Novel SLC7A7 large rearrangements in lysinuric protein intolerance patients involving the same AluY repeat

Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y(+)LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y(+)LAT-1 protein, is mutated in LPI patients. Mutation analysis of the promoter localized in intron 1 and all exons of the SLC7A7 gene was performed in 11 patients from 9 unrelated LPI families. Point mutation screening was performed by exon direct sequencing and a new multiplex ligation probe amplification (MLPA) assay was set up for large rearrangement analysis. Eleven SLC7A7-specific mutations were identified, seven of them were novel: p.L124P, p.C425R, p.R468X, p.Y274fsX21, c.625+1G>C, DelE4-E11 and DelE6-E11. The novel large deletions originated by the recombination of Alu repeats at introns 3 and 5, respectively, with the same AluY sequence localized at the SLC7A7 3' region. The novel MLPA assay is robust and valuable for LPI molecular diagnosis. Our results suggest that genomic rearrangements of SLC7A7 play a more important role in LPI than has been reported, increasing the detection rate from 5.1 to 21.4%. Moreover, the 3' region AluY repeat could be a recombination hot spot as it is involved in 38% of all SLC7A7 rearranged chromosomes described so far.     

Evaluation of an Array-Based Method for Human Papillomavirus Detection and Genotyping in Comparison with Conventional Methods Used in Cervical Cancer Screening

Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with ≥2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting.Cervical cancer is the second most prevalent type of cancer in women worldwide. A total of 500,000 new cases are diagnosed each year and cause more than 270,000 deaths (15). Since the 1940s, screening programs for cervical cancer prevention, mainly based on the Papanicolau smear test, have been implemented in resource-rich countries, resulting in a remarkable decrease in its incidence and related mortality (16). However, this test has a limited sensitivity, especially for detecting precancerous lesions (1, 6).Genital human papillomavirus (HPV) is a highly common sexually transmitted infection. Although most HPV infections are transient and asymptomatic, epidemiological studies worldwide have demonstrated that persistent infection with certain genotypes is the necessary cause for the development of cervical cancer and its precursor lesions (3, 19, 24). More than 100 HPV types have been described and classified into high-risk types (HR-HPV) and low-risk types (LR-HPV) according to the probability of developing cervical cancer (14). Therefore, in addition to the Papanicolau smear test, HPV detection assays have been implemented in many countries to improve cervical cancer screening. These assays have a higher sensitivity than the Papanicolau smear test for the detection of women at risk of developing precancerous lesions (12).Since HPV cannot be grown in conventional cell cultures and serological assays are unreliable, molecular techniques constitute the best choice to diagnose HPV infection. Currently, the only assay that has been approved by the U.S. Food and Drug Administration for the detection of HPV-DNA is the Hybrid Capture II system (HC-II; Digene Corp., Gaithersburg, MD). This signal amplification assay was designed to detect LR-HPV and HR-HPV genotypes in two different kits but does not provide genotype information.The interest of HPV genotyping has increased in light of the recently licensed HPV bivalent and tetravalent vaccines (9, 23). Genotyping also allows clinicians to monitor patients according to the oncogenic risk of the HPV types identified. Several genotyping assays have been developed over the last years with a variety of amplification and detection strategies (reviewed in references 4 and 13). Methods based on consensus PCR and reverse hybridization of PCR products provide high sensitivity and extensive typing information, including identification of multiple infections. Recently, an assay based on amplification and array hybridization has been commercialized for the detection and genotyping of HPV in routine clinical specimens (Clinical Arrays Papillomavirus Humano [CAPH]; Genomica S.A.U., Madrid, Spain). This assay provides the possibility to detect simple or mixed-type infections with 35 HPV types (20 HR-HPV and 15 LR-HPV).The aim of the present study was to assess the usefulness of introducing this new array-based HPV detection and genotyping method in the cervical cancer screening algorithm in our center, a reference hospital with 600,000-habitant coverage. With this goal, we compared the results obtained using this method with those obtained by HC-II and the cytology findings.     

A somatic NLRP3 mutation as a cause of a sporadic case of chronic infantile neurologic,cutaneous, articular syndrome/neonatal‐onset multisystem inflammatory disease: Novel evidence of the role of low‐level mosaicism as the pathophysiologic mechanism underlying mendelian inherited diseases

Objective

Chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal‐onset multisystem inflammatory disease (NOMID), is a severe, early‐onset autoinflammatory disease characterized by an urticaria‐like rash, arthritis/arthropathy, variable neurologic involvement, and dysmorphic features, which usually respond to interleukin‐1 blockade. CINCA/NOMID has been associated with dominant Mendelian inherited NLRP3 mutations. However, conventional sequencing analyses detect true disease‐causing mutations in only ∼55–60% of patients, which suggests the presence of genetic heterogeneity. We undertook the current study to assess the presence of somatic, nongermline NLRP3 mutations in a sporadic case of CINCA/NOMID.

Methods

Clinical data, laboratory results, and information on treatment outcomes were gathered through direct interviews. Exhaustive genetic studies, including Sanger method sequencing, subcloning, restriction fragment length polymorphism assay, and pyrosequencing, were performed.

Results

The patient's CINCA/NOMID was diagnosed based on clinical features (early onset of the disease, urticaria‐like rash, knee arthropathy, and dysmorphic features). The patient has exhibited a successful response to anakinra within the last 28 months. Analysis of NLRP3 identified a novel heterozygous variant (p.D303H) that was detected in ∼30–38% of circulating leukocytes. The absence of this variant in healthy controls and in the patient's parents suggested a de novo true disease‐causing mutation. Additional analyses showed that this novel mutation was present in both leukocyte subpopulations and epithelial cells.

Conclusion

Our findings identify the novel p.D303H NLRP3 variant in a Spanish patient with CINCA/NOMID as a new disease‐causing mutation, which was detected as a somatic, nongermline mutation in hematopoietic and nonhematopoietic cell lineages. Our data provide new insight into the role of low‐level mosaicism in NLRP3 as the pathophysiologic mechanism underlying cryopyrin‐associated periodic syndrome.

     

Titration of Mechanical Insufflation–Exsufflation Optimal Pressure Combinations in Neuromuscular Diseases by Flow/Pressure Waveform Analysis

Introduction

The aim of this study was to assess several air-pressure settings for MI–E to determine their effect on peak cough flow (PCF), and to compare the best pressures with those are more common used in the literature (±40 cmH₂O) in patients with neuromuscular disorders (NMD).

Oral supplementation with physiological doses of leptin during lactation in rats improves insulin sensitivity and affects food preferences later in life

We have previously described that neonate rats supplemented with physiological doses of oral leptin during lactation become more protected against overweight in adulthood. The purpose of this study was to characterize further the long-term effects on glucose and leptin homeostasis and on food preferences. Neonate rats were supplemented during lactation with a daily oral dose of leptin or the vehicle. We followed body weight and food intake of animals until the age of 15 months, and measured glucose, insulin, and leptin levels under different feeding conditions: ad libitum feeding, 14-h fasting, and 3-h refeeding after fasting. An oral glucose tolerance test and a leptin resistance test were performed. Food preferences were also measured. Leptin-treated animals were found to have lower body weight in adulthood and to eat fewer calories than their controls. Plasma insulin levels were lower in leptin-treated animals than in their controls under the different feeding conditions, as was the increase in insulin levels after food intake. The homeostatic model assessment for insulin resistance index was significantly lower in leptin-treated animals, and the oral glucose tolerance test also indicated higher insulin sensitivity in leptin-treated animals. In addition, these animals displayed lower plasma leptin levels under the different feeding conditions and were also more responsive to exogenous leptin administration. Leptin-treated animals also showed a lower preference for fat-rich food than their controls. These observations indicate that animals supplemented with physiological doses of oral leptin during lactation were more protected against obesity and metabolic features of the metabolic syndrome.     

Similarity between human and chicken Escherichia coli isolates in relation to ciprofloxacin resistance status

BACKGROUND: The food supply is suspected to be a source of fluoroquinolone-resistant Escherichia coli that cause disease in humans, but supporting molecular data are lacking. METHODS: We performed a molecular-epidemiological comparison, in Barcelona, Spain (1996-1998), of 117 contemporaneous, geographically matched E. coli isolates from humans (35 blood isolates and 33 fecal) or chickens (49 fecal) that were either susceptible (n = 57) or resistant (n = 60) to ciprofloxacin and analyzed them by phylogenetic group, virulence genotype, and O antigens using random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). RESULTS: When analyzed by phylogenetic distribution, virulence profiles, and O antigens, resistant human isolates were distinct from susceptible human isolates but were largely indistinguishable from chicken isolates, whereas resistant and susceptible chicken isolates were similar. Susceptible human isolates contained more virulence-associated genes and more frequently expressed virulence-associated O antigens than did resistant human or any chicken isolates. Certain resistant human isolates closely resembled chicken isolates by RAPD and PFGE analysis. CONCLUSIONS: Ciprofloxacin-resistant E. coli may arise de novo in poultry from susceptible progenitors, be transmitted to humans via the food supply, and go on to cause potentially life-threatening infections. If confirmed, these findings would mandate efforts to eliminate this reservoir of drug-resistant pathogens and/or to block their transmission to humans.     

Large‐conductance calcium‐activated potassium channels modulate vascular tone in experimental cirrhosis

Background: Large‐conductance calcium‐activated potassium (BK_Ca) channels regulate vascular tone in different vascular systems. Moreover, activated hepatic stellate cells (HSC) contain BK_Ca channels. The aim of this study was to evaluate the role of BK_Ca channels in the regulation of vascular tone in control (CT) and carbon tetrachloride‐cirrhotic (CH) rat livers. Methods: Changes in intrahepatic vascular resistance were assessed by evaluating the portal perfusion pressure (PP) response to methoxamine (Mtx) in the presence of Iberiotoxin (Ibtx; a BK_Ca channel blocker), NS1619 (a BK_Ca channel opener), Ibtx plus the nitric oxide (NO) synthase inhibitor, N^G‐nitro‐l ‐arginine (l ‐NNA) or l ‐NNA alone. In addition, in CH livers, PP dose–response curves to the NO donor, S‐nitroso‐N‐acetyl‐d,l ‐penicillamine (SNAP), were performed after pre‐incubation with Ibtx or its vehicle. BK_Ca mRNA expression was assessed in liver homogenates, and BK_Ca protein expression in HSC isolated from CT and CH livers. Results: In CH livers, Ibtx significantly increased baseline PP and exacerbated the PP response to Mtx. Conversely, NS1619 induced a mild nonsignificant decrease of baseline PP and attenuated the hyperresponse to Mtx. CH livers exhibited an upregulation of both mRNA and protein of the α‐subunit of BK_Ca. Conclusion: Large‐conductance calcium‐activated potassium channels are overexpressed in CH livers and might represent a compensatory mechanism modulating the increased hepatic vascular tone of cirrhosis.     

Effects of aging and methionine restriction applied at old age on ROS generation and oxidative damage in rat liver mitochondria

It is known that a global decrease in food ingestion (dietary restriction, DR) lowers mitochondrial ROS generation (mitROS) and oxidative stress in young immature rats. This seems to be caused by the decreased methionine ingestion of DR animals. This is interesting since isocaloric methionine restriction in the diet (MetR) also increases, like DR, rodent maximum longevity. However, it is not known if old rats maintain the capacity to lower mitROS generation and oxidative stress in response to MetR similarly to young immature animals, and whether MetR implemented at old age can reverse aging-related variations in oxidative stress. In this investigation the effects of aging and 7 weeks of MetR were investigated in liver mitochondria of Wistar rats. MetR implemented at old age decreased mitROS generation, percent free radical leak at the respiratory chain and mtDNA oxidative damage without changing oxygen consumption. Protein oxidation, lipoxidation and glycoxidation increased with age, and MetR in old rats partially or totally reversed these age-related increases. Aging increased the amount of SIRT1, and MetR decreased SIRT1 and TFAM and increased complex IV. No changes were observed in the protein amounts of PGC1, Nrf2, MnSOD, AIF, complexes I, II and III, and in the extent of genomic DNA methylation. In conclusion, treating old rats with isocaloric short-term MetR lowers mitROS production and free radical leak and oxidative damage to mtDNA, and reverses aging-related increases in protein modification. Aged rats maintain the capacity to lower mitochondrial ROS generation and oxidative stress in response to a short-term exposure to restriction of a single dietary substance: methionine.