Marcelo J. Pando

Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis

Monozygotic or ‘identical’ twins have been widely studied to dissect the relative contributions of genetics and environment in human diseases. In multiple sclerosis (MS), an autoimmune demyelinating disease and common cause of neurodegeneration and disability in young adults, disease discordance in monozygotic twins has been interpreted to indicate environmental importance in its pathogenesis. However, genetic and epigenetic differences between monozygotic twins have been described, challenging the accepted experimental model in disambiguating the effects of nature and nurture. Here we report the genome sequences of one MS-discordant monozygotic twin pair, and messenger RNA transcriptome and epigenome sequences of CD4+ lymphocytes from three MS-discordant, monozygotic twin pairs. No reproducible differences were detected between co-twins among ∼3.6 million single nucleotide polymorphisms (SNPs) or ∼0.2 million insertion-deletion polymorphisms. Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and insertion-deletion genotypes, or the expression of ∼19,000 genes in CD4+ T cells. Only 2 to 176 differences in the methylation of ∼2 million CpG dinucleotides were detected between siblings of the three twin pairs, in contrast to ∼800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or between normal and cancerous tissues. In the first systematic effort to estimate sequence variation among monozygotic co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first, to our knowledge, female, twin and autoimmune disease individual genome sequences reported.

Expansion of a unique CD57+NKG2Chi natural killer cell subset during acute human cytomegalovirus infection

During human CMV infection, there is a preferential expansion of natural killer (NK) cells expressing the activating CD94–NKG2C receptor complex, implicating this receptor in the recognition of CMV-infected cells. We hypothesized that NK cells expanded in response to pathogens will be marked by expression of CD57, a carbohydrate antigen expressed on highly mature cells within the CD56dimCD16+ NK cell compartment. Here we demonstrate the preferential expansion of a unique subset of NK cells coexpressing the activating CD94–NKG2C receptor and CD57 in CMV+ donors. These CD57+NKG2Chi NK cells degranulated in response to stimulation through their NKG2C receptor. Furthermore, CD57+NKG2Chi NK cells preferentially lack expression of the inhibitory NKG2A receptor and the inhibitory KIR3DL1 receptor in individuals expressing its HLA-Bw4 ligand. Moreover, in solid-organ transplant recipients with active CMV infection, the percentage of CD57+NKG2Chi NK cells in the total NK cell population preferentially increased. During acute CMV infection, the NKG2C+ NK cells proliferated, became NKG2Chi, and finally acquired CD57. Thus, we propose that CD57 might provide a marker of “memory” NK cells that have been expanded in response to infection.

Different NK Cell Surface Phenotypes Defined by the DX9 Antibody Are Due to KIR3DL1 Gene Polymorphism

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.

The Protein Made from a Common Allele of KIR3DL1 (3DL1*004) Is Poorly Expressed at Cell Surfaces due to Substitution at Positions 86 in Ig Domain 0 and 182 in Ig Domain 1

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.

KIR3DL1 Polymorphisms That Affect NK Cell Inhibition by HLA-Bw4 Ligand

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the first two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an inhibitory response to HLA-Bw4 ligands.

A multi-site study using high-resolution HLA genotyping by next generation sequencing

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Co-evolution of Human Leukocyte Antigen (HLA) Class I Ligands with Killer-Cell Immunoglobulin-Like Receptors (KIR) in a Genetically Diverse Population of Sub-Saharan Africans

Interactions between HLA class I molecules and killer-cell immunoglobulin-like receptors (KIR) control natural killer cell (NK) functions in immunity and reproduction. Encoded by genes on different chromosomes, these polymorphic ligands and receptors correlate highly with disease resistance and susceptibility. Although studied at low-resolution in many populations, high-resolution analysis of combinatorial diversity of HLA class I and KIR is limited to Asian and Amerindian populations with low genetic diversity. At the other end of the spectrum is the West African population investigated here: we studied 235 individuals, including 104 mother-child pairs, from the Ga-Adangbe of Ghana. This population has a rich diversity of 175 KIR variants forming 208 KIR haplotypes, and 81 HLA-A, -B and -C variants forming 190 HLA class I haplotypes. Each individual we studied has a unique compound genotype of HLA class I and KIR, forming 1–14 functional ligand-receptor interactions. Maintaining this exceptionally high polymorphism is balancing selection. The centromeric region of the KIR locus, encoding HLA-C receptors, is highly diverse whereas the telomeric region encoding Bw4-specific KIR3DL1, lacks diversity in Africans. Present in the Ga-Adangbe are high frequencies of Bw4-bearing HLA-B*53:01 and Bw4-lacking HLA-B*35:01, which otherwise are identical. Balancing selection at key residues maintains numerous HLA-B allotypes having and lacking Bw4, and also those of stronger and weaker interaction with LILRB1, a KIR-related receptor. Correspondingly, there is a balance at key residues of KIR3DL1 that modulate its level of cell-surface expression. Thus, capacity to interact with NK cells synergizes with peptide binding diversity to drive HLA-B allele frequency distribution. These features of KIR and HLA are consistent with ongoing co-evolution and selection imposed by a pathogen endemic to West Africa. Because of the prevalence of malaria in the Ga-Adangbe and previous associations of cerebral malaria with HLA-B*53:01 and KIR, Plasmodium falciparum is a candidate pathogen.

Dimorphic motifs in D0 and D1+D2 domains of killer cell Ig-like receptor 3DL1 combine to form receptors with high, moderate, and no avidity for the complex of a peptide derived from HIV and HLA-A*2402.

Comparison of mutant killer cell Ig-like receptor (KIR) 3DL1*015 substituted at natural positions of variation showed that tryptophan/leucine dimorphism at position 283 uniquely changes receptor conformation and can strongly influence binding of the A24nef tetramer. Dimorphic motifs at positions 2, 47, and 54 in D0 and 182 and 283 in D1+D2 distinguish the two 3DL1 lineages, typified by 3DL1*005 and 3DL1*015. The interlineage recombinant, KIR3DL1*001, combines D0 of 3DL1*005 with D1+D2 of 3DL1*015 and binds A24nef more strongly than either parent. In contrast, the reciprocal recombinant with D0 from 3DL1*015 and D1+D2 from 3DL1*005 cannot bind A24nef. Thus, D0 polymorphism directly affects the avidity of the KIR3DL1 ligand binding site. From these observations, multiple sequence alignment, and homology modeling, we constructed structural models for KIR3DL1 and its complex with A24nef. In these models, D0, D1, and D2 come together to form a binding surface for A24nef, which is contacted by all three Ig-like domains. A central pocket binds arginine 83, the only Bw4 motif residue essential for KIR3DL1 interaction, similar to the binding of lysine 80 in HLA-C by KIR2DL1. Central to this interaction is a salt bridge between arginine 83 of Bw4 and glutamate 282 of 3DL1, which juxtaposes the functionally influential dimorphism at position 283. Further 3DL1 mutants were tested and shown to have A24nef-binding properties consistent with the models. A24nef was not bound by KIR3DS1, the activating counterpart of KIR3DL1. Moreover, introducing any one of three residues specific to KIR3DS1, serine 163, arginine 166, or leucine 199, into 3DL1*015, abrogated A24nef binding.

Genes encoding human killer-cell Ig-like receptors with D1 and D2 extracellular domains all contain untranslated pseudoexons encoding a third Ig-like domain

Abstract. Human killer-cell immunoglobulin-like receptors (KIR) show three types of organization of their extracellular domains: D0-D1-D2 in KIR3D, D1-D2 in the majority of KIR2D, and D0-D2 in KIR2DL4 and the novel KIR2DL5. The gene for a KIR2DL3 variant, which has a D1-D2 structure, has been shown previously to have a nonexpressed region (pseudoexon 3) that is paralogous to the exon encoding the D0 domain of other KIR. This pseudoexon is not expressed because it is skipped during splicing of pre-mRNA. In this study, we demonstrate that all eight genes encoding human KIR with D1-D2 configuration (KIR2DL1-KIR2DL3, KIR2DS1-KIR2DS5) have similarly untranslated pseudoexons. Whereas the pseudoexons of four of these KIR genes bear non-sense mutations and/or altered splicing sites, the pseudoexons in the other four KIR genes have no major structural abnormalities, indicating that other mechanisms are responsible for inactivation of their exons 3. A comparison of the sequences on pseudoexons 3 with the paralogous expressed exons suggests that an exonic splicing enhancer may be necessary for the expression of exon 3 in KIR genes.

Genetic risk variants in African Americans with multiple sclerosis

Objectives: To assess the association of established multiple sclerosis (MS) risk variants in 3,254 African Americans (1,162 cases and 2,092 controls). Methods: Human leukocyte antigen (HLA)-DRB1, HLA-DQB1, and HLA-A alleles were typed by molecular techniques. Single nucleotide polymorphism (SNP) genotyping was conducted for 76 MS-associated SNPs and 52 ancestry informative marker SNPs selected throughout the genome. Self-declared ancestry was refined by principal component analysis of the ancestry informative marker SNPs. An ancestry-adjusted multivariate model was applied to assess genetic associations. Results: The following major histocompatibility complex risk alleles were replicated: HLA-DRB1*15:01 (odds ratio [OR] = 2.02 [95% confidence interval: 1.54–2.63], p = 2.50e-07), HLA-DRB1*03:01 (OR = 1.58 [1.29–1.94], p = 1.11e-05), as well as HLA-DRB1*04:05 (OR = 2.35 [1.26–4.37], p = 0.007) and the African-specific risk allele of HLA-DRB1*15:03 (OR = 1.26 [1.05–1.51], p = 0.012). The protective association of HLA-A*02:01 was confirmed (OR = 0.72 [0.55–0.93], p = 0.013). None of the HLA-DQB1 alleles were associated with MS. Using a significance threshold of p < 0.01, outside the major histocompatibility complex region, 8 MS SNPs were also found to be associated with MS in African Americans. Conclusion: MS genetic risk in African Americans only partially overlaps with that of Europeans and could explain the difference of MS prevalence between populations.