Abstract
Background: The authors report a family in which two boys had severe neonatal encephalopathy of unknown origin. They both presented with the same condition and died of severe apnea before they were 1 year old. Their sister has a classic form of Rett syndrome.
Methods: Because mutations in the methyl-CpG-binding protein 2 (MECP2) gene have been identified in 70 to 80% of the sporadic cases of Rett syndrome, the authors looked for a mutation in the MECP2 gene in this family.
Results: The authors identified a missense mutation (T158M) in the affected girl and subsequently showed that one of her affected brothers, for whom DNA was available, carried the same mutation. The mother of the patients is a carrier of the T158M mutation. X-chromosome inactivation studies showed that the mother has a completely skewed X-chromosome inactivation pattern that favors the expression of the normal allele; this explains why she does not exhibit any phenotypic manifestation. In addition, the MECP2 mutation appeared on the grandpaternal X chromosome in this family.
Conclusions: An MECP2 mutation can be identified in boys, even though they do not present a Rett syndrome phenotype.
Rett syndrome (RTT, MIM 312750) is a severe neurologic disorder exclusively affecting girls.1 Its prevalence is about one in 15,000 live born girls. Rett patients stop developing at about 1 year old, and have a series of clinical signs indicative of a neurodegenerative process: arrest of brain development, regression of acquisitions, and behavioral troubles (stereotypic hand movement, autism).2 Most cases are sporadic (99.5%), although a few familial cases have been reported.
Mutations in the methyl-CpG binding protein 2 (MECP2) gene located in Xq28 were recently reported in up to 80% of sporadic Rett syndrome cases.3-9⇓⇓⇓⇓⇓⇓ The MeCP2 protein binds to symmetrically methylated CpG dinucleotides and represses the transcription of genes located in a methylated environment. Two functional domains were identified in the MeCP2 protein10,11⇓: a short amino acid domain is responsible for the binding to CpG dinucleotides (the methyl-binding domain or MBD) and a larger domain interacts with the transcriptional repressor mSin3A (the transcription repression domain or TRD). To date, most of the mutations in MECP2 were reported in either one of these two domains or are truncating mutations expected to lead to the loss of function of one or the other domain.3-9⇓⇓⇓⇓⇓⇓
In addition to the mutations identified in the sporadic cases of Rett syndrome, three mutations have been identified in familial cases of RTT.4,5⇓ The first mutation (R168X)4 is present in a mother with a completely skewed X-chromosome inactivation pattern who has transmitted this mutation to two daughters with Rett syndrome. The second mutation (R133C)5 was found in two sisters with a classic form of the disease and their unaffected mother; no data on X-chromosome inactivation are available for this family. The third mutation (806delG)4 is a frame-shift mutation identified in a two-generation family previously reported in the literature.12,13⇓ Two sisters are carriers of the mutation. One sister has the syndrome whereas the other presents a milder phenotype. This latter individual was shown to have a partly skewed pattern of X-chromosome inactivation favoring the expression of the normal MECP2 allele.4 Interestingly, this carrier woman transmitted the MECP2 mutation to one daughter with Rett syndrome and to one son who died in infancy from neonatal encephalopathy. The findings reported for this unique family showed for the first time that a MECP2 mutation can be found in a newborn boy. This fact is of importance because Rett syndrome was thought to be restricted to females and male lethality had been proposed to explain the absence of boys with the syndrome. In addition, the fact that the mouse Mecp2 knock-out established using XY embryonic stem cells does not accomplish normal embryonic development14 seemed to indicate that no males with the syndrome would be identified.
We have studied a family in which two boys died in infancy from severe encephalopathy in a sibship where a girl was affected with a classic form of Rett syndrome. We report the detailed clinical findings obtained for one of the affected boys together with the identification of a mutation in the coding region of MECP2 in this family. We show that one of the affected boys carries the same missense mutation as his affected sister and that this mutation originates from their grandfather. We also show that the unaffected carrier mother has a completely skewed pattern of X-chromosome inactivation, which probably explains recurrence in this family.
Patients and methods.
Patients.
A detailed clinical description of the patients is given in Results. A pedigree of the family is provided in figure 1. Appropriate informed consent was obtained from each human subject before biological samples were used.
Figure 1. Pedigree of the family. Affected subjects are indicated by filled symbols. The woman II-2 is a carrier of the T158M mutation as indicated by the black dot inside her pedigree symbol.
DNA preparation.
DNA was prepared from all individuals except II-2 from fresh blood using standard procedures. For Patient III-2, DNA was prepared from formalin-preserved brain tissue as follows: 0.5 cm2 of tissue was dissected into small pieces and placed into 1 mL of GTE buffer (100 mM glycine, 10 mM TrisCl pH 8.0, 1 mM ethylenediaminetetraacetic acid [EDTA]) for 3 days at room temperature with buffer replacement every 24 hours. Tissue pieces were then incubated at 65 °C for 3 additional days in 500 μL of 1% sodium dodecyl sulfate, 25 mM TrisCl pH 7.5, and 100 mM EDTA, to which 20 μL of 1 M dithiothreitol and 100 μL of proteinase K (10 mg/mL) were added. Every 24 hours, 20 μL of proteinase K (10 mg/mL) were added in the sample. The sample was then extracted three times with phenol, twice with phenol/chloroform/isoamylalcohol (25:24:1), and twice with chloroform/isoamylalcohol (24:1). DNA was precipitated with 2.5 volumes of ice cold EtOH 100%. After a single washing step with ice cold 70% EtOH, the DNA pellet was resuspended in 100 μL Tris–EDTA 10:1 pH 8.0 and quantitated.
PCR reactions.
PCR reactions were done in a final volume of 50 μL using 15 pmol of each primer and 200 ng of genomic DNA as a template. All the MECP2 primers that we have designed work at an annealing temperature of 60 °C under standard conditions.
Mutation screening.
Single-stranded conformation polymorphism (SSCP) experiments were conducted as follows. The coding region of the MECP2 gene was subdivided into eight fragments of approximately 300 base pairs (bp) in length (table). Several primers were designed according to the genomic sequence of the MECP2 locus (GenBank accession number AF031078). All PCR reactions were carried out using genomic DNA as a template.
Sequence of the primers used for single-stranded conformation polymorphism analysis of the MECP2 gene
The PCR products were mixed with an equal volume of freshly prepared 1 N NaOH. After denaturation for 4 minutes at 94 °C, the samples were quickly cooled on ice and immediately loaded onto a 6% acrylamide gel. Electrophoresis was carried out with a constant current of 10 mA overnight at 18 °C. The experiment was repeated at 4 °C to detect a potentially abnormally migrating fragment that would behave normally at 18 °C.
X-chromosome inactivation studies.
Primers were designed in the (CAG)n flanking sequences within the first intron of the androgen receptor (HUMARA) gene. The forward primer AR-P1 (5′ TCC AGA ATC TGT TCC AGA GCG TGC 3) was 5′ labeled (IRD800) and the reverse primer AR-P2 (5′ GCT GTG AAG GTT GCT GTT CCT CAT 3′) was unlabeled. Four hundred nanograms of DNA were digested by HpaII and ethanol precipitated. One hundred nanograms of both HpaII digested DNA and total uncut DNA from each individual was used as a template for PCR. One-fifteeth of the PCR product was diluted in loading buffer and directly loaded onto a Li-Cor (Lincoln, NE) automated sequencer. Analysis of the relative amount of each allele was done using the One-D-Scan software (Scanalytics, Fairfax, VA).
DNA sequencing.
Nucleotides, buffer, and unincorporated primers were removed from the PCR products using the Qiaquick PCR purification kit (Qiagen, Valencia, CA). The PCR products were directly sequenced using IRD800-labeled MECP2 specific primers and analyzed on a LiCor automated sequencer.
Genotyping.
The DXS markers used in this study are based on the Genethon human genetic linkage map. One primer of each PCR primer pair was labeled with the IRD800 infrared dye. PCR reactions were done in a 50 μL reaction volume for 30 cycles consisting of 45 seconds denaturation at 94 °C, 45 seconds annealing at 55 °C, and 45 seconds extension at 72 °C. PCR products were directly loaded onto a LiCor automated sequencer to determine the sizes of the alleles.
Results.
Clinical findings.
Individual III-1 is a normal 18-year-old man currently attending university.
Patient III-2 was born after an uneventful pregnancy. Neonatal measurements were within normal limits (length 49 cm, weight 3070 g, occipital-frontal circumference [OFC] 34 cm). Apgar scores were 9 at 1 minute and 10 at 5 minutes. Soon after birth, severe mental retardation with hypotonia and very slow milestone achievement were observed. At age 9 months, the baby had poor head control but could smile and follow with his eyes. He could be bottle-fed. No morphologic anomaly was noticed except for the peculiar (silvery-grayish) color of his hair, which remains unexplained. His condition deteriorated after 9 months and he died at 11 months of age, a few days after an episode of severe apnea. pH, pCO2, pO2, sodium, potassium, calcium, glucose, urea, protids, creatinin, liver enzymes, blood cell count, and tests related to mitochondrial or peroxysomal diseases were normal, as were EEG and visual evoked responses. Nuclear MR studies did not reveal any visible brain malformation. Postmortem gross examination and anatomic studies of lung, heart, kidney, liver, thymus, and pancreas were normal. Macroscopic examination of the brain (brain weight 810 g) revealed normal gyration without any visible malformation. Paraffin sections of the brain revealed that the right Ammon horn was normal. Neurons of the grey nucleus were normal. The only visible anomaly was a perivascular calcification in the thalamus. No cortical malformation or alteration of brainstem was detected.
Patient III-3 is a 17-year-old girl with typical Rett syndrome. She fulfilled eight of the nine international criteria: normal pregnancy and birth, normal early development, normal head circumference at birth, later slowing of head growth, loss of purposeful hand skills between ages 6 and 30 months associated with deterioration in communication and social withdrawal, development of severely impaired expressive and receptive language and presence of apparent severe psychomotor retardation, stereotypic hand movements, and diagnosis tentative until age 2 to 5 years. The missing criterion was the absence of gait apraxia and truncal apraxia/ataxia between ages 1 and 4 years. Among the additional neurologic abnormalities reported for Rett syndrome patients, she has no epilepsy or vasomotor disturbances of the lower limbs.
Patient III-4 died at the age of 9 months (9 years earlier than Patient III-2). No medical files are available. The clinical course of the disease was very similar to that of his younger brother, according to his parents. He also had the same peculiar hair color.
Identification of a mutation in MECP2.
DNA samples from the affected girl and her mother were analyzed using SSCP experiments designed to scan the entire coding region of the MECP2 gene (see Methods and table). The experiments conducted at 18 °C revealed an abnormal pattern of migration for one of the amplification products, which was subsequently sequenced. Sequencing revealed the presence of a mutation (C to T nucleotide change resulting in the T158M amino acid change) both in the mother’s and in her affected daughter’s DNA. The presence of the mutation was confirmed by restriction analysis of genomic DNA (figure 2A). The mutation was also shown to be absent from 100 unrelated X chromosomes, thus ruling out the possibility that it represents a common polymorphism. In the next step, we looked for the mutation in the rest of the family. Formalin-preserved brain tissue was available for Patient III-2. DNA was prepared from this tissue sample and the T158M mutation was also found (figure 2B). No biological sample was available for the second affected boy in the family (III-4), but it is likely that III-4 also carried the T158M mutation, as the clinical findings were identical in the two brothers (see above). The mutation was not found in the DNA of the other members of the family (I-1, I-2, II-1, and III-1).
Figure 2. Determination of the carrier status of individuals II-2, II-3, III-3, and III-2. The nucleotide change resulting in the T158M missense mutation present in the RTT patient introduces a restriction site for the NlaIII restriction endonuclease. The presence of the mutation was checked by restriction digest of the PCR product obtained after amplification of genomic DNA. (A) Amplification products (1217 bp) obtained using primers flanking the mutation for individuals II-2, II-3, and III-3 were analyzed for the presence of the mutation by comparing the size of the undigested DNA (UC lanes) to NlaIII digested DNA (NlaIII lanes). Abnormal restriction fragments (arrows) indicate that individuals II-2 and III-3 are carriers of the mutation. A molecular weight marker is loaded on lane MWM. (B) The same experiment was performed on DNA from Patient III-2. In this case, because fragments larger than 600 bp cannot be amplified by PCR starting from DNA prepared from formalin-preserved tissue, we have used a different primer pair that yields a 507 bp PCR product. The DNA from Patient III-2 contains the mutation whereas a normal control does not.
Analysis of X-chromosome inactivation.
Because the mother was a carrier of the T158M mutation, we wanted to determine if a favorably skewed pattern of X-chromosome inactivation could explain why she was clinically unaffected. In order to assess the X-chromosome inactivation status of the women in this family, we assessed the methylation status of the androgen receptor alleles by digestion of the DNA with the HpaII methylation sensitive restriction endonuclease. We found that the carrier mother (II-2) has a completely biased pattern of X-chromosome inactivation (figure 3). This finding is consistent with the fact that she is clinically normal. The ratio of the two alleles was scored to 99:1, as almost no second allele could be amplified from HpaII digested genomic DNA. The same analysis conducted using DNA from her phenotypically normal sister (II-1) and mother (I-1) showed that neither woman has this nonrandom pattern of X-chromosome inactivation (their inactivation ratios are 60:40 and 75:25) (see figure 3). Because the Rett syndrome patient (III-3) is homozygous at the androgen receptor locus, we could not determine her X-chromosome inactivation pattern.
Figure 3. Results of the X-chromosome inactivation analysis for individuals I-1, II-1, and II-2. The polymorphic repeated sequence at the androgen receptor locus was PCR amplified using HpaII digested or undigested genomic DNA as a template. The two alleles are numbered in each case together with the replicates associated with a given allele (1′ and 1″ for allele 1, 2′ for allele 2, and 3′ for allele 3). The amplification of allele 1 on HpaII digested DNA for individual II-2 gave out-of-scale values that result in a truncated peak in the graphic representation. In each case, the relative amount of each allele in the amplification reaction was analyzed with quantification software (see Methods) and the obtained values are given below each peak.
Determination of the parental origin of the mutation.
To determine the parental origin of the MECP2 mutation identified in this family, we genotyped all the individuals for whom DNA was available. For this purpose, 32 X-chromosome specific polymorphic markers were used. The resulting haplotypes are presented in figure 4. The chromosome carrying the MECP2 mutation is of grandpaternal origin; the active X chromosome in individual II-2 was found, as expected, to be the grandmaternal chromosome (i.e., the chromosome that does not carry the MECP2 mutation). Individual II-1 does not carry the mutation (see above) but carries the same haplotype in Xq28 as her carrier sister. Thus, it is likely that either mosaicism is present in the germline of individual I-2 or the mutation occurred de novo in individual II-2.
Figure 4. Pedigree of the family shows the X-chromosome haplotypes determined by PCR amplification of polymorphic microsatellite markers. The MECP2 gene is located between DXS8084 and DXS8103. The grandmaternal and grandpaternal X chromosome haplotypes are shaded with different greys and the corresponding chromosome segments transmitted to their offspring are shaded accordingly. The mutation arose on the grandpaternal haplotype. The active X chromosome in the woman with a completely skewed X-chromosome inactivation pattern (II-2) is the maternal X. The unaffected boy (III-1) has not received the chromosome segment carrying the mutation in his mother. Patient III-2 was genotyped only in the critical region and he was found to carry the morbid haplotype, as expected based on the mutation analysis.
Discussion.
Since its first description by Rett in 1966, Rett syndrome (RTT) has been thought to affect women exclusively. Several hypotheses have been postulated to explain the specificity of this disorder. The first hypothesis was that male lethality would be caused by a mutation in an X-linked gene. This hypothesis has been supported by the recent finding of mutations in the methyl-CpG-binding protein 2 (MECP2) gene in 70 to 80% of RTT patients. A nonfunctional MECP2 gene was shown to cause embryonic lethality in the knockout mouse.14 However, a recent report did not show any sex-ratio distortion in RTT families,15 although a de novo mutation leading to early embryonic lethality in a boy will probably remain undetected.
An alternative hypothesis has been proposed16 that postulates that de novo mutations of the RTT gene would be male-specific and thus could only be transmitted to females.
We report the second example of a boy presenting a mutation in MECP2 in a relatively short period. This finding raises the question of the frequency of MECP2 mutations in males, and the phenotype presented by boys carrying an MECP2 mutation.
Only two boys (possibly three, given that Patient III-4 in our study is likely to be affected with the same condition as Patient III-2) with an identified MECP2 mutation have been reported. However, if MECP2 mutations occur with the same frequency in boys and girls, it can be assumed that MECP2 mutations will frequently be found in boys. A possible reason why only a few cases have been described could be that the phenotype associated with MECP2 mutations in boys is not a Rett phenotype (see below). Accordingly, MECP2 mutations have been identified in the two cases reported so far because an RTT girl was diagnosed in the family and an MECP2 mutation was found in the index case. According to this first hypothesis, the frequency of MECP2 mutations in boys could be the same as in girls (1/15,000).
An alternative hypothesis, supported by another study4 and this report, is that MECP2 mutations only occur in the male germline and thus can only be transmitted to a boy by his mother. In this case, MECP2 mutations will occur in the grandpaternal germline and will subsequently be transmitted to the daughters. If the daughter has a normal X-chromosome inactivation pattern, then she will have Rett syndrome. However, if the daughter has a skewed X-chromosome inactivation pattern, she will be able to transmit the mutation either to a daughter or to a son who will then be affected with the disorder. This situation was observed in the two reported cases of MECP2 mutation identified in boys (this report and reference 4). If this hypothesis is true, we can expect to find MECP2 mutations in a boy only if his mother displays a skewed pattern of X-chromosome inactivation. Given that a skewed X-chromosome inactivation pattern is observed in between 1 and 10% of the normal population, the expected frequency of boys with MECP2 mutations could be calculated based on these figures (1/1,500,000 at least to 1/150,000 at most).
Concerning the phenotype of the boys presenting a mutation in MECP2, definitive conclusions are impossible to draw, as only two clinically documented cases have been reported. However, in both cases, the affected boys were presenting a severe neonatal encephalopathy and died in early childhood due to breathing failure. A large screening for MECP2 mutations in boys with severe neonatal encephalopathy of unknown origin (no chromosomal anomaly, no known syndrome, normal pregnancy) is necessary and is currently being conducted in our laboratory. This study will show if the observed phenotype associated with a MECP2 mutation in boys in the two documented cases is typical or if it is variable. Surprisingly, the postmortem analysis data reported here show that although the mutated MECP2 allele is the only one present, the brain anatomy is normal, thus indicating that the mutation does not impair normal organogenesis. This seems to contradict the data obtained in the mouse where proper development cannot be completed in the absence of a functional MeCP2 protein. However, it should be noted that the T158M mutation identified in our study may not completely abolish the MeCP2 activity as does the knockout of the mouse gene. Whatever the results of this study, MECP2 mutation screening in boys is a promising route for the exploration of the causes of sporadic mental handicap in males.
Acknowledgments
Supported by the Association Française du Syndrome de Rett and the INSERM Progres network.
Acknowledgment
The authors thank the family for its cooperation during the course of this study.
- Received April 27, 2000.
- Accepted August 2, 2000.
References
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