Publications by authors named "Keith Nykamp"

28 Publications

  • Page 1 of 1

Systematic use of phenotype evidence in clinical genetic testing reduces the frequency of variants of uncertain significance.

Am J Med Genet A 2022 May 16. Epub 2022 May 16.

Invitae Corporation, San Francisco, California, USA.

Guidelines for variant interpretation include criteria for incorporating phenotype evidence, but this evidence is inconsistently applied. Systematic approaches to using phenotype evidence are needed. We developed a method for curating disease phenotypes as highly or moderately predictive of variant pathogenicity based on the frequency of their association with disease-causing variants. To evaluate this method's accuracy, we retrospectively reviewed variants with clinical classifications that had evolved from uncertain to definitive in genes associated with curated predictive phenotypes. To demonstrate the clinical validity and utility of this approach, we compared variant classifications determined with and without predictive phenotype evidence. The curation method was accurate for 93%-98% of eligible variants. Among variants interpreted using highly predictive phenotype evidence, the percentage classified as pathogenic or likely pathogenic was 80%, compared with 46%-54% had the evidence not been used. Positive results among individuals harboring variants with highly predictive phenotype-guided interpretations would have been missed in 25%-37% of diagnostic tests and 39%-50% of carrier screens had other approaches to phenotype evidence been used. In summary, predictive phenotype evidence associated with specific curated genes can be systematically incorporated into variant interpretation to reduce uncertainty and increase the clinical utility of genetic testing.
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http://dx.doi.org/10.1002/ajmg.a.62779DOI Listing
May 2022

The global prevalence and ethnic heterogeneity of primary ciliary dyskinesia gene variants: a genetic database analysis.

Lancet Respir Med 2022 May 17;10(5):459-468. Epub 2022 Jan 17.

Herman Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address:

Background: Primary ciliary dyskinesia (PCD) is a motile ciliopathy characterised by otosinopulmonary infections. Inheritance is commonly autosomal recessive, with extensive locus and allelic heterogeneity. The prevalence is uncertain. Most genetic studies have been done in North America or Europe. The aim of the study was to estimate the worldwide prevalence and ethnic heterogeneity of PCD.

Methods: We calculated the allele frequency of disease-causing variants in 29 PCD genes associated with autosomal recessive inheritance in 182 681 unique individuals to estimate the global prevalence of PCD in seven ethnicities (African or African American, Latino, Ashkenazi Jewish, Finnish, non-Finnish European, east Asian, and south Asian). We began by aggregating variants that had been interpreted by Invitae, San Francisco, CA, USA, a genetics laboratory with PCD expertise. We then determined the allele frequency of each variant (pathogenic, likely pathogenic, or variant of uncertain significance [VUS]) in the Genome Aggregation Database (gnomAD), a publicly available next-generation sequencing database that aggregates exome and genome sequencing information from a wide variety of large-scale projects and stratifies allele counts by ethnicity. Using the Hardy-Weinberg equilibrium equation, we were able to calculate a lower-end prevalence of PCD for each ethnicity by including only pathogenic and likely pathogenic variants; and upper-end prevalence by also including VUS. This approach was similar to previous work on Li-Fraumeni (TP53 variants) prevalence. We were not diagnosing PCD, but rather estimating prevalence based on known variants.

Findings: The overall minimum global prevalence of PCD is calculated to be at least one in 7554 individuals, although this is likely to be an underestimate because some variants currently classified as VUS might be disease-causing and some pathogenic variants might not be detected by our methods. In the overall cohort, Invitae data could be included for variants without gnomAD data for a primary ethnicity. When using only gnomAD allele frequencies to calculate prevalence in individual ethnicities, the estimated prevalence of PCD was lower in each ethnicity compared with the overall cohort. This is because the overall cohort includes additional data from the Invitae database such as copy number variants and other variants not present in gnomAD. With gnomAD we found the expected PCD frequency to be higher in individuals of African ancestry than in most other populations (excluding VUS: 1 in 9906 in African or African American vs 1 in 10 388 in non-Finnish European vs 1 in 14 606 in east Asian vs 1 in 16 309 in Latino; including VUS: 1 in 106 in African or African American vs 1 in 178 in non-Finnish European vs 1 in 196 in Latino vs 1 in 188 in east Asian). In addition, we found that the top 5 genes most commonly implicated in PCD differed across ethnic ancestries and contrasted commonly published findings.

Interpretation: PCD appears to be more common than has been recognised, particularly in individuals of African ancestry. We identified gene distributions that differ from those in previous European and North American studies. These results could have an international impact on case identification. Our analytic approach can be expanded as more PCD loci are identified, and could be adapted to study the prevalence of other inherited diseases.

Funding: None.
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http://dx.doi.org/10.1016/S2213-2600(21)00453-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9064931PMC
May 2022

Fumarate hydratase variant prevalence and manifestations among individuals receiving germline testing.

Cancer 2022 02 1;128(4):675-684. Epub 2021 Nov 1.

Institute of Urologic Oncology, University of California Los Angeles, Los Angeles, California.

Background: Germline variants in fumarate hydratase (FH) are associated with autosomal dominant (AD) hereditary leiomyomatosis and renal cell cancer (HLRCC) and autosomal recessive (AR) fumarase deficiency (FMRD). The prevalence and cancer penetrance across different FH variants remain unclear.

Methods: A database containing 120,061 records from individuals undergoing cancer germline testing was obtained. FH variants were classified into 3 categories: AD HLRCC variants, AR FMRD variants, and variants of unknown significance (VUSs). Individuals with variants from these categories were compared with those with negative genetic testing.

Results: FH variants were detected in 1.3% of individuals (AD HLRCC, 0.3%; AR FMRD, 0.4%; VUS, 0.6%). The rate of AD HLRCC variants discovered among reportedly asymptomatic individuals without a clear indication for HLRCC testing was 1 in 2668 (0.04%). In comparison with those with negative genetic testing, the renal cell carcinoma (RCC) prevalence was elevated with AD HLRCC variants (17.0% vs 4.5%; P < .01) and VUSs (6.4% vs 4.5%; P = .02) but not with AR FMRD variants.

Conclusions: The prevalence of HLRCC discovered incidentally on germline testing is similar to recent population carrier estimates, and this suggests that this is a relatively common cancer syndrome. Compared with those with negative genetic testing, those with VUSs had an elevated risk of RCC, whereas those with AR FMRD variants did not.
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http://dx.doi.org/10.1002/cncr.33997DOI Listing
February 2022

Elucidating clinical phenotypic variability associated with the polyT tract and TG repeats in CFTR.

Hum Mutat 2021 09 10;42(9):1165-1172. Epub 2021 Jul 10.

Invitae, San Francisco, California, USA.

Biallelic pathogenic variants in CFTR manifest as cystic fibrosis (CF) or other CFTR-related disorders (CFTR-RDs). The 5T allele, causing alternative splicing and reduced protein activity, is modulated by the adjacent TG repeat element, though previous data have been limited to small, selective cohorts. Here, the risk and spectrum of phenotypes associated with the CFTR TG-T5 haplotype variants (TG11T5, TG12T5, and TG13T5) in the absence of the p.Arg117His variant are evaluated. Individuals who received physician-ordered next-generation sequencing of CFTR were included. TG[11-13]T5 variant frequencies (biallelic or with another CF-causing variant [CFvar]) were calculated. Clinical information reported by the ordering provider or the individual was examined. Among 548,300 individuals, the T5 minor allele frequency (MAF) was 4.2% (TG repeat distribution: TG11 = 68.1%, TG12 = 29.5%, TG13 = 2.4%). When present with a CFvar, each TG[11-13]T5 variant was significantly enriched in individuals with a high suspicion of CF or CFTR-RD (personal/family history of CF/CFTR-RD) compared to those with a low suspicion for CF or CFTR-RD (hereditary cancer screening, CFTR not requisitioned). Compared to CFvar/CFvar individuals, those with TG[11-13]T5/CFvar generally had single-organ involvement, milder symptoms, variable expressivity, and reduced penetrance. These data improve our understanding of disease risks associated with TG[11-13]T5 variants and have important implications for reproductive genetic counseling.
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http://dx.doi.org/10.1002/humu.24250DOI Listing
September 2021

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation.

Am J Hum Genet 2021 04 19;108(4):696-708. Epub 2021 Mar 19.

Invitae, 1400 16th St, San Francisco, CA 94103, USA. Electronic address:

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.
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http://dx.doi.org/10.1016/j.ajhg.2021.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059334PMC
April 2021

Prioritizing genes for systematic variant effect mapping.

Bioinformatics 2021 04;36(22-23):5448-5455

Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.

Motivation: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variants of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation.

Results: Here, we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes (i) by the number of unique missense VUS that had been reported to ClinVar; (ii) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS and (iii) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation toward greater impact on clinical variant interpretation.

Availability And Implementation: Source code available at: https://github.com/rothlab/mave-gene-prioritization.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa1008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016487PMC
April 2021

Frequency of Cystic Fibrosis Transmembrane Conductance Regulator Variants in Individuals Evaluated for Primary Ciliary Dyskinesia.

J Pediatr 2019 12 11;215:172-177.e2. Epub 2019 Oct 11.

Division of Pulmonology, Department of Pediatrics, Rainbow Babies and Children's Hospital, University Hospitals Cleveland Medical Center, Cleveland, OH.

Objective: To evaluate whether cystic fibrosis transmembrane conductance regulator (CFTR) variants are more common among individuals tested for primary ciliary dyskinesia (PCD) compared with controls.

Study Design: Data were studied from 1021 individuals with commercial genetic testing for suspected PCD and 91 777 controls with genetic testing at the same company (Invitae) for symptoms/diseases unrelated to PCD or CFTR testing. The prevalence of CFTR variants was compared between controls and each of 3 groups of individuals tested for PCD (PCD-positive, -uncertain, and -negative molecular diagnosis).

Results: The prevalence of 1 pathogenic CFTR variant was similar among the individual groups. When combining the PCD-uncertain and PCR-negative molecular diagnosis groups, there was a higher prevalence of single pathogenic CFTR variants compared with controls (P = .03). Importantly, >1% of individuals who had negative genetic testing results for PCD had 2 pathogenic CFTR variants (8 of 723), and the incidence of cystic fibrosis (CF) (2 pathogenic variants) is roughly 1 in 3000 individuals of Caucasian ethnicity (∼0.03%). This incidence was also greater than that of 2 pathogenic CFTR variants in the control population (0.09% [84 of 91 777]; P = 9.60 × 10). These variants correlate with mild CFTR-related disease.

Conclusions: Our results suggest that a single pathogenic CFTR variant is not likely to be a PCD-mimetic, but ongoing studies are needed in individuals in whom PCD is suspected and genetic testing results are uncertain or negative. Furthermore, CF may be misdiagnosed as PCD, reflecting phenotypic overlap. Among individuals evaluated for PCD, CF should be considered in the differential even in the CF newborn screening era.
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http://dx.doi.org/10.1016/j.jpeds.2019.08.039DOI Listing
December 2019

Possible precision medicine implications from genetic testing using combined detection of sequence and intragenic copy number variants in a large cohort with childhood epilepsy.

Epilepsia Open 2019 Sep 1;4(3):397-408. Epub 2019 Jul 1.

Invitae San Francisco California.

Objective: Molecular genetic etiologies in epilepsy have become better understood in recent years, creating important opportunities for precision medicine. Building on these advances, detailed studies of the complexities and outcomes of genetic testing for epilepsy can provide useful insights that inform and refine diagnostic approaches and illuminate the potential for precision medicine in epilepsy.

Methods: We used a multi-gene next-generation sequencing (NGS) panel with simultaneous sequence and exonic copy number variant detection to investigate up to 183 epilepsy-related genes in 9769 individuals. Clinical variant interpretation was performed using a semi-quantitative scoring system based on existing professional practice guidelines.

Results: Molecular genetic testing provided a diagnosis in 14.9%-24.4% of individuals with epilepsy, depending on the NGS panel used. More than half of these diagnoses were in children younger than 5 years. Notably, the testing had possible precision medicine implications in 33% of individuals who received definitive diagnostic results. Only 30 genes provided 80% of molecular diagnoses. While most clinically significant findings were single-nucleotide variants, ~15% were other types that are often challenging to detect with traditional methods. In addition to clinically significant variants, there were many others that initially had uncertain significance; reclassification of 1612 such variants with parental testing or other evidence contributed to 18.5% of diagnostic results overall and 6.1% of results with precision medicine implications.

Significance: Using an NGS gene panel with key high-yield genes and robust analytic sensitivity as a first-tier test early in the diagnostic process, especially for children younger than 5 years, can possibly enable precision medicine approaches in a significant number of individuals with epilepsy.
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http://dx.doi.org/10.1002/epi4.12348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698688PMC
September 2019

Correction: Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

Genet Med 2020 01;22(1):240

Invitae Corporation, San Francisco, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41436-019-0624-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944637PMC
January 2020

Recurring large deletion in DRC1 (CCDC164) identified as causing primary ciliary dyskinesia in two Asian patients.

Mol Genet Genomic Med 2019 08 4;7(8):e838. Epub 2019 Jul 4.

The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo, Japan.

Background: Primary ciliary dyskinesia (PCD) is a relatively rare autosomal recessive or X-linked disorder affecting ciliary function. In the set of causative genes, however, predominant pathogenic variants remain unknown in Asia.

Method: A diagnosis of PCD was made following a modern comprehensive testing including genetic analysis; targeted resequencing for screening variants, and Sanger sequencing for determination of the breakpoints, with an additional review of databases to calculate the deletion frequency. A multiplexed PCR-based detection method has also been developed.

Results: We ascertained a 50-year-old Japanese male who had been diagnosed with diffuse panbronchiolitis (DPB), but refractory to macrolide therapy. We reevaluated the case and identified a large homozygous deletion spanning exons 1 to 4 of the DRC1 and determined the breakpoints (NM_145038.4: c.1-3952_540 + 1331del27748-bp). In the PCD cohort at the University of North Carolina, we found a female PCD patient of Korean descent harboring the same homozygous deletion. From the Invitae testing cohort, we extracted four carriers of the same deletion among 965 Asian individuals, whereas no deletion was found in the 23,951 non-Asians.

Conclusion: We speculate that the DRC1 deletion is a recurrent or perhaps founder mutation in Asians. The simple PCR method could be a useful screening tool.
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http://dx.doi.org/10.1002/mgg3.838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687623PMC
August 2019

Nasal Nitric Oxide in Primary Immunodeficiency and Primary Ciliary Dyskinesia: Helping to Distinguish Between Clinically Similar Diseases.

J Clin Immunol 2019 02 26;39(2):216-224. Epub 2019 Mar 26.

Montreal Children's Hospital, McGill University Health Center, 1001 Boulevard Décarie, Montreal, Quebec, H4A 3J1, Canada.

Purpose: Primary ciliary dyskinesia (PCD) is a rare disorder of the mucociliary clearance leading to recurrent upper and lower respiratory tract infections. PCD is difficult to clinically distinguish from other entities leading to recurrent oto-sino-pulmonary infections, including primary immunodeficiency (PID). Nasal nitric oxide (nNO) is a sensitive and specific diagnostic test for PCD, but it has not been thoroughly examined in PID. Past publications have suggested an overlap in nNO levels among subjects with PCD and PID. We sought to determine if nNO measurements among patients diagnosed with PID would fall significantly above the established PCD diagnostic cutoff value of 77 nL/min.

Methods: Children > 5 years old and adults with definitive PID or PCD diagnoses were recruited from outpatient subspecialty clinics. Participants underwent nNO testing by standardized protocol using a chemiluminescence analyzer and completed a questionnaire concerning their chronic oto-sino-pulmonary symptoms, including key clinical criteria specific to diagnosed PCD (neonatal respiratory distress at term birth, year-round cough or nasal congestion starting before 6 months of age, any organ laterality defect).

Results: Participants included 32 patients with PID, 27 patients with PCD, and 19 healthy controls. Median nNO was 228.9.1 nL/min in the PID group, 19.7 nL/min in the PCD group, and 269.4 in the healthy controls (p < 0.0001). Subjects with PCD were significantly more likely to report key clinical criteria specific to PCD, but approximately 25% of PID subjects also reported at least 1 of these key clinical criteria (mainly year-round cough or nasal congestion).

Conclusions: While key clinical criteria associated with PCD often overlap with the symptoms reported in PID, nNO measurement by chemiluminescence technology allows for effective discrimination between PID and PCD.
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http://dx.doi.org/10.1007/s10875-019-00613-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6870987PMC
February 2019

Primary Ciliary Dyskinesia: Longitudinal Study of Lung Disease by Ultrastructure Defect and Genotype.

Am J Respir Crit Care Med 2019 01;199(2):190-198

12 Department of Pediatrics, Marsico Lung Institute, University of North Carolina School of Medicine, Chapel Hill, North Carolina; and.

Rationale: In primary ciliary dyskinesia, factors leading to disease heterogeneity are poorly understood.

Objectives: To describe early lung disease progression in primary ciliary dyskinesia and identify associations between ultrastructural defects and genotypes with clinical phenotype.

Methods: This was a prospective, longitudinal (5 yr), multicenter, observational study. Inclusion criteria were less than 19 years at enrollment and greater than or equal to two annual study visits. Linear mixed effects models including random slope and random intercept were used to evaluate longitudinal associations between the ciliary defect group (or genotype group) and clinical features (percent predicted FEV and weight and height z-scores).

Measurements And Main Results: A total of 137 participants completed 732 visits. The group with absent inner dynein arm, central apparatus defects, and microtubular disorganization (IDA/CA/MTD) (n = 41) were significantly younger at diagnosis and in mixed effects models had significantly lower percent predicted FEV and weight and height z-scores than the isolated outer dynein arm defect (n = 55) group. Participants with CCDC39 or CCDC40 mutations (n = 34) had lower percent predicted FEV and weight and height z-scores than those with DNAH5 mutations (n = 36). For the entire cohort, percent predicted FEV decline was heterogeneous with a mean (SE) decline of 0.57 (0.25) percent predicted/yr. Rate of decline was different from zero only in the IDA/MTD/CA group (mean [SE], -1.11 [0.48] percent predicted/yr; P = 0.02).

Conclusions: Participants with IDA/MTD/CA defects, which included individuals with CCDC39 or CCDC40 mutations, had worse lung function and growth indices compared with those with outer dynein arm defects and DNAH5 mutations, respectively. The only group with a significant lung function decline over time were participants with IDA/MTD/CA defects.
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http://dx.doi.org/10.1164/rccm.201803-0548OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353004PMC
January 2019

Sources of discordance among germ-line variant classifications in ClinVar.

Genet Med 2017 10 1;19(10):1118-1126. Epub 2017 Jun 1.

Invitae, San Francisco, California, USA.

PurposeClinVar is increasingly used as a resource for both genetic variant interpretation and clinical practice. However, controversies exist regarding the consistency of classifications in ClinVar, and questions remain about how best to use these data. Our study systematically examined ClinVar to identify common sources of discordance and thus inform ongoing practices.MethodsWe analyzed variants that had multiple classifications in ClinVar, excluding benign polymorphisms. Classifications were categorized by potential actionability and pathogenicity. Consensus interpretations were calculated for each variant, and the properties of the discordant outlier classifications were summarized.ResultsOur study included 74,065 classifications of 27,224 unique variants in 1,713 genes. We found that (i) concordance rates differed among clinical areas and variant types; (ii) clinical testing methods had much higher concordance than basic literature curation and research efforts; (iii) older classifications had greater discordance than newer ones; and (iv) low-penetrance variants had particularly high discordance.ConclusionRecent variant classifications from clinical testing laboratories have high overall concordance in many (but not all) clinical areas. ClinVar can be a reliable resource supporting variant interpretation, quality assessment, and clinical practice when factors uncovered in this study are taken into account. Ongoing improvements to ClinVar may make it easier to use, particularly for nonexpert users.
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http://dx.doi.org/10.1038/gim.2017.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632819PMC
October 2017

Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

Genet Med 2017 10 11;19(10):1105-1117. Epub 2017 May 11.

Invitae Corporation, San Francisco, California, USA.

PurposeThe 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines were a major step toward establishing a common framework for variant classification. In practice, however, several aspects of the guidelines lack specificity, are subject to varied interpretations, or fail to capture relevant aspects of clinical molecular genetics. A simple implementation of the guidelines in their current form is insufficient for consistent and comprehensive variant classification.MethodsWe undertook an iterative process of refining the ACMG-AMP guidelines. We used the guidelines to classify more than 40,000 clinically observed variants, assessed the outcome, and refined the classification criteria to capture exceptions and edge cases. During this process, the criteria evolved through eight major and minor revisions.ResultsOur implementation: (i) separated ambiguous ACMG-AMP criteria into a set of discrete but related rules with refined weights; (ii) grouped certain criteria to protect against the overcounting of conceptually related evidence; and (iii) replaced the "clinical criteria" style of the guidelines with additive, semiquantitative criteria.ConclusionSherloc builds on the strong framework of 33 rules established by the ACMG-AMP guidelines and introduces 108 detailed refinements, which support a more consistent and transparent approach to variant classification.
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http://dx.doi.org/10.1038/gim.2017.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632818PMC
October 2017

Pathogenic variant burden in the ExAC database: an empirical approach to evaluating population data for clinical variant interpretation.

Genome Med 2017 02 6;9(1):13. Epub 2017 Feb 6.

Invitae Corporation, 1400 16th St., San Francisco, CA, 94103, USA.

Background: The frequency of a variant in the general population is a key criterion used in the clinical interpretation of sequence variants. With certain exceptions, such as founder mutations, the rarity of a variant is a prerequisite for pathogenicity. However, defining the threshold at which a variant should be considered "too common" is challenging and therefore diagnostic laboratories have typically set conservative allele frequency thresholds.

Methods: Recent publications of large population sequencing data, such as the Exome Aggregation Consortium (ExAC) database, provide an opportunity to characterize with accuracy and precision the frequency distributions of very rare disease-causing alleles. Allele frequencies of pathogenic variants in ClinVar, as well as variants expected to be pathogenic through the nonsense-mediated decay (NMD) pathway, were analyzed to study the burden of pathogenic variants in 79 genes of clinical importance.

Results: Of 1364 BRCA1 and BRCA2 variants that are well characterized as pathogenic or that are expected to lead to NMD, 1350 variants had an allele frequency of less than 0.0025%. The remaining 14 variants were previously published founder mutations. Importantly, we observed no difference in the distributions of pathogenic variants expected to be lead to NMD compared to those that are not. Therefore, we expanded the analysis to examine the distributions of NMD expected variants in 77 additional genes. These 77 genes were selected to represent a broad set of clinical areas, modes of inheritance, and penetrance. Among these variants, most (97.3%) had an allele frequency of less than 0.01%. Furthermore, pathogenic variants with allele frequencies greater than 0.01% were well characterized in publications and included many founder mutations.

Conclusions: The observations made in this study suggest that, with certain caveats, a very low allele frequency threshold can be adopted to more accurately interpret sequence variants.
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http://dx.doi.org/10.1186/s13073-017-0403-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295186PMC
February 2017

Clinical Genetic Testing for the Cardiomyopathies and Arrhythmias: A Systematic Framework for Establishing Clinical Validity and Addressing Genotypic and Phenotypic Heterogeneity.

Front Cardiovasc Med 2016 27;3:20. Epub 2016 Jun 27.

Invitae Corporation , San Francisco, CA , USA.

Advances in DNA sequencing have made large, diagnostic gene panels affordable and efficient. Broad adoption of such panels has begun to deliver on the promises of personalized medicine, but has also brought new challenges such as the presence of unexpected results, or results of uncertain clinical significance. Genetic analysis of inherited cardiac conditions is particularly challenging due to the extensive genetic heterogeneity underlying cardiac phenotypes, and the overlapping, variable, and incompletely penetrant nature of their clinical presentations. The design of effective diagnostic tests and the effective use of the results depend on a clear understanding of the relationship between each gene and each considered condition. To address these issues, we developed simple, systematic approaches to three fundamental challenges: (1) evaluating the strength of the evidence suggesting that a particular condition is caused by pathogenic variants in a particular gene, (2) evaluating whether unusual genotype/phenotype observations represent a plausible expansion of clinical phenotype associated with a gene, and (3) establishing a molecular diagnostic strategy to capture overlapping clinical presentations. These approaches focus on the systematic evaluation of the pathogenicity of variants identified in clinically affected individuals, and the natural history of disease in those individuals. Here, we applied these approaches to the evaluation of more than 100 genes reported to be associated with inherited cardiomyopathies and arrhythmias including hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular dysplasia or cardiomyopathy, long QT syndrome, short QT syndrome, Brugada, and catecholaminergic polymorphic ventricular tachycardia, and to a set of related syndromes such as Noonan Syndrome and Fabry disease. These approaches provide a framework for delivering meaningful and accurate genetic test results to individuals with hereditary cardiac conditions.
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http://dx.doi.org/10.3389/fcvm.2016.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921949PMC
July 2016

Autosomal recessive MFN2-related Charcot-Marie-Tooth disease with diaphragmatic weakness: Case report and literature review.

Am J Med Genet A 2016 06 8;170(6):1580-4. Epub 2016 Mar 8.

Department of Pediatrics, Levine Children's Hospital/Carolinas Healthcare System, Charlotte, North Carolina.

Pathogenic variants in the mitofusin 2 gene (MFN2) are the most common cause of autosomal dominant Charcot-Marie-Tooth (CMT2) disease, which is typically characterized by axonal sensorimotor neuropathy. We report on a 7-month-old white female with hypotonia, motor delay, distal weakness, and motor/sensory axonal neuropathy in which next-generation sequencing analysis identified compound heterozygous pathogenic variants (c.2054_2069_1170del and c.392A>G) in MFN2. A review of the literature reveals that sporadic and familial cases of compound heterozygous or homozygous pathogenic MFN2 variants have been infrequently described, which indicates that MFN2 can also be inherited in a recessive manner. This case highlights several clinical findings not typically associated with MFN2 pathogenic variants, including young age of onset and rapidly progressing diaphragmatic paresis that necessitated tracheostomy and mechanical ventilation, and adds to the growing list of features identified in autosomal recessive MFN2-related CMT2. Our patient with MFN2-related CMT2 expands the clinical and mutational spectrum of individuals with autosomal recessive CMT2 and identifies a new clinical feature that warrants further observation. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajmg.a.37611DOI Listing
June 2016

The role of molecular genetic analysis in the diagnosis of primary ciliary dyskinesia.

Ann Am Thorac Soc 2014 Mar;11(3):351-9

1 Division of Clinical and Metabolic Genetics.

Rationale: Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder of motile cilia. The diagnosis of PCD has previously relied on ciliary analysis with transmission electron microscopy or video microscopy. However, patients with PCD may have normal ultrastructural appearance, and ciliary analysis has limited accessibility. Alternatively, PCD can be diagnosed by demonstrating biallelic mutations in known PCD genes. Genetic testing is emerging as a diagnostic tool to complement ciliary analysis where interpretation and access may delay diagnosis.

Objectives: To determine the diagnostic yield of genetic testing of patients with a confirmed or suspected diagnosis of PCD in a multiethnic urban center.

Methods: Twenty-eight individuals with confirmed PCD on transmission electron microscopy of ciliary ultrastructure and 24 individuals with a probable diagnosis of PCD based on a classical PCD phenotype and low nasal nitric oxide had molecular analysis of 12 genes associated with PCD.

Results: Of 49 subjects who underwent ciliary biopsy, 28 (57%) were diagnosed with PCD through an ultrastructural defect. Of the 52 individuals who underwent molecular genetic analysis, 22 (42%) individuals had two mutations in known PCD genes. Twenty-four previously unreported mutations in known PCD genes were observed. Combining both diagnostic modalities of biopsy and molecular genetics, the diagnostic yield increased to 69% compared with 57% based on biopsy alone.

Conclusions: The diagnosis of PCD is challenging and has traditionally relied on ciliary biopsy, which is unreliable as the sole criterion for a definitive diagnosis. Molecular genetic analysis can be used as a complementary test to increase the diagnostic yield.
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http://dx.doi.org/10.1513/AnnalsATS.201306-194OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028737PMC
March 2014

Broadening the ciliopathy spectrum: motile cilia dyskinesia, and nephronophthisis associated with a previously unreported homozygous mutation in the INVS/NPHP2 gene.

Am J Med Genet A 2013 Jul 24;161A(7):1792-6. Epub 2013 May 24.

Department of Pediatrics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.

Nephronophthisis associated ciliopathies (NPHP-AC) are a group of phenotypically related conditions that include Joubert syndrome, Meckel syndrome, nephronophthisis (NPHP), and Senior-Loken syndrome. We report on a male fetus with prenatal ultrasound findings at 24 weeks of gestation of anhydramnios, large and echogenic kidneys and situs inversus totalis. Histopathology revealed nephronophthisis and tracheal mucosa electron microscopy revealed ciliary dysgenesis. DNA analysis of the NPHP genes showed a previously unreported homozygous mutation, p.Arg603* (c.1078+1G>A), in the INVS/NPHP2 gene. This mutation is thought to abolish the splice donor site for exon 8, which likely disrupts the normal splicing of the INVS/NPHP2 gene.
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http://dx.doi.org/10.1002/ajmg.a.36036DOI Listing
July 2013

Differences in the clinical spectrum of two adolescent male patients with Alström syndrome.

Clin Dysmorphol 2013 Jan;22(1):7-12

Mother and Child Healthcare Institute of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia.

Alström syndrome is a rare disorder typified by early childhood obesity, neurosensory deficits, cardiomyopathy, progressive renal and hepatic dysfunction, and endocrinological features such as severe insulin resistance, type 2 diabetes, hyperlipidemia, and hypogonadism. Widespread fibrosis leads to multiple organ failure. Mutations in ALMS1 cause Alström syndrome. Two age-matched, unrelated adolescent males of Serbian descent with Alström syndrome underwent an extensive workup of blood chemistries, and ophthalmological, audiological, and genetic evaluations. Although both showed typical features of Alström syndrome in childhood, several differences were observed that have not been reported previously. Patient 1 was first studied at the age of 13 years for multisystemic disease and re-evaluated at the age of 15.5 years. Patient 2 is a 15-year-old boy who presented at birth with epilepsy and psychomotor developmental delay and generalized tonic-clonic seizures with severe cognitive impairment, features not documented previously in this syndrome. Sequencing analysis indicated two novel ALMS1 mutations in exon 8: p.E1055GfsX4 and p.T1386NfsX15. Metabolic and physiological similarities were observed in both patients, including severe insulin resistance, and truncal obesity with fat loss suggestive of partial lipodystrophy, supporting evidence for a role for ALMS1 in adipose tissue function. The unusual phenotypes of clonic-tonic seizures and severe cognitive abnormalities and lipodystrophy-like adiposity pattern have not been documented previously in Alström syndrome and may be an under-reported abnormality.
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http://dx.doi.org/10.1097/MCD.0b013e32835b9017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619948PMC
January 2013

Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span.

Biochem Biophys Res Commun 2011 Apr 23;407(3):495-500. Epub 2011 Mar 23.

Division of Hematology/Oncology, Department of Internal Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.
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http://dx.doi.org/10.1016/j.bbrc.2011.03.045DOI Listing
April 2011

Antagonism between GLD-2 binding partners controls gamete sex.

Dev Cell 2009 May;16(5):723-33

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Cytoplasmic polyadenylation is a key mechanism of gene control. In Caenorhabditis elegans, GLD-2 and GLD-3 provide the catalytic and RNA-binding subunits, respectively, of a major cytoplasmic poly(A) polymerase (PAP). Here, we identify RNP-8 as a second GLD-2 partner. RNP-8 binds GLD-2 and stimulates GLD-2 activity to form a functional PAP, much like GLD-3. Moreover, GLD-2/RNP-8 and GLD-2/GLD-3 exist as separate complexes that form selectively during development, and RNP-8 and GLD-3 appear to have distinct RNA-binding specificities. Therefore, GLD-2 can form either of two discrete PAPs. In C. elegans hermaphrodites, gamete production begins with spermatogenesis and transitions later to oogenesis. We suggest that the combinatorial use of GLD-2 contributes to this transition, as GLD-2/GLD-3 promotes spermatogenesis, whereas GLD-2/RNP-8 specifies oogenesis. Indeed, RNP-8 and GLD-3 antagonize each other, as evidenced by genetic cosuppression and molecular competition for GLD-2 binding. We conclude that GLD-2 and its binding partners control gamete identity.
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http://dx.doi.org/10.1016/j.devcel.2009.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728548PMC
May 2009

C. elegans La-related protein, LARP-1, localizes to germline P bodies and attenuates Ras-MAPK signaling during oogenesis.

RNA 2008 Jul 30;14(7):1378-89. Epub 2008 May 30.

Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

RNA regulators are critical for animal development, especially in the germ line where gene expression is often modulated by changes in mRNA stability, translation, and localization. In this paper, we focus on Caenorhabditis elegans LARP-1, a representative of one La-related protein (Larp) family found broadly among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus a second conserved motif, typical of LARP-1 homologs and therefore dubbed the LARP1 domain. LARP-1 appears to bind RNA in vitro via both the La motif and the LARP1 domain. larp-1 null mutants have an oogenesis defect reminiscent of hyperactive Ras-MAPK signaling; this defect is suppressed or enhanced by down- or up-regulating the Ras-MAPK pathway, respectively. Consistent with a role in down-regulating the Ras-MAPK pathway, larp-1 null mutants have higher than normal levels of selected pathway mRNAs and proteins. LARP-1 protein colocalizes with P bodies, which function in RNA degradation. We suggest that LARP-1 functions in P bodies to attenuate the abundance of conserved Ras-MAPK mRNAs. We also propose that the cluster of LARP-1 homologs may function generally to control the expression of key developmental regulators.
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http://dx.doi.org/10.1261/rna.1066008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441978PMC
July 2008

Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity.

J Biol Chem 2005 Jul 12;280(26):24532-8. Epub 2005 May 12.

CNRS UMR 5095, Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint-Saëns, F-33077 Bordeaux cedex, France.

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.
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http://dx.doi.org/10.1074/jbc.M504720200DOI Listing
July 2005

Toxic RNA in the nucleus: unstable microsatellite expression in neuromuscular disease.

Prog Mol Subcell Biol 2004 ;35:57-77

Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida 32610-0266, USA.

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http://dx.doi.org/10.1007/978-3-540-74266-1_3DOI Listing
May 2004

The alerting effects of dexamethasone.

Psychophysiology 2003 Mar;40(2):254-9

Henry Ford Hospital Sleep Disorders Center, Detroit, Michigan, USA.

The purpose of this study was to characterize the level of sleepiness/alertness following the nocturnal administration of dexamethasone. Thirteen healthy men participated in this study. Following the initial screening, dexamethasone (4 mg) or placebo was administered at 22:30 hr in a randomized double-blind procedure. Subjects were given nap opportunities at 23:00, 1:00, 3:00, 4:30, 5:30, 7:30, 9:30, 11:30, 13:30, 15:30, 17:30, and 19:30 hr. The administration of dexamethasone resulted in an overall lengthening of sleep latency. Although the two groups displayed comparable latencies to stage 1 for the 23:00-7:30 hr nap opportunities, the administration of dexamethasone resulted in significantly longer latencies on the 9:30-19:30 hr nap opportunities. Consistent with these results, participants reported significantly greater levels of alertness on the Stanford Sleepiness Scale. The results of this study revealed greater levels of daytime alertness following the nocturnal administration of dexamethasone.
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http://dx.doi.org/10.1111/1469-8986.00027DOI Listing
March 2003

Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export.

EMBO J 2002 Apr;21(7):1800-10

Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA.

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3'-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)(+) RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Delta hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.
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http://dx.doi.org/10.1093/emboj/21.7.1800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC125947PMC
April 2002
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