Search

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by cytogenetic abnormalities, such as t(11;14)(q13;q32), resulting in CCND1 overexpression. The rs9344 G allele within CCND1 is the most significant susceptibility allele for t(11;14). Sequencing data from 2 independent cohorts, CoMMpass (n = 698) and Mayo Clinic (n = 661), confirm the positive association between the G allele and t(11;14). Among 80% of individuals heterozygous for rs9344 with t(11;14), the t(11;14) event occurs on the G allele, demonstrating a biological preference for the G allele in t(11;14). Within t(11;14), the G allele is associated with higher CCND1 expression and elevated H3K27ac and H3K4me3. CRISPR/Cas9 mediated A to G conversion resulted in increased H3K27ac over CCND1 and elevated CCND1 expression. ENCODE ChIP-seq data supported a PAX5 binding site within the enhancer region covering rs9344, showing preferential binding to the G allele. Overexpression of PAX5 resulted in increased CCND1 expression. These results support the importance of rs9344 G enhancer in increasing CCND1 expression in MM., (© 2024. The Author(s).)

Pleomorphic xanthoastrocytomas (PXAs) harbor CDKN2A homozygous deletion in >90% of cases, resulting in loss of p16 expression by immunohistochemistry. Considering the proximity of MTAP to CDKN2A and their frequent concurrent deletions, loss of MTAP expression may be a surrogate for CDKN2A homozygous deletion. We evaluated p16 and MTAP expression in 38 patient PXAs (CNS WHO grade 2: n = 23, 60.5%; grade 3: n = 15, 39.5%) with available chromosomal microarray data to determine whether MTAP can be utilized independently or in combination with p16 to predict CDKN2A status. CDKN2A, CDKN2B, and MTAP homozygous deletion were present in 37 (97.4%), 36 (94.7%), and 25 (65.8%) cases, respectively. Expression of p16 was lost in 35 (92.1%) cases, equivocal in one (2.6%), and failed in 2 (5.3%), while MTAP expression was lost in 27 (71.1%) cases, retained in 10 (26.3%), and equivocal in one (2.6%). This yielded a sensitivity of 94.6% for p16 and 73.0% for MTAP in detecting CDKN2A homozygous deletion through immunohistochemistry. MTAP expression was lost in the 2 cases with failed p16 staining (combined sensitivity of 100%). Our findings demonstrate that combined p16 and MTAP immunostains correctly detect CDKN2A homozygous deletion in PXA, while MTAP expression alone shows reduced sensitivity., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Association of Neuropathologists, Inc.)

Objectives: Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting., Methods: The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed., Results: Fluorescence-activated cell sorting-FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs., Conclusions: Fluorescence-activated cell sorting-FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Competing Interests: Conflict of Interest The authors declare no conflicts of interest.

Competing Interests: Conflict of Interest C.J.Z.-M. is a director of a cytogenomics testing laboratory that offers cancer diagnostic testing. M.A. is a director of a molecular testing laboratory that offers carrier screening. K.B. is a genetic counselor of a molecular testing laboratory that offers carrier screening. F.Q.-R. is a director of a cytogenomics testing laboratory that offers cancer diagnostic testing. M.V. is a director of a molecular testing laboratory that offers carrier screening and sponsored panel testing. All other authors declare no conflicts of interest.

Acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21-ALL) represents a recurrent high-risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21-ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21-chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21-ALL. This study was a retrospective evaluation of iAMP21-ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21-ALL, 188 (91%) were classified as "typical" iAMP21-ALL, while 19 (9%) cases were classified as "unusual" iAMP21-ALL. The "unusual" iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the "unusual" iAMP21-ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21-chromosome. Nine percent of iAMP21-ALL cases fail to meet the FISH definition of iAMP21-ALL demonstrating that laboratories are at risk of misidentification of iAMP21-ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks., (© 2022 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

Purpose: Structural variants (SV) of the MYC gene region are common in multiple myeloma and influence disease progression. However, the prognostic significance of different MYC SVs in multiple myeloma has not been clearly established., Experimental Design: We conducted a retrospective study of multiple myeloma comparing MYC SV subtypes identified by next-generation sequencing (NGS) and FISH to MYC expression and disease survival using 140 cases from Mayo Clinic and 658 cases from the MMRF CoMMpass study., Results: MYC SVs were found in 41% of cases and were classified into nine subtypes. A correlation between the presence of a MYC SV and increased MYC expression was identified. Among the nine MYC subtypes, the non-immunoglobulin (non-Ig) insertion subtype was independently associated with improved outcomes, while the Ig insertion subtype, specifically involving the IgL gene partner, was independently associated with poorer outcomes compared with other MYC SV subtypes. Although the FISH methodology failed to detect approximately 70% of all MYC SVs, those detected by FISH were associated with elevated MYC gene expression and poor outcomes suggesting a different pathogenic role for FISH-detected MYC subtypes compared with other MYC subtypes., Conclusions: Understanding the impact of different MYC SVs on disease outcome is necessary for the reliable interpretation of MYC SVs in multiple myeloma. NGS approaches should be considered as a replacement technique for a more comprehensive evaluation of the multiple myeloma clone., (©2021 The Authors; Published by the American Association for Cancer Research.)

Phelan-McDermid syndrome (PMS) is a multi-system disorder characterized by significant variability in clinical presentation. The genetic etiology is also variable with differing sizes of deletions in the chromosome 22q13 region and types of genetic abnormalities (e.g., terminal or interstitial deletions, translocations, ring chromosomes, or SHANK3 variants). Position effects have been shown to affect gene expression and function and play a role in the clinical presentation of various genetic conditions. This study employed a topologically associating domain (TAD) analysis approach to investigate position effects of chromosomal rearrangements on selected candidate genes mapped to 22q13 in 81 individuals with PMS. Data collected were correlated with clinical information from these individuals and with expression and metabolic profiles of lymphoblastoid cells from selected cases. The data confirmed TAD predictions for genes encompassed in the deletions and the clinical and molecular data indicated clear differences among individuals with different 22q13 deletion sizes. The results of the study indicate a positive correlation between deletion size and phenotype severity in PMS and provide evidence of the contribution of other genes to the clinical variability in this developmental disorder by reduced gene expression and altered metabolomics., Competing Interests: The authors have declared that no competing interests exist.

Trichorhinophalangeal syndrome type I (TRPSI) is a rare disorder that causes distinctive ectodermal, facial, and skeletal features affecting the hair (tricho-), nose (rhino-), and fingers and toes (phalangeal) and is inherited in an autosomal dominant pattern. TRPSI is caused by loss of function variants in TRPS1 , involved in the regulation of chondrocyte and perichondrium development. Pathogenic variants in TRPS1 include missense mutations and deletions with variable breakpoints, with only a single instance of an intragenic duplication reported to date. Here we report an affected individual presenting with a classic TRPSI phenotype who is heterozygous for a de novo intragenic ∼36.3-kbp duplication affecting exons 2-4 of TRPS1 Molecular analysis revealed the duplication to be in direct tandem orientation affecting the splicing of TRPS1 The aberrant transcripts are predicted to produce a truncated TRPS1 missing the nuclear localization signal and the GATA and IKAROS-like zinc-finger domains resulting in functional TRPS1 haploinsufficiency. Our study identifies a novel intragenic tandem duplication of TRPS1 and highlights the importance of molecular characterization of intragenic duplications., (© 2019 Zepeda-Mendoza et al.; Published by Cold Spring Harbor Laboratory Press.)

Microphthalmia with brain and digital anomalies (MCOPS6, MIM# 607932) is an autosomal dominant disorder caused by loss-of-function variants or large deletions involving BMP4, which encodes bone morphogenetic protein 4, a member of the TGF-β protein superfamily. BMP4 has a number of roles in embryonic development including neurogenesis, lens induction, development of cartilage and bone, urogenital development, limb and digit patterning, hair follicle regeneration, as well as tooth formation. In addition to syndromic microphthalmia, BMP4 variants have been implicated in non-syndromic cleft lip with or without cleft palate and congenital healed cleft lip indicating different allelic presentations. MCOPS6 subjects may also lack some of the major phenotypic hallmarks of the disorder, including microphthalmia, indicating variable expressivity. As only a handful of individuals with MCOPS6 have been described, we review the clinical findings in previously reported cases with either deletions or loss-of-function variants in BMP4. We describe three new cases, including two subjects with novel deletions and one subject with a likely pathogenic de novo nonsense variant [c.1052C>G, p.(S351*)] in BMP4. One of the subjects had dual molecular diagnoses including a co-occurring microdeletion at 17q21.31 associated with Koolen de Vries syndrome, which has a partially overlapping disease phenotype. None of these individuals had clinically apparent microphthalmia or anopthalmia, which have been reported in a majority of previously described cases. One subject had exophthalmia and strabismus, while another had bilateral Peters anomaly and sclerocornea, thus expanding the phenotype associated with BMP4 loss-of-function variants.

Structural variation, composed of balanced and unbalanced genomic rearrangements, is an important contributor to human genetic diversity with prominent roles in somatic and congenital disease. At the nucleotide level, structural variants (SVs) have been shown to frequently harbor additional breakpoints and copy-number imbalances, a complexity predicted to emerge wholly as a single-cell division event. Chromothripsis, chromoplexy, and chromoanasynthesis, collectively referred to as chromoanagenesis, are three major mechanisms that explain the occurrence of complex germline and somatic SVs. While chromothripsis and chromoplexy have been shown to be key signatures of cancer, chromoanagenesis has been detected in numerous cases of developmental disease and phenotypically normal individuals. Such observations advocate for a deeper study of the polymorphic and pathogenic properties of complex germline SVs, many of which go undetected by traditional clinical molecular and cytogenetic methods. This review focuses on congenital chromoanagenesis, mechanisms leading to occurrence of these complex rearrangements, and their impact on chromosome organization and genome function. We highlight future applications of routine screening of complex and balanced SVs in the clinic, as these represent a potential and often neglected genetic disease source, a true "iceberg under water.", (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Great strides in gene discovery have been made using a multitude of methods to associate phenotypes with genetic variants, but there still remains a substantial gap between observed symptoms and identified genetic defects. Herein, we use the convergence of various genetic and genomic techniques to investigate the underpinnings of a constellation of phenotypes that include prostate cancer (PCa) and sensorineural hearing loss (SNHL) in a human subject. Through interrogation of the subject's de novo, germline, balanced chromosomal translocation, we first identify a correlation between his disorders and a poorly annotated gene known as lipid droplet associated hydrolase (LDAH). Using data repositories of both germline and somatic variants, we identify convergent genomic evidence that substantiates a correlation between loss of LDAH and PCa. This correlation is validated through both in vitro and in vivo models that show loss of LDAH results in increased risk of PCa and, to a lesser extent, SNHL. By leveraging convergent evidence in emerging genomic data, we hypothesize that loss of LDAH is involved in PCa and other phenotypes observed in support of a genotype-phenotype association in an n-of-one human subject.

Balanced and apparently balanced chromosome abnormalities (BCAs) have long been known to generate disease through position effects, either by altering local networks of gene regulation or positioning genes in architecturally different chromosome domains. Despite these observations, identification of distally affected genes by BCAs is oftentimes neglected, especially when predicted gene disruptions are found elsewhere in the genome. In this unit, we provide detailed instructions on how to run a computational pipeline that identifies relevant candidates of non-coding BCA position effects. This methodology facilitates quick identification of genes potentially involved in disease by non-coding BCAs and other types of rearrangements, and expands on the importance of considering the long-range consequences of genomic lesions.

Molecular characterization of balanced chromosomal abnormalities constitutes a powerful tool in understanding the pathogenic mechanisms of complex genetic disorders. Here we report a male with severe global developmental delay in the presence of a complex karyotype and normal microarray and exome studies. The subject, referred to as DGAP294, has two de novo apparently balanced translocations involving chromosomes 1 and 14, and chromosomes 4 and 10, disrupting several different transcripts of adhesion G protein-coupled receptor L2 (ADGRL2) and protocadherin 15 (PCDH15). In addition, a maternally inherited inversion disrupts peptidyl arginine deiminase types 3 and 4 (PADI3 and PADI4) on chromosome 1. None of these gene disruptions explain the patient's phenotype. Using genome regulatory annotations and chromosome conformation data, we predict a position effect ~370 kb upstream of a translocation breakpoint located at 14q12. The position effect involves forkhead box G1 (FOXG1), mutations in which are associated with the congenital form of Rett syndrome and FOXG1 syndrome. We believe the FOXG1 position effect largely accounts for the clinical phenotype in DGAP294, which can be classified as FOXG1 syndrome like. Our findings emphasize the significance of not only analyzing disrupted genes by chromosomal rearrangements, but also evaluating potential long-range position effects in clinical diagnoses.

Interpretation of variants of uncertain significance, especially chromosomal rearrangements in non-coding regions of the human genome, remains one of the biggest challenges in modern molecular diagnosis. To improve our understanding and interpretation of such variants, we used high-resolution three-dimensional chromosomal structural data and transcriptional regulatory information to predict position effects and their association with pathogenic phenotypes in 17 subjects with apparently balanced chromosomal abnormalities. We found that the rearrangements predict disruption of long-range chromatin interactions between several enhancers and genes whose annotated clinical features are strongly associated with the subjects' phenotypes. We confirm gene-expression changes for a couple of candidate genes to exemplify the utility of our analysis of position effect. These results highlight the important interplay between chromosomal structure and disease and demonstrate the need to utilize chromatin conformational data for the prediction of position effects in the clinical interpretation of non-coding chromosomal rearrangements., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Analysis of chromosomal rearrangements has been highly successful in identifying genes involved in many congenital abnormalities including hearing loss. Herein, we report a subject, designated DGAP242, with congenital hearing loss (HL) and a de novo balanced translocation 46,XX,t(1;5)(q32;q15)dn. Using multiple next-generation sequencing techniques, we obtained high resolution of the breakpoints. This revealed disruption of the orphan receptor ESRRG on chromosome 1, which is differentially expressed in inner ear hair cells and has previously been implicated in HL, and disruption of KIAA0825 on chromosome 5. Given the translocation breakpoints and supporting literature, disruption of ESRRG is the most likely cause for DGAP242's phenotype and implicates ESRRG in a monogenic form of congenital HL, although a putative contributory role for KIAA0825 in the subject's disorder cannot be excluded.

Background: Circular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2., Results: A significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed., Conclusions: Altogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function.

The relationship between chromatin organization and transcriptional regulation is an area of intense investigation. We characterized the spatial relationships between alleles of the Oct4, Sox2, and Nanog genes in single cells during the earliest stages of mouse embryonic stem cell (ESC) differentiation and during embryonic development. We describe homologous pairing of the Oct4 alleles during ESC differentiation and embryogenesis, and we present evidence that pairing is correlated with the kinetics of ESC differentiation. Importantly, we identify critical DNA elements within the Oct4 promoter/enhancer region that mediate pairing of Oct4 alleles. Finally, we show that mutation of OCT4/SOX2 binding sites within this region abolishes inter-chromosomal interactions and affects accumulation of the repressive H3K9me2 modification at the Oct4 enhancer. Our findings demonstrate that chromatin organization and transcriptional programs are intimately connected in ESCs and that the dynamic positioning of the Oct4 alleles is associated with the transition from pluripotency to lineage specification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

Background: Identical sequences with a minimal length of about 300 base pairs (bp) have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR) events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB) of the reference human genome., Results: The IRB sequences are distributed all over the genome in 66,600 regions, which correspond to approximately 2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs). By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes., Conclusions: In general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.