Recommendations for risk allele evidence curation, classification, and reporting from the ClinGen Low Penetrance/Risk Allele Working Group.

PURPOSE: Genetic variants at the low end of the penetrance spectrum have historically been challenging to interpret because their high population frequencies exceed the disease prevalence of the associated condition, leading to a lack of clear segregation between the variant and disease. There is currently substantial variation in the classification of these variants, and no formal classification framework has been widely adopted. The Clinical Genome Resource Low Penetrance/Risk Allele Working Group was formed to address these challenges and promote harmonization within the clinical community. METHODS: The work presented here is the product of internal and community Likert-scaled surveys in combination with expert consensus within the Working Group. RESULTS: We formally recognize risk alleles and low-penetrance variants as distinct variant classes from those causing highly penetrant disease that require special considerations regarding their clinical classification and reporting. First, we provide a preferred terminology for these variants. Second, we focus on risk alleles and detail considerations for reviewing relevant studies and present a framework for the classification these variants. Finally, we discuss considerations for clinical reporting of risk alleles. CONCLUSION: These recommendations support harmonized interpretation, classification, and reporting of variants at the low end of the penetrance spectrum.

Loss of heterozygosity leading to incorrect HLA typing for platelet-transfusion refractory patient.

BACKGROUND: Management of platelet-transfusion refractory (PR) patients due to anti-HLA antibodies includes the provision of HLA-matched (HLAm) platelets (PLT) or PLTs that are negative for HLA antigens corresponding to the recipient antibodies. Obtaining HLAm PLTs is predicated on accurate HLA antigen typing of the recipient and donor. Here, we present the clinical implications of a case involving loss of heterozygosity (LOH) in a patient presented for PR workup. STUDY DESIGN AND METHODS: HLA typing was performed by three methods: (1) Real-time PCR; (2) Sequence-specific oligonucleotide (SSO) typing test; and (3) Next-Generation Sequencing (NGS). Cytogenomic SNP microarray was utilized to assess LOH. RESULTS: A 30-year-old female with newly diagnosed acute myelogenous leukemia was found to be PR secondary to HLA sensitization. A peripheral blood (PB) sample, containing 93% myeloid blast cells, was sent for HLA typing for the provision of HLAm PLTs. HLA typing revealed homozygosity at the HLA-A locus but was heterozygous at the -B and -C loci. After chemotherapy, HLA typing on a new PB sample, devoid of blast cells, identified HLA-A locus heterozygosity, which was subsequently confirmed by real-time PCR and NGS. Cytogenomic SNP microarray analysis demonstrated LOH of the HLA-A locus on chromosome 6p in the pretreatment sample but not in the posttreatment sample. CONCLUSION: In hematologic patients with high tumor burden, HLA homozygosity should be viewed with suspicion for potential LOH. Therefore, HLA testing should be repeated, preferably with a non-hematological source (e.g., buccal swab) or following successful reduction of the tumor burden.

Clinical Validation and Diagnostic Utility of Optical Genome Mapping for Enhanced Cytogenomic Analysis of Hematological Neoplasms.

The current standard-of-care cytogenetic techniques for the analysis of hematological malignancies include karyotyping, fluorescence in situ hybridization, and chromosomal microarray, which are labor intensive and time and cost prohibitive, and they often do not reveal the genetic complexity of the tumor, demonstrating the need for alternative technology for better characterization of these tumors. Herein, we report the results from our clinical validation study and demonstrate the utility of optical genome mapping (OGM), evaluated using 92 sample runs (including replicates) that included 69 well-characterized unique samples (59 hematological neoplasms and 10 controls). The technical performance (quality control metrics) resulted in 100% first-pass rate, with analytical performance (concordance) showing a sensitivity of 98.7%, a specificity of 100%, and an accuracy of 99.2%. OGM demonstrated robust technical, analytical performance, and interrun, intrarun, and interinstrument reproducibility. The limit of detection was determined to be at 5% allele fraction for aneuploidy, translocation, interstitial deletion, and duplication. OGM identified several additional structural variations, revealing the genomic architecture in these neoplasms that provides an opportunity for better tumor classification, prognostication, risk stratification, and therapy selection. Overall, OGM has outperformed the standard-of-care tests in this study and demonstrated its potential as a first-tier cytogenomic test for hematologic malignancies.

Evaluating use of changing technologies for rapid next-generation sequencing in pediatrics.

BACKGROUND: Rapid next-generation sequencing (NGS) offers the potential to shorten the diagnostic process and improve the care of acutely ill children. The goal of this study was to report our findings, including benefits and limitations, of a targeted NGS panel and rapid genome sequencing (rGS) in neonatal and pediatric acute clinical care settings. METHODS: Retrospective analysis of patient characteristics, diagnostic yields, turnaround time, and changes in management for infants and children receiving either RapSeq, a targeted NGS panel for 4500+ genes, or rGS, at the University of Utah Hospital and Primary Children's Hospital, from 2015 to 2020. RESULTS: Over a 5-year period, 142 probands underwent rapid NGS: 66 received RapSeq and 76 rGS. Overall diagnostic yield was 39%. In the majority of diagnostic cases, there were one or more changes in clinical care management. Of note, 7% of diagnoses identified by rGS would not have been identified by RapSeq. CONCLUSIONS: Our results indicate that rapid NGS impacts acute pediatric care in real-life clinical settings. Although affected by patient selection criteria, diagnostic yields were similar to those from clinical trial settings. Future studies are needed to determine relative advantages, including cost, turnaround time, and benefits for patients, of each approach in specific clinical circumstances. IMPACT: The use of comprehensive Mendelian gene panels and genome sequencing in the clinical setting allows for early diagnosis of patients in neonatal, pediatric, and cardiac intensive care units and impactful change in management. Diagnoses led to significant changes in management for several patients in lower acuity inpatient units supporting further exploration of the utility of rapid sequencing in these settings. This study reviews the limitations of comparing sequencing platforms in the clinical setting and the variables that should be considered in evaluating diagnostic rates across studies.

Effective variant filtering and expected candidate variant yield in studies of rare human disease.

In studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

Targeted gene panel sequencing for the rapid diagnosis of acutely ill infants.

BACKGROUND: Exome/genome sequencing (ES/GS) have been recently used in neonatal and pediatric/cardiac intensive care units (NICU and PICU/CICU) to diagnose and care for acutely ill infants, but the effectiveness of targeted gene panels for these purposes remains unknown. METHODS: RapSeq, a newly developed panel targeting 4,503 disease-causing genes, was employed on selected patients in our NICU/PICU/CICU. Twenty trios were sequenced from October 2015 to March 2017. We assessed diagnostic yield, turnaround times, and clinical consequences. RESULTS: A diagnosis was made in 10/20 neonates (50%); eight had de novo variants (ASXL1, CHD, FBN1, KMT2D, FANCB, FLNA, PAX3), one was a compound heterozygote for CHAT, and one had a maternally inherited GNAS variant. Preliminary reports were generated by 9.6 days (mean); final reports after Sanger sequencing at 16.3 days (mean). In all positive infants, the diagnosis changed management. In a case with congenital myasthenia, diagnosis and treatment occurred at 17 days versus 7 months in a historical control. CONCLUSIONS: This study shows that a gene panel that includes the majority of known disease-causing genes can rapidly identify a diagnosis in a large number of tested infants. Due to simpler deployment and interpretation and lower costs, this approach might represent an alternative to ES/GS in the NICU/PICU/CICU.

ZMIZ1 Variants Cause a Syndromic Neurodevelopmental Disorder.

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.

Novel mutation in CCBE 1 as a cause of recurrent hydrops fetalis from Hennekam lymphangiectasia-lymphedema syndrome-1.

Whole exome sequencing (WES) was used to determine the etiology of recurrent hydrops fetalis in this case of Hennekam lymphangiectasia-lymphedema syndrome-1. WES is a useful approach for diagnosing rare single-gene conditions with nonspecific phenotypes and should be considered early in the diagnostic process of investigating fetal abnormalities.

Pathogenic ASXL1 somatic variants in reference databases complicate germline variant interpretation for Bohring-Opitz Syndrome.

The clinical interpretation of genetic variants has come to rely heavily on reference population databases such as the Exome Aggregation Consortium (ExAC) database. Pathogenic variants in genes associated with severe, pediatric-onset, highly penetrant, autosomal dominant conditions are assumed to be absent or rare in these databases. Exome sequencing of a 6-year-old female patient with seizures, developmental delay, dysmorphic features, and failure to thrive identified an ASXL1 variant previously reported as causative of Bohring-Opitz syndrome (BOS). Surprisingly, the variant was observed seven times in the ExAC database, presumably in individuals without BOS. Although the BOS phenotype fit, the presence of the variant in reference population databases introduced ambiguity in result interpretation. Review of the literature revealed that acquired somatic mosaicism of ASXL1 variants (including pathogenic variants) during hematopoietic clonal expansion can occur with aging in healthy individuals. We examined all ASXL1 truncating variants in the ExAC database and determined most are likely somatic. Failure to consider somatic mosaicism may lead to the inaccurate assumption that conditions like BOS have reduced penetrance, or the misclassification of potentially pathogenic variants.

Clinical utility of a next generation sequencing panel assay for Marfan and Marfan-like syndromes featuring aortopathy.

Aortopathy can be defined as aortic dilation, aneurysm, dissection, and tortuosity. Familial aortopathy may occur secondary to fibrillin-1 (FBN1) mutations in the setting of Marfan syndrome, or may occur as a result of other genetic defects with different, but occasionally overlapping, phenotypes. Because of the phenotypic overlap and genetic heterogeneity of disorders featuring aortopathy, we developed a next generation sequencing (NGS) assay and comparative genomic hybridization (CGH) array to detect mutations in 10 genes that cause thoracic aortic aneurysms (TAAs). Here, we report on the clinical and molecular findings in 175 individuals submitted for aortopathy panel testing at ARUP laboratories. Ten genes associated with heritable aortopathies were targeted using hybridization capture prior to sequencing. NGS results were analyzed, and variants were confirmed using Sanger sequencing. Array CGH was used to detect copy-number variation. Of 175 individuals, 18 had a pathogenic mutation and 32 had a variant of uncertain significance (VUS). Most pathogenic mutations (72%) were identified in FBN1. A novel large SMAD3 duplication and FBN1 deletion were identified. Over half who had TAAs or other aortic involvement tested negative for a mutation, suggesting that additional aortopathy genes exist. We anticipate that the clinical sensitivity of at least 10.3% will rise with VUS reclassification and as additional genes are identified and included in the panel. The aortopathy NGS panel aids in the timely molecular diagnosis of individuals with disorders featuring aortopathy and guides proper treatment.

Stress and coping in parents of children with Prader-Willi syndrome: Assessment of the impact of a structured plan of care.

Hyperphagia, developmental delays, and maladaptive behaviors are common in Prader-Willi syndrome (PWS) likely resulting in heightened parental stress. Objectives were to evaluate stress, describe usefulness of coping behaviors, and assess the impact of a structured Plan of Care (PC) on parents with children with PWS. Parents answered Perceived Stress Scale (PSS-14), Coping Health Inventory for Parents (CHIP), and narrative/demographic surveys. The PC was introduced to a cohort of parents after completion of the PSS-14 and CHIP and re-administered 4-6 month after the introduction of the PC. Higher parental stress (n = 57) was observed compared to the general population, and associated with parent's age, number of children living at home, and child's age and residential setting. "Maintaining family integration, cooperation, and an optimistic definition of the situation" was the most useful coping pattern. Thirty-eight parents answered the PSS-14 and CHIP after the PC. Parental stress decreased after the PC (P = 0.035). Coping behaviors related to "maintaining family integration" increased after the PC (P = 0.042). Women and men preferred different coping patterns before and after the PC. In conclusion, parental stress is increased in PWS, and a PC decreased stress and increased coping behaviors related to family stability for parents with children with PWS.

Genetic counselor review of genetic test orders in a reference laboratory reduces unnecessary testing.

Genetic tests are routinely ordered by health care providers (HCPs) within a wide range of medical specialties. Many providers have limited knowledge or experience with ordering and interpreting genetic tests; thus, test order errors are common. Rigorous review of genetic test orders by genetic counselors (GCs) can provide a direct financial benefit to medical institutions, patients and insurers. GCs at ARUP (Associated Regional University Pathologists) Laboratories routinely perform a preanalytic assessment of complex molecular genetic test orders that includes reviewing clinical and family history information and considering the clinical utility and cost-effectiveness of ordered tests. GCs contact the ordering institution and/or HCP as needed to collect additional clinical information and confirm the test order or suggest alternative testing based on the provided information. A retrospective review of the GC-facilitated test changes over a 21-month period at ARUP laboratories was performed. Approximately 26% of all requests for complex genetic tests assessing germ line mutations were changed following GC review. Testing fees associated with canceled tests were summed to estimate the cost-savings resulting from GC-facilitated test reviews. The test review process resulted in an average reduction in charges to the referring institutions of $48,000.00 per month. GC review of genetic test orders for appropriateness and clinical utility reduces healthcare costs to hospitals, insurers, and patients.