliva, captured using the SureSelect XT Human All Exon v5 Kit (50 Mb; Agilent Technologies, Santa Clara, CA, USA), and sequenced on an Illumina HiSeq2000 or 2500 (Illumina, San Diego, CA, USA) with 101 bp paired-end reads. Data processing, variant calling, annotation, and filtering of single nucleotide variants (SNVs) were performed as previously described (Iwama et al., 2018). The average read depth of the protein-coding regions ranged from 98.7× to 101×, and at least 96.3% of the target bases were sequenced by 10 or more reads. Copy number variation (CNV) was investigated using two bioinformatics tools (Tsuchida et al., 2018): eXome Hidden Markov Model (XHMM) (Fromer et al., 2012) and Nord's method, which combines the relative depth of coverage and scanning for partially mapped reads (Nord et al., 2011). The Nord program targeted more than 250 genes reported to cause neurodevelopmental disorders. The deletion was confirmed by polymerase chain reaction (PCR) and Sanger sequencing with an ABI PRISM 3500xl Autosequencer (Life Technologies, Carlsbad, CA, USA) using genomic DNA from the individuals and their family members as PCR template. 3.3. X-chromosome inactivation (XCI) analysis The X-chromosome inactivation pattern was investigated using the human androgen receptor (HUMARA) assay and fragile X methylation locus assay (FRAXA), as previously described (Kondo et al., 2012). Genomic DNA from the affected girl (II-3) and her healthy mother (I-2) was digested with two methylation-sensitive enzymes, HpaII and HhaI. PCR was performed using FAM-labeled primer sets (Supplementary Table S1). Fluorescent-labeled products were analyzed on an ABI PRISM 3130xl Genetic Analyzer using GeneMapper Software (Applied Biosystems, Foster City, CA, USA). 4. Results 4.1. Genetic testing No candidates were found by usual filtering for SNVs and XHMM for CNVs. Nord's method revealed that the affected sister (II-3) harbored a partial deletion in MECP2 (Fig. 1B). The integrative genomics viewer indicated some discordant read pairs with an abnormal insert size (Fig. 1C). Sanger sequencing revealed a 635 base pair deletion (NM_004992.3:c.559_1193del, p.Gly187Profs*6) in two affected individuals (II-3 and II-4) and their healthy mother (I-2) (Fig. 1D). 2
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Fig. 1. Copy number variations analyzed by whole-exome sequencing data. (A) Familial pedigree with phenotypes and genotypes shown. Right: gray symbols indicate a developmental delay, microcephaly and apnea, which appear regardless of gender. Left: gray symbols indicate sick sinus syndrome, tracheomalasia and gastroesophageal reflux (only males). WT and DEL indicate the wild-type allele and a 635-bp deletion, respectively. NA indicates “not analyzed” (II-1). (B) The relative coverage ratio with Nord's method revealed that the affected sister (II-3) harbored a partial deletion (red arrow) in MECP2 (highlighted in brown). (C) Integrative genomics viewer (IGV) revealed discordant read pairs with an abnormal insert size (burgundy thin line). IGV indicated that the deletion involved exon 4 of MECP2 (NM_004992.3) (D) Sanger sequencing revealed a 635-bp deletion (c.559_1193del, p.Gly187Profs*6) in the affected male (II-4, hemizygous), the affected female (II-3, heterozygous), and their healthy mother (I-2, heterozygous). The father (I-1) and a healthy sister (II-2) harbor the wild-type sequence. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
5. Discussion
HUMARA analysis revealed that the healthy mother and the affected female showed a markedly skewed (99:1) and random inactivation pattern (60:40), respectively (FRAXA analysis was not informative). This result suggests that favorable XCI skewing prevented the affected individual's mother from disease conditions.
We presented three familial cases with a 635 base pair deletion in exon 4 of MECP2. We predict that this deletion is pathological because the 635 base pair deletion causes a frameshift and loses nearly one half of coding sequence of MECP2, including two major domains, the TRD and the NLS. No individual met the diagnostic criteria of Rett syndrome (Neul et al., 2010). Both males developed apnea, gastroesophageal reflux, sick sinus syndrome, and tracheomalacia, some of which was serious. Some of these symptoms, including apnea, gastroesophageal reflux, sick sinus syndrome, and tracheomalacia, can occur in other congenital syndromes, including congenital central hypoventilation syndrome,
4.2. Deposition of genetic data The data obtained in this study were submitted to the Leiden Open Variation Database (LOVD), an online gene-centered collection and display of DNA variation (Patient ID 00238978 Variant ID 0000484081). 3
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atropine (Herrera et al., 2016). Based on these similarities, the sinus pauses in these cases were thought to result from the dysfunction of cholinergic neurons in central nervous system. Individuals with Rett syndrome have a high incidence of sudden unexpected death (26% of all deaths), which is believed to be of cardiac origin (Kerr et al., 1997). Although both animals and humans with MECP2 mutations often exhibit a long QTc (Guideri et al., 1999; McCauley et al., 2011; Herrera et al., 2015), more cases are needed to assess the significance of sinus dysfunction in the prognosis of Rett syndrome and MECP2 males. Both males had severe tracheomalacia, which required high PEEP and frequent sedation (Fraga et al., 2016). In II-1, frequent gastroesophageal reflux, vomiting and aspiration pneumonia was seen from infancy, and tracheomalacia emerged at 1 year. In II-4, although the nasojejunal tube prevented vomiting, gastroesophageal reflux, and frequent aspiration pneumonia, severe tracheomalacia emerged at 10 months. Because removal of the aggravating cause could not prevent tracheomalacia, it is plausible that a common congenital factor influenced this presentation. Several cases with MECP2 duplication developed tracheomalacia (Sanlaville et al., 2005; del Gaudio et al., 2006), which indicates that MECP2 dysfunction makes patients susceptible to tracheomalacia. In MECP2, many mutations are seen in either males or females (Neul et al., 2019), and some mutations show a range of clinical presentations (Schönewolf-Greulich et al., 2016). In our cases, two males showed a very similar clinical course; the female showed a milder presentation, and the mother, with skewed inactivation, had no symptoms. These results indicate that this mutation is pathological for both males and females, is more severe for males, and shows strong reproducibility. The MECP2 protein harbors three major domains: the MBD, NLS, and TRD domains. All three domains are essential for MECP2 function, and point mutations in these domains tend to cause typical Rett syndrome in females (Cuddapah et al., 2014). However, it is not clear whether deletions including major domains correlate with the phenotype. So far, only two other males with a large deletion in MECP2 have been reported. One case involved a deletion including the MBD, NLS, and TRD domains and showed neonatal encephalopathy. The other case involved a deletion from the from the C-terminus to the 3’ untranslated region and showed autism with muscle weakness (Table 1) (Hardwick et al., 2007; Wang et al., 2012). Given these facts, it is plausible that a deletion involving a major domain causes the severe phenotype and that the deletion of the NLS and TRD domains was correlated with severe ANSD in this case. In conclusion, this report describes the clinical aspects of individuals with a deletion involving major domains in MECP2. Autonomic dysregulation may be a unique presentation in this mutation, although further cases are required to confirm this finding.
Fig. 2. (A) Bronchoscopy finding of main carina and main bronchial branches of Ⅱ:1. The diagram represents the individual's trachea and bronchi. (1): Trachea from tracheal cannula. (2): Left main bronchi. (3) Left lower lobe bronchi. (4): Right main bronchi. Severe tracheomalacia and bronchomalacia are shown. (B) Electrocardiogram of Ⅱ:1. Sinus arrest for 6.0 s is shown. (C) Electrocardiogram of Ⅱ:4. Sinus arrest for 4.7 s is shown.
Pitt–Hopkins syndrome, and Prader–Willi syndrome as ANSD symptoms (Axelrod et al., 2006; Gallego, 2012). Our cases could be differentiated in terms of these syndromes by the following: [1] apnea that was present both awake and asleep, [2] no hyperventilation that proceeded apnea, [3] presentation with severe intellectual disability and microcephaly, and [4] mild presentation in the female sibling. No mutations were detected upon sequencing of PHOX2B, TCF4, and SNRPN. Because the syndromic sinus pauses were sustained more than 3 s, we diagnosed these males with sick sinus syndrome according to the 2018 ACC/AHA/HRS Guidelines (Kusumoto et al., 2018). Although bradycardia is often reported in both males and females with MECP2 mutations (Madan et al., 2004; Jülich et al., 2009), sick sinus syndrome is extremely rare (Shioda et al., 2018). Interestingly, however, the courses of these cases resembled that seen in Mecp2 mutant mice, with sudden sinus pauses, worsening with aging, and a transient response to
Conflicts of interest The authors declare no conflict of interest.
Table 1 Genetic testing and clinical presentation with a large deletion of MECP2 in males.
Age Deletion region Size Including exon(s) Deletion site Clinical feature
Case 1 Hardwick et al.)
Case 2 Wang et al.)
These cases
1y ChrX:153,296,172 −15,33,04,647 8.4 kbp Exons 3,4 MBD, NLS, TRD Neonatal encephalopathy
8y ChrX:153,294,275 −15,32,96,120 1.8 kbp Exon 4 C-terminus Autism
1y ChrX 153,296,086 −15,32,96,720 635 bp Exon 4 NLS,TRD Autosomal nervous system dysregulation
Deletion regions described based on GRCh37/hg19. Abbreviations: MBD, methyl-binding domain; MECP2, methyl-CpG-binding protein 2; NLS, nuclear localization sequence. TRD, transcriptional repression domain; y, year(s). 4
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Acknowledgments
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