Jungmin Choi

Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial kindreds with pulmonary fibrosis. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no previous connection to telomere biology or disease, with five new heterozygous damaging mutations in unrelated cases and none in controls (P = 1.3 × 10−8); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more new damaging and missense variants at conserved residues in cases than in controls (P = 1.6 × 10−6). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths, and we observed epigenetic inheritance of short telomeres in family members. Together, these genes explain ∼7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction.

Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles

Premature fusion of the cranial sutures (craniosynostosis), affecting 1 in 2000 newborns, is treated surgically in infancy to prevent adverse neurologic outcomes. To identify mutations contributing to common non-syndromic midline (sagittal and metopic) craniosynostosis, we performed exome sequencing of 132 parent-offspring trios and 59 additional probands. Thirteen probands (7%) had damaging de novo or rare transmitted mutations in SMAD6, an inhibitor of BMP – induced osteoblast differentiation (p<10−20). SMAD6 mutations nonetheless showed striking incomplete penetrance (<60%). Genotypes of a common variant near BMP2 that is strongly associated with midline craniosynostosis explained nearly all the phenotypic variation in these kindreds, with highly significant evidence of genetic interaction between these loci via both association and analysis of linkage. This epistatic interaction of rare and common variants defines the most frequent cause of midline craniosynostosis and has implications for the genetic basis of other diseases. DOI: http://dx.doi.org/10.7554/eLife.20125.001

Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial–mesenchymal transition

Significance Some cancers, termed carcinosarcomas (CSs), have mixed cell types, with either epithelial or mesenchymal features. Sequencing the genomes of uterine and ovarian CSs demonstrated that these different cell types derive from a common precursor cell that has many mutations typical of epithelial cancers. In addition, we find that these tumors have a significant burden of point mutations and amplification of histone genes, suggesting a potential role of these mutations in sarcomatous transformation. Consistent with this finding, expression of specific histone gene mutations in uterine carcinoma cells changed gene expression toward a mesenchymal state. These findings have potential implications for the treatment of these cancers. Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC. Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial–mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

Isolated polycystic liver disease genes define effectors of polycystin-1 function

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

Impaired HLA Class I Antigen Processing and Presentation as a Mechanism of Acquired Resistance to Immune Checkpoint Inhibitors in Lung Cancer

Mechanisms of acquired resistance to immune checkpoint inhibitors (ICI) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy-number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell-surface HLA Class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knockout of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo, proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer.Significance: As programmed death 1 axis inhibitors are becoming more established in standard treatment algorithms for diverse malignancies, acquired resistance to these therapies is increasingly being encountered. Here, we found that defective antigen processing and presentation can serve as a mechanism of such resistance in lung cancer. Cancer Discov; 7(12); 1420-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

CLCN2 chloride channel mutations in familial hyperaldosteronism type II

Primary aldosteronism, a common cause of severe hypertension1, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II)2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.Whole-exome sequencing identifies mutations in CLCN2 in individuals with familial hyperaldosteronism type II or early-onset primary aldosteronism. These gain-of-function mutations cause chloride channel opening and glomerulosa cell depolarization, showing a role for anion channels in aldosterone production.

Exceptional response to pembrolizumab in a metastatic, chemotherapy/radiation resistant ovarian cancer patient harboring a CD274/PD-L1-genetic rearrangement

Purpose: Ovarian carcinoma no longer responsive to surgery and chemotherapy remains an incurable disease. Alternative therapeutic options remain desperately needed. Patients and Methods: We describe a heavily pretreated patient with ovarian cancer with recurrent disease experiencing a remarkable clinical response to treatment with the anti-PD1 immune checkpoint inhibitor pembrolizumab. The clinical, pathological, and genomic characteristics of this exceptional ovarian cancer responder were carefully investigated using immunohistochemistry (IHC), quantitative multiplex fluorescence methods (i.e., automated quantitative analysis, AQUA) and whole-exome sequencing (WES) techniques. Results: The patient harbored a recurrent/metastatic radiation and chemotherapy-resistant high-grade ovarian carcinoma with clear cell features. While progressing on any standard treatment modality, she demonstrated a remarkable complete response to the anti-PD1 immune checkpoint inhibitor pembrolizumab. WES results were notable for the presence a relative low number of mutations (tumor mutation load/Mb = 4.31, total mutations = 164) and a peculiar structural variant disrupting the 3′ region of the PD-L1 gene causing aberrant PD-L1 surface expression as confirmed by IHC and AQUA technology. Heavy infiltration of the PD-L1–mutated and PD-L1–overexpressing tumor with T-cell lymphocytes (i.e., CD4+/CD8+ TIL), CD68+ macrophages, and CD20+ B cells was detected in the surgical specimen strongly suggesting immune evasion as a key mechanism of tumor growth and survival. Patients complete clinical responses remain unchanged at the time of the writing of this report with no significant side effects reported to date. Conclusions: Anti-PD1 inhibitors may represent a novel treatment option for recurrent/metastatic human tumors refractory to salvage treatment harboring PD-L1 gene structural variations causing aberrant PD-L1 expression. Clin Cancer Res; 24(14); 3282–91. ©2018 AACR. See related commentary by Lheureux, p. 3233

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Significance Craniosynostosis is a common congenital malformation resulting from premature fusion of the bones that comprise the cranial vault, requiring surgery in infancy to prevent adverse neurologic outcomes. Eighty-five percent of cases are non-syndromic and of unknown cause. By exome sequencing of families with non-syndromic midline craniosynostosis, we show that 5% of cases have de novo damaging mutations in negative regulators of the Wnt, bone morphogenetic protein (BMP), and Ras/ERK signaling pathways, developmental cascades that converge on common nuclear targets to promote bone formation. Another 5% have transmitted mutations in these pathways. Common variants near BMP2 show genetic interaction with these rare mutations. The results provide insight into pathophysiology and have immediate implications for the diagnosis and genetic counseling of families with craniosynostosis. Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Comprehensive Genetic Analysis of Follicular Thyroid Carcinoma Predicts Prognosis Independent of Histology

Context Follicular thyroid carcinoma (FTC) is classified into minimally invasive (miFTC), encapsulated angioinvasive (eaFTC), and widely invasive (wiFTC) subtypes, according to the 2017 World Health Organization guidelines. The genetic signatures of these subtypes may be crucial for diagnosis, prognosis, and treatment but have not been described. Objective Identify and describe the genetic underpinnings of subtypes of FTC. Methods Thirty-nine tumors, comprising 12 miFTCs, 17 eaFTCs, and 10 wiFTCs, were whole-exome sequenced and analyzed. Somatic mutations, constitutional sequence variants, somatic copy number alterations, and mutational signatures were described. Clinicopathologic parameters and mutational profiles were assessed for associations with patient outcomes. Results Total mutation burden was consistent across FTC subtypes, with a median of 10 (range 1 to 44) nonsynonymous somatic mutations per tumor. Overall, 20.5% of specimens had a mutation in the RAS subfamily (HRAS, KRAS, or NRAS), with no notable difference between subtypes. Mutations in TSHR, DICER1, EIF1AX, KDM5C, NF1, PTEN, and TP53 were also noted to be recurrent across the cohort. Clonality analysis demonstrated more subclones in wiFTC. Survival analysis demonstrated worse disease-specific survival in the eaFTC and wiFTC cohorts, with no recurrences or deaths for patients with miFTC. Mutation burden was associated with worse prognosis, independent of histopathological classification. Conclusions Though the number and variety of somatic variants are similar in the different histopathological subtypes of FTC in our study, mutational burden was an independent predictor of mortality and recurrence.

A noncoding variant in GANAB explains isolated polycystic liver disease (PCLD) in a large family

Expanded mutation detection and novel gene discovery for isolated polycystic liver disease (PCLD) are necessary as 50% of cases do not have identified mutations in the seven published disease genes. We investigated a family with five affected siblings for which no loss‐of‐function variants were identified by whole exome sequencing analysis. SNP genotyping and linkage analysis narrowed the candidate regions to ∼8% of the genome, which included two published PCLD genes in close proximity to each other, GANAB and LRP5. Based on these findings, we re‐evaluated the exome sequencing data and identified a novel intronic nine base pair deletion in the vicinity of the GANAB exon 24 splice donor that had initially been discarded by the sequence analysis pipelines. We used a minigene assay to show that this deletion leads to skipping of exon 24 in cell lines and primary human cholangiocytes. These findings prompt genomic evaluation beyond the coding region to enhance mutation detection in PCLD and to avoid premature implication of other genes in linkage disequilibrium.