Canonical and uncanonical pathogenic germline variants in colorectal cancer patients by next-generation sequencing in a European referral center.

BACKGROUND: Despite increasing use of next-generation sequencing (NGS), data concerning the gain in germline pathogenic variants (PVs) remain scanty, especially with respect to uncanonical ones. We aimed to verify the impact of different cancer predisposition genes (CPGs) on colorectal cancer (CRC) in patients referred for genetic evaluation. MATERIALS AND METHODS: We enrolled for NGS, by Illumina TruSight Cancer panel comprising 94 CPGs, 190 consecutive subjects referred for microsatellite instability (MSI) CRC, polyposis, and/or family history. RESULTS: Overall, 51 (26.8%) subjects carried 64 PVs; PVs coexisted in 4 (7.8%) carriers. PVs in mismatch repair (MMR) genes accounted for one-third of variant burden (31.3%). Four Lynch syndrome patients (20%) harbored additional PVs (HOXB13, CHEK2, BRCA1, NF1 plus BRIP1); such multiple PVs occurred only in subjects with PVs in mismatch syndrome genes (4/20 versus 0/31; P = 0.02). Five of 22 (22.7%) patients with MSI cancers but wild-type MMR genes harbored PVs in unconventional genes (FANCL, FANCA, ATM, PTCH1, BAP1). In 10/63 patients (15.9%) with microsatellite stable CRC, 6 had MUTYH PVs (2 being homozygous) and 4 exhibited uncanonical PVs (BRCA2, BRIP1, MC1R, ATM). In polyposis, we detected PVs in 13 (25.5%) cases: 5 (9.8%) in APC, 6 (11.8%) with biallelic PVs in MUTYH, and 2 (3.9%) in uncanonical genes (FANCM, XPC). In subjects tested for family history only, we detected two carriers (18.2%) with PVs (ATM, MUTYH). CONCLUSION: Uncanonical variants may account for up to one-third of PVs, underlining the urgent need of consensus on clinical advice for incidental findings in cancer-predisposing genes not related to patient phenotype.

Predictors of germline status for hereditary melanoma: 5 years of multi-gene panel testing within the Italian Melanoma Intergroup.

BACKGROUND: The incidence of cutaneous melanoma is increasing in Italy, in parallel with the implementation of gene panels. Therefore, a revision of national genetic assessment criteria for hereditary melanoma may be needed. The aim of this study was to identify predictors of susceptibility variants in the largest prospective cohort of Italian high-risk melanoma cases studied to date. MATERIALS AND METHODS: From 25 Italian centers, we recruited 1044 family members and germline sequenced 940 cutaneous melanoma index cases through a shared gene panel, which included the following genes: CDKN2A, CDK4, BAP1, POT1, ACD, TERF2IP, MITF and ATM. We assessed detection rate according to familial status, region of origin, number of melanomas and presence and type of non-melanoma tumors. RESULTS: The overall detection rate was 9.47% (5.53% analyzing CDKN2A alone), ranging from 5.14% in sporadic multiple melanoma cases (spoMPM) with two cutaneous melanomas to 13.9% in familial cases with at least three affected members. Three or more cutaneous melanomas in spoMPM cases, pancreatic cancer and region of origin predicted germline status [odds ratio (OR) = 3.23, 3.15, 2.43, P < 0.05]. Conversely, age > 60 years was a negative independent predictor (OR = 0.13, P = 0.008), and was the age category with the lowest detection rate, especially for CDKN2A. Detection rate was 19% when cutaneous melanoma and pancreatic cancer clustered together. CONCLUSIONS: Gene panel doubled the detection rate given by CDKN2A alone. National genetic testing criteria may need a revision, especially regarding age cut-off (60) in the absence of strong family history, pancreatic cancer and/or a high number of cutaneous melanomas.

Tapentadol prolonged release and the long-term management of chronic musculoskeletal pain in the elderly - focus on anxiety, depression, cognitive status and life quality: the TaPE study.

OBJECTIVE: The use of long-term opioids for the management of chronic musculoskeletal pain is a hot topic in the scientific community, especially when it concerns the elderly. This paper aimed at assessing the efficacy and tolerability of tapentadol prolonged release (PR), a molecule with a unique mechanism of action combining µ-opioid-receptor (MOR) agonism and noradrenaline reuptake inhibition (NRI), administered to patients aged ≥80 years with chronic persistent pain. The effect of this molecule on anxiety, depression, cognitive status, and overall quality of life were investigated. PATIENTS AND METHODS: This was a spontaneous, observational, open-label, prospective study, in 80 older patients aged ≥80 years, naïve to strong opioids, presenting moderate-to-severe chronic pain from different etiologies. Tapentadol PR was initially prescribed at the dose of 25-50 mg/day and increased gradually in case of insufficient analgesia. Pain intensity was assessed by a 10-point Numeric Rating Scale (NRS). Other endpoints were as follows: DN4 questionnaire for the evaluation of the neuropathic component of pain, SF12, HADS, and MMSE questionnaires to evaluate the quality of life, anxiety, and cognitive impairment, respectively. Safety evaluations were also performed through the assessment of the frequency and severity of adverse events. RESULTS: At T45, NRS score reduction was achieved in 86.0% of patients. On average, pain decreased by 55% from a mean of 8.2 to a mean of 3.6. At T90, tapentadol PR did not affect the psychophysical and cognitive abilities of older patients. CONCLUSIONS: The benefits with tapentadol PR in controlling pain have improved the quality of life of our patients, also showing a favorable effect on their cognitive performance.

A Novel Truncating FLAD1 Variant, Causing Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) in an 8-Year-Old Boy.

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is a clinically heterogeneous disorder affecting fatty acid and amino acid metabolism. Presentations range from a severe neonatal form with hypoglycemia, metabolic acidosis, and hepatomegaly with or without congenital anomalies to later-onset lipid storage myopathy. Genetic testing for MADD traditionally comprises analysis of ETFA, ETFB, and ETFDH. Patients may respond to pharmacological doses of riboflavin, particularly those with late-onset MADD due to variants in ETFDH. Increasingly other genes involved in riboflavin transport and flavoprotein biosynthesis are recognized as causing a MADD phenotype. Flavin adenine dinucleotide synthase (FADS) deficiency caused by biallelic variants in FLAD1 has been identified in nine previous cases of MADD. FLAD1 missense mutations have been associated with a riboflavin-responsive phenotype; however the effect of riboflavin with biallelic loss of function FLAD1 mutations required further investigation. Herein we describe a novel, truncating variant in FLAD1 causing MADD in an 8-year-old boy. Fibroblast studies showed a dramatic reduction in FADS protein with corresponding reduction in the FAD synthesis rate and FAD cellular content, beyond that previously documented in FLAD1-related MADD. There was apparent biochemical and clinical response to riboflavin treatment, beyond that previously reported in cases of biallelic loss of function variants in FLAD1. Early riboflavin treatment may have attenuated an otherwise severe phenotype.

Association of SULT1A1 Arg²¹³His polymorphism with male breast cancer risk: results from a multicenter study in Italy.

Male breast cancer (MBC) is rare and poorly understood. Like female breast cancer (FBC), MBCs are highly sensitive to hormonal changes, and hyperestrogenism, specifically, represents a major risk factor for MBC. MBC is considered similar to late-onset, post-menopausal estrogen/progesteron receptors positive FBC (ER+/PR+). Sulfotransferase 1A1 (SULT1A1) is an enzyme involved in the metabolism of estrogens. Recently, SULT1A1 common functional polymorphism Arg(213)His (638G>A) variant has been found to be associated with increased breast cancer (BC) risk, particularly in post-menopausal women. For this reason, we decided to explore whether SULT1A1 Arg(213)His could exert an effect on MBC development. The primary aim of this study was to evaluate the influence of the SULT1A1 Arg(213)His polymorphism on MBC risk. The secondary aim was to investigate possible associations with relevant clinical-pathologic features of MBC. A total of 394 MBC cases and 786 healthy male controls were genotyped for SULT1A1 Arg(213)His polymorphism by PCR-RFLP and high-resolution melting analysis. All MBC cases were characterized for relevant clinical-pathologic features. A significant difference in the distribution of SULT1A1 Arg(213)His genotypes was found between MBC cases and controls (P < 0.0001). The analysis of genotype-specific risk showed a significant increased MBC risk in individuals with G/A (OR 1.97, 95% CI 1.50-2.59; P < 0.0001) and A/A (OR 3.09, 95% CI 1.83-5.23; P < 0.0001) genotypes in comparison to wild-type genotype, under co-dominant model. A significant association between SULT1A1 risk genotypes and HER2 status emerged. Results indicate that SULT1A1 Arg(213)His may act as a low-penetrance risk allele for developing MBC and could be associated with a specific tumor subtype associated with HER2 overexpression.

Association of low-penetrance alleles with male breast cancer risk and clinicopathological characteristics: results from a multicenter study in Italy.

It is well-known that male breast cancer (MBC) susceptibility is mainly due to high-penetrance BRCA1/2 mutations. Here, we investigated whether common low-penetrance breast cancer (BC) susceptibility alleles may influence MBC risk in Italian population and whether variant alleles may be associated with specific clinicopathological features of MBCs. In the frame of the Italian Multicenter Study on MBC, we genotyped 413 MBCs and 745 age-matched male controls at 9 SNPs annotating known BC susceptibility loci. By multivariate logistic regression models, we found a significant increased MBC risk for 3 SNPs, in particular, with codominant models, for rs2046210/ESR1 (OR = 1.71; 95 % CI: 1.43-2.05; p = 0.0001), rs3803662/TOX3 (OR = 1.59; 95 % CI: 1.32-1.92; p = 0.0001), and rs2981582/FGFR2 (OR = 1.26; 95 % CI: 1.05-1.50; p = 0.013). Furthermore, we showed that the prevalence of the risk genotypes of ESR1 tended to be higher in ER- tumors (p = 0.062). In a case-case multivariate analysis, a statistically significant association between ESR1 and ER- tumors was found (OR = 1.88; 95 % CI: 1.03-3.49; p = 0.039). Overall, our data, based on a large and well-characterized MBC series, support the hypothesis that common low-penetrance BC susceptibility alleles play a role in MBC susceptibility and, interestingly, indicate that ESR1 is associated with a distinct tumor subtype defined by ER-negative status.

Polyomavirus BK replication in adult polycystic kidney disease post-renal transplant patients and possible role of cellular permissivity.

Cell division and differentiation but not proliferation seem to be necessary for BK virus (BKV) replication and reactivation of persistent infection. Only terminal differentiating cells are permissive to BKV replication. Renal tubular epithelial cells in human adult polycystic kidney disease (ADPKD) are characterized by increased proliferative activity leading to cyst growth with less cellular differentiation. The aim of this study was to evaluate the possibility of different BKV replication patterns in patients with polycystic kidney disease versus non-ADPKD patients. From May 2006 to April 2008, we performed renal transplantations in 47. Eleven patients were affected by ADPKD (Pc group) and 36 patients, non ADPKD (n-Pc group). BKV replication was evaluated by quantitative real-time polymerase chain reactions (PCR) on plasma and urine samples at 12 hours (T0/early) as well as 3 (T3) and 6 (T6) months after transplantation. BKV viremia occurred in 9%, 12.5%, and 20% among the Pc group versus 33.3%, 42.4%, and 50% among the n-Pc group at 12 hours as well as 3 and 6 months posttransplantation, respectively. A higher discordance (BKV-PCR blood -/urine +) was observed in plasma and urine BKV replication among Pc versus n-Pc groups. We hypothesized that the lower number of patients with active BKV plasma replication in the Pc group may be related to a lower cellular permissivity of the renal tubular epithelial cells in ADPKD.

Improvement of graft function after conversion to once daily tacrolimus of stable kidney transplant patients.

BACKGROUND: Chronic renal dysfunction is present in about one quarter of kidney transplant patients at 1 year and in about 90% by 10 years. Nephrotoxicity caused by calcineurin inhibitors is among the most common factors. Elevated tacrolimus levels have been correlated with worse control of side effects including acute and/or chronic nephrotoxicity. The aim of this study was to observe the effects on graft function of conversion from the twice daily to the once daily extended release tacrolimus formulation in stable kidney transplant recipients within 5 years of grafting. METHODS: Thirty-one stable kidney transplant patients were converted at the same dosage (1 mg:1 mg). Patients served as their own controls based on results before versus after conversion. RESULTS: The trough levels of tacrolimus showed a slight albeit significant reduction after the conversion. Serum creatinine and glomerular filtration rate showed a significant improvement without an association with the tacrolimus trough levels. CONCLUSION: We suggest that the immunosuppression with once daily tacrolimus may be a good option for kidney transplant patients.

Oral contraceptive use and breast or ovarian cancer risk in BRCA1/2 carriers: a meta-analysis.

BACKGROUND: Women with BRCA1 or BRCA2 mutations are at increased risk of breast and ovarian cancer. Oral contraceptives (OC) use has been associated with a reduction in ovarian cancer risk and with a moderately increased breast cancer risk, which tends to level off in the few years after stopping. The association between oral contraceptive and BRCA1 or BRCA2 gene mutations carriers is unclear. METHODS: We performed a comprehensive literature search updated to March 2010 of studies on the associations between OC users and breast or ovarian cancer for ascertained BRCA1/2 carriers. We obtained summary risk estimated for ever OC users, for duration of use and time since stopping. RESULTS: A total of 2855 breast cancer cases and 1503 ovarian cancer cases, carrying an ascertained BRCA1/2 mutation, were included in our meta-analyses, based on overall 18 studies. Use of OC was associated with a significant reduced risk of ovarian cancer for BRCA1/2 carriers (summary relative risk (SRR)=0.50; 95% confidence interval (CI), 0.33-0.75). We also observed a significant 36% risk reduction for each additional 10 years of OC use (SRR: 0.64; 95% CI, 0.53-0.78; P trend<0.01). We found no evidence of a significant association between OC and breast cancer risk in carriers (SRR: 1.13; 95% CI, 0.88-1.45) and with duration of use. OC formulations used before 1975 were associated with a significant increased risk of breast cancer (SRR: 1.47; 95% 1.06, 2.04), but no evidence of a significant association was found with use of more recent formulations (SRR: 1.17; 95% 0.74, 1.86). CONCLUSIONS: OC users carrying an ascertained BRCA1/2 mutation have a reduced risk of ovarian cancer, proportional to the duration of use. There is no evidence that recent OC formulations increase breast cancer risk in carriers.

Chronic kidney disease-epidemiology formula and model for end-stage liver disease score in the assessment of renal function in candidates for liver transplantation.

Assessment of renal function in patients with end-stage liver disease (ESLD) awaiting liver transplantation (OLT) is critical. Various conditions may cause renal damage in ESLD. Renal and liver functions are intertwined due to splanchnic hemodynamic relationships; renal failure rarely occurs in patients without advanced decompensated cirrhosis. The recent literature suggests that evaluation of renal function should include an assessment of liver function. The aim of this study was to evaluate different methods to estimate glomerular filtration rate (GFR) in patient among ESLD candidates for OLT over 1 year. We also correlated renal and hepatic functions. Fifty-two cirrhotic patients Model for End-Stage Liver Disease [MELD] > 10) were enrolled in the study. All patients were evaluated at baseline and every 4 months (T1-T4) thereafter for 1 year. The GFR was calculated by creatinine clearance, and estimated by Cockroft and Gault, Modified Diet Renal Disease (MDRD) 4 and 6 variable and Chronic Kidney Disease-Epidemiology (CKD-EPI) formulae. Hepatic functions were evaluated by MELD score, albumin, bilirubin, and International Normalized Ratio (INR). We observed not statistically significant increase mean value of MELD score, bilirubin, serum creatinine, and blood urea nitrogen and a reduced serum sodium. There were no significant differences among various methods to evaluate GFR at each time over 1 year. We did not observe any association between renal and hepatic function, except at T4 for MELD and GFR estimated with MDRD 4 (P = .009) and 6 (P = .008) parameters or CKD-EPI (P = .036), and MELD and sodium (P = .001). Our results showed that evaluation of renal function in cirrhosis should include an evaluation of hepatic function. In our case, MDRD and CKD-EPI seemed to be the more accurate formulae to evaluate renal function in relation to hepatic function.

Once daily tacrolimus formulation: monitoring of plasma levels, graft function, and cardiovascular risk factors.

BACKGROUND: Advagraf, an extended release formulation of tacrolimus, is administered once daily during the morning fast. Tacrolimus can be safely converted from the twice daily formulation (Prograf) to the same dose (1 mg:1 mg) of once daily dosing tacrolimus (m-Tac). The adverse effects of tacrolimus play important roles in posttransplant cardiovascular risk factors (CVR): hyperglycemia, posttransplant diabetes mellitus, dyslipidemia and hypertension. It has been suggested that avoiding high tacrolimus peak levels minimizes its diabetogenic effects leading to better glycemic control. The aim of our study was to observe the effects of conversion to m-Tac therapy on graft function and CVR among stable transplant kidney recipients. METHODS: We selected 2 groups of 20 patients with stable kidney transplantation, who had been treated with Prograf for >6 months with a triple regimen. Group 1 were converted to once daily tacrolimus at the same dose (1 mg:1 mg); whereas in group 2, the therapy was maintained as a twice daily regimen. Blood pressure, creatinine and glomerular filtration rate levels evaluated by the Modification of Diet in Renal Disease formula, as well as urea, total, high- and low-density lipoprotein remained stable between the 2 groups as well as inside group 1 before and after conversion. RESULTS: After conversion, glycemia and triglyceride values showed significant reductions in group 1 and between the 2 groups. These results were significant, as they may be associated with better long-term graft and patient survivals.

Meaning of early polyomavirus-BK replication post kidney transplant.

Polyomavirus BK (BKV) infection is ubiquitous in the human population. Under immunosuppression, BKV can undergo reactivation resulting in viral replication. What really happens in the early hours posttransplantation is not clearly defined; the meaning of early viremia and viruria is not clear. BKV viremia is considered a marker of infection. The aim of our study was to investigate the prevalence of early BKV infection in kidney transplant patients, to evaluate the relationship to infections at 3 and 6 months and the association with recipient, donor, and graft features. We enrolled 36 kidney transplanted patients from May 2006 to April 2007. BKV load was measured on plasma and urine samples by Q-PCR at 12 hours (T(0)/early) as well as 3 (T(3)) and 6 (T(6)) months thereafter. A high percentage of BKV infections were detectable in the first hours after transplantation (33.3%), which remained unchanged to month 6 post transplantation. Moreover, patients who were positive at T(0) had a high probability of remaining positive thereafter. The number of copies in plasma samples tended to increase at 3 months and to decrease thereafter, whereas the urine viral load tended to steadily increase. Among BKV-positive patients, we identified 2 groups according to viremic state at T(0): 9 patients (group A); who were already positive and remained so to T(6) 5 and 3 patients who turned positive at 3 or at 6 months, respectively (group B). Group A included 75% of positive patients at T(0) and 90% of positive patients at either T(3) or T(6) (P = .007). The most important contribution of our study was to highlight the presence of BKV infection in renal transplant recipients from the first hours posttransplantation. This condition seemed to be the most important risk factor for persistent infection in the first 6 months.

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA).

BACKGROUND: In this study we aimed to evaluate the role of a SNP in intron 1 of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers. METHODS: We have genotyped rs744154 in 9408 BRCA1 and 5632 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and assessed its association with breast cancer risk using a retrospective weighted cohort approach. RESULTS: We found no evidence of association with breast cancer risk for BRCA1 (per-allele HR: 0.98, 95% CI: 0.93-1.04, P = 0.5) or BRCA2 (per-allele HR: 0.97, 95% CI: 0.89-1.06, P = 0.5) mutation carriers. CONCLUSION: This SNP is not a significant modifier of breast cancer risk for mutation carriers, though weak associations cannot be ruled out.

Clinical trials with retinoids for breast cancer chemoprevention.

Retinoids have been studied as chemopreventive agents in clinical trials due to their established role in regulating cell growth, differentiation and apoptosis in preclinical models. Experimental evidence suggests that retinoids affect gene expression both directly, by activating and/or repressing specific genes, and indirectly, by interfering with different signal transduction pathways. Induction of apoptosis is a unique feature of fenretinide, the most widely studied retinoid in clinical trials on breast cancer chemoprevention due to its selective accumulation in breast tissue and to its favourable toxicological profile. In a phase III breast cancer prevention trial, fenretinide showed a durable trend to a reduction of second breast malignancies in premenopausal women. This pattern was associated with a favourable modulation of circulating IGF-I and its main binding protein (IGF-binding protein-3, IGFBP-3), which have been associated with breast cancer risk in premenopausal women in different prospective studies. In a subsequent biomarker study on premenopausal women who had participated in the phase III trial, high IGF-I and low IGFBP-3 baseline levels were found to predict second breast cancer risk, although the magnitude of their changes during treatment did not fulfil the requirements for suitable surrogate end-point biomarkers. In postmenopausal women, fenretinide did not reduce second breast cancer incidence, nor did it induce significant modulation of the IGF system. Similarly, fenretinide was not found to affect risk biomarkers significantly in early postmenopausal women on hormone replacement therapy, who are at increased risk of developing breast cancer. Biomarker studies of fenretinide alone or in combination with different nuclear receptor ligands are being conducted. In particular, clinical trials of fenretinide and tamoxifen have proved to be feasible, and this combination appears to be safe and well tolerated in high-risk women, especially when low-dose tamoxifen is employed. Novel retinoid X receptor-selective retinoids, or rexinoids, have been shown to suppress the development of breast cancer in several animal models with minimal toxicity, and are being intensively studied either alone or in combination with selective oestrogen receptor modulators, both in vitro and in vivo. The rexinoid, bexarotene, has recently been approved for the treatment of patients with cutaneous T-cell lymphoma, and a biomarker trial with bexarotene in women with high breast cancer risk is currently underway.

Molecular characterization of Italian nevoid basal cell carcinoma syndrome patients.

Mutations in the PTCH gene, the human homolog of the Drosophila patched gene, have been found to lead to the autosomal dominant disorder termed Nevoid Basal Cell Carcinoma Syndrome (NBCCS, also called Gorlin Syndrome). Patients display an array of developmental anomalies and are prone to develop a variety of tumors, with multiple Basal Cell Carcinomas occurring frequently. We provide here the results of molecular testing of a set of Italian Nevoid Basal Cell Carcinoma Syndrome patients. Twelve familial patients belonging to 7 kindreds and 5 unaffected family members, 6 non-familial patients and an additional set of 7 patients with multiple Basal Cell Carcinoma but no other criteria for the disease were examined for mutations in the PTCH gene. All of the Nevoid Basal Cell Carcinoma Syndrome patients were found to carry variants of the PTCH gene. We detected nine novel mutations (1 of which occurring twice): 1 missense mutation (c.1436T>G [p.L479R]), 1 nonsense mutation (c.1138G>T [p.E380X]), 6 frameshift mutations (c.323_324ins2, c.2011_2012dup, c.2535_2536dup, c.2577_2583del, c.3000_3005del, c.3050_3051del), 1 novel splicing variant (c.6552A>T) and 3 mutations that have been previously reported (c.3168+5G>A, c.1526G>T [p.G509V], and c.3499G>A [p.G1167R]). None of the patients with multiple Basal Cell Carcinoma but no other criteria for the syndrome, carried germline coding region mutations.

Bullous pemphigoid in Liguria: a 2-year survey.

BACKGROUND: The epidemiology of bullous pemphigoid (BP) is not clear because of the heterogeneity of the disease, and its possible association with internal malignancies has been under debate for many years. We report the findings of a 2-year study on incident BP cases in the Liguria region of Italy. SUBJECTS AND METHODS: Thirty-two patients with BP were collected over the 2-year period. Diagnosis was made based on clinical findings and confirmed by histology, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) with salt-split skin and monkey oesophagus, and immunoblotting (IB). All patients were thoroughly investigated for possible malignancies and all were followed up for 6 months to monitor the response to treatment. RESULTS: DIF showed linear deposits at the dermoepidermal junction in all but one patient. IIF gave positive findings for 15 sera tested with monkey oesophagus and 20 tested with salt-split skin. IB gave positive findings in 19 cases. There was a malignancy in six cases, but no clinical or immunological features that could be considered to predict this occurrence. CONCLUSIONS: The findings of this study are in accordance with most of the data found in the literature, including the fact that IgG serum levels did not predict the course of the disease. Contrary to previous indications, IgE levels were not indicative of disease course either. Mucosal lesions, erythema multiform-like lesions, negative IIF findings and antibodies to AgPB2 were not a prediction for the development of malignancy.

A phenylalanine residue at segment D3-S6 in Nav1.4 voltage-gated Na(+) channels is critical for pyrethroid action.

Mammalian voltage-gated Na(+) channels were less sensitive to pyrethroids than their insect counterparts by 2 to 3 orders of magnitude. Deltamethrin at 10 microM elicited weak gating changes in rat skeletal muscle alpha-subunit Na(+) channels (Nav1.4) after > 30 min of perfusion. About 10% of the peak current was maintained during the 8-ms, +50-mV pulse and, upon repolarization to -140 mV, the amplitude of the slow tail current corresponded to less than 3% of total Na(+) channels modified by deltamethrin. A background mutation, Nav1.4-I687M (within D2:S4-S5 cytoplasmic linker), enhanced the deltamethrin-induced maintained current by approximately 2.5-fold, whereas Nav1.4-I687T, a homologous superkdr mutation, reduced it by approximately 2-fold. Repetitive pulses at 2 Hz further augmented the effects of deltamethrin on Nav1.4-I687M mutant channels so that approximately 75% of peak currents were maintained. A second mutation, Nav1.4-I687M/F1278I at the middle of D3-S6, rendered the channel greatly resistant to deltamethrin. This double mutant channel remained sensitive to batrachotoxin (BTX), even though nearby residues S1276 and L1280 were critical for BTX action. We hypothesize that the deltamethrin receptor and the BTX receptor are situated at the middle but opposite surface of the D3-S6 alpha-helical structure. Another mutant, Nav1.4-I687M/N784K, exhibited a partial deltamethrin-resistant phenotype but was completely resistant to BTX. Consistent with the BTX-resistant phenotype of N784K and the known adjacent kdr mutation at position L785F, deltamethrin and BTX were probably situated next to each other upon binding at D2-S6. Evidently, distinct residues from multiple S6 segments were critical for deltamethrin and BTX actions.

Disparate role of Na(+) channel D2-S6 residues in batrachotoxin and local anesthetic action.

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.