Rejane Gus Kessler

Prenatal diagnosis of fetal chromosomal abnormalities: Report of an 18-year experience in a Brazilian public hospital

The study of the fetal karyotype became an important tool for the fetal diagnosis of genetic diseases in the 1970s. Although application of this test has remained very restricted in Brazil, we had 905 referrals for prenatal fetal karyotyping between 1989 and 2007. In 879 cases, a fetal karyotype was obtained. We detected 74 abnormal karyotypes (8.4%), the majority being found when the prior indication was fetal malformation. When obtaining amniotic fluid or chorionic villus samples was difficult, alternative fetal materials (urine, cystic hygroma, cystic lung, intreperitoneal and cerebrospinal fluids) were collected and we had success in obtaining karyotypes in all 13 cases. Although, the option of terminating abnormal pregnancies does not legally exist in Brazil, the information gained in assessing the prognosis of on-going pregnancies or estimating recurrence risks justifies prenatal diagnosis of chromosome abnormalities. We conclude that, in keeping with the policy in most other countries, prenatal cytogenetic analysis is strongly recommended in high-risk pregnancies for fetal abnormalities. However, the unique aspect of this type of study is not its rarity in world terms, but its rarity in Brazil. This argues that Brazilian health policy on prenatal diagnosis requires reforming to make it much more widely available within the public health care sector.

Hidropisia fetal não imune: experiência de duas décadas num hospital universitário

PURPOSE: To identify the etiology of nonimmune hydrops fetalis cases in pregnant women diagnosed and referred for prenatal care. METHODS: Retrospective analysis of cases with nonimmune hydrops fetalis that were monitored between March 1992 and December 2011. Diagnosis was confirmed by the presence of fetal subcutaneous edema (≥5 mm) with effusion in at least one serous cavity using obstetric ultrasound, and etiological investigation was conducted with cytogenetic (karyotype), infectious (syphilis, parvovirus B19, toxoplasmosis, rubella, cytomegalovirus, adenovirus and herpes simplex), hematologic and metabolic (inborn errors) analysis and fetal echocardiography. Twin pregnancies were excluded. Statistical analysis was performed using the χ2 test for adhesion (software R 2.11.1). RESULTS: We included 116 patients with nonimmune hydrops fetalis; the etiology was elucidated in 91 cases (78.5%), while 25 cases (21.5%) were classified as idiopathic. Most cases had a chromosomal etiology, for a total of 26 cases (22.4%), followed by lymphatic etiology with 15 cases (12.9% with 11 cases of cystic hygroma), and cardiovascular and infectious etiology with 14 cases each (12.1%). In the remaining cases, the etiology was thoracic in 6.9% (eight cases), malformation syndromes in 4.3% (five cases), extrathoracic tumors in 3.4% (four cases), metabolic in 1.7% (two cases), and hematologic, gastrointestinal and genitourinary in 0.9% (one case each). During the postnatal period, 104 cases were followed up until the 40th day of life, and 12 cases had intrauterine fetal death. The survival rate of these 104 newborns was 23.1% (24 survived). CONCLUSION: An attempt should be made to clarify the etiology of hydrops diagnosed during pregnancy since the condition is associated with a wide spectrum of diseases. It is especially important to determine whether a potentially treatable condition is present and to identify disease at risk for recurrence in future pregnancies for adequate pre-conception counseling.PURPOSE To identify the etiology of nonimmune hydrops fetalis cases in pregnant women diagnosed and referred for prenatal care. METHODS Retrospective analysis of cases with nonimmune hydrops fetalis that were monitored between March 1992 and December 2011. Diagnosis was confirmed by the presence of fetal subcutaneous edema (≥ 5 mm) with effusion in at least one serous cavity using obstetric ultrasound, and etiological investigation was conducted with cytogenetic (karyotype), infectious (syphilis, parvovirus B19, toxoplasmosis, rubella, cytomegalovirus, adenovirus and herpes simplex), hematologic and metabolic (inborn errors) analysis and fetal echocardiography. Twin pregnancies were excluded. Statistical analysis was performed using the χ² test for adhesion (software R 2.11.1). RESULTS We included 116 patients with nonimmune hydrops fetalis; the etiology was elucidated in 91 cases (78.5%), while 25 cases (21.5%) were classified as idiopathic. Most cases had a chromosomal etiology, for a total of 26 cases (22.4%), followed by lymphatic etiology with 15 cases (12.9% with 11 cases of cystic hygroma), and cardiovascular and infectious etiology with 14 cases each (12.1%). In the remaining cases, the etiology was thoracic in 6.9% (eight cases), malformation syndromes in 4.3% (five cases), extrathoracic tumors in 3.4% (four cases), metabolic in 1.7% (two cases), and hematologic, gastrointestinal and genitourinary in 0.9% (one case each). During the postnatal period, 104 cases were followed up until the 40th day of life, and 12 cases had intrauterine fetal death. The survival rate of these 104 newborns was 23.1% (24 survived). CONCLUSION An attempt should be made to clarify the etiology of hydrops diagnosed during pregnancy since the condition is associated with a wide spectrum of diseases. It is especially important to determine whether a potentially treatable condition is present and to identify disease at risk for recurrence in future pregnancies for adequate pre-conception counseling.

Pitfalls in the prenatal diagnosis of mucolipidosis II alpha/beta: A case report

Mucolipidosis II alpha/beta is an autosomal recessive disorder caused by deficient activity of GlcNAc-1-phosphotransferase. We report the prenatal diagnosis of a fetus who was found to exhibit normal levels of lysosomal enzymes in the amniotic fluid but low levels in amniocytes, and who was found to be heterozygous for the most common GNPTAB mutation. As in some carriers of Mucolipidosis II biochemical abnormalities may hinder prenatal diagnosis, we suggest DNA analysis should be performed whenever possible.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients

Niemann‐Pick type C (NP‐C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP‐C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP‐C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP‐C patients are arising as biomarkers for this disease screening: 7‐ketocholesterol and cholestane‐3β,5α,6β‐triol, both oxidized cholesterol products.

Elevation of glycosaminoglycans in the amniotic fluid of a fetus with mucopolysaccharidosis VII

The aim of this study was to quantify glycosaminoglycans (GAGs) in amniotic fluid (AF) from an MPS VII fetus compared with age‐matched fetuses obtained from normal pregnancies.

Alfafetoproteína: valores normais no líquido amniótico entre 14 e 21 semanas

OBJETIVO: Definir uma curva de normalidade dos valores de alfafetoproteina (AFP) no liquido amniotico em gestantes entre 14 e 21 semanas de gravidez no Hospital de Clinicas de Porto Alegre. MATERIAIS E METODOS: Nas 137 mulheres que procuraram o diagnostico pre-natal e tiveram indicacao de coleta de liquido amniotico. A alfafetoproteina foi dosada em todas as amostras por enzima imunoensaio. Foram selecionadas 109 gestacoes normais (sem malformacoes, cariotipo normal, nao-gemelares) e cujas amostras de liquido amniotico nao eram sanguinolentas. Essas foram divididas quanto a idade gestacional e tiveram calculadas as medianas dos valores de AFP e seus multiplos. RESULTADOS: As medianas da alfafetoproteina (KUI/ml) para cada idade gestacional foram as seguintes: 14 semanas:16,32; 15 semanas:14,36; 16 semanas: 13,43; 17 semanas:10,93; 18 semanas: 8,22; 19 semanas: 7,35; 20 semanas: 5,62; 21 semanas:4,47. CONCLUSAO: O estabelecimento de uma curva normal de AFP em nosso servico permite a utilizacao deste exame para pacientes em risco de defeitos de fechamento de tubo neural. Permite tambem que sejam analisadas amostras enviadas para estudos citogeneticos ou metabolicos de maneira a identificar fetos com niveis elevados de AFP que necessitarao de estudos ultrasonograficos mais detalhados pela possibilidade de defeitos morfologicos.BACKGROUND: To define the normal values of amniotic fluid alphafetoprotein in pregnant women, whose gestational ages range from 14 to 21 weeks, in the Hospital de Clinicas de Porto Alegre. MATERIAL AND METHOD: One hundred thirty seven women with indication for amniocentesis were studied. The alphafetoprotein was measured in all samples using enzyme immunoassay. One hundred and nine normal pregnancies were selected. All of these fetuses had normal cariotype and had no malformation. They were not twins and their amniotic fluid samples were not bloody. These samples were divided by their gestational ages. Then the medians of the alphafetoprotein values and their multiples were calculated. RESULTS: The medians of alphafetoprotein (KUI/ml) for each gestational age were as follows: 14 weeks: 16.32; 15 weeks: 14.36; 16 weeks: 13.43; 17 weeks: 10.93; 18 weeks: 8.22; 19 weeks: 7.35; 20 weeks: 5.62; 21 weeks: 4.47. CONCLUSION: The establishment of alphafetoprotein normal values in our service allows us to use this assay for patients at risk of neural tube defects. It also makes possible to analise samples sent for cytogenetic or metabolic studies, in order to identify elevated levels of alphafetoprotein, so that these fetuses could have a more detailed sonography study to look for malformations.

Monoolein-based nanoparticles for drug delivery to the central nervous system: A platform for lysosomal storage disorder treatment

&NA; Lysosomal Storage Disorders (LSDs) are characterized by an abnormal accumulation of substrates within the lysosome and comprise more than 50 genetic disorders with a frequency of 1:5000 live births. Nanotechnology may be a promising way to circumvent the drawbacks of the current therapies for lysosomal diseases. The blood circulation time and bioavailability of the enzymes or drugs could be improved by inserting them in nanocarriers, which could decrease and/or avoid the need of frequent intravenous infusions along with the minimization or elimination of associated immunogenic responses. Considering the exposed, we aimed to build monoolein‐based nanoparticles stabilized by polysorbate 80 as a smart platform able to reach the central nervous system (CNS) to deliver drugs or enzymes inside lysosomes. We developed and characterized the nanoparticles by dynamic light scattering (DLS), small‐angle X‐ray scattering (SAXS) and cryogenic transmission electron microscopy (Cryo‐TEM). The nanoparticles showed a diameter of 115 nm, which is compatible with in vivo application. The SAXS patterns of the formulations displayed a single broad correlation peak that was fitted to the Teubner‐Strey model confirming that disordered bicontinuous structures were obtained. Cryo‐TEM images corroborated this finding and showed nanoparticles with size values that are similar to those determined by DLS. Furthermore, the nanoparticles did not present cytotoxicity when they were incubated with human fibroblasts, and demonstrated hemolytic activity proportional to the negative control, proving to be safe for parenteral administration. Through the use of a fluorescent dye to track the nanoparticles inside the cell, we demonstrated that they reached lysosomes after 1 h of treatment. More interestingly, the fluorescent dye was detected in the CNS of mice just after 3 h of treatment. The nanoparticles show great potential to improve the treatment of LSDs with brain impairment, acting as a smart platform to targeted delivery of drugs or enzymes. Graphical abstract Figure. No caption available.

The Contribution of Molecular Techniques in Prenatal Diagnosis and Post mortem Fetus with Multiple Malformation

The development of conventional cytogenetic techniques in the 50’s leaded to a rapid increase of the knowledge on the etiology of malformation syndromes, being chromosomal anomalies reported as the most common genetic condition in humans (Pena, 1998). Around 2-3% of newborns may have congenital malformations, and from those, just 20% have an established etiology (genetic or environmental), being 80% of these multifactorial or unknown (Stevenson & Hall, 2006). But this is only the tip of the iceberg, as probably half of the human concepts may have some kind of chromosomal defect (A. Boue & J. Boue, 1973), indicating that cytogenetic analysis is fundamental for the investigation of these cases. Since the 70’s, prenatal diagnosis for detecting cytogenetic abnormalities has become a routine procedure in many countries, and an important tool for the prevention of birth of handicapped children (A. Milunsky & J. Milunsky, 1998). Cytogenetic analysis is an important component of invasive prenatal diagnosis as chromosomal abnormalities are detected in about 1 in 200 newborns and constitute a major cause of mental retardation and congenital malformations (Shaffer & Lupski, 2000). Microscopic chromosome analysis of cultured cells has been regarded as the gold standard method for prenatal diagnosis, since its first application to prenatal testing in 1966 by Steele and Breg (Steele & Breg, 1966) and the routine use of chromosome banding analysis in 1970s. Karyotyping has proved to be highly reliable for diagnosis of numerical chromosome abnormalities and structural rearrangements in fetal cells obtained invasively by either amniocentesis in the second trimester of pregnancy, or chorionic villus sampling (CVS) in the first trimester, since the early 1980s. The diagnostic accuracy of karyotyping fetal cells from cultured amniotic fluid (AF) has been found to be 99.4%-99.8%, and that of CVS 97.5-99.6%. However, the main limitation of karyotyping is the requirement of a cell culture, resulting in a period of 10-14 days for obtaining the final results (Bui, 2007). Furthermore, the success of cell culture depends on many factors: very good laboratory conditions for tissue culture, technician’s experience, and satisfactory cell growth with good quality of metaphases. Unfortunately, due to failure in one of these steps the whole process becomes jeopardized.