Report of the first cases
Two four-month-old male Labrador retriever littermates owned by a breeder were referred by their veterinarian to the neurology consultation of the Alfort school of veterinary medicine. The puppies presented with gait stiffness, exercise reluctance and marked palmigrade and plantigrade posture (Fig. 1A). At the clinical examination, normoreflexia and absence of proprioceptive defect were observed. Both puppies had markedly elevated serum creatine kinase (LRMD1: 83000 UI/L; LRMD2: 30000 UI/L), and the electrodiagnostic studies revealed normal nerve conduction velocities, but spontaneous activity was evidenced in all the tested muscles (mainly complex repetitive discharges, Fig. 1B). Biopsies from the biceps femoris muscle were sampled from both dogs and histological analysis revealed lesions of active necrosis, regeneration, endomysial fibrosis and a few inflammatory foci with calcifications (Fig. 1C). The phenotype of these two dogs, together with the high CK, the myopathic electrodiagnostic profile and the histological lesions were suggestive of a muscular dystrophy process. Immunostaining of the biopsies showed no reactivity with an anti-dystrophin antibody directed against the central rod domain (Dys 1, Fig. 1D). Analysis of muscle tissue by multiplex western blot (Fig. 1E) confirmed the absence of Dp 427 dystrophin and the presence of normal size dysferlin, γ-sarcroglycans, and calpain 3; proteins altered in human limb-girdle muscular dystrophies. A new litter born from the same parents yielded two additional affected males, reinforcing the hypothesis of an X-linked transmission. The breeder kindly gifted these two affected males (LRMD 3 and 4) as well as an unaffected female born from the same parents to our research unit. This allowed us to establish a LRMD colony in the same facilities in which a pre-existing GRMD colony had already been established.
LRMD colony establishment
A first litter was obtained by crossing the presumed female carrier and one of the affected males (LRMD 4). The subsequent litters were obtained by crossing the descendant dogs (carrier females x affected males). The inbreeding of the colony was high (ranging from 25 % for LRMD 5 and 6, to 41 % for LRMD 11 to 14). Over a period of 6 years, ten litters were obtained (Fig. S1). A total of 14 LRMD dogs (9 males and 5 females) survived the neonatal period and were followed-up.
Main phenotypic features
Neonatal period
At birth, the LRMD dogs had weights that were comparable to that of their healthy littermates. Shortly after birth these animals showed striking difficulties sucking and needed intensive nursing during their first days to ensure survival (feeding by oro-gastric gavage and keeping in incubator when required). Despite this care, around 50 % of the LRMD newborn puppies died within the 48 first hours (Fig. S2) following a paroxystic weakness episode associated with severe dyspnoea. Myoglobinuria was observed in some of these puppies, as well as very high serum CK values (>100000 UI/L) and hyperkalaemia. Rhabdomyolysis was confirmed by histological analysis. Selective involvement of some muscles such as the diaphragm, the tongue or the sartorius cranialis (Fig. S2) was observed, confirming that this neonatal syndrome reproduces the neonatal fulminating form well described in the GRMD dog model [58].
Clinical observations
Locomotor signs
After few days of intensive nursing, the surviving LRMD puppies became able to suck on their own but exhibited growth retardation compared to their healthy littermates (Fig S2). At the age of 2 months, the LRMD puppies showed stiff gait and difficulty jumping over an obstacle. In the subsequent months, they became stiffer, and rapidly developed posture abnormalities resembling those seen in GRMD dogs notably marked pelvic verticalisation, palmigrady and plantigrady. When the LRMD dogs reached six months of age, most of them were unable to run and showed abnormal gait while walking. Despite the abnormalities, most animals remained ambulant, except for LRMD 8, who completely lost ambulation at 6 months of age and was therefore euthanized. His littermate (LRMD 9) also developed a severely compromised locomotion.
Respiratory and digestive signs
All the LRMD dogs developed signs of dyspnoea at the age of 2-3 months, mainly paradoxical thoraco-abdominal movements. In the following months, the dogs developed moderate exercise-induced polypnoea, noisy breathing, and in some cases intermittent cyanosis and elevated serum bicarbonate concentration. Oro-pharyngeal dysphagia became a major feature from the age of 4 months onwards, with a prominent aggravation in the following months. This severe dysphagia probably resulted from markedly reduced jaw opening, together with prominent macroglossia, retraction of the tongue, and weakness of the pharyngeal muscles. The symptoms were observed in all LRMD dogs, and in some cases, they limited their ability to maintain their water intake; consequently the deterioration of 50 % of the animals was cause for humane euthanasia. The oro-pharyngeal dysphagia observed in the LRMD dogs was frequently complicated with aspiration bronchopneumonias that were successfully treated in most cases, but led to the death of 4 out of the 14 affected dogs. The chest radiographs showed usual abnormalities found in GRMD dogs: hiatal hernias, megaoesophagus, pectus excavatum, pulmonary hyperinflation associated with diaphragm flattening [59].
Survival
The mean survival of these 14 LRMD dogs was 21.6 months, with a first quartile at 10.8 months and a third quartile at 31.4 months. The LRMD dog with the longest survival (LRMD 7) died at the age of 103.5 months (8.6 years), probably following paroxystic cardiac arrhythmias (sudden death without significant findings in the necropsy in a dog known to have prominent ventricular arrhythmias). Despite this long survival, this dog exhibited impaired ambulation, dyspnoea, dysphagia and suffered many aspiration pneumonia episodes. Consequently, this animal cannot be considered a “phenotypic escaper”. A comparative survival analysis was performed between LRMD and GRMD dogs and showed that LRMD dogs tended to live longer (log-rank test p = 0.01) (Fig. 2).
Histological observations
The studied biopsies showed general trend towards necrosis-regeneration lesions in LRMD dogs younger than 1 year. Inflammatory foci and calcifications were observed in some biopsies from 4- and 6-month-old dogs. As the animals grew older, endomysial fibrosis became prominent and most animals beyond two years of age suffered adiposis (Fig. S3). In parallel, the CK values, although fluctuating, were markedly elevated during the first year, and decreased to lower values in older LRMD dogs. The most severely affected muscles were the diaphragm and the extensor carpi radialis with pathological indices > 60 %.
Functional evaluation and comparison with the GRMD model
The overall phenotypic characteristics of the LRMD dogs resembled those observed in GRMD dogs. In order to position the model in comparison to the “reference” GRMD dog model, a quantitative comparative study between both canine muscular dystrophies was performed, using the tools developed to evaluate GRMD dogs.
Clinical score
Clinical scoring rapidly increased during the first months in LRMD dogs; thereafter the score progressed at slower rate until the animals reached the age 7-8 months when it stabilised. The same type of evolution was seen in the GRMD population (Fig. 3A). However when comparing both colonies in detail, the LRMD dogs had a faster and more homogeneous disease progression within the first months. At the age of 4 months, the LRMD dogs tended to have higher scores (p = 0.07) and the coefficient of variation of the clinical score was lower than that of the GRMD colony (12 % vs 34 % in GRMD dogs). Thereafter, two subpopulations of LRMD dogs emerged, the first one formed by a group of two animals that were more severely affected from a locomotor point of view (LRMD 8 and 9). This suggests that, as previously described for the GRMD model, there may be a severe form leading to a loss of ambulation in LRMD dogs [40]. The less severely affected LRMD dogs tended to have higher clinical scores than GRMD dogs between the ages of 10-16 months. These observations were confirmed by comparing the colonies at an adult age (Fig. 3B); stage at which LRMD dogs had significantly higher clinical scores (mean 67.5 %, SD 12.1 %) than GRMD dogs (mean 48.1 %, SD 11.7 %; p = 0.005). It is noteworthy that LRMD7, the oldest survivor, had clinical scores among those measured in other LRMD dogs.
Gait analysis
Gait analysis of LRMD dogs using accelerometry indicated that both the LRMD and the GRMD models exhibited similar profiles compared to healthy dogs: decreased speed, stride length and frequency, decreased total power and increased medio-lateral relative power. Among the three LRMD dogs longitudinally followed-up (Fig. 3C and D), two showed values that were similar to those of more affected GRMD dogs, as well as a decrease in gait quality with age and dramatic increase in gait waddle. The third dog (LRMD 14) had mild gait impairment, attesting to the existence of inter-individual heterogeneity in LRMD dogs. Despite this heterogeneity and the low number of animals followed-up, the total power, a very discriminating variable, was found to be significantly decreased at all tested ages (p = 0.035 at 4 months of age, p = 0.0001 at 6 months of age, p = 0.002 at 9 months of age), similarly to GRMD dogs (no significant difference between both models). The four LRMD dogs examined at an adult age obtained gait index values overlapping with those usually measured in the GRMD model and significantly different from healthy dogs (p = 0.036 for the stride frequency to p < 0.0001 for the total power). No significant difference was found between LRMD and GRMD dogs, though the relative medio-lateral power tended to be higher in LRMD dogs (p = 0.059, mean LRMD = 42 % (SD = 10 %), mean GRMD = 29 % (SD = 13%)). When projecting these four dogs as supplementary individuals on a PCA plane constructed using healthy and GRMD adults as active individuals, the LRMD dogs projected in the GRMD cloud, attesting the gait characteristic similarities between both colonies (Fig. 3E).
Force measurement
Two dogs were subjected to muscle force tests, at 4 and 6 months of age. Muscle force was decreased compared to healthy dogs. The decrease in force observed in these animals was slightly higher than the one observed in GRMD dogs (Fig. 4A). Muscle relaxation impairment is a feature of GRMD, showcased by the presence of incomplete relaxation after a twitch or a tetanic contraction; these muscle relaxation impairment signs are variable and usually correlate with the severity of the phenotype. This feature was also found in LRMD dogs, with an increase of residual post-tetanic contraction at the age of 6 months (Fig 4B).
Respiratory function
Four adult LRMD dogs underwent respiratory tests and showed, as observed for other physiological functions, a similar pattern to the one seen in GRMD dogs. LRMD dogs showed a significant caudal retraction of the diaphragm measured by the angle index (p < 0.001); the retraction observed was similar to the one observed in GRMD dogs (no significant difference between both models) (Fig. 5A). The LRMD diaphragm was hypokinetic, with a decreased range of motion (p = 0.005), but slightly less affected than that of GRMD dogs (p = 0.02) (Fig. 5B). The flow-volume loop analysis showed a dramatic flow decrease at the end of expiration as indicated by a decrease of the EF75/PEF ratio p = 0.0004); statistically significant differences were found for this parameter between both DMD models (Fig. 5C). The PIF/PEF ratio was also dramatically decreased (p < 0.0001), as it was the case for GRMD dogs (no significant difference between both models), compared to healthy animals. It should be noted however, that the LRMD dogs tended to have ratios that overlap with the lowest values observed for GRMD dogs (Fig. 5D).
Echocardiographic findings
A dog examined at a young age (LRMD 4, 5 months) exhibited hyperechoic lesions on the left ventricular free wall, but the measures performed using conventional echocardiography were within normal range (shortening fraction 47.5 %). However, tissue Doppler imaging, used to analyse the radial motion of the left ventricular free wall, revealed a slightly decreased endo-epicardial gradient of velocity, a hallmark of early pre-symptomatic dilated cardiomyopathy; this alteration has also been reported in GRMD dogs [54]. Another dog examined at the age of 5.5 and 7.5 years (LRMD7) showed dilated cardiomyopathy with a marked decrease of the shortening fraction worsening with time (21.5 % at 5.5 years of age and 10.3 % at 7.5 years of age). This dog also showed frequent ventricular arrhythmias (left and right ventricular premature contractions), which were suspected to be the underlying cause for the sudden death of this animal one year after the last echocardiographic examination.
Identification of the causal mutation
Overlapping RT-PCRs covering the dystrophin cDNA led to the amplification of all regions of the DMD transcript in LRMD dogs, with the exception of the sequence encompassing exons 20 and 21. Indeed, RT-PCRs between exons 10 and 20 and between exons 21 and 26 amplified normal products while no product could be obtained between exons 15 and 22 (Fig. 6A). The presence of a normal size exon 10-20 RT-PCR amplicon ruled out the possibility that the LRMD dogs carried the same mutation as the one reported in another strain of Labrador retrievers (pseudoexon inserted in the intron 19 leading to a premature stop codon) [6, 37].
A Southern blot of the EcoRI-digested genomic DNA of LRMD dogs and using cDNA probes encompassing exons 18-24, exon 21 or exon 20 revealed abnormal bands (Fig. 6B), confirming a putative remodelled spot at the DNA level, mapped between exons 20 and 21 of the DMD gene.
The complete intron 20 (4.5 kb) was thereafter explored by PCR using four overlapping couples of primers (Fig. 6 C). The three first pairs covered the 3.2‑kb 5’ segment of intron 20; the PCR products amplified using these primers were of the expected size. By contrast, the fourth PCR using the F4 - R4 primers yielded no amplicon in LRMD dogs (Fig. 6C), pinpointing that a sequence remodeling had occurred in the 1.6 kb of the 3’ region of the intron. New primers designed to amplify the putative mutation site allowed to correctly amplify the 640 bp segment covering the 3’ end of the intron; subsequent sequencing of the amplicon confirmed a normal acceptor splice site in LRMD dogs. However, a 700 bp region (X:27,623,229 to X: 27,622,528) remained non-amplifiable even when using long-range PCR conditions. Altogether, these molecular data confirmed a gross genomic DNA rearrangement involving the nearly 3’ end of intron 20. In addition, when compared to a healthy unrelated Labrador retriever, a slight size difference was seen in the amplicon using the F1 – R1 pair of primers, which appeared to be longer in dogs belonging to the LRMD colony regardless of their clinical status (data not shown). Sanger sequencing showed a normal donor splice site in LRMD dogs, but revealed an insertion of 36 bp, 960 bp downstream the 5’ end of intron 20, including a 21 bp polyA and a repetition of 15 bp of the adjacent normal sequence. This insertion explained the shift in size of the smallest band on the southern blot using the exon 20 probe (Fig. 6B). We identified this insertion in all animals of the colony, including healthy dogs and thus concluded that this insertion was a DMD-unrelated polymorphism that segregates in this line of Labradors.
The LRMD-causing mutation involving the 3’ region of intron 20 was then studied by RNA-sequencing with the aim of quantifying DMD transcription levels from both exons and introns using strand-specific libraries prepared from total RNA. This experiment revealed that the minus strand transcription from the Dp427m promoter abruptly decreased within the F4 - R4 PCR region located between exons 20 and 21 (Fig. 7A). Ectopic transcription was observed in a region normally located 2 Mb farther away, towards the centromere on the plus strand 100 kb proximal to the TMEM47 gene, where this novel transcription continued for ~0.3 Mb (X:29,824,000-30,122,000) into a region of the X chromosome containing no annotated element. The level and pattern of transcription across this ~0.3 Mb region was similar to DMD large introns, suggesting that the causal mutation in LRMD dogs may be a 2.2 Mb inversion disrupting the DMD gene and involving TMEM47 (Fig. 7A).
In light of these results, a new PCR experiment was designed with the aim of sequencing the breakpoint flanking regions, and developing a LRMD mutation diagnostic test able to identify carriers, healthy and diseased dogs. A pair of primers (mutF - mutR) flanking the presumed distant breakpoint was designed, and the absence of an amplification product when used in the LRMD genome confirmed the location of a breakpoint between these primers. The use of these primers in combination with the primers flanking the intron 20 breakpoint (F4 – R4) amplified a PCR product in LRMD but not in healthy dogs. The sequencing of these PCR products indicated that the LRMD mutation was a 2.2 Mb inversion of a region encompassing nucleotides 27,622,834 to 29,823,785 and which can be annotated: chrX:g.27,622,834_29,823,788 inv. This inverted region included the entire TMEM47 gene without disrupting it. The sequencing of the two breakpoints revealed a 27,622,834 to 29,823,789 junction on the one hand (PCR F4-MutR), and a 27,622,824 to 29,823,785 junction on the other hand (PCR MutF-R4). This indicated that the inversion is associated with a 9 nt loss in intron 20 and a 3 nt loss in the distant region, most likely suggesting that the LRMD mutation probably occurred after two double strand breaks 2.2 Mb apart, followed by a process of DNA repair through non-homologous end joining after inversion of the released fragment. Based on PCR confirmation of the breakpoint from genomic DNA, close inspection of the RNA-seq data revealed several intronic reads that mapped across the 27,622,834 to 29,823,789 breakpoint and several spliced exonic reads that mapped from DMD exon 20 to multiple locations beyond the inversion breakpoint, confirming transcription and splicing from the DMD region. Finally, a multiplex PCR was designed to generate a genetic test able to reliably identify LRMD, carrier, and healthy dogs, and we confirmed its ability to discriminate between the three genotypes (Fig. 7B).
Characterization of the dystrophin expression in muscles
Dystrophin immunostainings performed on LRMD muscle biopsies using an antibody directed against the C-terminal part of the protein (Dys2) showed a faint but undoubtful signal with normal subsarcolemmal localization (Fig. 8A). The percentage of Dys2-positive myofibres was evaluated on 32 skeletal muscle biopsies from 7 LRMD dogs and was found to range from 0.2 % (LRMD6, sartorius cranialis muscle) and 44.1 % (LRMD1, tibialis cranialis), with a mean of 11.6 % (SD = 12.3 %). In order to better characterise this protein, monoclonal antibodies cross-reacting with different regions of the protein were used on serial sections. No Dys2 positive fibres were seen using any of the antibodies cross-reacting with upstream regions of the dystrophin protein: the staining was negative for the N-terminal part (MANEX 1A), the first repeat of the rod domain (MANEX 1011 C), or for parts of the rod domain located downstream of the mutation (Dys1, MANDYS 107 (Fig. S3)) (Fig. 8A). In some of the biopsies, a few fibres that were positive for the N-terminal part of the protein were found Manex 1A+ and Manex1011C+; these fibres were however not recognised by the antibody specific for the C-terminal part of the protein (Dys2) (Fig. S4).
In order to investigate the size of the LRMD N-terminal truncated dystrophin, a western blot analysis was performed using the Dys2 antibody (C-terminal portion). The analysis of the two biopsies tested indicated the presence of a protein of around 70 kD (Fig. 8B). These results led us to speculate that the protein expressed in these muscles could be the Dp71 isoform. The expression of this isoform was confirmed with RT-PCR using a forward primer designed to bind the specific first exon of the aforementioned isoform (Fig. 8C); this result was consistent with RNA-seq coverage data observed in the Dp71 region (Fig. 7A). No expression of this isoform was found in a biopsy originating from a GRMD dog, suggesting that this Dp71 expression is not a compensatory mechanism in a context of canine dystrophinopathy, but rather specific of the LRMD context. Finally, we wanted to know if there was a correlation between the percentage of Dys2 positive fibres and the muscle lesions observed in the H&E stained sections. For this purpose, a correlation study was performed on 28 muscle biopsies and indicated that there was no correlation between the two phenomena (Pearson’s R = -0.34, p = 0.069), suggesting this isoform expression has no major influence on muscle pathology (Fig. S4).
Investigation of known disease modifiers: Jagged1, Pitpna and LTBP4
In order to asses if the inter-individual heterogeneity observed in the LRMD colony could be related to polymorphisms in known modifier genes, the presence of the described mutation in the Jagged1 gene promoter leading to ‘escaper’ phenotypes in dogs [41] was investigated in 5 LRMD dogs with diverse phenotypes, including both the animal with the mildest phenotype (LRMD 14) and the oldest survivor (LRMD 7). The results showed that the sequence of this promoter was identical in all the LRMD dogs tested and that none of these dogs harboured the G>T point mutation described in GRMD escapers. Furthermore, the Pitpna mRNA expression levels in skeletal muscle were assessed in four LRMD dogs. The levels of Pitpna mRNA have been found to be decreased in escapers compared to severely affected GRMD dogs [42]. For this assessment, we used two severely affected animals (LRMD 8 and 9) and the two less severely affected LRMD dogs (LRMD 7 and 14). No differences in expression levels were seen among the studied dogs (p = 0.921), suggesting that the phenotype heterogeneity is not associated with fluctuations in Pitpna mRNA expression in LRMD dogs.
Polymorphisms in the coding sequence of LTBP4 have been associated with modulation of the phenotype in mdx mice and in human DMD patients [60, 61]. Eleven SNPs were found in the canine LTBP4 coding sequence, 4 of these SNPs were responsible for amino-acid changes (amino acids number 425, 439, 545 and 668). The study of these polymorphisms in 5 LRMD dogs showed that all the animals were homozygous and identical for each of the SNPs. Particular attention was put on the 4 SNPs that could potentially lead to amino acid changes. The results obtained showed that amino acid 425 was a Threonine, 439 and 545 were Prolines, and 668 an Alanine. In conclusion, the analysis performed showed that none of the modulators analysed were responsible for the inter-individual heterogeneity. The homozygosity observed in the LRMD colony is most likely related to the high inbreeding rate of the colony; this fact probably makes this LRMD model a favourable context to discover new DMD phenotype modifiers.