Importance Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. Objective To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. Design, Setting, and Participants Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. Main outcomes and Measures Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. Results Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. Conclusions and Relevance In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.

We examined the immunoglobulin (Ig) heavy chain variable region genes (V H genes) used by leukemia cells of 1220 unrelated patients with chronic lymphocytic leukemia (CLL). We found 1188 (97%) expressed Ig encoded by a single Ig V H subgroup, the most common of which was V H 3 (571 or 48.1%), followed by V H 1 (319 or 26.8%) and V H 4 (241 or 20.2%). Using allele-specific primers, we found 13.8% of all samples (n ‫؍‬ 164) used one major V H 1-69 allele, designated 51p1, 163 of which were not somatically mutated. For these cases, there was marked restriction in the structure of the Ig third complementarity determining regions (CDR3s), which were encoded by a small number of unmutated D and J H gene segments. Strik IntroductionThe mutational status of the immunoglobulin (Ig) genes expressed in B-cell chronic lymphocytic leukemia (CLL) can be used to segregate patients into 2 subsets that have significantly different tendencies for disease progression. Those patients with leukemia cells that express unmutated Ig heavy chain variable region genes (V H genes) have a greater tendency for disease progression and shorter survival than those who have leukemia cells that express Ig V H genes with less than 98% nucleic acid sequence homology with their germ-line counterparts. 1-7 Generally, the Ig V H genes expressed by any leukemia cell population do not display significant intraclonal diversity or tendency to accumulate additional somatic mutations over time. 8 As such, the leukemia cells that express mutated Ig genes apparently do not evolve from cases that originally expressed unmutated Ig genes. Because of this, the presence or absence of Ig somatic mutations was thought by some to reflect 2 subtypes of CLL, each with distinctive cytogenesis. 1,2,9 The subtype that expressed unmutated Ig V H genes was considered to be derived from naive or pre-germinal center B cells, whereas the CLL cells expressing mutated receptors were thought likely to be derived from a post-germinal center or "memory-type" B cells. 10 However, molecular analyses of the Ig V H genes expressed by CLL B cells suggest that neither subtype is derived from a naive, nonselected B cell. CLL B cells express an Ig repertoire that appears more restricted than that of adult blood B cells. Several genes, such as the 51p1 allele of V H 1-69, are expressed at high frequency, 11 are rarely mutated, 12 and constitute a large proportion (eg, Ϸ 20%) of the cases that lack Ig somatic mutation. In addition, prior studies found that 51p1-expressing CLL B cells preferentially use certain diversity (D) and J H gene segments with restricted reading frames (RFs), encoding relatively long third complementarity determining regions (CDR3s) with conserved amino acid motifs. 12 The CDR3s of 51p1-expressing CLL B cells contrasted with those of the Ig heavy chains expressed by CLL B cells that used other Ig V H 1 genes or by nonneoplastic tonsillar or blood B cells that used 51p1. [12][13][14] As the CDR3 is the most variable region of the heavy chain and is directly involved ...

IntroductionSeveral studies have demonstrated that leukemic B cells of patients with chronic lymphocytic leukemia (CLL) express a restricted immunoglobulin (Ig) heavy chain repertoire and that expression of Ig variable heavy chain (IGHV) genes in CLL is not random. [1][2][3][4][5] One particular IGHV gene, the 51p1 allele of IGHV1-69, generally is expressed with little or no somatic mutation in 10 to 20% of all cases of this disease. 6 Furthermore, CLL B cells that express 51p1 have a preferential use of certain diversity (IGHD) and junctional (IGHJ) gene segments with restricted reading frames (RF) that encode relatively long third complementarity determining regions (CDR3), which have conserved amino acid motifs that are not characteristic of the CDR3s of the Ig expressed by nonneoplastic tonsilar or blood B cells that use 51p1. 3,7,8 The CDR3 is typically the most variable region of the Ig heavy chain and is generally directly involved in binding to conventional antigens. As such, the expression of such conserved Ig with restricted CDR3 argues that the Ig repertoire expressed in this disease is highly selected, suggesting a potential role for antigen in the development and/or progression of CLL.More recently we identified 15 unrelated CLL cases that expressed nearly identical Ig, with Ig heavy and light chain variable regions encoded by 51p1 and IGKV3-20 (A27), respectively. 9 The heavy and light chains of these 15 samples were virtually identical to SMI, a previously characterized CLL that expressed a polyreactive IgM/ autoantibody with low-affinity-binding activity for a variety of self-antigens. 10 The CDR3 regions of both the heavy and light chains together comprise the Ig binding site for antigen. The prevalent use of the 51p1 allele of IGHV1-69 in CLL with little or no somatic mutation allowed us to examine the effect of different Ig heavy chain CDR3 (HCDR3) motifs on Ig light chain pairing in CLL cases that otherwise expressed nearly identical Ig heavy chain variable regions. For this, we examined the IGKV and IGLV gene usage and CDR3 structure of 258 CLL cases that expressed unmutated Ig heavy chains encoded by 51p1 that were identified in a large cohort of 1846 nonselected CLL cases followed by the CLL Research Consortium (CRC). Methods Patient materialBlood was collected from consenting patients who satisfied the diagnostic and immunophenotypic criteria for B-cell CLL 11 and who presented for evaluation at the referral centers of the CRC. Institutional review board approval from each participating institution and informed consent were obtained in all cases, in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque 1077 (Sigma-Aldrich, St Louis, MO), washed twice, and analyzed directly or suspended in fetal calf serum containing 10% dimethylsulfoxide (DMSO; Sigma-Aldrich) for storage in liquid nitrogen. All samples contained more than 90% CLL B cells as Submitted February 8, 2007; accepted June 18, 2007. Pr...