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-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

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.

Diagnostic cytogenetic testing following positive noninvasive prenatal screening results: a clinical laboratory practice resource of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: ACMG Clinical Laboratory Practice Resources are developed primarily as an educational tool for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these practice resources is voluntary and does not necessarily assure a successful medical outcome. This Clinical Laboratory Practice Resource should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this Clinical Laboratory Practice Resource. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Noninvasive prenatal screening (NIPS) using cell-free DNA has been rapidly adopted into prenatal care. Since NIPS is a screening test, diagnostic testing is recommended to confirm all cases of screen-positive NIPS results. For cytogenetics laboratories performing confirmatory testing on prenatal diagnostic samples, a standardized testing algorithm is needed to ensure that the appropriate testing takes place. This algorithm includes diagnostic testing by either chorionic villi sampling or amniocentesis samples and encompasses chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray.

Chromosomal microarray testing identifies a 4p terminal region associated with seizures in Wolf-Hirschhorn syndrome.

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion syndrome involving variable size deletions of the 4p16.3 region. Seizures are frequently, but not always, associated with WHS. We hypothesised that the size and location of the deleted region may correlate with seizure presentation. METHODS: Using chromosomal microarray analysis, we finely mapped the breakpoints of copy number variants (CNVs) in 48 individuals with WHS. Seizure phenotype data were collected through parent-reported answers to a comprehensive questionnaire and supplemented with available medical records. RESULTS: We observed a significant correlation between the presence of an interstitial 4p deletion and lack of a seizure phenotype (Fisher's exact test p=3.59e-6). In our cohort, there were five individuals with interstitial deletions with a distal breakpoint at least 751 kbp proximal to the 4p terminus. Four of these individuals have never had an observable seizure, and the fifth individual had a single febrile seizure at the age of 1.5 years. All other individuals in our cohort whose deletions encompass the terminal 751 kbp region report having seizures typical of WHS. Additional examples from the literature corroborate these observations and further refine the candidate seizure susceptibility region to a region 197 kbp in size, starting 368 kbp from the terminus of chromosome 4. CONCLUSIONS: We identify a small terminal region of chromosome 4p that represents a seizure susceptibility region. Deletion of this region in the context of WHS is sufficient for seizure occurrence.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy.

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon-level duplications, including so-called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof-of-concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.

Mosaic deletion of 20pter due to rescue by somatic recombination.

We report on a unique case of a mosaic 20pter-p13 deletion due to a somatic repair event identified by allele differentiating single nucleotide polymorphism (SNP) probes on chromosomal microarray. Small terminal deletions of 20p have been reported in a few individuals and appear to result in a variable phenotype. This patient was a 24-month-old female who presented with failure to thrive and speech delay. Chromosomal microarray analysis (CMA) performed on peripheral blood showed a 1.6 Mb deletion involving the terminus of 20p (20pter-20p13). This deletion appeared mosaic by CMA and this suspicion was confirmed by fluorescence in situ hybridization (FISH) analysis. Additionally, the deletion interval at 20p was directly adjacent to 15 Mb of mosaic copy-neutral loss of heterozygosity (LOH). The pattern of SNP probes was highly suggestive of a somatic repair event that resulted in rescue of the deleted region using the non-deleted homologue as a template. Structural mosaicism is rare and most often believed to be due to a postzygotic mechanism. This case demonstrates the additional utility of allele patterns to help distinguish mechanisms and in this case identified the possibility of either a post-zygotic repair of a germline deletion or a post-zygotic deletion with somatic recombination repair in a single step.

Braddock-Carey syndrome: A 21q22 contiguous gene syndrome encompassing RUNX1.

In 1994, Braddock and Carey first reported two unrelated girls with a new multiple malformation syndrome. The primary features included Pierre Robin sequence, persistent neonatal-onset thrombocytopenia, agenesis of the corpus callosum, a distinctive facies, enamel hypoplasia, and severe developmental delay. Since that time, there have been multiple other reported patients with a similar phenotype. In addition, several reports of thrombocytopenia and developmental delay have been documented in association with deletions in the Down syndrome critical region at 21q22. The similarity of the reported cases with deletions involving 21q22 with the clinical presentation of the two patients with Braddock-Carey syndrome resulted in a reinvestigation of the genetic etiology of these two patients 20 years after the original study. This investigation provides evidence that the etiology of this and other "Fanconi-like" disorders represent a newly recognized contiguous gene deletion syndrome involving 21q22 and specifically, the RUNX1 gene. © 2016 Wiley Periodicals, Inc.

Wolf-Hirschhorn syndrome: A review and update.

Since 4p- was first described in 1961, significant progress has been made in our understanding of this classic deletion disorder. We have been able to establish a more complete picture of the WHS phenotype associated with distal 4p monosomy, and we are working to delineate the phenotypic effects when each gene on distal 4p is hemizygous. Our aim is to provide genotype-specific anticipatory guidance and recommendations to families of individuals with a diagnosis of WHS. In addition, establishing the molecular underpinnings of the disorder will potentially suggest targets for molecular treatments. Thus, the next step is to determine the precise effects of specific gene deletions. As we look forward to deepening our understanding of distal 4p deletion, our focus will continue to be on the establishment of robust genotype-phenotype correlations and the penetrance of these phenotypes. We will continue to follow our WHS cohort closely as they age to determine the presence or absence of some of these comorbidities, including hepatic neoplasms, hematopoietic dysfunction, and recurrence of seizures. We will also continue to refine the critical regions for other phenotypes as we enroll additional (hopefully informative) participants into the research study and as the mechanisms of the genes in these regions are elucidated. New animal models will also be developed to further our understanding of the effects of hemizygosity as well as to serve as models for treatment development.

Turner Syndrome in Girls Presenting with Coarctation of the Aorta.

OBJECTIVE: To evaluate the frequency of Turner syndrome in a population-based, statewide cohort of girls with coarctation of the aorta. STUDY DESIGN: The Utah Birth Defects Network was used to ascertain a cohort of girls between 1997 and 2011 with coarctation of the aorta. Livebirths with isolated coarctation of the aorta or transverse arch hypoplasia were included and patients with complex congenital heart disease not usually seen in Turner syndrome were excluded. RESULTS: Of 244 girls with coarctation of the aorta, 77 patients were excluded, leaving a cohort of 167 girls; 86 patients (51%) had chromosomal studies and 21 (12.6%) were diagnosed with Turner syndrome. All patients were diagnosed within the first 4 months of life and 5 (24%) were diagnosed prenatally. Fifteen patients (71%) had Turner syndrome-related findings in addition to coarctation of the aorta. Girls with mosaicism were less likely to have Turner syndrome-associated findings (3/6 mosaic girls compared with 12/17 girls with non-mosaic 45,X). Twelve girls (57%) diagnosed with Turner syndrome also had a bicommissural aortic valve. CONCLUSION: At least 12.6% of girls born with coarctation of the aorta have karyotype-confirmed Turner syndrome. Such a high frequency, combined with the clinical benefits of an early diagnosis, supports genetic screening for Turner syndrome in girls presenting with coarctation of the aorta.

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

Observation and prediction of recurrent human translocations mediated by NAHR between nonhomologous chromosomes.

Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ∼359-kb and ∼215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24 bp within interchromosomal paralogous LCRs of ∼130 kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal translocations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55 bp in ∼7.8-kb paralogous subunits of 95.3% sequence identity located in the ∼579-kb (chr 8) and ∼287-kb (chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide a computationally determined genome-wide "recurrent translocation map."

Clinical utility of chromosomal microarray analysis of DNA from buccal cells: detection of mosaicism in three patients.

Mosaic chromosomal abnormalities are relatively common. However, mosaicism may be missed due to multiple factors including failure to recognize clinical indications and order appropriate testing, technical limitations of diagnostic assays, or sampling tissue (s) in which mosaicism is either not present, or present at very low levels. Blood leukocytes have long been the "gold standard" sample for cytogenetic analysis; however, the culturing process for routine chromosome analysis can complicate detection of mosaicism since the normal cell line may have a growth advantage in culture, or may not be present in the cells that produce metaphases (the lymphocytes). Buccal cells are becoming increasingly utilized for clinical analyses and are proving to have many advantages. Buccal swabs allow for simple and noninvasive DNA collection. When coupled with a chromosomal microarray that contains single nucleotide polymorphic probes, analysis of buccal cells can maximize a clinician's opportunity to detect cytogenetic mosaicism. We present three cases of improved diagnosis of mosaic aberrations using buccal specimens for chromosomal microarray analysis. In each case, the aberration was either undetectable in blood or present at such a low level it likely could have gone undetected. These cases highlight the limitations of certain laboratory methodologies for identifying mosaicism. We also present practice implications for genetic counselors, including clinic workflow changes and counseling approaches based on increasing use of buccal samples.

ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013.

Microarray methodologies, including array comparative genomic hybridization and single-nucleotide polymorphism-detecting arrays, are accepted as an appropriate first-tier test for the evaluation of imbalances associated with intellectual disability, autism, and multiple congenital anomalies. This technology also has applicability in prenatal specimens. To assist clinical laboratories in validation of microarray methodologies for constitutional applications, the American College of Medical Genetics and Genomics has produced the following revised professional standards and guidelines.

Chromosomal loss of 3q26.3-3q26.32, involving a partial neuroligin 1 deletion, identified by genomic microarray in a child with microcephaly, seizure disorder, and severe intellectual disability.

Neuroligin 1 (NLGN1) is one of five members of the neuroligin gene family and may represent a candidate gene for neurological disorders, as members of this family are involved in formation and remodeling of central nervous system synapses. NLGN1 is expressed predominantly in the central nervous system, where it dimerizes and then binds with ß-neurexin to form a functional synapse. Mutations in neurexin 1 (NRXN1) as well as two other members of the neuroligin family, NLGN3 and NLGN4, have been associated with autism and mutations in NLGN4 have also been associated with intellectual disability, seizures, and EEG abnormalities. Genomic microarray is recommended for the detection of chromosomal gains or losses in patients with intellectual disability and multiple congenital anomalies. Results of uncertain significance are not uncommon. Parental studies can provide additional information by demonstrating that the imbalance is either de novo or inherited, and therefore is more or less likely to be causative of the clinical phenotype. However, the possibility that even inherited deletions and duplications may play a role in the phenotype of the proband cannot be excluded as many copy number variants associated with neurodevelopmental conditions show incomplete penetrance and may be inherited from an unaffected parent. Here, we report on a patient with a 2.2 Mb deletion at 3q26.3-3q26.32-encompassing the terminal end of NLGN1 and the entire NAALADL2 gene-detected by genomic microarray, and confirmed by FISH and real-time quantitative PCR. The same size deletion was subsequently found in her healthy, asymptomatic, adult mother.