A novel hypomorphic MECP2 point mutation is ... - The LaSalle Lab

Hum Genet (2009) 124:615–623 DOI 10.1007/s00439-008-0585-6

ORIGINAL INVESTIGATION

A novel hypomorphic MECP2 point mutation is associated with a neuropsychiatric phenotype Abidemi A. Adegbola · Michael L. Gonzales · Andrew Chess · Janine M. LaSalle · Gerald F. Cox

Received: 8 September 2008 / Accepted: 28 October 2008 / Published online: 7 November 2008 © Springer-Verlag 2008

Abstract The MECP2 gene on Xq28 encodes a transcriptional repressor, which binds to and modulates expression of active genes. Mutations in MECP2 cause classic or preserved speech variant Rett syndrome and intellectual disability in females and early demise or marked neurodevelopmental handicap in males. The consequences of a hypomorphic Mecp2 allele were recently investigated in a mouse model, which developed obesity, motor, social, learning, and behavioral deWcits, predicting a human neurobehavioral syndrome. Here, we describe mutation analysis of a nondysmorphic female proband and her father who

A. A. Adegbola and M. L. Gonzales contributed equally to this study. A. A. Adegbola · A. Chess Center for Human Genetic Research, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

presented with primarily neuropsychiatric manifestations and obesity with relative sparing of intelligence, language, growth, and gross motor skills. We identiWed and characterized a novel missense mutation (c.454C>G; p.P152A) in the critical methyl-binding domain of MeCP2 that disrupts MeCP2 functional activity. We show that a gradient of impairment is present when the p.P152A mutation is compared with an allelic p.P152R mutation, which causes classic Rett syndrome and another Rett syndrome-causing mutation, such that protein–heterochromatin binding observed by immunoXuorescence and immunoblotting is wild-type > P152A > P152R > T158 M, consistent with the severity of the observed phenotype. Our Wndings provide evidence for very mild phenotypes in humans associated with partial reduction of MeCP2 function arising from subtle variation in MECP2.

Introduction A. A. Adegbola · A. Chess · G. F. Cox Harvard Medical School, Boston, MA, USA A. A. Adegbola · G. F. Cox Division of Genetics, Department of Medicine, Children’s Hospital Boston, Boston, MA, USA M. L. Gonzales · J. M. LaSalle Medical Microbiology and Immunology, Rowe Program in Human Genetics, School of Medicine, University of California, Davis, CA, USA G. F. Cox Department of Clinical Research, Genzyme Corporation, Cambridge, MA, USA A. A. Adegbola (&) Harvard-Partners Center for Genetics and Genomics, 77 Avenue Louis Pasteur, NRB 250, Boston, MA 02115, USA e-mail: [email protected]

MECP2-related conditions constitute a broad neurodevelopmental spectrum and are considered to act as X-linked dominant disorders. In females, the range of phenotypes ascribed to MECP2 mutations include classic and variant forms of Rett syndrome (RTT; MIM #312750) as well as rare cases of isolated learning disabilities, while in males MECP2 mutations generally lead to severe neonatal encephalopathy (SNE) and marked intellectual disability (ID) (Erlandson and Hagberg 2005). Classic RTT is generally characterized by normal early development, regression at 6–18 months of age, absent or minimal speech, severe cognitive impairment, and in approximately 40–50% of cases, loss of motor functioning that results in children becoming wheelchair-bound. Atypical RTT is diagnosed in individuals who fulWll some, but not all, necessary diagnostic criteria for classic RTT,

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according to speciWed parameters (Hagberg and Skjeldal 1994). These include ‘forme fruste’ RTT, Zappella variant (preserved speech variant), and early seizure variants. Males meeting diagnostic criteria for RTT have been identiWed in association with a 47,XXY karyotype or with a postzygotic MECP2 mutation resulting in somatic mosaicism (ClaytonSmith et al. 2000; Leonard et al. 2001; Topçu et al. 2002). Males with a normal 46,XY karyotype and a RTT-causing MECP2 mutation are severely aVected and usually die during infancy (Wan et al. 1999). Males with milder missense MECP2 mutations not found in females with RTT present with mild to severe ID (Couvert et al. 2001; Dotti et al. 2002; Orrico et al. 2000). Males having a duplication of the entire MECP2 gene typically present with a severe phenotype of absent speech, facial dysmorphies, and recurrent infections (del Gaudio et al. 2006). MECP2-related phenotypes are inXuenced by the type and location of the mutation, the degree of X chromosome inactivation (XCI) in females, and other undetermined factors (HoVbuhr et al. 2001). Truncating mutations, particularly early truncating mutations, have been associated with higher clinical severity scores in some studies, but not invariably so (Charman et al. 2005; Huppke et al. 2002; Neul et al. 2008; Weaving et al. 2003; Zappella et al. 2001). Some of these discrepant Wndings may be attributable to small sample sizes in a number of studies. A recent large multicenter analysis established correlations between speciWc common recurring mutations and individual phenotypic characteristics such as hand use, ambulation, and language (Bebbington et al. 2008). Still, patients with identical missense mutations and discordant phenotypes have been described, implicating the presence of disease modiWers (Amir et al. 2000; Auranen et al. 2001; Huppke et al. 2000; Nielsen et al. 2001). Although XCI appears to be an important modiWer (Archer et al. 2007; Villard et al. 2000; Wan et al. 1999; Weaving et al. 2003), the XCI pattern in peripheral lymphocytes does not always fully explain the clinical diVerences observed between patients with identical mutations or correlate with disease severity (Auranen et al. 2001; Bao et al. 2008; Nielsen et al. 2001; Scala et al. 2007). MeCP2 binds to methylated DNA and associates predominantly with nuclear heterochromatin (Nan et al. 1996). While Wrst described as a transcriptional repressor, more recent studies have demonstrated that MeCP2 binds to and modulates expression of active genes (Chahrour et al. 2008; Yasui et al. 2007). MeCP2 contains multiple functional domains including a methyl CpG-binding domain (MBD) essential for heterochromatin association (Nan et al. 1998). In this report, we have identiWed a novel p.P152A mutation in the MBD of MeCP2 in a girl and her father who had a mild neurobehavioral phenotype. We performed cellular functional assays to determine the likely pathogenicity of this mutation and demonstrate partially reduced heterochro-

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matin association of p.P152A–MeCP2 that is less severe than RTT-causing MBD mutations, implicating a potential role for hypomorphic alleles such as this in the etiology of neuropsychiatric disease.

Materials and methods Phenotypic characterization The proband, a 10-year old nondysmorphic female, is the only child of her parents. After a period of generally normal development, slowing of motor skills and hypotonia began at 12 months of age without documented regression. Her weight and height have tracked along the 98th and 50th percentiles, respectively. Her head circumference measurements dropped from the 90th percentile to the 75th percentile at 18 months of age, increased again, and have since tracked at the 98th percentile. Current body mass index is 35.7 (morbidly obese). She has purposeful hand movements, with occasional hand wringing stereotypies. She is prone to frequent episodic aggressive outbursts. She was diagnosed with pervasive developmental disorder-not otherwise speciWed (PDD-NOS) at age 7 because of her insistence on sameness, preference for rituals, chronic inXexibility, inability to sustain conversations, and poor recognition of verbal and nonverbal cues. Her electroencephalogram (EEG) is abnormal with multifocal sharp and spike wave discharges, but no visualized seizure activity. She was treated with valproic acid between 4 and 8 years of age for mood stabilization, and since then has taken aripripazole, an antipsychotic. Her behavior and socialization have greatly improved over the past 1–2 years, although she continues to have some adherence to Wxed routines and inXexibility. Her only other medical problems are genu valgum (knock-knees) and pes planus (Xat feet). The proband’s father is 28 years old and is in good health. His early childhood and midadolescence were characterized by severe episodic behavioral dyscontrol, hyperactivity, anxiety, and apathy. He had socialization and cognitive diYculties and received special schooling for the duration of his education. His various childhood diagnoses included DSM-III Attention DeWcit Hyperactivity Disorder, Developmental Articulation Disorder with a residual chronic stutter in adulthood, Developmental Expressive Language Disorder, Developmental Coordination Disorder, and Developmental Reading Disorder. He was treated with methylphenidate through his childhood and abruptly discontinued schooling in the tenth grade. His height is Wfth percentile, weight is 90–95th percentile, and BMI is 33.7 (obese). He has never had an EEG. His behavioral diYculties improved in his early teens. He now lives independently and has a full-time job and full custody of his daughter. He has

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a learning disability. For instance, he is able to write and read simple sentences, but has diYculty reading and spelling longer words. The proband’s father has one sibling, a nondysmorphic brother who is 25 years old. This individual is in good health and, in fact, has a history of being underweight as a child. He did not require special education. He completed college, has a job, and lives independently. His mother is 47 years of age, and has short stature, obesity, and cognitive problems suggestive of executive dysfunction. The latter two individuals did not consent for testing. The proband’s mother has a history of substance abuse and is not involved in her care. The proband has four maternal half-siblings with no further information available. None of these maternal relatives were available for evaluation. Neuromotor, neuropsychological, and neuropsychiatric assessments were carried out. The proband was administered the Bruininks–Oseretsky test of motor proWciency (BOT-2) at 6 years and 4 months, which revealed a Wne motor composite score of 20 (less than the Wrst percentile), consistent with grossly diminished Wne motor abilities. The Wechsler intelligence scale for children, fourth edition (WISC-IV) revealed a full-scale intelligence quotient (FSIQ) in the mild ID range (Table 1). Neuropsychiatric assessment conWrmed the diagnosis of PDD-NOS. The father had previously been assessed with the Wechsler intelligence scales, and the Bender–Gestalt test of visual-motor functioning. Cognitive testing on two occasions during childhood revealed low average intellectual functioning with a signiWcant verbal learning disability (Table 2), consistent with current adaptive functioning. The Bender–Gestalt neuromotor assessment completed at Table 1 Summary of WISC-IV scores for proband, ages 6 and 8 years

WISC-IV Wechsler intelligence scale for children, 4th edition

Scale

8 years and 7 months revealed a Koppitz developmental score of 10 (Wfth percentile), consistent with perceptualmotor diYculties. IdentiWcation of a novel missense mutation in MECP2 and exclusion of other contributing genes Cytogenetic and molecular analyses were performed to determine the cause of the neuropsychiatric phenotypes in the proband and her father. The proband had a normal 46,XX female karyotype in peripheral blood. Fragile X testing by Southern blotting was normal with FMR1 alleles containing approximately 27 § 3 and 29 § 3 CGG repeats. Testing for Prader–Willi and Angelman syndromes by chromosome 15q11–13 methylation analysis using MLPA/PCR was normal (normal maternal and paternal bands). Melanocortin-4 receptor (coding regions and Xanking splice sites) and the PTEN (exons 1–9, Xanking splice sites, and core promoter) gene sequences were normal. Whole genome microarray analysis revealed no copy number variants on a 10-kb oligonucleotide-based microarray (Agilent). Two copies of the MECP2 gene were present by MLPA analysis. MECP2 gene sequencing (coding regions and Xanking splice sites) revealed a single heterozygous nucleotide variant, c.454C>G in exon 4 (Genbank Accession Number NM_004992.2), resulting in the change of a proline to an alanine residue, p.P152A. The same sequence variant was present in the father, who had a 46,XY karyotype in peripheral blood. This is a novel missense mutation that has not been reported in the literature or in the RTT mutation database (Christodoulou et al. 2003; http://mecp2.chw. edu.au). Furthermore, it has not been observed in 100

Age 6 years 11 months

Age 8 years

Composite score

Percentile rank

Composite score

Percentile rank

Verbal comprehension

83

13

81

10

Perceptual reasoning

69

2

57

0.2

Working memory

91

27

65

1

Processing speed

56

0.2

59

0.3

Full-scale IQ

70

2

58

0.3

Table 2 Summary of WISC-R and WISC-III scores for father, ages 8 years and 11 years Scale

WISC-R (age 8 years 4 months)

WISC-III (age 11 years 3 months)

Composite score

Percentile rank

Composite score

Percentile rank

Verbal IQ

75

4.5

74

4

Performance IQ

96

38

103

58

Full-scale IQ

84

14

85

16

WISC-R Wechsler intelligence scale for children, revised edition; WISC-III Wechsler intelligence scale for children, 3rd edition

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normal individuals tested by the DNA Laboratory at Children’s Hospital, Boston. X-inactivation analysis using a polymorphic marker in the androgen receptor locus revealed a random X-inactivation pattern (ratio 57:43). Online computational analysis The potential eVect of the p.P152A substitution was evaluated using the PolyPhen (Polymorphism Phenotyping) (http:// genetics.bwh.harvard.edu/cgi-bin/pph/polyphen.cgi) and SIFT (Sorting Intolerant From Tolerant) (http://blocks. fhcrc.org/ sift/SIFT.html) programs (Ng and HenikoV 2001; Ramensky et al. 2002). The PolyPhen program predicts the signiWcance of a missense mutation by analysis of protein sequence homology and mapping of the amino acid substitution to known three-dimensional structures. The p.P152A change was predicted to be “probably damaging.” P is invariant at position 152 in seven reference species. The shift in amino acid properties is measured in the positionspeciWc independent counts (PSIC) score (P score +1.917; A score ¡0.357; PSIC score diVerence 2.274). By comparison, p.P152R, a diVerent amino acid substitution at the same position that causes classic RTT, also is predicted to be “probably damaging,” with a PSIC score diVerence of 2.499 (P +1.917, A ¡0.582). The SIFT program predicted that both the p.P152A and p.P152R changes are not tolerated. Subcellular and biochemical characterization of the p.P152A mutation

P152R_fw 5⬘-CGACACATCCCTGGACCGTAA TGATTTTGACTTCAC-3⬘ P152R_rv 5⬘-GTGAAGTCAAAATCATTACGGTCC AGGGATGTGTCG-3⬘ T158M_fw 5⬘-GGACCCTAATGATTTTGACTTC ATGGTAACTGGGAGAG-3⬘ T158M_rv 5⬘-CTCTCCCAGTTACCATGAAGTCA AAATCATTAGGGTCC-3⬘ Cell culture and transfection Mouse BALB/c 3T3 Wbroblasts were obtained from ATCC and maintained in DMEM with 10% FBS at 37°C in 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and allowed to express the Mecp2e1 cDNA for 24 h before Wxation. ImmunoXuorescence 3T3 cells were plated on glass slides and transfected with MeCP2–FLAG constructs. The slides were washed twice in 1£ PBS, Wxed in Histochoice for 15 min at 25°C, washed twice in 1£ PBS and then incubated with anti-FLAG M2 (Sigma-Aldrich), detected with Alexa 488-conjugated antimouse antibodies and counterstained with DAPI. Images were captured using a Sensys CCD camera (Photometrics) attached to a Zeiss Axioplan 2 Xuorescence microscope. Nuclear fractionation

To further characterize the potential pathogenic eVects of this novel amino acid substitution, we compared the subcellular localization patterns of wild-type MeCP2 and MeCP2 containing p.P152A and two mutations that cause classic RTT, p.P152R and p.T158M. The mutations were generated in a mammalian expression vector encoding an MeCP2e1–FLAG fusion protein. Wild-type and mutant Mecp2 constructs were transiently transfected into cultured BALB/c 3T3 mouse Wbroblasts, and their nuclear localization was examined by immunoXuorescent detection with the anti-FLAG M2 monoclonal antibody. Generation of FLAG expression constructs The open reading frame of mouse Mecp2e1 was subcloned into the pCMV Tag4 c-terminal FLAG expression vector (Stratagene). Point mutants were generated by PCR-based mutagenesis using the following primers: P152A_fw 5⬘-CGACACATCCCTGGACGCTAATG ATTTTGACTTC-3⬘ P152A_rv 5⬘-GAAGTCAAAATCATTAGCGTCCA GGGATGTGTCG-3⬘

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3T3 cells transiently transfected with MeCP2–FLAG constructs were collected in 1£ PBS + 1.0 mM EDTA and washed twice with ice cold 1£ PBS. Cells were swollen in hypotonic buVer and lysed using a Dounce homogenizer. Nuclei were pelletted at 3300 g at 4°C. PuriWed nuclei were lysed in RIPA buVer for 20 min on ice and soluble and insoluble material were separated by centrifugation at 16,000 g for 15 min. 5£ SDS sample buVer was added to the resulting supernatant, representing the soluble fraction, to a Wnal concentration of 2£. The pellet, representing the insoluble fraction, was resuspended in an equal volume of 2£ sample buVer. Samples were boiled, separated by SDSPAGE and analyzed by immunoblotting with an anti-FLAG (M2) antibody (Sigma-Aldrich).

Results Biochemical Wndings In mouse cells, endogenous MeCP2 is concentrated in nuclear heterochromatin characterized by dense (punctate)

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Fig. 1 MeCP2 P152A has less severely impaired association with insoluble heterochromatin than RTT MBD mutations. a Subnuclear localization of MeCP2e1–FLAG wild type and mutants in 3T3 cells stained with anti-FLAG M2 (green, top) to detect MeCP2–FLAG and counterstained with DAPI (blue, lower) to visualize heterochromatic regions. b Immunoblot analysis using anti-FLAG M2 antibody to

detect protein samples isolated from the four transfected cell lines shown in (a). Comparison of soluble (S) and insoluble (I) fractions are shown to distinguish diVerences in binding to insoluble heterochromatin. c The ratio of insoluble to soluble MeCP2–FLAG was calculated for three independent experiments, and the results are shown as the mean percent decrease from wild-type control + SEM

DAPI staining. However, many RTT mutations can disrupt this organization to varying extents (Kudo et al. 2003). Wild-type MeCP2–FLAG fusion protein was found exclusively in the nucleus, where it was concentrated in DAPIlabeled heterochromatic foci (Fig. 1a) in a pattern identical to endogenous MeCP2. The MeCP2 mutants also showed some colocalization with DAPI-stained heterochromatin. However, they also showed varying levels of diVuse nuclear staining outside of the heterochromatic foci. The classic RTT mutations, p.P152R and p.T158M, showed the most pronounced increase in the level of nonheterochromatic nuclear staining (Fig. 1a), whereas p.P152A showed only subtly more diVuse MeCP2 localization than wildtype. Expression of the MeCP2 mutants had no detectable eVect on overall nuclear morphology or on formation of DAPI-stained heterochromatin (Fig. 1a). Biochemically, heterochromatin exists in an insoluble form following fractionation of puriWed nuclei. To assess the biochemical consequences of diVerential heterochromatin association, nuclei were puriWed and fractionated from 3T3 cells expressing wild-type or mutant MeCP2. Wildtype MeCP2 was primarily associated with the insoluble fraction (Fig. 1b), conWrming its association with the insoluble heterochromatin (Fig. 1a). The p.P152A mutation showed an approximate 40% decrease in its association with insoluble heterochromatin compared to wild-type. The classic RTT mutations, p.P152R and p.T158 M, showed an

even larger decrease, 70–80%, in insoluble MeCP2 (Fig. 1b, c).

Discussion Several lines of evidence point toward the p.P152A missense mutation in MECP2 as being pathogenic rather than incidental to this family’s phenotype. First, to the best of our knowledge, p.P152A has not been previously observed in the general population, indicating that is not a reported polymorphism. Second, this proline appears to be critical to the function of MeCP2, based on its conservation across seven species and the Wnding that another missense mutation involving the same amino acid, p.P152R, causes classic RTT (Huppke et al. 2000; Cheadle et al. 2000). The p.P152A missense mutation is located in a critical MBD of MeCP2 that associates with heterochromatin. Third, the PolyPhen and SIFT software programs predict that both p.P152A and p.P152R are “probably damaging” and not tolerated. Finally, overexpression of MeCP2–p.P152A shows approximately 40% reduction in heterochromatin binding when overexpressed in cultured 3T3 cells. This partial reduction in heterochromatin binding is less than the 70–80% reduction observed with two classic RTT mutations (p.P152R and T158M) and would be consistent with the milder phenotype in this family. The apparent diVerence

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in clinical severity observed between the two amino acid substitutions at the same position is probably related to the less conservative nature of the p.P152R mutation, with R being basic and A being nonpolar. While nuclear heterochromatin binding is one global measure of MeCP2 function shown to be aVected by the p.P152R mutation, additional functional studies of the eVects of this novel mutation on speciWc MeCP2 target genes would be of interest. Many RTT-associated MBD mutations reduce the aYnity of MeCP2 for methylated DNA (Ballestar et al. 2000; Kumar et al. 2008; Yusufzai and WolVe 2001). Transgenic mice with a hypomorphic Mecp2 allele that expresses 50% of the wild-type level of MeCP2 were found to be heavier than control animals and had social, behavioral, and learning deWcits (Samaco et al. 2008; Kerr et al. 2008). These Wndings demonstrate the importance of precise control of MeCP2 levels and function and predict that human neurodevelopmental disorders may result from a decrease of MeCP2 levels by as little as 50%. The importance of regulatory precision is also consistent with the development of seizures and premature demise in transgenic mice overexpressing Mecp2 (Collins et al. 2004). Notably, the deWcits associated with partial reduction in MeCP2 expression were sensitive to modiWer eVects in two diVerent mouse strains. Samaco et al. predict that this observation is likely to extend to humans and suggest that the array of phenotypes associated with subtle reduction in MeCP2 levels is likely to be broad and that interindividual clinical variability will be common. Assuming that a 50% reduction in MeCP2 expression in mice is comparable to the reduction in the level of heterochromatin association observed with the p.P152A mutation, the mouse phenotype is remarkably similar to that observed in our family. Males with milder missense MECP2 mutations not found in females with RTT present with mild to severe ID. In this family, p.P152A was associated with intellectual dysfunction ranging from learning disabilities on a background of normal, although low average IQ (father) to mild ID (daughter). Both individuals had large diVerences between verbal and performance function of 20 points or greater, although the areas of relative strength and weakness were diVerent. The proband’s verbal abilities were superior to those of her father, while this Wnding was reversed for the performance measures. The highest fullscale IQ reported in a male with a MBD mutation is 60 (WAIS-R, age 9 years) (Gendrot et al. 1999; Couvert et al. 2001) with moderate to severe ID being the rule in the majority of cases. Neuropsychiatric manifestations have been reported in the context of severe neuromotor disease, primarily psychosis, pyramidal signs, and macro-orchidism (PPMX) in one family (Klauck et al. 2002). Both of our cases had intermittent explosive mood dysregulation, subtle

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Wne motor dysfunction, and obesity. In both cases, behavioral and adaptive functioning improved over time. The father has not had Xorid aggressive outbursts since his midteens, lives independently, holds down a regular job, and maintains full custody and oversight of his daughter. After a period of apparently worsening function, the proband has shown recent behavioral progress in the last year. Therefore, the p.P152A phenotype observed in this family appears to be more attenuated than the Zappella variant of RTT (Renieri et al. 2008; Zappella et al. 2001). As with some Zappella RTT-variant girls, she is overweight. She is also macrocephalic with a mild kyphosis and substantially improving pervasive developmental features. A comparison of the patient to typical Zappella variant patients is described in Table 3, using an ordering of the criteria suggested by the Zappella group (Renieri et al. 2008). There are distinct diVerences that emerge, such as the absence of regression, that distinguish the proband from individuals with RTT, the Zappella variant inclusive. Notably, the proband would not fulWll the consensus criteria for variant RTT (Hagberg et al. 2002). We have excluded mutations in several other known genes that could have contributed to the proband’s phenotype. Our analysis also showed that intrinsic methylation abnormalities at the SNRPN locus were not responsible for the obesity observed in our family. However, given the previous description of obesity in some Zappella variant RTT cases and the strong binding of MeCP2 to the SNRPN ICR and other 15q11–13 genes (Yasui et al., 2007), further studies to investigate potential 15q11–13 dysregulation may be informative. Given the proband’s random XCI pattern in blood, it is not clear why heterozygosity for a MECP2 mutation in the daughter is associated with a more severe phenotype than in her hemizygous father. Testing of the father’s blood showed no evidence of mosaicism for the MECP2 mutation or a 47,XXY karyotype, but somatic mosaicism in brain cannot be excluded as a potential explanation for the milder phenotype of the father. It is possible that behavioral problems associated with the proband’s diagnosis of PDD-NOS prevented valid IQ testing, though adaptive behaviors as reported by teachers and parents are consistent with mild ID. The lower adaptive functioning found in patients with autism spectrum disorders may possibly increase the severity of her clinical presentation to a greater degree than in the absence of a non-PDD background. Other possible explanations include inheritance of maternal deleterious risk genes, or environmental eVects such as in utero exposure to nicotine or substance abuse. Another environmental factor could be the long-term treatment of the proband with divalproex sodium (valproate), a histone deacetylase (HDAC) inhibitor, for mood stabilization. Given that MeCP2 is thought to recruit HDAC to the heterochromatin, it is possible that the addition of the

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Table 3 Comparison of proband to typical Zappella variant Rett syndrome phenotype Main criteria

Proband

Language recovered at a mean age of 6 years

Present

Hand stereotypies as in Rett syndrome

Rare hand stereotypies

Milder intellectual disabilities (IQ up to 50)

IQ of 70 and 58 on separate tests 2 years apart

Normal head circumference, weight and height in most patients

Present; obesity

Milder reduction of hand skills allowing quite good hand use

Present

Rett syndrome disease proWle with regression after 3 years and a prolonged Stage 3

Stage 2 not noted

Supportive criteria

Proband

Rarer epilepsy

No epilepsy; abnormal EEG

Rarer neurovegetative dysfunctions

No neurovegetative dysfunction

Milder scoliosis and kyphosis

No scoliosis Task-related kyphosis

Presence of autistic behavior

Mild; diagnosis of PDD-NOS

Presence of late-truncating or missense mutations in MECP2

Missense mutation in MECP2

HDAC inhibitor may have exacerbated the MeCP2 eVect, aVecting phenotypic severity. No controlled studies of the long-term eVects of valproate on longitudinal severity scores in RTT have been performed, although the potential interaction has been noted. Hints to this interaction include the diminished eYcacy of valproic acid in controlling RTTassociated seizures relative to other antiepileptic drugs (Huppke et al. 2007) and observed alterations in MeCP2 mRNA and Class I HDAC isoforms (HDAC 1, 2, and 3) in response to clinically signiWcant levels of valproic acid (Kim et al. 2008). In conclusion, we have identiWed a milder phenotype relative to RTT-spectrum phenotypes, characterized by learning and cognitive disabilities, obesity and behavioral dyscontrol, in association with a novel mutation in the MBD of MeCP2. This MECP2 mutation is predicted to cause a partial (40%) reduction in heterochromatin binding. Therefore, it would appear that subtle variation, which causes limited functional impairment in MeCP2 may present with attenuated neurological manifestations such as, in this family, primarily neuropsychiatric debility. Because the threshold for phenotypic manifestations appears to lie on a gradient of functional impairment, the presence of a nucleotide change in a male with normal intelligence does not necessarily determine that the change is benign. In addition, MECP2 may escape consideration because the pedigree may not appear X-linked and the somatic and motor phenotype is dissimilar to that of classic RTT. Individuals with neurobehavioral debility and the peculiar somatic phenotype of macrocephaly and obesity observed in some patients with preserved speech variant RTT, and in our described cases, may prove to be the most initial appropriate candidates for mutation screening in a broader population. More population-based research concerning the role

of hypomorphic MECP2 point mutations in the etiology of intellectual disability needs to be undertaken. Acknowledgments We would like to thank Jennifer Gentile and Susan Waisbren for neuropsychological testing and Yiping Shen for his help with MECP2 controls. Abidemi Adegbola was supported by an NIH (NIGMS) grant (grant number T32 GM007748). Michael Gonzales was supported by an NIH (NIMH) grant (grant number T32MH073124). Janine Lasalle was supported by NIH (NICHD) grants (grants numbers R01HD041462 and R01HD048799).

References 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 (2000) InXuence of mutation type and X chromosome inactivation on Rett syndrome phenotypes. Ann Neurol 47:670–679 Archer H, Evans J, Leonard H, Colvin L, Ravine D, Christodoulou J, Williamson S, Charman T, Bailey ME, Sampson J, de Klerk N, Clarke A (2007) Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation. J Med Genet 44:148–152 Auranen M, Vanhala R, Vosman M, Levander M, Varilo T, Hietala M, Riikonen R, Peltonen L, Järvelä I (2001) MECP2 gene analysis in classical Rett syndrome and in patients with Rett-like features. Neurology 56:611–617 Ballestar E, Yusufzai TM, WolVe AP (2000) EVects of Rett syndrome mutations of the methyl-CpG binding domain of the transcriptional repressor MeCP2 on selectivity for association with methylated DNA. Biochemistry 39:7100–7106 Bao X, Jiang S, Song F, Pan H, Li M, Wu XR (2008) X chromosome inactivation in Rett Syndrome and its correlations with MECP2 mutations and phenotype. J Child Neurol 23:22–25 Bebbington A, Anderson A, Ravine D, Fyfe S, Pineda M, de Klerk N, Ben-Zeev B, Yatawara N, Percy A, Kaufmann WE, Leonard H (2008) Investigating genotype–phenotype relationships in Rett syndrome using an international data set. Neurology 70:868– 875

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622 Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, Zoghbi HY (2008) MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320:1224–1229 Charman T, Neilson TC, Mash V, Archer H, Gardiner MT, Knudsen GP, McDonnell A, Perry J, Whatley SD, Bunyan DJ, Ravn K, Mount RH, Hastings RP, Hulten M, Orstavik KH, Reilly S, Cass H, Clarke A, Kerr AM, Bailey ME (2005) Dimensional phenotypic analysis and functional categorisation of mutations reveal novel genotype–phenotype associations in Rett syndrome. Eur J Hum Genet 13:1121–1130 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 (2000) Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum Mol Genet 12:1119–1129 Christodoulou J, Grimm A, Maher T, Bennetts B (2003) RettBASE: the IRSA MECP2 variation database—a new mutation database in evolution. Hum Mutat 21:466–472 Clayton-Smith J, Watson P, Ramsden S, Black GC (2000) Somatic mutation in MECP2 as a non-fatal neurodevelopmental disorder in males. Lancet 356:830–832 Collins AL, Levenson JM, Vilaythong AP, Richman R, Armstrong DL, Noebels JL, David Sweatt J, Zoghbi HY (2004) Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Hum Mol Genet 13:2679–2689 Couvert P, Bienvenu T, Aquaviva C, Poirier K, Moraine C, Gendrot C, Verloes A, Andrès C, Le Fevre AC, Souville I, SteVann J, des Portes V, Ropers HH, Yntema HG, Fryns JP, Briault S, Chelly J, Cherif B (2001) MECP2 is highly mutated in X-linked mental retardation. Hum Mol Genet 10:941–946 del Gaudio D, Fang P, Scaglia F, Ward PA, Craigen WJ, Glaze DG, Neul JL, Patel A, Lee JA, Irons M, Berry SA, Pursley AA, Grebe TA, Freedenberg D, Martin RA, Hsich GE, Khera JR, Friedman NR, Zoghbi HY, Eng CM, Lupski JR, Beaudet AL, Cheung SW, Roa BB (2006) Increased MECP2 gene copy number as the result of genomic duplication in neurodevelopmentally delayed males. Genet Med 8:784–792 Dotti MT, Orrico A, De Stefano N, Battisti C, Sicurelli F, Severi S, Lam CW, Galli L, Sorrentino V, Federico A (2002) A Rett syndrome MECP2 mutation that causes mental retardation in men. Neurology 22:226–230 Erlandson A, Hagberg B (2005) MECP2 abnormality phenotypes: clinicopathologic area with broad variability. J Child Neurol 20:727–732 Gendrot C, Ronce N, Raynaud M, Ayrault AD, Dourlens J, Castelnau P, Muh JP, Chelly J, Moraine C (1999) X-linked nonspeciWc mental retardation (MRX16) mapping to distal Xq28: linkage study and neuropsychological data in a large family. Am J Med Genet 23:411–418 Hagberg BA, Skjeldal OH (1994) Rett variants: a suggested model for inclusion criteria. Pediatr Neurol 11:5–11 Hagberg B, Hanefeld F, Percy A, Skjeldal O (2002) An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001. Eur J Paediatr Neurol 6:293–297 HoVbuhr 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, HoVman EP, Naidu S (2001) MeCP2 mutations in children with and without the phenotype of Rett syndrome. Neurology 56:1486–1495 Huppke P, Laccone F, Krämer N, Engel W, Hanefeld F (2000) Rett syndrome: analysis of MECP2 and clinical characterization of 31 patients. Hum Mol Genet 9:1369–1375

123

Hum Genet (2009) 124:615–623 Huppke P, Held M, Hanefeld F, Engel W, Laccone F (2002) InXuence of mutation type and location on phenotype in 123 patients with Rett syndrome. Neuropediatrics 33:63–68 Huppke P, Köhler K, Brockmann K, Stettner GM, Gärtner J (2007) Treatment of epilepsy in Rett syndrome. Eur J Pediatr Neurol 11:10–16 Kerr B, Alvarez-Saavedra M, Sáez MA, Saona A, Young JI (2008) Defective body-weight regulation, motor control and abnormal social interactions in Mecp2 hypomorphic mice. Hum Mol Genet 17:1707–1717 Kim B, Rincón Castro LM, Jawed S, Niles LP (2008) Clinically relevant concentrations of valproic acid modulate melatonin MT(1) receptor, HDAC and MeCP2 mRNA expression in C6 glioma cells. Eur J Pharmacol 589:45–48 Klauck SM, Lindsay S, Beyer KS, Splitt M, Burn J, Poustka A (2002) A mutation hot spot for nonspeciWc X-linked mental retardation in the MECP2 gene causes the PPM-X syndrome. Am J Hum Genet 70:1034–1037 Kudo S, Nomura Y, Segawa M, Fujita N, Nakao M, Schanen C, Tamura M (2003) Heterogeneity in residual function of MeCP2 carrying missense mutations in the methyl CpG binding domain. J Med Genet 40:487–493 Kumar A, Kamboj S, Malone BM, Kudo S, Twiss JL, Czymmek KJ, Lasalle JM, Schanen NC (2008) Analysis of protein domains and Rett syndrome mutations indicate that multiple regions inXuence chromatin-binding dynamics of the chromatin-associated protein MECP2 in vivo. J Cell Sci 121:1128–1137 Leonard H, Silberstein J, Falk R, Houwink-Manville I, Ellaway C, RaVaele LS, Engerström IW, Schanen C (2001) Occurrence of Rett syndrome in boys. J Child Neurol 16:333–338 Nan X, Tate P, Li E, Bird A (1996) DNA methylation speciWes chromosomal localization of MeCP2. Mol Cell Biol 16:414–421 Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A (1998) Transcriptional repression by the methyl-CpGbinding protein MeCP2 involves a histone deacetylase complex. Nature 393:386–389 Neul JL, Fang P, Barrish J, Lane J, Caeg EB, Smith EO, Zoghbi H, Percy A, Glaze DG (2008) SpeciWc mutations in methyl-CpG-binding protein 2 confer diVerent severity in Rett syndrome. Neurology 70:1313–1321 Ng PC, HenikoV S (2001) Predicting deleterious amino acid substitutions. Genome Res 11:863–874 Nielsen JB, Henriksen KF, Hansen C, Silahtaroglu A, Schwartz M, Tommerup N (2001) MECP2 mutations in Danish patients with Rett syndrome: high frequency of mutations but no consistent correlations with clinical severity or with the X chromosome inactivation pattern. Eur J Hum Genet 3:178–184 Orrico A, Lam C, Galli L, Dotti MT, Hayek G, Tong SF, Poon PM, Zappella M, Federico A, Sorrentino V (2000) MECP2 mutation in male patients with non-speciWc X-linked mental retardation. FEBS Lett 481:285–288 Ramensky V, Bork P, Sunyaev S (2002) Human non-synonymous SNPs: server and survey. Nucleic Acids Res 30:3894–3900 Renieri A, Mari F, Mencarelli MA, Scala E, Ariani F, Longo I, Meloni I, Cevenini G, Pini G, Hayek G, Zappella M (2008) Diagnostic criteria for the Zappella variant of Rett syndrome (the preserved speech variant). Brain Dev. doi:10.1016/j.braindev.2008.04.007 Samaco RC, Fryer JD, Ren J, FyVe S, Chao HT, Sun Y, Greer JJ, Zoghbi HY, Neul JL (2008) A partial loss of function allele of methyl-CpG-binding protein 2 predicts a human neurodevelopmental syndrome. Hum Mol Genet 17:1718–1727 Scala E, Longo I, Ottimo F, Speciale C, Sampieri K, Katzaki E, Artuso R, Mencarelli MA, D’Ambrogio T, Vonella G, Zappella M, Hayek G, Battaglia A, Mari F, Renieri A, Ariani F (2007) MECP2 deletions and genotype–phenotype correlation in Rett syndrome. Am J Med Genet A 143A:2775–2784

Hum Genet (2009) 124:615–623 Topçu M, Akyerli C, Sayi A, Törüner GA, Koçoflu SR, Cimbio M, Ozçelik T (2002) Somatic mosaicism for a MECP2 mutation associated with classic Rett syndrome in a boy. Eur J Hum Genet 10:77–81 Villard L, Kpebe A, Cardoso C, Chelly PJ, Tardieu PM, Fontes M (2000) Two aVected boys in a Rett syndrome family: clinical and molecular Wndings. Neurology 55:1188–1193 Wan M, Lee SS, Zhang X, Houwink-Manville I, Song HR, Amir RE, Budden S, Naidu S, Pereira JL, Lo IF, Zoghbi HY, Schanen NC, Francke U (1999) Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots. Am J Hum Genet 65:1520–1529 Weaving LS, Williamson SL, Bennetts B, Davis M, Ellaway CJ, Leonard H, Thong MK, Delatycki M, Thompson EM, Laing N,

623 Christodoulou J (2003) EVects of MECP2 mutation type, location and X-inactivation in modulating Rett syndrome phenotype. Am J Med Genet A 118A:103–114 Yasui DH, Peddada S, Bieda MC, Vallero RO, Hogart A, Nagarajan RP, Thatcher KN, Farnham PJ, Lasalle JM (2007) Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes. Proc Natl Acad Sci USA 104:19416–19421 Yusufzai TM, WolVe AP (2001) Functional consequences of Rett syndrome mutations on human MeCP2. Nucleic Acids Res 28:4172– 4179 Zappella M, Meloni I, Longo I, Hayek G, Renieri A (2001) Preserved speech variants of the Rett syndrome: molecular and clinical analysis. Am J Med Genet 104:14–22

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