Association of the LILRA3 Deletion with B-NHL and Functional Characterization of the Immunostimulatory Molecule

LILRA3 is the sole soluble member of the LILR family. Previous studies from our group had shown that a 6.7 kb genetic deletion of LILRA3 is associated with MS and Sjögren’s syndrome. An impairment of the immune response leads to a predisposition for B-NHL, so we wanted to study whether the deletion of LILRA3 is also a risk factor for B-NHL, as well as the function of LILRA3. We discovered that the frequency of the homozygous LILRA3 deletion was significantly higher in B-NHL (6%) than in blood donors (3%) (P = 0.03). We detected binding of fluorochrome-conjugated recombinant LILRA3 to monocytes and B-cells. Incubation of PBMCs with recombinant LILRA3 induced proliferation of CD8⁺ T-cells and NK cells, as determined by CFSE staining. Using a transwell system, we demonstrated that LILRA3-stimulated lymphocyte proliferation was mediated by monocytes and required both cell contact and soluble factors. Secretion of IL-6, IL-8, IL-1β and IL-10 in the cell supernatant was stimulated by LILRA3. We conclude that LILRA3 is an immunostimulatory molecule, whose deficiency is associated with higher frequency of B-NHL.     

Long-term persistence of both functional and non-functional alleles at the leukocyte immunoglobulin-like receptor A3 (LILRA3) locus suggests balancing selection

The leukocyte immunoglobulin-like receptor (LILR) family consists of 13 loci, and a number of variations have been identified in these genes. Some polymorphisms of the LILR genes are reported to be associated with susceptibility to diseases such as rheumatoid arthritis and multiple sclerosis. LILRA3, one of the LILR genes, exhibits a presence or absence variation due to a 6.7-kb deletion in various populations. In this study, variation screening of the LILRA3 gene revealed high allele frequency of the 6.7-kb LILRA3 deletion (71%) in Japanese, in contrast to the frequency reported for the other populations. In addition, we identified a splice acceptor mutation in intron 1 with allele frequency of 19%, resulting in three alternatively spliced isoforms. Surprisingly, all of these isoforms were found to contain premature termination codons (PTCs) in the exon 3. Taken together, approximately 80% of Japanese lack functional LILRA3 alleles. The maximum likelihood coalescent analysis suggested that two major lineages, functional alleles and PTC-containing alleles, have been maintained for 2.75 million years in humans. These results prompted us to hypothesize that balancing selection had maintained both the functional and non-functional alleles at the LILRA3 locus. This hypothesis is consistent with the observation that the 6.7-kb LILRA3 deletion is detected worldwide in the presence of functional LILRA3 alleles.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB236941, AB236942, AB236943, AB236944, AB236945, AB236946, AB236947, AB236948, AB236949, AB236950.     

Influence of the LILRA3 Deletion on Multiple Sclerosis Risk: Original Data and Meta-Analysis

Background

Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system. Genome-wide association studies (GWAS) have identified over hundred polymorphisms with modest individual effects in MS susceptibility and they have confirmed the main individual effect of the Major Histocompatibility Complex. Additional risk loci with immunologically relevant genes were found significantly overrepresented. Nonetheless, it is accepted that most of the genetic architecture underlying susceptibility to the disease remains to be defined. Candidate association studies of the leukocyte immunoglobulin-like receptor LILRA3 gene in MS have been repeatedly reported with inconsistent results.

Objectives

In an attempt to shed some light on these controversial findings, a combined analysis was performed including the previously published datasets and three newly genotyped cohorts. Both wild-type and deleted LILRA3 alleles were discriminated in a single-tube PCR amplification and the resulting products were visualized by their different electrophoretic mobilities.

Results and Conclusion

Overall, this meta-analysis involved 3200 MS patients and 3069 matched healthy controls and it did not evidence significant association of the LILRA3 deletion [carriers of LILRA3 deletion: p = 0.25, OR (95% CI) = 1.07 (0.95–1.19)], even after stratification by gender and the HLA-DRB1*15:01 risk allele.     

DNA sequence variation and molecular genotyping of natural killer leukocyte immunoglobulin-like receptor,LILRA3

Leukocyte immunoglobulin-like receptors (LILRs) resemble killer cell immunoglobulin-like receptors (KIR) in structure and function and the KIR and LILR gene families form the major part of the leukocyte receptor cluster (LRC) of human chromosome 19q13.4. Unlike KIR, the LILR gene clusters do not vary in gene number. However, some individuals lack expression of LILRA3. This null allele has a 6.7-kb deletion, which encompasses the first six translated exons. This haplotype enabled unambiguous direct sequencing of LILRA3 alleles using genomic DNA from individuals heterozygous for the deletion. We have performed nucleotide sequencing of a 2.5-kb region within LILRA3 and identified eight bi-allelic substitutions, four of which were non-synonymous. Two from four previously identified LILRA3 cDNA sequences were confirmed and a further six alleles characterised, of which four will encode unique peptides. At least one of the polymorphic positions identified (encoding residue 84 of the first Ig domain) is likely to directly influence ligand binding. A PCR-SSP molecular genotyping system was developed and used to describe a panel of 172 Caucasoid individuals from South-East England. Six alleles were present in this group but they were unevenly distributed, as three alleles accounted for 88% of the studied chromosomes.     

LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.     

LILRA2 activation inhibits dendritic cell differentiation and antigen presentation to T cells

The differentiation of monocytes into dendritic cells (DC) is a key mechanism by which the innate immune system instructs the adaptive T cell response. In this study, we investigated whether leukocyte Ig-like receptor A2 (LILRA2) regulates DC differentiation by using leprosy as a model. LILRA2 protein expression was increased in the lesions of the progressive, lepromatous form vs the self-limited, tuberculoid form of leprosy. Double immunolabeling revealed LILRA2 expression on CD14+, CD68+ monocytes/macrophages. Activation of LILRA2 on peripheral blood monocytes impaired GM-CSF induced differentiation into immature DC, as evidenced by reduced expression of DC markers (MHC class II, CD1b, CD40, and CD206), but not macrophage markers (CD209 and CD14). Furthermore, LILRA2 activation abrogated Ag presentation to both CD1b- and MHC class II-restricted, Mycobacterium leprae-reactive T cells derived from leprosy patients, while cytokine profiles of LILRA2-activated monocytes demonstrated an increase in TNF-alpha, IL-6, IL-8, IL-12, and IL-10, but little effect on TGF-beta. Therefore, LILRA2 activation, by altering GM-CSF-induced monocyte differentiation into immature DC, provides a mechanism for down-regulating the ability of the innate immune system to activate the adaptive T cell response while promoting an inflammatory response.     

CTNS mutations in an American-based population of cystinosis patients.

Nephropathic cystinosis is an autosomal recessive lysosomal storage disease characterized by renal failure at 10 years of age and other systemic complications. The gene for cystinosis, CTNS, has 12 exons. Its 2.6-kb mRNA codes for a 367-amino-acid putative cystine transporter with seven transmembrane domains. Previously reported mutations include a 65-kb "European" deletion involving marker D17S829 and 11 small mutations. Mutation analysis of 108 American-based nephropathic cystinosis patients revealed that 48 patients (44%) were homozygous for the 65-kb deletion, 2 had a smaller major deletion, 11 were homozygous and 3 were heterozygous for 753G-->A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 derived from Germany, 28 from the British Isles, and 4 from Iceland. Eighteen new mutations were identified, including the first reported missense mutations, two in-frame deletions, and mutations in patients of African American, Mexican, and Indian ancestry. CTNS mutations are spread throughout the leader sequence, transmembrane, and nontransmembrane regions. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and for W138X have average disease, whereas mutations involving the first amino acids prior to transmembrane domains are associated with mild disease. By northern blot analysis, CTNS was not expressed in patients homozygous for the 65-kb deletion but was expressed in all 15 other patients tested. These data demonstrate the origins of CTNS mutations in America and provide a basis for possible molecular diagnosis in this population.     

Evidence for natural selection on leukocyte immunoglobulin-like receptors for HLA class I in Northeast Asians

Human leukocyte antigen (HLA) plays a critical role in innate and adaptive immunity and is a well-known example of genes under natural selection. However, the genetic aspect of receptors recognizing HLA molecules has not yet been fully elucidated. Leukocyte immunoglobulin (Ig)-like receptors (LILRs) are a family of HLA class I-recognizing receptors comprising activating and inhibitory forms. We previously reported that the allele frequency of the 6.7 kb LILRA3 deletion is extremely high (71%) in the Japanese population, and we identified premature termination codon (PTC)-containing alleles. In this study, we observed a wide distribution of the high deletion frequency in Northeast Asians (84% in Korean Chinese, 79% in Man Chinese, 56% in Mongolian, and 76% in Buryat populations). Genotyping of the four HapMap populations revealed that LILRA3 alleles were in strong linkage disequilibrium with LILRB2 alleles in Northeast Asians. In addition, PTC-containing LILRA3 alleles were detected in Northeast Asians but not in non-Northeast Asians. Furthermore, flow-cytometric analysis revealed that the LILRB2 allele frequent in Northeast Asians was significantly associated with low levels of expression. F(ST) and extended-haplotype-homozygosity analysis for the HapMap populations provided evidence of positive selection acting on the LILRA3 and LILRB2 loci. Taken together, our results suggest that both the nonfunctional LILRA3 alleles and the low-expressing LILRB2 alleles identified in our study have increased in Northeast Asians because of natural selection. Our findings, therefore, lead us to speculate that not only HLA class I ligands but also their receptors might be sensitive to the local environment.     

Diversity of the human LILRB3/A6 locus encoding a myeloid inhibitory and activating receptor pair

Leukocyte immunoglobulin-like receptor (LILR)B3 and LILRA6 represent a pair of inhibitory/activating receptors with identical extracellular domains and unknown ligands. LILRB3 can mediate inhibitory signaling via immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail whereas LILRA6 can signal through association with an activating adaptor molecule, FcRγ, which bears a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif. The receptors are encoded by two highly polymorphic neighboring genes within the leukocyte receptor complex on human chromosome 19. Here, we report that the two genes display similar levels of single nucleotide polymorphisms with the majority of polymorphic sites being identical. In addition, the LILRA6 gene exhibits copy number variation (CNV) whereas LILRB3 does not. A screen of healthy Caucasians indicated that 32 % of the subjects possessed more than two copies of LILRA6, whereas 4 % have only one copy of the gene per diploid genome. Analysis of mRNA expression in the major fractions of PBMCs showed that LILRA6 is primarily expressed in monocytes, similarly to LILRB3, and its expression level correlates with copy number of the gene. We suggest that the LILRA6 CNV may influence the level of the activating receptor on the cell surface, potentially affecting signaling upon LILRB3/A6 ligation.     

Crystal Structure of Myeloid Cell Activating Receptor Leukocyte Ig-like Receptor A2 (LILRA2/ILT1/LIR-7) Domain Swapped Dimer: Molecular Basis for Its Non-binding to MHC Complexes

The leukocyte Ig-like receptor (LILR/ILT/LIR) family comprises 13 members that are either activating or inhibitory receptors, regulating a broad range of cells in the immune responses. LILRB1 (ILT2), LILRB2 (ILT4) and LILRA1 (LIR6) can recognize MHC (major histocompatibility complex) class I or class I-like molecules, and LILRB1/HLA-A2, LILRB1/UL18 and LILRB2/HLA-G complex (extracellular domains D1D2) structures have been solved recently. The details of binding to MHC have been described. Despite high levels of sequence similarity among LILRA1, LILRA2 (ILT1), LILRA3 (ILT6) and LILRB1/B2, all earlier experiments showed that LILRA2 does not bind to MHC, but the reason is unknown. Here, we report the LILRA2 extracellular D1D2 domain crystal structure at 2.6 Å resolution, which reveals structural shifts of the corresponding MHC-binding amino acid residues in comparison with LILR B1/B2, explaining its non-binding to MHC molecules. We identify some key residues with great influence on the local structure, which exist only in the MHC-binding receptors. Moreover, we show that LILRA2 forms a domain-swapped dimer. Further work with these key swapping residues yields a monomeric form, confirming that the domain-swapping is primarily amino acid sequence-specific. The structure described here supports the dimer conformation in solution observed earlier, and implies a stress-induced regulation by dimerization, consistent with its function as a heat shock promoter.     

Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs)

Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families.     

Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP-mediated, site-specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P-FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP-mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.     

Assessment of the Olfactory Function in Italian Patients with Type 3 von Willebrand Disease Caused by a Homozygous 253 Kb Deletion Involving VWF and TMEM16B/ANO2

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability.     

Proportion of the GSTM 10/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases

A homozygous gene deletion at the glutathioneS-transferase M1 (GSTM1) locus of genomic DNA from blood spots was studied by PCR in the group of Slavic populations from the north-western and central-eastern regions of European Russia and in patients with lung cancer (LC), other tumors (OT), endometriosis (E), alcoholic cirrhosis (AC), cystic fibrosis (CF) and chronic bronchitis (CB). The frequencies of the GSTMI 0/0 genotype were 38.8% and 67.5% for both population groups, respectively. The proportion of the GSTM1 gene deletion geno-type was estimated as significantly increased in LC (81%), OT (65%), E (81%), AC (77.3%), and in CB (73.6%) patients with symptoms of CB confirmed by X-ray but not in CB patients without X-ray evidence of disease (40.9%). A definite preponderance of GSTM1-0 homozygotes (51.1%) has been registered in CF patients of the pancreatic sufficient group with clear-cut pulmonological manifestations but not in those of the pancreatic insufficient group with predominantly intestinal or mixed clinical symptoms (41.2% and 37.5%, respectively). Earlier clinical manifestations and death before the age of 5 years are typical for GSTM1-deleted CF patients. These data support the notion that GSTM1 deletion should be considered as a convenient genetic marker for the early detection of groups at higher risk of many diseases caused by environmental and genetic factors, where manifestation depends on the lack of detoxification. High levels of GSTM1 0/0 genotypes in E patients favor the substantial contribution of certain environmental toxins in the pathogenesis of this widespread disease.     

Identification of an anti-aldolase autoantibody as a diagnostic marker for diabetic retinopathy by immunoproteomic analysis

Circulating autoantibodies specific for retinal proteins are associated with retinal destruction in patients with diabetic retinopathy (DR). In this study, we screened diabetic sera for the presence of anti-retinal autoantibodies with an aim of developing diagnostic markers for DR. Immunoblot analysis of DR patients' sera with human retinal cytosolic proteins revealed a higher incidence of anti-retinal autoantibodies, compared to normal blood donors or diabetic patients without DR. Anti-retinal protein autoantibody profiles of DR patient sera were obtained by 2-DE immunoblot analysis. Specifically, 20 protein spots reactive with DR patient sera were identified by ESI-MS/MS. Of these spots, 14 were specific for DR patients, and 4 reacted with both non-proliferative DR (non-PDR) and PDR sera. The anti-aldolase autoantibody was selected as a DR marker candidate, and specific reactivity of DR patient sera was confirmed by immunoblot analysis with rabbit aldolase. The serum anti-aldolase autoantibody level was measured by ELISA. DR patients showed significantly higher autoantibody levels than normal donors or diabetic patients without retinopathy. However, no significant differences were observed between non-PDR and PDR patients, suggesting that the level of anti-aldolase autoantibody is not determined by the severity of retinopathy in diabetic patients. Our data collectively demonstrate that the anti-aldolase autoantibody serves as a useful marker for DR diagnosis.     

Interleukin (IL)-1 gene polymorphisms: relevance of disease severity associated alleles with IL-1beta and IL-1ra production in multiple sclerosis

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disorder, with a considerable genetic influence on susceptibility and disease course. Cytokines play an important role in MS pathophysiology, and genes encoding various cytokines are logical candidates to assess possible associations with MS susceptibility and disease course. We previously reported an association of a combination of polymorphisms in the interleukin (IL)-1B and IL-1 receptor antagonist (IL-1RN) genes (i.e. IL-1RN allele 2+/IL-1B(+3959)allele 2-) with disease severity in MS. Extending this observation, we investigated whether IL-1beta and IL-1ra production differed depending on carriership of this gene combination. METHODS: Twenty MS patients and 20 controls were selected based upon carriership of the specific combination. In whole blood, in vitro IL-1beta and IL-1ra production was determined by enzyme-linked immunosorbent-assay after 6 and 24 h of stimulation with lipopolysaccharide. RESULTS: Carriers of the specific combination produced more IL-1ra, especially in MS patients, although not significantly. IL-1ra production was significantly higher in individuals homozygous for IL-1RN allele 2. In patients, Il-1ra production was higher and IL-1beta production lower compared with controls. In primary progressive patients, the IL-1beta /IL-1ra ratio was significantly lower than in relapsing-remitting patients. CONCLUSION: Our results suggest higher in vitro IL-1ra production in carriers of IL-1RN allele 2, with an indication of an allelic dose-effect relationship.     

RNT4 3'-UTR insertion/deletion polymorphisms are not associated with atrial septal defect in Chinese Han population: a brief communication

Atrial septal defect (ASD) is a common type of congenital heart disease, which is defined as any communication through atrial septum. Several studies have revealed that genetic factors may influence the susceptibility of ASD. Recent studies have shown that reticulon 4 (RTN4) gene might be involved in some processes relevant to heart development, such as regulation of cell migration and vascular remodeling. This study aimed to evaluate RTN4 gene polymorphisms of CAA and TATC insertion/deletion in relation to the risk of ASD in Chinese Han population. A total of 175 ASD patients and 308 unrelated healthy controls were successfully investigated. The polymorphisms of patients were determined by polymerase chain reaction-polyacrylamide gel electrophoresis. There was no significant difference in the allele frequencies of CAA and TATC insertion/deletion in RNT4 gene between ASD patients and controls. The same results were seen in their genotypes. The present study suggests that CAA and TATC insertion/deletion polymorphisms of RNT4 gene may not be a useful marker to predict the susceptibility of ASD in Chinese Han population.     

Identification of complement C3a as a candidate biomarker in human chronic hepatitis C and HCV-related hepatocellular carcinoma using a proteomics approach

Although the significant risk factors for hepatocellular carcinoma (HCC) are well known from epidemiological studies, diagnosis of this disease at an early stage is difficult, and HCC remains one of the leading causes of cancer death worldwide. Thus, to identify any useful HCC-related biomarkers is still a need. We performed SELDI-TOF MS to identify differentially expressed proteins in HCC serum using weak cation exchange protein chips. Protein characterization was performed by 2-DE separation and nano flow LC-MS/MS. A total of 55 sera were collected from HCC patients and compared with those from 48 patients with chronic hepatitis and 9 healthy individuals. A candidate marker of about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus (HCV)-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. The combination of SELDI-TOF MS and 2-DE provides a solution to identify disease-associated serum biomarkers.     

Recurrent Muscle Weakness with Rhabdomyolysis,Metabolic Crises,and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3–9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3–9. Additionally, a homozygous exons 4–6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3–9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.