Utilizing ClinGen gene-disease validity and dosage sensitivity curations to inform variant classification.

Understanding whether there is enough evidence to implicate a gene's role in a given disease, as well as the mechanisms by which variants in this gene might cause this disease, is essential to determine clinical relevance. The National Institutes of Health-funded Clinical Genome Resource (ClinGen) has developed evaluation frameworks to assess both the strength of evidence supporting a relationship between a gene and disease (gene-disease validity), and whether loss (haploinsufficiency) or gain (triplosensitivity) of individual genes or genomic regions is a mechanism for disease (dosage sensitivity). ClinGen actively applies these frameworks across multiple disease domains, and makes this information publicly available via its website (https://www.clinicalgenome.org/) for use in multiple applications, including clinical variant classification. Here, we describe how the results of these curation processes can be utilized to inform the appropriate application of pathogenicity criteria for both sequence and copy number variants, as well as to guide test development and inform genomic filtering pipelines.

Interpretation and reporting of large regions of homozygosity and suspected consanguinity/uniparental disomy, 2021 revision: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Genomic testing, including single-nucleotide variation (formerly single-nucleotide polymorphism)-based chromosomal microarray and exome and genome sequencing, can detect long regions of homozygosity (ROH) within the genome. Genomic testing can also detect possible uniparental disomy (UPD). Platforms that can detect ROH and possible UPD have matured since the initial American College of Medical Genetics and Genomics (ACMG) standard was published in 2013, and the detection of ROH and UPD by these platforms has shown utility in diagnosis of patients with genetic/genomic disorders. The presence of these segments, when distributed across multiple chromosomes, may indicate a familial relationship between the proband's parents. This technical standard describes the detection of possible consanguinity and UPD by genomic testing, as well as the factors confounding the inference of a specific parental relationship or UPD. Current bioethical and legal issues regarding detection and reporting of consanguinity are also discussed.

Int22h1/Int22h2-mediated Xq28 duplication syndrome: de novo duplications, prenatal diagnoses, and additional phenotypic features.

Int22h1/Int22h2-mediated Xq28 duplication syndrome is a relatively new X-linked intellectual disability syndrome, arising from duplications of the subregion flanked by intron 22 homologous regions 1 and 2 on the q arm of chromosome X. Its primary manifestations include variable cognitive deficits, distinct facial dysmorphia, and neurobehavioral abnormalities that mainly include hyperactivity, irritability, and autistic behavior. Affected males are hemizygous for the duplication, which explains their often more severe manifestations compared with heterozygous females. In this report, we describe the cases of nine individuals recently identified having the syndrome, highlighting unique and previously unreported findings of this syndrome. Specifically, we report for the first time in this syndrome, two cases with de novo duplications, three receiving prenatal diagnosis with the syndrome, and three others having atypical versions of the duplication. Among the latter, one proband has a shortened version spanning only the centromeric half of the typical duplication, while the other two cases have a nearly identical length duplication as the classical duplication, with the exception that their duplication's breakpoints are telomerically shifted by about 0.2 Mb. Finally, we shed light on two new manifestations in this syndrome, vertebral anomalies and multiple malignancies, which possibly expand the phenotypic spectrum of the syndrome.

Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen).

PURPOSE: Copy-number analysis to detect disease-causing losses and gains across the genome is recommended for the evaluation of individuals with neurodevelopmental disorders and/or multiple congenital anomalies, as well as for fetuses with ultrasound abnormalities. In the decade that this analysis has been in widespread clinical use, tremendous strides have been made in understanding the effects of copy-number variants (CNVs) in both affected individuals and the general population. However, continued broad implementation of array and next-generation sequencing-based technologies will expand the types of CNVs encountered in the clinical setting, as well as our understanding of their impact on human health. METHODS: To assist clinical laboratories in the classification and reporting of CNVs, irrespective of the technology used to identify them, the American College of Medical Genetics and Genomics has developed the following professional standards in collaboration with the National Institutes of Health (NIH)-funded Clinical Genome Resource (ClinGen) project. RESULTS: This update introduces a quantitative, evidence-based scoring framework; encourages the implementation of the five-tier classification system widely used in sequence variant classification; and recommends "uncoupling" the evidence-based classification of a variant from its potential implications for a particular individual. CONCLUSION: These professional standards will guide the evaluation of constitutional CNVs and encourage consistency and transparency across clinical laboratories.

Pediatric acute myeloid leukemia with t(7;21)(p22;q22).

The t(7;21)(p22;q22) resulting in RUNX1-USP42 fusion, is a rare but recurrent cytogenetic abnormality associated with acute myeloid leukemia (AML) and myelodysplastic syndromes. The prognostic significance of this translocation has not been well established due to the limited number of patients. Herein, we report three pediatric AML patients with t(7;21)(p22;q22). All three patients presented with pancytopenia or leukopenia at diagnosis, accompanied by abnormal immunophenotypic expression of CD7 and CD56 on leukemic blasts. One patient had t(7;21)(p22;q22) as the sole abnormality, whereas the other two patients had additional numerical and structural aberrations including loss of 5q material. Fluorescence in situ hybridization analysis on interphase cells or sequential examination of metaphases showed the RUNX1 rearrangement and confirmed translocation 7;21. Genomic SNP microarray analysis, performed on DNA extracted from the bone marrow from the patient with isolated t(7;21)(p22;q22), showed a 32.2 Mb copy neutral loss of heterozygosity (cnLOH) within the short arm of chromosome 11. After 2-4 cycles of chemotherapy, all three patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). One patient died due to complications related to viral reactivation and graft-versus-host disease. The other two patients achieved complete remission after HSCT. Our data displayed the accompanying cytogenetic abnormalities including del(5q) and cnLOH of 11p, the frequent pathological features shared with other reported cases, and clinical outcome in pediatric AML patients with t(7;21)(p22;q22). The heterogeneity in AML harboring similar cytogenetic alterations may be attributed to additional uncovered genetic lesions.

A Novel Homozygous Deletion within the FRY Gene Associated with Nonsyndromic Developmental Delay.

The role of autosomal recessive (AR) variants in clinically heterogeneous conditions such as intellectual disability and developmental delay (ID/DD) has been difficult to uncover. Implication of causative pathogenic AR variants often requires investigation within large and consanguineous families, and/or identifying rare biallelic variants in affected individuals. Furthermore, detection of homozygous gene-level copy number variants during first-line genomic microarray testing in the pediatric population is a rare finding. We describe a 6.7-year-old male patient with ID/DD and a novel homozygous deletion involving the FRY gene identified by genomic SNP microarray. This deletion was observed within a large region of homozygosity on the long arm of chromosome 13 and in a background of increased low-level (2.6%) autosomal homozygosity, consistent with a reported common ancestry in the family. FRY encodes a protein that regulates cell cytoskeletal dynamics, functions in chromosomal alignment in mitosis in vitro, and has been shown to function in the nervous system in vivo. Homozygous mutation of FRY has been previously reported in 2 consanguineous families from studies of autosomal recessive ID in Middle Eastern and Northern African populations. This report provides additional supportive evidence that deleterious biallelic mutation of FRY is associated with ID/DD and illustrates the utility of genomic SNP microarray detection of low-level homozygosity.

Genetic evaluation of juvenile xanthogranuloma: genomic abnormalities are uncommon in solitary lesions, advanced cases may show more complexity.

Juvenile xanthogranuloma is a rare histiocytic proliferation primarily affecting infants and young children, characterized by aberrant infiltration of histiocyte-derived cells in the skin, soft tissues and more rarely, visceral organs. Juvenile xanthogranuloma is generally considered to be a benign disorder; most lesions are solitary cutaneous nodules that resolve spontaneously without treatment. However, cases with extracutaneous involvement, multiple lesions, and/or systemic disease often require aggressive therapy. Though molecular studies have provided evidence of clonality in juvenile xanthogranuloma, in support of a neoplastic process, little is known about the genetic profile of juvenile xanthogranuloma. We used molecular inversion probe array technology to evaluate the genomic characteristics (copy number alterations or copy neutral-loss of heterozygosity) of 21 archived cases of juvenile xanthogranuloma (19 solitary, 1 diffuse cutaneous, 1 systemic). Four cases (19%) showed acquired, clonal alterations. Two lesions from a case of diffuse cutaneous juvenile xanthogranuloma showed distinct profiles: JXG-1a contained trisomy 5 and 17 and JXG-1b contained loss of heterozygosity in 5q. The systemic juvenile xanthogranuloma (JXG-2) showed multiple genomic alterations. Only two of 19 solitary juvenile xanthogranulomas showed abnormal genomic profiles: JXG-3 showed gains on 1q and 11q and JXG-4 showed a 7.2 Mb loss in 3p. No recurrent abnormalities were observed among these cases. The presence of non-recurrent copy number alterations in a subset of samples implies that copy number changes are unlikely driving pathogenesis in juvenile xanthogranuloma, but may be acquired during disease progression. The presence of genomic abnormalities in more advanced cases (ie, systemic and diffuse cutaneous juvenile xanthogranuloma) supports this notion, particularly as the advanced cases of juvenile xanthogranuloma presented more genomic complexity.

Genomic analysis of follicular dendritic cell sarcoma by molecular inversion probe array reveals tumor suppressor-driven biology.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies.

Xq28 duplication overlapping the int22h-1/int22h-2 region and including RAB39B and CLIC2 in a family with intellectual and developmental disability.

Duplications involving terminal Xq28 are a known cause of intellectual disability (ID) in males and in females with unfavorable X-inactivation patterns. Within Xq28, functional disomy of MECP2 causes a severe ID syndrome, however the dosage sensitivity of other Xq28 duplicated genes is less certain. Duplications involving the int22h-1/int22h-2 LCR-flanked region in distal Xq28 have recently been linked to a novel ID-associated phenotype. While evidence for the dosage sensitivity of this region is emerging, the phenotypic contribution of individual genes within the int22h-1/int22h-2-flanked region has yet to be determined. We report a familial case of a novel 774 kb Xq28-qter duplication, detected by cytogenomic microarray analysis, that partially overlaps the int22h-1/int22h-2-flanked region. This duplication and a 570 kb Xpter-p22.33 loss within the pseudoautosomal region were identified in three siblings, one female and two males, who presented with developmental delays/intellectual disability, mild dysmorphic features and short stature. Although unconfirmed, these results are suggestive of maternal inheritance of a recombinant X. We compare our clinical findings to patients with int22h-1/int22h-2-mediated duplications and discuss the potential pathogenicity of genes within the duplicated region, including those within the shared region of overlap, RAB39B and CLIC2.

Genomic landscape of cutaneous follicular lymphomas reveals 2 subgroups with clinically predictive molecular features.

Primary cutaneous follicle center lymphomas (PCFCLs) are indolent B-cell lymphomas that predominantly remain skin restricted and manageable with skin-directed therapy. Conversely, secondary cutaneous involvement by usual systemic follicular lymphoma (secondary cutaneous follicular lymphoma [SCFL]) has a worse prognosis and often necessitates systemic therapy. Unfortunately, no histopathologic or genetic features reliably differentiate PCFCL from SCFL at diagnosis. Imaging may miss low-burden internal disease in some cases of SCFLs, leading to misclassification as PCFCL. Whereas usual systemic FL is well characterized genetically, the genomic landscapes of PCFCL and SCFL are unknown. Herein, we analyzed clinicopathologic and immunophenotypic data from 30 cases of PCFCL and 10 of SCFL and performed whole-exome sequencing on 18 specimens of PCFCL and 6 of SCFL. During a median follow-up of 7 years, 26 (87%) of the PCFCLs remained skin restricted. In the remaining 4 cases, systemic disease developed within 3 years of diagnosis. Although the SCFLs universally expressed BCL2 and had BCL2 rearrangements, 73% of the PCFCLs lacked BCL2 expression, and only 8% of skin-restricted PCFCLs had BCL2 rearrangements. SCFLs showed low proliferation fractions, whereas 75% of PCFCLs had proliferation fractions >30%. Of the SCFLs, 67% had characteristic loss-of-function CREBBP or KMT2D mutations vs none in skin-restricted PCFCL. Both SCFL and skin-restricted PCFCL showed frequent TNFRSF14 loss-of-function mutations and copy number loss at chromosome 1p36. These data together establish PCFCL as a unique entity with biological features distinct from usual systemic FL and SCFL. We propose 3 criteria based on BCL2 rearrangement, chromatin-modifying gene mutations (CREBBP, KMT2D, EZH2, and EP300), and proliferation index to classify cutaneous FL specimens based on the likelihood of concurrent or future systemic spread.

Alternative ESC and ESC-like subunits of a polycomb group histone methyltransferase complex are differentially deployed during Drosophila development.

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. Here we describe a novel fly PcG repressor, called ESC-Like (ESCL), whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds.

Identification of two 14q32 deletions involving DICER1 associated with the development of DICER1-related tumors.

DICER1 encodes an RNase III endonuclease protein that regulates the production of small non-coding RNAs. Germline mutations in DICER1 are associated with an autosomal dominant hereditary cancer predisposition syndrome that confers an increased risk for the development of several rare childhood and adult-onset tumors, the most frequent of which include pleuropulmonary blastoma, ovarian sex cord-stromal tumors, cystic nephroma, and thyroid gland neoplasia. The majority of reported germline DICER1 mutations are truncating sequence-level alterations, suggesting that a loss-of-function type mechanism drives tumor formation in DICER1 syndrome. However, reports of patients with germline DICER1 whole gene deletions are limited, and thus far, only two have reported an association with tumor development. Here we report the clinical findings of three patients from two unrelated families with 14q32 deletions that encompass the DICER1 locus. The deletion identified in Family I is 1.4 Mb and was initially identified in a 6-year-old male referred for developmental delay, hypotonia, macrocephaly, obesity, and behavioral problems. Subsequent testing revealed that this deletion was inherited from his mother, who had a clinical history that included bilateral multinodular goiter and papillary thyroid carcinoma. The second deletion is 5.0 Mb and was identified in a 15-year-old female who presented with autism, coarse facial features, Sertoli-Leydig cell tumor, and Wilms' tumor. These findings provide additional supportive evidence that germline deletion of DICER1 confers an increased risk for DICER1-related tumor development, and provide new insight into the clinical significance of deletions involving the 14q32 region.

Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes.

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Genomic Analyses Identify Recurrent Alterations in Immune Evasion Genes in Diffuse Large B-Cell Lymphoma, Leg Type.

Cutaneous diffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas with a poor prognosis. To elucidate their genetic bases, we analyzed exome sequencing of 37 cutaneous DLBCLs, including 31 DLBCLs, leg type (DLBCL-LT) and 6 cutaneous DLBCLs-not otherwise specified (DLBCL-NOS). As reported previously, 77% of DLBCL-LT harbor NF-κB-activating MYD88 mutations. In nearly all MYD88-wild-type DLBCL-LT, we found cancer-promoting mutations that either activate the NF-κB pathway through alternative genes (NFKBIE or REL) or activate other canonical cancer pathways (BRAF, MED12, PIK3R1, and STAT3). After NF-κB, the second most commonly mutated pathway putatively enables immune evasion via mutations predicted to downregulate antigen processing (B2M, CIITA, HLA) or T-cell co-stimulation (CD58). DLBCL-LT have little genetic overlap with the genetically heterogeneous DLBCL-NOS. Instead, they resemble primary central nervous system and testicular large B-cell lymphomas (primary central nervous system lymphomas and primary testicular lymphomas). Like primary central nervous system lymphomas/primary testicular lymphomas, 40% of DLBCL-LT (vs. 0% of DLBCLs-not otherwise specified) harbored PDL1/PDL2 translocations, which lead to overexpression of PD-L1 or PD-L2 in 50% of the cases. Collectively, these data broaden our understanding of cutaneous DLBCLs and suggest novel therapeutic approaches (e.g., BRAF or PI3K inhibitors). Additionally, they suggest novel treatment paradigms, wherein DLBCL-LT can be targeted with strategies (e.g., immune checkpoint blockers) currently being developed for genomically similar primary central nervous system lymphomas/primary testicular lymphomas.

Deletions involving genes WHSC1 and LETM1 may be necessary, but are not sufficient to cause Wolf-Hirschhorn Syndrome.

Wolf-Hirschhorn syndrome (WHS) is a complex genetic disorder caused by the loss of genomic material from the short arm of chromosome 4. Genotype-phenotype correlation studies indicated that the loss of genes within 4p16.3 is necessary for expression of the core features of the phenotype. Within this region, haploinsufficiency of the genes WHSC1 and LETM1 is thought to be a major contributor to the pathogenesis of WHS. We present clinical findings for three patients with relatively small (<400 kb) de novo interstitial deletions that overlap WHSC1 and LETM1. 3D facial analysis was performed for two of these patients. Based on our findings, we propose that hemizygosity of WHSC1 and LETM1 is associated with a clinical phenotype characterized by growth deficiency, feeding difficulties, and motor and speech delays. The deletion of additional genes nearby WHSC1 and LETM1 does not result in a marked increase in the severity of clinical features, arguing against their haploinsufficiency. The absence of seizures and typical WHS craniofacial findings in our cohort suggest that deletion of distinct or additional 4p16.3 genes is necessary for expression of these features. Altogether, these results show that although loss-of-function for WHSC1 and/or LETM1 contributes to some of the features of WHS, deletion of additional genes is required for the full expression of the phenotype, providing further support that WHS is a contiguous gene deletion disorder.