Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

Eighteen females presenting a mosaic pattern of dystrophin expression under immunohistochemical analysis were enrolled in the study, which was approved by the local ethics committee (document n.9/2005).

Females were recruited in the course of regular NM clinic in different collaborating centers in the period between 2004 and 2010, and they underwent muscular biopsy due to persistent high CK or other signs and symptoms of the disease. All the analysed females were unrelated cases.

According to the literature, female carriers were classified as (1) symptomatic, when presenting with muscle weakness and/or dilated cardiomyopathy or (2) asymptomatic, when characterized by the presence of high CK levels and/or minor myopathic signs like muscle cramps and myalgia, but no muscle weakness or dilated cardiomyopathy [5, 19].

Genomic DNA was extracted from peripheral blood by Biorobot Qiagen (Qiagen, Chatsworth, CA), after informed consent.

Mutation analysis was performed by Multiplex Ligation-dependent Probe Amplification (MLPA), to detect deletions and duplications, and direct sequencing, to identify point mutations. MLPA analysis was carried out with SALSA probemix 034 and 035 according to the manufacturer’s recommendations (MRC Holland, Amsterdam, Netherlands), thereby consenting the copy-number screening of all 79 dystrophin exons. PCR products were analysed on an ABI 3130 automated sequencer using Genescan software (Applied Biosystems). The dosage quotient was calculated as previously described [22].

For sequence analysis, dystrophin exons, including 3’ and 5’ intron boundaries, were PCR amplified. Amplification reactions were performed as previously described [23]. All PCR products were purified and sequenced on an ABI 3130 automated sequencer.

Comparative Genomic Hybridization (CGH) analysis by DMD-CGH microarray was performed as previously described [24].

Genomic DNA was extracted from muscle tissue using the QIAblood Kit procedure (Qiagen, Chatsworth, CA). The (CAG)_n repeat at the AR locus was PCR-amplified in a reaction containing: 200 ng DNA, 10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM of each dNTP, 175 ng of each primer (forward: 5’-TCCAGAATCTGTTCCAGAGCGTG-3’; reverse: 5’-CTGTGAAGGTTGCTGTTCCTCAT-3’), 1.25U Taq polymerase (Invitrogen), under the following conditions: 94°C for 5 min; 28 cycles of 94°C for 45 sec, 60°C for 30 sec, 72°C for 30 sec. Amplification products were analyzed on an ABI 3130 automated sequencer and AR alleles were determined by Genemapper software (Applied Biosystems). The X-inactivation pattern was investigated in carriers informative for the (CAG)_n repeat. Analysis was performed in duplicates by semi-quantification of the relative inactivation of AR alleles [15]. 1 μg of DNA was digested with 10U HpaII and 10U CfoI (Boehringer Mannheim) for 2 h at 37°C. Enzymes were heat-inactivated and digested DNA samples were purified with Microcon columns; 200 ng were amplified as described above. PCR products from undigested and digested samples were run in parallel on ABI 3130 sequencer. The ratio between the two alleles of the undigested sample was calculated and the resulting correction factor was applied to allele values in the digested samples. The degree of X-inactivation was calculated by normalizing the sum of the two alleles in the digested sample to 100%. Results obtained from either peak height or peak area were compared and very similar values were obtained. X-inactivation pattern was considered as random for ratios ≤80:20, moderately skewed for ratios >80:20 and ≤90:10, and highly skewed for ratios >90:10, according to Amos-Landgraf et al., 2006 [25].

Total RNA was isolated from skeletal muscle biopsy samples using the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. Reverse transcription and cDNA amplification (RT-PCR) was performed as previously described [23]. The dystrophin transcript was analysed using appropriate pairs of oligonucleotides (sequences are available upon request). All PCR fragments were purified and sequenced.

In order to assess the proportion of wild-type vs. deleted/duplicated transcripts, a relative quantification was performed using an Agilent High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA), which allows the sizing and quantification of cDNA samples in the 5–500 pg/μL concentration range. Different numbers of amplification cycles were tested: 25 cycles reactions were performed in order to avoid quantification during the plateau phase of PCR. Amplification products were analysed by an Agilent 2100 Bioanalyzer. The relative percentages were calculated as the ratio between each type of transcript (wild-type or mutated) and the sum of all transcripts (wild-type + mutated).

A relative quantification of dystrophin transcript was performed by real-time PCR. Taqman probes specific for dystrophin exon 12 and exon 55 and a commercially available Taqman expression assay for beta actin (ACTB Endogenous Control) (Applied Biosystems) were used. Samples were analyzed in triplicate on the Applied Biosystems Prism 7900 system. The relative quantification of the target sequences in respect to beta actin was performed by the comparative CT method (ΔΔCT Method) (Applied Biosystems User Bullettin #2); cDNAs from two control females were used as calibrators. Each experiment was performed in duplicate.

17 out of 18 carriers were genotyped for the SPP1 (osteopontin) intragenic variant T/G in the promoter region (dbSNP: rs28357094). The polymerase chain reaction was performed on genomic DNA using a specific pair of primers (forward: 5’-AAGTGCTCTTCCTGGATGCTG-3’; reverse: 5’-CTCCTGCTGCTGCTGACAAC-3’). All PCRs were run in a final volume of 25 μl, containing 100 ng genomic DNA, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l dNTPs, 5U Taq DNA Polymerase (Invitrogen) and 0.4 μl of each primer.

20-μm thick frozen muscle sections from each patient and a control female were homogenized for 2 hours at 4°C with a RIPA lysis buffer containing: 50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 0.5% deoxycholic acid, and an EDTA-free protease inhibitor cocktail tablet (Roche), and then centrifuged at 1500 g for 10 min. Protein content was determined by the bicinchoninic acid method (Bicinchoninic protein assay, Pierce) [26]. Equal amounts of protein (30 μg) were subjected to gel electrophoresis (150 V for 1 h) on a 6% gel, and then electrophoretically transferred to a nitrocellulose membrane (100 V for 2 h). After transfer, the gel was stained with Coomassie brilliant blue solution.

Membrane was blocked with non-fat dried milk for 1 h at room temperature and incubated overnight at 4°C with the specific antibodies DYS2 (a mouse monoclonal antibody to the carboxy terminal region of dystrophin, 1:200, NovoCastra, Newcastle, U.K.) and DYS1 (a mouse monoclonal antibody to the rod domain of dystrophin, 1:500, NovoCastra, Newcastle, U.K.). After washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse 1:40000, for 1 h at room temperature. Immunocomplexes were visualized by means of the ECL Advance Western Blotting Detection Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK); exposure time on X-ray film was from less than 10 seconds to 1 minute. Densitometric analysis of autoradiographic bands was performed with a Bio-Rad densitometer GS700 and expressed as absorbance (A).

Genomic analysis consented detection of the causative DMD mutation in all subjects analysed (Table 2). 10 deletions and 5 duplications of one or more exons were identified by MLPA. Most deletions were clustered in the distal hot-spot region of the gene; 8 were out-of-frame and 2 extended beyond exon 79 (carriers 15 and 16). For female 16, carrying an exon-44-to-79 deletion, CGH-array allowed us to determine the 3’ breakpoint, and a large 3.5 Mb deletion, also including IL1RAPL-1 gene, was identified (Figure 2a).

Duplications were primarily located at the 5’-region of the gene; 3 were out-of-frame and 1 represented by a large in-frame duplication spreading from exon 3 to 44. A non-contiguous duplication of exons 1P-7 and exons 13–42 was also identified by MLPA and confirmed by CGH-array analysis (Figure 2b).

In two females negative for deletions and duplications, nonsense mutations, occurring at exons 14 and 69, were identified by sequencing.

17 out of the 18 female carriers were found to be heterozygous at the AR locus, and the X-inactivation pattern was assessed (Table 2); two independent experiments were performed with reproducible results. 4 symptomatic females showed clearly unbiased X-inactivation (carriers 2, 3, 6, and 7), 1 featured a moderately skewed pattern (carrier 4), and 1 highly skewed X-inactivation (carrier 1); among asymptomatic females, 6 showed random X-inactivation (carriers 11, 12, 13, 16, 17, and 18), 4 a moderately skewed pattern (carriers 9, 10, 14, and 15), and 1 highly skewed X-inactivation (carrier 8). The pattern obtained in the muscle DNA of the two highly skewed samples (carriers 1 and 8) was compared with that observed in their blood DNA. In carrier 1, bearing the X;9 translocation, a total skewing of 100:0 was confirmed in blood, while the pattern observed in the muscle of carrier 8 contrasted with the random inactivation observed in lymphocytes.

Transcriptional analysis was performed on muscle biopsy samples from all subjects (Table 2).

All females, irrespective of their clinical status, showed the same transcriptional behaviour, with the co-existence of both wild-type and mutated transcripts.

The presence of biallelic transcripts was demonstrated by RT-PCR in all subjects carrying deletions, with the exception of females 15 and 16, who carry deletions extending into the 3’-UTR. In these cases, the expression of common polymorphisms of the DMD gene, found to be heterozygous at genomic level, was evaluated: in carrier 15 (del 50–79) the biallelic expression of c.5234G>A polymorphism in exon 37 demonstrated the presence of a biallelic transcript; carrier 16 (del 44–79) manifested biallelic expression of c.2645G>A polymorphism in exon 21 and monoallelic expression of c.5234G>A polymorphism in exon 37. This unexpected transcriptional behaviour could suggest the occurrence of splicing with an unknown gene downstream of the deletion breakpoint and the consequent generation of a chimeric transcript.

RNA studies in females 17 and 18, both carrying nonsense mutations, showed the presence of both wild-type and mutated transcripts.

Semi-quantitative analysis (Table 2) revealed a prevalence of the wild-type transcript in 3 symptomatic (carriers 3, 5 and 6) and 3 asymptomatic (carriers 9, 13, and 14) females and equal proportions of wild-type and mutated mRNAs in 1 symptomatic (carrier 4) and 2 asymptomatic (carriers 11 and 12) females.

In the majority of cases, no relationship was found between the X-inactivation pattern and the transcriptional level of dystrophin alleles (Figure 3 and Figure 4).

Eight carriers were further investigated by real-time PCR in order to quantify the level of dystrophin transcription, in respect to control females. Total transcript quantification was performed by two different real-time assays, on dystrophin exon 12 and exon 55 (Figure 5a). The selected exons are not implicated in deletion or duplication in any of the carriers analyzed; the level of wild-type transcript was derived from the relative ratio of wild-type and mutated transcripts previously calculated (Figure 5b). No relationship was identified between phenotype and dystrophin transcription level. Among symptomatic carriers, the percentage of wild-type transcript ranged between 9% and 96% (exon 12 assay) and between 6% and 69% (exon 55 assay), in respect to control females; similar levels were observed among asymptomatic carriers, with wild-type transcript levels comprised between 17% and 58% (exon 12 assay) and between 25% and 63% (exon 55 assay), in respect to controls.

Genotype analysis of the SPP1 variant was performed in 17 subjects; T allele homozygosity was revealed in 5 out of 6 symptomatic carriers and T allele heterozygosity was detected in only one. In the asymptomatic group, T allele homozygosity was detected in 7 out of 11 subjects and heterozygosity in 3 out of 11; the presence of an homozygous G allele was found only in one asymptomatic carrier.

For carriers 1 and 8, both presenting skewed X-inactivation but different phenotypes, we performed a precise quantification of dystrophin protein content in quadriceps muscles via immunoblot using DYS2 and DYS1 antibodies. This revealed the presence of a protein of the correct size in both patient samples, but relative quantification by densitometric analysis demonstrated that muscles from subjects 1 (manifesting) and 8 (asymptomatic) contained, respectively, 15% and 70% of normal, as compared to the control sample (Figure 6a). These proportions were confirmed with DYS1 antibody, which revealed 11% and 87% of normal protein levels for carriers 1 and 8, respectively (Figure 6b).