Monogenic Human Skin Disorders

Human genodermatoses represent a broad and partly confusing spectrum of countless rare diseases with confluent and overlapping phenotypes often impeding a precise diagnosis in an affected individual. High-throughput sequencing techniques have expedited the identification of novel genes and have dramatically simplified the establishment of genetic diagnoses in such heterogeneous disorders. The precise genetic diagnosis of a skin disorder is crucial for the appropriate counselling of patients and their relatives regarding the course of the disease, prognosis and recurrence risks. Understanding the underlying pathophysiology is a prerequisite to understanding the disease and developing specific, targeted or individualized therapeutic approaches. We aimed to create a comprehensive overview of human genodermatoses and their respective genetic aetiology known to date. We hope this may represent a useful tool in guiding dermatologists towards genetic diagnoses, providing patients with individual knowledge on the respective disorder and applying novel research findings to clinical practice.

Probabilistic reasoning with a bayesian DNA device based on strand displacement

We present a computing model based on the DNA strand displacement technique which performs Bayesian inference. The model will take single stranded DNA as input data, representing the presence or absence of a specific molecular signal (evidence). The program logic encodes the prior probability of a disease and the conditional probability of a signal given the disease playing with a set of different DNA complexes and their ratios. When the input and program molecules interact, they release a different pair of single stranded DNA species whose relative proportion represents the application of Bayes? Law: the conditional probability of the disease given the signal. The models presented in this paper can empower the application of probabilistic reasoning in genetic diagnosis in vitro.

Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.

Análise exômica em pacientes portadores de cardiomiopatia hipertrófica

A cardiomiopatia hipertrófica (CMH) é uma doença geneticamente determinada, caracterizada por hipertrofia ventricular primária, com prevalência estimada de 0.2% na população geral. Qualquer portador tem 50% de chance de transmitir esta doença para seus filhos, o que torna cada vez mais relevante a importância do estudo genético dos indivíduos acometidos e de seus familiares. Já foram descritas diversas mutações genéticas causadoras de CMH, a maioria em genes que codificam proteínas do sarcômero, e algumas mutações mais raras em genes não sarcoméricos. O objetivo desse estudo é sequenciar as regiões exônicas de genes candidatos, incluindo os principais envolvidos na hipertrofia miocárdica, utilizando o sequenciamento de nova geração (Generation Sequencing); testar a aplicabilidade e viabilidade deste sistema para identificar mutações já confirmadas e propor as prováveis novas mutações causadoras de CMH. Métodos e resultados: 66 pacientes não aparentados portadores de CMH foram estudados e submetidos à coleta de sangue para obtenção do DNA para analisar as regiões exômicas de 82 genes candidatos, utilizando a plataforma MiSeq (Illumina). Identificou-se 99 mutações provavelmente patogênicas em 54 pacientes incluídos no estudo (81,8%) relacionadas ou não a CMH, e distribuídas em 42 genes diferentes. Destas mutações 27 já haviam sido publicadas, sendo que 17 delas descritas como causadoras de CMH. Em 28 pacientes (42,4%) identificou-se mutação nos três principais genes sarcoméricos relacionados à CMH (MYH7, MYBPC3, TNNT2). Encontrou-se também um grande número de variantes não sonôminas de efeito clínico incerto e algumas mutações relacionadas a outras enfermidades. Conclusão: a análise da sequencia dos exônos de genes candidatos, demonstrou ser uma técnica promissora para o diagnóstico genético de CMH de forma mais rápida e sensível. A quantidade de dados gerados é o um fator limitante até o momento, principalmente em doenças geneticamente complexas com envolvimento de diversos genes e com sistema de bioinformática limitado.

Construction of a New Familial Hypercholesterolemia Variant Data Base. A Systematic Review for a 2015 Update

Aims: Familial hypercholesterolemia (FH) is an autosomal dominant disorder with increased cardiovascular risk, caused by mutations in LDLR, APOB and PCSK 9 genes. Although it is described that over 1700 variants have been found, none of the existing databases are completely updated. The aim of this work is to construct a FH database in order to provide a unique source of verified information about variants associated with FH for a more accurate genetic diagnosis.

Análise das mutações C288Y, S65C e H63D e frequência alélica do gene HFE em pacientes com hiperferritinemia, em uma cidade do Nordeste, Brasil

Background & Aims: HFE-associated Hereditary Hemochromatosis (HH) is one of the most frequent autosomal recessive disease in the caucasian population, caused by the high absorption and deposition of iron in several organs. This accumulation results in several clinical complications such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. Although most of the cases are homozygous individuals for the C282Y mutation, another two mutations, H63D and S65C, have been reported to be associated with milder forms of the disease. The objective is to avaluate the distribution of C282Y, H63D and S65C mutations in the HFE gene in patients with suspected HH in the state of Rio Grande do Norte, Brazil. Methods: Samples of peripheral blood were taken from 335 patients originating from Natal-RN, a city in northeastern Brazil with suspected of HH and which were screened for the HFE gene C282Y, H63D and S65C mutations, using molecular genetics assays (Polymerase Chain Reaction- Restriction Fragments Length Polymorphism). The main criterion for including such patients in the study was the increasing of persistent serum ferritin in individuals aged between 18 and 70 or older, both males and females. As to the exclusion criteria, individuals holding hemolytical anemia, talassemy and previously report of blood transfusion did not take part of the study. Results: Out of the 335 patients studied, 143 patients showed absence of mutation and 195 showed some kind of mutation in the HFE gene: 07/335 (2,08%) were homozigous C282Y, 25/335 heterozygous C282Y, 25/335 (7,46%) were homozigous H63D, 115/335 (34,32%) heterozygous H63D, 5/335 (1,48%) heterozygous S65D, 11/ 335 (3,28%) and were double heterozygous (H63D/C282Y). None patients were Homozygous S65D and S65D heterozygous (S65D/H63D and S65D/C282Y). Conclusions. The distribution of the HFE gene C282Y, H63D and S65C mutations found in our group matches the tendencies observed in other European countries. Due to the high prevalence of hemochromatosis, its seriousness and easy treatment, the genetic diagnosis of HH has become a dream, especially in the high risk group.

A Rare Cause of Myoclonus: A Cupric Conundrum

A woman aged 22 years presented with a 3-year history of jerks when brushing her teeth and a tremor when carrying drinks. Examination revealed a bilateral jerky tremor, stimulus-sensitive myoclonus, and difficulty with tandem gait. Thyroid and liver function test results were normal, but she had rapidly progressive renal failure. Serum copper, ceruloplasmin, and manganese levels were normal, but her urinary copper level was elevated on 2 occasions. Pathological findings on organ biopsy prompted genetic testing to confirm the diagnosis. The differential diagnosis, tissue biopsy findings, and final genetic diagnosis are discussed.

Network analysis approach to find new candidate genes and pathways involved in the pathophysiology of familial hypercholesterolemia

Still a big gap exists between clinical and genetic diagnosis of dyslipidemic disorders. Almost the 60% of the patients with a clinical diagnosis of Familial hypercholesterolemia (FH) still lack of a genetic diagnosis. Here we present the preliminary results of an integrative approach intended to identify new candidate genes and to dissect pathways that can be dysregulated in the disease. Interesting hits will be subsequently knocked down in vitro in order to evaluate their functional role in the uptake of fluorescently-labeled LDL and free cell cholesterol using automated microscopy.

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.

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Prevalence of GJB2 (Connexin-26) and GJB6 (Connexin-30) Mutations in a Cohort of 300 Brazilian Hearing-Impaired Individuals: Implications for Diagnosis and Genetic Counseling

Objective: Hereditary nonsyndromic deafness is an autosomal recessive condition in about 80% of cases, and point mutations in the GJB2 gene (connexin 26) and two deletions in the GJB6 gene (connexin 30), del(GJB6-D13S1830) and del(GJB6-D13S1854), are reported to account for 50% of recessive deafness, Aiming at establishing the frequencies of GJB2 mutations and GJB6 deletions in the Brazilian population, we screened 300 unrelated individuals with hearing impairment, who were not affected by known deafness related syndromes. Methods: We firstly screened the most frequently reported mutations, c.35delG and c.167delT in the GJB2 gene, and del(GJB6-D13S1830) and del(GJB6-D13S1854) in the GJB6 gene, through specific techniques. The detected c.35delG and c.167delT mutations were validated by sequencing. Other mutations in the GJB2 gene were screened by single-strand conformation polymorphism and the coding region was sequenced when abnormal patterns were found. Results: Pathogenic mutations in GJB2 and GJB6 genes were detected in 41 individuals (13.7%), and 80.5% (33/41) presented these mutations in homozygosis or compound heterozygosis, thus explaining their hearing defect. The c.35delG in the GJB2 gene was the most frequent mutation (37/300; 12.4%), detected in 23% familial and 6.2% the sporadic cases. The second most frequent mutation (1%; 3/300) was the del(GJB6- D13S1830), always found associated with the c.35delG mutation. Nineteen different sequence variations were found in the GJB2 gene. In addition to the c.35delG mutation, nine known pathogenic alterations were detected 0 67delT, p.Trp24X, p.Val37lle, c.176_191del16, c.235delC, p.Leu90Pro, p.Arg127His, c.509insA, and p.Arg184Pro, Five substitutions had been previously considered benign polymorphisms: c.-15C>T, p.Val27lle, p.Met34hr, p.Ala40Ala, and p.Gly160Ser. Two previously reported Mutations of unknown pathogenicity were found (p.Lys168Arg, and c.684C>A), and two novel substitutions, p.Leu81Val (c.G241C) and p.Met195Val (c.A583G), both in heterozygosis without an accompanying mutation in the other allele. None of these latter four variants of undefined status was present in a sample of 100 hearing controls. Conclusions: The present study demonstrates that Mutations in the GJB2 gene and del(GJB6 D13S1830) are important causes of hearing impairment in Brazil, thus justifying their screening in a routine basis. The diversity of variants in our sample reflects the ethnic heterogeneity of the Brazilian population.