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RSK2 gene mutations in Coffin–Lowry syndrome with drop episodes
Brain & Development 27 (2005) 114–117 www.elsevier.com/locate/braindev
Original article
RSK2 gene mutations in Coffin–Lowry syndrome with drop episodesq Miki Nakamura, Takanori Yamagata, Masato Mori, Mariko Y. Momoi* Department of Pediatrics, Jichi Medical School, 3311-1 Minamikawachi-machi, Tochigi 329-0498, Japan Received 19 August 2003; received in revised form 23 February 2004; accepted 23 February 2004
Abstract Coffin –Lowry syndrome is an X-linked mental retardation disorder with dysmorphism caused by mutation of the ribosomal S6 kinase (RSK2) gene. Coffin– Lowry syndrome patients can experience unusual drop episodes whereby an abrupt loss of muscle tone and falling down can be induced by sudden, unexpected tactile or auditory stimuli. We detected a C913T (R305X) mutation in a female Coffin– Lowry syndrome patient with drop episodes. All mutations in our patient and those previously reported in patients with drop episodes result in premature truncation of the RSK2 protein in the N-terminal kinase domain or upstream of this domain. q 2004 Elsevier B.V. All rights reserved. Keywords: Coffin–Lowry syndrome; RSK2 gene; Drop episodes; Cataplexy
1. Introduction Coffin –Lowry syndrome (CLS) was reported independently by Coffin et al. [1] and Lowry et al. [2], and recognized as a novel syndrome by Temtamy et al. in 1975 [3]. CLS is transmitted in an X-linked dominant manner. Symptomatic men present with severe to moderate mental retardation, a dysmorphic face, puffy proximal digits, thick fingers with tapered ends and hyperextensibility of joints. Most female carriers show partial manifestation of the disease. In 1998, Crow et al. [4] and our group [5] reported ‘drop episodes’ or ‘cataplexy’ in CLS whereby a sudden loss of muscle tone was induced by unexpected tactile or auditory stimuli. Subsequently, several CLS patients with this feature or other types of movement disorder have been reported [6]. These movement disorders have been labeled as drop episodes, cataplexy, exaggerated startle responses, hyperekplexia or stimulus-induced drop episodes. These movement disorders have been recognized as symptoms of CLS, although they are difficult to distinguish from each other and their mechanisms remain unclear. The prevalence of movement disorders in CLS patients is estimated at more than 15%, and their onset ranges from 4 to 17 years, q
The paper is based on the lecture given at the 6th annual meeting of the Infantile Seizure Society, Tokyo, March 15–16, 2003. * Corresponding author. Fax: þ 81-285-44-6123. E-mail address: [email protected] (M.Y. Momoi). 0387-7604/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2004.02.015
with a mean age of 8.6 years [6] (Stephenson, JB. The 6th Infantile Seizure Society Meeting, 2003). RSK2 on chromosome Xp22.2 is a growth-factorregulated serine-threonine protein kinase acting in the rasmitogen-activated protein kinase signaling pathway, and has been identified as the causative gene for CLS [7]. The human RSK2 gene has an open reading frame of 22 exons encoding a protein of 740 amino acids containing two nonidentical kinase catalytic domains [8]. RSK2 mutations have been detected in approximately 50% of CLS patients, and over 80 different mutations have been identified thus far [9,10]. Although some missense mutations have been associated with a mild phenotype [11], no obvious correlation between genotype and phenotype such as mental retardation and dysmorphism has been observed. However, an association between drop episodes and RSK2 mutations type were not investigated. We performed mutational analysis of the RSK2 gene and surveyed the literature to investigate whether there is an association between RSK2 genotype and drop episodes.
2. Methods 2.1. Patient The patient is a 24-year-old female with full manifestation of CLS. Her clinical and electrophysiological date
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
was reported previously [5]. Briefly, she developed frequent drop attacks in response to unexpected tactile or auditory stimuli at 12 years of age. At 16 years of age, we examined her drop attacks using electrophysiological procedures. Immediately after the unexpected touch on her shoulder, she dropped to the ground with her knees flexed. Her consciousness was considered to be preserved and she raised her arms as if to protect herself from falling. Her arms maintained normal tension, while her legs seemed to have lost their muscle tone. She stood up after a few seconds. EEGs were unremarkable before and during the attacks. The electrostatic condition lasted for 30 ms and was recorded 60 ms following the stimulus in her lower limb-girdle muscles. No myoclonic discharges were observed in the myograms, and the EEG showed no paroxysmal activity. Valproic acid, phenobarbital and carbamazepine were ineffective. Then we administrated clonazepam (2 mg/day) that successfully prevented her drop attacks. The severity and frequency of attack did worsen after time, but improved following an increase in clonazepam dosage. The dosage of clonazepam has been repeatedly increased during the last 8 years and is now at 6 mg/day.
115
and the ratio of methylated and unmethylated DNA was calculated by measuring each peak.
3. Results 3.1. Mutation analysis of the RSK2 gene DHPLC analysis of the patient’s DNA showed a heteroduplex in exon 11 of the RSK2 gene. Direct sequencing of the exon 11 showed a C913T heterozygous mutation (Fig. 1a). The C913T base substitution at the end of the N-terminal kinase domain resulting in a premature truncation of the RSK2 protein (codon R305X) has been previously reported in a patient with CLS (Fig. 1b) [8]. 3.2. X-chromosome inactivation analysis The patient had skewed X-inactivation pattern. The ratio of X-chromosome inactivation was 89:11.
4. Discussion 2.2. Molecular analysis Lymphocytes were obtained from the patient with informed consent from the parents, and genomic DNA was extracted. We used the primers reported by Jacquot et al. [8] to amplify all 22 exons of the RSK2 gene. PCRs were performed under conditions optimized for each primer pair. PCR products were screened for molecular variants by denaturing high performance liquid chromatography (DHPLC) using WAVEe DNA-fragment analysis system (Transgenomic, Inc., Omaha, NB, USA). Heteroduplex formation was induced by heat denaturation of PCR products at 94 8C for 5 min, followed by gradual reannealing from 94 to 25 8C over 45 min. PCR products were eluted at a flow rate of 0.9 ml/min with a linear acetonitrile gradient. Heterozygous profiles were detected as distinct elution peaks from homozygous wild-type peaks. PCR products forming heteroduplexes were subjected to direct sequence analysis using ABI PRISMe 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 2.3. X-chromosome inactivation study The X-chromosome inactivation study was done using the analysis based on methylation-specific PCR [12]. Genomic DNA was treated with sodium bisulfate using CpGenome DNA modification kit (Serologicals, Co., Norcross, GA, USA). PCR for methylated and unmethylated DNA was performed using treated DNA as template and the primers were the same with those Kubota described [12]. PCR products were separated by the WAVEe DNA-fragment analysis system (Transgenomic, Inc., Omaha, NB, USA),
RSK2, a member of the growth-factor-regulated protein kinase family, regulates transcriptional activation through various cytosolic substrates such as c-AMP responsive element-binding protein [13]. Thus, inactivating mutations in the RSK2 gene could lead to dysfunction of the MAPK/ RSK signaling pathway. To date, over 80 RSK2 mutations have been reported in CLS patients. Of these, missense mutations account for approximately 40%, nonsense mutations 20%, splicing errors 20%, and short deletions or insertions 20% [9,10]. Thus far, no significant relationship between mutation type or location and clinical features, including severity and phenotype, has been observed. It is possible that other proteins with a similar function to RSK2 may influence the CLS phenotype. Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders, and probably reflects selection against those cells in which the mutation is on the active chromosome [14]. Currently, there is only one paper describing the inactivation pattern of CLS [15]. This study examined the mental level and ratio of X-inactivation in carrier females. The X-inactivation ratio of carrier females was 96:4 –75:25 (mean, 88:12) and did not correlate with the mental retardation level (mean IQ 65.0). Other features of CLS were not described. The patient in our study had severe mental retardation and the full manifestations of CLS but the skewed X-inactivation level (89:11) was only moderate. Our result confirms that X-inactivation occurs in CLS, and that the X-inactivation pattern in lymphocytes may not reflect the severity of mental retardation and other clinical features. The C913T mutation detected in our patient has been previously reported in a male patient with moderate mental
116
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
Fig. 1. RSK2 sequence from the DNA of the patient and mutation spectrum of the RSK2 gene. (a) C913T (R305X) mutation in the RSK2 gene. Direct sequencing of the RSK2 gene identified an exon 11 heterozygous mutation. (b) The positions of reported mutations on RSK2 gene in CLS patients were indicated. And also the position of the mutation site with and without drop episode were indicated. All of the mutations reported in the patients with drop episode were in N-terminal and induced early termination. PDK1, 3-phosphoinositide-dependent protein kinase-1; ERK, extracellular signal-regulated kinase; DE, drop episode/cataplexy.
retardation [8]. Our patient was female and had severe mental retardation. Therefore, our results are compatible with the previous report that mental retardation level is not related to genotype. However, both patients had cataplexy/ drop episodes. There are no reports describing an association between drop episodes and RSK2 genotype. We have listed all reported cases of drop attacks or cataplexy with RSK2 gene mutations to examine the relationship between drop attack events and genotype [8,16,17] (Table 1). RSK2 gene
mutations identified in CLS patients were found throughout the gene (Fig. 1b). However, the mutations reported in patients presenting with drop attacks or cataplexy were either nonsense, frameshift, or splice-site mutations in 50 site of the gene, that induced early termination of the translation of the RSK2 N-terminal domain. Although cases 9 and 12 have the same splice-site mutation, only case 9 had a drop episode. Since case 12 is 4 years of age, he may present with the movement disorders at a later age. Because the mean onset age is 8.6 years and the oldest onset age is 17 years,
Table 1 Mutation and clinical data of CLS with or without drop episode or cataplexy in literature Case no.
Authors, year
Drop episode or cataplexy
Mental retardation
Sex
Age (years)
Onset (years)
Exon or intron
Nucleotide change
Mutations
1 2 3 4 5 6 7 8 9
Jacquot et al., 1998 Zeniou et al., 2002 Zeniou et al., 2002 Zeniou et al., 2002 Jacquot et al., 1998 Zeniou et al., 2002 Jacquot et al., 1998 Present study, 2003 Fryssira et al., 2002
þ þ þ þ þ þ þ þ þ
Mo Mo S S Mi S Mo S Mi
M ··· ··· ··· F ··· M F F
21 7 21 30 38 7 12 24 13
··· ··· ··· ··· ··· ··· ··· 12 9
3 3 4 6 8 10 11 11 12
244 -2A ! G IVS4-11 A ! G 262 insA IVS6 þ 3 A ! G 632 -1G ! T IVS10 þ 5 G ! A 913 C ! T 913 C ! T 1000 -2A ! G
Splice site Unusual splice site Frameshift Unusual splice site Splice site Unusual splice site Nonsense (R305X) Nonsense (R305X) Splice site
10 11 12 13 14
Zeniou et al., 2002 Jacquot et al., 1998 Jacquot et al., 1998 Jacquot et al., 1998 Jacquot et al., 1998
2 2 2 2 2
Mo Mo S S S
··· M M M M
10 10 4 17 33
··· ··· ··· ··· ···
4 5 12 18 20
IVS4 þ 3 insT 379 T ! G 1000 -2A ! G 1672 C ! T 1934 G ! A
Unusual splice site Missense (H127Q) Splice site Nonsense (R558X) Nonsense (W645X)
Note. þ , Positive; 2, negative; ‘Mental retardation’ Mi, mild; Mo, moderate; Si, Severe; · · ·, no information.
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
patient age should be considered when investigating genotype/phenotype relationships. All reported mutations that induce truncation of the protein in patients over 17 years of age without drop episodes are in the RSK2 C-terminal domain. None of the patients with an RSK2 missense mutation had drop episodes [8,16,17]. It was speculated that truncating mutations resulting in a protein containing the RSK2 N-terminal kinase domain may prevent drop episodes whereas truncating mutations resulting in smaller RSK2 proteins and disrupting this domain may induce drop episode events. The mechanisms of drop attacks seen in CLS remains unclear. Drop episodes have been considered as reflex events rather than epileptic events, because no epileptic discharges are observed during the episodes and consciousness is preserved with the voluntary motion of her arms during the attack. This involvement of both legs only was difficult to explain by cortical event. Addition to that, our previous study showed that drop attacks induced by electrostatic condition to certain muscle groups resulted in the sudden loss of muscle tone for the involved muscles [5]. On the other side, startle epilepsy is characterized by tonic or myoclonic seizures, that are muscle contraction, associated with paroxysmal discharges revealed by electroencephalogram [18]. Although drop episodes are not the hallmark feature of CLS, it is a unique neurological feature typical in some CLS patients. Determining the mechanism of drop episodes may contribute to understanding brain function. Mutational analysis of the RSK2 gene in a greater number of CLS patients is required to determine the relationship between disease genotype and phenotype. We believe that the accumulation of genotype data will provide clues for elucidating the association between RSK2 function and drop episodes. Further analysis of RSK2 function should be carried out, including testing for alternate splicing that may induce truncated protein to be expressed in the brain.
References [1] Coffin GS, Siris E, Wegienka LC. Mental retardation with osteocartilaginous anomalies. Am J Dis Child 1966;112:205– 13.
117
[2] Lowry RB, Miller JR, Fraser FC. A new dominant gene mental retardation syndrome: associated with small stature, tapering fingers, characteristic facies, and possible hydrocephalus. Am J Dis Child 1971;121:496 –500. [3] Temtamy SA, Miller JD, Hussels-Maumenee I. The Coffin –Lowry syndrome: an inherited facio-digital mental retardation syndrome. J Pediatr 1975;86:724 –31. [4] Crow Y, Zuberi S, McWilliam R, Tolmie JL, Hollman A, Pohl K, et al. Cataplexy and muscle ultrasound abnormalities in Coffin –Lowry syndrome. J Med Genet 1998;35:94–8. [5] Nakamura M, Yamagata T, Momoi M, Yamazaki T. Drop episodes in Coffin– Lowry syndrome: exaggerated startle responses treated with clonazepam. Pediatr Neurol 1998;19:148–50. [6] Nelson GB, Hahn JS. Stimulus-induced drop episodes in Coffin – Lowry syndrome. Pediatrics 2003;111:e197–e202. [7] Trivier E, De Cesare D, Jacquot S, Pannetier S, Zackai E, Young I, et al. Mutations in the kinase Rsk-2 associated with Coffin –Lowry syndrome. Nature 1996;384:567–70. [8] Jacquot S, Merienne K, De Cesare D, Pannetier S, Mandel JL, Sassone-Corsi P, et al. Mutation analysis of the RSK2 gene in Coffin – Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations. Am J Hum Genet 1998;63:1631 –40. [9] Touraine RL, Zeniou M, Hanauer A. A syndromic form of X-linked mental retardation: the Coffin–Lowry syndrome. Eur J Pediatr 2002; 161:179–87. [10] Hanauer A, Young ID. Coffin – Lowry syndrome: clinical and molecular features. J Med Genet 2002;39:705–13. [11] Delaunoy J, Abidi F, Zeniou M, Jacquot S, Merienne K, Pannetier S, et al. Mutations in the X-linked RSK2 gene (RPS6KA3) in patients with Coffin– Lowry syndrome. Hum Mutat 2001;17:103– 16. [12] Kubota T, Nonoyama S, Tonoki H, Masuno M, Imaizumi K, Kojima M, et al. A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR. Hum Genet 1999;104:49– 55. [13] Blenis J. Signal transduction via the MAP kinase; proceed at your own RSK. Proc Natl Acad Sci USA 1993;90:5889 –92. [14] Plenge RM, Stevenson RA, Lubs HA, Schwartz CE, Willard HF. Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders. Am J Hum Genet 2002;71: 168–73. [15] Simensen RJ, Abidi F, Collins J, Schwartz CE, Stevenson RE. Cognitive function in Coffin –Lowry syndrome. Clin Genet 2002;61: 299–304. [16] Fryssira H, Kountoupi S, Delaunoy JP, Thomaidis L. A female with Coffin – Lowry syndrome and cataplexy. Genet Couns 2002;13: 405–9. [17] Zeniou M, Pannetier S, Fryns JP, Hanauer A. Unusual splice-site mutations in the RSK2 gene and suggestion of genetic heterogeneity in Coffin–Lowry syndrome. Am J Hum Genet 2002;70:1421–33. [18] Saenz-Lope E, Herranz FJ, Masdeu JC. Startle epilepsy: a clinical study. Ann Neurol 1984;16:78–81.
Original article
RSK2 gene mutations in Coffin–Lowry syndrome with drop episodesq Miki Nakamura, Takanori Yamagata, Masato Mori, Mariko Y. Momoi* Department of Pediatrics, Jichi Medical School, 3311-1 Minamikawachi-machi, Tochigi 329-0498, Japan Received 19 August 2003; received in revised form 23 February 2004; accepted 23 February 2004
Abstract Coffin –Lowry syndrome is an X-linked mental retardation disorder with dysmorphism caused by mutation of the ribosomal S6 kinase (RSK2) gene. Coffin– Lowry syndrome patients can experience unusual drop episodes whereby an abrupt loss of muscle tone and falling down can be induced by sudden, unexpected tactile or auditory stimuli. We detected a C913T (R305X) mutation in a female Coffin– Lowry syndrome patient with drop episodes. All mutations in our patient and those previously reported in patients with drop episodes result in premature truncation of the RSK2 protein in the N-terminal kinase domain or upstream of this domain. q 2004 Elsevier B.V. All rights reserved. Keywords: Coffin–Lowry syndrome; RSK2 gene; Drop episodes; Cataplexy
1. Introduction Coffin –Lowry syndrome (CLS) was reported independently by Coffin et al. [1] and Lowry et al. [2], and recognized as a novel syndrome by Temtamy et al. in 1975 [3]. CLS is transmitted in an X-linked dominant manner. Symptomatic men present with severe to moderate mental retardation, a dysmorphic face, puffy proximal digits, thick fingers with tapered ends and hyperextensibility of joints. Most female carriers show partial manifestation of the disease. In 1998, Crow et al. [4] and our group [5] reported ‘drop episodes’ or ‘cataplexy’ in CLS whereby a sudden loss of muscle tone was induced by unexpected tactile or auditory stimuli. Subsequently, several CLS patients with this feature or other types of movement disorder have been reported [6]. These movement disorders have been labeled as drop episodes, cataplexy, exaggerated startle responses, hyperekplexia or stimulus-induced drop episodes. These movement disorders have been recognized as symptoms of CLS, although they are difficult to distinguish from each other and their mechanisms remain unclear. The prevalence of movement disorders in CLS patients is estimated at more than 15%, and their onset ranges from 4 to 17 years, q
The paper is based on the lecture given at the 6th annual meeting of the Infantile Seizure Society, Tokyo, March 15–16, 2003. * Corresponding author. Fax: þ 81-285-44-6123. E-mail address: [email protected] (M.Y. Momoi). 0387-7604/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2004.02.015
with a mean age of 8.6 years [6] (Stephenson, JB. The 6th Infantile Seizure Society Meeting, 2003). RSK2 on chromosome Xp22.2 is a growth-factorregulated serine-threonine protein kinase acting in the rasmitogen-activated protein kinase signaling pathway, and has been identified as the causative gene for CLS [7]. The human RSK2 gene has an open reading frame of 22 exons encoding a protein of 740 amino acids containing two nonidentical kinase catalytic domains [8]. RSK2 mutations have been detected in approximately 50% of CLS patients, and over 80 different mutations have been identified thus far [9,10]. Although some missense mutations have been associated with a mild phenotype [11], no obvious correlation between genotype and phenotype such as mental retardation and dysmorphism has been observed. However, an association between drop episodes and RSK2 mutations type were not investigated. We performed mutational analysis of the RSK2 gene and surveyed the literature to investigate whether there is an association between RSK2 genotype and drop episodes.
2. Methods 2.1. Patient The patient is a 24-year-old female with full manifestation of CLS. Her clinical and electrophysiological date
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
was reported previously [5]. Briefly, she developed frequent drop attacks in response to unexpected tactile or auditory stimuli at 12 years of age. At 16 years of age, we examined her drop attacks using electrophysiological procedures. Immediately after the unexpected touch on her shoulder, she dropped to the ground with her knees flexed. Her consciousness was considered to be preserved and she raised her arms as if to protect herself from falling. Her arms maintained normal tension, while her legs seemed to have lost their muscle tone. She stood up after a few seconds. EEGs were unremarkable before and during the attacks. The electrostatic condition lasted for 30 ms and was recorded 60 ms following the stimulus in her lower limb-girdle muscles. No myoclonic discharges were observed in the myograms, and the EEG showed no paroxysmal activity. Valproic acid, phenobarbital and carbamazepine were ineffective. Then we administrated clonazepam (2 mg/day) that successfully prevented her drop attacks. The severity and frequency of attack did worsen after time, but improved following an increase in clonazepam dosage. The dosage of clonazepam has been repeatedly increased during the last 8 years and is now at 6 mg/day.
115
and the ratio of methylated and unmethylated DNA was calculated by measuring each peak.
3. Results 3.1. Mutation analysis of the RSK2 gene DHPLC analysis of the patient’s DNA showed a heteroduplex in exon 11 of the RSK2 gene. Direct sequencing of the exon 11 showed a C913T heterozygous mutation (Fig. 1a). The C913T base substitution at the end of the N-terminal kinase domain resulting in a premature truncation of the RSK2 protein (codon R305X) has been previously reported in a patient with CLS (Fig. 1b) [8]. 3.2. X-chromosome inactivation analysis The patient had skewed X-inactivation pattern. The ratio of X-chromosome inactivation was 89:11.
4. Discussion 2.2. Molecular analysis Lymphocytes were obtained from the patient with informed consent from the parents, and genomic DNA was extracted. We used the primers reported by Jacquot et al. [8] to amplify all 22 exons of the RSK2 gene. PCRs were performed under conditions optimized for each primer pair. PCR products were screened for molecular variants by denaturing high performance liquid chromatography (DHPLC) using WAVEe DNA-fragment analysis system (Transgenomic, Inc., Omaha, NB, USA). Heteroduplex formation was induced by heat denaturation of PCR products at 94 8C for 5 min, followed by gradual reannealing from 94 to 25 8C over 45 min. PCR products were eluted at a flow rate of 0.9 ml/min with a linear acetonitrile gradient. Heterozygous profiles were detected as distinct elution peaks from homozygous wild-type peaks. PCR products forming heteroduplexes were subjected to direct sequence analysis using ABI PRISMe 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 2.3. X-chromosome inactivation study The X-chromosome inactivation study was done using the analysis based on methylation-specific PCR [12]. Genomic DNA was treated with sodium bisulfate using CpGenome DNA modification kit (Serologicals, Co., Norcross, GA, USA). PCR for methylated and unmethylated DNA was performed using treated DNA as template and the primers were the same with those Kubota described [12]. PCR products were separated by the WAVEe DNA-fragment analysis system (Transgenomic, Inc., Omaha, NB, USA),
RSK2, a member of the growth-factor-regulated protein kinase family, regulates transcriptional activation through various cytosolic substrates such as c-AMP responsive element-binding protein [13]. Thus, inactivating mutations in the RSK2 gene could lead to dysfunction of the MAPK/ RSK signaling pathway. To date, over 80 RSK2 mutations have been reported in CLS patients. Of these, missense mutations account for approximately 40%, nonsense mutations 20%, splicing errors 20%, and short deletions or insertions 20% [9,10]. Thus far, no significant relationship between mutation type or location and clinical features, including severity and phenotype, has been observed. It is possible that other proteins with a similar function to RSK2 may influence the CLS phenotype. Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders, and probably reflects selection against those cells in which the mutation is on the active chromosome [14]. Currently, there is only one paper describing the inactivation pattern of CLS [15]. This study examined the mental level and ratio of X-inactivation in carrier females. The X-inactivation ratio of carrier females was 96:4 –75:25 (mean, 88:12) and did not correlate with the mental retardation level (mean IQ 65.0). Other features of CLS were not described. The patient in our study had severe mental retardation and the full manifestations of CLS but the skewed X-inactivation level (89:11) was only moderate. Our result confirms that X-inactivation occurs in CLS, and that the X-inactivation pattern in lymphocytes may not reflect the severity of mental retardation and other clinical features. The C913T mutation detected in our patient has been previously reported in a male patient with moderate mental
116
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
Fig. 1. RSK2 sequence from the DNA of the patient and mutation spectrum of the RSK2 gene. (a) C913T (R305X) mutation in the RSK2 gene. Direct sequencing of the RSK2 gene identified an exon 11 heterozygous mutation. (b) The positions of reported mutations on RSK2 gene in CLS patients were indicated. And also the position of the mutation site with and without drop episode were indicated. All of the mutations reported in the patients with drop episode were in N-terminal and induced early termination. PDK1, 3-phosphoinositide-dependent protein kinase-1; ERK, extracellular signal-regulated kinase; DE, drop episode/cataplexy.
retardation [8]. Our patient was female and had severe mental retardation. Therefore, our results are compatible with the previous report that mental retardation level is not related to genotype. However, both patients had cataplexy/ drop episodes. There are no reports describing an association between drop episodes and RSK2 genotype. We have listed all reported cases of drop attacks or cataplexy with RSK2 gene mutations to examine the relationship between drop attack events and genotype [8,16,17] (Table 1). RSK2 gene
mutations identified in CLS patients were found throughout the gene (Fig. 1b). However, the mutations reported in patients presenting with drop attacks or cataplexy were either nonsense, frameshift, or splice-site mutations in 50 site of the gene, that induced early termination of the translation of the RSK2 N-terminal domain. Although cases 9 and 12 have the same splice-site mutation, only case 9 had a drop episode. Since case 12 is 4 years of age, he may present with the movement disorders at a later age. Because the mean onset age is 8.6 years and the oldest onset age is 17 years,
Table 1 Mutation and clinical data of CLS with or without drop episode or cataplexy in literature Case no.
Authors, year
Drop episode or cataplexy
Mental retardation
Sex
Age (years)
Onset (years)
Exon or intron
Nucleotide change
Mutations
1 2 3 4 5 6 7 8 9
Jacquot et al., 1998 Zeniou et al., 2002 Zeniou et al., 2002 Zeniou et al., 2002 Jacquot et al., 1998 Zeniou et al., 2002 Jacquot et al., 1998 Present study, 2003 Fryssira et al., 2002
þ þ þ þ þ þ þ þ þ
Mo Mo S S Mi S Mo S Mi
M ··· ··· ··· F ··· M F F
21 7 21 30 38 7 12 24 13
··· ··· ··· ··· ··· ··· ··· 12 9
3 3 4 6 8 10 11 11 12
244 -2A ! G IVS4-11 A ! G 262 insA IVS6 þ 3 A ! G 632 -1G ! T IVS10 þ 5 G ! A 913 C ! T 913 C ! T 1000 -2A ! G
Splice site Unusual splice site Frameshift Unusual splice site Splice site Unusual splice site Nonsense (R305X) Nonsense (R305X) Splice site
10 11 12 13 14
Zeniou et al., 2002 Jacquot et al., 1998 Jacquot et al., 1998 Jacquot et al., 1998 Jacquot et al., 1998
2 2 2 2 2
Mo Mo S S S
··· M M M M
10 10 4 17 33
··· ··· ··· ··· ···
4 5 12 18 20
IVS4 þ 3 insT 379 T ! G 1000 -2A ! G 1672 C ! T 1934 G ! A
Unusual splice site Missense (H127Q) Splice site Nonsense (R558X) Nonsense (W645X)
Note. þ , Positive; 2, negative; ‘Mental retardation’ Mi, mild; Mo, moderate; Si, Severe; · · ·, no information.
M. Nakamura et al. / Brain & Development 27 (2005) 114–117
patient age should be considered when investigating genotype/phenotype relationships. All reported mutations that induce truncation of the protein in patients over 17 years of age without drop episodes are in the RSK2 C-terminal domain. None of the patients with an RSK2 missense mutation had drop episodes [8,16,17]. It was speculated that truncating mutations resulting in a protein containing the RSK2 N-terminal kinase domain may prevent drop episodes whereas truncating mutations resulting in smaller RSK2 proteins and disrupting this domain may induce drop episode events. The mechanisms of drop attacks seen in CLS remains unclear. Drop episodes have been considered as reflex events rather than epileptic events, because no epileptic discharges are observed during the episodes and consciousness is preserved with the voluntary motion of her arms during the attack. This involvement of both legs only was difficult to explain by cortical event. Addition to that, our previous study showed that drop attacks induced by electrostatic condition to certain muscle groups resulted in the sudden loss of muscle tone for the involved muscles [5]. On the other side, startle epilepsy is characterized by tonic or myoclonic seizures, that are muscle contraction, associated with paroxysmal discharges revealed by electroencephalogram [18]. Although drop episodes are not the hallmark feature of CLS, it is a unique neurological feature typical in some CLS patients. Determining the mechanism of drop episodes may contribute to understanding brain function. Mutational analysis of the RSK2 gene in a greater number of CLS patients is required to determine the relationship between disease genotype and phenotype. We believe that the accumulation of genotype data will provide clues for elucidating the association between RSK2 function and drop episodes. Further analysis of RSK2 function should be carried out, including testing for alternate splicing that may induce truncated protein to be expressed in the brain.
References [1] Coffin GS, Siris E, Wegienka LC. Mental retardation with osteocartilaginous anomalies. Am J Dis Child 1966;112:205– 13.
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