Supplemental Data

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Myotonic Dystrophy Type 2 Caused by a CCTG Expansion in Intron 1 of ZNF9
Christina L. Liquori, Kenneth Ricker, Melinda L. Moseley, Jennifer F. Jacobsen, Wolfram Kress, Susan L. Naylor, John W. Day, and Laura P. W. Ranum

Supplementary Material

Polymorphic Marker Development

BACs spanning the DM2 region were identified and ordered by STS content mapping. Additional polymorphic STR markers were developed using di-, tri-, and tetranucleotide repeat sequences that mapped to the region (1). PCR primers for the following markers are as follows: CL3N49 (CL3N49 F 5'-GTGTGTGTGCATTTGTGTGC-3', CL3N49 R 5'-GAGGTTGCAGTGAGCTGAATC-3'); CL3N88 (CL3N88 F 5'-AGCTGACCCTTGTCTTCCAG-3', CL3N88 R 5'-CAAACAAACCCAGTCCTCGT-3'); CL3N58 (CL3N58-D F 5'-GCCTAGGGGACAAAGTGAGA-3', CL3N58-D R 5'-GGCCTTATAACCATGCAAATG-3'); CL3N59 (CL3N59 F 5'-GCTGGCACCTTTTACAGGAA-3', CL3N59 R 5'-ATTTGCCACATCTTCCCATC-3'); CL3N83 (CL3N83 F 5'-GTGTGTAAGGGGGAGACTGG-3', CL3N83 R 5'-AAGCCCAAGTGGCATTCTTA-3'); CL3N84 (CL3N84 F 5'-TCATTCCCAGACGTCCTTTC-3', CL3N84 R 5'-AATCGCTTGAACCTGGAAGA-3'); CL3N99 (CL3N99 F 5'-CTGCCGGTGGGTTTTAAGT-3', CL3N99 R 5'-TGCAAGACGGTTTGAAGAGA-3'); CL3N9 (CL3N9 F 5'-AGACACTCAACCGCTGACCT-3', CL3N9 R 5'-GATCTGGAAGTGGAGCCAAC-3').

Linkage Disequilibrium Analysis

Transmission disequilibrium testing (TDT) (2) was performed using the GENEHUNTER program (version 1.0) (3).

Southern Analysis

BsoBI digests are more useful as a screening tool to identify individuals with DM2 expansions, as the bands are stronger and more discretely focused at the top of the gel. EcoRI digests work better for accurate sizing of large alleles, but the bands are often present as smears and can be less intense.

Supplemental Table 1. Parent-offspring transmissions of the expanded allele in blood. Allele sizes are given in Kb. Multiple expansion sizes indicative of somatic instability are found in some individuals.
Male TransmissionsFemale Transmissions
Parental Alleles (Kb)Offspring Alleles (Kb)Parental Alleles (Kb)Offspring Alleles (Kb)

Confirmation of Monozygous Twins

Bayesian statistics were used on 6 STR markers from different chromosomes {D3S3684, SCA1 [CAG-a & CAG-b (4)], SCA2 [SCA2-A & SCA2-B (5)], SCA3 [MJD52 & MJD25 (6)], SCA6 [S-5-F1 & S-5-R1 (7)], SCA8 [SCA8 F3 & SCA8 R2 (8)]}, sex, and disease status to confirm that the twins described in Fig. 4d were monozygotic (P > 0.001). DNA from both parents and the twins were used to establish haplotypes.

Assembly of ZNF9 Genomic Sequence

Unordered sequence contigs from BAC RP11-814L21 (AC022944) spanning the ZNF9 gene were connected by sequencing from the ends of the known sequence contigs using the following primers: 77 3' (5'-CCTGACCTTGTGATCCGACT-3'), 66 3' (5'-TGCTTTATTATAGATTGGAATCCTCA-3'), 66B 3' (5'-AAGACACCTGTCCCCCTAGAA-3'), 39-5' (5'-GGGTGACAGAGCAAGACTCC-3'), 52 3' (5'-TTTTAAACAATGCTACTTAGAATTTCA-3'), 52 5' (5'-GCCGAATTCTTTGTTTTTGC-3'), 59 5' (5'-TTGCTGCAGTTGATGGCTAC-3'), 59B 3' (5'-TGAATTTACTAAGGCCCTTCCA-3'), and 59C 3' (5'- GTGCTCACCTCTCCAAGCTC-3'). These connections were also verified by overlap with sequence from Celera (x2HTBKUAD8C) (9). To confirm the genomic organization of the ZNF9 gene, NsiI-digested genomic DNA (5 mug) was hybridized with an exon 5 probe generated by PCR using the primers ZNF9-E5 F (5'-GTAGCCATCAACTGCAGCAA-3') and ZNF9-E5 R (5'-TAATACGACTCACTATAGGGAGGACGGGCTTACTGGTCTGACTC- 3' (note: T7 RNA polymerase promotor sequence is underlined) (10).

Supplemental Figure 1. Northern analysis of ZNF9 RNA expression. A human multiple-tissue northern (Clontech, Palo Alto, CA) blot was hybridized at 68°C in UltraHyb hybridization buffer (Ambion, Austin, TX) with a 423 bp riboprobe that included exon 5 of ZNF9 (upper panel). A PCR product which included a T7 RNA polymerase promoter was generated using the primers ZNF9-E5 F and ZNF9-E5 R and used for in vitro transcription. Actin (upper band, lower panel) was used as a loading control. The lower molecular weight band observed in heart and muscle is represents the alpha or gamma isoforms of actin. Sizes are given in Kb.

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