Genomic Sequencing for Newborn Screening: Results of the NC NEXUS Project.

Newborn screening (NBS) was established as a public health program in the 1960s and is crucial for facilitating detection of certain medical conditions in which early intervention can prevent serious, life-threatening health problems. Genomic sequencing can potentially expand the screening for rare hereditary disorders, but many questions surround its possible use for this purpose. We examined the use of exome sequencing (ES) for NBS in the North Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS) project, comparing the yield from ES used in a screening versus a diagnostic context. We enrolled healthy newborns and children with metabolic diseases or hearing loss (106 participants total). ES confirmed the participant's underlying diagnosis in 15 out of 17 (88%) children with metabolic disorders and in 5 out of 28 (∼18%) children with hearing loss. We discovered actionable findings in four participants that would not have been detected by standard NBS. A subset of parents was eligible to receive additional information for their child about childhood-onset conditions with low or no clinical actionability, clinically actionable adult-onset conditions, and carrier status for autosomal-recessive conditions. We found pathogenic variants associated with hereditary breast and/or ovarian cancer in two children, a likely pathogenic variant in the gene associated with Lowe syndrome in one child, and an average of 1.8 reportable variants per child for carrier results. These results highlight the benefits and limitations of using genomic sequencing for NBS and the challenges of using such technology in future precision medicine approaches.

Correction: Common-variant associations with fragile X syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Common-variant associations with fragile X syndrome.

Fragile X syndrome is rare but a prominent cause of intellectual disability. It is usually caused by a de novo mutation that occurs on multiple haplotypes and thus would not be expected to be detectible using genome-wide association (GWA). We conducted GWA in 89 male FXS cases and 266 male controls, and detected multiple genome-wide significant signals near FMR1 (odds ratio = 8.10, P = 2.5 × 10-10). These findings withstood robust attempts at falsification. Fine-mapping yielded a minimum P = 1.13 × 10-14, but did not narrow the interval. Comprehensive functional genomic integration did not provide a mechanistic hypothesis. Controls carrying a risk haplotype had significantly longer FMR1 CGG repeats than controls with the protective haplotype (P = 4.75 × 10-5), which may predispose toward increases in CGG number to the premutation range over many generations. This is a salutary reminder of the complexity of even "simple" monogenetic disorders.

CSF1R mosaicism in a family with hereditary diffuse leukoencephalopathy with spheroids.

Mutations in the colony stimulating factor 1 receptor (CSF1R) have recently been discovered as causal for hereditary diffuse leukoencephalopathy with axonal spheroids. We identified a novel, heterozygous missense mutation in CSF1R [c.1990G > A p.(E664K)] by exome sequencing in five members of a family with hereditary diffuse leukoencephalopathy with axonal spheroids. Three affected siblings had characteristic white matter abnormalities and presented with progressive neurological decline. In the fourth affected sibling, early progression halted after allogeneic haematopoietic stem cell transplantation from a related donor. Blood spot DNA from this subject displayed chimerism in CSF1R acquired after haematopoietic stem cell transplantation. Interestingly, both parents were unaffected but the mother's blood and saliva were mosaic for the CSF1R mutation. Our findings suggest that expression of wild-type CSF1R in some cells, whether achieved by mosaicism or chimerism, may confer benefit in hereditary diffuse leukoencephalopathy with axonal spheroids and suggest that haematopoietic stem cell transplantation might have a therapeutic role for this disorder.

FNA smears as a potential source of DNA for targeted next-generation sequencing of lung adenocarcinomas.

BACKGROUND: Diff-Quik-stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. This study describes the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and reports the DNA quality and success rates of FNA smears versus other specimens from 1 year of clinical use. METHODS: A validation set of 10 slides from 9 patients with prior clinical epidermal growth factor receptor (EGFR) Sanger sequencing and KRAS pyrosequencing (5 KRAS-positive/EGFR-negative and 4 KRAS-negative/EGFR-negative) underwent DNA extraction, quality assessment, and targeted NGS. Subsequently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in 1 calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, and 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. RESULTS: All 10 validation samples met the DNA quality threshold (delta Ct threshold < 8; range, -2.2 to 4.9) and yielded 0.5 to 22 µg of DNA. The KRAS and EGFR mutation status from FNA smears according to NGS was concordant with previous clinical testing for all 10 samples. In the 1-year review, FNA smears were 100% successful, and this suggested a performance equivalent to or better than the performance of established specimen types, including FNA cell blocks. DNA quality according to ΔCt was significantly better with FNA smears versus biopsies, resections, and FNA cell blocks. CONCLUSIONS: FNA smears of lung adenocarcinomas are high-quality alternative specimens for a targeted NGS panel with a high success rate in clinical practice. Cancer Cytopathol 2016;124:406-14. © 2016 American Cancer Society.

A semiquantitative metric for evaluating clinical actionability of incidental or secondary findings from genome-scale sequencing.

PURPOSE: As genome-scale sequencing is increasingly applied in clinical scenarios, a wide variety of genomic findings will be discovered as secondary or incidental findings, and there is debate about how they should be handled. The clinical actionability of such findings varies, necessitating standardized frameworks for a priori decision making about their analysis. METHODS: We established a semiquantitative metric to assess five elements of actionability: severity and likelihood of the disease outcome, efficacy and burden of intervention, and knowledge base, with a total score from 0 to 15. RESULTS: The semiquantitative metric was applied to a list of putative actionable conditions, the list of genes recommended by the American College of Medical Genetics and Genomics (ACMG) for return when deleterious variants are discovered as secondary/incidental findings, and a random sample of 1,000 genes. Scores from the list of putative actionable conditions (median = 12) and the ACMG list (median = 11) were both statistically different than the randomly selected genes (median = 7) (P < 0.0001, two-tailed Mann-Whitney test). CONCLUSION: Gene-disease pairs having a score of 11 or higher represent the top quintile of actionability. The semiquantitative metric effectively assesses clinical actionability, promotes transparency, and may facilitate assessments of clinical actionability by various groups and in diverse contexts.Genet Med 18 5, 467-475.

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.

PURPOSE: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. METHODS: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. RESULTS: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. CONCLUSION: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823-832.

Sensorineural hearing loss in a pediatric population: association of congenital cytomegalovirus infection with intracranial abnormalities.

OBJECTIVES: To examine the incidence of congenital cytomegalovirus (CMV) infection relative to common genetic etiologies of hearing loss in a pediatric population with sensorineural hearing loss (SNHL), and to characterize intracranial radiological abnormalities in patients with CMV-associated hearing loss. DESIGN: Retrospective study. SETTING: Academic tertiary care center. PATIENTS: A total of 112 pediatric patients with confirmed SNHL. MAIN OUTCOME MEASURES: The association of congenital CMV infection status with abnormal brain magnetic resonance imaging (MRI) scans and the frequencies of congenital CMV infection, gap junction ß-2 (GJB2) mutations, and the mitochondrial DNA (mtDNA) 1555A>G mutation in children with SNHL. RESULTS: Of 109 patients, 11 (10%) had positive results for CMV DNA; 10 of the 11 had normal GJB2 sequence and had negative test results for the mtDNA 1555A>G mutation. Brain MRI scans for 97 patients demonstrated a higher proportion of abnormalities in patients with positive CMV test results (80%) compared with those with no detectable CMV DNA (33%) (P = .006). GJB2 mutations and the mtDNA 1555A>G mutation were seen in 10 of 88 patients (11%) and 1 of 97 patients (1%) with SNHL, respectively. CONCLUSIONS: The presence of brain abnormalities in most patients with congenital CMV infection suggests that neurological damage in otherwise asymptomatic patients may not be limited to SNHL. Congenital CMV infection accounted for a significant proportion of patients with SNHL, with an incidence rate comparable with that of GJB2-related SNHL.

Validation of clinical testing for warfarin sensitivity: comparison of CYP2C9-VKORC1 genotyping assays and warfarin-dosing algorithms.

Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 -1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses.

High levels of Epstein-Barr virus DNA in latently infected gastric adenocarcinoma.

Gastric adenocarcinoma is the second leading cause of cancer death worldwide. Epstein-Barr virus (EBV) is present in the malignant cells of approximately 10% of cases. It is unclear whether EBV is being missed in some gastric adenocarcinomas due to insensitive test methods or partial EBV genome loss. In this study, we screened 113 gastric adenocarcinomas from low- and high-incidence regions (United States and Central America) for the presence of EBV using a battery quantitative real-time PCR (Q-PCR) assays targeting disparate segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, BZLF1, EBER1) and histochemical stains targeting EBV-encoded RNA (EBER), the latent proteins LMP1 and LMP2, and the lytic proteins BMRF1 and BZLF1. EBV DNA was detected by Q-PCR in 48/75 United States cancers (64%) and in 38/38 Central American cancers (100%), which was a significant difference. EBER was localized to malignant epithelial cells in 8/48 (17%) United States and 3/38 (8%) Central American cancers. Viral loads were considerably higher for EBER-positive vs EBER-negative cancers (mean 162 986 vs 62 EBV DNA copies per 100,000 cells). A viral load of 2000 copies per 100,000 cells is recommended as the threshold distinguishing EBER-positive from EBER-negative tumors. One infected cancer selectively failed to amplify the LMP2 gene because of a point mutation, whereas another cancer had an atypical pattern of Q-PCR positivity suggesting deletion of large segments of the EBV genome. Three different viral latency profiles were observed in the cancers based on constant expression of EBER and focal or variable expression of LMP1 or LMP2, without lytic protein expression. We conclude that EBV DNA levels generally reflect EBER status, and a panel of at least two Q-PCR assays is recommended for sensitive identification of infected cancers.

Increased uptake of BRCA1/2 genetic testing among African American women with a recent diagnosis of breast cancer.

PURPOSE: Studies suggest that African American women are less likely to pursue BRCA1/2 genetic testing than white women. However, such studies are often confounded by unequal access to care. METHODS: Data from 132 African American and 636 white women, obtained from a clinical database at the University of North Carolina (Chapel Hill, NC) between 1998 and 2005, were analyzed to assess BRCA1/2 genetic testing uptake. Importantly, the clinical setting minimized barriers of both cost and access. Race and time of new breast cancer diagnosis (recent v > 1 year before genetic evaluation) were assessed for association with BRCA1/2 testing uptake using multivariable logistic regression models. RESULTS: Both race (P = .0082) and a recent diagnosis of breast cancer (P = .014) were independently associated with testing uptake. African American women had a lower estimated odds of pursuing testing than white women (odds ratio [OR], 0.54; 95%CI, 0.34 to 0.85), and women with a recent diagnosis had a higher OR than those with a remote diagnosis (OR, 1.58; 95% CI, 1.10 to 2.29). In a race-stratified analysis, there was no statistical evidence for association between recent status and testing uptake in the larger white stratum (OR, 1.38, P = .13) while there was for the smaller African American sample (OR, 2.77, P = .018). The test of interaction between race and remote status was not significant (P = .15). CONCLUSION: African American race was associated with an overall decreased uptake of BRCA1/2 genetic testing, even when barriers of ascertainment and cost were minimized. However, among African American women, a recent diagnosis of breast cancer was associated with substantially increased uptake of testing.

A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort.

PURPOSE: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening. METHODS: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort. RESULTS: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants. CONCLUSIONS: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.

The Prothrombin 20209C>T Sequence Variant: To Test or Not to Test.

Only one mutation in the prothrombin gene (Factor II), 20210G>A, has been definitively associated with an increased risk for venous thrombosis. Using hybridization probe analysis for mutation detection on the LightCycler (Roche Molecular Biochemicals), we identified seven patient samples with atypical melt curve patterns. Sequence analyses of each of these samples revealed heterozygosity for a C to T transition at position 20209. As in other reported cases, each of the apparently unrelated patients was of African-American descent, suggesting that the variant is population specific. Two patients were referred for testing due to a history of stroke, one with a right major coronary artery embolic stroke and the other with a right cerebellar stroke. A third patient had chronic renal failure secondary to hypertension with a reported family history of renal failure. Three patients had a history of multiple pregnancy losses. The last patient had a kidney transplant for end stage renal disease secondary to glomerulonephritis. She was being evaluated for a second transplant, but to our knowledge, had a negative history of venous thrombosis. The prevalence and the clinical significance of the prothrombin 20209C>T mutation is unknown. Recently reported functional studies revealed conflicting results. The clinical utility of testing for and reporting this variant remains unresolved.

Detection of common disease-causing mutations in mitochondrial DNA (mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes MTTL1 3243 A>G and myoclonic epilepsy associated with ragged-red fibers MTTK 8344A>G) by real-time polymerase chain reaction.

The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.

False-negative factor V Leiden genetic testing in a patient with recurrent deep venous thrombosis.

False-negative genetic testing of the factor V Leiden (fVL) mutation is unusual. We report a case of a young woman with a history of deep venous thrombosis tested for the fVL at four separate laboratories on four separate dates. Two laboratories reported the patient to be heterozygous for the fVL, while the other two reported no evidence of a mutation. Testing methods of the various laboratories were reviewed, and additional testing was performed on stored and newly drawn DNA samples, including sequencing of the fVL gene segment. The preponderance of evidence indicates the patient to be heterozygous for the fVL mutation. Dissection of data suggests that either sample misidentification or faulty allele specific amplification methods could have led to false-negative results in two laboratories. In one of the two laboratories, misinterpretation of results and clerical error could not be excluded. There is a need for standardization of optimized fVL genetic testing methods. Further education of ordering physicians on the limitations of genetic testing is necessary.

Glutaric acidemia type 1 in patients of Lumbee heritage from North Carolina.

Glutaric acidemia type I (GA-I) is an autosomal recessive disorder of the catabolism of lysine, hydroxylysine, and tryptophan caused by deficiency of glutaryl-CoA dehydrogenase (GCD). Among our patients with GA-I, we noted a prevalence of Lumbee individuals. The Lumbee are a close-knit Native American tribe of eastern North Carolina. Five Lumbee individuals with GA-I had homozygous 1240G>A mutations in GCD. This is a rare, known mutation that was likely introduced by a Lumbee founder.

Mosaicism for an FMR1 gene deletion in a fragile X female.

Most cases of fragile X syndrome result from expansion of CGG repeats in the FMR1 gene; deletions and point mutations of FMR1 are much less common. Mosaicism for an FMR1 full mutation with a deletion or with a normal allele has been reported in fragile X males. Here we report on a fragile X female who is mosaic for an FMR1 full mutation and an intragenic deletion. The patient is a 4-year-old girl with developmental delay, autistic-like behaviors, and significant speech and language abnormalities. Southern blotting demonstrated the presence of a methylated full mutation, a normal allele in methylated and unmethylated forms, and an additional fragment smaller than the normal methylated allele. This result indicates that the patient is mosaic for a full mutation and a deletion, in the presence of a normal allele. By DNA sequence analysis, we mapped the 5' breakpoint 63/65 bp upstream from the CGG repeat region and the 3' breakpoint 86/88 bp downstream of the CGG repeats within the FMR1 gene. The deletion removed 210 bp, including the entire CGG repeat region. The full mutation was inherited from a premutation in the patient's mother. The deletion, which remained methylated at the Eag I and Nru I sites, was probably derived from the full mutation allele. Mosaicism of this type is rare in females with a fragile X mutation but should be kept in mind in the interpretation of Southern blots.