mechanism of deletion removing all dystrophin exons in canine model for dmd implicates concerted evolution of x chromosomal pseudogenes

Accepted Manuscript Mechanism of Deletion Removing All Dystrophin Exons in Canine Model for DMD Implicates Concerted Evolution of X-Chromosomal Pseudogenes D Jake VanBelzen, Alock S Malik, Paula S Henthorn, Joe N Kornegay, Hansell H Stedman PII: S2329-0501(16)30134-6 DOI: 10.1016/j.omtm.2016.12.001 Reference: OMTM To appear in: Molecular Therapy: Methods & Clinical Development Received Date: 27 October 2016 Revised Date: December 2016 Accepted Date: December 2016 Please cite this article as: VanBelzen DJ, Malik AS, Henthorn PS, Kornegay JN, Stedman HH, Mechanism of Deletion Removing All Dystrophin Exons in Canine Model for DMD Implicates Concerted Evolution of X-Chromosomal Pseudogenes, Molecular Therapy: Methods & Clinical Development (2017), doi: 10.1016/j.omtm.2016.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Mechanism of Deletion Removing All Dystrophin Exons in Canine Model for DMD Implicates Concerted Evolution of X-Chromosomal Pseudogenes SC RI PT D Jake VanBelzen1, Alock S Malik1, Paula S Henthorn2, Joe N Kornegay3, Hansell H Stedman1,4 M AN U Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America TE D Section of Medical Genetics, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas, United States of America Corporal Michael Crescenz Veterans Administration Medical Center, Philadelphia, Pennsylvania, United Stated of America EP 22 AC C 10 11 12 13 14 15 16 17 18 19 20 21 23 Correspondence should be addressed to H.H.S (hstedman@mail.med.upenn.edu) 24 422 Curie BLVD, 709A Stellar Chance, Philadelphia, PA 19104, USA Tel: +1 25 2158981432 Fax: +1 2156140398 26 27 Short Title: Characterization of Deletion in Dog Model for DMD ACCEPTED MANUSCRIPT Abstract 29 Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder 30 caused by mutations in the large, 2.4 Mb dystrophin gene The majority of DMD-causing 31 mutations are sporadic, multi-exon, frameshifting deletions, with the potential for 32 variable immunological tolerance to the dystrophin protein from patient to patient While 33 systemic gene therapy holds promise in the treatment of DMD, immune responses to 34 vectors and transgenes must first be rigorously evaluated in informative preclinical 35 models to ensure patient safety A widely used canine model for DMD, golden retriever 36 muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to 37 alternative splicing around an intronic point mutation, thereby confounding the 38 interpretation of immune responses to dystrophin-derived gene therapies Here, we 39 characterize a naturally occurring deletion in a dystrophin-null canine, the German 40 shorthaired pointer The deletion spans 5.6 Mb of the X chromosome and encompasses 41 all coding exons of the DMD and TMEM47 genes The sequences surrounding the 42 deletion breakpoints are virtually identical, suggesting that the deletion occurred through 43 a homologous recombination event Interestingly, the deletion breakpoints are within 44 loci that are syntenically conserved among mammals, yet the high homology among this 45 subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific 46 concerted evolution of these atypical sequence elements AC C EP TE D M AN U SC RI PT 28 ACCEPTED MANUSCRIPT Introduction 48 Recent progress in vector-mediated gene therapy shows promise in the treatment of 49 Duchenne muscular dystrophy (DMD)1 However, in the case of many genetic diseases, 50 a protein is mutated or altogether absent, preventing the establishment of immunological 51 tolerance to its wild type form Thus, gene therapies that deliver a transgene modeled 52 after a wild type protein may contain epitopes to which the patient’s immune system 53 lacks central tolerance, and therefore risk inciting a deleterious host immune response2, 54 In addition, the sporadic and highly varied dystrophin mutations within the DMD patient 55 population make evaluation of immune responses following treatment exceptionally 56 challenging, as each patient’s immune system may react differently to the peptide product 57 of a recombinant transgene Therefore, an animal model void of immunological 58 tolerance to all dystrophin epitopes should provide the most sensitive prediction of 59 immune responses to gene therapies SC M AN U TE D 60 RI PT 47 Preclinical development of gene therapies for DMD has centered on the use of two 62 naturally occurring animal models, the mdx mouse and the golden retriever muscular 63 dystrophy (GRMD) dog, which are caused, respectively, by a nonsense mutation within 64 exon 234 and a point mutation within the splice acceptor site of intron 65 These 65 mutations represent only a small portion of those seen in the DMD patient population and 66 therefore cannot accurately predict the potential human immune responses to DMD gene 67 therapies, and there are currently no primate models for DMD Furthermore, naturally 68 occurring exon skipping and stop codon read-through can result in ‘leaky’ dystrophin 69 expression, as evidenced by revertant fibers6-8, and may allow for the establishment of AC C EP 61 ACCEPTED MANUSCRIPT immunological tolerance to dystrophin during development in these animal models 71 Alternatively, dystrophin expression in revertant fibers could result in a primed immune 72 response to dystrophin peptides, as was shown in humans9 Both of these outcomes 73 convolute the interpretation of immune responses to newly produced proteins acting as 74 neoantigens RI PT 70 75 The German shorthaired pointer-muscular dystrophy (GSHPMD) is a recently described, 77 naturally occurring dog model of DMD10 Western blot and fluorescence in situ 78 hybridization (FISH) analyses suggest that the deletion in this model may encompass the 79 entirety of the dystrophin gene11 Here, we used a PCR approach to precisely define the 80 deletion endpoints, and confirm the complete absence of the DMD gene by sequencing 81 across the deletion We found that the deletion spans 5.6 Mb and is remarkably similar to 82 that of patient B.B., a boy whose deletion dramatically accelerated the characterization of 83 the DMD locus (Figure 1) 12-16 Interestingly, the GSHPMD deletion breakpoints are 84 within highly homologous DNA loci that are conserved on the mammalian X 85 chromosome The GSHPMD model, lacking the entirety of the dystrophin gene and 86 therefore void of any possible level of immunological tolerance or sensitivity to wild type 87 dystrophin-epitopes, could provide a much needed platform for the prediction of immune 88 responses to gene therapies for DMD AC C EP TE D M AN U SC 76 ACCEPTED MANUSCRIPT Results 90 Despite 100 million years of evolution17, the regions of the human and canine X 91 chromosomes that encompass DMD, including exon-intron spacing and size, are 92 conserved (Figure 1), perhaps indicative of the vital role its encoded peptide, dystrophin, 93 plays in muscle biology For clarity in the presentation of our results, we reference the 94 default orientation and numbering of the X chromosome delegated by the National Center 95 for Biotechnology Information (NCBI), which assigns DMD to the antisense strand in the 96 dog genome assembly (Figure 1) M AN U 97 SC RI PT 89 98 Genetic mapping of the GSHPMD deletion breakpoints 99 To map the deletion in the GSHPMD model, we designed a PCR-based strategy to locate the breakpoints on the X chromosome Using previously reported FISH data from a 101 GSHPMD carrier female11, we estimated the location of the deletion and designed primer 102 pairs that broadly spanned this region of the X chromosome (Figure 2) Gel 103 electrophoresis was used to compare PCR results from wild type (WT) dog or GSHPMD 104 DNA to broadly map the deleted region of the X chromosome Following a similar 105 approach, additional primer pairs were designed to finely map the telomeric (TBP) and 106 centromeric (CBP) breakpoints In this way, we employed 74 unique primer pairs and 107 mapped the TBP to be within base pairs 26237921-26239551 and the CBP to be within 108 base pairs 31867082-31871007 of the dog X chromosome, proving that the deletion 109 spans 5.6 Mb and encompasses the entirety of the DMD and TMEM47 genes AC C EP TE D 100 110 ACCEPTED MANUSCRIPT Further attempts to more finely map the deletion breakpoints repeatedly failed (data not 112 shown), perhaps due to a 711 bp genome assembly gap at base pairs 26238732-26239442 113 within the TBP region (Figure S1a) To determine the DNA sequence of the assembly 114 gap, we used primer pair 74 to PCR-amplify the respective genomic region from a BAC 115 clone from the library used in the dog genome-assembly project (Figure S1b) 18 116 Amplification of this region was difficult and dependent on the addition of betaine, a 117 PCR enhancer, which may explain why the region was not sequenced in the dog genome 118 assembly Following amplification, we gel-purified and sequenced the 1.5 kb PCR 119 product and assembled the individual Sanger reads into a single DNA contig (GenBank 120 accession no KR907258 and Figure S1c) A Pustell DNA matrix was used to compare 121 the assembled contig to the region of the X chromosome that harbors the assembly gap 122 (Figure S1d and File S1) This comparison revealed >97% homology between our 123 sequenced PCR-product and the chromosomal sequence flanking the genome assembly 124 gap, suggesting that the internal 650 bp region of the PCR-product sequence is 125 representative of the previously unknown assembly gap sequence We therefore replaced 126 base pairs 26238718-26239484 of the dog X chromosome with base pairs 338-1045 of 127 our BAC-derived, PCR-product and used the resulting sequence in our subsequent 128 analyses SC M AN U TE D EP AC C 129 RI PT 111 130 The GSHPMD deletion spans 5.6 Mb and is contiguous 131 To confirm the absence of the entire 5.6 Mb DNA fragment from the X chromosome, we 132 designed a primer pair that flanks the predicted deletion The primer pair sequences 133 encompass 5.6 Mb, which is far outside the limits of PCR However, unique to PCRs ACCEPTED MANUSCRIPT 134 containing DNA from affected GSHPMD males and carrier females, a kb amplicon was 135 generated, suggesting that the entire 5.6 Mb region is deleted from the X chromosome in 136 the GSHPMD model (Figure 3a) RI PT 137 To confirm its identity, we gel-purified and sequenced the GSHPMD-specific amplicon 139 and subsequently assembled the individual Sanger reads into a 1.7 kb contig (GenBank 140 accession no KR907259) A Pustell DNA matrix comparing the deletion-spanning 141 contig and base pairs 26-33 Mb of the dog X chromosome revealed >90% sequence 142 homology between the 5’ portion of the contig and the TBP region, with an eventual, 143 subtle shift in homology to favor the CBP region (Figure 3b), consistent with the 144 sequenced amplicon-spanning deletion A short break in homology near the TBP was 145 present in our original Pustell matrix, but was found to result from three indels not 146 accommodated by the matrix (Figure S2a) In addition, a break in homology attributable 147 to a TAAA tandem repeat was present between 400-500 bp of the Pustell matrix The 148 abundance of this repeat became apparent when specificity parameters of the Pustell 149 matrix were reduced (Fig S2b.) M AN U TE D EP 150 SC 138 To our surprise, our Pustell matrix (Figure 3b) also revealed that the TBP and CBP 152 regions were highly homologous to each other, raising questions as to the mechanism of 153 the deletion Importantly, both the 5’ and 3’ ends of the deletion-spanning contig 154 extended into regions unique to the TBP and CBP, respectively, confirming that, despite 155 the homology surrounding both breakpoints, the deletion-spanning contig indeed spans 156 the GSHPMD deletion (Figure 3c) Taken together, these findings demonstrate that the AC C 151 ACCEPTED MANUSCRIPT 157 entirety of the mapped 5.6 Mb region, encompassing the DMD and TMEM47 genes, is 158 deleted from the X chromosome in the GSHPMD model and that the deletion breakpoints 159 are highly homologous to each other RI PT 160 Identification of homologous FTHL loci present on the dog X chromosome 162 To further investigate the identified homology between the deletion breakpoints, we 163 generated a Pustell matrix comparing the DNA sequence of the deletion-spanning contig 164 and 26-33 Mb base pairs of the dog X chromosome, allowing for comparison of DNA in 165 both orientations This revealed four additional locations of the X chromosome with 166 >90% homology to the deletion-spanning contig, although in opposite orientation to the 167 previously identified deletion breakpoints (Figure 4a) M AN U SC 161 168 To determine the identity of the six homologous regions, we approximated their locations 170 on the X chromosome and searched for available annotations within the dog genome 171 assembly via NCBI Five of the six homologous regions were annotated in the dog 172 genome, and surprisingly, all five regions were designated as ferritin heavy chain-like 173 (FTHL), two being protein coding genes and three being pseudogenes (Figure 4b) 174 Importantly, the unidentified, sixth region overlaps with the TBP and extends into the 175 aforementioned assembly gap, likely accounting for its lack of annotation in the dog 176 genome assembly We found that while the shared homology spanned nearly the entire 177 length of the pseudogenes, it was unique to only a portion of the protein-coding genes, 178 specifically exon (Figure 4c) Using the encoded peptide sequence of exon of these 179 genes, we queried reference proteomes using HMMER19, which uses hidden Markov AC C EP TE D 169 ACCEPTED MANUSCRIPT modeling to search for homologous proteins, and found that exon is similar to the 181 ferritin-like domain (File S2) Intriguingly, a DNA sequence alignment of the six 182 homologous regions identified in our Pustell matrix revealed that >96% identities are 183 conserved between the pseudogenes and exon of the protein coding genes (File S3), 184 raising questions as to the mechanism responsible for such high sequence conservation 185 among FTHL genes and pseudogenes alike SC 186 RI PT 180 Of the identified ferritin loci, two have NCBI descriptions that link them to the protein- 188 coding, ferritin heavy polypeptide gene (FTH1) (Figure 4b) Of note, we identified two 189 copies of FTH1 in the dog, one with introns located on chromosome 18 and another 190 lacking introns located on chromosome 11 (Figure S3a) and demonstrated that the cDNA 191 sequences of the dog FTH1 genes are 100% identical Further examination revealed a 192 polyadenylation (polyA) signal, AATAAA, followed closely by a string of forty adenine 193 residues, both well characterized hallmarks of processed mRNAs, uniquely in the intron- 194 lacking copy of the dog FTH1 gene on chromosome 11 (Figure S3) 20-22 A brief search 195 of NCBI suggested that the additional, intron-lacking copy of FTH1 is unique to the dog, 196 despite the fact that the intron-containing gene is evolutionarily ancient, as marked by the 197 presence of an ortholog in a wide range of species, including the elephant shark, chicken, 198 cat, and human (data not shown) These findings provide strong evidence to suggest that 199 the intron-lacking copy of FTH1 on dog chromosome 11 is a processed pseudogene, 200 known to arise through reverse transcription and integration of an mRNA, and further, 201 that it is of recent origin AC C EP TE D M AN U 187 202 ACCEPTED MANUSCRIPT 473 15.0, extend gap penalty of 6.7, and delay divergence of 30% DNA sequences used in 474 the alignments can be retrieved from NCBI using the coordinates provided in Figure 4B 475 and File S5 RI PT 476 tBLASTn search 478 A tBLASTn search26 was performed under default conditions using the dog FTH1 479 peptide sequence as query against many species’ genome assemblies Search results 480 were exported as csv files, which were then filtered by length and quality parameters 481 (Figure S4) Individual hit information for each species is provided in File S7 482 M AN U SC 477 Genome assembly builds 484 The following genome assembly builds were accessed via NCBI and used in our analysis 485 The described species are organized as follows: Common Name, Scientific Name, 486 Genome Assembly Build Dog, Canis lupus familiaris, CanFam3.1; Human, Homo 487 sapiens, GRCh38.p3; Chimpanzee, Pan troglodytes, Pan_troglodytes-2.1.4; Mouse, Mus 488 musculus, GRCm38.p4; Cat, Felis catus, Felis_catus_8.0; Pig, Sus scrofa, Sscrofa10.2; 489 Opossum, Monodelphis domestica, MonDom5; Chicken, Gallus gallus, Gallus_gallus- 490 4.0 It should be noted that much of the gene and locus information provided herein are 491 predicted model sequences produced by NCBI’s eukaryotic genome annotation pipeline 492 and therefore are subject to change due to the dynamic nature of genome assemblies and 493 annotation software48 AC C EP TE D 483 23 ACCEPTED MANUSCRIPT 494 Conflict of Interest Statement 495 The authors declare no conflicts of interest Author Contributions 498 Conceptualization: DJV, PSH, JNK, HHS 499 Methodology: DJV, JNK, HHS 500 Investigation: DJV, ASM 501 Resources: JNK 502 Writing – Original Draft Preparation: DJV 503 Writing – Review & Editing: ASM, PSH, JNK, HHS 504 Visualization: DJV, ASM, JNK, HHS 505 Supervision: PSH, HHS 506 Funding Acquisition: JNK, HHS M AN U TE D 507 SC 497 RI PT 496 Acknowledgements 509 This work was supported by: The National Institute of Neurological Disorders and Stroke 510 [R01NS094705 and R01NS042874 to H.H.S.]; The National Center for Research 511 Resources [S10RR028027 to H.H.S.]; The National Institute of Arthritis and 512 Musculoskeletal and Skin Diseases [P30AR050950 and T32AR053461]; and The 513 Muscular Dystrophy Association [276601 to H.H.S and 86447 to J.N.K.] AC C EP 508 24 ACCEPTED MANUSCRIPT References RI PT Ramos, J, and Chamberlain, JS (2015) Gene Therapy for Duchenne muscular dystrophy Expert Opin Orphan Drugs 3: 1255-1266 Potter, MA, and Chang, PL (1999) Review the use of immunosuppressive agents to prevent neutralizing antibodies against a transgene product Ann N Y Acad Sci 875: 159-174 Yang, Y, Jooss, KU, Su, Q, Ertl, HC, and Wilson, JM (1996) Immune responses to viral antigens 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DMD, dystrophin; TMEM47, transmembrane 665 protein 47; XK, X-linked Kx blood group; CYBB, cytochrome b-245 beta polypeptide; 666 RPGR, retinitis pigmentosa GTPase regulator; M, million M AN U SC RI PT 659 667 Figure Deletion map of the GSHPMD model by PCR The X chromosome of male 669 wild type GSHP and affected GSHPMD dogs are compared The locations of primer 670 pairs are labeled alphabetically The results of each PCR experiments are shown, with + 671 indicating successful amplification, and – indicating failed amplification The mapped 672 deletion in the GSHPMD model is depicted inferiorly XWT/Y, wild type male; XMT/Y, 673 mutant male; PCR, polymerase chain reaction; TSPAN7, tetraspan 7; Mb, million base 674 pairs; M, million EP AC C 675 TE D 668 676 Figure PCR amplification across the 5.6 Mb deletion in the GSHPMD model (a) 677 Schematic showing primers that are spaced over 5.6 Mb apart in wild type dog PCR 678 products generated from this primer pair using DNA from affected males, carrier 679 females, and wild type male are displayed on an agarose gel following electrophoresis 29 ACCEPTED MANUSCRIPT A positive control using an unrelated primer pair is provided in the rightmost lane for the 681 wild type dog (b) Pustell DNA matrix comparing the sequenced deletion-spanning 682 amplicon from a GSHPMD male to the indicated region of wild type dog X chromosome 683 Black lines indicate homology between the compared sequences (c) Macroscale DNA 684 sequence comparison of the telomeric and centromeric deletion breakpoints Shaded 685 region indicates >96% homology between the deletion breakpoints XMT/Y, affected 686 male; XMT/XWT, carrier female; XWT/Y, wild type male; TBP, telomeric breakpoint; CBP, 687 centromeric breakpoint; WT, wild type M AN U 688 SC RI PT 680 Figure Identification of homologous DNA segments on dog X chromosome as 690 members of the ferritin-like superfamily (a) Pustell DNA matrix comparing both 691 orientations of the sequenced deletion-spanning amplicon from a GSHPMD male to the 692 indicated region of wild type dog X chromosome from the dog reference genome Strand 693 homology is provided in the subsequent table (b) Regions of homology identified in 694 Pustell matrix and corresponding gene annotations for these regions of the dog genome 695 from NCBI Loci are group and labeled based on chromosomal location (c) Schematic 696 of identified ferritin-like genes and pseudogenes Arrows indicate the location of the 697 genes and pseudogenes in the Pustell matrix Area of shared homology is shown with a 698 grey bar above each respective locus *Gene inferred from mammalian FTHL17 699 ortholog EP AC C 700 TE D 689 701 Figure Quantification of results from tBLASTn search of dog FTH1 peptide in 702 multiple species (a) Bar graph quantifying tBLASTn search results of dog FTH1 30 ACCEPTED MANUSCRIPT peptide sequence against the genomes of the indicated species Number at the top of each 704 bar signifies hits returned prior to applying filtering criteria Number internal to bottom 705 section of each bar represents hits remaining after applying all filtering criteria (b) Bar 706 graph quantifying the number of filtered tBLASTn hits present on the chromosome that 707 harbors the DMD gene for the indicated species CHR, chromosome RI PT 703 708 Figure Phylogenetic analysis of identified FTHL loci from tBLASTn search in 710 human, chimpanzee, dog, and cat (a) Comparison of syntenic portion of X 711 chromosome from each species showing the grouping of FTHL loci to four regions, 712 labeled A, B, C, and D Number of pseudogenes present in reach region is indicated in 713 parenthesis (b) Phylogeny of identified FTHL loci The human FTH1 CDS is used as an 714 outgroup Branches with values less than 0.05 are not displayed Tree-building 715 parameters are provided as text in figure CDS, coding DNA sequence (c) ClustalW 716 multiple DNA sequence alignment of identified FTHL loci Note extreme sequence 717 homology among dog FTHL loci AC C EP TE D M AN U SC 709 31 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ...ACCEPTED MANUSCRIPT Mechanism of Deletion Removing All Dystrophin Exons in Canine Model for DMD Implicates Concerted Evolution of X- Chromosomal Pseudogenes SC RI PT D Jake VanBelzen1,... a naturally occurring deletion in a dystrophin- null canine, the German 40 shorthaired pointer The deletion spans 5.6 Mb of the X chromosome and encompasses 41 all coding exons of the DMD and... prediction of immune responses to gene therapies for DMD Sequence confirmation 271 of the deletion breakpoint insures that no coding exons of DMD remain, precluding 272 expression of any dystrophin- derived