Inherited variants at 3q13.33 and 3p24.1 are associated with risk of diffuse large B-cell lymphoma and implicate immune pathways.

We previously identified five single nucleotide polymorphisms (SNPs) at four susceptibility loci for diffuse large B-cell lymphoma (DLBCL) in individuals of European ancestry through a large genome-wide association study (GWAS). To further elucidate genetic susceptibility to DLBCL, we sought to validate two loci at 3q13.33 and 3p24.1 that were suggestive in the original GWAS with additional genotyping. In the meta-analysis (5662 cases and 9237 controls) of the four original GWAS discovery scans and three replication studies, the 3q13.33 locus (rs9831894; minor allele frequency [MAF] = 0.40) was associated with DLBCL risk [odds ratio (OR) = 0.83, P = 3.62 × 10-13]. rs9831894 is in linkage disequilibrium (LD) with additional variants that are part of a super-enhancer that physically interacts with promoters of CD86 and ILDR1. In the meta-analysis (5510 cases and 12 817 controls) of the four GWAS discovery scans and four replication studies, the 3p24.1 locus (rs6773363; MAF = 0.45) was also associated with DLBCL risk (OR = 1.20, P = 2.31 × 10-12). This SNP is 29 426-bp upstream of the nearest gene EOMES and in LD with additional SNPs that are part of a highly lineage-specific and tumor-acquired super-enhancer that shows long-range interaction with AZI2 promoter. These loci provide additional evidence for the role of immune function in the etiology of DLBCL, the most common lymphoma subtype.

Association of polygenic risk score with the risk of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

Inherited loci have been found to be associated with risk of chronic lymphocytic leukemia (CLL). A combined polygenic risk score (PRS) of representative single nucleotide polymorphisms (SNPs) from these loci may improve risk prediction over individual SNPs. Herein, we evaluated the association of a PRS with CLL risk and its precursor, monoclonal B-cell lymphocytosis (MBL). We assessed its validity and discriminative ability in an independent sample and evaluated effect modification and confounding by family history (FH) of hematological cancers. For discovery, we pooled genotype data on 41 representative SNPs from 1499 CLL and 2459 controls from the InterLymph Consortium. For validation, we used data from 1267 controls from Mayo Clinic and 201 CLL, 95 MBL, and 144 controls with a FH of CLL from the Genetic Epidemiology of CLL Consortium. We used odds ratios (ORs) to estimate disease associations with PRS and c-statistics to assess discriminatory accuracy. In InterLymph, the continuous PRS was strongly associated with CLL risk (OR, 2.49; P = 4.4 × 10-94). We replicated these findings in the Genetic Epidemiology of CLL Consortium and Mayo controls (OR, 3.02; P = 7.8 × 10-30) and observed high discrimination (c-statistic = 0.78). When jointly modeled with FH, PRS retained its significance, along with FH status. Finally, we found a highly significant association of the continuous PRS with MBL risk (OR, 2.81; P = 9.8 × 10-16). In conclusion, our validated PRS was strongly associated with CLL risk, adding information beyond FH. The PRS provides a means of identifying those individuals at greater risk for CLL as well as those at increased risk of MBL, a condition that has potential clinical impact beyond CLL.

Cancer risk in oil refinery workers: a pooled mortality study in Italy.

BACKGROUND: Oil refinery workers are exposed to several well-established carcinogens and working in this type of industry has been classified by IARC as probable carcinogen to humans (Group 2A). OBJECTIVES: To examine the mortality experience of workers employed in four Italian oil refineries. METHODS: The cohort included 5112 male workers ever employed between 1949 and 2011. The average follow-up period was 49 years. Standardized mortality ratios (SMR) and 95% Confidence Intervals (CI) were calculated using as reference age-gender-calendar specific regional rates. Analyses by duration of employment and latency were performed. RESULTS: In the whole cohort, pleural cancer (6 deaths, SMR 1.59; 95% CI 0.71-3.53), brain cancer (14 deaths, SMR 1.47; 95% CI 0.87-2.49) and lymphatic leukemia (LL) (8 deaths, SMR 1.81; 95% CI 0.91-3.62) showed increased risks. All pleural cancers occurred after 10 years of latency and the highest risk was observed among workers with duration ≥20 years; the brain cancer excess was confined in the shortest duration and latency. The LL (and chronic lymphatic leukemia in particular) excess regarded workers with latency and duration longer than 20 years. Four deaths from acute myeloid leukemia (AML) were observed and all occurred after 20 years of latency (SMR 1.55, 95% CI 0.58-4.12); a two-fold-increased risk was observed in the longest duration. No increased risk for skin cancer has been observed in our study population. CONCLUSION: Our findings are consistent with recent evidence of an increased mortality from pleural and hematopoietic malignancies (AML and LL) among oil refinery workers. However, the lack of individual quantitative exposure data and the small number of observed events prevent the identification of the possible causal role of individual chemicals, including benzene, especially at the current very low exposure levels.

Position Paper on Asbestos of the Italian Society of Occupational Medicine.

The Position Paper (PP) on asbestos of the Italian Society of Occupational Medicine (SIML) aims at providing a tool to the occupational physician to address current diagnostic criteria and results of epidemiological studies, and their consequences in terms of preventive and evaluation actions for insurance, compensation and litigation. The PP was based on an extensive review of the scientific literature and was compiled by a Working Group comprising researchers who have contributed to the international literature on asbestos-related diseases, as well as occupational physicians with extensive experience in the evaluation of risks and the medical surveillance of workers currently and formerly exposed to asbestos. The PP was drafted and reviewed between 2017 and 2018; its final version was prepared according to the guidelines of AGREE Reporting Checklist. All the members of the Working Group subscribed to the document, which was eventually approved by SIML's Executive Committee. The first section addresses industrial hygiene issues, such as methods for environmental monitoring, advantages and limitations of different microscopy techniques, the potential role of microfibers and approaches for retrospective assessment of exposure, in particular in epidemiological studies. The second section reviews the biological effects of asbestos with particular attention to the diagnostic aspects of asbestosis, pleural changes, mesothelioma and lung cancer. In the following section the criteria of causal attribution are discussed, together with different hypotheses on the form of the risk functions, with a comparison of the opinions prevalent in the literature. In particular, the models of the risk function for mesothelioma were examined, in the light of the hypothesis of an acceleration or anticipation of the events in relation to the dose. The last section discusses topics of immediate relevance for the occupational physician, such as health surveillance of former exposed and of workers currently exposed in remediation activities.

Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease.

BACKGROUND: Alzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives. METHODS: Blood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year-old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean +/- standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test. RESULTS: Data reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients. CONCLUSION: We suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease.

Birth order, allergies and lymphoma risk: results of the European collaborative research project Epilymph.

Lymphomas are a heterogeneous group of malignancies of the immune system. Recent studies suggest that immunological conditions which are modulated by lifestyle-dependent environmental determinants might affect lymphoma risk. We used data from Epilymph, a European multi-centric case-control study with 2,480 cases and 2,540 controls, to analyse the relationship between lifestyle-dependent immunological determinants and risk of lymphomas. We found an inverse relationship between risk of lymphoma and allergies, mainly respiratory (OR=0.86, CI=0.89-1.01) and food allergies (OR=0.67, CI=0.52-0.85), a slightly elevated lymphoma risk for first-born children (OR=1.17, CI=0.99-1.39) and only children (OR=1.10, CI=0.86-1.39). The inverse relationship between atopic disorders and risk of lymphomas is consistent with earlier observations. Our findings on birth order and lymphoma increase the inconsistency of findings across studies and suggest a critical reappraisal of potential underlying mechanisms.

Statin use and risk of lymphoid neoplasms: results from the European Case-Control Study EPILYMPH.

BACKGROUND: Statins, drugs used to treat dyslipidemia, may have anticancer properties. We have evaluated lymphoma risk associated with regular statin use in an international case-control study. METHODS: This case-control study included 2,362 cases of incident B- and T-cell lymphoma from Czech Republic, France, Germany, Ireland, Italy, and Spain and 2,206 hospital or population controls. Information on drug use, diagnosis at admission (for hospital controls), and putative risk factors for lymphoma was collected with personal interviews. Hospital controls admitted for diseases possibly entailing use of statins were excluded from the analysis. RESULTS: The odds ratio for regular statin use was 0.61 (95% confidence interval, 0.45-0.84); all major lymphoma subtypes showed similarly decreased risks. Decreased risks were observed in all centers. Duration of statin use was not associated with a greater reduction in the risk of lymphoma. Use of other lipid lowering drugs, such as fibrates, did not significantly modify the risk of lymphoma (odds ratio, 0.75; 95% confidence interval, 0.44-1.27). CONCLUSION: Statin use was associated with an important reduction in lymphoma risk, adding to the growing evidence of anticancer properties of this group of drugs. These results are reassuring for the increasing number of patients taking statins on a regular basis.

Medical history and risk of lymphoma: results of a European case-control study (EPILYMPH).

INTRODUCTION: Lymphomas are a heterogeneous group of immune-cell malignancies. Immunology-related conditions are among the few factors for which consistent evidence exists relating them to lymphoma risk. MATERIALS AND METHODS: We used the data from the European case-control study Epilymph on 2,362 lymphoma cases and 2,458 controls to investigate associations between a medical history of infectious and non-infectious diseases with overall and subentity-specific lymphoma risk. RESULTS: As key results, we observed an increased odds ratio (OR) for self-reported infections with hepatitis B virus (HBV, OR = 1.91, 95% CL = 1.24-2.94) and a null result for rheumatoid arthritis. Additionally, we found an increased OR for infectious mononucleosis (OR = 1.68, 95% CL = 1.14-2.48), an inverse association to frequency of sickness in childhood (OR = 0.68, 95% CL = 0.55-0.84), and-as casual finding-an increased OR with acetaminophen intake (OR = 2.29, 95% CL = 1.49-3.51). CONCLUSION: Our results are consistent with the current knowledge about the association with mononucleosis as indicator of Epstein-Barr-virus infection, suggest serological study of the association to HBV infection and do not support the view of a positive association between rheumatoid arthritis and lymphoma risk.

Altered cholesterol ester cycle in skin fibroblasts from patients with Alzheimer's disease.

Intracellular cholesterol metabolism was reported to modulate amyloid-beta (Abeta) generation in Alzheimer's disease (AD). Results presented herein demonstrated that, like brain cells, cultured skin fibroblasts from AD patients contained more cholesterol esters than fibroblasts from healthy subjects. Particularly, Oil Red-O, Nile Red, and filipin staining highlighted higher levels of neutral lipids which responded to inhibitors of acyl-coenzyme A:cholesterol acyl-transferase (ACAT-1), associated with an increase in free cholesterol. ACAT-1 mRNA levels increased significantly in AD fibroblasts, whereas those of sterol regulatory element binding protein-2, neutral cholesterol ester hydrolase, and ATP-binding cassette transporter member 1 were markedly down-regulated. Instead, mRNA levels of low-density lipoprotein receptor, hydroxy-methyl-glutaryl-coenzyme A reductase, caveolin-1, and amyloid-beta protein precursor (AbetaPP) were virtually unchanged. Notably, mRNA levels of both beta-site AbetaPP-cleaving enzyme 1 (BACE1) and neprilysin were significantly down-regulated. An increase in Abeta(40) and Abeta(42) immunostaining and a decrease in BACE1 active form were also found in AD versus control fibroblasts. Altogether, these findings support the hypothesis that the derangement of cholesterol homeostasis is a systemic alteration involving central but also peripheral cells of AD patients, and point to cholesterol ester levels in AD fibroblasts as an additional metabolic hallmark useful in the laboratory and clinical practice.

Exposure to ultraviolet radiation and risk of malignant lymphoma and multiple myeloma--a multicentre European case-control study.

BACKGROUND: Three recent studies have reported a decreased risk of non-Hodgkin lymphoma (NHL) for high ultraviolet (UV) radiation exposure. METHODS: We conducted a multicentre case-control study during 1998-2004 in France, Germany, Ireland, Italy and Spain, comprising 1518 cases of NHL, 268 cases of Hodgkin lymphoma, 242 cases of multiple myeloma and 2124 population or hospital controls. We collected information on sensitivity to sun and personal exposure to UV radiation in childhood and adulthood via interview, and assessed occupational exposure to UV radiation from the occupational history. RESULTS: The risk of Hodgkin and NHL was increased for increasing skin sensitivity to the sun [odds ratio (OR) for no suntan vs very brown 2.35, 95% CI 0.94-5.87 and 1.39, 95% CI 1.03-1.87, respectively]. The risk of diffuse large B-cell lymphoma was reduced for increasing adult personal (OR for highest vs lowest quartile of exposure in free days 0.62, 95% CI 0.44-0.87) and for occupational exposure to UV radiation (OR for highest vs lowest exposure tertile 0.63, 95% CI 0.37-1.04). The risk of multiple myeloma was increased for personal exposure to UV radiation during adulthood (OR for highest vs lowest quartile of exposure in free days 1.49, 95% CI 0.88-2.50). A protective effect was observed for use of sun lamps for diffuse large B-cell lymphoma (OR for 25+ times vs never 0.63, 95% CI 0.38-1.03). CONCLUSIONS: The hypothesis of a protective effect of UV radiation on lymphoma is supported by our results. The underlying mechanisms might differ from those operating in skin carcinogenesis. The increased risk of multiple myeloma is worth replication.

Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls.

Epstein-Barr Virus (EBV) is consistently associated with distinct lymphoproliferative malignancies and aberrant EBV antibody patterns are found in most EBV cancer patients. We evaluate the detection of an abnormal reactive serological pattern to EBV (ab_EBV) infection and the risk of lymphoma in a multicentric case-control study. Serum samples were collected at study entry from 1,085 incident lymphoma cases from Spain, France, Germany, Czech Republic, Italy and 1,153 age, sex and country matched controls. EBV immunoglobulin G (IgG) serostatus was evaluated through a peptide-based ELISA combining immunodominant epitopes of EBNA1 (BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody diversity patterns to EBV early antigens (EA), besides EBNA1, VCA-p18, VCA-p40 (BdRF1) and Zebra (BZLF1). Patients with chronic active EBV infection and aberrant EBV activity were characterized as having an abnormal reactive pattern (ab_EBV). Ab_EBV was observed in 20.9% of 2,238 included subjects with an increased proportion of cases presenting ab_EBV as compared to the control population (23.9% vs. 18.0% p = 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] = 1.42, 95% confidence interval [CI]=1.15-1.74), and specifically for chronic lymphocytic leukaemia (OR = 2.96, 95%CI = 2.22-3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR = 0.38, 95%CI = 0.15-0.98). EBV may be involved in a larger subset of lymphomas among clinically immunocompetent subjects than previously thought, probably explained by an underlying loss of immune control of EBV latent infection. Ab_EBV is a useful tool to explore EBV imbalances preceding or paralleling possible EBV associated oncogenic events.

Variation in DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma.

Cytogenetic analysis in myeloma reveals marked chromosomal instability. Both widespread genomic alterations and evidence of aberrant class switch recombination, the physiological process that regulates maturation of the antibody response, implicate the DNA repair pathway in disease pathogenesis. We therefore assessed 27 SNPs in three genes (XRCC3, XRCC4 and XRCC5) central to DNA repair in patients with myeloma and controls from the EpiLymph study and from an Irish hospital registry (n = 306 cases, 263 controls). For the haplotype-tagging SNP (htSNP) rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4 versus 80.8%; odds ratio 1.51; 95% confidence interval 1.10-2.08; P = 0.0133), as was the AA genotype (74 versus 65%) (P = 0.026). Haplotype analysis was performed using Unphased for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A-T haplotype from rs963248-rs2891980 (P = 0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only one control (P = 0.015). This SNP is located in the 3'-UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.

Family history of hematopoietic malignancies and risk of non-Hodgkin lymphoma (NHL): a pooled analysis of 10 211 cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph).

A role for genetic susceptibility in non-Hodgkin lymphoma (NHL) is supported by the accumulating evidence of common genetic variations altering NHL risk. However, the pattern of NHL heritability remains poorly understood. We conducted a pooled analysis of 10 211 NHL cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph) to evaluate NHL risk among those with hematopoietic malignancies in first-degree relatives. Odds ratios (ORs) and 95% confidence intervals (CIs) of NHL and its subtypes were estimated from unconditional logistic regression models with adjustment for confounders. NHL risk was elevated for individuals who reported first-degree relatives with NHL (OR = 1.5; 95% CI = 1.2-1.9), Hodgkin lymphoma (OR = 1.6; 95% CI = 1.1-2.3), and leukemia (OR = 1.4; 95% CI = 1.2-2.7). Risk was highest among individuals who reported a brother with NHL (OR = 2.8; 95% CI = 1.6-4.8) and was consistent for all NHL subtypes evaluated. If a first-degree relative had Hodgkin lymphoma, NHL risk was highest if the relative was a parent (OR = 1.7; 95% CI = 1.0-2.9). If a first-degree relative had leukemia, NHL risk was highest among women who reported a sister with leukemia (OR = 3.0; 95% CI = 1.6-5.6). The pattern of NHL heritability appeared to be uniform across NHL subtypes, but risk patterns differed by specific hematopoietic malignancies and the sex of the relative, revealing critical clues to disease etiology.

Association between personal use of hair dyes and lymphoid neoplasms in Europe.

Hair dyes have been evaluated as possibly being mutagenic and carcinogenic in animals. Studies of the association between human cancer risk and use of hair dyes have yielded inconsistent results. The authors evaluated the risk of lymphoid malignancies associated with personal use of hair dyes. The analysis included 2,302 incident cases of lymphoid neoplasms and 2,417 hospital- or population-based controls from the Czech Republic, France, Germany, Ireland, Italy, and Spain (1998-2003). Use of hair dyes was reported by 74% of women and 7% of men. Lymphoma risk among dye users was significantly increased by 19% in comparison with never use (odds ratio (OR) = 1.19, 95% confidence interval (CI): 1.00, 1.41) and by 26% among persons who used hair dyes 12 or more times per year (OR = 1.26, 95% CI: 1.00, 1.60; p for linear trend = 0.414). Lymphoma risk was significantly higher among persons who had started coloring their hair before 1980 (OR = 1.37, 95% CI: 1.09, 1.72) and persons who had used hair dyes only before 1980 (OR = 1.62, 95% CI: 1.10, 2.40). Personal use of hair dyes is associated with a moderate increase in lymphoma risk, particularly among women and persons who used dyes before 1980. Specific compounds associated with this risk remain to be elucidated.

Hepatitis C and risk of lymphoma: results of the European multicenter case-control study EPILYMPH.

BACKGROUND & AIMS: Increasing evidence points toward a role of hepatitis C virus (HCV) infection in the etiology of malignant lymphomas. However, previous epidemiologic studies were limited in size to establish an association between HCV infection and specific lymphoma subtypes. We performed a large, multicenter, case-control study to address this question. METHODS: The study comprised 5 European countries and included newly diagnosed cases of any lymphoid malignancy recruited between 1998 and 2004. Controls were matched to cases by 5-year age group, sex, and study center. In-person interviews were conducted to collect data on demographic, medical, and family history as well as environmental exposures. Serum samples of 1807 cases and 1788 controls (excluding human immunodeficiency virus-positive and organ-transplantation subjects) were screened for HCV infection using an enzyme immunoassay. Positive as well as randomly selected negative samples were subjected to HCV RNA detection and HCV genotyping. RESULTS: HCV infection was detected in 53 (2.9%) lymphoma cases and in 41 (2.3%) control subjects (odds ratio [OR], 1.42; 95% confidence interval [CI]: 0.93-2.15). Restricted to individuals who tested positive for HCV-RNA (indicating persistent infection and active viral replication), the OR was 1.82 (95% CI: 1.13-2.91). In subtype-specific analyses, HCV prevalence was associated with diffuse large B-cell lymphoma (OR, 2.19; 95% CI: 1.23-3.91) but not with chronic lymphocytic leukemia or follicular, Hodgkin's, or T-cell lymphoma. The sample size was not sufficient to derive any conclusions for rare lymphoma entities such as splenic marginal zone lymphoma. CONCLUSIONS: These results support a model that chronic HCV replication contributes to lymphomagenesis and establish a specific role of HCV infection in the pathogenesis of diffuse large B-cell lymphoma.

Changes in GABA(A) receptor gene expression induced by withdrawal of, but not by long-term exposure to, ganaxolone in cultured rat cerebellar granule cells.

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA(A) receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl- currents evoked by GABA at recombinant human GABA(A) receptors (comprising alpha1beta2gamma2L or alpha2beta2gamma2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 microM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the alpha1, alpha2, alpha3, alpha4, alpha5, gamma2L, or gamma2S subunits of the GABA(A) receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of alpha2, alpha4, and alpha5 subunit mRNAs and a decrease in the amounts of alpha1, gamma2L, and gamma2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA(A) receptor to several positive modulators. Nevertheless, the reduction in the amounts of the alpha1 and gamma2 subunit mRNAs together with the increase in the abundance of the alpha4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.