Antibody repertoire diversity, potentially as high as 10 11 unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 ؋ 10 6 reads, including more than 1.9 ؋ 10 5 high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 ؋ 10 4 sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 ؋ 10 10 . The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.HMM ͉ phage display ͉ pyrosequencing ͉ CDRs

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V H and D H segment use is strongly determined by an individual's germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.heritable variation | next generation sequencing | V-gene S pecific biases in the antibody repertoire have been found in many diseases, from viral infections to cancers to autoimmune disorders (1-15). Although it is possible that heritable variation in the composition of the antibody repertoire could alter inherent risk to specific diseases, the diversity of the antibody repertoire has hindered direct characterization of heritable influences.Early twin studies provided some evidence of genetic variation affecting reactive titers from the antibody repertoire. Multiple studies observed both total Ig and antigen-specific titers to be more correlated in monozygotic twins than dizygotic twins or unrelated individuals (16)(17)(18). In some cases of monozygotic twins discordant for autoimmune diseases, the healthy twin often shared high autoantibody reactive titers with their affected twin (16,19,20).Early sequencing studies were able to identify some systematic biases in the antibody repertoire with limited sampling depth. The first sequencing studies to characterize V(D)J diversification mechanisms identified the gene segment recombination process, but also implied a repertoire too diverse to exhaustively interrogate by traditional sequencing technologies (21). Complete characterization of V-segment loci established ∼50 V H , 40 V κ , and 30 V λ segments in an individual, with a number of allelic variants for the majority of segments (22)(23)(24). Evaluation of use across individuals revealed biased V-gene representation that preceded selection (25)(26)(27). Quantitative PCR of V-gene families showed family use largely stable over time, with fluctuations in use correlated to antigen-specific responses (28). In the T-cell receptor (TCR) repertoire, TCRB-V use was more highly correlated in healthy monozygotic twins than unrelated individua...

Multiple myeloma is defined as a malignant proliferation of a single clone of plasma cells resulting in monoclonal immunoglobulin production. It is often associated with extensive skeletal destruction, infections, anemia, hypercalcemia, and renal failure. Due to its multi-system consequences, it is important to be able to identify patients at risk and those who present with classical and non-classical signs and symptoms. It is equally as important to understand the pathophysiology of the disease to be able to treat specific disease processes adequately. EpidemiologyMultiple myeloma (MM) accounts for nearly 1% of all cancers and for approximately 13% of all hematologic malignancies. The annual incidence in the United States ranges from 4 to 6 cases per 100,000 persons. 1 The incidence of myeloma increases with age, with the median age of diagnosis being 66 years old. It is very rare in individuals under the age of 40 years. Males are more commonly affected than females and the incidence in African-Americans is 2-3 times that in Caucasians. 1 Renal dysfunction in multiple myeloma is also quite common. In two large studies, it was found that 43% of patients had a plasma creatinine concentration 41.5 mg/dL. 2 Renal disease often plays an important role in the survival of these patients. 2 One report found that the one-year survival in patients with plasma creatinine concentrations less than 1.5 mg/dL was upwards of 80% versus 50% in those with plasma creatinine concentrations greater than 2.3 mg/dL. 2 There is a general association between the severity of renal disease in MM and the long-term prognosis for patients. PathophysiologyMultiple myeloma (MM) is thought to develop from the malignant transformation of postgerminal center plasma cells. Multiple genetic and environmental changes lead to the transformation of these progenitor cells into a malignant neoplasm. 3 It appears that the majority of cases of MM are preceded by monoclonal gammopathy of unknown significance (MGUS). It is thought to be the result of an abnormal plasma cell response to

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