Genomic evolution and polymorphism: segmental duplications and haplotypes at 108 regions on 21 chromosomes.

We describe here extensive, previously unknown, genomic polymorphism in 120 regions, covering 19 autosomes and both sex chromosomes. Each contains duplication within multigene clusters. Of these, 108 are extremely polymorphic with multiple haplotypes. We used the genomic matching technique (GMT), previously used to characterise the major histocompatibility complex (MHC) and regulators of complement activation (RCA). This genome-wide extension of this technique enables the examination of many underlying cis, trans and epistatic interactions responsible for phenotypic differences especially in relation to individuality, evolution and disease susceptibility. The extent of the diversity could not have been predicted and suggests a new model of primate evolution based on conservation of polymorphism rather than de novo mutation.

Genomic evolution in domestic cattle: ancestral haplotypes and healthy beef.

We have identified numerous Ancestral Haplotypes encoding a 14-Mb region of Bota C19. Three are frequent in Simmental, Angus and Wagyu and have been conserved since common progenitor populations. Others are more relevant to the differences between these 3 breeds including fat content and distribution in muscle. SREBF1 and Growth Hormone, which have been implicated in the production of healthy beef, are included within these haplotypes. However, we conclude that alleles at these 2 loci are less important than other sequences within the haplotypes. Identification of breeds and hybrids is improved by using haplotypes rather than individual alleles.

Almost total protection from age-related macular degeneration by haplotypes of the Regulators of Complement Activation.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It has been proposed that the polymorphism encoding Y402H (T1277C) in the complement factor H gene (CFH) is one of the main determinants of disease. We genotyped the polymorphism at a number of loci in the region encompassing the Regulators of Complement Activation (RCA) on chromosome 1, including T1277C SNP, in 187 patients and 146 controls. Haplotypes have been classified as protective (P) or susceptible (S) with respect to AMD. This included the identification of an S haplotype with a T at 1277. The results show that no single locus should be assumed to be directly responsible for AMD, but rather argue for the existence of RCA haplotypes, which can be assigned meaningful predictive values for AMD. We conclude that the critical sequences are within a region 450 kb centromeric to 128 kb telomeric of CFH.

MHC Genomics and Disease: Looking Back to Go Forward.

Ancestral haplotypes are conserved but extremely polymorphic kilobase sequences, which have been faithfully inherited over at least hundreds of generations in spite of migration and admixture. They carry susceptibility and resistance to diverse diseases, including deficiencies of CYP21 hydroxylase (47.1) and complement components (18.1), as well as numerous autoimmune diseases (8.1). The haplotypes are detected by segregation within ethnic groups rather than by SNPs and GWAS. Susceptibility to some other diseases is carried by specific alleles shared by multiple ancestral haplotypes, e.g., ankylosing spondylitis and narcolepsy. The difference between these two types of association may explain the disappointment with many GWAS. Here we propose a pathway for combining the two different approaches. SNP typing is most useful after the conserved ancestral haplotypes have been defined by other methods.

Novel sequence elements define ancestral haplotypes of the region encompassing complement factor H.

The genomic region encompassing complement factor H (CFH) is thought to be important in determining susceptibility to inflammatory diseases such as age-related macular degeneration, but only limited polymorphism has been described. After applying the genomic matching technique to three-generation families and an ethnically diverse reference panel we have demonstrated that the polymorphism resembles that found in the major histocompatibility complex. The different ancestral haplotypes carry either T or C at T1277C but also other more polymorphic alleles over a region of 2 Mb. Thus the association between age-related macular degeneration and T1277 or Y402 actually reflects multiple linked polymorphisms including an indel that cannot be dissected from any direct effect of Y402 and may be more important. We show for the first time that simple algorithms can identify genomic sequence elements that appear to be more useful haplospecific markers than single nucleotide polymorphism or microsatellites.

Genetics of Marbling in Wagyu Revealed by the Melting Temperature of Intramuscular and Subcutaneous Lipids.

Extreme marbling or intramuscular deposition of lipid is associated with Wagyu breeds and is therefore assumed to be largely inherited. However, even within 100% full blood Wagyu prepared under standard conditions, there is unpredictable scatter of the degree of marbling. Here, we evaluate melting temperature (Tm ) of intramuscular fat as an alternative to visual scores of marbling. We show that "long fed" Wagyu generally has Tm below body temperature but with a considerable range under standardized conditions. Individual sires have a major impact indicating that the variation is genetic rather than environmental or random error. In order to measure differences of lower marbling breeds and at shorter feeding periods, we have compared Tm in subcutaneous fat samples from over the striploin. Supplementary feeding for 100 to 150 days leads to a rapid decrease in Tm of 50% Red Wagyu (Akaushi) : 50% European crosses, when compared to 100% European. This improvement indicates that the genetic effect of Wagyu is useful, predictable, and highly penetrant. Contemporaneous DNA extraction does not affect the measurement of Tm . Thus, provenance can be traced and substitution can be eliminated in a simple and cost-effective manner.

Haplotypes for Type, Degree, and Rate of Marbling in Cattle Are Syntenic with Human Muscular Dystrophy.

Traditional analyses of a QTL on Bota 19 implicate a surfeit of candidates, but each is of marginal significance in explaining the deposition of healthy, low melting temperature fat within marbled muscle of Wagyu cattle. As an alternative approach, we have used genomic, multigenerational segregation to identify 14 conserved, ancestral 20 Mb haplotypes. These determine the degree and rate of marbling in Wagyu and other breeds of cattle. The melting temperature of intramuscular fat is highly heritable and traceable by haplotyping. Fortunately, for the production of healthy beef, some of these haplotypes are sufficiently penetrant to be expressed in heterozygous crossbreds, thereby allowing selection of sires which will improve the healthiness of beef produced under even harsh climatic conditions. The region of Bota 19 is syntenic to a region of Hosa 17 known to be important in muscle metabolism and in determining susceptibility to a form of human muscular dystrophy.

In polymorphic genomic regions indels cluster with nucleotide polymorphism: Quantum Genomics.

Previously, we have described polymorphic frozen blocks (PFBs) within the Major Histocompatibility Complex (MHC) as regions of several hundred kilobases characterised by high nucleotide diversity, little or no recombination, duplicated segments, disease susceptibility, and human endogenous retroviruses. The nucleotide diversity profile within these PFBs shows peaks and troughs outside of the Class I genes, reflecting other important genes (or sequences) in the region. Here we show that indel density is also clustered with similar peaks and troughs. In fact, SNPs and indels are co-located within PFBs.

Genesis of ancestral haplotypes: RNA modifications and reverse transcription-mediated polymorphisms.

Understanding the genesis of the block haplotype structure of the genome is a major challenge. With the completion of the sequencing of the Human Genome and the initiation of the HapMap project the concept that the chromosomes of the mammalian genome are a mosaic, or patchwork, of conserved extended block haplotype sequences is now accepted by the mainstream genomics research community. Ancestral Haplotypes (AHs) can be viewed as a recombined string of smaller Polymorphic Frozen Blocks (PFBs). How have such variant extended DNA sequence tracts emerged in evolution? Here the relevant literature on the problem is reviewed from various fields of molecular and cell biology particularly molecular immunology and comparative and functional genomics. Based on our synthesis we then advance a testable molecular and cellular model. A critical part of the analysis concerns the origin of the strand biased mutation signatures in the transcribed regions of the human and higher primate genome, A-to-G versus T-to-C (ratio ∼ 1.5 fold) and C-to-T versus G-to-A (≥ 1.5 fold). A comparison and evaluation of the current state of the fields of immunoglobulin Somatic Hypermutation (SHM) and Transcription-Coupled DNA Repair focused on how mutations in newly synthesized RNA might be copied back to DNA thus accounting for some of the genome-wide strand biases (e.g., the A-to-G vs T-to-C component of the strand biased spectrum). We hypothesize that the genesis of PFBs and extended AHs occurs during mutagenic episodes in evolution (e.g., retroviral infections) and that many of the critical DNA sequence diversifying events occur first at the RNA level, e.g., recombination between RNA strings resulting in tandem and dispersed RNA duplications (retroduplications), RNA mutations via adenosine-to-inosine pre-mRNA editing events as well as error prone RNA synthesis. These are then copied back into DNA by a cellular reverse transcription process (also likely to be error-prone) that we have called "reverse transcription-mediated long DNA conversion." Finally we suggest that all these activities and others can be envisaged as being brought physically under the umbrella of special sites in the nucleus involved in transcription known as "transcription factories."

Mannose binding lectin (MBL) copy number polymorphism in Zebrafish (D. rerio) and identification of haplotypes resistant to L. anguillarum.

We describe a novel extension of the Genomic Matching Technique (GMT) that defines haplotypes of the mannose binding lectin (MBL) region in Zebrafish (D. rerio). Four ancestral haplotypes have been identified to date, with at least one of these demonstrating a significant increase in resistance to L. anguillarum. MBL activates the lectin pathway of the complement system and stimulates the development of the complement cascade and the Membrane Attack Complex. Polymorphisms in humans have been associated with increased susceptibility and severity to a number of pathogenic organisms. As teleosts have a relatively immature acquired immune system, polymorphisms within MBL and other innate defence genes are likely to be critical in defining their susceptibility/resistance to various pathogenic organisms. We report multiple copies of MBL-like genes in D. rerio, with up to three copies tightly linked within a cluster spanning approximately 15 kb on chromosome 2. Genomic analysis suggests that duplication, retroviral insertion and possibly gene mutation and/or deletion have been key factors in the evolution of this cluster. Molecular analysis has revealed extensive polymorphism, including at least five distinct amplicons and haplospecific gene copy number variation. This study demonstrates polymorphism within a critical component of the teleost innate immune system. The polymorphisms and the haplotypes encoding the unique variants are likely to be informative in defining susceptibility/resistance to infectious agents commonly encountered within aquatic environments. Future investigations will define other important haplotypes and transfer the knowledge to other finfish species, thereby enabling selection of broodstock for the aquaculture industry.

Extensive genomic and functional polymorphism of the complement control proteins.

Using combinations of genomic markers, we describe more than 20 distinct ancestral haplotypes (AH) of complement control proteins (CCPs), located within the regulators of complement activation (RCA) alpha block at 1q32. This extensive polymorphism, including functional sites, is important because CCPs are involved in the regulation of complement activation whilst also serving as self and viral receptors. To identify haplotypes, we used the genomic matching technique (GMT) based on the pragmatic observation that extreme nucleotide polymorphism is packaged with duplicated sequences as polymorphic frozen blocks (PFB). At each PFB, there are many alternative sequences (haplotypes) which are inherited faithfully from very remote ancestors. We have compared frequencies of RCA haplotypes and report differences in recurrent spontaneous abortion (RSA) and psoriasis vulgaris (PV).

Amino acid patterns within short consensus repeats define conserved duplicons shared by genes of the RCA complex.

Complement control proteins (CCPs) contain repeated protein domains, short consensus repeats (SCRs), which must be relevant to diverse functions such as complement activation, coagulation, viral binding, fetal implantation, and self-nonself recognition. Although SCRs share some discontinuous and imperfect motifs, there are many variable positions and indels making classification in subfamilies extremely difficult. Using domain-by-domain phylogenetic analysis, we have found that most domains can be classified into only 11 subfamilies, designated a, b, c, d, e, f, g, h, i, j, or k and identified by critical residues. Each particular CCP is characterized by the order of representatives of the subfamilies. Human complement receptor 1 (CR1) has ajefbkd repeated four times and followed by ch. The classification crosses CCPs and indicates that a particular CCP is a function of the mix of SCRs. The aje set is a feature of several CCPs including human CR1 and DAF and murine Crry and appears to be associated with the success or failure of implantation inter alia. This approach facilitates genomic analysis of available sequences and suggests a framework for the evolution of CCPs. Units of duplication range from single SCRs, to septamers such as efbkdaj, to extensive segments such as MCP-CR1L. Imperfections of duplication with subsequent deletion have contributed to diversification.