A STR mutation in a heteropaternal twin case

A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.

Combined mutation differential evolution to solve systems of nonlinear equations

This paper presents a differential evolution heuristic to compute a solution of a system of nonlinear equations through the global optimization of an appropriate merit function. Three different mutation strategies are combined to generate mutant points. Preliminary numerical results show the effectiveness of the presented heuristic.

Utilização de origens de água com qualidade inferior a A3 na produção de água para consumo humano

A eutrofização das massas hídricas superficiais constitui um dos mais significativos problemas, a nível de planeamento e gestão dos recursos hídricos. Uma das consequências consiste no bloom de organismos fitoplanctónicos, como as cianobactérias, produzindo um risco para a saúde, quando presentes na água para consumo humano. Muitas das tecnologias convencionais utilizadas no tratamento de água têm-se demonstrado pouco eficazes na sua remoção. Estações de tratamento de águas (ETAs) que utilizam águas com presença destes organismos tóxicos, têm que possuir barreiras eficientes para a sua remoção. Assim, torna-se necessário munir as ETAs de tecnologia apropriada à sua eliminação. Deste modo, este trabalho consiste, com base em investigação bibliográfica, avaliar a capacidade de remoção de cianobactérias, e das toxinas a elas associadas, ao longo de um sistema de tratamento e prever a possibilidade de introdução de um processo de separação por membranas, como uma barreira segura e eficaz no tratamento de águas com este tipo de problemas. Para tal, utilizou-se como caso de estudo a ETA de S. Domingos (sistema de tratamento convencional), responsável por parte do abastecimento de água da cidade de Peniche, cuja origem de água, albufeira de S. Domingos, se encontra eutrofizada e, a presença destes organismos é praticamente constante ao longo de todo o ano. Prevê-se que a ETA de S. Domingos consiga alguma remoção de cianobactérias e cianotoxinas intracelulares, mas a principal incerteza prende-se com o desempenho da ETA na remoção de toxinas extra celulares. Concluiu-se então, que desde que o sistema de tratamento já existente seja totalmente optimizado, e por introdução de um sistema de filtração por membranas (Nanofiltração) é possível reter com sucesso estas toxinas.

HLA and malaria in four colombian ethnic groups

HLA antigens and their relationship with malaria infection were studied in four different ethnic groups in Colombia (South America): two groups of indians (Kunas and Katios), one of negroes and a group of mixed ancestry. A total of 965 persons were studied, 415 with malaria and 550 as controls. HLA-A,B, and C antigen frequencies in the four groups are reported. The association of each HLA antigen with malaria infection due to P. vivax and to P. falciparum was evaluated. Negroes, Kunas and Katios indians variously lack from 6 to 9 of the HLA antigens found in the mixed group. In the designated ethnic groups, antigens B5, B13, B15, Cw2 and Cw4 showed borderline association with malaria infection. However, in the mixed ethnic group, statistically significant associations were found with malaria infection and the presence of A9, Aw19, B17, B35, and Z98 (a B21-B45: crossreacting determinant) with few differences when P. vivax infection and P. falciparum infection were considered individually. This finding may represent a lack of general resistance to malaria in the group that harbors antigens of Caucasian origin. These individuals have been in direct and permanent contact with malaria only in the past 65 years. In contrast, indians, both Kunas and Katios, and Negroes have lived for centuries in malaria endemic areas, and it is possible that a natural selection system has developed through which only those individuals able to initiate an acute immune response to malaria have survived.

Análise do polimorfismo HLA/DR-DQ em Angolanos portadores de Diabetes Mellitus tipo I

A diabetes mellitus do tipo 1 (DM1) é o distúrbio endócrino-metabólico mais frequente em crianças. É uma doença autoimune resultado da destruição selectiva das células beta pancreáticas. A velocidade de destruição das células beta pode ser rápida em algumas pessoas e lenta em outras; esta última é típica de adulto é conhecido como diabetes autoimune latente em adulto (LADA). A sua etiologia envolve factores ambientais e genéticos, dos genes envolvidos a maior contribuição vem da região do genoma onde estão localizados os genes do Complexo Principal de Histocompatibilidade (MHC). A combinação de diferentes alelos do sistema de antigénio leucocitário humano tipo II (HLAII) esta associada a susceptibilidade da doença e as principais molécula envolvidas são a DQ e DR. O estágio pré-clínico da doença se caracteriza pela presença de auto-anticorpos, sendo o anti-GAD o mais sensível nesta patologia. OBJECTIVO: Analizar a frequência dos polimorfismos HLA-DR/DQ em angolanos portadores de diabetes tipo 1, residentes em Luanda. O tipo de estudo adotado foi casocontrolo num universo de voluntários provenientes da consulta externa de hospitais e clínicas locais no período de outubro de 2012 a outubro 2013. A amostra biológica utilizada foi sangue total, tendo sido processada no laboratório LABGENE, da Faculdade de Medicina (FM) da Universidade Agostinho Neto (UNA). Os auto-anticorpos, anti-GAD, foram doseados pelo método de ELISA. O ADN genómico foi extraído à partir de sangue total periférico e tipagem genética foi realizada mediante PCR-SSP.O alelo DQB1*02 (DQ2/DQ2) (p=0,033, OR= 4, IC (1,2-13,3) foi associado a susceptibilidade da DM1; os alelos DQB1*06 (DQ6/DQ6) (p=0,000, OR=0,30, IC (0,17-0,54); *11 (p=0,011, OR=0,14, IC=0,032-0,62); *13 (p=0,006, OR=0,13, IC=0,049-0,588) e *15 (p=0,001, OR=0,044, IC=0,005-0,39) foram associados a proteção. Foi encontrado 29,2% de positividade para o anti-GAD, não houve associação significativa (p=0,69) entre a resposta positiva do anti-GAD e a idade. Não foi encontrada associação significativa (p=0,39) entre o tempo de diagnóstico e resposta humoral. Observou-se associação significativa entre os alelos de risco DQB1*02 (p=0.000) e resposta positiva para anti-GAD; da mesma maneira houve uma associação significativa para os alelos DQB1*06 (p=0,002), DRB1*11 (p=0,048); *13 (p=0,004) e *15 (p=0,021) e a resposta negativa do anti-GAD.Os dados demostram o forte envolvimentos do gene HLA-II (DQ) com a suceptibilidade a DM1 e sugere que a autoimunidade se desenvolve na presença de susceptibilidade genética, quer dizer, em associação com alelos HLA-II específicos.

Specificity and Sensitivity of Screening for Anti-HLA Antibodies in Kidney Allograft Dysfunction

BACKGROUND: Prospective testing for posttransplant circulating anti-HLA antibodies seems to be a critical noninvasive tool, but confirmatory data are lacking. MATERIALS AND METHODS: Over the last 3 years, peritubular capillary (PTC) C4d deposition was prospectively sought by an immunofluorescence technique applied to frozen tissue in biopsies obtained for allograft dysfunction. Screening for circulating anti-HLA class I/II alloantibodies (AlloAb) by the flow cytometric test was performed simultaneously. RESULTS: We evaluated 132 sets of biopsies and simultaneous serum samples. PTC C4d deposition was demonstrated in 15.9% (21/132) of biopsies. Circulating anti-HLA I/II AlloAb were detected in 25% (33/132) of serum samples. Employing receiver-operator characteristic (ROC) curves for all C4d-positive biopsies, screening for AlloAb showed a global specificity of 82% and sensitivity of 61.9%. When this analysis was restricted to biopsies obtained in the first month posttransplantation, the sensitivity increased to 81.8%, but the specificity decreased to 76.9%. After the first month posttransplantation, we observed sensitivity of 40.0% and a specificity of 86.4%. In the first month posttransplantation, all patients with a diagnosis of acute antibody-mediated rejection displayed circulating anti-HLA class I/II, but not always at the same time as the C4d-positive biopsy. CONCLUSIONS: In the first month posttransplantation, prospective monitoring of anti-HLA antibodies may be useful. The high sensitivity allows the identification of patients at risk, affording an earlier diagnosis of antibody-mediated rejection. After the first month, the test can be used to evaluate allograft dysfunction episodes, since positivity is highly suggestive of an antibody-mediated process.

Peritubular Capillaries C4d Deposits in Renal Allograft Biopsies and Anti HLA I/II Alloantibodies Screening. Incidence and Clinical Importance

Aim: To characterise clinically the patients with C4d in peritubular capillaries deposits (C4dPTCD) and/or circulating anti-HLA class I/II alloantibodies. To determine the correlation between positive C4dPTCD and circulating anti-HLA class I/II alloantibodies during episodes of graft dysfunction. Subjects and Methods: C4d staining was performed in biopsies with available frozen tissue obtained between January 2004 and December 2006. The study was prospective from March 2005, when a serum sample was obtained at the time of biopsy to detect circulating anti-HLA class I/II alloantibodies. Results: We studied 109 biopsies in 86 cadaver renal transplant patients. Sixteen of these (14.7%) presented diffuse positive C4dPTCD. There was a 13.5% rate of +C4dPTCD incidence within the first six months of transplantation and 16% after six months (p>0.05). Half of the +C4dPTCD in the first six months was associated with acute humoral rejection. After six months, the majority of +C4dPTCD (n=7/8) was present in biopsies with evidence of interstitial fibrosis/tubular atrophy and/or transplant glomerulopathy. The C4dPTCD was more frequent in patients with positive anti-HCV antibodies(p<0.0001), a previous renal transplant (p=0.007), and with a panel reactivity antibody (PRA) ≥ 50%(p=0.0098). The anti-HCV+ patients had longer time on dialysis (p=0.0019) and higher PRA(p=0.005). Circulating anti-HLA I/II alloantibodies were screened in 46 serum samples. They were positive in 10.9% of samples, all obtained after six months post transplant. Circulating alloantibodies were absent in 92.5% of the C4d negative biopsies. Conclusion: We found an association between the presence of C4dPTCD and 2nd transplant recipients,higher PRA and the presence of anti-HCV antibodies. The presence of HCV antibodies is not a risk factor for C4dPTCD per se, but appears to reflect longer time on dialysis and presensitisation. In renal dysfunction a negative alloantibody screening is associated with a reduced risk of C4dPTCD (<10%).

SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring.

Caucasian Familial Moyamoya Syndrome With Rare Multisystemic Malformations

Moyamoya disease is an idiopathic progressive steno-occlusive disorder of the intracranial arteries located at the base of the brain. It is associated with the development of compensatory extensive network of fine collaterals. Moyamoya disease is considered syndromic when certain genetic or acquired disorders such as polycystic kidney disease, neurofibromatosis, or meningitis are also present. Although the genetic contribution in moyamoya is indisputable, its cause and pathogenesis remain under discussion. Herein, we report a rare occurrence of moyamoya syndrome in two European Caucasian siblings in association with unusual multisystemic malformations (polycystic kidney disease in one, and intestinal duplication cyst in the other). The karyotype was normal. No mutation in the RFN213 gene was found, and none of the HLA types linked to moyamoya disease or described in similar familial cases were identified. By describing these multisystemic associations, polycystic kidney disease for the second time, and intestinal malformation for the first time in the literature, our report expands the phenotypic variability of moyamoya syndrome. The coexistence of disparate malformations among close relatives suggests an underlying common genetic background predisposing to structural or physiological abnormalities in different tissues and organs.

Adaptation from standing genetic variation and from mutation

Dissertation presented to obtain the Ph.D degree in Evolutionary Biology

A Novel Autosomal Recessive GJA1 Missense Mutation Linked to Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a rare sclerosing skeletal disorder with progressive hyperostosis of craniofacial bones. CMD can be inherited in an autosomal dominant (AD) trait or occur after de novo mutations in the pyrophosphate transporter ANKH. Although the autosomal recessive (AR)form of CMD had been mapped to 6q21-22 the mutation has been elusive. In this study, we performed whole-exome sequencing for one subject with AR CMD and identified a novel missense mutation (c.716G>A, p.Arg239Gln) in the C-terminus of the gap junction protein alpha-1 (GJA1) coding for connexin 43 (Cx43). We confirmed this mutation in 6 individuals from 3 additional families. The homozygous mutation cosegregated only with affected family members. Connexin 43 is a major component of gap junctions in osteoblasts, osteocytes, osteoclasts and chondrocytes. Gap junctions are responsible for the diffusion of low molecular weight molecules between cells. Mutations in Cx43 cause several dominant and recessive disorders involving developmental abnormalities of bone such as dominant and recessive oculodentodigital dysplasia (ODDD; MIM #164200, 257850) and isolated syndactyly type III (MIM #186100), the characteristic digital anomaly in ODDD. However, characteristic ocular and dental features of ODDD as well as syndactyly are absent in patients with the recessive Arg239Gln Cx43 mutation. Bone remodeling mechanisms disrupted by this novel Cx43 mutation remain to be elucidated.

Hyper-IgD and Periodic Fever Syndrome: a New MVK Mutation (p.R277G) Associated with a Severe Phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM#260920) is a rare recessively-inherited autoinflammatory condition caused bymutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Herewe report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, where as anakinra showed positive responses only at high doses. The p.R277Gmutation here described is a novel missense MVK mutation, and it has been detected in this casewith a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.

Mitchell-Riley Syndrome: A Novel Mutation in RFX6 Gene

A novel RFX6 homozygous missense mutation was identified in an infant with Mitchell-Riley syndrome. The most common features of Mitchell-Riley syndrome were present, including severe neonatal diabetes associated with annular pancreas, intestinal malrotation, gallbladder agenesis, cholestatic disease, chronic diarrhea, and severe intrauterine growth restriction. Perijejunal tissue similar to pancreatic tissue was found in the submucosa, a finding that has not been previously reported in this syndrome. This case associating RFX6 mutation with structural and functional pancreatic abnormalities reinforces the RFX6 gene role in pancreas development and β-cell function, adding information to the existent mutation databases.

Frequency of human leukocyte antigen (hla) in patients with malaria and in the general population of Humaitá county, Amazonas state, Brazil

In August 1983,85 inhabitants of the municipality of Humaitá, Amazonas State, Brazil were studied to determine the prevalence of antigens to HLA-A, -B, -C and DR. Thirty-eight were sick with malaria due to Plasmodium falciparum. All subjects were examined for splenomegaly, blood parasitaemia and antibodies to malaria. They constituted three groups: 1) 25 subjects native to the Amazon region who had never had malaria; 2) 38 Amazonian subjects who had malaria in the past or currently had an infection; 3) 22 patients with malaria who had acquired the infection in the Amazon Region but came from other regions of Brazil. Blood was taken from each person, the lymphocytes were separated and typed by the test of microlymphocytotoxicity. There was a high frequency of antigens that could not be identified in the groups studied which suggests the existence of a homozygote or phenotype not identified in the population. There was a high frequency of the phenotype Ag(W24) (44.7%) in group 2 when compared with group 1 (32%) or group 3 (9%). Also the individuals in group 2 showed an elevated frequency of antigen DR(4)80%) when compared with group 1 (36.6%) or group 3 (16.6%). These observations suggest the possibility of a genetic susceptibility to malaria among Amazonian residents and indicate a necessity for more extensive studies of the frequency of HLA antigens among inhabitants of this endemic malarial zone.