There are seven confirmed hepatitis C virus (HCV) genotypes, with whole-genome nucleotide sequences differing by Ͼ30%, and each can be further subdivided into related subtypes (67 confirmed), with nucleotide sequence divergence of between 15% and 30% (1).Genotype identification has long been used in clinical practice, because major genotypes have different response rates and require different doses and durations of pegylated interferon and ribavirin (PR) treatment. In contrast, until recently, subtype identification was mainly used in epidemiological studies. However, both in vitro studies and clinical trials with different classes of direct-acting antiviral (DAA) agents (NS3 protease, NS5A-, and nucleos[t]ide and nonnucleos[t]ide NS5B-polymerase inhibitors), given with PR or in interferon-free combinations, have shown lower response rates for HCV genotype 1a than for HCV genotype 1b (2-8). Moreover, at least for HCV genotype 1, both the frequency and the pattern of resistance to different DAA classes are subtype specific (9). A striking example is the NS3-Q80K polymorphism, naturally found in Ͼ30% of naive subtype 1a patients but in Ͻ1% of subtype 1b patients (10), which conveys 30%-to-40%-lower sustained-virologic-response (SVR) rates to the macrocyclic protease inhibitor simeprevir (2). Similarly, all subtype 1g sequences identified naturally carry a mutation conferring resistance to linear NS3 protease inhibitors (11).Subtype-specific differences in the genetic barrier to resistance appear to correlate to the RNA-dependent RNA polymerase mu-

IntroductionCOPD has complex etiologies involving both genetic and environmental determinants. Among genetic determinants, the most recognized is a severe PiZZ (Glu342Lys) inherited alpha1-antitrypsin deficiency (AATD). Nonetheless, AATD patients present a heterogeneous clinical evolution, which has not been completely explained by sociodemographic or clinical factors. Here we performed the gene expression profiling of blood cells collected from mild and severe COPD patients with PiZZ AATD. Our aim was to identify differences in messenger RNA (mRNA) and microRNA (miRNA) expressions that may be associated with disease severity.Materials and methodsPeripheral blood mononuclear cells from 12 COPD patients with PiZZ AATD (6 with severe disease and 6 with mild disease) were used in this pilot, high-throughput microarray study. We compared the cellular expression levels of RNA and miRNA of the 2 groups, and performed functional and enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene-ontology (GO) terms. We also integrated the miRNA and the differentially expressed putative target mRNA. For data analyses, we used the R statistical language R Studio (version 3.2.5).ResultsThe severe and mild COPD–AATD groups were similar in terms of age, gender, exacerbations, comorbidities, and use of augmentation therapy. In severe COPD–AATD patients, we found 205 differentially expressed genes (DEGs) (114 upregulated and 91 downregulated) and 28 miRNA (20 upregulated and 8 downregulated) compared to patients with mild COPD–AATD disease. Of these, hsa-miR-335-5p was downregulated and 12 target genes were involved in cytokine signaling, MAPK/mk2, JNK signaling cascades, and angiogenesis were much more highly expressed in severe compared with mild patients.ConclusionsDespite the small sample size, we identified downregulated miRNA (hsa-miR-335) and the activation of pathways related to inflammation and angiogenesis on comparing patients with severe vs mild COPD–AATD. Nonetheless, our findings warrant further validation in large studies.

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The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

The objective of this clinical study was to determine the real frequency and clinical importance of partial obstruction of the seminal path in patients with oligozoospermia. We have designed a prospective clinical study including men with oligozoospermia seen at an andrological consultation in both private and institutional hospitals. A testicular biopsy was done on all patients under local anaesthesia. A complete study for sterility was also done [hormonal determinations: follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, testicular ultrasound, semen analysis, testicular Doppler ultrasound, etc.]. We have made a quantitative and qualitative evaluation of testicular biopsy (percentage of tubules with Sertoli cell only or with hyalinization; mean tubular diameter; number of spermatogonia, primary spermatocytes, young spermatids, mature spermatids and Sertoli cells; and evaluation of testicular interstitium: number of Leydig cell clusters, presence of angiectasis, presence of perivascular inflammation). Sixty one per cent of all oligozoospermia cases were obstructive. The principal cause of obstructive oligozoospermia was the presence of testicular varicocele. In obstructive oligozoospermia, the tubular diameter and number of mature spermatids are statistically significantly higher than in non-obstructive oligozoospermia. Obstructive oligozoospermia is a frequent condition caused by partial obstruction of seminal path. A quantitative analysis of the testicular biopsy is the only method of diagnosis.

Screening of liver disease in alpha-1 antitrypsin deficiency (AATD) is usually carried out with liver enzymes, with low sensitivity. We conducted a multicenter cross-sectional study aiming to describe the utility of transient elastography for the identification of liver disease in patients with AATD. A total of 148 AATD patients were included. Among these, 54.7% were Pi*ZZ and 45.3% were heterozygous for the Z allele. Between 4.9% and 16.5% of patients had abnormal liver enzymes, without differences among genotypes. Liver stiffness measurement (LSM) was significantly higher in Pi*ZZ individuals than in heterozygous Z (5.6 vs. 4.6 kPa; p = 0.001). In total, in 8 (5%) individuals LSM was >7.5 kPa, considered significant liver fibrosis, and ≥10 kPa in 3 (1.9%) all being Pi*ZZ. Elevated liver enzymes were more frequently observed in patients with LSM > 7.5 kPa, but in 5 out of 8 of these patients all liver enzymes were within normal range. In patients with AATD, the presence of abnormal liver enzymes is frequent; however, most of these patients do not present significant liver fibrosis. Transient elastography can help to identify patients with liver fibrosis even with normal liver enzymes and should be performed in all Z-allele carriers to screen for liver disease.

AIMTo determine the variability/conservation of the domain of hepatitis B virus (HBV) preS1 region that interacts with sodium-taurocholate cotransporting polypeptide (hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism (S267F, NTCP variant) in a Spanish population.METHODSSerum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection (CHB) (n = 41, 73% Caucasians), patients with resolved HBV infection (n = 100, 100% Caucasians) and an HBV-uninfected control group (n = 105, 100% Caucasians). Variability/conservation of the amino acid (aa) sequences of the NTCP-interacting domain, (aa 2-48 in viral genotype D) and a highly conserved preS1 domain associated with virion morphogenesis (aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTSThe HBV preS1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21 (in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCP-interacting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies (25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms (34% vs 27.3%), according to consensus sequences from GenBank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable (limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSIONIn our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.

AbstractαWe tested the applicability of this new technique for the identification of the Mmalton allele in AATD screening using whole blood, dried blood spot (DBS) and serum samples. Mmalton heterozygote and homozygote samples and samples without this allele were included in the study.This new assay is able to detect homozygous and heterozygous genotypes in the same reaction and in a single step, giving matching results with those obtained byThis technology is optimal for working with small amounts of DNA, such as in DBS and even with residual DNA present in serum samples, allowing improvement in routine algorithms of AATD diagnosis or large-scale screening. This method will be useful for obtaining more in depth knowledge of the real incidence of the Mmalton variant.