Whole‐genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness

White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white‐spotting phenotype were deaf. We obtained whole‐genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63‐kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR‐based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader–Willi syndrome mouse models

Prader–Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11–13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%–100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p^30PUb deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p^30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p^30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p^30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.     

Delineating the role of MITF isoforms in pigmentation and tissue homeostasis

MITF, a gene that is mutated in familial melanoma and Waardenburg syndrome, encodes multiple isoforms expressed from alternative promoters that share common coding exons but have unique amino termini. It is not completely understood how these isoforms influence pigmentation in different tissues and how the expression of these independent isoforms of MITF is regulated. Here, we show that melanocytes express two isoforms of MITF, MITF‐A and MITF‐M. The expression of MITF‐A is partially regulated by a newly identified retinoid enhancer element located upstream of the MITF‐A promoter. Mitf‐A knockout mice have only subtle changes in melanin accumulation in the hair and reduced Tyr expression in the eye. In contrast, Mitf‐M‐null mice have enlarged kidneys, lack neural crest‐derived melanocytes in the skin, choroid, and iris stroma, yet maintain pigmentation within the retinal pigment epithelium and iris pigment epithelium of the eye. Taken together, these studies identify a critical role for MITF‐M in melanocytes, a minor role for MITF‐A in regulating pigmentation in the hair and Tyr expression in the eye, and a novel role for MITF‐M in size control of the kidney.     

Genome‐wide association study for pigmentation traits in Chinese Holstein population

With the Illumina BovineSNP50K BeadChip, we performed a genome‐wide association study (GWAS) for two pigmentation traits in a Chinese Holstein population: proportion of black (PB) and teat colour (TC). A case–control design was used. Cases were the cows with PB <0.30 (n = 129) and TC <2 points (n = 140); controls were those with PB >0.90 (n = 58) and TC >4 points (n = 281). The RM test of roadtrips (version 1.2) was applied to detect SNPs for the two traits with 42 883 and 42 741 SNPs respectively. A total of nine and 12 genome‐wide significant (P < 0.05) SNPs associated with PB and TC respectively were identified. Of these, two SNPs for PB were located within the KIT and IGFBP7 genes, and the other four SNPs were 23~212 kb away from the PDGFRA gene on BTA6; nine SNPs associated with TC were located within or 21~78.8 kb away from known genes on chromosomes 4, 11, 22, 23 and 24. By combing through our GWAS results and the biological functions of the genes, we suggest that the KIT, IGFBP7, PDGFRA, MITF, ING3 and WNT16 genes are promising candidates for PB and TC in Holstein cattle, providing a basis for further investigation on the genetic mechanism of pigmentation formation.     

Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes

White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re‐investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes’ individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9‐kb deletion spanning exons 10–13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

Screening for signatures of selection of Tianzhu white yak using genome‐wide re‐sequencing

The Tianzhu white yak, a domestic yak indigenous to the Qilian Mountains, migrated inland from the Qinghai‐Tibet Plateau. Specific ecological and long‐term artificial selection influenced the evolution of its pure white coat and physiological characteristics. Therefore, it is not only a natural population that represents a genomic selective region of environmental adaptability but is also an animal model for studying the pigmentation of the yak coat. A total of 24 261 829 variants, including 22 445 252 SNPs, were obtained from 29 yaks by genome‐wide re‐sequencing. According to the results of a selective sweep analysis of Tianzhu white yak in comparison to Tibetan yaks, nine candidate genes under selection in Tianzhu white yak were identified by combining π, Tajima's D, πA/πB and F_ST statistics, with threshold standards of 5%. These genes include PDCD1, NUP210, ABCG8, NEU4, LOC102287650, D2HGDH, COL4A1, RTP5 and HDAC11. Five of the nine genes were classified into 12 molecular signaling pathways, and most of these signaling pathways are involved in environmental information processing, organismal systems and metabolism. A majority of these genes has not been implicated in previous studies of yak coat color and high‐altitude animals. Our findings are helpful not only for explaining the molecular mechanism of yak coat pigmentation but also for exploring the genetic changes in Tianzhu white yak due to environmental adaptation.     

A novel KIT deletion variant in a German Riding Pony with white‐spotting coat colour phenotype

White spotting phenotypes in horses may be caused by developmental alterations impairing melanoblast differentiation, survival, migration and/or proliferation. Candidate genes for white‐spotting phenotypes in horses include EDNRB, KIT, MITF, PAX3 and TRPM1. We investigated a German Riding Pony with a sabino‐like phenotype involving extensive white spots on the body together with large white markings on the head and almost completely white legs. We obtained whole genome sequence data from this horse. The analysis revealed a heterozygous 1273‐bp deletion spanning parts of intron 2 and exon 3 of the equine KIT gene (Chr3: 79 579 925–79 581 197). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. Knowledge of the functional impact of similar KIT variants in horses and other species suggests that this deletion represents a plausible candidate causative variant for the white‐spotting phenotype. We propose the designation W28 for the mutant allele.     

Altered Ultrasonic Vocalization and Impaired Learning and Memory in Angelman Syndrome Mouse Model with a Large Maternal Deletion from Ube3a to Gabrb3

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11–q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11–q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m−/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m−/p+), but not paternal (m+/p−), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome

Background  

Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice.