Fetal Type Morphologies Suggest the Presence of DICER1 Hotspot Mutations in Non-small Cell Lung Cancer.

Germline and somatic pathogenic variants (PVs) in DICER1 , encoding a miRNA biogenesis protein, are associated with a wide variety of highly specific pathologic entities. The lung tumors pleuropulmonary blastoma, pulmonary blastoma (PB), and well-differentiated fetal lung adenocarcinoma (WDFLAC) are all known to harbor DICER1 biallelic variants (loss of function and/or somatic hotspot missense mutations), and all share pathologic features reminiscent of the immature lung. However, the role of DICER1 PVs in non-small cell lung cancer (NSCLC) is relatively unknown. Here, we aimed to establish the spectrum of lung pathologies associated with DICER1 hotspot PVs and to compare the mutational landscape of DICER1 -mutated NSCLC with and without hotspots. We queried DNA sequencing data from 12,146 NSCLCs featuring somatic DICER1 variants. 235 (1.9%) cases harboring ≥ 1 DICER1 PV were found and 9/235 (3.8%) were DICER1 hotspot-positive cases. Histologic review of DICER1 hotspot-positive cases showed that all but one tumor were classified as within the histologic spectrum of PB/WDFLAC, whereas all the DICER1 non-hotspot double variants were classified as lung adenocarcinomas, not otherwise specified. Comparison between the mutational landscape of DICER1 hotspot-positive and hotspot-negative cases revealed a higher frequency of CTNNB1 mutations in the hotspot-positive cases (5/9 vs. 2/225; P <0.00001). We conclude that DICER1 somatic hotspots are not implicated in the most common forms of NSCLC but rather select for morphologic features of lung tumor types such as PB and WDFLAC. As a corollary, cases showing this tumor morphology should undergo testing for DICER1 variants, and if positive, genetic counseling should be considered.

BRCA1/2 Reversion Mutations in Patients Treated with Poly ADP-Ribose Polymerase (PARP) Inhibitors or Platinum Agents.

Background: Reversion mutations in BRCA1/2, resulting in restoration of the open reading frame, have been identified as a mechanism of resistance to platinum-based chemotherapy or PARP inhibition. We sought to explore the incidence of BRCA1/2 reversion mutations in different tumor types. Methods: We retrospectively analyzed molecular profiling results from primary and/or metastatic tumor samples submitted by multiple institutions. The samples underwent DNA and RNA sequencing at a CLIA/CAP-certified clinical lab. Reversion mutations were called only in patients whose available clinical records showed the use of PARP inhibitors or platinum agents prior to tumor profiling. Results: Reversion mutations were identified in 75 of 247,926 samples profiled across all tumor types. Among patients carrying pathogenic or likely pathogenic BRCA1/2 mutations, reversion mutations in BRCA1/2 genes were seen in ovarian cancer (OC) (30/3424), breast cancer (BC) (27/1460), endometrial cancer (4/564), pancreatic cancer (2/340), cholangiocarcinoma (2/178), prostate cancer (5/461), cervical cancer (1/117), cancer of unknown primary (1/244), bladder cancer (1/300), malignant pleural mesothelioma (1/10), and a neuroendocrine tumor of the prostate. We identified 22 reversion mutations in BRCA1 and 8 in BRCA2 in OC. In BC, we detected 6 reversion mutations in BRCA1 and 21 in BRCA2. We compared molecular profile results of 14 high-grade serous ovarian cancers (HGSOC) with reversion mutations against 87 control HGSOC with pathogenic BRCA1/2 mutations without reversion mutations. Tumors with reversion mutations trended to have had lower ER expression (25% vs. 64%, p = 0.024, q = 0.82) and higher KDM6A mutation rate (15% vs. 0, p = 0.016, q = 0.82). Conclusions: We present one of the largest datasets reporting reversion mutations in BRCA1/2 genes across various tumor types. These reversion mutations were rare; this may be because some patients may not have had repeat profiling post-treatment. Repeat tumor profiling at times of treatment resistance can help inform therapy selection in the refractory disease setting.

Transcriptional Profiling of Malignant Melanoma Reveals Novel and Potentially Targetable Gene Fusions.

Invasive melanoma is the deadliest type of skin cancer, with 101,110 expected cases to be diagnosed in 2021. Recurrent BRAF and NRAS mutations are well documented in melanoma. Biologic implications of gene fusions and the efficacy of therapeutically targeting them remains unknown. Retrospective review of patient samples that underwent next-generation sequencing of the exons of 592 cancer-relevant genes and whole transcriptome sequencing for the detection of gene fusion events and gene expression profiling. Expression of PDL1 and ERK1/2 was assessed by immunohistochemistry (IHC). There were 33 (2.6%) cases with oncogenic fusions (14 novel), involving BRAF, RAF1, PRKCA, TERT, AXL, and FGFR3. MAPK pathway-associated genes were over-expressed in BRAF and RAF1 fusion-positive tumors in absence of other driver alterations. Increased expression in tumors with PRKCA and TERT fusions was concurrent with MAPK pathway alterations. For a subset of samples with available tissue, increased phosphorylation of ERK1/2 was observed in BRAF, RAF1, and PRKCA fusion-positive tumors. Oncogenic gene fusions are associated with transcriptional activation of the MAPK pathway, suggesting they could be therapeutic targets with available inhibitors. Additional analyses to fully characterize the oncogenic effects of these fusions may support biomarker driven clinical trials.

Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer.

PURPOSE: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies. EXPERIMENTAL DESIGN: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings. RESULTS: The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1-RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. CONCLUSIONS: This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Prognostic Values of G-Protein Mutations in Metastatic Uveal Melanoma.

Uveal melanoma is the most common primary ocular malignancy in adults, characterized by gene mutations in G protein subunit alpha q (GNAQ) and G protein subunit alpha 11 (GNA11). Although they are considered to be driver mutations, their role in MUM remains elusive. We investigated key somatic mutations of MUM and their impact on patients' survival after development of systemic metastasis (Met-to-Death). Metastatic lesions from 87 MUM patients were analyzed by next generation sequencing (NGS). GNA11 (41/87) and GNAQ (39/87) mutations were most predominantly seen in MUM. Most GNA11 mutations were Q209L (36/41), whereas GNAQ mutations comprised Q209L (14/39) and Q209P (21/39). Epigenetic pathway mutations BAP1 (42/66), SF3B1 (11/66), FBXW7 (2/87), PBRM1 (1/66), and SETD2 (1/66) were found. No specimen had the EIF1AX mutation. Interestingly, Met-to-Death was longer in patients with GNAQ Q209P compared to GNAQ/GNA11 Q209L mutations, suggesting the difference in mutation type in GNAQ/GNA11 might determine the prognosis of MUM. Structural alterations of the GNAQ/GNA11 protein and their impact on survival of MUM patients should be further investigated.

Tumour mutational burden, microsatellite instability, and actionable alterations in metastatic colorectal cancer: Next-generation sequencing results of TRIBE2 study.

BACKGROUND: We performed a comprehensive genomic profiling of tumour samples from metastatic colorectal cancer (mCRC) patients enrolled in the TRIBE2 study to assess the concordance among different techniques to evaluate mismatch repair (MMR) and microsatellite instability (MSI) status, to characterize tumours according to the tumour mutational burden (TMB) and explore the clinical relevance of different TMB cutpoints, and to investigate the prevalence of alterations actionable with targeted approaches or immune checkpoint inhibitors. MATERIAL AND METHODS: Tumour samples of 296 (44%) of 679 enrolled patients underwent 592-gene DNA next-generation sequencing (NGS). MMR status was assessed by immunohistochemistry (MMR-IHC), and MSI status was assessed by NGS (MSI-NGS). TMB was defined as low, intermediate, or high if <7, 7-16, or ≥17 mutations/megabase (mut/Mb) were found. The performance of TMB to predict MSI status was tested by receiver operating characteristic (ROC) curve. Actionable alterations included BRAF V600E, KRAS G12C, POLE mutations, HER2 amplification and mutations, and MSI-H. RESULTS: Of 216 paired cases, concordance between MMR-IHC and MSI-NGS was 98.6%. Among 11 TMB-high tumours, eight (73%) were MSI-H and three (27%) were microsatellite stable and harboured POLE or MSH6 mutations. High TMB had a trend for a better outcome than low/intermediate TMB (hazard ratio for overall survival 0.45, 95% confidence interval 0.28-1.33; P = 0.106). No interaction effect between TMB and treatment arm was observed. Seventeen mut/Mb was identified as the optimal threshold of TMB for predicting MSI status. Actionable alterations were found in 62 (21%) of 296 patients. CONCLUSIONS: Genomic profiling provides an overview of the genomic landscape of mCRC in a single analysis, including actionable targets and markers of immune sensitivity.

Population bias in somatic measurement of microsatellite instability status.

Microsatellite instability (MSI) is a key secondary effect of a defective DNA mismatch repair mechanism resulting in incorrectly replicated microsatellites in many malignant tumors. Historically, MSI detection has been performed by fragment analysis (FA) on a panel of representative genomic markers. More recently, using next-generation sequencing (NGS) to analyze thousands of microsatellites has been shown to improve the robustness and sensitivity of MSI detection. However, NGS-based MSI tests can be prone to population biases if NGS results are aligned to a reference genome instead of patient-matched normal tissue. We observed an increased rate of false positives in patients of African ancestry with an NGS-based diagnostic for MSI status utilizing 7317 microsatellite loci. We then minimized this bias by training a modified calling model that utilized 2011 microsatellite loci. With these adjustments 100% (95% CI: 89.1% to 100%) of African ancestry patients in an independent validation test were called correctly using the updated model. This poses not only a significant technical improvement but also has an important clinical impact on directing immune checkpoint inhibitor therapy.

Relationship between protein biomarkers of chemotherapy response and microsatellite status, tumor mutational burden and PD-L1 expression in cancer patients.

Chemotherapy and checkpoint inhibitor immunotherapies are increasingly used in combinations. We determined associations between the presence of anti-PD-1/PD-L1 therapeutic biomarkers and protein markers of potential chemotherapy response. Data were extracted from a clinical-grade testing database (Caris Life Sciences; February 2015 through November 2017): immunotherapy response markers (microsatellite instability-high [MSI-H], tumor mutational burden-high [TMB-H], and PD-L1 protein expression) and protein chemotherapy response markers (excision repair complementation group 1 [ERCC1], topoisomerase 1 [TOPO1], topoisomerase 2 [TOP2A], thymidylate synthase [TS], tubulin beta 3 [TUBB3], ribonucleotide reductase regulatory subunit M1 [RRM1] and O-6-methyl guanine DNA methyltransferase [MGMT]). Relationships were determined by the Mantel-Haenszel chi-squared test or Fischer's exact tests. Overall, 28,034 patients representing a total of 40 tumor types were assessed. MSI-H was found in 3.3% of patients (73% were also TMB-H), TMB-H, 8.4% (28.3% were also MSI-H) and PD-L1 expression in 11.0% of patients (5.1% were also MSI-H; 16.4% were also TMB-H). Based on concurrent biomarker expression, combinations of immunotherapy with platinum (ERCC1 negativity) or with doxorubicin, epirubicin or etoposide (TOP2A positivity) have a higher probability of response, whereas combinations with irinotecan or topotecan (TOPO1 positivity), with gemcitabine (RRM1 negativity), and fluorouracil, pemetrexed or capecitabine (TS negativity) may be of less benefit. The potential for immunotherapy and taxane (TUBB3 negativity) combinations is present for MSI-H but not TMB-H or PD-L1-expressing tumors; for temozolomide and dacarbazine (MGMT negative), PD-L1 is frequently coexpressed, but MSI-H and TMB-H are not associated. Protein markers of potential chemotherapy response along with next-generation sequencing for immunotherapy response markers can help support rational combinations as part of an individualized, precision oncology approach.

Spindle Epithelial Tumor with Thymus-Like Differentiation (SETTLE): A Next-Generation Sequencing Study.

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a malignant biphasic neoplasm of the thyroid or neck with propensity for late metastasis. Unlike synovial sarcoma, its main morphologic mimic, SETTLE lacks synovial sarcoma-associated translocations. A single case of SETTLE has shown a KRAS mutation but to date no comprehensive next generation sequencing studies of this rare neoplasm have been undertaken. Herein, we subjected 5 well defined cases of SETTLE to direct sequence analysis of 592 genes and fusion gene analysis of 52 genes frequently rearranged in human cancers. We identified one case with two pathogenic variants in the KMT2D gene, one being in an intron splice site (c.674-1A>G) and the other being a frameshift variant (p.M2829fs). This same case also had a pathogenic nonsense variant in the KMT2C gene (p.R1237*). A second case of SETTLE carried a pathogenic NRAS missense variant, Q61R. No other molecular alterations, microsatellite instability, gene fusions or amplifications were identified.

Tumor Mutational Burden Is Site Specific in Non-Small-Cell Lung Cancer and Is Highest in Lung Adenocarcinoma Brain Metastases.

PURPOSE: Tumor mutational burden (TMB) is a developing biomarker in non-small-cell lung cancer (NSCLC). Little is known regarding differences between TMB and sample location, histology, or other biomarkers. METHODS: A total of 3,424 unmatched NSCLC samples, including 2,351 lung adenocarcinomas (LUADs) and 1,073 lung squamous cell carcinomas (LUSCs), underwent profiling, including next-generation sequencing of 592 cancer-related genes, programmed death ligand 1 immunohistochemistry, and TMB. The rate TMB of 10 mutations per megabase (Mb) or greater was compared between primary and metastatic LUAD and LUSC. Molecular alteration frequency was compared at a cutoff of 10 mutations/Mb. RESULTS: LUAD metastases were more likely to have a TMB of 10 mutations/Mb or greater compared with primary LUADs (38% v 25%; P < .001), and this difference was most pronounced with brain metastases (61% v 35% for other metastases; P < .001). The median TMB for LUAD brain metastases was 13 mutations/Mb compared with six mutations/Mb for primary LUADs. Variability existed for other LUAD metastasis sites, with adrenal metastases most likely to meet the cutoff of 10 mutations/Mb (51%) and bone metastases least likely to meet the cutoff (19%). TMB was more commonly 10 mutations/Mb or greater for LUSC primary tumors than for LUAD primary tumors (35% v 25%, respectively; P < .001). LUSC metastases were more likely to have a TMB of 10 mutations/Mb or greater than LUSC primary tumors. Poorly differentiated disease was more likely have a TMB of 10 mutations/Mb or greater when stratified by histology and primary tumor or metastasis. Site-specific molecular differences existed at this TMB cutoff including programmed death ligand 1 positivity and STK11 and KRAS mutation rate. CONCLUSION: TMB is a site-specific biomarker in NSCLC with important spatial and histologic differences. TMB is more frequently 10 mutations/Mb or greater in LUAD and LUSC metastases and highest in LUAD brain metastases. Along this TMB cutoff, clinically informative distinctions exist in other tumor profiling characteristics. Further investigation is needed to expand on these findings.

Towards Molecular Profiling in Multiple Myeloma: A Literature Review and Early Indications of Its Efficacy for Informing Treatment Strategies.

Multiple myeloma (MM), the second most common hematologic malignancy, is characterized by the clonal expansion of plasma cells. Despite dramatic improvements in patients' survival over the past decade due to advances in therapy exploiting novel molecular targets (immunomodulatory drugs, proteasome inhibitors and monoclonal antibodies), the treatment of relapsed and refractory disease remains challenging. Recent studies confirmed complex, dynamic, and heterogeneous genomic alterations without unifying gene mutations in MM patients. In the current review, we survey recent therapeutic strategies, as well as molecular profiling data on MM, with emphasis on relapsed and refractory cases. A critical appraisal of novel findings and of their potential therapeutic implications will be discussed in detail, along with the author's own experiences/views.

Comprehensive analysis of cancers of unknown primary for the biomarkers of response to immune checkpoint blockade therapy.

BACKGROUND: Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Avoiding immune destruction is a major cancer characteristic and therapies aimed at immune checkpoint blockade are in use for several specific cancer types. A comprehensive survey of predictive biomarkers to immune checkpoint blockade in CUP were explored in this study. METHODS: About 389 cases of CUP were analysed for mutations in 592 genes and 52 gene fusions using a massively parallel DNA sequencing platform (next-generation sequencing [NGS]). Total mutational load (TML) and microsatellite instability (MSI) were calculated from NGS data. PD-L1 expression was explored using immunohistochemistry (with 5% cutoff value). RESULTS: High TML was seen in 11.8% (46/389) of tumours. MSI-high (MSI-H) was detected in 7/384 (1.8%) of tumours. Tumour PD-L1 expression was detected in 80/362 CUP (22%). A small proportion of CUP cases harboured genetic alterations of negative predictive biomarkers to immune checkpoint inhibitors (predictors to hyperprogression) including MDM2 gene amplification (2%) and loss of function JAK2 gene mutations (1%). Amplifications of CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes were also rare (1.4% and 0.8%, respectively). The most frequently mutated genes were TP53 (54%), KRAS (22%), ARID1A (13%), PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%) and PBRM1, STK11, APC, RB1 (5%, respectively). CONCLUSIONS: Using a multiplex testing approach, 28% of CUP carried one or more predictive biomarkers (MSI-H, PD-L1 and/or TML-H) to the immune checkpoint blockade, providing a novel option for treatment in patients with CUP.

Mutational burden, immune checkpoint expression, and mismatch repair in glioma: implications for immune checkpoint immunotherapy.

BACKGROUND: Despite a multiplicity of clinical trials testing immune checkpoint inhibitors, the frequency of expression of potential predictive biomarkers is unknown in glioma. METHODS: In this study, we profiled the frequency of shared biomarker phenotypes. To clarify the relationships among tumor mutational load (TML), mismatch repair (MMR), and immune checkpoint expression, we profiled patients with glioma (n = 327), including glioblastoma (GBM) (n = 198), whose samples had been submitted for analysis from 2009 to 2016. The calculation algorithm for TML included nonsynonymous mutation counts per tumor, with germline mutations filtered out. Immunohistochemical analysis and next-generation sequencing were used to determine tumor-infiltrating lymphocyte expression positive for programmed cell death protein 1 (PD-1), PD ligand 1 (PD-L1) expression on tumor cells, MMR (MLH1, MSH2, MSH6, and PMS2) protein expression and mutations, and DNA polymerase epsilon (POLE) mutations. RESULTS: High TML was only found in 3.5% of GBM patients (7 of 198) and was associated with the absence of protein expression of mutL homolog 1 (MLH1) (P = .0345), mutS homolog 2 (MSH2) (P = .0099), MSH6 (P = .0022), and postmeiotic segregation increased 2 (PMS2) (P = .0345) and the presence of DNA MMR mutations. High and moderate TML GBMs did not have an enriched influx of CD8+ T cells, PD-1+ T cells, or tumor-expressed PD-L1. IDH1 mutant gliomas were not enriched for high TML, PD-1+ T cells, or PD-L1 expression. CONCLUSIONS: To clarify the relationships among TML, MMR, and immune checkpoint expression, we profiled the frequency of shared biomarker phenotypes. On the basis of a variety of potential biomarkers of response to immune checkpoints, only small subsets of glioma patients are likely to benefit from monotherapy immune checkpoint inhibition.

The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Confirmation of the HPCX prostate cancer predisposition locus in large Utah prostate cancer pedigrees.

Several genetic predisposition loci for prostate cancer have been identified through linkage analysis, and it is now generally recognized that no single gene is responsible for more than a small proportion of prostate cancers. However, published confirmations of these loci have been few, and failures to confirm have been frequent. The genetic etiology of prostate cancer is clearly complex and includes significant genetic heterogeneity, phenocopies, and reduced penetrance. Powerful analyses that involve robust statistics and methods to reduce genetic heterogeneity are therefore necessary. We have performed linkage analysis on 143 Utah pedigrees for the previously published Xq27-28 (HPCX) prostate cancer susceptibility locus. We employed a robust multipoint statistic (TLOD) and a novel splitting algorithm to reduce intra-familial heterogeneity by iteratively removing the top generation from the large Utah pedigrees. In a dataset containing pedigrees having no more than five generations, we observed a multipoint TLOD of 2.74 (P=0.0002), which is statistically significant after correction for multiple testing. For both the full-structure pedigrees (up to seven generations) and the smaller sub-pedigrees, the linkage evidence was much reduced. This study thus represents the first significant confirmation of HPCX (Xq27-28) and argues for the continued utility of large pedigrees in linkage analyses for complex diseases.

Characterization of linkage disequilibrium structure, mutation history, and tagging SNPs, and their use in association analyses: ELAC2 and familial early-onset prostate cancer.

In association analyses, it is critical that informative single-nucleotide polymorphisms (SNPs) be selected for study and utilized appropriately. We sequenced 38 kb, including exons of ELAC2, promoter region and conserved upstream intergenic sequences. A comprehensive characterization of linkage disequilibrium (LD) structure and mutation history was performed using our principal components analysis (PCA) method and a phylogenetic analysis. We identified a complex pattern of LD structure consistent with the occurrence of both recombination and mutation events within ELAC2. Four overlapping and noncontiguous LD groups were defined. Eight tagging SNPs (tSNPs) were identified, accounting for over 90% of the genetic variation of the 19 total variants. We tested associations between familial early-onset prostate cancer (PRCA) and each variant independently and in haplotypes. We performed these tests using all 19 variants and the 8 tSNPs; the results using tSNP haplotypes accurately represent the association evidence for the full haplotypes. We observed increased evidence for association when SNPs were analyzed in haplotypes. The phylogenetic analysis indicated three haplotypes, clustered farthest from the root-node, all of which were found more often in cases than controls. These three haplotypes together showed the best evidence of association with familial, early-onset PRCA (P=0.0024; odds ratio=2.23; 95% CI, 1.33-3.74), indicating possible allelic heterogeneity. Our results suggest that 8 tSNPs are required to comprehensively assess associations in ELAC2, and that haplotypes should be considered for analysis, and that a knowledge of mutation history may be helpful in parsing allelic heterogeneity and suggesting combinations of haplotypes to be tested.

Association of a Trp16Ser variation in the gonadotropin releasing hormone signal peptide with bone mineral density, revealed by SNP-dependent PCR typing.

Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone mineral density (BMD). Among those factors, adequate estrogen is essential for achievement of peak bone mass as well as for postmenopausal maintenance of skeletal homeostasis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus is the primary determinant in the hypothalamic-pituitary-gonadal feedback system. In genetic studies of 384 postmenopausal Japanese women, we found a significant association between BMD and an amino acid variation (Trp16Ser) located within the signal peptide of GnRH (r = 0.143, P = 0.005). These results were achieved by genotyping all subjects using a newly developed SNP-dependent PCR method. This automated, high-throughput, and inexpensive procedure is suitable for typing large numbers of samples. BMD was lowest among 16Ser/Ser homozygotes, highest among 16Trp/Trp homozygotes, and intermediate among heterozygotes. A case-control study involving 125 osteoporosis patients and 92 healthy controls revealed a significant association between the presence of a 16Ser GnRH allele and affected status (chi(2) = 4.74, P = 0.041). The results suggested that variation of the GnRH signal peptide may be an important risk factor for postmenopausal osteoporosis.

Association of common missense changes in ELAC2 ( HPC2) with prostate cancer in a Japanese case-control series.

The recently identified prostate cancer susceptibility gene ELAC2 ( HPC2) harbors two common missense variants, a serine to leucine substitution at residue 217 (Leu217) and an alanine to threonine substitution at residue 541 (Thr541). We genotyped the two variants in a Japanese cohort consisting of 350 prostate cancer patients 242 male population controls, and 114 male low-risk controls. Both missense alleles, Leu217 and Thr541, were carried at higher frequency in Japanese patients than in the controls (Leu217, P= 0.0012; Thr541, P = 0.0145), and the odds ratios associated with carrying these sequence variants were higher in Japanese than in Caucasians. Although the Leu217 and Thr541 variants of ELAC2 are less common in Japanese than in Caucasians, both variants confer significantly increased risk of prostate cancer in Japanese. Carriage of these variants was not associated with age at diagnosis, tumor stage, or tumor grade in these Japanese prostate cancer patients. The allele-specific pattern of risk observed in Japanese and familial Caucasian patients was qualitatively similar; however, the magnitude of that risk was considerably greater in Japanese than in Caucasians.