Genetic testing for cannabinoid use / Testes genéticos para uso de canabinoides

ABSTRACT BACKGROUND AND OBJECTIVES: Cannabis is the most popular and consumed illicit drug in the world, it has about 540 bioactive phytocannabinoids, including tetrahydrocarbinol (THC) and cannabidiol (CBD). The therapeutic potential of phytocannabinoids has been the subject of many studies in recent decades for many medical situations, including the management of chronic pain. The advent of pharmacogenetics currently allows the indication of the Cannabis dose to be evaluated individually. The objective of this work was to carry out a survey of the literature on the medicinal use of Cannabis and the application of pharmacogenetics in this therapy. CONTENTS: THC and CBD phytocannabinoids are the most abundant and researched. In the endocannabinoid system there are compounds similar to phytocannabinoids, cell receptors and metabolism enzymes. All these molecules are secreted from genes, which may have individual genetic polymorphisms that determine the modulation of the endocannabinoid system, and consequently impact the patients' therapeutic response. CONCLUSION: The existence of genetic tests for the prior assessment of the patients genetic profile in order to avoid side effects and to have more assertiveness in the indication of the cannabis product is an important tool to increase adherence to cannabis treatment.

RESUMO JUSTIFICATIVA E OBJETIVOS: A cannabis é a droga ilícita mais popular e consumida no mundo, possuindo cerca de 540 fitocanabinoides bioativos, entre eles o tetra-hidrocarbinol (THC) e o canabidiol (CBD). O potencial terapêutico dos fitocanabinoides tem sido alvo de muitos estudos nas últimas décadas para muitas situações médicas, incluindo o manejo da dor crônica. O advento da farmacogenética permite que atualmente a indicação da dose de cannabis seja avaliada individualmente. O objetivo deste estudo foi realizar um levantamento da literatura sobre o uso medicinal da cannabis e a aplicação da farmacogenética nessa terapia. CONTEÚDO: Os fitocanabinoides THC e CBD são os mais abundantes e pesquisados. No sistema endocanabinoide, existem compostos similares aos fitocanabinoides, receptores celulares e enzimas de metabolismo. Todas essas moléculas são secretadas a partir de genes que podem possuir polimorfismos genéticos individuais determinantes para a modulação do sistema endocanabinoide e, consequentemente, impactam a resposta terapêutica do paciente. CONCLUSÃO: A existência de testes genéticos para avaliação prévia do perfil genético do paciente a fim de evitar efeitos colaterais e ter mais assertividade na indicação do produto de cannabis é uma importante ferramenta para aumentar a aderência ao tratamento com cannabis.

Case report of Zika virus during controlled ovarian hyperstimulation: results from follicular fluid, cumulus cells and oocytes.

We describe a case of a 37-year-old female, indicated for in vitro fertilisation. She developed skin rash on her trunk and limbs, during the treatment. RT-PCR results were positive in her blood and negative in her husband's blood and semen. Oocyte aspiration was performed, retrieving 7 oocytes, follicular fluid, and cumulus cells. RT-PCR results for the follicular fluid and cumulus cells were negative for ZIKV, and positive for only 2 oocytes. This is the first report in the literature analysing ZIKV in the follicular fluid, cumulus cells, and oocytes, and will contribute to the understanding of ZIKV infection and transmission.

Estimation of age-dependent decrease in blastocyst euploidy by next generation sequencing: development of a novel prediction model.

BACKGROUND: We developed a model to estimate the female age-dependent decrease in blastocyst euploidy and the impact of blastocyst cohort size on the likelihood of having at least one euploid blastocyst for transfer. METHODS: Retrospective analysis of 1296 trophectoderm biopsies by next-generation sequencing analysis from 436 infertile couples undergoing intracytoplasmic sperm injection and preimplantation genetic testing for aneuploidy. A logistic regression model was fit to the data. The dependent and independent variables were embryo genetic status and female age, respectively. The method of fitting was quadratic on age, and the model was validated with cross validation by a data splitting technique. RESULTS: The decrease in the probability of blastocyst euploidy follows an age-dependent binomial distribution, progressing with every year of female age, from 1.2% to 24.5% in 28-44 years-old women (P<0.0001). The minimum number of blastocysts needed to obtain at least one euploid blastocyst for transfer was computed for different probabilities and female ages. At the age of 28 years, a total of three blastocysts is required to obtain at least one euploid blastocyst with 90% probability, whereas it is 4, 5, 6, 9, 16 and 29 for ages 35, 37, 39, 41, 43, and 45, respectively. CONCLUSIONS: A novel prediction model estimates the probability of blastocyst euploidy and the number of blastocysts required to obtain at least one euploid embryo for transfer. This new resource based on f emale age and blastocyst cohort size will aid clinicians counsel and plan treatment of infertile couples undergoing IVF/ICSI.

The first South American case of pre-implantation genetic diagnosis to select compatible embryo for cord blood transplantation as treatment for sickle cell anemia.

Sickle cell anemia is an inherited systemic hemoglobinopathy that affects hemoglobin production in red blood cells, leading to early morbidity and mortality. It is caused by a homozygous nucleotide substitution (c.20A>T) in the ß-globin gene (HBB) that changes a glutamic acid to a valine in the protein. We present a case report of a fertile couple, both carriers of the sickle cell anemia mutation, with one affected daughter. Six cycles of assisted reproductive techniques were performed, resulting in 53 embryos in cleavage stage. Each embryo was biopsied and analyzed for pre-implantation genetic diagnosis (PGD) by fluorescent polymerase chain reaction, using polymorphic markers of the region of interest followed by capillary electrophoresis in an automated genetic analyzer. HLA Compatible and normal embryos for the mutation represented 3 (5.66%); while the carriers and compatible 6 (11.32%); therefore, embryos matching those of the affected daughter represented 9 (16.98%). A selected embryo in blastocyst stage was transferred, resulting in a healthy male newborn, who had the umbilical cord blood cells collected and stored. The affected daughter was immunosuppressed and received transplanted cells from the umbilical cord blood of her brother; the treatment was successful. Embryo selection using PGD technologies represent the most effective treatment plan for parents who want to have a healthy child, and it could cure another child already affected by inherited hemoglobinopathy.

Preimplantation genetic diagnosis for a patient with multiple endocrine neoplasia type 1: case report.

Preimplantation genetic diagnosis was carried out for embryonic analysis in a patient with multiple endocrine neoplasia type 1 (MEN1). This is a rare autosomal-dominant cancer syndrome and the patients with MEN1 are characterized by the occurrence of tumors in multiple endocrine tissues, associated with germline and somatic inactivating mutations in the MEN1 gene. This case report documents a successful preimplantation genetic diagnosis (PGD) involving a couple at-risk for MEN1 syndrome, with a birth of a healthy infant. The couple underwent a cycle of controlled ovarian stimulation and intracytoplasmic sperm injection (ICSI). Embryos were biopsied at the blastocyst stage and cryopreserved; we used PCR-based DNA analysis for PGD testing. Only one of the five embryos analyzed for MEN1 syndrome was unaffected. This embryo was thawed and transferred following endometrial preparation. After positive ßHCG test; clinical pregnancy was confirmed by ultrasound, and a healthy infant was born. PGD for single gene disorders has been an emerging therapeutic tool for couples who are at risk of passing a genetic disease on to their offspring.

Chemokine (C-C motif) ligand 25 expressed by trophoblast cells and leukocytes bearing its receptor Ccr9: An alliance during embryo implantation?

PROBLEM: We hypothesized that trophoblast expression of Ccl25 attracts a specific leukocyte cell population to the implantation site for local regulation. METHOD OF STUDY: Mice blastocysts, ectoplacental cones, and decidua at gestational days 3.5-7.5 were evaluated for Ccl25 and Ccr9 expressions. Peripheral availability and characterization of Ccr9+ leukocytes were determined by flow cytometry. Leukocyte chemotaxis was assessed in the presence of Ccl25 recombinant protein and embryos using antisense oligomers (ODNs) to Ccl25 and Ccr9 neutralizing antibody. RESULTS: Ccl25 was expressed by embryonic cells, whereas Ccr9 expression was strong at the maternal compartment and in PBMC. Immunolocalization confirmed this expression. In vitro, chemotaxis assays showed that the embryonic Ccl25 signals to Ccr9+ PBMCs. Maternal Ccr9+α4ß7+ monocytes switch from an anti-inflammatory phenotype (F4/80+11b+Ly6C-TGF-ß+ cells, pre-implantation) to an inflammatory profile (F4/80+11b+Ly6C+TNF-α+ cells, post-implantation). CONCLUSION: Our data support the establishment of a CCL25/CCR9-axis at the maternal-fetal interface in mice, which may be involved in immune regulatory mechanisms during embryo implantation.

Characterization of vitamin K-dependent carboxylase mutations that cause bleeding and nonbleeding disorders.

Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix γ-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo.

Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

Assessment of free fetal DNA concentration in maternal plasma during the first trimester of pregnancy: comparative study between EDTA and PPT tubes - pilot study.

OBJECTIVE: To compare ethylenediamine tetraacetic acid (EDTA) tubes and plasma preparation tubes (PPT) for evaluating maternal plasma during the first trimester of pregnancy. METHODS: A cross-sectional study was conducted on 24 male fetuses in women between 6 and 14 weeks of pregnancy. Blood samples (10 mL) were collected and stored in EDTA and PPT tubes. Subsequently, the samples were centrifuged and sent for free fetal DNA extraction by means of the polymerase chain reaction (PCR) technique. The reactions were performed in a real time PCR machine for detecting the amplification products. The genome region chosen for performing the PCR reactions was a target specific for the Y chromosome, in which the DYS-14 marker was amplified only when the DNA was of male sex. The free fetal DNA concentration was given by the threshold cycle (TC). To compare the tubes, the paired Student t-test was used. RESULTS: The mean gestational age was 11.08 ± 2.30 weeks (range: 6-14). The mean TC for PPT was 30.08 ± 1.05 (range: 27.08-32.61) and for EDTA, 30.23 ± 0.96 (range: 28.01-32.09), but without statistical significance (p=0.357). CONCLUSION: We did not observe any statistically significant difference in free fetal DNA concentration between the EDTA and PPT tubes.

Biópsia embrionária: qual a melhor escolha? / Embryo biopsy: what is the best choice?

Introdução: a biópsia embrionária tem como objetivo selecionar embriões geneticamente normais. Essa seleção ocorre por meio de testes genéticos pré-implantacionais. Espera-se, com isso, uma diminuição dos riscos de doenças genéticas e um aumento das taxas de implantação em fertilização in vitro. Objetivo: verificar, por meio de revisão bibliográfica, qual técnica de biópsia embrionária é considerada mais apropriada para feitura de testes genéticos pré-implantacionais. Método: pesquisa bibliográfica, na forma de revisão de publicações científicas, por meio das redes US National Library of Medicine (Pubmed), Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs), Google Acadêmico e Biblioteca Virtual em Saúde (BVS). Resultados e conclusão: existem três maneiras de efetuar a biópsia para reprodução humana assistida. A primeira consiste em retirar o primeiro e/ou o segundo corpúsculo polar estruído pelo oócito. Também se pode fazer a biópsia a partir de um blastômero do embrião em estágio de clivagem ou usar cinco a dez células do trofoectoderma de blastocisto. Normalmente as técnicas usadas para o diagnóstico são PCR, Fish, CGH array e SNP array, entre outras. Acredita-se que a biópsia de blastocistos é a melhor técnica para manter o potencial de implantação embrionária. Essa tendência se justifica por causa da maior quantidade de material genético disponível em fase avançada de desenvolvimento embrionário. Admite-se que nessa fase a incidência de mosaicismo seja menor em relação à biópsia de blastômeros, com consequente aumento na eficácia dos testes genéticos. Outra questão importante é que na biópsia de blastocistos as células são retiradas do trofoectoderma, enquanto que na biópsia em estágio de clivagem a remoção de um blastômero pode prejudicar o desenvolvimento embrionário.

Introduction: the embryo biopsy aims to select genetically normal embryos. This selection occurs through pre- implantation genetic testing. It is expected the reduction of risk ofgenetic disorders and increase implantation rates in IVF.Objective: to verify, through bibliographical revision, which embryo biopsy technique is considered more suitable for pre-implantation genetic diagnosis. Method: bibliographical research, in the form of literary review of scientific publications via networks, US National Library of Medicine (Pubmed), Latin-American Literature and Caribbean Health Sciences (Lilacs), Google Scholar and Virtual Health Library. Results and conclusion: there are three ways to perform the biopsy on assisted human reproduction.The first one consists in removing the 1st and/or 2nd polar body (if there wasfertilization). You can also perform the biopsy from the one blastomere of embryo cleavage stage or use 5-10 trophoectoderm cells blastocyst. Usually the techniques used for diagnosticpurpose are PCR, Fish, CGH array, SNP array and others. Nowadays it is believed that blastocyst biopsy is the best technique in order to maintain the embryonic implantation. This tendency is justified by the larger amount of genetic material available in an advancedstage of embryonic development. It is assumed that in this stage the incidence of mosaicism is reduced with the consequent increase in the effectiveness of genetic testing. Another important question is that the blastocyst biopsy cells are removed from the trophoectoderm while inbiopsy incleavage stage, the removal of one blastomere can impair embryonicdevelopment.

A chromosome 19 locus positively influences the number of retrieved oocytes during stimulated cycles in Brazilian women.

PURPOSE: To evaluate if several genetic loci that are associated with variation in normal menopause age and early menopause can account for a poor response to controlled ovarian stimulation. METHODS: A total of 71 patients age ≤35 years old who were undergoing intracytoplasmic sperm injection were genotyped for four genetic variants that are associated with normal variation in menopausal age and early menopause. The patients were divided into two groups based upon treatment response: a poor responder group (PR group, n = 21) and a normal responder group (NR group, n = 50). The genetic variants rs244715, rs9379896, rs4806660 and rs16991615 were analyzed. RESULTS: There was no significant difference in the incidence of the genetic variants between the NR and PR group. The risk allele for the chromosome 19 variant (rs4806660) demonstrated a protective effect for a poor ovarian response. The presence of a risk allele was associated with an increased response to COS, which resulted in an elevated number of follicles (Coef: 2.54, P = 0.041) and retrieved oocytes (Coef: 1.41, P = 0.041). CONCLUSIONS: The genetic variants rs244715, rs9379896, rs4806660 and rs16991615 are not risk factors for poor ovarian response in Brazilian women. In contrast, rs4806660 is associated with higher number of follicles and retrieved oocytes. rs4806660 may be associated with an increased response to gonadotrophin stimulation in this population.

Sperm organelle morphologic abnormalities: contributing factors and effects on intracytoplasmic sperm injection cycles outcomes.

OBJECTIVE: To (1) analyze possible relationships between motile sperm organelle morphology examination (MSOME) and sperm chromatin status, aneuploidy incidence, and patient's age; (2) determine the effects of sperm morphologic abnormalities on intracytoplasmic sperm injection (ICSI) outcomes; and (3) identify the benefits of intracytoplasmic morphologically selected sperm injection (IMSI) in patients with high DNA fragmentation rate. METHODS: The study was performed in 50 patients undergoing ICSI cycles. The MSOME, sperm DNA fragmentation, and sperm aneuploidy incidence were performed in 200 sperm cells of each patient. Regression models were used to assess the relationships among sperm morphology and sperm aneuploidy, sperm DNA fragmentation, patient's age, and ICSI outcomes. In cycles with patients showing a high incidence of DNA fragmentation, oocytes were split into 2 groups according to the sperm selection method: Standard-ICSI (n = 82) and IMSI (n = 79). Fertilization and high-quality embryo rates were compared between the groups. RESULTS: A close relationship between sperm DNA fragmentation and the presence of vacuoles in the MSOME was noted. The patient's age was correlated to the presence of vacuoles. No correlation between sperm aneuploidy and IMSI was observed. Vacuolated cells were negatively correlated with fertilization, pregnancy, and implantation. In patients with a high incidence of sperm DNA fragmentation, fertilization and high-quality embryo rates were similar when comparing IMSI and Standard-ICSI. CONCLUSIONS: Our data demonstrate a correlation between paternal age and the incidence of nuclear vacuoles, as well as an effect of large and small vacuoles on late embryo development.

Alterações no cariótipo que podem gerar infertilidade ou abortamento de repetição / Karyotype alterations that may lead to infertility or repetitive abortion

A infertilidade pode ser definida como a incapacidade de se conseguir uma gravidez dentro de um determinado período ou a falha repetida em levar uma gravidez a termo. Os fatores mais comuns associados com o abortamento habitual são de ordem genética, hormonal ou anatômica. O aumento da heterocromatina encontrado nos cromossomos humanos autossomos 1, 9, 16 e no cromossomo sexual Y tem sido comumente definido como sendo uma variação da normalidade. Todavia, têm-se alguns relatos da frequência aumentada dessas alterações em pais de crianças com anomalias cromossômicas, casais com abortos recorrentes e em conceptos cromossomicamente anormais. Sabe-se que os genes responsáveis pela fertilidade e viabilidade são encontrados presentes na heterocromatina, sugerindo que tais variantes não podem ser ignoradas.(AU)

Infertility can be defined as the inability to achieve pregnancy within certain time frame, or the repetitive failure to carry pregnancy through completion. The most common factors associated with habitual abortion have a genetic, hormonal, or anatomical cause. The increase found in heterochromatin at chromosomes 1, 9 and 16 as well as in the sex chromosome Y has been implicated as a variation of normality. However, some reports have highlighted cases of children having chromosomal abnormalities from parents carrying a higher alteration frequency, cases of repetitive abortions, and also some chromosomal abnormal conceptions. It is known that genes for fertility and viability are now thought to reside in heterochromatin, which suggests that variants should not be ignored.(AU)

Tratamento da endometriose associada à infertilidade - revisão da literatura / Treatment of endometriosis associated with infertility - a literature review

A endometriose é uma desordem estrogênio-dependente definida pela presença de tecido endometrial fora da cavidade uterina, e é uma das principais causas de infertilidade feminina. A melhor escolha para seu tratamento, associado à infertilidade, ainda permanece obscura. As evidências científicas disponíveis indicam que a supressão da função ovariana, apenas com o tratamento hormonal, não melhora as taxas de gravidez. O tratamento cirúrgico é, entretanto, provavelmente eficaz em todos os estágios da doença. Nos casos de endometriose mínima e leve, corrigida cirurgicamente e com anatomia pélvica normal, a inseminação intrauterina (IIU), acompanhada de hiperestímulo ovariano controlado, é recomendada. Em casos de endometriose avançada, especialmente se estiver associada a alterações tubárias, fatores masculinos ou falha de tratamento prévio, após laparoscopia, a melhor opção é a Fertilização in vitro (FIV). O sucesso pode ser maior com o tratamento de análogos de gonadotropina (GnRH) de três a seis meses antes da FIV. Finalmente, é importante ressaltar que as recomendações acima deverão ser revistas à medida que estudos clínicos randomizados e controlados mostrarem evidências mais concretas e confiáveis dessa enigmática doença.

Endometriosis is an estrogen-dependent disorder defined as the presence of endometrial tissue outside of the uterine cavity, and is a leading cause of female infertility. The optimal choice for its treatment associated with infertility remains obscure. The available scientific evidence indicates that suppression of ovarian function with hormonal treatment is not effective and should not be offered for this indication alone, and it does not get better pregnancy rates. Surgery treatment is probably efficacious for all stages of the disease. Intrauterine insemination (IUI) with controlled ovarian stimulation is effective in endometriosis minimal-mild and surgically corrected when the pelvic anatomy is normal. In advanced endometriosis cases, especially if tubal function is compromised, if there is also male factor infertility, and other treatments have failed, in vitro fertilization (IVF) is the appropriate treatment. The success may increase with gonadotropin-releasing hormone analog treatment (GnRH) for three to six months before IVF. Finally, it is important to emphasize that the recommendation above should be revised as randomized controlled clinical trials with more concrete and reliable evidence of this enigmatic disease.

Noninvasive determination of fetal rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population.

We evaluated the diagnostic accuracy of Rh blood group, D antigen (RHD) fetal genotyping, using real-time polymerase chain reaction in maternal blood samples, in a racially mixed population. We performed a prospective study conducted between January 2006 and December 2007, analyzing fetal RHD genotype in the plasma of 102 D- pregnant women by real-time polymerase chain reaction, targeting exons 7 and 10 of the RHD gene. Genotype results were compared with cord blood phenotype obtained after delivery or before the first intrauterine transfusion when necessary. Most of the participants (75.5%) were under 28 weeks of pregnancy, and 87.5% had at least one relative of black ancestry. By combining amplification of two exons, the accuracy of genotyping was 98%, sensitivity was 100%, and specificity was 92%. The positive likelihood ratio was 12.5, and the negative likelihood ratio was 0. The two false-positive cases were confirmed to be pseudogene RHD by real-time polymerase chain reaction. There were no differences between the patients with positive or negative Coombs test ( P = 0.479). Determination of fetal RHD status in maternal peripheral blood was highly sensitive in this racially mixed population and was not influenced by the presence of antierythrocyte antibodies.

Diagnóstico genético pré-implantacional: uma ferramenta importante para a rotina de fertilização in vitro? / Preimplantation genetic diagnosis: an important tool for the in vitro fertilization routine?

Erros cromossômicos numéricos são comuns durante as primeiras etapas do desenvolvimento embrionário humano, contribuem significativamente com processos de falta de implantação e são causadores da perda gestacional recorrente em pelo menos 50 por cento dos abortos ocorridos no primeiro trimestre. Tradicionalmente, a prevenção das anomalias genéticas cromossômicas em pacientes de alto risco é realizada por exames pré-natais, como a biópsia do vilo coriônico, aminiocentese e a cordocentese. Uma vez diagnosticada a anomalia, não existe tratamento eficaz para portadores de aberrações genéticas e a interrupção da gestação nestes casos ainda é ética e legalmente questionável. O diagnóstico genético pré-implantacional (DGPI) representa uma ferramenta valiosa aos casais de alto risco, por permitir a seleção de embriões saudáveis obtidos através de programas de fertilização in vitro antes de estes serem transferidos para um útero materno. Os primeiros relatos da utilização do DGPI datam da década de 90, quando eram utilizadas metodologias da reação em cadeia da polimerase para determinação do sexo do embrião, permitindo desta maneira transferir embriões que não fossem portadores de doenças ligadas ao cromossomo X. O DGPI é uma técnica extremamente eficaz, que analisa uma única célula do embrião que é biopsiado a partir do terceiro dia de desenvolvimento. Esta metodologia tem como finalidade identificar embriões gerados por processos de reprodução assistida, os quais sejam portadores de aberrações cromossômicas numéricas que envolvam os cromossomos X, Y, 13, 16, 18, 21 e 22. A metodologia mais utilizada para a realização do DGPI é a técnica de hibridização in situ, utilizando-se sondas fluorescentes para os cromossomos citados. Este é um método eficiente e que deve ser discutido com casais cuja idade da mulher seja acima dos 39 anos, casais com cariótipo alterado ou ainda casais com histórico familiar de presença de portadores de cromossomopatias. A eficiência...

The potential transmission of genetic disorders to the offspring has been a major problem for many couples when contemplating pregnancy. Numerical chromosomes errors are common during the first stages of the human embryonic development and contribute significantly with processes of implantation failure and it is also related to recurrent miscarriage, in at least 50 percent of the abortions occurred in the first trimester. Traditionally, the prevention of genetic chromosomal abnormalities in high risk patients is achieved by pre-natal examinations such as biopsy of the chorionic villi, aminiocentese of cordocentesis. Once diagnosed the genetic error, there is no effective treatment for patients with genetic aberrations and the interruption of pregnancy in these cases are ethically and legally questionable. The risk has been greatly decreases by the evaluation of the family history of the age of the mother, and the implementation of prenatal diagnosis in those couples in which the risk was increased compared to the general population. An alternative approach that is available with assisted reproductive technologies is the preimplantation genetic diagnosis (PGD), which it is possible to observe X, Y, 13, 16, 18, 21 and 22 chromosomes or to perform gene screen by PCR for knowing genetic diseases before the corresponding embryo is transferred to the uterus of the mother. PGD was first clinically applied in the early nineties, and was initially used in sexing cases for couples who were at risk of transmitting an X-linked recessive disorder. PGD involves the analysis of either polar bodies, extruded from oocytes during meiosis, or single cells (blastomeres) biopsied from embryos after fertilization. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization and polymerase chain reaction. The efficiency of the method is about 98 percent and its extremely invasive technique demands high level professional.

Autosomal dominant Alport syndrome: molecular analysis of the COL4A4 gene and clinical outcome.

BACKGROUND: Alport syndrome is a clinically and genetically heterogeneous nephropathy characterized by glomerular basement membrane lesions often associated with hearing loss and ocular anomalies. While the X-linked and the autosomal recessive forms are well known, the autosomal dominant form is not well acknowledged. METHODS: We have clinically investigated 38 patients with a diagnosis of autosomal dominant Alport syndrome belonging to eight different families. The analysis of the COL4A4 gene was performed by denaturing high performance liquid chromatography and automated DNA sequencing. RESULTS: In our cohort of patients, only 24.3% (9/37) reached end-stage renal disease, at the mean age of 51.2 years. Four patients had hearing loss (13.3%) and none ocular changes. Molecular analysis revealed eight novel private COL4A4 gene mutations: three frameshift, three missense and two splice-site mutations. CONCLUSIONS: These data indicate autosomal dominant Alport syndrome as a disease with a low risk of ocular and hearing anomalies but with a significant risk to develop renal failure although at an older age than the X-linked form. We were unable to demonstrate a genotype-phenotype correlation. Altogether, these data make difficult the differential diagnosis with the benign familial haematuria due to heterozygous mutations of COL4A4 and COL4A3, especially in young patients, and with the X-linked form of Alport syndrome in families where only females are affected. A correct diagnosis and prognosis is based on a comprehensive clinical investigation in as many family members as possible associated with a broadly formal genetic analysis of the pedigree.

Absolute quantification of gene E7 in cervical samples of women infected by HPV using real time PCR

Objective: High grade oncogenic types of human papillomavirus (HPV), especially HPV16 and HPV18, possess a gene called E7, which acts on genes that regulate cell growth, promoting development of pre-neoplastic lesions that can lead to invasive carcinomas. The absolute quantification of this gene in cervical samples of HPV-infected women may contribute for better understanding the evolution of these lesions induced by the virus. Methods: We collected 60 cervicovaginal smears of women infected by HPV with or without uterine cervical squamous intraepithelial lesion, SIL) and 10 samples of women with no HPV infection or SIL. The absolute quantification of gene E7 was performed by Realtime PCR using specific primers and probes. Results: Samples infected by HPV16 have a higher number of gene E7 copies when compared to samples infected by HPV18. In the HPV18 group it was observed that those obtained from patients with low or high grade squamous intraepithelial lesions (HSIL) or invasive cervical cancer presented significantly higher concentrations of gene E7 when compared to patients with no cervical lesions. The number of gene E7 copies was higher in the group infected by HPV16 than by HPV18. In spite of that, there was no difference in the number of gene E7 copies in samples infected by HPV16 with or without SIL. Conclusions: Among the samples with HPV18, the number of copies of gene E7 was higher in the group with cervical lesions, and no differences were found for SIL, HSIL or invasive cancer patients.