Methods

DNA extraction

We received peripheral blood samples (5 to 10 mL) from patients and, whenever possible, their parents. Genomic DNA was extracted from leucocytes, using the Puregene DNA isolation system (Gentra, Minneapolis, MN).

Single strand conformation polymorphism (SSCP) and sequencing

The coding region and exon/intron boundaries of the MECP2 gene (RefSeq ID:

NM_004992) were amplified by PCR, using one pair of primers for exon 2, three pairs of primers for exon 3, and five pairs of primers for exon 4 (table S2.1 in appendix I). DNA amplification was performed in a final volume of 25 µl of a PCR mixture, which consisted of 1X enzyme buffer, 0.5 mM MgCl2, 0.2 mM dNTP, 0.8 µM of each primer, 10% DMSO, 1.5U Taq DNA Polymerase (Fermentas), and approximately 100 ng of genomic DNA. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation step for 5 min, at 95ºC, followed by 35 cycles of denaturation, for 1 min, at 95ºC, annealing for 1 min, at TaºC (specific for each pair of primers, table 2.1.1 in appendix I), elongation for 1 min, at 72ºC, and a final extension step for 5 min, at 72ºC.

The total PCR product was denaturated for 5 min at 95ºC, chilled on ice and loaded in a non-denaturating 15% polyacrylamide gel electrophoresis (PAGE), and fragments

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were allowed to migrate in a DCODE system (BioRad), at 20ºC and 300V, over approximately 14 hours.

The pattern of migration was visualized by silver staining (0.2%), and the gel was transferred to a 3MM Whatman paper and allowed to dry, at 80ºC, for 30 min.

The samples in which a different pattern of migration was detected were re-amplified by PCR, using the same primers and conditions described above (table S2.1 in appendix I). The sequencing reaction consisted of 1X enzyme buffer, 1 µM of primer forward/reverse, 4 µL of Pre-mix Big dye (Applied Biossystems), and 3 µL of the PCR product, in a final volume of 10 µL. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation step for 3 min, at 94ºC, followed by 25 cycles of denaturation for 10 sec, at 96ºC, annealing for 5 sec, at 58ºC, and elongation for 4 sec, at 60ºC.

After purification of the sequencing reaction, the pellet was ressuspended in template sample ressuspension, and ran in an ABI model 377 automatic sequencer (Perkin-Elmer, Norwalk, CT), for the identification of the possible variants.

Detection of small deletions and insertions

PCR mixtures and conditions were used as described under “single strand conformation polymorphism (SSCP) and sequencing”. The total PCR product was denaturated for 5 min, at 95ºC, chilled on ice, and loaded in a denaturating 8%

sequencing PAGE; fragments were allowed to migrate at 1200/1500V, over approximately 3 hours.

Allele-specific PCR

Allele-specific PCR was optimized for the detection of each of the five recurrent mutations, and for one variant of unknown function (K305R) identified in the MECP2 gene (table S2.2 in appendix I).

Two PCR mixtures for each variant were prepared in order to amplify either the normal or the mutated allele, using three primers: one that is common to both reactions;

and the other two, one for each PCR mixture, specific either for the normal or for the

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mutated allele. In a final volume of 25 µl: 1X enzyme buffer, 0.5 mM MgCl₂, 0.2 mM dNTP, 0.8 µM of each primer pair (either for the normal allele or for the mutated allele), 10%

DMSO and 1.5U Taq DNA Polymerase (Fermentas), and 100 ng DNA. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation step for 5 min, at 95ºC, followed by 35 cycles of denaturation for 1 min, at 95ºC, annealing for 1 min, at TaºC (specific for each pair of primers, table S2.2 in appendix I), elongation for 1 min, at 72ºC, and then a final extension step for 5 min, at 72ºC.

PCR products were electrophoresed in a 2% agarose gel, and visualized under UV light.

Direct sequencing

The coding region and exon-intron boundaries of the MECP2 gene were amplified by PCR, using one pair of primers for exons 1, 2 and 3, and three pairs of primers for exon 4 (table S2.3 in appendix I). DNA amplification of exon 1 was performed in a final volume of 25 µl. After an initial denaturation step of the DNA eluted in TE buffer (60 ng) for 10 min, at 97ºC; the PCR mixture was added (0.5 mM MgCl₂, 0.133 mM dNTP, 0.2 µM of each primer, 5% DMSO and 2.5U Taq DNA Polymerase (Fermentas)). The thermal cycling profile (My Cycler, BioRad) consisted of 40 cycles of a denaturation for 1 min, at 95ºC, annealing for 2 min, at 63ºC, elongation for 3 min, at 72ºC, and then a final extension step for 5 min, at 72ºC.

For the DNA amplification of exons 2 to 4, a final volume of 30 µl PCR mixture (0.8 mM MgCl₂, 0.133 mM dNTP, 0.2 µM of each primer and 2U of Taq DNA Polymerase (Fermentas)) was used. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation for 5 min, at 95ºC, 35 cycles of a denaturation for 1 min, at 95ºC, annealing for 1 min, at TaºC (specific for each pair of primers, see table S2.3 in appendix I), extension for 1 min, at 72ºC, and a final extension for 5 min, at 72ºC.

After PCR amplification the different fragments were automatically sequenced, using the same primers used for the PCR, in a ABI 377 model (Perkin Elmer), and the sequences analysed for point mutations or small rearrangements (deletions and duplications). Sequence changes were confirmed by re-amplification of genomic DNA and sequencing in the opposite direction.

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Detection Of Virtually All Mutations – SSCP (DOVAM-S)

Several non-coding regions of the MECP2 3’UTR were selected, based on their conservation among human and mouse species (almost 100% conservation at the nucleotide level), in a total of 9 blocks (NM_004992: c.1607-c.1956, c.2561-c.2891, c.3551-c.3805, c.3768-c.4128, c.6851-c.7029, c.7116-c.7436. c.8372-c.8645, c.8607-c.8872 and c.9844-c.10182), which also included the regulatory regions around three of the four polyadenylation signals. These 3’UTR blocks were scanned for mutations with DOVAM-S (Shibayama et al. 2004). The different blocks were first amplified robotically, pooled, denatured and electrophoresed, under five nondenaturing conditions, varying in gel matrix, buffer, temperature, and additive: (1) 10% PAGE+/30 mMTricine/Triethanolamine, at 20°C, (2) 10% HR1000/ 30mM Tricine/Triethanolamine, at 4°C, (3) 10% PAGE+/TBE/5% glycerol, at 20°C, (4) 10 % HR1000/TBE/2.5% glycerol, at 4°C, and (5) 10% PAGE+/30mM Capso, at 4°C. PCR prod ucts with mobility shifts were sequenced with the ABI 377 (Perkin-Elmer, Norwalk, CT), and nucleotide alterations were analyzed. Sequence changes were confirmed by re-amplification with genomic DNA and sequencing in the opposite direction.

For the DNA amplification of the different 3’UTR blocks, a final volume of 25 µl PCR mixture (2.5 mM MgCl₂, 0.2 mM dNTP, 2.5 µM of each primer and 2U of AmpliTaq Gold (Gibco)) was used. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation for 10 min, at 94ºC, 35 cycles of a denaturation for 15 sec, at 94ºC, annealing for 30 sec, at 55 ºC, extension for 1 min, at 72ºC, and a final extension for 10 min, at 72ºC (table S2.4 in appendix I).

Detection of large rearrangements by robust dosage-PCR (RD-PCR)

The DNA concentrations were measured in a UV spectrophotometer, at 260 nm, and adjusted to a working concentration of 30 ng/µL in TE buffer. Genomic DNA samples were incubated at 90°C in TE buffer, for 10 minutes . Four RD-PCR assays for three coding exons of MECP2 gene were designed according to (Shi et al. 2005) (table S2.5 in appendix I). These assays were divided into two groups: group I, which included amplification of exons 2 and 3 of MECP2 and exon 12 of ataxia telangiectasia mutated (ATM) gene, was used as an autosomal internal control segment; group II included the two other assays for exon 4, subdivided in two fragments (4I and 4II) of MECP2, and was used as the internal autosomal control segment the fucosyltransferase 2 (FUT) gene.

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The RD-PCR conditions were slightly modified from the original report (Shi et al.

2005), and adapted to our laboratory conditions.

For group I, 60 ng of DNA were used in a PCR mixture, which consisted of 1X buffer

#3 (Roche), 4.5 mM MgCl₂, 0.2 mM of each dNTP, 0.2 µM of each pair of primers, 0.5 µg BSA, 1U of Platinum Taq DNA Polymerase (Invitrogen), 1U of Platinum Taq DNA polymerase HiFi (Invitrogen), in a final volume of 25 µl. For Group II, 60 ng of DNA were used in a PCR mixture consisting of 1X buffer #3 (Roche), 3 mM MgCl₂, 0.2 mM dNTP-G, 0.05 mM/0.15 mM of 7-deaza GTP/dGTP, 0.2 µM of each pair of primers, 10% DMSO, 0.5 µg BSA, 1U of Platinum Taq DNA Polymerase (Invitrogen), 1U of Platinum Taq DNA polymerase HiFi (Invitrogen), in a final volume of 25 µl. The thermal cycling profile (My Cycler, BioRad) consisted of 25 or 30 cycles (for groups I and II, respectively) of denaturation of 15 sec, at 94ºC, annealing for 30 sec, at TaºC (specific for each pair of primers, see table S2.5 in appendix I), and elongation for 1 min, at 72ºC.

For validation of the four assays covering the coding region of the MECP2 gene, a blinded analysis was performed with 48 blinded genomic DNA samples, where either the sex status, or the number of each status, was unknown. The male sample was functionally equivalent to a RTT patient with a large heterozygous deletion.

In order to characterize the deletion junction of patient P3, ten more RD-PCR assays were developed, in the 3’ and 5’ flanking regions of the MECP2 gene (Shi et al.

2005).

The PCR product (12 µl) was electrophoresed in a 2% agarose gel (0.2 µg/mL ethidium bromide) for 2 hours, at 120V, and scanned in a AlphaImager (BioRad).

Spotdenso software was used to quantify the PCR yield. The ratio of yields (ROY) was calculated by dividing the target net signal by the internal control net signal. For normalization, the ROY of patient samples were divided by the average ROY of control normal females.

Southern blotting analysis

Southern blotting was performed using probes RTT2, RTT3 and p(A)10, hybridizing with exon 2, exon 3 and the end of the 3’UTR (figure 2.3, and table S2.6 in appendix I).

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Probes were generated by PCR from genomic DNA, purified from 1% agarose gel by QIAEX II (QIAGEN, Valencia, CA), and labeled with ³²P dCTP by Prime-It II Random primer (Stratagene, Cedar Creek, TX). The genomic DNAs (8 µg) of female control, male control and patient P3 were digested with Hind III and Pst I for probe RTT2, Sac I for probe RTT3 and Hind III and Sac I for probe p(A)10. Digested DNA fragments were separated in a 1.5% agarose gel, and blotted into a nylon membrane (Hybond H-N+;

Amersham Pharmacia Biotech, Buckinghamshire, England). Hybridization was performed overnight, at 65ºC, and washings were carried out in a series of SSC/SDS solutions (0.1%SDS, 2%-0.1% SSC). Membranes were exposed to storage phosphor screen, scanned by Typhoon 9410 Imager (Amersham, Molecular Dynamics, Sunnyvale, CA).

ImageQuant™ software was used to quantify the signals.

Figure 2.3. Schematic representation of the MECP2 gene. Regions analyzed by Southern blotting and probes used in the assay (figure is not to scale).

Determination of X chromosome inactivation (XCI) pattern

XCI assays were performed in genomic DNA isolated from leukocytes of peripheral blood, to assess the pattern of XCI. The assay was based on a previously described method (Allen et al. 1992), which allows the determination of the X-inactivation status, using a polymorphic trinucleotide repeat polymorphism in the androgen receptor gene (AR, RefSeq ID: NM_000044.2), flanked by two methylation-sensitive restriction enzyme sites.

Two µg of genomic DNA were digested with the endonuclease Hha I (1x enzime buffer #4, 1XBSA and 2U Hha I (NEB), in a final volume of 20 µL), at 37°C, ove rnight. The restriction enzyme hydrolyzed only the unmethylated alleles.

Two µL of the digestion mixture were used for the amplification of exon 1 of AR gene (table S2.1.7 in appendix I). A final volume of 30 µl PCR mixture (1X enzyme buffer,

3’UTR

2750 bp Hind III Pst I

3578 bp Hind III Sac I 3593 bp

Sac I

RTT2 RTT3 P(A)10

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0.625 mM MgCl₂, 0.2 mM dNTP-A, 0.032 mM ³⁵S dATP, 0.8 µM of each primer, 2%

formamide and 1.8U of Taq DNA Polymerase (Fermentas)) was used. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation step for 5 min, at 95ºC, 34 cycles of a denaturation for 45 sec, at 95ºC, annealing for 30 sec, at 60ºC, extension for 30 sec, at 72ºC, and a final extension for 5 min, at 72ºC.

After PCR amplification, 6 µL of each sample were loaded in a denaturating 6%

PAGE, and the fragments were allowed to separate at 1200/1700V. The gel was transferred to a 3MM Whatman paper, allowed to dry, and then exposed to an X-ray film (Kodak) for 3 days, at room temperature.

Films were scanned by Typhoon 9410 Imager (Amersham); scoring of the XCI pattern was made by densitometry of the amplified DNA bands, with the ImageQuant software.

Identification of reported mutations in neuroligin 3 (NLGN3) and neuroligin 4 (NLGN4) genes

Exon 6 of the NLGN3 and exon 5 of NLGN4 genes were amplified by PCR (primers in table S2.8 in appendix I). For the DNA amplification, a final volume of 30 µl PCR mixture (0.8 mM MgCl₂, 0.133 mM dNTP, 0.2 µM of each primer and 2U of Taq DNA Polymerase (Fermentas)) was used. The thermal cycling profile (My Cycler, BioRad) consisted of an initial denaturation step for 5 min, at 95ºC, 35 cycles of a denaturation for 1 min, at 95ºC, annealing for 1 min, at Ta ºC (specific for each pair of primers, see table S2.1.8 in appendix I), extension for 1 min, at 72ºC, and a final extension for 5 min, at 72ºC.

After PCR amplification the different fragments were automatically sequenced in an ABI 377 model (Perkin Elmer) and the sequences analysed for the reported mutations (NLNG3: c.1186insT and R451C; NLGN4: c.1253delAG) or others, eventually. Sequence changes were confirmed by re-amplification of genomic DNA and sequencing in the opposite direction.

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