Diffuse pleural mesotheliomas with genomic near-haploidization: a newly recognized subset with distinct clinical, histologic, and molecular features.

PURPOSE: Diffuse pleural mesotheliomas (DPMs) with genomic near-haploidization (GNH) represent a novel subtype first recognized by the TCGA project; however, its clinicopathologic and molecular features remain poorly defined. EXPERIMENTAL DESIGN: We analyzed clinical genomic profiling data from 290 patients with DPM using the MSK-IMPACT assay. Allele-specific copy number analysis was performed using the FACETS algorithm. RESULTS: 210 patients were evaluable for LOH analysis using FACETS. In this cohort, GNH was detected in 10 cases (4.8%). Compared to non-GNH tumors, GNH DPMs were associated with younger age and less frequent self-reported history of occupational asbestos exposure. Histologically, GNH DPMs were enriched in biphasic subtype (80% vs. 14.5%) and showed abundant tumor infiltrating lymphocytes (TILs). Genomic analysis revealed a higher frequency of TP53 alterations, while SETDB1 mutations were present in nearly all and only in this subset. The clinicopathologic and molecular findings were further validated in a separate cohort. Despite the younger age, patients with GNH DPMs had a shorter overall survival (10.9 vs. 25.4 months, p=0.004); the poor prognostic impact of GNH remained significant after controlling for biphasic histology. Out of three patients with GNH DPMs who received immune checkpoint blockade (ICB), two achieved a clinician assessed partial response. CONCLUSIONS: GNH defines an aggressive subtype of mainly biphasic DPMs in younger patients with recurrent alterations in SETDB1 and TP53. The enrichment in biphasic histology and TILs, together with our preliminary ICB response data and anecdotal clinical trial data, suggests that further evaluation of immunotherapy may be warranted in this subset.

Cell-free DNA from nail clippings as source of normal control for genomic studies in hematologic malignancies.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patient management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs. In contrast to solid tumors, conventional sources of normal control (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we find nail cfDNA is a robust source of germline control for paired genomic studies. In a subset of patients, nail DNA may have tumor DNA contamination, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1482) compared to lymphoid diseases (5.4%; 61/1128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. For nails collected after allogeneic stem-cell transplantation, donor DNA was identified in 22% (11/50). In this cohort, an association with recent history of graft-vs-host disease was identified. These findings should be considered as a potential limitation for the use of nail as normal control but could also provide important diagnostic information regarding the disease process.

Microsatellite Instability and Mismatch Repair Deficiency Define a Distinct Subset of Lung Cancers Characterized by Smoking Exposure, High Tumor Mutational Burden, and Recurrent Somatic MLH1 Inactivation.

INTRODUCTION: Microsatellite instability (MSI) and mismatch repair (MMR) deficiency represent a distinct oncogenic process and predict response to immune checkpoint inhibitors (ICIs). The clinicopathologic features of MSI-high (MSI-H) and MMR deficiency (MMR-D) in lung cancers remain poorly characterized. METHODS: MSI status from 5171 patients with NSCLC and 315 patients with SCLC was analyzed from targeted next-generation sequencing data using two validated bioinformatic pipelines. RESULTS: MSI-H and MMR-D were identified in 21 patients with NSCLC (0.41%) and six patients with SCLC (1.9%). Notably, all patients with NSCLC had a positive smoking history, including 11 adenocarcinomas. Compared with microsatellite stable cases, MSI-H was associated with exceptionally high tumor mutational burden (37.4 versus 8.5 muts/Mb, p < 0.0001), MMR mutational signatures (43% versus 0%, p < 0.0001), and somatic biallelic alterations in MLH1 (52% versus 0%, p < 0.0001). Loss of MLH1 and PMS2 expression by immunohistochemistry was found in MLH1 altered and wild-type cases. Similarly, the majority of patients with MSI-H SCLC had evidence of MLH1 inactivation, including two with MLH1 promoter hypermethylation. A single patient with NSCLC with a somatic MSH2 mutation had Lynch syndrome as confirmed by the presence of a germline MSH2 mutation. Among patients with advanced MSI-H lung cancers treated with ICIs, durable clinical benefit was observed in three of eight patients with NSCLC and two of two patients with SCLC. In NSCLC, STK11, KEAP1, and JAK1 were mutated in nonresponders but wild type in responders. CONCLUSIONS: We present a comprehensive clinicogenomic landscape of MSI-H lung cancers and reveal that MSI-H defines a rare subset of lung cancers associated with smoking, high tumor mutational burden, and MLH1 inactivation. Although durable clinical benefit to ICI was observed in some patients, the broad range of responses suggests that clinical activity may be modulated by co-mutational landscapes.

Predicting tumour content of liquid biopsies from cell-free DNA.

BACKGROUND: Liquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample's tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy. RESULTS: We used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma. CONCLUSIONS: This proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies.

Associations Between Cancer Predisposition Mutations and Clonal Hematopoiesis in Patients With Solid Tumors.

PURPOSE: Clonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors. METHODS: We used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants. RESULTS: We observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH comutation patterns in ATM, TP53, and CHEK2 suggested biallelic inactivation as a potential mediator of clonal expansion. Moreover, we observed that CH-PPM1D mutations, similar to somatic tumor-associated PPM1D mutations, were depleted in patients with P/LP germline mutations in the DNA damage response (DDR) genes ATM, CHEK2, and TP53. Patients with solid tumors and harboring P/LP germline mutations, CH mutations, and mosaicism chromosomal alterations might be at an increased risk of developing secondary leukemia while germline variants in TP53 were identified as an independent risk factor (hazard ratio, 36; P < .001) for secondary leukemias. CONCLUSION: Our results suggest a close relationship between inherited variants and CH mutations within the DDR genes in patients with solid tumors. Associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities in patients with cancer harboring these germline mutations.

Matched Molecular Profiling of Cell-Free DNA and Tumor Tissue in Patients With Advanced Clear Cell Renal Cell Carcinoma.

PURPOSE: The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. MATERIALS AND METHODS: We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. RESULTS: Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. CONCLUSION: cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

Rb Tumor Suppressor in Small Cell Lung Cancer: Combined Genomic and IHC Analysis with a Description of a Distinct Rb-Proficient Subset.

PURPOSE: RB1 mutations and loss of retinoblastoma (Rb) expression represent consistent but not entirely invariable hallmarks of small cell lung cancer (SCLC). The prevalence and characteristics of SCLC retaining wild-type Rb are not well-established. Furthermore, the performance of targeted next-generation sequencing (NGS) versus immunohistochemistry for Rb assessment is not well-defined. EXPERIMENTAL DESIGN: A total of 208 clinical SCLC samples were analyzed by comprehensive targeted NGS, covering all exons of RB1, and Rb IHC. On the basis of established coordination of Rb/p16/cyclinD1 expression, p16-high/cyclinD1-low profile was used as a marker of constitutive Rb deficiency. RESULTS: Fourteen of 208 (6%) SCLC expressed wild-type Rb, accompanied by a unique p16-low/cyclinD1-high profile supporting Rb proficiency. Rb-proficient SCLC was associated with neuroendocrine-low phenotype, combined SCLC with non-SCLC (NSCLC) histology and aggressive behavior. These tumors exclusively harbored CCND1 amplification (29%), and were markedly enriched in CDKN2A mutations (50%) and NSCLC-type alterations (KEAP1, STK11, FGFR1). The remaining 194 of 208 SCLC were Rb-deficient (p16-high/cyclinD1-low), including 184 cases with Rb loss (of which 29% lacked detectable RB1 alterations by clinical NGS pipeline), and 10 cases with mutated but expressed Rb. CONCLUSIONS: This is the largest study to date to concurrently analyze Rb by NGS and IHC in SCLC, identifying a 6% rate of Rb proficiency. Pathologic-genomic data implicate NSCLC-related progenitors as a putative source of Rb-proficient SCLC. Consistent upstream Rb inactivation via CDKN2A/p16↓ and CCND1/cyclinD1↑ suggests the potential utility of CDK4/6 inhibitors in this aggressive SCLC subset. The study also clarifies technical aspects of Rb status determination in clinical practice, highlighting the limitations of exon-only sequencing for RB1 interrogation. See related commentary by Mahadevan and Sholl, p. 4603.

Clonal hematopoiesis is associated with risk of severe Covid-19.

Acquired somatic mutations in hematopoietic stem and progenitor cells (clonal hematopoiesis or CH) are associated with advanced age, increased risk of cardiovascular and malignant diseases, and decreased overall survival. These adverse sequelae may be mediated by altered inflammatory profiles observed in patients with CH. A pro-inflammatory immunologic profile is also associated with worse outcomes of certain infections, including SARS-CoV-2 and its associated disease Covid-19. Whether CH predisposes to severe Covid-19 or other infections is unknown. Among 525 individuals with Covid-19 from Memorial Sloan Kettering (MSK) and the Korean Clonal Hematopoiesis (KoCH) consortia, we show that CH is associated with severe Covid-19 outcomes (OR = 1.85, 95%=1.15-2.99, p = 0.01), in particular CH characterized by non-cancer driver mutations (OR = 2.01, 95% CI = 1.15-3.50, p = 0.01). We further explore the relationship between CH and risk of other infections in 14,211 solid tumor patients at MSK. CH is significantly associated with risk of Clostridium Difficile (HR = 2.01, 95% CI: 1.22-3.30, p = 6×10-3) and Streptococcus/Enterococcus infections (HR = 1.56, 95% CI = 1.15-2.13, p = 5×10-3). These findings suggest a relationship between CH and risk of severe infections that warrants further investigation.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor patients.

BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.

Clonal hematopoiesis is associated with risk of severe Covid-19.

Acquired somatic mutations in hematopoietic stem and progenitor cells (clonal hematopoiesis or CH) are associated with advanced age, increased risk of cardiovascular and malignant diseases, and decreased overall survival. 1-4 These adverse sequelae may be mediated by altered inflammatory profiles observed in patients with CH. 2,5,6 A pro-inflammatory immunologic profile is also associated with worse outcomes of certain infections, including SARS-CoV-2 and its associated disease Covid-19. 7,8 Whether CH predisposes to severe Covid-19 or other infections is unknown. Among 515 individuals with Covid-19 from Memorial Sloan Kettering (MSK) and the Korean Clonal Hematopoiesis (KoCH) consortia, we found that CH was associated with severe Covid-19 outcomes (OR=1.9, 95%=1.2-2.9, p=0.01). We further explored the relationship between CH and risk of other infections in 14,211 solid tumor patients at MSK. CH was significantly associated with risk of Clostridium Difficile (HR=2.0, 95% CI: 1.2-3.3, p=6×10 -3 ) and Streptococcus/Enterococcus infections (HR=1.5, 95% CI=1.1-2.1, p=5×10 -3 ). These findings suggest a relationship between CH and risk of severe infections that warrants further investigation.

Drosophila models of pathogenic copy-number variant genes show global and non-neuronal defects during development.

While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

PURPOSE: Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. EXPERIMENTAL DESIGN: We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). RESULTS: Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. CONCLUSIONS: These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect.

Extremely low-coverage whole genome sequencing in South Asians captures population genomics information.

BACKGROUND: The cost of Whole Genome Sequencing (WGS) has decreased tremendously in recent years due to advances in next-generation sequencing technologies. Nevertheless, the cost of carrying out large-scale cohort studies using WGS is still daunting. Past simulation studies with coverage at ~2x have shown promise for using low coverage WGS in studies focused on variant discovery, association study replications, and population genomics characterization. However, the performance of low coverage WGS in populations with a complex history and no reference panel remains to be determined. RESULTS: South Indian populations are known to have a complex population structure and are an example of a major population group that lacks adequate reference panels. To test the performance of extremely low-coverage WGS (EXL-WGS) in populations with a complex history and to provide a reference resource for South Indian populations, we performed EXL-WGS on 185 South Indian individuals from eight populations to ~1.6x coverage. Using two variant discovery pipelines, SNPTools and GATK, we generated a consensus call set that has ~90% sensitivity for identifying common variants (minor allele frequency ≥ 10%). Imputation further improves the sensitivity of our call set. In addition, we obtained high-coverage for the whole mitochondrial genome to infer the maternal lineage evolutionary history of the Indian samples. CONCLUSIONS: Overall, we demonstrate that EXL-WGS with imputation can be a valuable study design for variant discovery with a dramatically lower cost than standard WGS, even in populations with a complex history and without available reference data. In addition, the South Indian EXL-WGS data generated in this study will provide a valuable resource for future Indian genomic studies.

A Rare Variant in CACNA1D Segregates with 7 Bipolar I Disorder Cases in a Large Pedigree.

Whole-genome sequencing was performed on 3 bipolar I disorder (BPI) cases from a multiplex pedigree of European ancestry with 7 BPI cases. Within CACNA1D, a gene implicated by genome-wide association studies, a G to C nucleotide transversion at 53,835,340 base pairs (bps) was found predicting the substitution of proline for alanine at amino acid position 1751 (A1751P). Using Sanger sequencing, the DNA variant was shown to co-segregate with the remaining 4 BPI cases within the pedigree. A high-resolution DNA denaturing curve method was then used to screen for the presence of the A1751P change in 4,150 BPI cases from the NIMH Genetics Initiative. The A1751P variant was found in 4 BPI cases. A second variant within exon 43, a C to T nucleotide transition, was found in 1 case at 53,835,355 bps, predicting the substitution of tryptophan for arginine at amino acid position 1771 (R1771W). In the NHLBI Exome Sequencing Project database, the heterozygous A1751P variant was present in 3 of 4,300 subjects of European ancestry, and the R1771W change was not present in any subject. Given the rarity of these variants, large-scale case/control rare variant sequencing studies will be required for definitive conclusions.

Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are a group of fatal, mostly recessive neurodegenerative lysosomal storage diseases. While clinically similar, they are genetically distinct and result from mutations in at least twelve different genes. Estimates of NCL incidence range from 0.6 to 14 per 100,000 live births but vary widely between populations and are influenced by whether patients are classified based upon clinical or genetic criteria. We investigated mutations in twelve NCL genes in ~61,000 individuals represented in the Exome Aggregation Consortium (ExAC) whole exome sequencing database. Variants were extracted from ExAC and pathogenic alleles were differentiated from neutral polymorphisms using annotated variant databases and missense mutation prediction tools. Carrier frequency was dependent on ethnicity, with the highest (1/75) observed for PPT1 in the Finnish. When data are adjusted for ethnic diversity within the USA, PPT1, TPP1 and CLN3 carrier frequencies were found to be the highest of the NCLs, each at ~1/500. Carrier frequencies calculated from ExAC correlated well with incidence estimated from numbers of living NCL patients in the US. In addition, the analysis identified numerous variants that are annotated as pathogenic in public repositories but have a predicted frequency that is not consistent with patient studies. These variants appear to be neutral polymorphisms that are reported as pathogenic without validation. Based upon literature reports, such alleles may be annotated in public databases as pathogenic and this propagates errors that can have clinical consequences.