GENETIC TESTINGVolume 5, Number 4, 2001© Mary Ann Liebert, Inc.

Mutation Analysis in Rett Syndrome

JEFF M. MILUNSKY, ROGER V. LEBO, TOHRU IKUTA, THOMAS A. MAHER, CARRIE E. HAVERTY, and AUBREY MILUNSKY

ABSTRACT

Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2gene. Mutations have been demonstrated in more than 80% of females with typical features of Rett syndrome.We identified mutations in the MECP2 gene and documented the clinical manifestations in 65 Rett syndromepatients to characterize the genotype–phenotype spectrum. Bidirectional sequencing of the entire MECP2 cod-ing region was performed. We diagnosed 65 patients with MECP2 mutations. Of these, 15 mutations had beenreported previously and 13 are novel. Two patients have multiple deletions within the MECP2 gene. Eightcommon mutations were found in 43 of 65 patients (66.15%). The majority of patients with identified muta-tions have the classic Rett phenotype, and several had atypical phenotypes. MECP2 analysis identified muta-tions in almost all cases of typical Rett syndrome, as well as in some with atypical phenotypes. Eleven (20.4%)of the 54 patients with defined mutations and in whom phenotypic data were obtained did not develop ac-quired microcephaly. Hence, microcephaly at birth or absence of acquired microcephaly does not obviate theneed for MECP2 analysis. We have initiated cascade testing starting with PCR analysis for common muta-tions followed by sequencing, when necessary. Analysis of common mutations before sequencing the entiregene is anticipated to be the most efficacious strategy to identify Rett syndrome gene mutations.

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INTRODUCTION

RETT SYNDROME is an X-linked dominant neurodevelop-mental disorder, first described by Andreas Rett (Rett,

1966), and affecting about 1 in 12,000 females (Hagberg andHagberg, 1997). Criteria for the clinical diagnosis of Rett syn-drome have been delineated previously (Clarke, 1996). Afteran initial normal period of development (6–18 months), femaleswith classic Rett syndrome have neurodevelopmental regres-sion, seizures, acquired microcephaly, loss of speech, and pur-poseful hand use. Additional features described include autis-tic behavior, gait apraxia, stereotypic hand movements,respiratory dysfunction, difficulties with swallowing, scoliosis,growth failure, autonomic dysfunction, and a prolonged QTcinterval as seen on electrocardiograms (Hagberg and Aicardi,1983; Glaze et al., 1987; Sekul et al., 1994).

Rett syndrome results from mutations in the 4 exon methyl-CpG-binding protein 2 (MECP2) gene, localized to chromo-some Xq28 (Amir et al., 1999; Vilain et al., 1996). MECP2 ap-pears to repress transcription from methylated promoters of

other genes by binding to methylated DNA (Nan et al., 1997).About 99.5% of all Rett cases are sporadic, whereas familialcases of Rett syndrome are rare and are due to X-chromosomalinheritance from a carrier mother (Girard et al., 2001; Trappeet al., 2001). Several large studies have now identified MECP2mutations routinely in over 80% of classic Rett syndrome pa-tients (Cheadle et al., 2000; Vacca et al., 2001). A broader phe-notype is now emerging in those with MECP2 mutations in-cluding affected males, females with atypical presentations, andthose with Angelman syndrome-like phenotypes (Meloni et al.,2000; Villard et al., 2000; Imessaoudene et al., 2001; Inui etal., 2001; Watson et al., 2001; Williams et al., 2001). Somegenotype/phenotype correlations are emerging with the type andlocation of MECP2 mutation and the involvement of skewedX-inactivation (Amir et al., 2000; Hoffbuhr et al., 2001; Neil-son et al., 2001; Villard et al., 2001; Webb and Latif, 2001).Although more than 100 mutations have been described in theMECP2 gene, several recurrent mutations appear to be com-mon, especially in exon 4 (Wan et al., 1999; Amir et al., 2000;Buyse et al., 2000; Dragich et al., 2000; Laccone et al., 2001).

Center for Human Genetics and the Department of Pediatrics, Boston University School of Medicine, Boston, MA 02118.

Various laboratories have chosen different methodologies forscreening the MECP2 gene. A two-tiered approach has beenproposed using either single-strand conformation polymor-phism (SSCP) or denatured high-performance liquid chro-matography (dHPLC), followed by confirmatory sequencinganalysis (Buyse et al., 2000; Vacca et al., 2001). Our Centerhas implemented analysis of common mutations by PCR anal-ysis followed by bidirectional sequencing of the entire MECP2coding region, when necessary.

We report our clinical and molecular experience with 65cases of Rett syndrome and discuss common mutation analysisof the MECP2 gene.

MATERIALS AND METHODS

Clinical data

Our experience includes 64 female patients and the af-fected brother of 1 patient. All subjects have a mutation inthe MECP2 gene identified by common mutation or sequenceanalysis. The referring providers for 54 of the patients re-sponded to requests for clinical data. The information re-quested included the patient’s speech/communication abili-ties, gross motor function including ability to walk,age-achieved and persistence of ambulation, as well as sei-zure activity, type, and age of onset. In addition, we ascer-tained if the patient had acquired microcephaly, respiratorydysfunction such as hyperventilation, loss of purposeful handmovements, scoliosis, self-abuse, screaming fits, develop-mental regression, and excessive drooling or swallowingproblems. Clinical summaries and neurology evaluation re-ports were also requested.

DNA analysis

DNA extraction: Blood samples were received and processedat the Center for Human Genetics, Boston University Schoolof Medicine. Genomic DNA was extracted using the Puregenekit (Gentra Systems, Minneapolis, MN), according to manu-facturer’s instructions.

Amplification and analysis of genomic DNA: The currentmutation detection strategy is two-fold. The samples wereinitially screened for five common Rett mutations (T158M,R168X, R255X, R294X, R306C/H) using restriction enzymeanalysis based on their frequency of detection in previousstudies (Amir et al., 1999, 2000; Wan et al., 1999; Buyse etal., 2000; Cheadle et al., 2000). The amplicons generatedused either primer sequences that annealed perfectly to takeadvantage of naturally occurring restriction sites or containeda mismatched base to create a restriction enzyme site. If nomutations were detected, the same amplicons were then sub-jected to sequencing. PCR reactions were performed in 25-ml reaction volumes under the following conditions: 95°Cfor 5 min for one cycle, 30 cycles (94°C for 30 sec, 55°Cfor 30 sec, 72°C for 30 sec), and one cycle of 72°C for 5min. Amplifications were performed in either a PTC100 ther-mocycler (MJ Research, Watertown, MA.) or a Primus 96thermocycler (MWG-Biotech). Primer sequences for gener-ating amplicons and sequencing derived from NCBI (acces-

sion number, AF030876) are available upon request. Then10 ml of the amplicons were digested overnight with the ap-propriate enzyme and electrophoresed on an agarose gel un-til the different-length fragments were clearly resolved. Thegel was stained with ethidum bromide and photographed un-der UV excitation.

Sequence analysis: Simultaneous bidirectional cycle se-quencing (SBS) using IRD-labeled nested primers was com-pleted on the amplicons with the Taq FS sequencing kit (Ap-plied Biosystems) using the above PCR conditions with theelimination of the final 72°C/5 min extension cycle. The se-quences were analyzed using the Li-Cor automated sequenc-ing system (Li-Cor; Lincoln, NE). The patient’s sequence wascompared to the normal MECP2 sequence in the database(NCBI). Any mismatch found by using BLAST to comparethe derived sequence against the database sequences was con-firmed in the simultaneously sequenced complementarystrand. Maternal blood samples were requested on all patientswith detected MECP2 mutations to ascertain their carrier status.

RESULTS

The 65 patients we studied had a total of 28 mutations in theMECP2 gene. Of these, 15 mutations have been reported pre-viously, and 13 are novel (Table 1). Among the 28 mutationsfound, 8 mutations recurred in more than 3 unrelated individ-uals in our series. These 8 most common mutations comprised43 (66.15%) of the total mutations detected (Table 2).

Examination of the clinical data received on 54 of the pa-tients revealed that the vast majority have the classic Rett syn-drome phenotype. Nevertheless, several had been labeled pre-viously as having nonspecific mental retardation or mentalretardation with spastic cerebral palsy. Many patients had pre-viously undergone extensive, unrevealing evaluations. Thosepatients with mutations in the amino-terminus of MECP2tended to have more severe clinical manifestations comparedwith those patients with mutations at the carboxyl-terminus ofthe gene.

Atypical cases

We observed several atypical molecular and clinical cases.Previously, we reported a female with 5 tandem deletions anda second unrelated female with 3 tandem deletions within theMECP2 exon 4 that encode truncated protein products result-ing in typical Rett syndrome (Lebo et al., 2001).

A 754insG frameshift mutation was detected in a 15-year-old female with classic Rett syndrome and her 6-year-oldbrother with static encephalopathy. Her brother was born withcentral hypoventilation and hypotonia, and subsequently de-veloped a significant seizure disorder and a facial dyskinesia.He is now ventilator dependent. Their mother does not sharethis mutation in blood lymphocytes, implying germ-line mo-saicism. We also found a normal mother whose 4-year-olddaughter has typical Rett syndrome, with both possessing theP388L MECP2 mutation. X-inactivation studies on the motherare being done.

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Two of the common mutations (R168X and R255X) werenoted several times in females with abnormal development be-fore 6 months of age. Ten mutations, including 5 common mu-tations (T158M, R106W, R168X, R133C/P, and R255X), wereseen in patients greater than 5 years of age without acquiredmicrocephaly. In our sequence analysis, we detected 10 poly-morphisms, 8 of which were the S411S common MECP2 poly-morphism reported previously (Amir et al., 1999).

DISCUSSION

The clinical and molecular spectrum of Rett syndrome con-tinues to be elucidated. Guidelines for reporting clinical fea-tures in cases with MECP2 mutations have now emerged froman international group (Kerr et al., 2001). Although no clearmutation-specific genotype–phenotype correlations haveemerged, the type of mutation (i.e., missense vs. truncation)and location of mutation within the MECP2 gene may indeedinfluence the resultant phenotype. Truncating mutations andthose occurring in the amino-terminus of MECP2 appear tohave more severe clinical manifestations (Hoffbuhr et al.,2001).

The majority of patients in our study with identified muta-tions have the classic Rett phenotype, but several had atypicalphenotypes. Several of the common mutations that we foundwere in females with abnormal development before 6 monthsof age and in others with either normocephaly after age 5 ormicrocephaly at birth. The male patient described again pointsto the rare familial cases as well as the potential for germ-linemosaicism in Rett syndrome. X-inactivation patterns may ex-plain some of the clinical diversity seen within and betweenmutations. Further studies are needed to document the clinicalspectrum associated with MECP2 mutations in patients withatypical phenotypes.

The indications for molecular testing have clearly gonebeyond the female with classic Rett syndrome. Males with static encephalopathy, males and females with mental retardation, those with spasticity, Angelman-like phe-notypes, and now those with abnormal development frombirth are potential candidates for this testing. A total of 11(20.4%) of the 54 patients with defined mutations and phe-notypic data obtained in this study did not develop acquiredmicrocephaly. Hence, microcephaly at birth or absence ofacquired microcephaly does not obviate the need for MECP2analysis.

In this study, we describe common mutations in theMECP2 gene that accounted for two-thirds of those affectedwith Rett syndrome. This percentage of common recurrentmutations found is similar to previous studies (Wan et al.,1999; Amir et al., 2000; Buyse et al., 2000; Dragich et al.,2000; Laccone et al., 2001). We implemented cascade test-ing starting with PCR analysis of common mutations fol-lowed by sequencing, when necessary. We currently test for5 MECP2 mutations (T158M, R306C/H, R294X, R168X,and R255X) and have added 2 additional mutations (R106Wand R133C/P) to our initial screen, increasing our detectionrate to greater than 60%. This approach has been efficientand less costly than SSCP, dHPLC, or sequencing alone. Itis also useful when testing other family members. Analysisof common mutations before sequencing the entire gene isanticipated to be the most efficacious strategy to identify Rettsyndrome gene mutations.

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TABLE 1. NOVEL MUTATIONS IN THE MECP2 GENE

Mutation Exon Type Phenotype

N126S 4 Missense Microcephaly before 6 m.o., neonatal seizuresP127L 4 Missense ClassicK135E 4 Missense ClassicG161V 4 Missense ClassicQ262X 4 Nonsense NDA378G 4 Missense ClassicP388L 4 Missense Classic495delC 4 Deletion ND753insG 4 Insertion See text; sister, classic; brother, severe816delG 4 Deletion ND1126del52 4 Deletion ND1161delCC 4 Deletion ND1163del39 4 Deletion No acquired microcephaly, no speech, seizures, drooling

ND, Not determined.

TABLE 2. COMMON MUTATIONS IN THE MECP2 GENE

Common Number of % of totalmutation cases mutations

T158M 8 12.31R306C/H 7 10.77R294X 6 9.23R106W 5 7.69R168X 5 7.69R133C/P 5 7.69R255X 4 6.15R270X 3 4.62

Total 43 66.15

ACKNOWLEDGMENTS

We would like to thank Masamichi Ito, Ph.D., for his assis-tance. We would also like to thank all of the clinicians who re-ferred these cases to us.

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Address reprint requests to:Dr. Jeff Milunsky

Center for Human GeneticsBoston University School of Medicine

715 Albany Street, Suite W408Boston, MA 02118

E-mail: jmilunsk@bu.edu

Received for publication September 28, 2001; accepted December 14, 2001.

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