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.

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.

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.

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)

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.

Malformações congênitas e a fertilização in vitro / Congenital malformations and in vitro fertilization

Nas últimas décadas a fertilização in vitro (FIV) tornou-se uma alternativa eficaz e largamente difundida para tratamento de casais inférteis. Contudo, nesses anos de aplicação clínica tem havido preocupação com a saúde das crianças concebidas através da técnica, em particular quanto à possibilidade de malformação congênita. Diferentes trabalhos foram publicados para avaliar as crianças de FIV. Contudo, de modo geral o resultado foi considerado satisfatório, não tendo sido conclusivos os estudos sobre o aumento na incidência de malformações congênitas. Por outro lado, o advento da inseminação intracitoplasmática de espermatozóides (ICSI), dando possibilidde de ter filhos a indivíduos com diminuição acentuada na contagem de espermatozóides, ou mesmo com azoospermia, exacerbou os questionamentos sobre a segurança. Os primeiros trabalhos visando avaliar malformações congênitas nas crianças nascidas por ICSI não conseguiram mostrar incidência maior. No entanto, estudos mais recentes, com casuística maior, têm apontado um risco superior na ocorrência de malformação, porém sem conclusão definitiva. Até o momento, na literatura não há dados suficientes que contra-indiquem a aplicação das técnicas de reprodução assistida, embora persistam preocupações quanto ao bem-estar das crianças concebidas por ICSI e por FIV.

Accuracy of fetal gender determination in maternal plasma at 5 and 6 weeks of pregnancy.

OBJECTIVE: To assess the viability of the early diagnosis of fetal gender in maternal plasma before 7 weeks of pregnancy by real-time polymerase chain reaction (real-time PCR), starting at 5 weeks of pregnancy. METHOD: Peripheral blood was collected from pregnant women, starting at 5 weeks of gestation. After centrifugation, plasma was separated for fetal DNA extraction. DNA was analyzed by quantitative real-time PCR for two genomic regions, one on the Y chromosome (DYS-14) and the other shared by both sexes (ss-globin), by the TaqMan Minor Groove Binder (MGB) probe assay. The results of the examinations were compared to fetal gender determined after delivery. RESULTS: A total of 79 examinations of fetal DNA in maternal plasma were performed for 52 pregnant women. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks, and 95.6% (22 of 23 cases) at 6 weeks. These results also demonstrate that fetal DNA is present at low concentrations in maternal plasma at 5 weeks (8.5 genome equivalents (GE)/mL) and 6 weeks (34.1 GE/mL) of pregnancy. CONCLUSION: Quantitative real-time PCR and TaqMan MGB probes specific for the detection of fetal gender in maternal plasma starting at 5 weeks of gestation have good sensitivity and excellent specificity.

Determinação precoce do sexo fetal pela análise do DNA no plasma materno / Early determination of gender by analysis of fetal DNA in maternal plasma

OBJETIVO: avaliar a possibilidade do diagnóstico precoce do sexo fetal no plasma materno pela técnica da reação em cadeia da polimerase em tempo real (PCR em tempo real) a partir da 5ª semana de gestação. MÉTODOS: nesse estudo prospectivo foi coletado sangue periférico de gestantes com feto único a partir da 5ª semana de gestação. Após centrifugação do sangue, 0,4 mL de plasma foi separado para extração de DNA fetal. O DNA foi analisado em duplicata por PCR em tempo real para duas regiões genômicas (uma do cromossomo Y e outra comum a ambos os sexos) pelo método de TaqMan®, o qual utiliza um par de primers e uma sonda fluorescente. Foram excluídos da amostragem os casos que evoluíram para aborto. Para o cálculo da sensibilidade e especificidade, usamos o método de comparação com padrão-ouro, que foi o sexo ao nascimento. RESULTADOS: foram realizados 79 exames de DNA fetal no plasma materno de 52 gestantes. O resultado dos exames foi comparado com o sexo da criança após o parto. O índice de acerto conforme a idade gestacional foi de 92,6 por cento (25 de 27 casos) na 5ª semana, conferindo sensibilidade de 87 por cento e 95,6 por cento (22 de 23 casos) na 6ª semana, com sensibilidade de 92 por cento. A partir da 7ª semana de gestação o acerto foi em 100 por cento (29 de 29 casos). A especificidade foi de 100 por cento independente da idade gestacional. CONCLUSÕES: a técnica de PCR em tempo real para detecção do sexo fetal a partir da 5ª semana no plasma materno possui boa sensibilidade e excelente especificidade. Houve concordância do resultado em 100 por cento dos casos em que o diagnóstico foi masculino, independente da idade gestacional, e no caso de feminino, a partir da 7ª semana de gestação.

PURPOSE: to verify the viability of early diagnosis of fetal gender in maternal plasma by the real-time polymerase chain reaction (real-time PCR) starting at the 5th week of pregnancy. METHODS: peripheral blood was collected from pregnant women with single fetus starting at the 5th week of gestation. After centrifugation, 0.4 mL plasma was separated for fetal DNA extraction. The DNA was analyzed in duplicate by real-time PCR for two genomic regions, one of the Y chromosome and the other common to both sexes, through the TaqMan® method, which uses a pair of primers and a fluorescent probe. Patients who aborted were excluded. RESULTS: a total of 79 determinations of fetal DNA in maternal plasma were performed in 52 pregnant women. The results of the determinations were compared to fetal gender after delivery. Accuracy according to gestational age was 92.6 percent (25 of 27 cases) at 5 weeks with 87 percent sensitivity, and 95.6 percent (22 of 23 cases) at 6 weeks with 92 percent sensitivity. Starting at the 7th week of pregnancy, accuracy was 100 percent (29 of 29 cases). Specificity was 100 percent regardless of gestational age. CONCLUSION: real-time PCR for the detection of fetal gender in maternal plasma starting at the 5th week of gestation has good sensitivity and excellent specificity. There was agreement of the results in 100 percent of the cases in which male gender was diagnosed, regardless of gestational age, and from the 7th week of gestation for female gender diagnosis.

Incidência de malformações congênitas em crianças concebidas através de injeção intracitoplasmática de espermatozóides / Incidence of congenital malformations in children conceived through intracytoplasmic sperm injection

OBJETIVO: avaliar a incidência e tipos de malformações congênitas maiores (MCM) em crianças concebidas por injeção intracitoplasmática de espermatozóides (ICSI) e nascidas vivas. MÉTODOS: um total de 680 crianças nasceram vivas de 511 casais submetidos à ICSI no período de janeiro de 1999 a dezembro de 2002. A coleta de dados das crianças foi procedida por meio de questionário padronizado e exame clínico. Dos 511 casais, 366 foram contatados para amostragem de 371 gestações. Das 680 crianças nascidas vivas, 520 foram avaliadas, 250 delas (48,1 por cento) por meio de questionário e 270 (51,9 por cento) por questionário e exame físico. Duzentas e cinqüenta crianças foram de gestação única e 270 de gestação múltipla. Na análise das malformações congênitas foi empregada a 10ª Revisão da Classificação Internacional de Doenças. Nesse estudo foram analisadas apenas as MCM. A incidência de MCM foi comparada à da população geral obtida pelo Estudo Colaborativo Latino-Americano de Malformações Congênitas. A análise estatística foi feita usando o teste do chi2 (nível de significância p<0,05). RESULTADOS: das 520 crianças observadas, 15 apresentaram MCM, dando incidência de 2,9 por cento. Não houve diferença significativa em relação ao grupo controle (p>0,05), que teve 2,6 por cento de incidência de MCM. As malformações mais freqüentes foram as de origem cardíaca (quatro isoladas e duas associadas), correspondendo a 40 por cento do total. Os outros tipos de MCM foram: renal (três), defeito de fechamento do tubo neural (dois), defeito do crânio (um), lábio leporino (um), genital (um), síndrome de Down (associada à cardiopatia) (dois) e músculo-esquelética (um). Seis MCM ocorreram em crianças provenientes de gestações únicas e nove de gestações múltiplas. CONCLUSÃO: as crianças concebidas por ICSI e nascidas vivas apresentaram incidência de malformações congênitas maiores (2,9 por cento) próximo ao esperado para a população geral (2,6 por cento). Entretanto, para estabelecer com precisão os riscos de MCM é necessária continuidade na avaliação das crianças concebidas por ICSI.

Splenopancreatic field abnormality is not unique to trisomy 13.

Splenopancreatic fusion is an uncommon finding, usually only seen as part of the splenopancreatic field abnormality associated with trisomy 13. It may present itself either as ectopic splenic tissue in the cauda pancreatis, as ectopic pancreatic tissue in the spleen or accessory spleen, or as fusion of the cauda pancreatis and splenic hilum. In this study, we report four unrelated congenital anomaly cases presenting trisomy 21, osteocraniostenosis syndrome, isolated congenital heart defect, and oligohydramnios sequence due to prune belly syndrome, in which fusion was observed. This demonstrates that, although it may be more common in trisomy 13, this phenomenon should not be interpreted as pathognomonic to that syndrome.