Towards solving the genetic diagnosis odyssey in Iranian patients with congenital anomalies.

Understanding the underlying causes of congenital anomalies (CAs) can be a complex diagnostic journey. We aimed to assess the efficiency of exome sequencing (ES) and chromosomal microarray analysis (CMA) in patients with CAs among a population with a high fraction of consanguineous marriage. Depending on the patient's symptoms and family history, karyotype/Quantitative Fluorescence- Polymerase Chain Reaction (QF-PCR) (n = 84), CMA (n = 81), ES (n = 79) or combined CMA and ES (n = 24) were performed on 168 probands (66 prenatal and 102 postnatal) with CAs. Twelve (14.28%) probands were diagnosed by karyotype/QF-PCR and seven (8.64%) others were diagnosed by CMA. ES findings were conclusive in 39 (49.36%) families, and 61.90% of them were novel variants. Also, 64.28% of these variants were identified in genes that follow recessive inheritance in CAs. The diagnostic rate (DR) of ES was significantly higher than that of CMA in children from consanguineous families (P = 0·0001). The highest DR by CMA was obtained in the non-consanguineous postnatal subgroup and by ES in the consanguineous prenatal subgroup. In a population that is highly consanguineous, our results suggest that ES may have a higher diagnostic yield than CMA and should be considered as the first-tier test in the evaluation of patients with congenital anomalies.

Clinical whole-genome sequencing and FISH identify two different fusion partners for NUP98 in a patient with acute myeloid leukemia: A case report.

BACKGROUND: Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. CASE PRESENTATION: Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the likely underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. CONCLUSIONS: NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

High Coverage Highly Accurate Long-Read Sequencing of a Mouse Neuronal Cell Line Using the PacBio Revio Sequencer.

Recently, Pacific Biosciences released a new highly accurate long-read sequencer called the Revio System that is projected to generate 30× HiFi whole-genome sequencing for the human genome within one sequencing SMRT Cell. Mouse and human genomes are similar in size. In this study, we sought to test this new sequencer by characterizing the genome and epigenome of the mouse neuronal cell line Neuro-2a. We generated long-read HiFi whole-genome sequencing on three Revio SMRT Cells, achieving a total coverage of 98×, with 30×, 32×, and 36× coverage respectively for each of the three Revio SMRT Cells. We performed several tests on these data including single-nucleotide variant and small insertion detection using GPU-accelerated DeepVariant, structural variant detection with pbsv, methylation detection with pb-CpG-tools, and generating de novo assemblies with the HiCanu and hifiasm assemblers. Overall, we find consistency across SMRT Cells in coverage, detection of variation, methylation, and de novo assemblies for each of the three SMRT Cells.

High-depth next-generation sequencing panel testing in the evaluation of arteriovenous malformations.

Arteriovenous malformations (AVMs) are vascular lesions in which an overgrowth of blood vessels of varying sizes develops with one or more direct connections between the arterial and venous circulation. We performed a retrospective review of a cohort of 54 patients with AVMs referred to our clinical genomic laboratory for high-depth next-generation sequencing (NGS) panel of Disorders of Somatic Mosaicism (DoSM). Thirty-seven of 54 patients were female (68.5%). Among the 54 cases, 37 (68.5%) cases had pathogenic and/or likely pathogenic (P/LP) variants identified, two cases (3.7%) had variants of uncertain clinical significance, and the remaining 15 cases (27.8%) had negative results. MAP2K1 variants were found in 12 cases, followed by eight cases with KRAS variants and seven with TEK variants, and the remainder being identified in several other genes on the panel. Among the 37 positive cases, 32 cases had somatic alterations only; the remaining five cases had at least one germline P/LP variant, including four cases with PTEN and one with RASA1. Of note, two cases had the unexpected co-existence of two P/LP variants. In summary, this study illustrated the molecular diagnostic yield (68.5%) of this cohort of patients with a clinical indication of AVMs by our high-depth DoSM NGS panel.

Comparing Gene Panels for Non-Retinal Indications: A Systematic Review.

IMPORTANCE: The options for genetic testing continue to grow for ocular conditions, including optic atrophy, anterior segment dysgenesis, cataracts, corneal dystrophy, nystagmus, and glaucoma. Gene panels can vary in content and coverage, as we and others have evaluated in inherited retinal disease (IRD). OBJECTIVE: To describe gene panel testing options for inherited eye disease phenotypes and their differences. This review is important for making diagnostic decisions. EVIDENCE REVIEW: A licensed, certified genetic counselor (RP) used Concert Genetics and the search terms optic atrophy, corneal dystrophy, cataract, glaucoma, anterior segment dysgenesis, microphthalmia/anophthalmia, and nystagmus to identify available testing options performed by CLIA-certified commercial genetic testing laboratories. Other co-authors were surveyed with respect to genetic panels used for the indications of interest. Ophthalmic panels were then compared using Concert Genetics in addition to their own websites. FINDINGS: Panels from each clinical category were included and summarized. This comparison highlighted the differences and similarities between panels so that clinicians can make informed decisions. CONCLUSIONS: Access to genetic testing is increasing. The diagnostic yield of genetic testing is increasing. Each panel is different, so phenotyping or characterizing clinical characteristics that may help predict a specific genotype, as well as pre-test hypotheses regarding a genotype, should shape the choice of panels.

A comparative analysis of RAS variants in patients with disorders of somatic mosaicism.

PURPOSE: RAS genes (HRAS, KRAS, and NRAS) are commonly found to be mutated in cancers, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed. METHODS: A total of 938 individuals with suspected DoSM underwent high-sensitivity clinical next-generation sequencing-based testing. We investigated the mutational spectrum and genotype-phenotype associations of mosaic RAS variants. RESULTS: In this article, we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Furthermore, we present 12 individuals with HRAS and KRAS in-frame duplication/insertion (dup/ins) variants in the switch II domain. Among the 18.3% individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations of these. The median age at testing was higher and the variant allelic fraction was lower in individuals with in-frame dup/ins variants than those in individuals with mosaic RAS hotspots. CONCLUSION: Our work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in nonmalignant conditions.

Modifications to student quarantine policies in K-12 schools implementing multiple COVID-19 prevention strategies restores in-person education without increasing SARS-CoV-2 transmission risk, January-March 2021.

OBJECTIVE: To determine whether modified K-12 student quarantine policies that allow some students to continue in-person education during their quarantine period increase schoolwide SARS-CoV-2 transmission risk following the increase in cases in winter 2020-2021. METHODS: We conducted a prospective cohort study of COVID-19 cases and close contacts among students and staff (n = 65,621) in 103 Missouri public schools. Participants were offered free, saliva-based RT-PCR testing. The projected number of school-based transmission events among untested close contacts was extrapolated from the percentage of events detected among tested asymptomatic close contacts and summed with the number of detected events for a projected total. An adjusted Cox regression model compared hazard rates of school-based SARS-CoV-2 infections between schools with a modified versus standard quarantine policy. RESULTS: From January-March 2021, a projected 23 (1%) school-based transmission events occurred among 1,636 school close contacts. There was no difference in the adjusted hazard rates of school-based SARS-CoV-2 infections between schools with a modified versus standard quarantine policy (hazard ratio = 1.00; 95% confidence interval: 0.97-1.03). DISCUSSION: School-based SARS-CoV-2 transmission was rare in 103 K-12 schools implementing multiple COVID-19 prevention strategies. Modified student quarantine policies were not associated with increased school incidence of COVID-19. Modifications to student quarantine policies may be a useful strategy for K-12 schools to safely reduce disruptions to in-person education during times of increased COVID-19 community incidence.

Clinical whole-genome sequencing in cancer diagnosis.

Characterizing the genomic landscape of cancers is a routine part of clinical care that began with the discovery of the Philadelphia chromosome and has since coevolved with genomic technologies. Genomic analysis of tumors at the nucleotide level using DNA sequencing has revolutionized the understanding of cancer biology and identified new molecular drivers of disease that have led to therapeutic advances and improved patient outcomes. However, the application of next-generation sequencing in the clinical laboratory has generally been limited until very recently to targeted analysis of selected genes. Recent technological innovations and reductions in sequencing costs are now able to deliver the long-promised goal of tumor whole-genome sequencing as a practical clinical assay.

Acceptance of Saliva-Based Specimen Collection for SARS-CoV-2 Testing Among K-12 Students, Teachers, and Staff.

OBJECTIVE: Saliva specimens collected in school populations may offer a more feasible, noninvasive alternative to nasal swabs for large-scale COVID-19 testing efforts in kindergarten through 12th grade (K-12) schools. We investigated acceptance of saliva-based COVID-19 testing among quarantined K-12 students and their parents, teachers, and staff members who recently experienced a SARS-CoV-2 exposure in school. METHODS: We surveyed 719 participants, in person or by telephone, who agreed to or declined a free saliva-based COVID-19 reverse-transcription polymerase chain reaction test as part of a surveillance investigation about whether they would have consented to testing if offered a nasal swab instead. We conducted this investigation in 6 school districts in Greene County (n = 3) and St. Louis County (n = 3), Missouri, from January 25 through March 23, 2021. RESULTS: More than one-third (160 of 446) of K-12 students (or their parents or guardians), teachers, and staff members who agreed to a saliva-based COVID-19 test indicated they would have declined testing if specimen collection were by nasal swab. When stratified by school level, 51% (67 of 132) of elementary school students or their parents or guardians would not have agreed to testing if a nasal swab was offered. CONCLUSIONS: Some students, especially those in elementary school, preferred saliva-based COVID-19 testing to nasal swab testing. Use of saliva-based testing might increase voluntary participation in screening efforts in K-12 schools to help prevent the spread of SARS-CoV-2.

SARS-CoV-2 screening testing in schools for children with intellectual and developmental disabilities.

BACKGROUND: Transmission of SARS-CoV-2 in schools primarily for typically developing children is rare. However, less is known about transmission in schools for children with intellectual and developmental disabilities (IDD), who are often unable to mask or maintain social distancing. The objectives of this study were to determine SARS-CoV-2 positivity and in-school transmission rates using weekly screening tests for school staff and students and describe the concurrent deployment of mitigation strategies in six schools for children with IDD. METHODS: From November 23, 2020, to May, 28, 2021, weekly voluntary screening for SARS-CoV-2 with a high sensitivity molecular-based saliva test was offered to school staff and students. Weekly positivity rates were determined and compared to local healthcare system and undergraduate student screening data. School-based transmission was assessed among participants quarantined for in-school exposure. School administrators completed a standardized survey to assess school mitigation strategies. RESULTS: A total of 59 students and 416 staff participated. An average of 304 school staff and students were tested per week. Of 7289 tests performed, 21 (0.29%) new SARS-CoV-2 positive cases were identified. The highest weekly positivity rate was 1.2% (n = 4) across all schools, which was less than community positivity rates. Two cases of in-school transmission were identified, each among staff, representing 2% (2/103) of participants quarantined for in-school exposure. Mitigation strategies included higher than expected student mask compliance, reduced room capacity, and phased reopening. CONCLUSIONS: During 24 weeks that included the peak of the COVID-19 pandemic in winter 2020-21, we found lower rates of SARS-CoV-2 screening test positivity among staff and students of six schools for children with IDD compared to community rates. In-school transmission of SARS-CoV-2 was low among those quarantined for in-school exposure. However, the impact of the emerging SARS-CoV-2 Delta variant on the effectiveness of these proven mitigation strategies remains unknown. TRIAL REGISTRATION: Prior to enrollment, this study was registered at ClinicalTrials.gov on September 25, 2020, identifier NCT04565509 , titled Supporting the Health and Well-being of Children with Intellectual and Developmental Disability During COVID-19 Pandemic.

SARS-CoV-2 Screening Testing in Schools for Children with Intellectual and Developmental Disabilities.

BACKGROUNDTransmission of SARS-CoV-2 in schools primarily for typically developing children is rare. However, less is known about transmission in schools for children with intellectual and developmental disabilities (IDD), who are often unable to mask or maintain social distancing. The objectives of this study were to determine SARS-CoV-2 positivity and in-school transmission rates using weekly screening tests for school staff and students and describe the concurrent deployment of mitigation strategies in six schools for children with IDD.METHODSFrom 11/23/20 to 5/28/21, weekly voluntary screening for SARS-CoV-2 with a high sensitivity molecular-based saliva test was offered to school staff and students. Weekly positivity rates were determined and compared to local healthcare system and undergraduate student screening data. School-based transmission was assessed among participants quarantined for in-school exposure. School administrators completed a standardized survey to assess school mitigation strategies.RESULTSA total of 59 students and 416 staff participated. An average of 304 school staff and students were tested per week. Of 7,289 tests performed, 21 (0.29%) new SARS-CoV-2 positive cases were identified. The highest weekly positivity rate was 1.2% (n = 4) across all schools, which was less than community positivity rates. Two cases of in-school transmission were identified, each among staff, representing 2% (2/103) of participants quarantined for in-school exposure. Mitigation strategies included higher than expected student mask compliance, reduced room capacity, and phased reopening.CONCLUSIONSDuring 24 weeks that included the peak of the COVID-19 pandemic, we found no evidence for elevated SARS-CoV-2 screening test positivity among staff and students of six schools for children with IDD compared to community rates. In-school transmission of SARS-CoV-2 was low among those quarantined for in-school exposure.Clinical Trial RegistryPrior to enrollment, this study was registered at ClinicalTrials.gov on 9/25/2020, identifier NCT04565509, titled Supporting the Health and Well-being of Children with Intellectual and Developmental Disability During COVID-19 Pandemic (https://clinicaltrials.gov/ct2/show/NCT04565509?term=NCT04565509).

Pilot Investigation of SARS-CoV-2 Secondary Transmission in Kindergarten Through Grade 12 Schools Implementing Mitigation Strategies - St. Louis County and City of Springfield, Missouri, December 2020.

Many kindergarten through grade 12 (K-12) schools offering in-person learning have adopted strategies to limit the spread of SARS-CoV-2, the virus that causes COVID-19 (1). These measures include mandating use of face masks, physical distancing in classrooms, increasing ventilation with outdoor air, identification of close contacts,* and following CDC isolation and quarantine guidance† (2). A 2-week pilot investigation was conducted to investigate occurrences of SARS-CoV-2 secondary transmission in K-12 schools in the city of Springfield, Missouri, and in St. Louis County, Missouri, during December 7-18, 2020. Schools in both locations implemented COVID-19 mitigation strategies; however, Springfield implemented a modified quarantine policy permitting student close contacts aged ≤18 years who had school-associated contact with a person with COVID-19 and met masking requirements during their exposure to continue in-person learning.§ Participating students, teachers, and staff members with COVID-19 (37) from 22 schools and their school-based close contacts (contacts) (156) were interviewed, and contacts were offered SARS-CoV-2 testing. Among 102 school-based contacts who received testing, two (2%) had positive test results indicating probable school-based SARS-CoV-2 secondary transmission. Both contacts were in Springfield and did not meet criteria to participate in the modified quarantine. In Springfield, 42 student contacts were permitted to continue in-person learning under the modified quarantine; among the 30 who were interviewed, 21 were tested, and none received a positive test result. Despite high community transmission, SARS-CoV-2 transmission in schools implementing COVID-19 mitigation strategies was lower than that in the community. Until additional data are available, K-12 schools should continue implementing CDC-recommended mitigation measures (2) and follow CDC isolation and quarantine guidance to minimize secondary transmission in schools offering in-person learning.

Diagnostic Utility of Next-Generation Sequencing for Disorders of Somatic Mosaicism: A Five-Year Cumulative Cohort.

Disorders of somatic mosaicism (DoSM) are a diverse group of syndromic and non-syndromic conditions caused by mosaic variants in genes that regulate cell survival and proliferation. Despite overlap in gene space and technical requirements, few clinical labs specialize in DoSM compared to oncology. We adapted a high-sensitivity next-generation sequencing cancer assay for DoSM in 2014. Some 343 individuals have been tested over the past 5 years, 58% of which had pathogenic and likely pathogenic (P/LP) findings, for a total of 206 P/LP variants in 22 genes. Parameters associated with the high diagnostic yield were: (1) deep sequencing (∼2,000× coverage), (2) a broad gene set, and (3) testing affected tissues. Fresh and formalin-fixed paraffin embedded tissues performed equivalently for identification of P/LP variants (62% and 71% of individuals, respectively). Comparing cultured fibroblasts to skin biopsies suggested that culturing might boost the allelic fraction of variants that confer a growth advantage, specifically gain-of-function variants in PIK3CA. Buccal swabs showed high diagnostic sensitivity in case subjects where disease phenotypes manifested in the head or brain. Peripheral blood was useful as an unaffected comparator tissue to determine somatic versus constitutional origin but had poor diagnostic sensitivity. Descriptions of all tested individuals, specimens, and P/LP variants included in this cohort are available to further the study of the DoSM population.

Outcome of infants diagnosed with 3-methyl-crotonyl-CoA-carboxylase deficiency by newborn screening.

INTRODUCTION: 3-Methyl CoA carboxylase (3-MCC) deficiency is an inborn error of metabolism in the catabolism of the amino acid leucine. Original reports suggested this disorder was associated with significant neurological and biochemical effects. However newborn screening has identified a higher than expected incidence of this disorder with apparent normal outcome in most cases. METHOD: A retrospective analysis of thirty-five cases of 3-MCC deficiency identified by newborn screening and diagnosed by enzyme or molecular analysis. RESULTS: There was a strong inverse correlation between initial C5OH level and residual enzyme activity. A few reports of hypoglycemia, ketosis, poor feeding/failure to thrive or fasting intolerance were reported, but there was no clear relationship between symptoms and residual enzyme activity. Developmental outcome included several children with mental retardation (including one with Down syndrome and one with schizencephaly) and two with Autism Spectrum disorders but there was no apparent relationship to residual enzyme activity. Free carnitine deficiency was relatively common. DISCUSSION: Although residual enzyme activity was clearly related to metabolite elevation, there was no apparent relationship with other measures of outcome. The number of reports of neurologic abnormalities or metabolic symptoms (poor feeding, hypoglycemia, fasting intolerance, etc.) is concerning, but the significance is unclear in this retrospective sample.

Improving accuracy of Tay Sachs carrier screening of the non-Jewish population: analysis of 34 carriers and six late-onset patients with HEXA enzyme and DNA sequence analysis.

The purpose of this study was to determine whether combining different testing modalities namely beta-hexosaminidase A (HEXA) enzyme analysis, HEXA DNA common mutation assay, and HEXA gene sequencing could improve the sensitivity for carrier detection in non-Ashkenazi (AJ) individuals. We performed a HEXA gene sequencing assay, a HEXA DNA common mutation assay, and a HEXA enzyme assay on 34 self-reported Tay-Sachs disease (TSD) carriers, six late-onset patients with TSD, and one pseudodeficiency allele carrier. Sensitivity of TSD carrier detection was 91% for gene sequencing compared with 91% for the enzyme assay and 52% for the DNA mutation assay. Gene sequencing combined with enzyme testing had the highest sensitivity (100%) for carrier detection. Gene sequencing detected four novel mutations, three of which are predicted to be disease causing [118.delT, 965A-->T (D322V), and 775A-->G (T259A)]. Gene sequencing is useful in identifying rare mutations in patients with TSD and their families, in evaluating spouses of known carriers for TSD who have indeterminate enzyme analysis and negative for common mutation analysis, and in resolving ambiguous enzyme testing results.

Asymptomatic maternal combined homocystinuria and methylmalonic aciduria (cblC) detected through low carnitine levels on newborn screening.

A symptom-free woman gave birth to a girl with a low carnitine level on newborn screening. The baby was unaffected, but the mother had biochemical abnormalities and mutations characteristic of the cblC defect of vitamin B(12) metabolism (late-onset form). This patient with cblC was detected through her infant's newborn screening.