Sunday, December 9, 2012

The peptide binding site accounts for the MHC link to Rheumatoid Arthritis

That genes within the major histocompatibility complex (MHC, human HLA) influence susceptibility to rheumatoid arthritis (RA) has been known for over 40 years, even before HLA nomenclature was well established (e.g., Dick et al. 1975). However, the few “classical” HLA genes constitute only a small fraction of the hundreds genes within the MHC, which include the inflammatory cytokine tumor necrosis factor (TNF), a key player in RA. Which genes are responsible for the association?

The authors investigated 5,018 “cases” of RA, all with antibodies against cyclic citrullinated peptides, CCP (i.e, seropositive, accounting for 70% of RA patients [review]) and 14,974 health, ethnically matched controls.

First they tested their ability to “impute” HLA alleles from their SNP data using a reference panel of 2,767 individuals. Conclusion, not bad: 98% accuracy for “two digit” mapping and >80% for 4 digit (allele). Then they found the most significant nucleotide (p<10^-526!) is part of a codon for amino acid 11 of the HLA-DR beta 1, and thus not part of the “shared epitope” (a 5-amino acid sequence and antibody epitope linked to RA [review]). A valine at this position confers a 3.8-fold higher risk whereas a (polar) serine is protective (the converse of risk). Comparing cases and controls shows a clear difference (shown here, from fig 3). Adding amino acids at positions 71 and 74 improved significance slightly, and alleles with these amino acids were independently shown to confer risk.

The authors conclude “These results are consistent with a disease model in which classical HLA genes and proteins are the dominant factors in rheumatoid arthritis pathogenesis, with only a minor contribution from non-HLA loci in the MHC”. It seems that someone might also explore whether these variants bind CCP better!

Raychaudhuri S, Sandor C, Stahl EA, Freudenberg J, Lee HS, Jia X, Alfredsson L, Padyukov L, Klareskog L, Worthington J, Siminovitch KA, Bae SC, Plenge RM, Gregersen PK, de Bakker PI. “Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis.” Nat Genet. 2012 Jan 29;44(3):291-6

Sunday, October 7, 2012

ENCODE salvages “junk” DNA

The “ENCyclopedia Of DNA Elements”, ENCODE, founded in 2003 with grants from the NIH Genome Institute, seeks to identify all the functional parts of the human genome, assessed by DNA and histone modifications, chromatin looping, transcription factor binding, chromatin compaction (DNAse accessibility), and transcripts.  The collaboration of ~37 groups, first developed technology. Recently they published their first salvo of 30 research papers, several published in Nature along with a News & Views.

The paper by Djebali and scores of colleagues offers “a genome-wide catalogue of human transcripts”, together with their location (nucleus or cytoplasm), and whether they have a 7mG cap 5’ or a poly-A tail 3’.  They prepared RNA from 15 human cells lines after fractionation (whole cell, nucleus and cytosol) and separation of RNA into short and long (>200 nucleotides).  Long RNAs were further separated into +/- polyA tails.  They sequenced these RNAs and determined their initiation sites and their 5’ and 3’ termini (using technologies felicitously named CAGE and PET).  Then they did bioinformatics: compared to annotated genome (GENCODE) statistics, etc.,  All these data are available for your perusal using the RNA Dashboard
They made many interesting observations; e.g., they conclude there is very little “junk” DNA.  Nearly 75% of the genome is transcribed in at least one of the cell lines, though only a little over 50% in any given line.  (This is similar to previous findings, albeit not as “encyclopedic”).  Only 28% of the 7,053 small RNAs (including snRNAs, snoRNAs, miRNAs, and tRNAs) annotated by GENCODE are found in any of these cell lines, suggesting the expression of many annotated small RNAs is cell type specific.   
They also find that protein-coding transcripts are more abundant than long non-coding RNAs (lncRNAs) and that the same genes are transcribed in different cells.  Figure 3, shown here, plots the number of transcripts (r.p.k.m., reads per kilobase per million reads) on the x axis vs. the ratio of nuclear/cytoplasmic for protein-coding (orange), which are abundant (right) in the cytoplasm (down),  non-coding (blue), and novel intergenic (green), which tend to be expressed at lower levels (left) and mostly nuclear (up).  A few individual transcripts are also identified, giving appreciation for the range of expression.
Also not for the first time, they suggest that shrinking “intergenic” regions  “prompts the reconsideration of the definition of a gene”.   They “propose that the transcript be considered as the basic atomic unit of inheritance” and that “gene … denote … all those transcripts …. that contribute to a given phenotypic trait".  Mendel would approve. 
PubMed. Djebali et al. "Landscape of transcription in human cells." 2012 Nature Sep 6;489(7414):101-8.

Saturday, March 31, 2012

Monkey’s Uncle? Your HLA might be Neanderthal

Humans’ genomes are extremely similar to those of other primates because the species diverged relatively recently, approximately 6 million years ago in the case of our nearest cousins, Chimpanzees. Modern humans and Denisovans separated 250,000 years ago (10,000 generations). With the recent sequencing of extinct, ancient hominids, such as Neanderthals and their Denisovan relatives, it was realized that up to 6% of the genomes of humans now in Europe and Asia derive from these older lineages.

HLA genes are by far the most polymorphic within the human genome, with thousands of variants (alleles). Here, investigators first identified one particular HLA allele, HLA-B*73:01, as being more similar to homologous Chimpanzee alleles than other human HLA-B alleles. This allele diverged from other HLA-B alleles 16 million years ago, before the separation of humans and Chimps, and was lost from the majority of modern humans. Its reappearance in the human genome was most likely, they reckoned, a result of “introgression”, introduction from ancient humans such as Neanderthal. An alternative model, which computer simulations indicate is 100 times less probable, is that this allele came out of Africa late

They also simply “typed” (sequenced and matched) the most important HLA loci, HLA-A, -B and –C from 1 Denisovan and 2 Neanderthal subjects. Surprisingly, most of these archaic HLA alleles were identical to common HLA types of modern humans. HLA-A2, the most widespread allele at the HLA-A locus, was shared with and might have been acquired from Denisovans. Putative archaic HLA-A alleles are now more common in China and Europe than in Africa (Figure, from fig. 4d). The authors conclude that although a small minority of our genomes overall derived from archaic humans, about half of our HLA was acquired through interbreeding between modern humans migrating out of Africa and locally established archaic humans. These archaic alleles conferred fitness in the new environment, e.g., pathogen and allergen resistance, and so outcompeted and displaced previous human HLA alleles.

The shaping of modern human immune systems by multiregional admixture with archaic humans. Abi-Rached et al. Science. 2011 Oct 7;334(6052):89-94.

Sunday, February 26, 2012

Gut Feeling – intestinal microbes influence immune system tolerance of central nervous system

Multiple Sclerosis (MS: Wikipedia, PubMedHealth) is an autoimmune disease wherein lymphocytes attack the central nervous system (CNS), including the brain and spinal cord, leading to relapsing, progressive loss of neurons. Lesions containing B and T lymphocytes develop in the CNS. The cause of MS is unknown.

A mouse model of MS, called experimental autoimmune encephalomyelitis (EAE), can be induced when mice of certain strains are immunized with spinal cord proteins, or it can occur spontaneously in genetically engineered strains in which many CD4+ “helper” T cells express a transgenic T cell receptor specific for myelin oligodendrocyte glycoprotein (MOG), a protein abundant on the surface of key non-neuronal cells of the CNS.

These authors observed that depending on the animal housing facility, between 35-90% of MOG-specific-TCR-transgenic mice spontaneously develop EAE at between 3-8 months of age. The wide range in the disease incidence reminded the authors of a 1993 report by Goverman that mice with T cells expressing transgenic antigen receptors specific for another nerve protein, MBP, developed EAE ‘spontaneously’ in non-sterile housing but not in sterile housing.

They compared EAE incidence in mice that possess normal gut microbes but harbor no known pathogens, termed Specific Pathogen Free (SPF), and mice that possess no microbes at all, termed “germ free” (GF), and found that GF mice were protected (Fig 1a, shown, left panel).

Gut microbes are known to contribute to lymphocyte maturation, stimulated by , e.g., segmented filamentous bacteria) or polysaccharides of Bacteroides fragilis. However, the authors argue this does not explain protection because GF mice colonized with “conventional commensal” microbes developed EAE “promptly”, starting about a month later (Fig 1b, shown, right panel). They add that colonization with segmented filamentous bacteria – shown to trigger autoimmunity in another model – conferred EAE susceptibility only inefficiently. They also argue that GF mice immunized with MOG in complete adjuvant develop EAE (though again with a delay of about a month) and produce specific antibodies (though measured crudely, not titered), demonstrating that their lymphocytes are mature.

Instead, the authors argue that some lymphocyte activities are reduced in GF mice, particularly T cell production of the pro-inflammatory interleukin-17 and spontaneous B cell production of MOG-specific antibodies (which is also “promptly” albeit only partially corrected by colonization, Fig 3a). Moreover, MOG-specific B cells – but not polyclonal normal B cells – transferred into MOG-specific-TCR-transgenic mice – but not MOG-deficient mice – home to germinal centers where they mature and make antibodies that are IgG2a class-switched, and therefore implicitly effective in cooperating with specific T cells to induce EAE. They conclude that commensal gut microbes activate autoreactive T cells that recruit autoreactive B cells, which together mediate disease.

Berer K, Mues M, Koutrolos M, Rasbi ZA, Boziki M, Johner C, Wekerle H, Krishnamoorthy G. Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination. Nature. 2011 Oct 26;479(7374):538-41. doi: 10.1038/nature10554. PubMed PMID: 22031325

Sunday, January 22, 2012

Would you like some E. coli with that?

An epidemic of bloody stools and failing kidneys, some with hemolytic uremic syndrome (HUS) appeared in Germany in May 2011 and subsequently 15 other countries. By late July when the epidemic had subsided, a total of 3,816 cases - including 54 deaths - were reported in Germany, 845 of which included HUS. Rasko and colleagues cultured E. coli bacteria isolated from a 64 year old woman from Hamburg, Germany, who did not develop HUS. They characterized this bacterium, designated C227-11, as enteroaggregative, which means a gut pathogen that aggregates and forms “biofilms” that are resistant to treatment.

They sequenced the bacterium’s genome and found it was a unique strain of the O104:H4 serotype of E. coli bacteria, distinguished by possession of a prophage ( ) producing the Shiga toxin. Shiga toxin binds to cells, inhibits protein synthesis, and kills by inducing apoptosis [review]. The O104 serotype is rare; the most frequent cause of HUS worldwide is the shiga-toxin–producing E. coli O157 (Tarr 2005).
Although they isolate only one strain themselves, they analyzed also 3 additional sequences from the current outbreak that had been made public (that’s data mine-ing!) together with 7 other O104:H4 serotype isolates, all from Africa, and 4 other reference strains. The authors conclude that the outbreak was caused by a difficult (enteroaggregative) strain made more virulent by its acquisition of the Shiga toxin gene in addition to antibiotic-resistance and “additional virulence and antibiotic-resistance factors”. Rohde and colleagues reached the same conclusion using "rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses".

Where did the E. coli O104:H4 come from? A subsequent publication reported the results of trace-back and –forward investigations by Buchholz and colleagues who analyzed 26 HUS patients and 81 healthy controls. They concluded that despite only about a quarter of the patients recalling in exploratory interviews having eaten bean sprouts during the 14 days before the onset of illness, 100% of these illnesses were attributable to the consumption of sprouts – and not other raw foods such as tomatoes or cucumbers or lettuce – at a particular restaurant, and for other patients, sprouts obtained from a single, common supplier (figure).

N Engl J Med. 2011 Aug 25;365(8):709-17. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS, Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank D, Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP, Schadt EE, Waldor MK.