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A novel stop mutation in the EDNRB gene in a family with Hirschsprung’s disease associated with Multiple Sclerosis
Journal of Pediatric Surgery 49 (2014) 622–625
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
A novel stop mutation in the EDNRB gene in a family with Hirschsprung’s disease associated with Multiple Sclerosis Anna Löf Granström a, b, Ellen Markljung b, Katharina Fink c, d, Edvard Nordenskjöld b, Daniel Nilsson e, f, Tomas Wester a, b,⁎, Agneta Nordenskjöld a, b a
Division for Pediatric Surgery, Astrid Lindgren Children's Hospital, Karolinska, University Hospital, Stockholm, Sweden Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden d Department of Neurology Huddinge, Karolinska University Hospital, Stockholm, Sweden e Science for Life Laboratory, Department of Cell and Molecular Biology, Karolinska, Institutet, Stockholm, Sweden f Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden b c
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 16 September 2013 Accepted 11 October 2013 Key words: Hirschsprung’s disease Multiple sclerosis EDNRB gene
a b s t r a c t Purpose: We identified a girl with Hirschsprung’s disease (HSCR) whose mother and grandmother had HSCR associated with multiple sclerosis (MS). The aim of this study was to outline mutations in HSCR-related genes and MS susceptibility alleles in these three individuals. Methods: The phenotypes were reviewed based on medical records. The three subjects had rectosigmoid HSCR verified with histopathology. The mother and grandmother fulfilled the McDonald criteria for MS. DNA was isolated from EDTA-preserved blood according to standard procedures. Exome sequencing aiming mainly at analyzing HSCR associated genes as well as Sanger sequencing for confirmation was performed. Results: All affected individuals carry a novel heterozygous nonsense mutation in the EDNRB gene (c.C397T, p.R133X,refNM_000115), changing an arginine at position 133 into a premature stop codon. None of the subjects were homozygous for the HLA risk alleles for MS. Conclusion: We report a novel non-sense EDNRB gene mutation in a girl with HSCR and her mother and grandmother with HSCR and MS. We propose that this EDNRB gene mutation plays a role in the etiology of HSCR and also makes the subjects susceptible to MS. © 2014 Elsevier Inc. All rights reserved.
Hirschsprung’s disease (HSCR, OMIM 142623) is a developmental defect characterized by the absence of ganglion cells in the distal hindgut. The etiology of HSCR is not fully understood but premature arrest of the craniocaudal migration of vagal neural crest cells along the gastrointestinal tract between the fifth and 12th week of gestation may explain the condition [1]. Isolated HSCR appears to be of complex non-mendelian inheritance with low sex-dependent penetrance and variable expression with respect to the length of the aganglionic segment, probably involving one or more genes with low penetrance [2,3]. There are two major signaling pathways involved in the enteric nervous system (ENS) formation, which are known to be involved in HSCR: the rearranged during Transfection (RET)-cell line-derived neurotrophic factor gene and the endothelin-3 (EDN3)/endothelin receptor B (EDNRB) genes [4,5]. The majority of HSCR mutations are identified in the RET gene; 50% of familial and 15%–20% of sporadic cases of HSCR [6]. In addition several other HSCR susceptibility genes have been identified; GDNF (HSCR3, 5p13.2), HSCR6 (3p21), HSCR7 (19q12), HSCR5 (9q31), HSCR9 (4q31.3-q32.3, NKX2-1 (14q13.3), ⁎ Corresponding author at: Division of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Q3:03, Karolinska University Hospital, Solna, SE-17176 Stockholm, Sweden. E-mail address: [email protected] (T. Wester). 0022-3468/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2013.10.027
HSCR8 (16q23), PHOX2B (4p13, SOX10 (22q13.1), NRTN (19p13.3), WS4A (13q22.3), GFRA2 (8p21.3), GFRA1 (10q25.3), NRG1 (8p12), NRG3 (10q23.1) [7,8]. Multiple sclerosis (MS) is believed to be an acquired, chronic autoimmune disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration in young adults. The overall prevalence of MS in Sweden in December 2008 was approximately 189 per 100,000 individuals [9]. Classical studies of twins, adoptees and families have led to the widely accepted concept that MS is a complex genetic disease that is triggered by environmental factors in genetically susceptible individuals [10]. Several environmental factors, including smoking and sunlight exposure have been implicated to influence the risk to develop MS [11,12]. The human leukocyte antigen (HLA) region is considered the most important genetic region for MS susceptibility [13,14]. In a large genetic study with over 9000 cases and 17,000 controls, almost all previously suggested associations could be replicated and 29 new susceptibility loci could be identified; in close distance to those loci, immunologically relevant genes are overrepresented [15]. We identified a family with HSCR associated with MS. The purpose of this study was to look for mutations in this family in order to establish a molecular link between these two disorders.
A.L. Granström et al. / Journal of Pediatric Surgery 49 (2014) 622–625
1. Materials/Methods Three related Caucasian subjects, a girl (III:2) with HSCR and her mother (II:2) and grandmother (I:2) with HSCR and associated MS, were enrolled in the study (Fig. 1). The phenotypes were reviewed based on medical records. DNA was extracted from EDTApreserved blood and isolated according to standard procedures. The Regional Ethics Review Board approved the study. Informed consent was obtained. 1.1. Clinical data The grandmother was operated for recto-sigmoid HSCR as an infant. She has no associated malformations and there were no known relatives with HSCR. She was diagnosed with MS at 37 years of age but had symptoms since her mid-twenties. Since then she has had two relapses over ten years ago and has since terminated her immunemodulating treatment with interferon-beta (IFN-beta). Recently, she was in a secondary progressive phase of the disease with symptoms of fatigue and cognitive impairment, but able to walk up to 500 m without aid and had an Expanded Disability Status Scale (EDSS) point of at least 4.0 [16]. The mother presented with isolated recto-sigmoid HSCR as a neonate and underwent surgery. At 25 years of age, she presented with brain-stem symptoms (vertigo). After a relapse at 26 years of age, she fulfilled the McDonald criteria [17]. Due to an ongoing pregnancy with twins and hypothyroidism postpartum, continuous IFN-beta treatment was postponed. Since then she has had two relapses. During her last visit in the clinics she presented with an EDSS of 1.5. The girl is a dizygotic twin, born prematurely at 31 weeks of gestation. She was diagnosed with rectosigmoid HSCR at 45 days of age. She underwent a transanal endorectal pull-through procedure at 2 months of age. Recently she was investigated with magnetic resonance imaging of the central nervous system due to neurological symptoms suggesting cerebral palsy. The findings were normal, without signs of white matter lesions. 1.2. Exome-sequencing Exome sequencing: Libraries for sequencing on Illumina HiSeq2000 (Illumina) were prepared from DNA samples and exome sequences enriched with Agilent SureSelect Human All Exon 50 M (Agilent), according to the manufacturer’s instructions. Postcapture libraries were sequenced as 2 × 100 bp paired end reads on the Illumina sequencer. Reads were base-called using offline CASAVA (v 1.7, Illumina). Sample library preparation, sequencing, and initial bioinformatics up to base-calling and de-multiplexing were performed by the genomics core facility at the Science for Life Laboratory, Stockholm.
Fig. 1. Pedigree of the family with three generations (I–III) with squares indicating males and circles indicating females. Filled symbols are affected persons.
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An in-house pipeline, which is freely available under a GPL license (http://github.com/dnil/etiologica), was used to process reads and arrive at candidate genes. Briefly, reads are mapped to the human reference genome (hg19) using Mosaik (v1.0.1388) [Michael Strömberg, unpublished, http://code.google.com/p/mosaik-aligner/]. Duplicate read pairs were removed using MosaikDupSnoop. Variants were called using the samtools package (v.0.1.18) [18]. These were quality filtered (Q N = 20), and annotated using ANNOVAR (version 2011 June 18) [19]. Variants were further filtered using ANNOVAR to remove those found at a 1000 genomes minor allele frequency of 2% and above, as well as variants found in “dispensable” genes, truncated at a MAF of more than 1% in any 1000 genomes subpopulation, and variants not predicted damaging by PolyPhen2 (P threshold of 0.85) [20]. Non-synonymous variants, indels and putative splice site variants were retained. The patients were compared, assuming a dominant mode of inheritance. A further filtering against dbSNP131 was performed. 1.3. DNA sequencing of the EDNRB gene The exon 2 of the EDNRB gene was amplified by PCR using standard protocol. Primers and PCR condition are available on request. The PCR product was purified by Exo I/SAP treatment (Fermentas) prior to direct sequencing using the BigDye Terminator v3.1 kit (Applied Biosystems) and analyzed using the 3730 DNA Analyzer (Applied Biosystem). 2. Results Exome sequencing, especially focusing on all described HSCR genes as well as the SOX8 gene due to the interaction between Sox8 and the ednrb in enteric nervous system in mice, revealed a novel heterozygote nonsense mutation in the EDNRB gene (c.C397T, p.R133X, ref NM_000115) changing an arginine at position 133 into a premature stop codon in the proband and her grandmother. The mutation was confirmed by traditional Sanger sequencing in all three affected family members. We also manually analysed the results for the risk allele in HLA, the class II allele HLA-DRB1*15:01, and could not confirm that they were carriers of this allele. Nor did any of the SOX-genes show any unusual and shared alleles. Further, we compared regions implicated in the GWAS for MS with all identified genes or chromosomal regions for HSCR and found no overlaps. 3. Discussion This is the first description of HSCR associated with MS. In the three subjects, HSCR was confirmed by histopathology on the resected distal hindgut, showing an absence of ganglion cells and hypertrophied nerve trunks. MS was diagnosed based on the McDonald Criteria in the mother and grandmother. We showed a novel heterozygote non-sense mutation in the EDNRB gene in the girl with HSCR and in her mother and grandmother with both HSCR and MS. More than 40 mutations in the EDNRB gene have previously been reported in HSCR, but no association between mutations in the EDNRB gene and MS has been shown and thus our finding could be a coincidence. The EDNRB gene was located to chromosome 13q22 in 1993. It spans 24 kilobases and consists of seven exons and six introns [21]. The EDNRB gene is coding for a subtype of G protein-coupled receptors, which have an essential role for development of both enteric neurons and epidermal melanocytes [22,23]. In 1994, the first disruption of the EDNRB gene that leads to aganglionosis in mice was reported [24]. Mutations in the EDNRB gene are mainly inherited from unaffected parents, and usually associated with short segment aganglionosis [25].
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A.L. Granström et al. / Journal of Pediatric Surgery 49 (2014) 622–625
EDNRB gene mutations have been found in both isolated HSCR and Shah–Waardenburg syndrome (WS4) (OMIM). There are only a few reports in the English literature describing EDNRB gene mutations in humans with clinical neurological symptoms. All of them are associated with WS4 that comprise sensorineural deafness, depigmentation and HSCR. A more severe phenotype of WS4 includes peripheral demyelinating neuropathy and central dysmyelinating leukodystrophy [26]. Some patients have predominantly neurological symptoms, including peripheral neuropathy, mental retardation, cerebellar ataxia and spasticity [27–30]. WS4 can also be caused by a mutation in the endothelin 3 gene (EDN3) or the SOX10 gene [29]. Mutations in the EDNRB or EDN3 genes are inherited according to an autosomal recessive trait, while SOX10 gene mutations are inherited in an autosomal dominant way [30]. The association between HSCR and demyelinating disorders has previously mainly been related to mutations in the SOX10 gene. The SOX10 gene (22q13) is a member of the sex determining factor (SRY) like group of high mobility group (HMG) DNA binding proteins [31]. The expression of SOX10 is essential for the development of neural crest cells including melanocytes and enteric neurons [32,33]. Canrell et al. described that interaction between the SOX10 and EDNRB genes influenced penetrance and severity of the aganglionosis in a sox10 Dom mouse model of HSCR [34]. A heterozygous mutation in the SOX10 gene causing peripheral amyelination and disturbance of axonal developmental was recently reported in a full-term male with almost complete absence of myelinated fibers and severe reduction in the total numbers of axons [35]. In another study, Shimotake et al. reported brain hypomyelination, peripheral dysmyelinating neuropathy, and enteric glial deficiency associated with a SOX10 gene mutation [36]. No mutation was detected in the SOX10 gene in our patients. The etiology of MS is still unknown but an interplay between neuroinflammation and degeneration leads to relapses with neurological deficits and results in accumulation of disability. It is a complex disorder, more common in Scandinavia that is affected by several minor risk alleles mainly in the HLA-regions [9,13,14]. An association between a mutation in the EDNRB gene and MS has not been described previously. In the article from the International Multiple Sclerosis Genetics Consortium (IMSGC) and the Welcome Trust Case Control Consortium 2 (WTCCC2), no association between a mutation in a gene on chromosome 13, where the EDNRB gene is located, and MS could be shown or replicated [15]. SPG20, ATP7B, GWA, GPC5 and GPC6 on chromosome 13 have been earlier associated to MS in smaller studies but could not be replicated in the later article [37]. The pathway analysis indicates that MS genes are involved in T-Helper cell differentiation, lymphocyte trafficking, vitamin D metabolism and only a few regions have been identified to contain genes that influence neuronal function: KIF21B, GALC and SOX8 [15]. The SOX8gene belongs to the SOX E group and is a transcriptional regulator that is involved in oligodendrocyte maturation. Further on, it has been shown that SOX8 functions as a modifier of the HSCR phenotype in mice with an altered SOX10 gene [38]. SOX8 has some functional redundancy with SOX10 and considering this it could potentially bind EDNRB and influence its function [39]. HSCR is a neurocristopathy and results from abnormal migration, proliferation, survival, and differentiation of neuroblasts originating from the vagal neural crest cells during embryonic development. Neurocristopathies are a diverse group of disorders including pigmentary disorders, mesectodermal syndromes, Waardenburg’s syndrome, neuroblastoma, neurofibromatosis, pheochromocytoma and multiple endocrine neoplasia [40]. Interestingly, it was recently proposed that MS also could be considered a neurocristopathy due to an association between MS and other disorders of glial cell proliferation and differentiation [41]. Earlier a SOX10 gene structure–function analysis in the chicken neural tube concluded the importance of SOX10 during the development of neurocristopathies
[42]. If MS, as well as HSCR, is a neurocristopathy there could be a similar pathophysiology with the EDNRB gene playing a role. Recent studies have shown that EDNRB and endothelin 2 may be important for remyelination in MS. This is interesting since this may be a link between EDNRB, HSCR and MS [43]. We report a novel non-sense EDNRB gene mutation that is most likely the cause of HSCR in this family and it may also be a contributing factor in MS as well. Mutations in the EDNRB gene may have an influence on the central, peripheral and enteric nervous system. We suggest that this EDNRB gene mutation may play a role in the etiology of both HSCR and MS. References [1] Bolande RP. The neurocristopathies; a unifying concept of disease arising in neural crest maldevelopment. Hum Pathol 1974;5:409–20. [2] Bodian M, Carter C. A family study of Hirschsprung disease. Ann Hum Genet 1963;26:261. [3] Badner JA, Charkravarti A. Waardenburg syndrome and Hirschsprung disease: evidence for pleiotropic effects of a single dominant gene. Am J Med Genet 1990;35:100–4. [4] Amiel J, Sproat-Emison E, Garcia-Barcelo M, et al. Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet 2008;45:1–14. [5] Tam PKH, Garcia-Barcelo AM. Genetic basis of Hirschsprung’s disease. Pediatr Surg Int 2009;25:543–58. [6] Angrist M, Bolk S, Thiel B, et al. Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease. Hum Mol Genet 1995;4:821–30. [7] Tang CS, Cheng G, So MT, et al. Genome-wide copy number analysis uncovers a new HSCR gene: NRG3. PLoS Genet 2012;8(5):e1002687. [8] Garcia-Barcelo MM, Tang CS, Ngan ES, et al. Genomwide association study identifies NRG1 as a susceptibility locus for Hirschsprung’s disease. Proc Natl Acad Sci U S A 2009;24(106):2694–9. [9] Ahlgren C, Odén A, Lycke J. High nationwide prevalence of multiple sclerosis in Sweden. Mult Scler 2011;17:901–8. [10] Dyment DA, Ebers GC, Sadovnick AD. Genetics of multiple sclerosis. Hum Mol Genet 1997;6:1693–8. [11] Hedström AK, Bäärnhielm M, Olsson T, et al. Tobacco smoking, but nor Swedish snuff use, increases the risk of multiple sclerosis. Neurology 2009;73:696–701. [12] Bäärnhielm M, Hedström AK, Kockum I, et al. Sunlight is associated with decreased multiple sclerosis risk: no interaction with human leukocyte antigenDRB1*15. Eur J Neurol 2012;19:955–62. [13] Jersild C, Fog T, Hansen GS, et al. Histocompatibility determinants in multiple sclerosis, with special reference to clinical course. Lancet 1973;2:1221–5. [14] Fogdell-Hahn A, Ligers A, Gronning M, et al. Multiple sclerosis: a modifying influence of HLA class I genes in an HLA class II associated autoimmune disease. Tissue Antigens 2000;55:140–8. [15] International Multiple Sclerosis Genetics Consortium, Welcome Trust Case Control Consortium 2, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 2011;476:214–9. [16] Kurtzke JF. Historical and clinical perspectives of the expanded disability status scale. Neuroepidemiology 2008;31:1–9. [17] Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011;69:292–302. [18] Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009;25:2078–9. [19] Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010;38:164. [20] Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–9. [21] Arai H, Nakao K, Takaya K, et al. The human endothelin-B receptor gene: structural organization and chromosomal assignment. J Biol Chem 1993;268: 3463–70. [22] Baynash AG, Hosada K, Giaid A, et al. Interaction of endothelin-3 with endothelinB receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 1994;79:1277–85. [23] Hosoda K, Hammer RE, Richardson JA, et al. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 1994;79:1267–76. [24] Puffenberger EG, Hosoda K, Washington SS, et al. A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease. Cell 1994;79: 1257–66. [25] Tam P, Garcia-Barcelo M. Molecular genetics of Hirschsprung’s disease. Semin Pediatr Surg 2004;13:236–48. [26] Read AP, Newton VE. Waardenburg syndrome. J Med Genet 1997;34:656–65. [27] Pingault V, Ente D, Dastot-Le Moal F, et al. Review and update of mutations causing Waardenburg syndrome. Hum Mutat 2010;31:391–406. [28] Bondurand N, Dastot-Le Moal F, Stanchina L, et al. Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4. Am J Hum Genet 2007;81: 1169–85. [29] Verheij JB, Sival DA, van der Hoeven JH, et al. Shah–Waardenburg syndrome and PCWH associated with SOX10 mutations: a case report and review of the literature. Eur J Paediatr Neurol 2006;10:11–7.
A.L. Granström et al. / Journal of Pediatric Surgery 49 (2014) 622–625 [30] Touraine RL, Attié-Bitach T, Manceau E, et al. Neurological phenotype in Waardenburg syndrome type 4 correlates with novel SOX10 truncating mutations and expression in developing brain. Am J Hum Genet 2000;66: 1496–503. [31] Herbarth B, Pingault V, Bondurand N, et al. Mutations of the sryrelated SOX10 gene in Dominant megacolon, a mouse model for human Hirchsprung disease. Proc Natl Acad Sci U S A 1998;95:5161–5. [32] Wegner M. From head to toes: the multiple facts of Sox proteins. Nucleic Acids Res 1999;27:1409–20. [33] Kuhlbrodt K, Herbarth B, Sock E, et al. SOX10, a novel transcriptional modulator in glial cells. J Neurosci 1998;18:237–50. [34] Cantrell VA, Owens SE, Chandler RL, et al. Interactions between Sox10 and EDNRB modulate penetrance and severity of aganglionosis in the Sox10Dom mouse model of Hirschsprung disease. Hum Mol Genet 2004;13:2289–301. [35] Parthey K, Kornhuber M, Kunze C, et al. SOX10 mutation with peripheral amyelination and developmental disturbance of axons. Muscle Nerve 2012;45: 284–90. [36] Shimotake T, Tanaka S, Fukui R, et al. Neuroglial disorders of central and peripheral nervous systems in a patient with Hirschsprung's disease carrying allelic SOX10 truncating mutation. J Pediatr Surg 2007;42:725–31.
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[37] Cavanillas ML, Fernández O, Comabella M, et al. Replication of top markers of a genome-wide association study in multiple sclerosis in Spain. Genes Immun 2011;12:110–5. [38] Maka M, Stolt CC, Wegner M. Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect. Dev Biol 2005;277:155–69. [39] Stolt CC, Lommes P, Friedrich RP, et al. Transcription factors Sox8 and Sox10 perform non-equivalent roles during oligodendrocyte development despite functional redundancy. Development 2004;131:2349–58. [40] Bolande RP. Neurocristopathy: its growth and development in 20 years. Pediatr Pathol Lab Med 1997;17:1–25. [41] Behan PO, Chaudhuri A. The sad plight of multiple sclerosis research (low on fact, high on fiction): critical data to support it being a neurocristopathy. Inflammopharmacology 2010;18:265–90. [42] Cossais F, Wahlbuhl M, Kriesch J, et al. SOX10 structurefunction analysis in the chicken neural tube reveals important insights into its role in human neurocristopathies. Hum Mol Genet 2010;19:2409–20. [43] Yuen TJ, Johnson KR, Miron VE, et al. Identification of endothelin 2 as an inflammatory factor that promotes central nervous system remyelination. Brain 2013;136:1035–47.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
A novel stop mutation in the EDNRB gene in a family with Hirschsprung’s disease associated with Multiple Sclerosis Anna Löf Granström a, b, Ellen Markljung b, Katharina Fink c, d, Edvard Nordenskjöld b, Daniel Nilsson e, f, Tomas Wester a, b,⁎, Agneta Nordenskjöld a, b a
Division for Pediatric Surgery, Astrid Lindgren Children's Hospital, Karolinska, University Hospital, Stockholm, Sweden Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden d Department of Neurology Huddinge, Karolinska University Hospital, Stockholm, Sweden e Science for Life Laboratory, Department of Cell and Molecular Biology, Karolinska, Institutet, Stockholm, Sweden f Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden b c
a r t i c l e
i n f o
Article history: Received 5 July 2013 Received in revised form 16 September 2013 Accepted 11 October 2013 Key words: Hirschsprung’s disease Multiple sclerosis EDNRB gene
a b s t r a c t Purpose: We identified a girl with Hirschsprung’s disease (HSCR) whose mother and grandmother had HSCR associated with multiple sclerosis (MS). The aim of this study was to outline mutations in HSCR-related genes and MS susceptibility alleles in these three individuals. Methods: The phenotypes were reviewed based on medical records. The three subjects had rectosigmoid HSCR verified with histopathology. The mother and grandmother fulfilled the McDonald criteria for MS. DNA was isolated from EDTA-preserved blood according to standard procedures. Exome sequencing aiming mainly at analyzing HSCR associated genes as well as Sanger sequencing for confirmation was performed. Results: All affected individuals carry a novel heterozygous nonsense mutation in the EDNRB gene (c.C397T, p.R133X,refNM_000115), changing an arginine at position 133 into a premature stop codon. None of the subjects were homozygous for the HLA risk alleles for MS. Conclusion: We report a novel non-sense EDNRB gene mutation in a girl with HSCR and her mother and grandmother with HSCR and MS. We propose that this EDNRB gene mutation plays a role in the etiology of HSCR and also makes the subjects susceptible to MS. © 2014 Elsevier Inc. All rights reserved.
Hirschsprung’s disease (HSCR, OMIM 142623) is a developmental defect characterized by the absence of ganglion cells in the distal hindgut. The etiology of HSCR is not fully understood but premature arrest of the craniocaudal migration of vagal neural crest cells along the gastrointestinal tract between the fifth and 12th week of gestation may explain the condition [1]. Isolated HSCR appears to be of complex non-mendelian inheritance with low sex-dependent penetrance and variable expression with respect to the length of the aganglionic segment, probably involving one or more genes with low penetrance [2,3]. There are two major signaling pathways involved in the enteric nervous system (ENS) formation, which are known to be involved in HSCR: the rearranged during Transfection (RET)-cell line-derived neurotrophic factor gene and the endothelin-3 (EDN3)/endothelin receptor B (EDNRB) genes [4,5]. The majority of HSCR mutations are identified in the RET gene; 50% of familial and 15%–20% of sporadic cases of HSCR [6]. In addition several other HSCR susceptibility genes have been identified; GDNF (HSCR3, 5p13.2), HSCR6 (3p21), HSCR7 (19q12), HSCR5 (9q31), HSCR9 (4q31.3-q32.3, NKX2-1 (14q13.3), ⁎ Corresponding author at: Division of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Q3:03, Karolinska University Hospital, Solna, SE-17176 Stockholm, Sweden. E-mail address: [email protected] (T. Wester). 0022-3468/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2013.10.027
HSCR8 (16q23), PHOX2B (4p13, SOX10 (22q13.1), NRTN (19p13.3), WS4A (13q22.3), GFRA2 (8p21.3), GFRA1 (10q25.3), NRG1 (8p12), NRG3 (10q23.1) [7,8]. Multiple sclerosis (MS) is believed to be an acquired, chronic autoimmune disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration in young adults. The overall prevalence of MS in Sweden in December 2008 was approximately 189 per 100,000 individuals [9]. Classical studies of twins, adoptees and families have led to the widely accepted concept that MS is a complex genetic disease that is triggered by environmental factors in genetically susceptible individuals [10]. Several environmental factors, including smoking and sunlight exposure have been implicated to influence the risk to develop MS [11,12]. The human leukocyte antigen (HLA) region is considered the most important genetic region for MS susceptibility [13,14]. In a large genetic study with over 9000 cases and 17,000 controls, almost all previously suggested associations could be replicated and 29 new susceptibility loci could be identified; in close distance to those loci, immunologically relevant genes are overrepresented [15]. We identified a family with HSCR associated with MS. The purpose of this study was to look for mutations in this family in order to establish a molecular link between these two disorders.
A.L. Granström et al. / Journal of Pediatric Surgery 49 (2014) 622–625
1. Materials/Methods Three related Caucasian subjects, a girl (III:2) with HSCR and her mother (II:2) and grandmother (I:2) with HSCR and associated MS, were enrolled in the study (Fig. 1). The phenotypes were reviewed based on medical records. DNA was extracted from EDTApreserved blood and isolated according to standard procedures. The Regional Ethics Review Board approved the study. Informed consent was obtained. 1.1. Clinical data The grandmother was operated for recto-sigmoid HSCR as an infant. She has no associated malformations and there were no known relatives with HSCR. She was diagnosed with MS at 37 years of age but had symptoms since her mid-twenties. Since then she has had two relapses over ten years ago and has since terminated her immunemodulating treatment with interferon-beta (IFN-beta). Recently, she was in a secondary progressive phase of the disease with symptoms of fatigue and cognitive impairment, but able to walk up to 500 m without aid and had an Expanded Disability Status Scale (EDSS) point of at least 4.0 [16]. The mother presented with isolated recto-sigmoid HSCR as a neonate and underwent surgery. At 25 years of age, she presented with brain-stem symptoms (vertigo). After a relapse at 26 years of age, she fulfilled the McDonald criteria [17]. Due to an ongoing pregnancy with twins and hypothyroidism postpartum, continuous IFN-beta treatment was postponed. Since then she has had two relapses. During her last visit in the clinics she presented with an EDSS of 1.5. The girl is a dizygotic twin, born prematurely at 31 weeks of gestation. She was diagnosed with rectosigmoid HSCR at 45 days of age. She underwent a transanal endorectal pull-through procedure at 2 months of age. Recently she was investigated with magnetic resonance imaging of the central nervous system due to neurological symptoms suggesting cerebral palsy. The findings were normal, without signs of white matter lesions. 1.2. Exome-sequencing Exome sequencing: Libraries for sequencing on Illumina HiSeq2000 (Illumina) were prepared from DNA samples and exome sequences enriched with Agilent SureSelect Human All Exon 50 M (Agilent), according to the manufacturer’s instructions. Postcapture libraries were sequenced as 2 × 100 bp paired end reads on the Illumina sequencer. Reads were base-called using offline CASAVA (v 1.7, Illumina). Sample library preparation, sequencing, and initial bioinformatics up to base-calling and de-multiplexing were performed by the genomics core facility at the Science for Life Laboratory, Stockholm.
Fig. 1. Pedigree of the family with three generations (I–III) with squares indicating males and circles indicating females. Filled symbols are affected persons.
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An in-house pipeline, which is freely available under a GPL license (http://github.com/dnil/etiologica), was used to process reads and arrive at candidate genes. Briefly, reads are mapped to the human reference genome (hg19) using Mosaik (v1.0.1388) [Michael Strömberg, unpublished, http://code.google.com/p/mosaik-aligner/]. Duplicate read pairs were removed using MosaikDupSnoop. Variants were called using the samtools package (v.0.1.18) [18]. These were quality filtered (Q N = 20), and annotated using ANNOVAR (version 2011 June 18) [19]. Variants were further filtered using ANNOVAR to remove those found at a 1000 genomes minor allele frequency of 2% and above, as well as variants found in “dispensable” genes, truncated at a MAF of more than 1% in any 1000 genomes subpopulation, and variants not predicted damaging by PolyPhen2 (P threshold of 0.85) [20]. Non-synonymous variants, indels and putative splice site variants were retained. The patients were compared, assuming a dominant mode of inheritance. A further filtering against dbSNP131 was performed. 1.3. DNA sequencing of the EDNRB gene The exon 2 of the EDNRB gene was amplified by PCR using standard protocol. Primers and PCR condition are available on request. The PCR product was purified by Exo I/SAP treatment (Fermentas) prior to direct sequencing using the BigDye Terminator v3.1 kit (Applied Biosystems) and analyzed using the 3730 DNA Analyzer (Applied Biosystem). 2. Results Exome sequencing, especially focusing on all described HSCR genes as well as the SOX8 gene due to the interaction between Sox8 and the ednrb in enteric nervous system in mice, revealed a novel heterozygote nonsense mutation in the EDNRB gene (c.C397T, p.R133X, ref NM_000115) changing an arginine at position 133 into a premature stop codon in the proband and her grandmother. The mutation was confirmed by traditional Sanger sequencing in all three affected family members. We also manually analysed the results for the risk allele in HLA, the class II allele HLA-DRB1*15:01, and could not confirm that they were carriers of this allele. Nor did any of the SOX-genes show any unusual and shared alleles. Further, we compared regions implicated in the GWAS for MS with all identified genes or chromosomal regions for HSCR and found no overlaps. 3. Discussion This is the first description of HSCR associated with MS. In the three subjects, HSCR was confirmed by histopathology on the resected distal hindgut, showing an absence of ganglion cells and hypertrophied nerve trunks. MS was diagnosed based on the McDonald Criteria in the mother and grandmother. We showed a novel heterozygote non-sense mutation in the EDNRB gene in the girl with HSCR and in her mother and grandmother with both HSCR and MS. More than 40 mutations in the EDNRB gene have previously been reported in HSCR, but no association between mutations in the EDNRB gene and MS has been shown and thus our finding could be a coincidence. The EDNRB gene was located to chromosome 13q22 in 1993. It spans 24 kilobases and consists of seven exons and six introns [21]. The EDNRB gene is coding for a subtype of G protein-coupled receptors, which have an essential role for development of both enteric neurons and epidermal melanocytes [22,23]. In 1994, the first disruption of the EDNRB gene that leads to aganglionosis in mice was reported [24]. Mutations in the EDNRB gene are mainly inherited from unaffected parents, and usually associated with short segment aganglionosis [25].
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EDNRB gene mutations have been found in both isolated HSCR and Shah–Waardenburg syndrome (WS4) (OMIM). There are only a few reports in the English literature describing EDNRB gene mutations in humans with clinical neurological symptoms. All of them are associated with WS4 that comprise sensorineural deafness, depigmentation and HSCR. A more severe phenotype of WS4 includes peripheral demyelinating neuropathy and central dysmyelinating leukodystrophy [26]. Some patients have predominantly neurological symptoms, including peripheral neuropathy, mental retardation, cerebellar ataxia and spasticity [27–30]. WS4 can also be caused by a mutation in the endothelin 3 gene (EDN3) or the SOX10 gene [29]. Mutations in the EDNRB or EDN3 genes are inherited according to an autosomal recessive trait, while SOX10 gene mutations are inherited in an autosomal dominant way [30]. The association between HSCR and demyelinating disorders has previously mainly been related to mutations in the SOX10 gene. The SOX10 gene (22q13) is a member of the sex determining factor (SRY) like group of high mobility group (HMG) DNA binding proteins [31]. The expression of SOX10 is essential for the development of neural crest cells including melanocytes and enteric neurons [32,33]. Canrell et al. described that interaction between the SOX10 and EDNRB genes influenced penetrance and severity of the aganglionosis in a sox10 Dom mouse model of HSCR [34]. A heterozygous mutation in the SOX10 gene causing peripheral amyelination and disturbance of axonal developmental was recently reported in a full-term male with almost complete absence of myelinated fibers and severe reduction in the total numbers of axons [35]. In another study, Shimotake et al. reported brain hypomyelination, peripheral dysmyelinating neuropathy, and enteric glial deficiency associated with a SOX10 gene mutation [36]. No mutation was detected in the SOX10 gene in our patients. The etiology of MS is still unknown but an interplay between neuroinflammation and degeneration leads to relapses with neurological deficits and results in accumulation of disability. It is a complex disorder, more common in Scandinavia that is affected by several minor risk alleles mainly in the HLA-regions [9,13,14]. An association between a mutation in the EDNRB gene and MS has not been described previously. In the article from the International Multiple Sclerosis Genetics Consortium (IMSGC) and the Welcome Trust Case Control Consortium 2 (WTCCC2), no association between a mutation in a gene on chromosome 13, where the EDNRB gene is located, and MS could be shown or replicated [15]. SPG20, ATP7B, GWA, GPC5 and GPC6 on chromosome 13 have been earlier associated to MS in smaller studies but could not be replicated in the later article [37]. The pathway analysis indicates that MS genes are involved in T-Helper cell differentiation, lymphocyte trafficking, vitamin D metabolism and only a few regions have been identified to contain genes that influence neuronal function: KIF21B, GALC and SOX8 [15]. The SOX8gene belongs to the SOX E group and is a transcriptional regulator that is involved in oligodendrocyte maturation. Further on, it has been shown that SOX8 functions as a modifier of the HSCR phenotype in mice with an altered SOX10 gene [38]. SOX8 has some functional redundancy with SOX10 and considering this it could potentially bind EDNRB and influence its function [39]. HSCR is a neurocristopathy and results from abnormal migration, proliferation, survival, and differentiation of neuroblasts originating from the vagal neural crest cells during embryonic development. Neurocristopathies are a diverse group of disorders including pigmentary disorders, mesectodermal syndromes, Waardenburg’s syndrome, neuroblastoma, neurofibromatosis, pheochromocytoma and multiple endocrine neoplasia [40]. Interestingly, it was recently proposed that MS also could be considered a neurocristopathy due to an association between MS and other disorders of glial cell proliferation and differentiation [41]. Earlier a SOX10 gene structure–function analysis in the chicken neural tube concluded the importance of SOX10 during the development of neurocristopathies
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