NTRK3-Rearranged Prostatic Acinar Adenocarcinoma: Report of a Patient and Review of the Literature.

Molecular investigations have led to increased therapeutic options for prostatic adenocarcinoma. A single case report of a PRPSAP1::NTRK3 gene fusion occurring in prostate cancer was previously reported. A review of the literature revealed that NTRK gene rearrangements are exceedingly rare molecular events in prostate cancer. NTRK gene fusions can be oncogenic drivers or develop as resistance mechanisms. The tumor-agnostic approvals of TRK inhibitors by the FDA provide additional rationale for molecular investigations of aggressive prostatic adenocarcinomas. This may prove to be an additional therapeutic option for patients with aggressive prostatic carcinomas refractory to initial therapy. We report a case of an aggressive castrate-resistant prostatic adenocarcinoma with a BMP6::NTRK3 gene fusion.

CAMTA1-related disorder: Phenotypic and molecular characterization of 26 new individuals and literature review.

Calmodulin-binding transcriptional activator 1 (CAMTA1) is highly expressed in the brain and plays a role in cell cycle regulation, cell differentiation, regulation of long-term memory, and initial development, maturation, and survival of cerebellar neurons. The existence of human neurological phenotypes, including cerebellar dysfunction with variable cognitive and behavioral abnormalities (CECBA), associated with CAMTA1 variants, has further supported its role in brain functions. In this study, we phenotypically and molecularly characterize the largest cohort of individuals (n = 26) with 23 novel CAMTA1 variants (frameshift-7, nonsense-6, splicing-1, initiation codon-1, missense-5, and intragenic deletions-3) and compare the findings with all previously reported cases (total = 53). We show that the most notable phenotypic findings are developmental delay/intellectual disability, unsteady or uncoordinated gait, hypotonia, behavioral problems, and eye abnormalities. In addition, there is a high incidence of dysarthria, dysgraphia, microcephaly, gastrointestinal abnormalities, sleep difficulties, and nonspecific brain MRI findings; a few of which have been under-reported. More than one third of the variants in this cohort were inherited from an asymptomatic or mildly affected parent suggesting reduced penetrance and variable expressivity. Our cohort provides a comprehensive characterization of the spectrum of phenotypes and genotypes among individuals with CECBA and the large data will facilitate counseling and formulating management plans and surveillance recommendations for these individuals.

Intragenic CNTN4 copy number variants associated with a spectrum of neurobehavioral phenotypes.

Deletions and duplications involving the CNTN4 gene, which encodes for the contactin 4 protein, have been reported in children with autism spectrum disorder (ASD) and other neurodevelopmental phenotypes. In this study, we performed clinical and genetic characterization of three individuals from unrelated families with copy number variants (CNV) (one deletion and two duplications) within CNTN4. The patients exhibited cognitive delay (3/3), growth restriction (3/3), motor delay (2/3), and febrile seizure/epilepsy (2/3). In contrast to previous reports, all probands presented with speech apraxia or delay with no diagnosis of ASD. Parental studies for the proband with the deletion and one of the 2 probands with the duplication revealed paternal origin of the CNTN4 CNV. Interestingly, previously documented CNV involving this gene were mostly inherited from unaffected fathers, raising questions regarding reduced penetrance and potential parent-of-origin effect. Our findings are compared with previously reported patients and patients in the DECIPHER database. The speech impairment in the three probands suggests a role for CNTN4 in language development. We discuss potential factors contributing to phenotypic heterogeneity and reduced penetrance and attempt to find possible genotype-phenotype correlation. Larger cohorts are needed for comprehensive and unbiased phenotyping and molecular characterization that may lead to better understanding of the underlying mechanisms of reduced penetrance, variable expressivity, and potential parent-of-origin effect of copy number variants encompassing CNTN4.

Future of Personalized Therapy Targeting Aberrant Signaling Pathways in Multiple Myeloma.

Multiple myeloma (MM) is a genetically complex disease. Identification of mutations and aberrant signaling pathways that contribute to the progression of MM and drug resistance has potential to lead to specific targets and personalized treatment. Aberrant signal pathways include RAS pathway activation due to RAS or BRAF mutations (targeted by vemurafenib alone or combined with cobimetinib), BCL-2 overexpression in t(11:14) (targeted by venetoclax), JAK2 pathway activation (targeted by ruxolitinib), NF-κB pathway activation (treated with DANFIN combined with bortezomib), MDM2 overexpression, and PI3K/mTOR pathway activation (targeted by BEZ235). Cyclin D1 (CCND1) and MYC are also emerging as key potential targets. In addition, histone deacetylase inhibitors are already in use for the treatment of MM in combination therapy, and targeted inhibition of FGFR3 (AZD4547) is effective in myeloma cells with t(4;14) translocation. Bromodomain and extra terminal (BET) protein antagonists decrease the expression of MYC and have displayed promising antimyeloma activity. A better understanding of the alterations in signaling pathways that promote MM progression will further inform the development of precision therapy for patients.

Unexpected favorable outcome in a patient with high grade B-cell lymphoma with abnormalities of MYC, BCL6 and BCL2 loci.

High grade B-cell lymphoma (HGBCL) by WHO 2016 classification requires rearrangements of MYC and BCL2 and/or BCL6, practically covering the so called "double-hit" or "triple hit" lymphomas. We report a case of HGBCL "triple-hit" lymphoma in a 64-year old female. Cytogenetic and fluorescence in situ hybridization (FISH) studies revealed complex karyotype including rearrangement of MYC to a novel, non-IG partner on chromosome 18, and rearrangement of BCL2, BCL6 and IGH as well as ins(3)(q21q27.3q25.1) among other abnormalities. FISH studies showed five copies of MYC and 3-8 copies of BCL2. Gene expression analysis by RNA sequencing showed that MYC, BCL2 and MECOM genes were overexpressed whereas BCL6 was under-expressed. BCL6 was fused to MBNL1 gene due to complex structural rearrangement. MYC was expressed in >70% of cells and BCL2 was diffusely but highly expressed by immunohistochemistry. No pathogenic mutations were identified by sequencing a 26-gene panel including TP53. The patient has unexpectedly been in complete remission for 12 months after diagnosis after intensive chemotherapy including DA-EPOCH regimen despite having HGBCL. The prognostication of HGBCL patients may further be improved by the sub-categorization of these lymphomas on the basis of more detailed genomic markers than merely the WHO 2016 classification.

A Phase II Trial of Neoadjuvant MK-2206, an AKT Inhibitor, with Anastrozole in Clinical Stage II or III PIK3CA-Mutant ER-Positive and HER2-Negative Breast Cancer.

Purpose: Hyperactivation of AKT is common and associated with endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The allosteric pan-AKT inhibitor MK-2206 induced apoptosis in PIK3CA-mutant ER+ breast cancer under estrogen-deprived condition in preclinical studies. This neoadjuvant phase II trial was therefore conducted to test the hypothesis that adding MK-2206 to anastrozole induces pathologic complete response (pCR) in PIK3CA mutant ER+ breast cancer.Experimental Design: Potential eligible patients with clinical stage II/III ER+/HER2- breast cancer were preregistered and received anastrozole (goserelin if premenopausal) for 28 days in cycle 0 pending tumor PIK3CA sequencing. Patients positive for PIK3CA mutation in the tumor were eligible to start MK-2206 (150 mg orally weekly, with prophylactic prednisone) on cycle 1 day 2 (C1D2) and to receive a maximum of four 28-day cycles of combination therapy before surgery. Serial biopsies were collected at preregistration, C1D1 and C1D17.Results: Fifty-one patients preregistered and 16 of 22 with PIK3CA-mutant tumors received study drug. Three patients went off study due to C1D17 Ki67 >10% (n = 2) and toxicity (n = 1). Thirteen patients completed neoadjuvant therapy followed by surgery. No pCRs were observed. Rash was common. MK-2206 did not further suppress cell proliferation and did not induce apoptosis on C1D17 biopsies. Although AKT phosphorylation was reduced, PRAS40 phosphorylation at C1D17 after MK-2206 persisted. One patient acquired an ESR1 mutation at surgery.Conclusions: MK-2206 is unlikely to add to the efficacy of anastrozole alone in PIK3CA-mutant ER+ breast cancer and should not be studied further in the target patient population. Clin Cancer Res; 23(22); 6823-32. ©2017 AACR.

Neratinib Efficacy and Circulating Tumor DNA Detection of HER2 Mutations in HER2 Nonamplified Metastatic Breast Cancer.

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut).Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31-74) years and three (2-10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%-55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8-31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%-94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%-100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687-95. ©2017 AACR.

NeoPalAna: Neoadjuvant Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, and Anastrozole for Clinical Stage 2 or 3 Estrogen Receptor-Positive Breast Cancer.

Purpose: Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor-positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the antiproliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies.Experimental Design: Eligible patients with clinical stage II/III ER+/HER2- breast cancer received anastrozole 1 mg daily for 4 weeks (cycle 0; with goserelin if premenopausal), followed by adding palbociclib (125 mg daily on days 1-21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67 > 10%, in which case patients went off study due to inadequate response. Anastrozole was continued until surgery, which occurred 3 to 5 weeks after palbociclib exposure. Later patients received additional 10 to 12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression, and mutation profiles. The primary endpoint was complete cell cycle arrest (CCCA: central Ki67 ≤ 2.7%).Results: Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs. C1D1 26%, P < 0.001). Palbociclib enhanced cell-cycle control over anastrozole monotherapy regardless of luminal subtype (A vs. B) and PIK3CA status with activity observed across a broad range of clinicopathologic and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with nonluminal subtypes and persistent E2F-target gene expression.Conclusions: Palbociclib is an active antiproliferative agent for early-stage breast cancer resistant to anastrozole; however, prolonged administration may be necessary to maintain its effect. Clin Cancer Res; 23(15); 4055-65. ©2017 AACR.

Association of Li-Fraumeni Syndrome With Small Cell Carcinoma of the Ovary, Hypercalcemic Type and Concurrent Pleomorphic Liposarcoma of the Cervix.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare, highly lethal malignancy predominantly affecting young adult females. We report a patient with widely metastatic SCCOHT and concurrent uterine cervical pleomorphic liposarcoma. Clinical targeted next-generation sequencing was performed on both neoplasms and demonstrated hemizygous stop-gain TP53 mutations (p.R196*), and wild-type SMARCA4 in both tumors. Microarray analyses of both tumors revealed similar but not identical widespread loss of heterozygosity over most chromosomes associated with loss of chromosomal copy number in the SCCOHT and pleomorphic liposarcoma tumors, amplification of FGFR1 in both tumors, and amplification of MYC in the SCCOHT. Immunohistochemistry demonstrated that SMARCA4 and SMARCB1 were retained in both tumors, and that SMARCA2 expression was retained but TP53 expression was lost in the SCCOHT. Germline testing using Sanger sequencing showed heterozygous TP53 mutation, confirming the diagnosis of Li-Fraumeni syndrome. These findings are novel and for the first time associate SCCOHT with Li-Fraumeni syndrome.

Gliosarcomas lack BRAFV600E mutation, but a subset exhibit ß-catenin nuclear localization.

Gliosarcoma (GS) is a rare subtype of glioblastoma (GBM) characterized by both glial and mesenchymal components. Unlike GBM, there are no specific prognostic markers, and optimized treatments for patients with GS do not exist. Recent reports describe BRAFV600E mutation in malignant peripheral nerve sheath tumors, and aberrant Wnt signaling and CTNNB1 (ß-catenin gene) mutations have been described in GBM. We sought to determine whether GS tumors harbor BRAFV600E mutations or aberrant Wnt signaling, as indicated by nuclear localization of ß-catenin, by immunohistochemical detection. Forty-eight (48) cases of primary and secondary adult GS (including recurrent ones) were evaluated by immunohistochemical techniques for the presence of nuclear ß-catenin and the BRAFV600E mutation. A small subset (6/46, 13%) showed nuclear localization of ß-catenin. None of the cases harbored BRAFV600E mutations (0/48). These results are the first to describe the presence of Wnt signaling pathway abnormalities, manifested by nuclear ß-catenin, in a subset, as well as the lack of BRAFV600E mutation in GS. We propose a potential role for Wnt pathway alterations in the pathogenesis of a subset of GS.

Identification of major factors associated with failed clinical molecular oncology testing performed by next generation sequencing (NGS).

PURPOSE: DNA analysis by NGS has become important to direct the clinical care of cancer patients. However, NGS is not successful in all cases, and the factors responsible for test failures have not been systematically evaluated. MATERIALS AND METHODS: A series of 1528 solid and hematolymphoid tumor specimens was tested by an NGS comprehensive cancer panel during 2012-2014. DNA was extracted and 2×101 bp paired-end sequence reads were generated on cancer-related genes utilizing Illumina HiSeq and MiSeq platforms. RESULTS: Testing was unsuccessful in 343 (22.5%) specimens. The failure was due to insufficient tissue (INST) in 223/343 (65%) cases, insufficient DNA (INS-DNA) in 99/343 (28.9%) cases, and failed library (FL) in 21/343 (6.1%) cases. 87/99 (88%) of the INS-DNA cases had below 10 ng DNA available for testing. Factors associated with INST and INS-DNA failures were site of biopsy (SOB) and type of biopsy (TOB) (both p < 0.0001), and clinical setting of biopsy (CSB, initial diagnosis or recurrence) (p < 0.0001). Factors common to INST and FL were age of specimen (p ≤ 0.006) and tumor viability (p ≤ 0.05). Factors common to INS-DNA and FL were DNA purity and DNA degradation (all p ≤ 0.005). In multivariate analysis, common predictors for INST and INS-DNA included CSB (p = 0.048 and p < 0.0001) and TOB (both p ≤ 0.003), respectively. SOB (p = 0.004) and number of cores (p = 0.001) were specific for INS-DNA, whereas TOB and DNA degradation were associated with FL (p = 0.04 and 0.02, respectively). CONCLUSIONS: Pre-analytical causes (INST and INS-DNA) accounted for about 90% of all failed cases; independent of test design. Clinical setting; site and type of biopsy; and number of cores used for testing all correlated with failure. Accounting for these factors at the time of tissue biopsy acquisition could improve the analytic success rate.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

BACKGROUND: A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC). METHODS: Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS: Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS: NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.

Detection of gene rearrangements in targeted clinical next-generation sequencing.

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Scoliosis and vertebral anomalies: additional abnormal phenotypes associated with chromosome 16p11.2 rearrangement.

The typical chromosome 16p11.2 rearrangements are estimated to occur at a frequency of approximately 0.6% of all samples tested clinically and have been identified as a major cause of autism spectrum disorders, developmental delay, behavioral abnormalities, and seizures. Careful examination of patients with these rearrangements revealed association with abnormal head size, obesity, dysmorphism, and congenital abnormalities. In this report, we extend this list of phenotypic abnormalities to include scoliosis and vertebral anomalies. We present detailed characterization of phenotypic and radiological data of 10 new patients, nine with the 16p11.2 deletion and one with the duplication within the coordinates chr16:29,366,195 and 30,306,956 (hg19) with a minimal size of 555 kb. We discuss the phenotypical and radiological findings in our patients and review 5 previously reported patients with 16p11.2 rearrangement and similar skeletal abnormalities. Our data suggest that patients with the recurrent 16p11.2 rearrangement have increased incidence of scoliosis and vertebral anomalies. However, additional studies are required to confirm this observation and to establish the incidence of these anomalies. We discuss the potential implications of our findings on the diagnosis, surveillance and genetic counseling of patients with 16p11.2 rearrangement.

Validation of a next-generation sequencing assay for clinical molecular oncology.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Clinical genomicist workstation.

The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.

Molecular characterization of a novel, de novo, cryptic interstitial deletion on 19p13.3 in a child with a cutis aplasia and multiple congenital anomalies.

We report on a de novo constitutional deletion within G-band region 19p13.3 in a girl with cutis aplasia of the scalp, facial anomalies, structural heart abnormalities, hypotonia, mild mental retardation and conductive hearing loss which we characterized with chromosomal microarray, fluorescence in situ hybridization (FISH), and SNP analyses. Initial microarray analysis revealed a 6-BAC-clone deletion covering an approximately 1.612 Mb region within 19p13.3. Subsequent BAC FISH studies delineated the proximal deletion breakpoint to within BAC clone RP11-125C3 and the distal deletion breakpoint to within BAC clone RP11-648B14. SNP analysis showed the deletion to be of paternal origin and further refined its distal breakpoint to within a 20 kb region between rs11666694 and novel SNP2 that we identified at g.2,924,845, and its proximal deletion breakpoint to within a 22 kb region between rs35280644 and rs262562. Accordingly, the size of the deletion was revised to 1.89-1.932 Mb in length. We identified many Alu, L1, and L2 repeats, as well as SINE and LINE sequences at both deletion breakpoints. We found the deletion to encompass 71 genes, two of which appear to be good candidates for the patient's observed craniofacial and cardiac anomalies: guanine nucleotide binding protein (G protein), alpha 11 (Gq class)(GNA11), and Transducin-like Enhancer of Split 2 (E(sp1) homolog, Drosophila)(TLE2).

Multiple superoxide dismutase 1/splicing factor serine alanine 15 variants are associated with the development and progression of diabetic nephropathy: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Genetics study.

BACKGROUND: Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes. RESEARCH DESIGN AND METHODS: We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,362 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome. RESULTS: We observed association between rs17880135 in the 3' region of superoxide dismutase 1 (SOD1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional SOD1 variants, including rs202446, rs9974610, and rs204732, which were also associated (P < 10(-3)) with persistent microalbuminuria, whereas rs17880135 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum SOD activity, serum SOD1 mass, or SOD1 mRNA expression in lymphoblastoid cell lines. CONCLUSIONS: Multiple variations in SOD1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

Polymorphisms within the protein tyrosine phosphatase 1B (PTPN1) gene promoter: functional characterization and association with type 2 diabetes and related metabolic traits.

BACKGROUND: Protein tyrosine phosphatase 1B (PTPN1) dephosphorylates insulin receptors and attenuates insulin signaling. Polymorphisms in the coding sequence of PTPN1 have been variably associated with type 2 diabetes (T2D). We hypothesized that variations within the PTPN1 promoter might contribute to the development of T2D and related metabolic traits. METHODS: We screened 2.0 kb of PTPN1 promoter in 174 T2D patients and 412 controls using PCR and denaturing HPLC. Association analysis was performed between diabetes and related traits and single-nucleotide polymorphism genotypes. We functionally tested 2 variants (-1023C>A and -51delA) by measuring their influence on luciferase activity in HepG2 cells and performing the electrophoretic mobility shift assay (EMSA). RESULTS: One common (-1023C>A) and 6 rare (-51delA, -451A>G, -467T>C, -1045G>A, -1286-3bp-del, and -1291-9bp-del) variants were identified in the PTPN1 promoter. The -1023(C) allele had significant association with T2D that disappeared after we adjusted for established diabetes risk factors. The alleles of -1023C>A and -51delA variants did not show significant effects on the biochemical markers after adjustment for established diabetes risk factors in the nondiabetic and diabetic groups separately. The -51delA variant decreased luciferase gene expression in HepG2 cells by 2-fold. EMSA revealed a weaker binding of -51delA to specific protein family proteins compared with the A allele. The -1023C>A variant had no influence in either experiment. CONCLUSIONS: The PTPN1 promoter variants -1023C>A and -51delA (which appears to be functional) were not associated with T2D or related traits in this study but must be investigated in a larger population to reveal any potential metabolic association.