Case Report: A Novel Mutation in the MECP2 Gene in a Korean ...

Available online at www.annclinlabsci.org

93

Annals of Clinical & Laboratory Science, vol. 41, no. 1, 2011

Case Report: A Novel Mutation in the MECP2 Gene in a Korean Patient with Rett Syndrome

Eun Young Lee,1,2 Hee-Jung Chung,3 Chang-Seok Ki,4 Jong-Ha Yoo,1,2 and Jong Rak Choi1 1Department

of Laboratory Medicine, Yonsei University College of Medicine, Seoul; Departments of 2Laboratory Medicine and 3Pediatrics, National Health Insurance Corporation Ilsan Hospital, Goyang; 4Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Abstract. Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder. Mutations in the MECP2 gene on chromosome Xq28 have been shown to be the cause of RTT. Using DNA samples from a RTT patient and her parents, we sequenced three exons and flanking intron regions of the MECP2 gene using the polymerase chain reaction. Sequencing of the MECP2 gene in the proband revealed a novel 41-base pair deletion in exon 4 (c.1152_1192del41). This mutation resulted in premature termination of the 487 amino acid protein at the 390th codon, predicting a partial loss of the C-terminal domain. We did not observe this mutation in either parent of the RTT patient, but further studies are needed to evaluate the possibility of germline mosaicism. Introduction Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder characterized by apparently normal psychomotor development during the first 6 months of life, followed by gradual loss of acquired skills, deceleration of head growth, initiation of stereotypic hand movements, and autistic behavior by the age of 3-4 years [1]. RTT affects about 1 in 10,000 to 15,000 new-born females [2], and there is wide variability in the rate of progression and severity of the disease. Clinically, the disease is present in either classical or variant forms [3]. Mutations in the methyl-CpGbinding protein 2 (MECP2) gene, which is located on chromosome X (Xq28), have been found in the majority of patients with RTT [4]. MECP2 is thought to selectively

bind methyl-CpG islands in the mammalian genome, functioning as a regulator (mostly a repressor) of gene expression [4]. MECP2 seems to function in neuronal maturation and synaptic transmission [5-8], but it is unknown how the loss of MECP2 functions causes the RTT phenotype [9]. Sporadic cases of RTT are the rule; 99.5% of cases are single occurrences within the family [10]. To date, more than 400 different mutations in the MECP2 gene have been reported to the Human Gene Mutation Database (HGMD) (http://www.hgmd.cf.ac.uk/ac/all.php)

and most are gross deletions or missense/nonsense mutations. We report a Korean RTT patient with a novel MECP2 mutation. According to molecular genetic testing of the MECP2 gene, the patient had a deletion of 41 base

pairs in exon 4, causing a premature stop codon. Case Report The proband was a 30-month-old Korean female delivered by normal spontaneous vaginal delivery after 38 weeks gestation. Prenatal history, perinatal history, and head circumference at birth were all normal. Psychomotor development was normal until the age of 18 months, when the patient’s parents noticed gait delay and speech delay. At the time of the study, speech delay and stereotypic hand movements were present, but loss of hand skills and acquired speech, and deceleration of head circumference growth were not observed. She was diagnosed clinically with suspicious RTT. After obtaining informed consent, blood samples were collected from the patient and her parents (Fig. 1).

Address correspondence to Jong-Ha Yoo, M.D., Ph.D., Department of Laboratory Medicine, National Health Insurance Corporation Ilsan Hospital, 1232 Baekseok-dong, Ilsandong-gu, Goyang, Korea; e-mail [email protected]; or to Jong Rak Choi, M.D., Ph.D., Department of Laboratory Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemungu, Seoul 120-752, Korea; e-mail: [email protected]. 0091-7370/11/0093-0096. $2.00. © 2011 by the Association of Clinical Scientists, Inc.

94

Annals of Clinical & Laboratory Science, vol. 41, no. 1, 2011

The institutional review board approved this study. Materials and Methods Genomic DNA was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit according to the manufacturer’s protocol (Promega, Madison, WI, USA). The MECP2 gene was amplified via PCR using the appropriate primers as designed by the authors and a thermal cycler (Applied Biosystems, Foster City, CA, USA). Bi-directional sequencing of all three coding exons along with the flanking intron regions of the MECP2 gene was performed twice with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) in conjunction with an ABI Prism 3100 automated genetic analyzer (Applied Biosystems). Results Direct sequencing analysis of the proband revealed a heterozygous 41bp deletion (c.1152_1192del41) in exon 4 of the MECP2 gene, which resulted in a frameshift leading to premature termination of the 487 amino acid protein at the 390th codon (Fig. 2). This mutation was absent in the proband’s parents and

Fig. 1. Pedigree of the patient’s family. Circle, female; square, male; black symbol, affected. Asterisk indicates that the family member was available for genetic analysis.

in 100 control chromosomes. We also identified a missense mutation (c.201C>T) previously described as a polymorphism (rs61748381) in exon 4 of the MECP2 gene. Discussion Mutations in the MECP2 gene on chromosome Xq28 have been shown to be the cause of RTT [4]. According to the literature, mutations are found in more than 80% of classical RTT cases and in more than 40% of variant cases [11]. Human MECP2 consists of 4 exons, and the majority of causative alterations (including missense, nonsense, and frameshift mutations) occur in exons 3 and 4 [12]. Exons 3

and 4 encode the MECP2 functional domains: MBD (methyl-CpG binding domain), capable of recognizing and physically associating with methylated CpG islands, which are known to be abundant in gene promoter regions; and TRD (transcription repression domain), responsible for the interaction with co-repressor mSin3A and histone deacetylases. The C-terminal region located downstream of the TRD is prone to deletions of various sizes resulting in the production of truncated proteins [13]. The patient described in this study appeared to have suspicious phenotypes of RTT according to international criteria [3] so we sequenced her MECP2 gene. We identified a novel heterozygous 41bp deletion (c.1152_1192del41) and a missense mutation (c.201C>T) previously described as a polymorphism (rs61748381) in exon 4 of the MECP2 gene. The novel mutation resulted in a frameshift leading to premature termination of the 487 amino acid protein at the 390th codon. The c.1152_1192del41 mutation is located in the C-terminal region of the MECP2 gene, which is in the C-terminal segment downstream of the TRD [14]. The C-terminal segment shares

Fig. 2. Identification of the MECP2 gene mutation. Direct sequencing of the proband demonstrated a 41-bp deletion (c.1152_1192del41) in exon 4 of the MECP2 gene, which resulted in frameshift leading to premature termination of the 487 amino acid protein at the 390th codon. The proband’s parents did not have the mutation. The location of the deletion is indicated by the arrow.

MECP2 gene mutation in a patient with Rett sundrome homology with neuronal-specific transcription factors, suggesting that the protein may have additional, more complex, and possibly neuronspecific functions [15]. This region also facilitates binding to the nucleosome core [16]. The C-terminal region is prone to deletions of various sizes, and deletions in this region represent about 10% of RTT genotypes [17,18]. The association between the severity of RTT phenotype and X-chromosome inactivation (XCI) has been studied. XCI is the process by which one of the two X chromosomes carried by females is inactivated to achieve gene expression patterns similar to those found in males, who carry only one copy of the X chromosome [19]. The RTT phenotypes in females are different according to the degree of inactivation between the mutant and wildtype alleles [19-22]. However, recent studies show that skewed XCI is insufficient to explain the phenotypic manifestations of RTT [19,2324]. Another factor affecting the severity of phenotype is the mutation site. Hoffbuhr et al [25] reported that patients with missense mutation within the MBD and mutations truncating the entire TRD had more severe clinical presentations compared with patients with missense and nonsense mutations within the TRD and frameshift deletions within the C-terminus. A recent study showed that overall severity of C-terminal cases appears to be in the middle of the range, significantly more severe than those with the mildest mutation, p. R133C, and yet significantly milder than the most severe mutations, p. R270X [26]. Smeets et al [17] studied 10 RTT female patients with deletions in the C-terminal region and found that despite an atypical or “milder” course, these patients eventually develop symptoms clinically recognizable as RTT.

The main symptom is gradual decline of gross motor ability and rapidly progressive spine deformation despite preservation of simple communicative and cognitive abilities. Recently, Bebbington et al [26] studied 832 RTT patients and determined that the cases with C-terminal deletions were more likely to have normal head circumference, weight, and ambulation. The mutation types are also associated with the severity of RTT phenotype. Patients with truncating mutations have higher incidence of awake respiratory dysfunction [27] and severe disease [28] compared to patients with missense mutations. However, extremely late truncating mutations (1152 del44bp and 1157 del41bp) had been excluded prior to this analysis [28]. Bebbington et al [26] reported that the frame shift C-terminal deletions were less severe than the in-frame C-terminal deletion. So correlation of phenotype and extremely late truncating mutation due to frame shift C-terminal deletion, as in this case, remains uncertain. In order to confirm the presence of germline mosaicism, DNA analysis must be done on the parents’ sperm or oocyte. We do not know whether the mutation in our patient is de novo or not, because we could not test the sperm or oocyte due to lack of specimens. Further studies are needed to evaluate the possibility of germline mosaicism in order to determine whether this mutation is de novo or inherited.

4.

5.

6.

7.

8. 9.

10.

11.

References 1.

Williamson SL, Christodoulou J. Rett syndrome: new clinical and molecular insights. Eur J Hum Genet 2006;14:896-903. 2. Hagberg B. Rett syndrome: Swedish approach to analysis of prevalence and cause. Brain Dev 1985;7:276-280. 3. Hagberg B, Hanefeld F, Percy A, Skjeldal O. An update on clinically applicable diagnostic criteria in

12.

13.

95

Rett syndrome. Eur J Paediatr Neurol 2002;6:293-297. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpGbinding protein 2. Nat Genet 1999;23:185-188. Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 2003;302:885-889. Harikrishnan KN, Chow MZ, Baker EK, Pal S, Bassal S, Brasacchio D, Wang L, Craig JM, Jones PL, Sif S, El-Osta A. Brahma links the SWI/SNF chromatinremodeling complex with MeCP2dependent transcriptional silencing. Nat Genet 2005;37:254-264. Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, Fan G, Sun YE. DNA methylation-related chromatin remodeling in activitydependent BDNF gene regulation. Science 2003;302:890-893. Na ES, Monteggia LM. The role of MeCP2 in CNS development and function. Horm Behav 2010. Chahrour M, Zoghbi HY. The story of Rett syndrome: from clinic to neurobiology. Neuron 2007;56: 422-437. Jedele KB. The overlapping spectrum of Rett and Angelman syndromes: a clinical review. Semin Pediatr Neurol 2007;14: 108-117. Archer H, Clarke A. Rett syndrome. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine, 1st ed (Ganten D, Ruckpaul K, Eds), Springer, New York, 2006; vol 1, pp 1655-1660. Van den Veyver IB, Zoghbi HY. Genetic basis of Rett syndrome. Ment Retard Dev Disabil Res Rev 2002;8:82-86. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone

96 14.

15.

16.

17.

18.

Annals of Clinical & Laboratory Science, vol. 41, no. 1, 2011 deacetylase complex. Nature 1998; 393:386-389. Laccone F, Huppke P, Hanefeld F, Meins M. Mutation spectrum in patients with Rett syndrome in the German population: Evidence of hot spot regions. Hum Mutat 2001;17:183-190. Vacca M, Filippini F, Budillon A, Rossi V, Mercadante G, Manzati E, Gualandi F, Bigoni S, Trabanelli C, Pini G, Calzolari E, Ferlini A, Meloni I, Hayek G, Zappella M, Renieri A, D’Urso M, D’Esposito M, MacDonald F, Kerr A, Dhanjal S, Hulten M. Mutation analysis of the MECP2 gene in British and Italian Rett syndrome females. J Mol Med 2001;78:648-655. Chandler SP, Guschin D, Landsberger N, Wolffe AP. The methylCpG binding transcriptional repressor MeCP2 stably associates with nucleosomal DNA. Biochemistry 1999;38:7008-7018. Smeets E, Terhal P, Casaer P, Peters A, Midro A, Schollen E, van Roozendaal K, Moog U, Matthijs G, Herbergs J, Smeets H, Curfs L, Schrander-Stumpel C, Fryns JP. Rett syndrome in females with CTS hot spot deletions: a disorder profile. Am J Med Genet A 2005; 132A:117-120. Lee SS, Wan M, Francke U. Spectrum of MECP2 mutations in Rett syndrome. Brain Dev 2001;23 (Suppl 1):S138-143.

19. Gonzales ML, LaSalle JM. The role of MeCP2 in brain development and neurodevelopmental disorders. Curr Psychiatry Rep 2010;12:127-134. 20. Villard L, Kpebe A, Cardoso C, Chelly PJ, Tardieu PM, Fontes M. Two affected boys in a Rett syndrome family: clinical and molecular findings. Neurology 2000;55:1188-1193. 21. Braunschweig D, Simcox T, Samaco RC, LaSalle JM. X-Chromosome inactivation ratios affect wild-type MeCP2 expression within mosaic Rett syndrome and Mecp2-/+ mouse brain. Hum Mol Genet 2004;13:1275-1286. 22. Shahbazian MD, Sun Y, Zoghbi HY. Balanced X chromosome inactivation patterns in the Rett syndrome brain. Am J Med Genet 2002;111:164-168. 23. Takahashi S, Ohinata J, Makita Y, Suzuki N, Araki A, Sasaki A, Murono K, Tanaka H, Fujieda K. Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome. Clin Genet 2008;73: 257-261. 24. Xinhua B, Shengling J, Fuying S, Hong P, Meirong L, Wu XR. X chromosome inactivation in Rett syndrome and its correlations with MECP2 mutations and phenotype. J Child Neurol 2008;23:22-25.

25. Hoffbuhr K, Devaney JM, LaFleur B, Sirianni N, Scacheri C, Giron J, Schuette J, Innis J, Marino M, Philippart M, Narayanan V, Umansky R, Kronn D, Hoffman EP, Naidu S. MeCP2 mutations in children with and without the phenotype of Rett syndrome. Neurology 2001;56:1486-1495. 26. Bebbington A, Percy A, Christodoulou J, Ravine D, Ho G, Jacoby P, Anderson A, Pineda M, Ben Zeev B, Bahi-Buisson N, Smeets E, Leonard H. Updating the profile of C-terminal MECP2 deletions in Rett syndrome. J Med Genet 2010;47:242-248. 27. Amir RE, Van den Veyver IB, Schultz R, Malicki DM, Tran CQ, Dahle EJ, Philippi A, Timar L, Percy AK, Motil KJ, Lichtarge O, Smith EO, Glaze DG, Zoghbi HY. Influence of mutation type and X chromosome inactivation on Rett syndrome phenotypes. Ann Neurol 2000;47:670-679. 28. Cheadle JP, Gill H, Fleming N, Maynard J, Kerr A, Leonard H, Krawczak M, Cooper DN, Lynch S, Thomas N, Hughes H, Hulten M, Ravine D, Sampson JR, Clarke A. Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum Mol Genet 2000;9: 1119-1129. .