MECP2 gene analysis in classical Rett syndrome and in patients with Rett-like features

Patients and methods.

Fifty-two Finnish patients whose disease phenotype fulfilled all or part of the diagnostic criteria of classical Rett syndrome participated in the study. The diagnostic criteria used were as following: 1) normal pre- and perinatal development and apparently normal development for the first 5 to 6 months; 2) developmental regression with onset between 6 months and 3 years; 3) normal head circumference growth following deceleration between 5 months and 4 years; 4) loss of purposeful hand movements and acquisition of hand movement stereotypies; and 5) marked developmental and cognitive delay. Altogether, 52/80 (65%) of the parents of the patients with Rett syndrome were available for the study; samples from both parents were obtained in 22 families and maternal samples alone in eight families. The birthplaces of the ancestors of the patients were traced back two to six generations by interviewing the parents and using church records.25

Results.

Fourteen different mutations were found in MECP2 in the Finnish patients, including one novel mutation, P127L ( table 1). When the clinical phenotypes of the patients were compared with the results of mutation analyses, it was evident that an MECP2 mutation was present in the 39 patients whose disease phenotype fulfilled the diagnostic criteria for classical Rett syndrome (see the Patients and methods section). One novel mutation, P127L, was found in a patient with preserved speech. No mutations were found in the 12 patients with developmental delay and some Rett-like features.

Mutation spectrum.

There were 19 patients with Rett syndrome with missense and 21 patients with frameshift or nonsense mutations that were scattered fairly evenly along the complementary DNA of the MECP2 gene. Eleven (25%) mutations were clustered in the NLS domain of 17 amino acid residues.29 The mutational hot spot previously found in 35 Rett families,17 R168X, was detected only once among this patient group. The most prevalent mutations were transitions R306C with six, and T158M and R255X with five occurrences (see table 1). The mother of one patient (R17) had epilepsy. However, she was not a carrier of the R270X mutation (data not shown). The second cousin of Patient R1 presented with features from the autism spectrum of the disorder. This prompted us to investigate the whole coding region of MECP2 for mutations in the cousin, but no sequence alterations were detected (data not shown). The sister of Patient R32 had mild learning disabilities, and her mother had dystonia in the neck (torticollis), strabismus, and myopia. However, no mutation could be detected in the DNA isolated from the peripheral blood of the mother (data not shown). The patient with PSV was found to carry a novel missense mutation 380C→T in the middle of the methyl-CPG-binding domain (MBD) region, which changes a conserved proline to a leucine, P127L ( figure). The mutation affects the first codon of exon 4 and was not detected in the parents or in 75 normal non-Rett X chromosomes. No mutations were detected in the parental DNA available from 52/80 (65%) of the parents. The genealogical studies revealed one family in which two patients with Rett syndrome (R3 and R5) were shown to be second and half-cousins. However, they carried different mutations: L386fsX403 and G269fsX288. No other consanguinities could be detected, even within the group carrying the same mutation (data not shown).

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Figure. The novel heterozygous mutation 380C→T causing P127L substitution in a patient with preserved speech variant.

Discussion.

After the identification of MECP2 as a causative gene for Rett syndrome, mutations have been detected in 35 to 90% of patients.13,15-23⇓⇓⇓⇓⇓⇓⇓⇓⇓ Recent studies have also shown that the spectrum of mutations of the MECP2 gene is wide and extends to phenotypes that are not easily recognized as Rett syndrome.13,18,24,31,32⇓⇓⇓⇓

We screened 52 Finnish patients who were diagnosed with classical or atypical Rett syndrome. An MECP2 mutation was found in all patients with classical Rett syndrome (39/39; 100%) and in one patient with PSV. This patient carries a novel missense mutation, P127L, that is the eighth mutation of the MECP2 gene found in a patient with PSV.12,18,20,23⇓⇓⇓ The clinical course of this patient fulfilled the diagnostic criteria for classical Rett syndrome in other respects apart from the longer preservation of speech. However, no mutations were found in 12 patients with developmental delay and some Rett-like features.

MECP2 contains two functional domains: an 85–amino acid MBD and a 104–amino acid transcriptional repression domain (TRD) that associates to a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases.33-36⇓⇓⇓ The majority of the known mutations underlying Rett syndrome are located in these regions. The MBD binds exclusively to the major groove of DNA that contains one or more methylated CpG.37 Mutations in the MBD, such as P127L found in this study, most probably prevent MECP2 from binding to methylated DNA or alter its affinity to heterochromatin. In addition to specific binding to methylated DNA, MECP2 also associates nonspecifically with nonmethylated DNA probably via short motifs that identify the minor groove of the AT-rich sequence, and are excluded from MBD.37,38⇓ Additional studies are needed to characterize the functional consequences of the different MECP2 mutations, and also to find the dysregulated genes affecting the early development of the brain.

When the clinical outcome of the current patient series and the type of mutation were compared, no clear correlation could be detected, even among the patients carrying the same mutation (see table 1). This is in agreement with previous findings,16,18-20⇓⇓⇓ although some indication for the difference in the phenotype between missense and truncating mutations has been reported.16-18⇓⇓ The fact that the same mutations that are associated with PSV (R133C, R168X, P302A, R306C, 1157del41, and 1159del44) also underlie classical Rett syndrome and are distributed along the whole coding region, including the two known domains (MBD and TRD) and the 3' end of the gene, supports the idea that factors other than the type of mutation play a role in the clinical severity of Rett syndrome.16,18-20⇓⇓⇓

Eight familial mutations have been reported in Rett syndrome so far.12,13,15-17,19⇓⇓⇓⇓⇓ Increased consanguinity has been suggested among Rett syndrome families.39,40⇓ This led us to address this issue among Finnish Rett syndrome families who originate from an isolated population41; these patients especially were found to carry identical mutations. No consanguinity was detected. Two girls with Rett syndrome (R3 and R5) carrying different MECP2 mutations were found to be second and half cousins. Also, different mutations in MECP2 have been found in another affected pair of second half cousins.17 No evidence for enrichment of any mutation was found, even in the isolated population of Finland. These results further support the sporadic nature of the MECP2 mutations in Rett syndrome.

The separation of variants that are caused by mutations in the MECP2 gene from those that are not is a major challenge for researchers and clinicians today. From the variants of Rett syndrome classified as: 1) milder or forme fruste variant, which is considered to be the most common variant of nonclassical Rett; 2) PSV; 3) congenital onset; 4) early seizures onset; 5) late regression; and 6) male Rett syndrome.10 Two different MECP2 mutations (R255X and T203M) have been detected in milder or forme fruste variants,24 and PSV has also been shown to be allelic to classical Rett syndrome. One mutation (R133C) has been described in a patient with normal pre- and perinatal development (criterion 1), marked developmental and cognitive delay (criterion 5), and typical hand movement stereotypies (criterion 4), but no developmental regression (criterion 2) or deceleration of head growth (criterion 3).16 One of our non-Rett patients had infantile spasms with hypsarrhythmia since the age of 7 months and the other two patients had onset of seizures before the age of 6 months. Similarly, no MECP2 mutations have been found in four patients with early seizures, such as infantile spasms.16 It is possible that the Rett variant of early-onset seizures may not belong to the clinical spectrum of Rett syndrome with an MECP2 mutation. Also, to our knowledge, no cases of the late-regression variant with an MECP2 mutation have been described. However, there exist congenital onset and male Rett syndrome variants with MECP2 mutations.13,18,24,31,32⇓⇓⇓⇓ A patient with congenital Rett syndrome without the initial period of normal development was found to carry a nonsense mutation 1364-1365insC.18 A male patient with congenital encephalopathy and respiratory arrest carrying a 806delG deletion has been described.13 Recent studies have identified MECP2 mutations (G406X and A140V) in familial cases in which adult males had severe mental retardation without features typical for Rett syndrome, and women with only mild mental retardation.24,31⇓

Random XCI has been found to be associated with most patients with Rett syndrome with MECP2 mutations and completely or partially skewed XCI with milder cases, such as carrier females.13,17,19⇓⇓ Therefore, it has been speculated that skewed XCI ameliorates the disease phenotype.17 The striking finding was that the XCI status of these mildly affected women with a G406X mutation was random; nevertheless a skewed pattern of XCI in relevant tissues (CNS) cannot be excluded. Also, a male patient having some features of Rett syndrome was shown to present somatic mosaicism for an early truncating mutation.32 In the current study 72% of patients with classical Rett syndrome, including the patient with PSV as well as all the atypical cases (100%), had random XCI. Two missense mutations (R133C and R306C), both with an overall mild clinical phenotype (all patients learned to walk, and developed normally before regression set in at ≥10 months) had a random XCI. In four patients (R10, R20, R21, R42) with R294X skewed XCI did not result in a milder outcome compared with random XCI (see table 1). The factors that delineate the clinical phenotype are complex, consisting partly of XCI pattern and the MECP2 mutation type.

We screened the coding region of MECP2 in 12 patients with developmental delay and one or two features of Rett syndrome (see table 2) and found no mutations in this small sample size. Future studies will show how common the MECP2 mutation is in these types of patients. The common feature of our atypical patients was that there was no clear regression of hand use. Similarly, patients with significantly milder impairment of hand use in whom an MECP2 gene mutation was not found have been reported.16 It has also been found that patients with an MECP2 mutation more frequently lose acquired purposeful hand movements than the patients without the mutation, although no significant p value was obtained.19 Breathing disorder and scoliosis, which are considered to be supportive criteria for Rett syndrome, were significantly less common in our atypical cases than in the classical ones. In atypical Patients R30, R39, and R202, developmental regression was associated with the onset of seizures. However, in Rett syndrome the regression often occurs clearly earlier than epileptic seizures. In Patients R29 and R43 the regression was of an autistic type, mainly involving the use of language.

Our data suggest that an MECP2 mutation can be found in almost every patient with classical Rett syndrome. The large number of mutations identified so far in the MECP2 gene makes the application of a mutation test laborious in clinical practice. However, if sequencing is considered, one should bear in mind that 85% (altogether 66 mutations) of all MECP2 mutations so far described are located in exon 4 (1084 bp). Only 12 mutations are so far found in exon 3. Two mutations involve the splicing site between exons 3 and 4. Considering the Rett syndrome variants, patients with PSV often have an MECP2 mutation; so far, eight cases with a mutation out of nine cases have been reported. Thus far, only one patient with Rett syndrome with an MECP2 mutation has been reported in the congenital-onset group. More patients need to be analyzed in order to clarify the mutation prevalence in these groups. Today, to our knowledge, no Rett syndrome variants of late regression or early-onset seizures with an MECP2 mutation have been characterized.

Furthermore, the spectrum of clinical features associated with an MECP2 mutation is wide, ranging from male patients with early-onset lethal encephalopathy to adult cases with severe mental retardation, and in female cases from asymptomatic or mildly mentally retarded to severe variant of Rett syndrome with congenital onset.13,24,31⇓⇓ More patients need to be analyzed in order to clarify the mutation prevalence in these groups.