Legacy Genetic Testing Results for Cancer Susceptibility: How Common are Conflicting Classifications in a Large Variant Dataset from Multiple Practices?

PURPOSE: The classification of germline variants may differ between labs and change over time. We apply a variant harmonization tool, Ask2Me VarHarmonizer, to map variants to ClinVar and identify discordant variant classifications in a large multipractice variant dataset. METHODS: A total of 7496 variants sequenced between 1996 and 2019 were collected from 11 clinical practices. Variants were mapped to ClinVar, and lab-reported and ClinVar variant classifications were analyzed and compared. RESULTS: Of the 4798 unique variants identified, 3699 (77%) were mappable to ClinVar. Among mappable variants, variants of unknown significance (VUS) accounted for 74% of lab-reported classifications and 60% of ClinVar classifications. Lab-reported and ClinVar discordances were present in 783 unique variants (21.2% of all mappable variants); 121 variants (2.5% of all unique variants) had within-practice lab-reported discordances; and 56 variants (1.2% of all unique variants) had lab-reported discordances across practices. The unmappable variants were associated with a higher proportion of lab-reported pathogenic classifications (50% vs. 21%, p < 0.0001) and a lower proportion of lab-reported VUS classifications (46% vs. 74%, p < 0.0001). CONCLUSIONS: Our study shows that discordant variant classification occurs frequently, which may lead to inappropriate recommendations for prophylactic treatments or clinical management.

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes.

Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNA-sequencing (RNA-seq) to generate a splicing profile of 18 TSGs (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53) in 345 whole-blood samples from healthy donors. We subsequently demonstrated that this approach can detect mis-splicing by comparing splicing profiles from the control dataset to profiles generated from whole blood of individuals previously identified with pathogenic germline splicing variants in these genes. To assess the utility of our TSG splicing profile to prospectively identify pathogenic splicing variants, we performed concurrent capture DNA and RNA-seq in a cohort of 1000 patients with suspected hereditary cancer syndromes. This approach improved the diagnostic yield in this cohort, resulting in a 9.1% relative increase in the detection of pathogenic variants, demonstrating the utility of performing simultaneous DNA and RNA genetic testing in a clinical context.

A study of over 35,000 women with breast cancer tested with a 25-gene panel of hereditary cancer genes.

BACKGROUND: As panel testing becomes more common in clinical practice, it is important to understand the prevalence and trends associated with the pathogenic variants (PVs) identified. This is especially true for genetically heterogeneous cancers, such as breast cancer (BC), in which PVs in different genes may be associated with various risks and cancer subtypes. The authors evaluated the outcomes of genetic testing among women who had a personal history of BC. METHODS: A total of 35,409 women with a single diagnosis of BC who underwent clinical genetic testing with a 25-gene panel were included in the current analysis. Women with multiple BCs and men with BC were excluded. The frequency and distribution of PVs were assessed for the overall cohort, among women with triple-negative BC (TNBC) (n = 4797), and by age at diagnosis. RESULTS: PVs were identified in 9.3% of women tested; 51.5% of PVs were identified in genes other than breast cancer 1 (BRCA1) and BRCA2, including checkpoint kinase 2 (CHEK2) (11.7%), ataxia telangiectasia mutated (ATM; ATM serine/threonine kinase) (9.7%), and partner and localizer of BRCA2 (PALB2) (9.3%). The prevalence of PVs in BRCA1, PALB2, BRCA1-associated RING domain 1 (BARD1), BRCA1-interacting protein C-terminal helicase 1 (BRIP1), and RAD51 paralog C (RAD51C) was statistically higher among women with TNBC. The PV rate was higher among women aged <40 years, lower among women aged >59 years, and relatively constant (8.5%-9.0%) among women who were diagnosed between ages 40 and 59 years. CONCLUSIONS: These results demonstrate that panel testing increased the number of women identified as carrying a PV in this cohort compared with BRCA testing alone. Furthermore, the proportion of women identified who carried a PV in this cohort did not decrease between ages 40 and 59 years. Cancer 2017;123:1721-1730. © 2017 American Cancer Society.

Dosage changes of a segment at 17p13.1 lead to intellectual disability and microcephaly as a result of complex genetic interaction of multiple genes.

The 17p13.1 microdeletion syndrome is a recently described genomic disorder with a core clinical phenotype of intellectual disability, poor to absent speech, dysmorphic features, and a constellation of more variable clinical features, most prominently microcephaly. We identified five subjects with copy-number variants (CNVs) on 17p13.1 for whom we performed detailed clinical and molecular studies. Breakpoint mapping and retrospective analysis of published cases refined the smallest region of overlap (SRO) for microcephaly to a genomic interval containing nine genes. Dissection of this phenotype in zebrafish embryos revealed a complex genetic architecture: dosage perturbation of four genes (ASGR1, ACADVL, DVL2, and GABARAP) impeded neurodevelopment and decreased dosage of the same loci caused a reduced mitotic index in vitro. Moreover, epistatic analyses in vivo showed that dosage perturbations of discrete gene pairings induce microcephaly. Taken together, these studies support a model in which concomitant dosage perturbation of multiple genes within the CNV drive the microcephaly and possibly other neurodevelopmental phenotypes associated with rearrangements in the 17p13.1 SRO.

Intragenic deletions of the IGF1 receptor gene in five individuals with psychiatric phenotypes and developmental delay.

Haploinsufficiency of the gene encoding the insulin-like growth factor 1 receptor (IGF1R), either caused by telomeric 15q26 deletions, or by heterozygous point mutations in IGF1R, segregate with short stature and various other phenotypes, including microcephaly and dysmorphic facial features. Psychomotor retardation and behavioral anomalies have been seen in some cases. Here we report small, intragenic deletions of IGF1R, identified by chromosome microarray analysis in two unrelated families affected primarily with neuropsychiatric phenotypes including developmental delay, intellectual disability and aggressive/autoaggressive behaviors. The deletions are in frame, and both wild-type and mutant mRNAs are expressed as measured by quantitative real-time PCR. While short stature is considered a phenotypic hallmark of IGF1R haploinsufficiency, the present report suggests that in frame exon deletions of IGF1R present predominantly with cognitive and neuropsychiatric phenotypes.

Mutations in SYNGAP1 cause intellectual disability, autism, and a specific form of epilepsy by inducing haploinsufficiency.

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.

Deletions in chromosome 6p22.3-p24.3, including ATXN1, are associated with developmental delay and autism spectrum disorders.

Interstitial deletions of the short arm of chromosome 6 are rare and have been associated with developmental delay, hypotonia, congenital anomalies, and dysmorphic features. We used array comparative genomic hybridization in a South Carolina Autism Project (SCAP) cohort of 97 subjects with autism spectrum disorders (ASDs) and identified an ~ 5.4 Mb deletion on chromosome 6p22.3-p23 in a 15-year-old patient with intellectual disability and ASDs. Subsequent database queries revealed five additional individuals with overlapping submicroscopic deletions and presenting with developmental and speech delay, seizures, behavioral abnormalities, heart defects, and dysmorphic features. The deletion found in the SCAP patient harbors ATXN1, DTNBP1, JARID2, and NHLRC1 that we propose may be responsible for ASDs and developmental delay.

Int22h-1/int22h-2-mediated Xq28 rearrangements: intellectual disability associated with duplications and in utero male lethality with deletions.

BACKGROUND: X linked intellectual disability (XLID) is common, with an estimated prevalence of 1/1000. The expanded use of array comparative genomic hybridisation (CGH) has led to the identification of several XLID-associated copy-number variants. METHODS: Array CGH analysis was performed using chromosomal microarray with ∼105 000 oligonucleotides covering the entire genome. Confirmatory fluorescence in situ hybridisation analyses were subsequently performed. Chromosome X-inactivation (XCI) was assessed using methylation-sensitive restriction enzyme digestion followed by PCR amplification. RESULTS: A novel ∼0.5 Mb duplication in Xq28 was identified in four cognitively impaired males who share behavioural abnormalities (hyperactivity and aggressiveness) and characteristic facial features (high forehead, upper eyelid fullness, broad nasal bridge and thick lower lip). These duplications were inherited from mothers with skewed XCI and are mediated by nonallelic homologous recombination between the low-copy repeat regions int22h-1 and int22h-2, which, in addition to int22h-3, are also responsible for inversions disrupting the factor VIII gene in haemophilia A. In addition, we have identified a reciprocal deletion in a girl and her mother, both of whom exhibit normal cognition and completely skewed XCI. The mother also had two spontaneous abortions. CONCLUSIONS: The phenotypic similarities among subjects with int22h-1/int22h-2-mediated Xq28 duplications suggest that such duplications are responsible for a novel XLID syndrome. The reciprocal deletion may not be associated with a clinical phenotype in carrier females due to skewed XCI, but may be lethal for males in utero. Advancements in array CGH technology have enabled the identification of such small, clinically relevant copy-number variants.

Detection of clinically relevant exonic copy-number changes by array CGH.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

A small recurrent deletion within 15q13.3 is associated with a range of neurodevelopmental phenotypes.

We report a recurrent 680-kb deletion within chromosome 15q13.3 in ten individuals, from four unrelated families, with neurodevelopmental phenotypes including developmental delay, mental retardation and seizures. This deletion likely resulted from nonallelic homologous recombination between low-copy repeats on the normal and inverted region of chromosome 15q13.3. Although this deletion also affects OTUD7A, accumulated data suggest that haploinsufficiency of CHRNA7 is causative for the majority of neurodevelopmental phenotypes in the 15q13.3 microdeletion syndrome.

Redefined genomic architecture in 15q24 directed by patient deletion/duplication breakpoint mapping.

We report four new patients with a submicroscopic deletion in 15q24 manifesting developmental delay, short stature, hypotonia, digital abnormalities, joint laxity, genital abnormalities, and characteristic facial features. These clinical features are shared with six recently reported patients with a 15q24 microdeletion, supporting the notion that this is a recognizable syndrome. We describe a case of an ~2.6 Mb microduplication involving a portion of the minimal deletion critical region in a 15-year-old male with short stature, mild mental retardation, attention deficit hyperactivity disorder, Asperger syndrome, decreased joint mobility, digital abnormalities, and characteristic facial features. Some of these features are shared with a recently reported case with a 15q24 microduplication involving the minimal deletion critical region. We also report two siblings and their mother with duplication adjacent and distal to this region exhibiting mild developmental delay, hypotonia, tapering fingers, characteristic facial features, and prominent ears. The deletion and duplication breakpoints were mapped by array comparative genomic hybridization and the genomic structure in 15q24 was analyzed further. Surprisingly, in addition to the previously recognized three low-copy repeat clusters (BP1, BP2, and BP3), we identified two other paralogous low-copy repeat clusters that likely mediated the formation of alternative sized 15q24 genomic rearrangements via non-allelic homologous recombination.