A de novo recessive mutation causative of Mandibulofacial Dysostosis in Hereford cattle
This video describes the process our lab completed to identify the genomic variant underlying a congenital defect in a breed of cattle. We investigated pedigrees to develop a hypothesized mode of inheritance. Then we used whole genome sequencing data to investigate our hypothesis. We identified and verified a candidate variant. I end this presentation by describing the role we believe this variant is playing in craniofacial development.
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[00:00:00.920]I'm Renae Sieck,
[00:00:01.990]and today I will present our group's research
[00:00:04.030]that was recently published in "Genes."
[00:00:06.810]In March and April of 2020,
[00:00:08.720]three cattle herds reported a total
[00:00:10.610]of six purebred Hereford calves
[00:00:12.590]born with unusual defects of the face and jaw.
[00:00:15.810]These were attributed to a condition we term
[00:00:18.121]Mandibulofacial Dysostosis, which we abbreviate as MD.
[00:00:22.560]A common bull was identified in the pedigree
[00:00:24.730]of all of the affected calves,
[00:00:26.440]indicating a plausible genetic cause of the condition.
[00:00:30.620]Throughout the project, five affected calves were sent
[00:00:33.240]to the UNL Veterinary Diagnostic Center
[00:00:35.710]for autopsy evaluation.
[00:00:37.810]This table presents a summary
[00:00:39.370]of the external pathologic findings in these calves.
[00:00:42.900]One defining characteristic of all calves evaluated
[00:00:45.830]was bilateral skin tags near the fusion site
[00:00:48.310]of the mandible and maxilla.
[00:00:50.510]All affected calves also had skin tags
[00:00:52.930]in variable locations at or below the ears.
[00:00:56.720]Multiple other pathologic characteristics were present
[00:00:59.290]in a subset of the observed calves at varying severity.
[00:01:03.810]Further autopsy evaluation revealed
[00:01:05.940]two additional internal hallmarks of MD.
[00:01:08.890]The skin tags near the commissure of the lips were found
[00:01:11.700]to be attached through a short dermal band
[00:01:13.840]to cartilage encased in a bony process.
[00:01:16.890]The bony process then extended
[00:01:18.430]from the skin tag toward the ear.
[00:01:20.950]When the bony process was fractured,
[00:01:22.690]it revealed a cartilage tube that was freely removed
[00:01:25.240]from the bone encasing it, as shown in the central image.
[00:01:28.820]This cartilage is quite abnormal in this region of the skull
[00:01:32.400]and was identified to be Meckel's cartilage,
[00:01:34.980]a remnant of cartilage normally present
[00:01:37.120]in the region early in development.
[00:01:40.320]Histologic evaluation of the bony process confirmed
[00:01:43.160]that this cartilage was sandwiched
[00:01:44.620]between plates of bone.
[00:01:47.440]This pedigree shows cattle
[00:01:48.900]displaying the MD phenotype in black
[00:01:51.000]and those that had a normal phenotype in gray.
[00:01:54.010]A potential founder bull was present
[00:01:55.790]in both the maternal and paternal sides of the pedigrees
[00:01:59.010]of all of the affected calves.
[00:02:01.510]Based on the inheritance pattern of the MD phenotype,
[00:02:04.460]we hypothesized that a de novo autosomal recessive mutation
[00:02:08.240]is causative of this novel condition in Hereford cattle.
[00:02:12.280]To evaluate this hypothesis,
[00:02:14.270]we began by whole genome sequencing three affected calves,
[00:02:17.620]10 parents of affected calves,
[00:02:19.390]and seven individuals that were related
[00:02:21.590]to the suspect founder bull.
[00:02:23.640]The sequencing yielded a list of 143 candidate variants
[00:02:27.960]that matched the hypothesized mode of inheritance.
[00:02:30.830]To further narrow down this list,
[00:02:32.570]we evaluated whole genome sequence data
[00:02:34.900]from other cattle projects in our lab,
[00:02:37.160]publicly available data on the Sequence Read Archive,
[00:02:40.190]and Sanger sequencing results from additional animals
[00:02:43.230]related to the presumed founder.
[00:02:45.690]Variants were removed from consideration
[00:02:47.920]that didn't match our hypothesized mode of inheritance.
[00:02:51.160]This left us with two candidates remaining.
[00:02:53.820]From this point forward, we pursued the missense variant
[00:02:56.690]in CYP26C1 because of the predicted deleterious impact
[00:03:00.860]of the mutation on protein function
[00:03:03.240]and because of the biological role of this gene.
[00:03:07.420]To further evaluate the predicted function
[00:03:09.350]of the CYP26C1 variant, we looked at its conservation
[00:03:13.340]of the altered amino acids sequence across multiple species.
[00:03:18.100]The leucine at position 188
[00:03:19.900]of the cattle amino acid sequence that we show here in bold
[00:03:23.500]was conserved across all species
[00:03:25.150]except for in the affected MD calves
[00:03:27.770]where the missense mutation resulted
[00:03:29.860]in a proline at this position.
[00:03:32.460]Conservation across many species
[00:03:34.530]indicates a biological importance
[00:03:36.450]of that portion of the protein.
[00:03:38.640]Therefore, this evaluation provides further evidence
[00:03:41.480]that this missense mutation is deleterious
[00:03:43.770]to protein function.
[00:03:46.590]We also performed protein modeling
[00:03:48.220]to show the predicted location
[00:03:49.770]of the changed amino acid sequence
[00:03:51.750]within the protein structure.
[00:03:53.680]Image A and B in this picture show that our mutation was not
[00:03:57.040]in the active binding side of this protein
[00:03:59.160]but instead is located in an alpha helix
[00:04:01.840]near that binding site.
[00:04:03.580]In C and D, the horizontal red bars
[00:04:06.360]indicate the predicted position of an alpha helix,
[00:04:10.260]and our confidence in that prediction is displayed
[00:04:12.760]by the vertical black bars.
[00:04:14.750]C represents the wild-type leucine,
[00:04:16.920]and D represents the amino acid changed to proline
[00:04:20.130]as a result of our candidate missense mutation.
[00:04:23.310]Proline is known to be a helix breaker.
[00:04:25.940]This predictor confirms that that could be the case
[00:04:28.390]with our candidate mutation
[00:04:30.050]by showing a break in the helix
[00:04:32.240]around amino acid position 188.
[00:04:35.990]In order to confirm the inheritance pattern of MD
[00:04:38.820]in this line of cattle,
[00:04:40.260]we genotyped an additional 782 Herefords.
[00:04:44.180]It's important to note that all of the animals
[00:04:46.700]with a homozygous recessive genotype
[00:04:49.080]displayed the MD phenotype,
[00:04:51.120]and no other animals we genotyped
[00:04:53.710]displayed the phenotypic characteristics of this condition.
[00:04:57.200]Also, all animals with no pedigree tie to the founder
[00:05:01.020]genotyped as homozygous for the reference allele.
[00:05:05.250]In order to discuss the biological role
[00:05:07.340]we believe CYP26C1 plays in the MD condition,
[00:05:10.820]I will first provide you with an overview
[00:05:13.110]of the process of mandible formation.
[00:05:15.980]Cranial neural crest cells migrate from the neural tube
[00:05:18.970]to the first pharyngeal arch of the developing embryo.
[00:05:22.360]Next, endothelin-1 signaling in the first pharyngeal arch
[00:05:26.280]results in activation of DLX5 and DLX6 genes.
[00:05:31.440]This activation initiates the formation
[00:05:33.580]of Meckel's cartilage, which eventually serves as a scaffold
[00:05:37.420]during the formation and elongation
[00:05:39.780]of the intramembranous mandible bone.
[00:05:42.490]Retinoic acid is important
[00:05:43.770]for understanding the role of CYP26C1
[00:05:46.390]in craniofacial development.
[00:05:48.650]Retinoic acid is a metabolite of vitamin A
[00:05:51.430]and is a repressor of endothelin-1.
[00:05:54.410]Therefore, an excess of retinoic acid can result
[00:05:56.860]in craniofacial abnormalities involving the mandible
[00:06:00.260]via changes in Meckel's cartilage formation.
[00:06:04.160]Retinoic acid in craniofacial development is regulated
[00:06:07.140]by two main families of genes.
[00:06:09.400]The Raldh family synthesizes retinoic acid,
[00:06:12.890]and the CYP26 family degrades it.
[00:06:15.970]Retinoic acid's effect is highly tissue
[00:06:18.300]and time point specific.
[00:06:20.420]CYP26C1 is expressed in the first pharyngeal arch
[00:06:23.420]during mandible development.
[00:06:25.770]The phenotype of the MD-affected calves would result
[00:06:28.620]in changes in retinoic acid level
[00:06:31.020]during approximately day 21 to 22 of gestation.
[00:06:36.750]Several human conditions exhibit similar pathologies
[00:06:39.740]to the MD calves.
[00:06:41.450]Focal facial dermal dysplasia type four is interesting
[00:06:44.920]because the skin lesions seen in the affected human patients
[00:06:48.700]are resulting from a loss of function mutation in CYP26C1.
[00:06:53.730]Interestingly, these skin lesions are in similar locations
[00:06:56.950]to the skin tags we observed in MD calves.
[00:07:00.110]Next, hemifacial microsomia in humans
[00:07:04.270]results in skin tags
[00:07:05.620]caudal to the commissure of the lips
[00:07:07.460]and near the ear and changes in mandible elongation.
[00:07:12.290]The skin tags and mandible development changes
[00:07:15.370]are strikingly similar to the pathologic characteristics
[00:07:18.590]we observed in the MD calves.
[00:07:21.570]Our study has wide implications within the Hereford breed
[00:07:24.680]because multiple highly-prolific sires are carriers
[00:07:27.460]of the condition.
[00:07:28.800]This resulted in over 40,000 cattle
[00:07:30.930]with pedigree ties to the founder bull.
[00:07:33.620]Additionally, the novel pathology presented
[00:07:36.930]could aid researchers studying similar conditions
[00:07:39.250]in other species.
[00:07:41.490]I would like to acknowledge
[00:07:42.760]the American Hereford Association
[00:07:44.570]and the Hereford breeders who reported cases
[00:07:46.760]for their assistance in sample collection
[00:07:48.700]and for providing pedigree records.
[00:07:51.000]I would also like to thank Leah Treffer and Anna Fuller
[00:07:54.450]for their contributions in the lab.
[00:07:56.810]Thank you for tuning in to my presentation.
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