Methods

The methods used in this study are summarized in Table 2. Methods are described in this section only briefly and more detail can be found in publications I-IV.

Table  2.  Methods  used  in  this  study.  

METHOD STUDY

DNA isolation I-IV

Allele sizing by PCR I-IV

Repeat-primed PCR (RP-PCR) I-IV

TaqMan 5′ nuclease assay I

Statistical analyses I, III

Single genome equivalent amplification III, IV

Sequencing III, IV

Genotyping of microsatellites and SNP III, IV

Histopathological analysis IV

Chromogenic and fluorescent in situ hybridization IV

SDS-PAGE and Western blotting IV

Long-range PCR repeat expansion assay and Southern blot IV

Allele specific expression analysis IV

Splice variant analysis IV

4.2.1 DNA isolation (I-IV)

DNA was isolated from peripheral blood and muscle samples with standard methods using Puregene DNA Blood Kit (Gentra Systems, Minneapolis, MN, USA) or UltraClean Blood DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA).

For isolating DNA from saliva samples, Oragene DNA saliva kits (DNA Genotek, Ottawa, Canada) were used according to manufacturer’s instructions.

4.2.2 Allele sizing PCR (I-IV)

PCR across the repeat regions in CNBP (DM2) and DMPK (DM1) genes was performed under conditions in which only normal-sized alleles are amplified (Liquori et al., 2001). Two normal-sized alleles exclude DM disease whereas one normal sized allele corresponds to either two normal alleles of the same size or one normal allele and one expanded mutant allele. Sizes of the amplification products were determined using fluorescently labeled (FAM) primers in capillary

electrophoresis on an ABI 3100 or 3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA). Results were analyzed with Genotyper or GeneMapper software (Applied Biosystems).

4.2.3 Repeat-primed PCR (RP-PCR) (I-IV)

Repeat expansion mutation in CNBP or DMPK genes can be detected with RP-PCR.

The reaction in which the repeat expansion is amplified uses three primers that are designed to flank the repeat region (primer 1), attach to the repeat itself (primer 2), and attach to the universal tail of primer 2 (primer 3) and further amplify the fragments generated with primers 1 and 2 (Bachinski et al., 2003). Results were obtained by capillary electrophoresis on an ABI 3100 or 3130 Genetic Analyzer (Applied Biosystems) using fluorescently labeled primer 3 and analyzed with Genotyper or GeneMapper software (Applied Biosystems).

4.2.4 TaqMan 5′ nuclease assay (I)

Mutation detection for three selected mutations in the CLCN1 gene, R894X, F413C and A531V, was performed using TaqMan 5′ nuclease assay (Sequence Detection System). Primers were designed to flank the mutation, and probes to attach to both the mutant allele and normal allele. The probes were labeled with different fluorescent dyes, and only the correct hybridization allowed the release of the corresponding dye and the separation of the mutant and normal allele. Primers and probes were designed using Primer Express Oligo Design software version 1.5 (Applied Biosystems, Foster City, CA, USA) and the results were obtained and analyzed with ABI Prism 7000 analyzer (Applied Biosystems).

4.2.5 Statistical analyses (I, III)

Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 15.0 (SPSS Inc., Chicago, IL, USA) statistical software package (Study I) or R version 2.11.1 (GNU Operating System, Free Software Foundation Inc.) statistical computing environment (Study III). One-sided Fisher’s Exact Test

was performed to calculate statistical significance in study I. For study III the statistical significance, P-value, was calculated in addition to computing both an equal-tailed (ET) 95% interval estimate (excluding the bottom and top 2.5% of all values) and a maximum likelihood (ML) 95% interval estimate.

4.2.6 Single Genome Equivalent Amplification (III, IV)

Single genome equivalent amplification (small-pool PCR, SP-PCR) was performed as previously described (Bachinski et al., 2009; Coolbaugh-Murphy et al., 2005) to determine the stability of the repeat region. DNA samples were diluted to levels of a single diploid genome (6 pg). Multiple SP-PCRs were performed in a three-primer reaction and labeled amplification products subjected to capillary electrophoresis (ABI 3100 Genetic Analyzer, Applied Biosystems).

4.2.7 Sequencing (III, IV)

The repeat regions in CNBP gene were sequenced from DNA isolated from peripheral blood leukocytes to characterize the repeat number and interruptions of the (CCTG)n repeat. The samples in which the RP-PCR resulted in a pattern consistent with a short repeat allele were sequenced. Alleles were amplified and the products cloned using the StrataClone PCR cloning kit (Agilent Technologies, Stratagene, La Jolla, CA, USA) according to manufacturer’s instructions. DNA was prepared from the clones using the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA) and plasmid DNA was sequenced directly using ABI BigDye terminator chemistry (Applied Biosystems). Sequences were visualized by capillary electrophoresis on an ABI 3100 Genetic Analyzer (Applied Biosystems) and analyzed using Sequencher software (Gene Codes, Ann Arbor, MI, USA).

4.2.8 Genotyping of microsatellites and SNP (III, IV)

Six microsatellite markers (D3S3606, D3S3607, 571C11_AG1, 221E20-GT1, 814L21-GT1 and 436B3-AG1) around the DM2 mutation locus were genotyped to characterize the haplotype of the short repeat family in comparison to previously

reported Finnish DM2-associated haplotype (Study IV). Markers were genotyped as previously described using PCR and fragment analysis (ABI 3100 or 3130 Genetic Analyzer, Applied Biosystems) (Bachinski et al., 2003; Bachinski et al., 2009).

Because the genotyping was performed in two different laboratories (in Tampere and in Houston) the primers used differ slightly between different laboratories.

However, the results obtained in one laboratory are comparable.

Single nucleotide polymorphism (SNP), rs1871922, which is in linkage-disequilibrium (LD) with the DM2 repeat expansion mutation, was genotyped for the samples with large, possibly unstable alleles in CNBP intron 1 (Study III) and for the samples of short repeat expansion family (Study IV). The SNP locus was amplified by PCR and the products digested with HaeIII (New England BioLabs, Ipswich, MA, USA). After digestion the samples were electrophoresed through 4%

MetaPhor agarose gels (FMC), and visualized by ethidium bromide staining as described previously (Bachinski et al., 2003).

4.2.9 Histopathological analysis (IV)

Muscle biopsy samples were obtained from two family members, the proband and his brother, of the short repeat expansion family. Both samples were from vastus lateralis muscle. Samples were snap frozen and 8-10 µm sections were cut and examined by using standard histochemical stainings. In addition, sections were immunostained according to established protocols for different myogenic antigens including myosin heavy chain isoforms (fetal, neonatal, slow and fast MyHC).

4.2.10 Chromogenic and fluorescent in situ hybridization (IV) Chromogenic in situ hybridization (CISH) was performed on frozen muscle sections as previously described (Sallinen et al., 2004). Sense probe (CCTG)8 was designed to attach to the DNA sequence of the CNBP repeat expansion and antisense probe (CAGG)8 to attach to the RNA aggregates. After hybridization with both digoxigenin end-labeled sense and antisense DNA oligonucleotide probes, Invitrogen Spot-Light^® CISH^TM Polymer Detection Kit (Invitrogen Corporation, CA, USA) was used for chromogenic detection. Sections were counterstained with

methyl green zinc chloride (Merck KGaA, Darmstadt, Germany), dehydrated, mounted and viewed under a bright field microscope.

RNA fluorescent in situ hybridization (FISH) to detect mutant (CCUG)DM2 RNAs in primary patient myoblast cultures was performed with an antisense Cy3-(CAGG)10. Briefly, cultures were fixed at 4 % PFA/PBS and washed in PBS, followed by permeabilization in 2 % chilled acetone/PBS. After pre-hybridization the pre-hybridization was performed with the (CCUG)DM2 LNA probe (2 ng/µl). Post-hybridization washing was done in 30 % formamide and 2x SSC followed by 1x SSC. Slides were mounted either in SlowFade^® Gold antifade reagent with DAPI (Molecular Probes) or used for subsequent IF. Images were acquired using a Nikon 2000U Deconvolution Microscope and processed with AutoQuant’s AutoDeBlur software (Silver Spring, MD, USA).

4.2.11 SDS-PAGE and Western blotting (IV)

Muscle biopsies were treated as previously described for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Haravuori et al., 2001). After SDS-PAGE, proteins were transferred onto a PVDF membrane and immunolabeled with a rabbit polyclonal antibody against CNBP (Chen et al., 2003). Anti-β-actin antibody C4 (Santa Cruz Biotechnology Inc., CA, USA) was used as a loading control. After HRP-conjugated goat rabbit and goat anti-mouse IgG secondary antibodies (Zymed Laboratories Inc., San Francisco, CA, USA) were incubated, the antibody reacting bands were visualized using an enhanced chemiluminescence detection kit (Pierce Biotechnology SuperSignal West femto maximum sensitivity substrate, Rockford, IL, USA).

4.2.12 Long-range PCR repeat expansion assay and Southern blot (IV)

Long-range PCR was performed with primers flanking the DM2 mutation locus.

PCR products were separated on 0.7 % agarose gel and transferred to Hybond N+

membranes (Amersham Biosciences, Amersham, UK). Radioactively end-labeled

oligo was used for visualization. The method has been described in detail by Schoser et al. (2004a).

4.2.13 Allele specific expression analysis (IV)

To verify that the short expansion allele of CNBP was expressed, reverse-transcriptase (RT) RP-PCR analysis was conducted on the cDNA sample of patient II-3, isolated from skeletal muscle as described previously (Raheem et al., 2010b). If pre-mRNA of mutant CNBP is present, a typical pattern in RP-PCR will be detected. To determine whether both CNBP alleles were transcribed to pre-mRNA the SNP rs1871922 A>C polymorphism was analyzed by quantitative allele-specific method as described previously (Raheem et al., 2010b).

4.2.14 Splice variant analysis (IV)

Alternative splicing of effector genes is part of DM2 disease pathogenesis. Aberrant splicing was analyzed for CLCN1, INSR, ATP2A1, RYR1, TNNT3 and TTN genes from cDNA of patient II-3, isolated from skeletal muscle. Splice variant analysis for all the studied genes was performed using the same protocol as previously described for TNNT3 (Vihola et al., 2010).