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Purpose: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). Patients and Methods: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. Results: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004). Conclusions: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

Supplementary Figure S4. Clinical impact of CD49d bimodal expression on durability of BTKi therapy.

Supplementary Table S6. DEGs for Baseline Comparison of CD49d+ and CD49d

Supplementary Methods

Purpose:To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL).Patients and Methods:In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients.Results:In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004).Conclusions:CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

Author(s): Sivakumaran, Theru A; Igo, Robert P; Kidd, Jeffrey M; Itsara, Andy; Kopplin, Laura J; Chen, Wei; Hagstrom, Stephanie A; Peachey, Neal S; Francis, Peter J; Klein, Michael L; Chew, Emily Y; Ramprasad, Vedam L; Tay, Wan-Ting; Mitchell, Paul; Seielstad, Mark; Stambolian, Dwight E; Edwards, Albert O; Lee, Kristine E; Leontiev, Dmitry V; Jun, Gyungah; Wang, Yang; Tian, Liping; Qiu, Feiyou; Henning, Alice K; LaFramboise, Thomas; Sen, Parveen; Aarthi, Manoharan; George, Ronnie; Raman, Rajiv; Das, Manmath Kumar; Vijaya, Lingam; Kumaramanickavel, Govindasamy; Wong, Tien Y; Swaroop, Anand; Abecasis, Goncalo R; Klein, Ronald; Klein, Barbara EK; Nickerson, Deborah A; Eichler, Evan E; Iyengar, Sudha K | Abstract: [This corrects the article DOI: 10.1371/journal.pone.0025598.].

We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 × 10 5, OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease. © 2010 Nature America, Inc. All rights reserved.

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

The most common recurrent copy-number variants associated with autism, developmental delay and epilepsy are flanked by segmental duplications. Complete genetic characterization of these events is challenging because their breakpoints often occur within high-identity, copy-number polymorphic paralogous sequences that cannot be specifically assayed using hybridization-based methods. Here we provide a protocol for breakpoint resolution with sequence-level precision. Massively parallel sequencing is performed on libraries generated from haplotype-resolved chromosomes, genomic DNA or molecular inversion probe (MIP)-captured breakpoint-informative regions harboring paralog-distinguishing variants. Quantification of sequencing depth over informative sites enables breakpoint localization, typically within several kilobases to tens of kilobases. Depending on the approach used, the sequencing platform, and the accuracy and completeness of the reference genome sequence, this protocol takes from a few days to several months to complete. Once established for a specific genomic disorder, it is possible to process thousands of DNA samples within as little as 3-4 weeks.

Item does not contain fulltext Recurrent deletions have been associated with numerous diseases and genomic disorders. Few, however, have been resolved at the molecular level because their breakpoints often occur in highly copy-number-polymorphic duplicated sequences. We present an approach that uses a combination of somatic cell hybrids, array comparative genomic hybridization, and the specificity of next-generation sequencing to determine breakpoints that occur within segmental duplications. Applying our technique to the 17q21.31 microdeletion syndrome, we used genome sequencing to determine copy-number-variant breakpoints in three deletion-bearing individuals with molecular resolution. For two cases, we observed breakpoints consistent with nonallelic homologous recombination involving only H2 chromosomal haplotypes, as expected. Molecular resolution revealed that the breakpoints occurred at different locations within a 145 kbp segment of >99% identity and disrupt KANSL1 (previously known as KANSL1). In the remaining case, we found that unequal crossover occurred interchromosomally between the H1 and H2 haplotypes and that this event was mediated by a homologous sequence that was once again missing from the human reference. Interestingly, the breakpoints mapped preferentially to gaps in the current reference genome assembly, which we resolved in this study. Our method provides a strategy for the identification of breakpoints within complex regions of the genome harboring high-identity and copy-number-polymorphic segmental duplication. The approach should become particularly useful as high-quality alternate reference sequences become available and genome sequencing of individuals' DNA becomes more routine.

Background: ABO(H) is a carbohydrate blood group system expressed in multiple tissues including red blood cells, blood vessels, and mucosal surfaces. ABO is the largest known genetic modifier of plasma VWF level (VWF:Ag). It has been hypothesized that the effect of ABO on VWF is mediated by H glycan density. FUT2, the gene underlying Secretor phenotype, encodes a glycosyltransferase synthesizing H antigen in mucosal tissues, and variation in the gene has also previously been associated with VWF:Ag, but past studies have been conflicting. To clarify these relationships, we studied the relationship between VWF:Ag, ABH glycans, and FUT2 genotype. Methods: The primary study group was a representative cohort of US blood donors from the Retrovirus Epidemiology Donor Study (REDS, N=499). A validation cohort of unrelated individuals was created from the Genes and Blood Clotting Study (GABC), healthy siblings between ages 14 and 35 years from University of Michigan, Ann Arbor, by choosing a random individual from each family (N=488). VWF:Ag was determined by ELISA in platelet poor plasma. Forensic techniques were adapted to detect ABH glycans in whole blood (REDS) or RBC-rich, frozen buffy coat (GABC). A and B glycans were detected using anti-A or anti-B (Immucor). A biotinylated Ulex europaeus agglutinin (UEA lectin, Vector Labs) was used to detect H. Relative A, B, and H antigen density was quantified on dot blots with ImageQuant (GE). In REDS, functional FUT2 alleles (Secretor) were determined by Sanger sequencing of FUT2 exon 2. FUT2 copy-number was assayed with real-time quantitative PCR. In GABC, FUT2 genotypes were determined through SNP genotyping (Illumina). In REDS, genotype data was phased (BEAGLE) to identify functional haplotypes. In GABC, functional alleles were inferred from the genotypes of two SNPs (rs601338, rs1047781) that determined secretor status in nearly all samples in REDS (see below). Multivariate regression was applied to VWF:Ag as a function H antigen density and O vs. non-O blood group, both separately and within the same model. All models were adjusted for age, gender, and self-reported ethnicity. Results: ABO blood group frequencies in both cohorts were similar to that of the US population. VWF:Ag differed significantly between ABO blood groups with lower values in blood group O versus non-O (REDS: ratio = 0.75, 95% CI [0.71, 0.80], p FUT2 sequencing in the REDS cohort identified 22 distinct FUT2 haplotypes. 99.5% (497/499) of individuals could be accurately assigned FUT2 genotypes based on the common W154* (rs601338) polymorphism alone. In GABC, this decreased to 94% (476/488) due to higher frequency of a hypomorphic allele (rs1047781) more commonly found in Asian individuals. In REDS, quantitative PCR did not identify copy-number variation at FUT2. There was no significant association between FUT2 genotype-predicted function and VWF:Ag or H glycan density in either cohort. Conclusion: H glycan density correlated with VWF:Ag in both cohorts. H glycan density could mediate the association between ABO blood group and VWF:Ag. If so, this suggests a non-linear, complex relationship between H and VWF and a separate ABO mechanism cannot be excluded. In both cohorts, there was no association of FUT2 (Secretor) genotype with VWF. Our data suggest prior focused approaches to FUT2 genotyping are at risk to undercall non-functional alleles, particularly in cohorts with non-European ancestries. Taken together, these indicate blood group H may be a significant modifier of VWF:Ag, and that this effect is not due to the influence of Secretor phenotype. Disclosures Johnsen: Octapharma: Other: Speaker; Biogen: Research Funding; CSL Behring: Other: Speaker.

While copy number variation (CNV) is an active area of research, de novo mutation rates within human populations are not well characterized. By focusing on large (>100 kbp) events, we estimate the rate of de novo CNV formation in humans by analyzing 4394 transmissions from human pedigrees with and without neurocognitive disease. We show that a significant limitation in directly measuring genome-wide CNV mutation is accessing DNA derived from primary tissues as opposed to cell lines. We conservatively estimated the genome-wide CNV mutation rate using single nucleotide polymorphism (SNP) microarrays to analyze whole-blood derived DNA from asthmatic trios, a collection in which we observed no elevation in the prevalence of large CNVs. At a resolution of ∼30 kb, nine de novo CNVs were observed from 772 transmissions, corresponding to a mutation rate of μ = 1.2 × 10−2 CNVs per genome per transmission (μ = 6.5 × 10−3 for CNVs >500 kb). Combined with previous estimates of CNV prevalence and assuming a model of mutation-selection balance, we estimate significant purifying selection for large (>500 kb) events at the genome-wide level to be s = 0.16. Supporting this, we identify de novo CNVs in 717 multiplex autism pedigrees from the AGRE collection and observe a fourfold enrichment (P = 1.4 × 10−3) for de novo CNVs in cases of multiplex autism versus unaffected siblings, suggesting that many de novo CNV mutations contribute a subtle, but significant risk for autism. We observe no parental bias in the origin or transmission of CNVs among any of the cohorts studied.

PUBLISHED, Background Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. Methods We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. Results We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10?7). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. Conclusions We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype., Supported in part by grants from the National Institutes of Health (NIH) (HD043569, to Dr. Eichler), the South Carolina Department of Disabilities and Special Needs (to Drs. Skinner, Stevenson, and Schwartz), the Wellcome Trust (061183, to Dr. Tassabehji), the Andre & Cyprien Foundation and the University Hospitals of Geneva (to Drs. Antonarakis, Bena, and Gallati), and the European Union (EU) (project 219250, to Dr. Sharp; AnEUploidy project 037627, to Drs. Leeuw, Armengol, Antonarakis, Estivill, Veltman, and de Vries). The Irish Autism Study was funded by the Wellcome Trust and the Health Research Board (a grant to Drs. Gallagher and Gill). Dr. Poot was supported by a grant from the Dutch Foundation for Brain Research (Hersenstichting grant 2008(1) 34); Drs. Regan and Knight, by the Oxford Partnership Comprehensive Biomedical Research Centre; Dr. Willatt, by the Cambridge Biomedical Research Centre, with funding from the United Kingdom Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme; Drs. Huang and Maloney, as part of the National Genetics Reference Laboratory (Wessex) by the United Kingdom Department of Health; Ms. Buysse, as a research assistant of the Research Foundation?Flanders (FWO?Vlaanderen); and Dr. Eichler, as an investigator of the Howard Hughes Medical Institute.

Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10(-109)); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10(-9)) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.

Copy number variants (CNVs) contribute to human genetic and phenotypic diversity. However, the distribution of larger CNVs in the general population remains largely unexplored. We identify large variants in approximately 2500 individuals by using Illumina SNP data, with an emphasis on "hotspots" prone to recurrent mutations. We find variants larger than 500 kb in 5%-10% of individuals and variants greater than 1 Mb in 1%-2%. In contrast to previous studies, we find limited evidence for stratification of CNVs in geographically distinct human populations. Importantly, our sample size permits a robust distinction between truly rare and polymorphic but low-frequency copy number variation. We find that a significant fraction of individual CNVs larger than 100 kb are rare and that both gene density and size are strongly anticorrelated with allele frequency. Thus, although large CNVs commonly exist in normal individuals, which suggests that size alone can not be used as a predictor of pathogenicity, such variation is generally deleterious. Considering these observations, we combine our data with published CNVs from more than 12,000 individuals contrasting control and neurological disease collections. This analysis identifies known disease loci and highlights additional CNVs (e.g., 3q29, 16p12, and 15q25.2) for further investigation. This study provides one of the first analyses of large, rare (0.1%-1%) CNVs in the general population, with insights relevant to future analyses of genetic disease.