Farbod Babrzadeh - Academia.edu (original) (raw)

Papers by Farbod Babrzadeh

To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predomi... more To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. Methods: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. Results: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. Conclusions: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.

Published Ahead of Print 31 October 2012. 10.1128/AAC.01601-12. 2013, 57(1):343. DOI: Antimicrob.... more Published Ahead of Print 31 October 2012. 10.1128/AAC.01601-12. 2013, 57(1):343. DOI: Antimicrob. Agents Chemother. Borroto-Esoda and Robert W. Shafer Pacold, Elizabeth C. Reuman, Susan P. Holmes, Katyna Farbod Babrzadeh, Ross Martin, Tommy F. Liu, Mary Severine Margeridon-Thermet, Evguenia S. Svarovskaia, Treatment Subjects with a Past History of Lamivudine Mutations in Hepatitis B Virus-Infected Low-Level Persistence of Drug Resistance

Journal of Immunology, Apr 1, 2010

Journal of Immunology, Apr 1, 2010

See next page for additional authors

Abstract: Problem statement: Human skin is a large ( ~ 1.75 m2) organ containing a large number o... more Abstract: Problem statement: Human skin is a large ( ~ 1.75 m2) organ containing a large number of ecologically distinct sites. These sites harbor a variety of distinct microbiomes. One challenge is to define the microbiome at every site. We chose two interesting sites: the base of the Front of the neck and the base of the Back of the neck (the nape) and enrolled forty volunteers. These two sites are interesting because the bacteria therein must interact with the skin and its microbiome and with clothing and hair and the external environment. Approach: The volunteers took their own neck swabs. Total DNA was prepared from each swab. That DNA was employed as a template in separate PCR reactions to amplify the V6 and V3 regions of the 16S ribosomal RNA gene. The V6 and V3 regions were pyrosequenced using Roche 454 Life Sciences technology. To identify the bacteria, the sequences were compared to the data in the Ribosomal Database Project. Results: From the sequences of the V6 region, i...

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MH... more Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appea...

See next page for additional authors

Applied Physics Letters, 2010

In this paper we present a scalable method based on the use of microfluidics and shear force spec... more In this paper we present a scalable method based on the use of microfluidics and shear force spectroscopy which can be used for determining the affinity between molecules. Our method involves the use of functionalization of the surface of microfluidic channels with ligand molecules, and the surface of microspheres with receptor molecules. Bound beads are detached from the surface of the microchannels using pressure driven flow. The drag force required to detach the beads is used to determine the affinity of the bond holding the two molecules together. The minimum force we are able to detect is 5 pN. We have used this method to determine the binding force between protein-protein interactions and DNA base-pair interactions. We also have shown the ability of this technique to distinguish between strong and weak protein-protein interactions. Using this approach, it may be possible to multiplex an array of these functionalized channels onto a chip and probe the interactions between large...

Proximal spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. I... more Proximal spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. It is characterised by degeneration of the anterior horn cells of the spinal cord, resulting in symmetrical limb muscle

Annals of the Academy of Medicine, Singapore

Introduction: Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessiv... more Introduction: Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran. Materials and Methods: Molecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR. Results: The homozygous-deletion freque...

Journal of Cancer Metastasis and Treatment, 2018

New insights into the role of intra-tumor genetic heterogeneity in carcinogenesis: identification... more New insights into the role of intra-tumor genetic heterogeneity in carcinogenesis: identification of complex single gene variance within tumors.

Human Immunology, 2019

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A bet... more Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5'UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.

Human immunology, Jan 18, 2018

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an... more The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01,...

The Journal of Immunology, 2017

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 a... more The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is ...

Human Immunology, 2016

Recent studies showed the existence of biologically relevant DPB1 expression variants that associ... more Recent studies showed the existence of biologically relevant DPB1 expression variants that associate with two 3′UTR SNPs of DPB1. The knowledge of sequence variation in DPA1 and DPB1 alleles is limited to a few exons. Only a few alleles are fully sequenced resulting in limitations for DP typing by NGS. We developed robust and efficient methods for long range amplification and molecular cloning that allowed us to examine and characterize numerous alleles. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated span exon 1–4 and 2–4 of DPA1 and DPB1. We characterized 16 DPA1 and 40 DPB1 alleles defined but their unique sequences in 63 samples. We obtained novel sequence information in 11 DPA1 alleles including 7 extended sequences, 3 novel intron variants and 1 novel exon variant. In DPB1 we extended the coverage in 20 allleles and identified 12 novel intron variants and 2 novel exon variants. In addition to the well known variations in exons, the long DPA1 intron1 (3556–3590 nt) showed 201 SNPs and 17 indels. DPA1 intron 2 (340 nt) and intron 3 (214 nt) are short, not variable in size with low polymorphism (12 and 9 SNP, respectively). In DPB1 intron 3 is conserved in length (547 nt) and presents only 17 SNPs. Intron 2 of DPB1 (3952–4025 nt) has the largest variability with 176 SNPs and 9 indels; in addition this intron has one variable STR region located at 44 nt before the beginning of exon 3 with 4–14 repeats of the tetramer AAGG. Two groups of alleles that have only 4 repeats (short STR) and 8–14 repeats (long STR) were identified. Also, phylogenetic trees define two distinct groups at each, DPA1 and DPB1 loci. The DPB1 clusters are tighter when exon 2 is excluded from the analysis and correlate well with the intron 2 STR short and long variants. We observed that the 3′ UTR variants of DPB1 that correlate with high and low expression, also associate strictly in a biunivocal fashion with the short and long intron 2 STR variants, respectively. Our studies provide useful information for mapping reads in NGS typing. In addition our findings identify potential regulatory elements that may affect gene expression possibly by altering the rate of mRNA splicing. In transplantation, the expression levels of the mismatched allele appear to determine both immunogenicity and outcome.

Human Immunology, 2016

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. More ... more Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. More complete sequence assessment can be achieved by the description of the entire common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of only one allele available. The haplotypes bearing DRB5 genes associate with susceptibility and resistance to many diseases; furthermore, the role of DRB5 mismatches in transplantation has not been utterly evaluated. In the present study, we cloned and sequenced DRB genes from 87 individuals including 18 subjects with DRB5 alleles. We developed robust and efficient methods for long range PCR and molecular cloning. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated overlapped and span exons 1–2 and 2–6 of DRB genes. We obtained DRB5 sequences spanning 12,638-12,681 of DRB5 ∗ 01:01, 01:02 and 02:01; partial information for the segment exon 2–6 was obtained for DRB5 ∗ 01:03, 01:08N and 02:03. Phylogenetic trees show that DRB5 alleles, group together and have distinctive differences with other DRB loci, with only 65, 73 and 83 percent homology with DRB1 ∗ 15, DRB4 and DRB3 alleles respectively. All DRB5 alleles are similar with at least 97.0 percent homology. Among 7 subjects we identified 4 intron variants of DRB5 ∗ 01:01, 3 differing in length in one intron2 STR and additional allele differing from the others by 1 SNP substitution. We identified 2 intron variants of DRB5 ∗ 01:02; with exception of the known exon substitutions one DRB5 ∗ 01:02 variant is identical to DRB5 ∗ 01:03 and DRB ∗ 01:08N differs in introns from DRB5 ∗ 01:02 by length in one STR. Similarly, we identified two DRB5 ∗ 02:02 variants differing in length at the same STR while DRB5 ∗ 02:03 is identical in the intron sequences to one of these alleles. The DRB5 intron variants of each DRB5 allele were found in subject carrying distinct associations with alleles of DRB1, B and/or ethnicity. Our studies provide useful information that can be applied in NGS based typing. Unexpectedly, we identified intron variation that may shed light on understanding the evolution of the DRB region. Examination of DRB5 variants may also lead to identify the disease susceptibility factors of DRB5 containing haplotypes.

American Journal of Microbiology, 2012

Human skin comprises a large number of distinguishable ecological niches. To describe fully the h... more Human skin comprises a large number of distinguishable ecological niches. To describe fully the human skin microbiome, it will be necessary to identify the bacteria in each niche and to distinguish the commensal bacteria from the temporary residents. To contribute to the description of the human skin microbiome and employing a gene-based technology, we have identified the bacteria in two niches: the front and back of the base of the neck and over the course of one year. There were 50 volunteers and a total of 232 neck skin swabs. Roche 454 Tag pyrosequencing was employed to sequence a short hypervariable sequence region (V6) of the 16S ribosomal RNA gene. To identify the bacteria corresponding to the front and back of the neck for each volunteer, the "Classifier" software in the "Pyrosequencing" section of the Ribosomal Database Project was employed. The bacteria on virtually all 232 neck skin swabs were classified into bacterial Class. The skin microbiome of these two niches was composed principally of a mixture of five Classes of bacteria: Actinobacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria and Gammaproteobacteria. The fraction of each Class could change over time. We could not distinguish the skin microbiome from the front of the base of the neck from the back of the base of the neck. At these two positions, we could not distinguish the male from the female skin microbiome. The principal variable was the time point. We concluded that the skin microbiome at the front and back of the base of the neck was composed principally of a mixture of five Classes of bacteria. The proportion of each Class could change over time.

To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predomi... more To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. Methods: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. Results: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. Conclusions: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.

Published Ahead of Print 31 October 2012. 10.1128/AAC.01601-12. 2013, 57(1):343. DOI: Antimicrob.... more Published Ahead of Print 31 October 2012. 10.1128/AAC.01601-12. 2013, 57(1):343. DOI: Antimicrob. Agents Chemother. Borroto-Esoda and Robert W. Shafer Pacold, Elizabeth C. Reuman, Susan P. Holmes, Katyna Farbod Babrzadeh, Ross Martin, Tommy F. Liu, Mary Severine Margeridon-Thermet, Evguenia S. Svarovskaia, Treatment Subjects with a Past History of Lamivudine Mutations in Hepatitis B Virus-Infected Low-Level Persistence of Drug Resistance

Journal of Immunology, Apr 1, 2010

Journal of Immunology, Apr 1, 2010

See next page for additional authors

Abstract: Problem statement: Human skin is a large ( ~ 1.75 m2) organ containing a large number o... more Abstract: Problem statement: Human skin is a large ( ~ 1.75 m2) organ containing a large number of ecologically distinct sites. These sites harbor a variety of distinct microbiomes. One challenge is to define the microbiome at every site. We chose two interesting sites: the base of the Front of the neck and the base of the Back of the neck (the nape) and enrolled forty volunteers. These two sites are interesting because the bacteria therein must interact with the skin and its microbiome and with clothing and hair and the external environment. Approach: The volunteers took their own neck swabs. Total DNA was prepared from each swab. That DNA was employed as a template in separate PCR reactions to amplify the V6 and V3 regions of the 16S ribosomal RNA gene. The V6 and V3 regions were pyrosequenced using Roche 454 Life Sciences technology. To identify the bacteria, the sequences were compared to the data in the Ribosomal Database Project. Results: From the sequences of the V6 region, i...

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MH... more Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appea...

See next page for additional authors

Applied Physics Letters, 2010

In this paper we present a scalable method based on the use of microfluidics and shear force spec... more In this paper we present a scalable method based on the use of microfluidics and shear force spectroscopy which can be used for determining the affinity between molecules. Our method involves the use of functionalization of the surface of microfluidic channels with ligand molecules, and the surface of microspheres with receptor molecules. Bound beads are detached from the surface of the microchannels using pressure driven flow. The drag force required to detach the beads is used to determine the affinity of the bond holding the two molecules together. The minimum force we are able to detect is 5 pN. We have used this method to determine the binding force between protein-protein interactions and DNA base-pair interactions. We also have shown the ability of this technique to distinguish between strong and weak protein-protein interactions. Using this approach, it may be possible to multiplex an array of these functionalized channels onto a chip and probe the interactions between large...

Proximal spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. I... more Proximal spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. It is characterised by degeneration of the anterior horn cells of the spinal cord, resulting in symmetrical limb muscle

Annals of the Academy of Medicine, Singapore

Introduction: Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessiv... more Introduction: Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran. Materials and Methods: Molecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR. Results: The homozygous-deletion freque...

Journal of Cancer Metastasis and Treatment, 2018

New insights into the role of intra-tumor genetic heterogeneity in carcinogenesis: identification... more New insights into the role of intra-tumor genetic heterogeneity in carcinogenesis: identification of complex single gene variance within tumors.

Human Immunology, 2019

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A bet... more Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5'UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.

Human immunology, Jan 18, 2018

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an... more The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01,...

The Journal of Immunology, 2017

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 a... more The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is ...

Human Immunology, 2016

Recent studies showed the existence of biologically relevant DPB1 expression variants that associ... more Recent studies showed the existence of biologically relevant DPB1 expression variants that associate with two 3′UTR SNPs of DPB1. The knowledge of sequence variation in DPA1 and DPB1 alleles is limited to a few exons. Only a few alleles are fully sequenced resulting in limitations for DP typing by NGS. We developed robust and efficient methods for long range amplification and molecular cloning that allowed us to examine and characterize numerous alleles. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated span exon 1–4 and 2–4 of DPA1 and DPB1. We characterized 16 DPA1 and 40 DPB1 alleles defined but their unique sequences in 63 samples. We obtained novel sequence information in 11 DPA1 alleles including 7 extended sequences, 3 novel intron variants and 1 novel exon variant. In DPB1 we extended the coverage in 20 allleles and identified 12 novel intron variants and 2 novel exon variants. In addition to the well known variations in exons, the long DPA1 intron1 (3556–3590 nt) showed 201 SNPs and 17 indels. DPA1 intron 2 (340 nt) and intron 3 (214 nt) are short, not variable in size with low polymorphism (12 and 9 SNP, respectively). In DPB1 intron 3 is conserved in length (547 nt) and presents only 17 SNPs. Intron 2 of DPB1 (3952–4025 nt) has the largest variability with 176 SNPs and 9 indels; in addition this intron has one variable STR region located at 44 nt before the beginning of exon 3 with 4–14 repeats of the tetramer AAGG. Two groups of alleles that have only 4 repeats (short STR) and 8–14 repeats (long STR) were identified. Also, phylogenetic trees define two distinct groups at each, DPA1 and DPB1 loci. The DPB1 clusters are tighter when exon 2 is excluded from the analysis and correlate well with the intron 2 STR short and long variants. We observed that the 3′ UTR variants of DPB1 that correlate with high and low expression, also associate strictly in a biunivocal fashion with the short and long intron 2 STR variants, respectively. Our studies provide useful information for mapping reads in NGS typing. In addition our findings identify potential regulatory elements that may affect gene expression possibly by altering the rate of mRNA splicing. In transplantation, the expression levels of the mismatched allele appear to determine both immunogenicity and outcome.

Human Immunology, 2016

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. More ... more Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. More complete sequence assessment can be achieved by the description of the entire common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of only one allele available. The haplotypes bearing DRB5 genes associate with susceptibility and resistance to many diseases; furthermore, the role of DRB5 mismatches in transplantation has not been utterly evaluated. In the present study, we cloned and sequenced DRB genes from 87 individuals including 18 subjects with DRB5 alleles. We developed robust and efficient methods for long range PCR and molecular cloning. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated overlapped and span exons 1–2 and 2–6 of DRB genes. We obtained DRB5 sequences spanning 12,638-12,681 of DRB5 ∗ 01:01, 01:02 and 02:01; partial information for the segment exon 2–6 was obtained for DRB5 ∗ 01:03, 01:08N and 02:03. Phylogenetic trees show that DRB5 alleles, group together and have distinctive differences with other DRB loci, with only 65, 73 and 83 percent homology with DRB1 ∗ 15, DRB4 and DRB3 alleles respectively. All DRB5 alleles are similar with at least 97.0 percent homology. Among 7 subjects we identified 4 intron variants of DRB5 ∗ 01:01, 3 differing in length in one intron2 STR and additional allele differing from the others by 1 SNP substitution. We identified 2 intron variants of DRB5 ∗ 01:02; with exception of the known exon substitutions one DRB5 ∗ 01:02 variant is identical to DRB5 ∗ 01:03 and DRB ∗ 01:08N differs in introns from DRB5 ∗ 01:02 by length in one STR. Similarly, we identified two DRB5 ∗ 02:02 variants differing in length at the same STR while DRB5 ∗ 02:03 is identical in the intron sequences to one of these alleles. The DRB5 intron variants of each DRB5 allele were found in subject carrying distinct associations with alleles of DRB1, B and/or ethnicity. Our studies provide useful information that can be applied in NGS based typing. Unexpectedly, we identified intron variation that may shed light on understanding the evolution of the DRB region. Examination of DRB5 variants may also lead to identify the disease susceptibility factors of DRB5 containing haplotypes.

American Journal of Microbiology, 2012

Human skin comprises a large number of distinguishable ecological niches. To describe fully the h... more Human skin comprises a large number of distinguishable ecological niches. To describe fully the human skin microbiome, it will be necessary to identify the bacteria in each niche and to distinguish the commensal bacteria from the temporary residents. To contribute to the description of the human skin microbiome and employing a gene-based technology, we have identified the bacteria in two niches: the front and back of the base of the neck and over the course of one year. There were 50 volunteers and a total of 232 neck skin swabs. Roche 454 Tag pyrosequencing was employed to sequence a short hypervariable sequence region (V6) of the 16S ribosomal RNA gene. To identify the bacteria corresponding to the front and back of the neck for each volunteer, the "Classifier" software in the "Pyrosequencing" section of the Ribosomal Database Project was employed. The bacteria on virtually all 232 neck skin swabs were classified into bacterial Class. The skin microbiome of these two niches was composed principally of a mixture of five Classes of bacteria: Actinobacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria and Gammaproteobacteria. The fraction of each Class could change over time. We could not distinguish the skin microbiome from the front of the base of the neck from the back of the base of the neck. At these two positions, we could not distinguish the male from the female skin microbiome. The principal variable was the time point. We concluded that the skin microbiome at the front and back of the base of the neck was composed principally of a mixture of five Classes of bacteria. The proportion of each Class could change over time.