The HIF1A Functional Genetic Polymorphism at Locus +1772 Associates with Progression to Metastatic Prostate Cancer and Refractoriness to Hormonal Castration

The hypoxia inducible factor 1 alpha (HIF1a) is a key regulator of tumour cell response to hypoxia, orchestrating mechanisms known to be involved in cancer aggressiveness and metastatic behaviour. In this study we sought to evaluate the association of a functional genetic polymorphism in HIF1A with overall and metastatic prostate cancer (PCa) risk and with response to androgen deprivation therapy (ADT). The HIF1A +1772 C>T (rs11549465) polymorphism was genotyped, using DNA isolated from peripheral blood, in 1490 male subjects (754 with prostate cancer and 736 controls cancer-free) through Real-Time PCR. A nested group of cancer patients who were eligible for androgen deprivation therapy was followed up. Univariate and multivariate models were used to analyse the response to hormonal treatment and the risk for developing distant metastasis. Age-adjusted odds ratios were calculated to evaluate prostate cancer risk. Our results showed that patients under ADT carrying the HIF1A +1772 T-allele have increased risk for developing distant metastasis (OR, 2.0; 95%CI, 1.1-3.9) and an independent 6-fold increased risk for resistance to ADT after multivariate analysis (OR, 6.0; 95%CI, 2.2-16.8). This polymorphism was not associated with increased risk for being diagnosed with prostate cancer (OR, 0.9; 95%CI, 0.7-1.2). The HIF1A +1772 genetic polymorphism predicts a more aggressive prostate cancer behaviour, supporting the involvement of HIF1a in prostate cancer biological progression and ADT resistance. Molecular profiles using hypoxia markers may help predict clinically relevant prostate cancer and response to ADT.

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

The HIF1A Functional Genetic Polymorphism at Locus +1772 Associates with Progression to Metastatic Prostate Cancer and Refractoriness to Hormonal Castration

The hypoxia inducible factor 1 alpha (HIF1a) is a key regulator of tumour cell response to hypoxia, orchestrating mechanisms known to be involved in cancer aggressiveness and metastatic behaviour. In this study we sought to evaluate the association of a functional genetic polymorphism in HIF1A with overall and metastatic prostate cancer (PCa) risk and with response to androgen deprivation therapy (ADT). The HIF1A +1772 C>T (rs11549465) polymorphism was genotyped, using DNA isolated from peripheral blood, in 1490 male subjects (754 with prostate cancer and 736 controls cancer-free) through Real-Time PCR. A nested group of cancer patients who were eligible for androgen deprivation therapy was followed up. Univariate and multivariate models were used to analyse the response to hormonal treatment and the risk for developing distant metastasis. Age-adjusted odds ratios were calculated to evaluate prostate cancer risk. Our results showed that patients under ADT carrying the HIF1A +1772 T-allele have increased risk for developing distant metastasis (OR, 2.0; 95%CI, 1.1-3.9) and an independent 6-fold increased risk for resistance to ADT after multivariate analysis (OR, 6.0; 95%CI, 2.2-16.8). This polymorphism was not associated with increased risk for being diagnosed with prostate cancer (OR, 0.9; 95%CI, 0.7-1.2). The HIF1A +1772 genetic polymorphism predicts a more aggressive prostate cancer behaviour, supporting the involvement of HIF1a in prostate cancer biological progression and ADT resistance. Molecular profiles using hypoxia markers may help predict clinically relevant prostate cancer and response to ADT.

Clinical Outcomes and Genetic Expression Profile in Human Liver Graft Dysfunction During Ischemia/Reperfusion Injury

Introduction. This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). Methods. We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-a, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. Results. Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P ¼ .013) and IL-1b at T0 (P ¼ .028) and early graft dysfunction. Conclusions. We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Analysis of Highly Conserved Regions of the 3'UTR of MECP2 Gene in Patients with Clinical Diagnosis of Rett Syndrome and Other Disorders Associated with Mental Retardation

In this work we explored the role of the 3'UTR of the MECP2 gene in patients with clinical diagnosis of RTT and mental retardation; focusing on regions of the 3'UTR with almost 100% conservation at the nucleotide level among mouse and human. By mutation scanning (DOVAM-S technique) the MECP2 3'UTR of a total of 66 affected females were studied. Five3'UTR variants in the MECP2 were found (c.1461+9G>A, c.1461+98insA, c.2595G>A, c.9961C>G and c.9964delC) in our group of patients. None of the variants found is located in putative protein-binding sites nor predicted to have a pathogenic role. Our data suggest that mutations in this region do not account for a large proportion of the RTT cases without a genetic explanation.

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

PROP1 Gene Analysis in Portuguese Patients with Combined Pituitary Hormone Deficiency

OBJECTIVE: Mutations of the PROP1 gene lead to combined pituitary hormone deficiency (CPHD), which is characterized by a deficiency of GH, TSH, LH/FSH, PRL and, less frequently, ACTH. This study was undertaken to investigate the molecular defect in a cohort of patients with CPHD. DESIGN, PATIENTS AND MEASUREMENTS: A multicentric study involving 46 cases of CPHD (17 familial cases belonging to seven kindreds and 29 sporadic cases) selected on the basis of clinical and hormonal evidence of GH deficiency, central hypothyroidism and hypogonadotrophic hypogonadism, in the absence of an identified cause of hypopituitarism. Mutations of PROP1 were investigated by DNA sequencing. Clinical, hormonal and neuroradiological data were collected at each centre. RESULTS: PROP1 mutations were identified in all familial cases: five kindreds presented a c. 301-302delAG mutation, one kindred presented a c. 358C --> T (R120C) mutation and one presented a previously unreported initiation codon mutation, c. 2T --> C. Of the 29 sporadic cases, only two (6.9%) presented PROP1 germline mutations (c. 301-302delAG, in both). Phenotypic variability was observed among patients with the same mutations, particularly the presence and age of onset of hypocortisolism, the levels of PRL and the results of pituitary imaging. One patient presented a sellar mass that persisted into adulthood. CONCLUSIONS: This is the first report of a mutation in the initiation codon of the PROP1 gene and this further expands the spectrum of known mutations responsible for CPHD. The low mutation frequency observed in sporadic cases may be due to the involvement of other unidentified acquired or genetic causes.

Mutational Analysis of Portuguese Families with Multiple Endocrine Neoplasia Type 1 Reveals Large Germline Deletions

OBJECTIVE: To determine the spectrum of MEN1 mutations in Portuguese kindreds, and identify mutation-carriers. PATIENTS, DESIGN AND RESULTS: Six unrelated MEN1 families were studied for MEN1 gene mutations by single-strand conformational polymorphism (SSCP) and DNA sequence analysis of the coding region and exon-intron boundaries of the MEN1 gene. These methods identified 4 different heterozygous mutations in four families: two mutations are novel (mt 1539 delG and mt 655 ims 11 bp) and two have been previously observed (mt 735 del 46p and mt 1656 del C) all resulting in a premature stop codon. In the remaining two families, in whom no mutations or abnormal MEN1 transcripts were detected, segregation studies of the 5' intragenic marker D11S4946 and codon 418 polymorphism in exon 9 revealed two large germline deletions of the MEN1 gene. Southern blot and tumour loss of heterozygosity analysis confirmed and refined the limits of these deletions, which spanned the MEN1 gene at least from: exon 7 to the 3' untranslated region, in one family, and the 5' polymorphic site D11S4946 to exon 9 (obliterating the initiation codon), in the other family. Twenty-six mutant-gene carriers were identified, 6 of which were asymptomatic. CONCLUSIONS: These results emphasize the importance of the detection of MEN1 germline deletions in patients who do not have mutations of the coding region. Important clues indicating the presence of such deletions may be obtained by segregation studies using the intragenic polymorphisms D11S4946 and at codon 418. The detection of these mutations will help in the genetic counselling of clinical management of the MEN1 families in Portugal.

LAMA2 Gene Analysis in a Cohort of 26 Congenital Muscular Dystrophy Patients

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-alpha2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin-alpha2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750-1713_7899-2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin-alpha2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.

Genetic Screening of LCA in Belgium: Predominance of CEP290 and Identification of Potential Modifier Alleles in AHI1 of CEP290-Related Phenotypes

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease.