Diagnostic gene sequencing panels: from design to report-a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Gene sequencing panels are a powerful diagnostic tool for many clinical presentations associated with genetic disorders. Advances in DNA sequencing technology have made gene panels more economical, flexible, and efficient. Because the genes included on gene panels vary widely between laboratories in gene content (e.g., number, reason for inclusion, evidence level for gene-disease association) and technical completeness (e.g., depth of coverage), standards that address technical and clinical aspects of gene panels are needed. This document serves as a technical standard for laboratories designing, offering, and reporting gene panel testing. Although these principles can apply to multiple indications for genetic testing, the primary focus is on diagnostic gene panels (as opposed to carrier screening or predictive testing) with emphasis on technical considerations for the specific genes being tested. This technical standard specifically addresses the impact of gene panel content on clinical sensitivity, specificity, and validity-in the context of gene evidence for contribution to and strength of evidence for gene-disease association-as well as technical considerations such as sequencing limitations, presence of pseudogenes/gene families, mosaicism, transcript choice, detection of copy-number variants, reporting, and disclosure of assay limitations.

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.

Assessing copy number aberrations and copy-neutral loss-of-heterozygosity across the genome as best practice: An evidence-based review from the Cancer Genomics Consortium (CGC) working group for chronic lymphocytic leukemia.

The prognostic role of cytogenetic analysis is well-established in B-cell chronic lymphocytic leukemia (CLL). Approximately 80% of patients have a cytogenetic aberration. Interphase FISH panels have been the gold standard for cytogenetic evaluation, but conventional cytogenetics allows detection of additional abnormalities, including translocations, complex karyotypes and multiple clones. Whole genome copy number assessment, currently performed by chromosomal microarray analysis (CMA), is particularly relevant in CLL for the following reasons: (1) copy number alterations (CNAs) represent key events with biologic and prognostic significance; (2) DNA from fresh samples is generally available; and (3) the tumor burden tends to be relatively high in peripheral blood. CMA also identifies novel copy number variants and copy-neutral loss-of-heterozygosity (CN-LOH), and can refine deletion breakpoints. The Cancer Genomics Consortium (CGC) Working Group for CLL has performed an extensive literature review to describe the evidence-based clinical utility of CMA in CLL. We provide suggestions for the integration of CMA into clinical use and list recurrent copy number alterations, regions of CN-LOH and mutated genes to aid in interpretation.

Coupling clinical exome sequencing with functional characterization studies to diagnose a patient with familial Mediterranean fever and MED13L haploinsufficiency syndromes.

Clinicians should consider that clinical exome sequencing provides the unique potential to disentangle complex phenotypes into multiple genetic etiologies. Further, functional studies on variants of uncertain significance are necessary to arrive at an accurate diagnosis for the patient.

Structural Chromosomal Rearrangements Require Nucleotide-Level Resolution: Lessons from Next-Generation Sequencing in Prenatal Diagnosis.

In this exciting era of "next-gen cytogenetics," integrating genomic sequencing into the prenatal diagnostic setting is possible within an actionable time frame and can provide precise delineation of balanced chromosomal rearrangements at the nucleotide level. Given the increased risk of congenital abnormalities in newborns with de novo balanced chromosomal rearrangements, comprehensive interpretation of breakpoints could substantially improve prediction of phenotypic outcomes and support perinatal medical care. Herein, we present and evaluate sequencing results of balanced chromosomal rearrangements in ten prenatal subjects with respect to the location of regulatory chromatin domains (topologically associated domains [TADs]). The genomic material from all subjects was interpreted to be "normal" by microarray analyses, and their rearrangements would not have been detected by cell-free DNA (cfDNA) screening. The findings of our systematic approach correlate with phenotypes of both pregnancies with untoward outcomes (5/10) and with healthy newborns (3/10). Two pregnancies, one with a chromosomal aberration predicted to be of unknown clinical significance and another one predicted to be likely benign, were terminated prior to phenotype-genotype correlation (2/10). We demonstrate that the clinical interpretation of structural rearrangements should not be limited to interruption, deletion, or duplication of specific genes and should also incorporate regulatory domains of the human genome with critical ramifications for the control of gene expression. As detailed in this study, our molecular approach to both detecting and interpreting the breakpoints of structural rearrangements yields unparalleled information in comparison to other commonly used first-tier diagnostic methods, such as non-invasive cfDNA screening and microarray analysis, to provide improved genetic counseling for phenotypic outcome in the prenatal setting.

An infant with MLH3 variants, FOXG1-duplication and multiple, benign cranial and spinal tumors: A clinical exome sequencing study.

A 4-month-old male infant presented with severe developmental delay, cerebellar, brainstem, and cutaneous hemangiomas, bilateral tumors (vestibular, hypoglossal, cervical, and lumbar spinal), and few café-au-lait macules. Cerebellar and lumbar tumor biopsies revealed venous telangiectasia and intraneural perineuroma, respectively. Sequencing NF1, NF2, and RASA1 (blood), and NF2 and SMARCB1 (lumbar biopsy) was negative for pathogenic mutations. Clinical exome sequencing (CES), requested for tumor syndrome diagnosis, revealed two heterozygous missense variants, c.359T>C;p.Phe120Ser and c.3344G>A;p.Arg1115Gln, in MLH3 (NM_001040108.1), a DNA mismatch repair (MMR) gene, Polyphen-predicted as probably damaging, and benign, respectively. Sanger sequencing confirmed both variants in the proband, and their absence in the mother; biological father unavailable. Both biopsied tissues were negative for microsatellite instability, and expressed MLH1, MSH2, PMS2, MSH6, and MLH3 immunohistochemically. Chromosomal microarray showed a 133 kb segment copy number duplication of 14q12 region encompassing FOXG1, possibly explaining the developmental delay, but not the tumors. The presence of MLH3 variants with multiple benign neural and vascular tumors was intriguing for their possible role in the pathogenesis of these neoplasms, which were suspicious for, but not diagnostic of, constitutional MMR deficiency. However, functional assays of non-neoplastic patient-derived cells showed intact base-base MMR function. Also, no previous FOXG1-aberrant patient was reported with tumors. We now report a 3-year-old FOXG1-duplicated patient with a yet undescribed tumor syndrome with clinical features of neurofibromatosis types I and II, where several validation studies could not ascertain the significance of CES findings; further studies may elucidate precise mechanisms and diagnosis for clinical management, including tumor surveillance.

De novo nonsense mutations in KAT6A, a lysine acetyl-transferase gene, cause a syndrome including microcephaly and global developmental delay.

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.

Clinical exome sequencing for genetic identification of rare Mendelian disorders.

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% (2 of 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.

Exome sequencing in the clinical diagnosis of sporadic or familial cerebellar ataxia.

IMPORTANCE: Cerebellar ataxias are a diverse collection of neurologic disorders with causes ranging from common acquired etiologies to rare genetic conditions. Numerous genetic disorders have been associated with chronic progressive ataxia and this consequently presents a diagnostic challenge for the clinician regarding how to approach and prioritize genetic testing in patients with such clinically heterogeneous phenotypes. Additionally, while the value of genetic testing in early-onset and/or familial cases seems clear, many patients with ataxia present sporadically with adult onset of symptoms and the contribution of genetic variation to the phenotype of these patients has not yet been established. OBJECTIVE: To investigate the contribution of genetic disease in a population of patients with predominantly adult- and sporadic-onset cerebellar ataxia. DESIGN, SETTING, AND PARTICIPANTS: We examined a consecutive series of 76 patients presenting to a tertiary referral center for evaluation of chronic progressive cerebellar ataxia. MAIN OUTCOMES AND MEASURES: Next-generation exome sequencing coupled with comprehensive bioinformatic analysis, phenotypic analysis, and clinical correlation. RESULTS: We identified clinically relevant genetic information in more than 60% of patients studied (n = 46), including diagnostic pathogenic gene variants in 21% (n = 16), a notable yield given the diverse genetics and clinical heterogeneity of the cerebellar ataxias. CONCLUSIONS AND RELEVANCE: This study demonstrated that clinical exome sequencing in patients with adult-onset and sporadic presentations of ataxia is a high-yield test, providing a definitive diagnosis in more than one-fifth of patients and suggesting a potential diagnosis in more than one-third to guide additional phenotyping and diagnostic evaluation. Therefore, clinical exome sequencing is an appropriate consideration in the routine genetic evaluation of all patients presenting with chronic progressive cerebellar ataxia.

X chromosome exome sequencing reveals a novel ALG13 mutation in a nonsyndromic intellectual disability family with multiple affected male siblings.

X-linked intellectual disability (XLID) is a heterogeneous condition associated with mutations in >100 genes, accounting for over 10% of all cases of intellectual impairment. The majority of XLID cases show nonsyndromic forms (NSXLID), in which intellectual disability is the sole clinically consistent manifestation. Here we performed X chromosome exome (X-exome) sequencing to identify the causative mutation in an NSXLID family with four affected male siblings and five unaffected female siblings. The X-exome sequencing at 88× coverage in one affected male sibling revealed a novel missense mutation (p.Tyr1074Cys) in the asparagine-linked glycosylation 13 homolog (ALG13) gene. Segregation analysis by Sanger sequencing showed that the all affected siblings were hemizygous and the mother was heterozygous for the mutation. Recently, a de novo missense mutation in ALG13 has been reported in a patient with X-linked congenital disorders of glycosylation type I. Our study reports the first case of NSXLID caused by a mutation in ALG13 involved in protein N-glycosylation.

De novo single exon deletion of AUTS2 in a patient with speech and language disorder: a review of disrupted AUTS2 and further evidence for its role in neurodevelopmental disorders.

The autism susceptibility candidate 2 (AUTS2) gene is suggested to play a critical role in early brain development, and its association with intellectual disability (ID), autism spectrum disorders, and other neurodevelopmental disorders (NDDs) has recently gained more attention. Genomic rearrangements and copy number variations (CNVs) involving AUTS2 have been implicated in a range of NDDs with or without congenital malformations and dysmorphic features. Here we report a 62 kb de novo deletion encompassing exon 6 of AUTS2 detected by chromosomal microarray analysis (CMA) in a 4.5 year-old female patient with severe speech and language disorder, history of tonic-clonic movements, and pes planus with eversion of the feet. This is one of the smallest de novo intragenic deletions of AUTS2 described in patients with NDDs. We reviewed previously reported small pathogenic CNVs (<300 kb) in 19 cases, and correlated their specific locations within AUTS2 as well as presence of enhancers, regulatory elements, and CpG islands with the clinical findings of these cases and our patient. Our report provides additional insight into the clinical spectrum of AUTS2 disruptions.

An unusual case of Philadelphia chromosome-positive chronic myelogenous leukemia with trisomy 19 presenting with megakaryoblastosis and myelofibrosis.

Initial identification of chronic myelogenous leukemia is very important since targeted therapy leads to life-saving remission. Rarely, chronic myelogenous leukemia presents with an unusual picture, making the diagnosis challenging. We describe such a case of chronic myelogenous leukemia in blast crisis in a previously healthy 61-year-old woman. The patient presented with fever, myalgias, and night sweats and was first worked up for an infectious etiology. Because of persistent anemia, a bone marrow biopsy was performed that revealed fibrosis with increased megakaryoblasts. Even though initial cytogenetic studies could not be performed because of "dry tap" aspirate, persistent efforts for cytogenetic studies were made, including a "squeeze preparation" from the core biopsy, which revealed t(9;22)(q34;q11.2) and trisomy 19. The patient was treated with tyrosine kinase inhibitors, chemotherapy, and subsequently an allogeneic stem cell transplant. She is in persistent remission. This case illustrates a complex presentation of chronic myelogenous leukemia and provides an overview of morphologic cues and the importance of performing cytogenetic studies that led to the diagnosis.

Atypical rearrangement involving 3'-IGH@ and a breakpoint at least 400 Kb upstream of an intact MYC in a CLL patient with an apparently balanced t(8;14)(q24.1;q32) and negative MYC expression.

The t(8;14)(q24.1;q32), the cytogenetic hallmark of Burkitt's lymphoma, is also found, but rarely, in cases of chronic lymphocytic leukemia (CLL). Such translocation typically results in a MYC-IGH@ fusion subsequently deregulating and overexpressing MYC on der 14q32. In CLL, atypical rearrangements resulting in its gain or loss, within or outside of IGH@ or MYC locus, have been reported, but their clinical significance remains uncertain. Herein, we report a 67 year-old male with complex cytogenetic findings of apparently balanced t(8;14) and unreported complex rearrangements of IGH@ and MYC loci. His clinical, morphological and immunophenotypic features were consistent with the diagnosis of CLL.Interphase FISH studies revealed deletions of 11q22.3 and 13q14.3, and an extra copy of IGH@, indicative of rearrangement. Karyotype analysis showed an apparently balanced t(8;14)(q24.1;q32). Sequential GPG-metaphase FISH studies revealed abnormal signal patterns: rearrangement of IGH break apart probe with the 5'-IGH@ on derivative 8q24.1 and the 3'-IGH@ retained on der 14q; absence of MYC break apart-specific signal on der 8q; and, the presence of unsplit 5'-MYC-3' break apart probe signals on der 14q. The breakpoint on 8q24.1 was found to be at least 400 Kb upstream of 5' of MYC. In addition, FISH studies revealed two abnormal clones; one with 13q14.3 deletion, and the other, with concurrent 11q deletion and atypical rearrangements. Chromosome microarray analysis (CMA) detected a 7.1 Mb deletion on 11q22.3-q23.3 including ATM, a finding consistent with FISH results. While no significant copy number gain or loss observed on chromosomes 8, 12 and 13, a 455 Kb microdeletion of uncertain clinical significance was detected on 14q32.33. Immunohistochemistry showed co-expression of CD19, CD5, and CD23, positive ZAP-70 expression and absence of MYC expression. Overall findings reveal an apparently balanced t(8;14) and atypical complex rearrangements involving 3'-IGH@ and a breakpoint at least 400 Kb upstream of MYC, resulting in the relocation of the intact 5'-MYC-3' from der 8q, and apposition to 3'-IGH@ at der 14q. This case report provides unique and additional cytogenetic data that may be of clinical significance in such a rare finding in CLL. It also highlights the utility of conventional and sequential metaphase FISH in understanding complex chromosome anomalies and their association with other clinical findings in patients with CLL. To the best of our knowledge, this is the first CLL reported case with such an atypical rearrangement in a patient with a negative MYC expression.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Human endometrial cells express elevated levels of pluripotent factors and are more amenable to reprogramming into induced pluripotent stem cells.

The human endometrium is a tissue with remarkable plasticity and regenerative capacity. Additionally, endometrial cells can be retrieved using minimally invasive procedures, which makes them an ideal source for reprogramming into a pluripotent state. Endometrial cells were obtained from donors in their fifth decade and reprogrammed into induced pluripotent stem (iPS) cells using retroviral transduction with SOX2, OCT4, KLF4, and MYC. The human endometrial cells displayed accelerated expression of endogenous NANOG and OCT4 during reprogramming compared with neonatal skin fibroblasts. As a result, iPS cell colonies that could be subcultured and propagated were established as early as 12 d after transduction rather than the usually reported 3-4 wk for other cell types. After 3 wk of reprogramming, the human endometrial cells also yielded significantly higher numbers of iPS colonies in comparison with the neonatal skin fibroblasts. Although the efficiency of iPS colony formation varied depending on the donor, the basal level of endogenous expression of the defined factors was positively correlated with reprogramming efficiency. The reprogramming resulted in an average colony-forming efficiency of 0.49 ± 0.10%, with a range from 0.31-0.66%, compared with the neonatal skin fibroblasts, resulting in an average efficiency of 0.03 ± 0.00% per transduction, with a range from 0.02-0.03%. Our studies show that the human endometrium expresses elevated levels of pluripotent factors, which with additional defined factors, results in significantly more efficient and accelerated generation of induced pluripotent stem cells compared with conventional somatic cells.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.