Genome-wide search for markers associated with osteochondrosis in Hanoverian warmblood horses

A genome-wide scan was performed to detect quantitative trait loci (QTLs) for osteochondrosis (OC) and osteochondrosis dissecans (OCD) in horses. The marker set comprised 260 microsatellites. We collected data from 211 Hanoverian warmblood horses consisting of 14 paternal half-sib families. Traits used were OC (fetlock and/or hock joints affected), OCD (fetlock and/or hock joints affected), fetlock OC, fetlock OCD, hock OC, and hock OCD. The first genome scan included 172 microsatellite markers. In a second step 88 additional markers were chosen to refine putative QTLs found in the first scan. Genome-wide significant QTLs were located on equine chromosomes 2, 4, 5, and 16. QTLs for fetlock OC and hock OC partly overlapped on the same chromosomes, indicating that these traits may be genetically related. QTLs reached the chromosome-wide significance level on eight different equine chromosomes: 2, 3, 4, 5, 15, 16, 19, and 21. This whole-genome scan was a first step toward the identification of candidate genome regions harboring genes responsible for equine OC. Further investigations are necessary to refine the map positions of the QTLs already identified for OC.

A radiation hybrid map of sheep chromosome 23 based on ovine BAC-end sequences

More than 375,000 BAC-end sequences (BES) of the CHORI-243 ovine BAC library have been deposited in public databases. blastn searches with these BES against HSA18 revealed 1806 unique and significant hits. We used blastn-anchored BES for an in silico prediction of gene content and chromosome assignment of comparatively mapped ovine BAC clones. Ovine BES were selected at approximately 1.3-Mb intervals of HSA18 and incorporated into a human-sheep comparative map. An ovine 5000-rad whole-genome radiation hybrid panel (USUoRH5000) was typed with 70 markers, all of which mapped to OAR23. The resulting OAR23 RH map included 43 markers derived from BES with high and unique BLAST hits to the sequence of the orthologous HSA18, nine EST-derived markers, 16 microsatellite markers taken from the ovine linkage map and two bovine microsatellite markers. Six new microsatellite markers derived from the 43 mapped BES and the two bovine microsatellite markers were linkage-mapped using the International Mapping Flock (IMF). Thirteen additional microsatellite markers were derived from other ovine BES with high and unique BLAST hits to the sequence of the orthologous HSA18 and also positioned on the ovine linkage map but not incorporated into the OAR23 RH map. This resulted in 24 markers in common and in the same order between the RH and linkage maps. Eight of the BES-derived markers were mapped using fluorescent in situ hybridization (FISH), to thereby align the RH and cytogenetic maps. Comparison of the ovine chromosome 23 RH map with the HSA18 map identified and localized three major breakpoints between HSA18 and OAR23. The positions of these breakpoints were equivalent to those previously shown for syntenic BTA24 and HSA18. This study presents evidence for the usefulness of ovine BES when constructing a high-resolution comprehensive map for a single sheep chromosome. The comparative analysis confirms and refines knowledge about chromosomal conservation and rearrangements between sheep, cattle and human. The constructed RH map demonstrates the resolution and utility of the newly constructed ovine RH panel.

The locus for bovine dilated cardiomyopathy maps to chromosome 18

Bovine dilated cardiomyopathy (BDCMP) is a severe and terminal disease of the heart muscle observed in Holstein-Friesian cattle over the last 30 years. There is strong evidence for an autosomal recessive mode of inheritance for BDCMP. The objective of this study was to genetically map BDCMP, with the ultimate goal of identifying the causative mutation. A whole-genome scan using 199 microsatellite markers and one SNP revealed an assignment of BDCMP to BTA18. Fine-mapping on BTA18 refined the candidate region to the MSBDCMP06-BMS2785 interval. The interval containing the BDCMP locus was confirmed by multipoint linkage analysis using the software loki. The interval is about 6.7 Mb on the bovine genome sequence (Btau 3.1). The corresponding region of HSA19 is very gene-rich and contains roughly 200 genes. Although telomeric of the marker interval, TNNI3 is a possible positional and a functional candidate for BDCMP given its involvement in a human form of dilated cardiomyopathy. Sequence analysis of TNNI3 in cattle revealed no mutation in the coding sequence, but there was a G-to-A transition in intron 6 (AJ842179:c.378+315G>A). The analysis of this SNP using the study's BDCMP pedigree did not conclusively exclude TNNI3 as a candidate gene for BDCMP. Considering the high density of genes on the homologous region of HSA19, further refinement of the interval on BTA18 containing the BDCMP locus is needed.

Functional Diversification of Dicer-like Proteins and Small RNAs Required for Genome Sculpting.

In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean Grey cattle.

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy.

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the illumina sequencing technology we obtained a whole genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared to the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single private non-synonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than one thousand control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. Based on the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy.

A RAB3GAP1 SINE Insertion in Alaskan Huskies with Polyneuropathy, Ocular Abnormalities and Neuronal Vacuolation (POANV) Resembling Human Warburg Micro Syndrome 1 (WARBM1)

We observed a hereditary phenotype in Alaskan Huskies, which was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV). The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier and an unrelated control revealed a 218 bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1), which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1 deficient mice have a much milder phenotype than either humans or dogs. Thus the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the non-intentional breeding of affected dogs.

New insights into the performance of human whole-exome capture platforms.

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variants.

Complete Genome Sequence of KPC-3- and CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 307.

Klebsiella pneumoniaesequence type (ST) 307, carryingblaKPC-3,blaCTX-M-15,blaOXA-1,aac(6')-Ib-cr, andqnrB1 genes, is replacing the predominant hyperepidemic ST258 clone in Italy. Whole-genome and complete plasmid sequencing of one ST307 strain was performed and new features were identified.

Genetic evaluation of short stature

After a proper medical history, growth analysis and physical examination of a short child, followed by radiological and laboratory screening, the clinician may decide to perform genetic testing. We propose several clinical algorithms that can be used to establish the diagnosis. GH1 and GHRHR should be tested in children with severe isolated growth hormone deficiency and a positive family history. A multiple pituitary dysfunction can be caused by defects in several genes, of which PROP1 and POU1F1 are most common. GH resistance can be caused by genetic defects in GHR, STAT5B, IGF1, IGFALS, which all have their specific clinical and biochemical characteristics. IGF-I resistance is seen in heterozygous defects of the IGF1R. If besides short stature additional abnormalities are present, these should be matched with known dysmorphic syndromes. If no obvious candidate gene can be determined, a whole genome approach can be taken to check for deletions, duplications and/or uniparental disomies.

Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of Necropsobacter gen. nov. and of Necropsobacter rosorum sp. nov. for organisms of the SP group

To allow classification of bacteria previously reported as the SP group and the Stewart-Letscher group, 35 isolates from rodents (21), rabbits (eight), a dog and humans (five) were phenotypically and genotypically characterized. Comparison of partial rpoB sequences showed that 34 of the isolates were closely related, demonstrating at least 97.4 % similarity. 16S rRNA gene sequence comparison of 20 selected isolates confirmed the monophyly of the SP group and revealed 98.5 %-100 % similarity between isolates. A blast search using the 16S rRNA gene sequences showed that the highest similarity outside the SP group was 95.5 % to an unclassified rat isolate. The single strain, P625, representing the Stewart-Letscher group showed the highest 16S rRNA gene similarity (94.9-95.5 %) to members of the SP group. recN gene sequence analysis of 11 representative strains resulted in similarities of 97-100 % among the SP group strains, which showed 80 % sequence similarity to the Stewart-Letscher group strain. Sequence similarity values based on the recN gene, indicative for whole genome similarity, showed the SP group being clearly separated from established genera, whereas the Stewart-Letscher group strain was associated with the SP group. A new genus, Necropsobacter gen. nov., with only one species, Necropsobacter rosorum sp. nov., is proposed to include all members of the SP group. The new genus can be separated from existing genera of the family Pasteurellaceae by at least three phenotypic characters. The most characteristic properties of the new genus are that haemolysis is not observed on bovine blood agar, positive reactions are observed in the porphyrin test, acid is produced from (+)-L-arabinose, (+)-D-xylose, dulcitol, (+)-D-galactose, (+)-D-mannose, maltose and melibiose, and negative reactions are observed for symbiotic growth, urease, ornithine decarboxylase and indole. Previous publications have documented that both ubiquinones and demethylmenaquinone were produced by the proposed type strain of the new genus, Michel A/76(T), and that the major polyamine of representative strains (type strain not included) of the genus is 1,3-diaminopropane, spermidine is present in moderate amounts and putrescine and spermine are detectable only in minor amounts. The major fatty acids of strain Michel A/76(T) are C(14 : 0), C(16 : 0), C(16:1)omega7c and summed feature C(14 : 0) 3-OH/iso-C(16 : 1) I. This fatty acid profile is typical for members of the family Pasteurellaceae. The G+C content of DNA of strain Michel A/76(T) was estimated to be 52.5 mol% in a previous investigation. The type strain is P709(T) ( = Michel A/76(T) = CCUG 28028(T) = CIP 110147(T) = CCM 7802(T)).

ENU-induced phenovariance in mice: inferences from 587 mutations

Background We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.