PYROSEQUENCING : ITS POTENTIAL AND LIMITATIONS IN DIAGNOSIS OF INHERITED DISEASES IN CATTLE

A large number of different groups of livestock diseases in which heredity plays a significant role are currently reported. In particular, more than 500 genetically determined morphological and functional disorders have been detected in cattle; for 150 of them, specific mutations are known. The sale of bovine genetic materials is associated with the spread of various diseases caused by mutations that occur in the prominent representatives of breeds. The abundance of lethal mutations in the populations requires a broader application of molecular diagnostic methods for detection of monogenic hereditary diseases. DNA pyrosequencing, being the most convenient technique for the rapid diagnosis of single nucleotide mutations in the bovine genome that are located in the regions with known nucleotide sequence, has a potential for meeting this need. Pyrosequencing-based methods for identification of the most common significant mutations in Holstein, Simmental, Brown Swiss and Aberdeen Angus cattle were developed, validated and approved at the FGBU “VGNKI”. Such mutations are associated with leukocyte adhesion deficiency, complex vertebral malformation, uridine monophosphate synthetase deficiency, citrullinemia, spinal muscular atrophy, spinal cord demyelination, Brown Swiss haplotype 2, Weaver syndrome, developmental duplications, α-mannosidosis, dwarfism, bovine male subfertility, trombocytopathya, arachnomelia syndrome, hypozincemia-like syndrome. These methods are intended to test semen in the breeding centres, scientific laboratories working in the field of biotechnology and animal reproduction, livestock reproduction centres. The use of the proposed genetic tests to detect mutant alleles, as well as reduced use of mutation-bearing animals in stock breeding will allow minimizing the occurrence of inherited diseases and thus improving the gene pool of cattle in the country.


INTRODUCTION
Intensive use of the world genetic pool and repro duction biotechnologies (artificial insemination, embryo transplantation) allows significant genetic improvement aimed at animal productivity increase by getting off spring from breeding animals being the breed leaders. However, such approach to selection for desirable cha racteristics of productivity in cattle breeds has resulted in accumulation of the mutations associated with vari ous pathologies [3,6]. This reduces reproductive perfor mance and fertility, newborn and young animal viability, duration of animal practical use resulting in significant economic losses. Therefore, development of methods for early detection of harbored mutations in breeding ani mals and rejection of genetically abnormal semen straws are of current importance. Animals carrying mutations associated with different diseases can be detected only with molecular and genetic methods.
As a rule, monogenic genetic disorders can be phe notypically identified only in homozygous recessive car riers at late postembryonic development stage or in early postnatal period. Such carriers are often nonviable. In heterozygous carriers such mutations are not phenotypi cally apparent. DNA testing of replacement young animals will allow latent carriers detection and inherited disease spread prevention in cattle populations. Alleles unde si rable for the population can be rapidly eliminated within one generation and selection process can be significantly accelerated.

IN DIAGNOSIS OF INHERITED DISEASES IN CATTLE
nucleotide sequences in genetic polymorphism region and known point mutations detection (singlenucleo tide substitutions, one or threenucleotide deletions/in sertions) by comparing to reference DNA sequence. The FGBU "VGNKI" developed pyrosequencingbased methods for identification of the most common mutations in Hol stein, Brown Swiss, Aberdeen Angus and Simmental cattle.

MATERIALS AND METHODS
Genetic materials (frozen semen, venous blood) derived from domestic and foreign Holstein, Brown Swiss, Aber deen Angus and Simmental bulls were used for testing. DNA was extracted from venous blood with adsorption method using DNAsorbСM kit (FGBI "Central Research Institute of Epidemiology", Rospotrebnadzor) according to the manufacturer instruction. DNA extraction from frozen semen was carried out with DNAsorbСM kit modified by the FGBU "VGNKI".

RESULTS AND DISCUSSION
Pyrosequencing-based methods for identification of the mutations associated with the most common mono genic inherited diseases in cattle were developed (Table 1).
At the first stage, analysis of the target gene nucleotide sequences from the NCBI OMIA database [10, 11] and li terature [4,7,8] was carried out. Genome fragments asso ciated with the particular inherited disease were selected. Oligonucleotide primers suitable for relevant genome fragment amplification in the mutation region as well as More than 500 genetically determined morphologi cal and functional disorders have been detected in cat tle; 150 of them are known to be associated with specific mutations [9,11]. Among six the most common breeds the highest number of such abnormalities were recorded in Holstein breed followed by Friesian, BlackandWhite, Simmental, Brown Swiss and Ayrshire breeds (45, 32, 26, 24, 20 and 19 abnormalities, respectively). Genetic nature of the majority of cattle inherited diseases is associated with point mutations that are commonly singlenucleo tide polymorphisms (SNP) in specific gene sites. A wide range of molecularbiological techniques based on poly merase chain reaction (PCR) is used for SNP detection in the genome [1]. The most common methods are as follows: restriction fragment length polymorphism ana lysis, allelespecific PCR, various variants of realtime PCR, DNAarray hybridization, massspectrometry analysis and DNA sequencing techniques. However, only sequencing being the direct method for nucleotide sequence determi nation allows unequivocally interpretation of the obtained results -nucleotide sequence of polymorphism region and homozygous or heterozygous nucleotide polymor phism.
Selection of the method for genetic polymorphism detection depends on tasks assigned to the laborato ry including number of tests to be performed as well as the nucleotide sequence of the analyzed genome region.
Since sequencing of small DNA fragment is sufficient for SNP detection use of Sanger sequencing technique or highthroughput genetic analysis systems is not always reasonable.
Pyrosequencing is the most convenient method for largescale screening for known polymorphisms associ ated with various monogenic inherited diseases in cattle. The method enables determination of shortfragment ORIGINAL ARTICLES | BOVINE DISEASES ОРИГИНАЛЬНЫЕ СТАТЬИ | БОЛЕЗНИ КРС primers for sequencing were selected using PyroMark As say Design Software. Forward and reverse sequencing can be performed de pending on the nucleotide sequence of the analyzed frag ment. Forward sequencing requires amplification reverse primer to be biotinlabelled at 5'end, reverse sequencing requires biotinlabelled forward primers ( Table 2).
Preparation of PCRproduct for pyrosequencing in cludes incubation of amplicon with streptavidincoated sepharose beads, alkaline denaturation for singlestrand DNA generation, several consecutive washings using vacuum filtering systems as well as sequencing primer annea ling in the analyzed genetic locus region.
When matrixcomplementary deoxynucleotide is gra dually added to the reaction mix, DNA polymerase cataly zes the nucleotide incorporation into growing DNA chain that is accompanied with the release of pyrophosphate in a quantity equimolar to the amount of incorporated nucleotides. ATPsulfurylase transforms pyrophosphate in ATF in the presence of adenosine5phosphosulfate. Resulting ATP triggers luciferin oxidation to oxyluciferin generating visible light emission proportional to the ATP amount. Chemiluminescent signal is detected and visible as peaks in pyrogram. The height of each peak is propor tional to number of nucleotides in the matrix. Nucleotides are gradually added to the reaction mix according to the specified nucleotide sequence [5]. Analysis of obtained data are carried out at final stage of pyrosequencing.
PyroMark Q96 MD genetic analyzer software can be used for automated processing of the results since cha racterized polymorphic loci in cattle genome are exa mined and position of SNP is known. Figure 1 shows re ference pyrograms for each of examined polymorphism variants in the TMEM95 gene. Mutation in the said gene is associated with bovine male subfertility (BMS) in Sim mental bulls. Figure 2 shows an example of pyrosequencing result interpretation. Polymorphism region is analyzed automati cally using software based on relative signal heights within polymorphism region (compared to the signals corres ponding to reference nucleotides). SNPRUN programme is used to determine peak readings accuracy.
Only pyrograms of appropriate quality are used for the polymorphism allele frequency estimation. The following criteria for pyrogram quality evaluation were established: 1) analyzed sequence shall comprise specified number of nonvariable reference nucleotides. Analyzed sequence is a short fragment of DNA sequence comprising one or several polymorphisms to be analyzed. During analysis, reference peaks are used for assessment of peak heights within the polymorphism region and as an internal control for quality assessment. For example, in Figure 2  2) absence of background signal at empty positions in the pyrogram automatically generated by the software programme. Empty positions indicate nonspecific nucleo tide insertion. Figure 2 shows that there are no nucleotides at positions 1 and 7 in the target nucleotide sequence, the nucleotides do not insert in generated chain and conse quently signal level is equal to zero; Figure 2); 4) sufficient signal intensity of target and reference peaks, a peak height shall be at least 30 relative light units (RLU) (value used in pyrosequencing for determination of peak heights in pyrogram). The peak height of 281 RLU is indicated in Figure 2a.

3) absence of background signal at variable positions (mutation region is indicated by colour in
Genotype is identified according to the analyzed gene tic locus based on relative height of target peaks at variable ORIGINAL ARTICLES | BOVINE DISEASES ОРИГИНАЛЬНЫЕ СТАТЬИ | БОЛЕЗНИ КРС  For example, results of analysis for BMSassociated mu tation identification are interpreted as follows (Fig. 1): -С/С -normal genotype (homozygous for normal allele, no mutation is identified); -С/А -the animal is a carrier of the bovine male sub fertility (BMS)associated mutation in the TMEM95 gene (heterozygous, with one mutant copy and one normal copy of the gene); -А/А -mutant genotype (homozygous for mutant allele).
Mutations associated with the following syndromes were detected in bulls tested with pyrosequencingbased techniques intended for singlenucleotide polymorphism monitoring: in Holstein cattle -2% of the bulls were car riers of the CVMassociated mutation and 0.5% of the bulls were BLADmutation carriers; in Simmental cattle -2% of the bulls were ASmutation carriers, 5% of the bulls were BMSmutation carriers and 7% of the bulls were TPmuta tion carriers; in Aberdeen Angus cattle -7.5% of the bulls were DDmutation carriers; in Brown Swiss cattle -5% of the bulls were SMAmutation carriers.
Thus, pyrosequencing has several advantages. It allows rapid detection of many short DNA sequences, single nuc leotide mutations, resequencing, etc. PyroMark Q96 MD genetic analysis system allows short DNA sequence iden tification, up to 96 samples within a short period. Simple chemical reactions and robust detection system make use of gels, electrophoresis and specific fluorescent labels un necessary for the said method that significantly simplifies sample preparation procedure. Therewith, the analysis time and cost are significantly reduced as compared to capillary electrophoresis technique [2].
Besides, analysis of pyrosequencing data is simple and very clear. It includes comparison of output graph to the reference pyrogram proposed by the software.
The abovementioned characteristics made the pyro sequencing technique the most suitable for rapid diagno sis of single nucleotide mutation in cattle genome located in the regions with known nucleotide sequence.
However, the technique has some disadvantages. Taking into account that DNA fragments intended for pyrosequencing should be no longer than 200 bp, the said technique is not suitable for diagnosis of the muta tions associated with extended deletions or insertions. Therefore, a method based on PCR with electrophoretic detection was developed for identification of brachyspina syndrome, osteopetrosis and arthrogryposis multiplex in Holstein and Aberdeen Angus cattle. The said diseases are associated with extended deletions (more than 2,000 bp).
Pyrosequencing does not allow precise determination of identical nucleotides in DNA sequence when there are position and conclusion on the animal genotype is issued. Identified genotype variants are as follows: homozygote for frequent allele (normal genotype), heterozygote, ho mozygote for rare allele (mutant genotype).  а b c more than 6 such consecutive nucleotides in the sequence. For example, mutation associated with arachnomyelitis and arthrogryposis in Brown Swiss cattle (SАA) is an in sertion of one nucleotide, guanine (c.363-364insG), at the specified position in the SOUX gene after the 7 guanine nucleotide sequence [GGGGGGG]. Therefore, Sanger se quencing of the mutation region is required for identifi cation of such SNP.
It should be noted that use of pyrosequencing tech nique for point mutation identification depends on the nucleotide sequence composition in the polymorphism region. In some cases, genotype identification based on tested genetic locus (normal genotype or carrier) is impossible. Thus, for example, reference pyrograms for trombocytopathy identification in Simmental cattle are shown in Figure 3. The disease develops when adenine is substituted to guanine at position 701 of RASGRP2 gene in 29 th chromosome. Correct identification of polymorphism variants based on relative height of target peaks at the substitution position with forward analysis is highly prob lematic ( Fig. 3а and 3b).
In such case, the sequencing direction was changed to simplify the result interpretation. Reverse analysis makes polymorphism variant identification easy (Fig. 4).

CONCLUSION
Control of genetic defects in cattle has become very im portant aspect of prevention in national veterinary prac tice and corrective selection in view of livestock breeding globalization and commercialization. Mandatory tests of breeding bulls for the breedspecific monogenic inherited diseases followed by recording of the mutation carriers in breed catalogues could further improve genetic potential of animals in the country.
Pyrosequencing technique allows mass screening for known polymorphisms associated with various mono genic inherited diseases in cattle. It enables identification of shortfragment nucleotide sequences in genetic poly morphism region and detection of known point mutations. Putting pyrosequencingbased genetic tests proposed by the FGBU "VGNKI" for identification of mutations associa ted with the most common inherited diseases in Holstein, Brown Swiss, Aberdeen Angus and Simmental cattle into practice as well as enhancing of farmers' awareness of the problem and of the possibilities of its solving could mini mize inherited disease spread and improve national cattle gene pool.