Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

Isochromosome 21q is overrepresented among false-negative cell-free DNA prenatal screening results involving Down syndrome.

False-negative cell-free DNA (cfDNA) screening results involving Down syndrome are rare, but have high clinical impact on patients and their healthcare providers. Understanding the biology behind these results may allow for improved diagnostic follow-up and counseling. In 5 different centers offering cfDNA prenatal screening, 9 false-negative results were documented in 646 confirmed cases of trisomy 21; a false-negative rate of 1.4% (95% CI, 0.7-2.6). False-negative results included 4 cases of classical trisomy 21 and 5 cases with a de novo 21q;21q rearrangement. Two out of five rearrangements had molecular studies and were confirmed as isochromosomes. When combined with reports from the cfDNA screening literature, 8 out of 29 (28%) Down syndrome cases with a false-negative "non-invasive prenatal test" (NIPT) were associated with a 21q;21q rearrangement, compared with 2% reported in live born children with Down syndrome. In our laboratory series, evidence for placental or fetal mosaicism was present in 3 out of 3 true-positive cases involving a 21q;21q rearrangement and was confirmed in one false-negative case where placental material was available for study. Isochromosome 21q rearrangements are thus overrepresented among false-negative cfDNA screening results involving Down syndrome. Postzygotic isochromosome formation leading to placental mosaicism provides a biological cause for the increased prevalence of these rearrangements among false-negative cases. For clinical practice, a low trisomic fraction (z-score or equivalent measure) relative to the fetal fraction suggests placental mosaicism. Care should be taken as these cases may not reflect confined placental mosaicism, but rather full trisomy in the presence of a placenta containing normal cells.

Integration of aerobic oxidation and intramolecular asymmetric aza-Friedel-Crafts reactions with a chiral bifunctional heterogeneous catalyst.

A new class of chiral bifunctional heterogeneous materials composed of Au/Pd nanoparticles and chiral phosphoric acids as active orthogonal catalysts was prepared by utilizing a facile pseudo-suspension co-polymerization method. It was found that this heterogeneous catalyst was capable of facilitating the sequential aerobic oxidation-asymmetric intramolecular aza-Friedel-Crafts reaction between benzyl alcohols and N-aminoethylpyrroles. Moreover, the designed chiral heterogeneous catalyst could be recovered and reused several times without significant loss of activity or enantioselectivity.

Metal-free, one-pot conversion of proline derivatives into 2-aryl-3-iodo pyrrolidines by a sequential scission-iodination-arylation process.

The metal-free, direct conversion of readily available proline derivatives into 2-aryl-3-iodopyrrolidines is carried out under mild conditions and in good yields, using a sequential radical decarboxylation-oxidation-iodination-arylation reaction. These iodinated pyrrolidines are valuable precursors of other compounds. For instance, they can be cyclized to tricyclic compounds or undergo dehalogenation to 2-aryl-2,5-dihydro-1H-pyrroles, which are iminosugar and 2-arylpyrrole precursors. This process provides a short pathway to a variety of alkaloid and drug analogues of potential pharmaceutical interest.

Simplification of antitumoral phenanthroindolizidine alkaloids: short synthesis of cytotoxic indolizidinone and pyrrolidine analogs.

Hydroxylated seco-analogs of cytotoxic phenanthroindolizidine alkaloids were prepared in good yields from inexpensive 4-hydroxyproline derivatives, in just two steps. Thus, a sequential oxidative radical scission-oxidation was used for the direct conversion of the proline derivative into a 2-(2-aryl-oxoethyl)pyrrolidine with a variety of aryl and heteroaryl groups. The 4R-stereogenic center allowed ready isomer separation, and stereocontrol in the introduction of new chains (interestingly, the 2,4-cis isomers predominated). In the second step, a cyclization reaction afforded alkaloid analogs with an indolizidinone core; a partial isomerization took place but the isomers were readily purified. Then the cytotoxic activity of the bicyclic indolizidinones and the simpler pyrrolidine derivatives was compared against tumorogenic human neuronal SHSY-5Y and breast cancer MCF7 cells. All the biphenyl derivatives displayed a potent activity (one derivative caused >80% cell death in both tumor lines at micromolar dosis), being comparable in the pyrrolidine and indolizidinone series.

Stereoselective conversion of sugar derivatives into C-nucleosides.

A two-step process for the transformation of readily available carbohydrate derivatives into acyclic C-nucleosides is described. The carbohydrate undergoes a scission process that is followed by the addition of aryl ketone derivatives, allowing the introduction of a variety of aryl rings. The resulting acyclic C-nucleosides are transformed into 2-deoxy cyclic pyranosides in good yield and excellent stereoselectivity.

Synthesis of indolizidinone analogues of cytotoxic alkaloids: monocyclic precursors are also active.

Readily available proline derivatives can be transformed in just two steps into analogues of cytotoxic phenanthroindolizidine alkaloids. The key step uses a sequential radical scission-oxidation-alkylation process, which yields 2-substituted pyrrolidine amides. A second process effects the cyclization to give the desired alkaloid analogues, which possess an indolizidine core. The major and minor isomers (dr 3:2 to 3:1) can be easily separated, allowing their use to study structure-activity relationships (SAR). The process is versatile and allows the introduction of aryl and heteroaryl groups (including biphenyl, halogenated phenyl, and pyrrole rings). Some of these alkaloid analogues displayed a selective cytotoxic activity against tumorogenic human neuronal and mammary cancer cells, and one derivative caused around 80% cell death in both tumor lines at micromolar doses. The cytotoxicity of some monocyclic precursors was also studied, being comparable or superior to the bicyclic derivatives.

Ultrasound detection rate of single umbilical artery in the first trimester of pregnancy.

OBJECTIVE: To determine accuracy of first trimester detection of single umbilical artery (SUA). METHODS: The number of vessels in the umbilical cord was examined in a prospective cohort of 779 singleton, low-risk, unselected pregnancies, in the first (11-13 weeks) and second (17-24 weeks) trimesters, using both power and color Doppler and after delivery, by placental histopathologic exam. Concordance between first and second trimester findings to postnatal diagnoses was compared by calculating kappa coefficients. RESULTS: There was medium concordance between the findings in the first trimester and the postnatal diagnoses (kappa = 0.52) and high concordance (kappa = 0.89) for the second trimester scan. Sensitivity, specificity, positive and negative predictive values for the findings in the first trimester were 57.1, 98.9, 50.0 and 99.2% and for the second trimester were 86.6, 99.9, 92.9 and 99.7%. CONCLUSION: Sensitivity and positive predictive value of first trimester scan to identify an isolated SUA in a prospective unselected population was poor. Diagnosis of isolated SUA as well as a definitive judgment about the presence of associated anomalies would still require a scan in the second trimester.

Relação entre a medida da translucência nucal no primeiro trimestre e a presença de marcadores ultrassonográficos para a Síndrome de Down no segundo trimestre da gestação / Second trimester soft markers: relation to first trimester nuchal translucency

A pesquisa de marcadores ultrassonográficos no segundo trimestre da gestação, após rastreamento combinado no primeiro, parece elevar substancialmente as taxas de detecção de Síndrome de Down, mas está amparada na assunção não comprovada de independência entre esses testes. O presente estudo investigou a relação entre a translucência nucal e uma série de marcadores ultrassonográficos no segundo trimestre. A medida da translucência nucal no primeiro trimestre era seguida pela realização da ultrassonografia morfológica entre 18 a 23 semanas e 6 dias de gestação, incluindo a pesquisa de três marcadores qualitativos (foco ecogênico intracardíaco, intestino hiperecogênico e defeito estrutural) e as medidas do osso nasal, da prega nucal, do comprimento do úmero, do comprimento do fêmur, do diâmetro anteroposterior das pelves renais e da espessura pré-nasal. Todas as variáveis contínuas foram expressas em múltiplos da mediana para a idade gestacional e os coeficientes de correlação entre a translucência nucal e essas variáveis (após transformação logarítmica) foram calculados. Em seguida, as frequências de marcadores clássicos no segundo trimestre, em casos com translucência nucal normal, foram comparadas àquelas com translucência nucal aumentada, usando pontos de corte definidos em múltiplos da mediana. Em população prospectiva de 1970 casos, a translucência nucal se correlacionou significativamente com todas as variáveis ultrassonográficas do segundo trimestre, em particular, com a prega nucal (r=0.10). Houve frequência significativamente maior de casos com prega nucal aumentada (10,7 versus 2,2%), definida como valor (em MoMs) acima do percentil 97,5, e intestino hiperecogênico (2,4% versus 0,1%) em casos com translucência nucal aumentada. Concluindo, a utilização de razões de verossimilhança baseadas na presença, ou ausência, de marcadores ultrassonográficos no segundo trimestre para modificar o risco calculado...

Genetic sonogram following first trimester combined screening appears to substantially increase detection rates for Down syndrome but it relies on the unproved assumption of independence between these tests. In this study we have investigated the relation of first trimester nuchal translucency to a series of secondtrimester soft markers. Nuchal translucency (NT) measurement in the first trimester was followed by second trimester scan (18-23w+6 days) including search for three categorical soft-markers (intracardiac echogenic foci, hyperechogenic bowel and structural defects) and measurements of nasal bone length, nuchal fold thickness, femur length, humerus length, renal pelvices diameter and prenasal thickness. All continuous variables were expressed in multiples of the medians for gestation (MoMs) and correlation coefficients between log-transformed NT and second trimester variables were calculated. In addition, frequencies of classical soft-markers in cases with increased NT were compared to those with normal NT, using MoMs cutoffs. In a dataset of 1970 cases, NT was significantly correlated (p<0.05) to all second trimester continuous variables, in particular to nuchal fold thickness (r=0.10). There was a higher frequency of cases with second trimester nuchal fold thickness above the 95th centile (10.7% versus 2.2%) and hyperechogenic bowel (2.4% versus 0.1%) in cases with increased NT. In conclusion, straightforward reassessment of risk using likelihood ratios derived from the classical genetic sonogram might lead to inaccurate estimates. Multivariate models using continuous second-trimester variables might be preferable in sequential screening strategies.

Model-predicted performance of second-trimester Down syndrome screening with sonographic prenasal thickness.

OBJECTIVE: The purpose of this study was to estimate the Down syndrome detection and false-positive rates for second-trimester sonographic prenasal thickness (PT) measurement alone and in combination with other markers. METHODS: Multivariate log Gaussian modeling was performed using numerical integration. Parameters for the PT distribution, in multiples of the normal gestation-specific median (MoM), were derived from 105 Down syndrome and 1385 unaffected pregnancies scanned at 14 to 27 weeks. The data included a new series of 25 cases and 535 controls combined with 4 previously published series. The means were estimated by the median and the SDs by the 10th to 90th range divided by 2.563. Parameters for other markers were obtained from the literature. RESULTS: A log Gaussian model fitted the distribution of PT values well in Down syndrome and unaffected pregnancies. The distribution parameters were as follows: Down syndrome, mean, 1.334 MoM; log(10) SD, 0.0772; unaffected pregnancies, 0.995 and 0.0752, respectively. The model-predicted detection rates for 1%, 3%, and 5% false-positive rates for PT alone were 35%, 51%, and 60%, respectively. The addition of PT to a 4-serum marker protocol increased detection by 14% to 18% compared with serum alone. The simultaneous sonographic measurement of PT and nasal bone length increased detection by 19% to 26%, and with a third sonographic marker, nuchal skin fold, performance was comparable with first-trimester protocols. CONCLUSIONS: Second-trimester screening with sonographic PT and serum markers is predicted to have a high detection rate, and further sonographic markers could perform comparably with first-trimester screening protocols.

Fetal tricuspid valve Doppler at 11-13 weeks and 6 days: reference values and reproducibility.

OBJECTIVE: To determine normal blood flow velocities across the fetal tricuspid valve (TV) at 11-13 weeks and 6 days of gestation and to examine the reproducibility of these measurements. METHODS: A prospective study involving 166 normal singleton pregnancies examined at 11-13 weeks and 6 days was carried out. Descriptive analysis of E- and A-waves' maximum velocities, E/A ratio, duration of the cardiac cycle (C) and diastole (D) and D/C ratio were calculated. Intraobserver and interobserver agreement analysis was performed in a subgroup of 12 cases. RESULTS: Average ( +/- SD) flow velocities were: E-wave, 25.0 ( +/- 4.6) cm/s; A-wave, 42.9 ( +/- 5.9) cm/s; E/A, 0.58 ( +/- 0.07); cardiac cycle, 390 ( +/- 21.1) ms; diastole, 147 ( +/- 18) ms and D/C, 0.38 ( +/- 0.04). Significant correlation was observed between all parameters (except A-wave) and gestational age but not with nuchal translucency (NT). Intraclass correlation coefficients (interobserver, intraobsever examiner 1 and intraobserver examiner 2) were: E-wave, 0.53, 0.53 and 0.64; A-wave, 0.45, 0.46 and 0.49; cardiac cycle, 0.70, 0.79 and 0.84 and diastole, 0.63, 0.85 and 0.82, respectively. CONCLUSIONS: The present study establishes normal Doppler parameters for blood flow across the TV at 11-13 weeks and 6 days and demonstrates that these parameters do not correlate with NT measurement and have good/moderate reproducibility.

Diagnóstico pré-natal das genodermatoses / Prenatal diagnosis of genodermatoses

O diagnóstico pré-natal está indicado para algumas genodermatoses graves, como a epidermólise bolhosa distrófica recessiva e a epidermólise bolhosa juncional. A biópsia de pele fetal foi introduzida em 1980, mas não pode ser realizada antes da 15a semana de gestação. A análise do DNA fetal é método preciso e pode ser realizado mais precocemente na gestação. No entanto, deve-se conhecer a base molecular da genodermatose, e é essencial determinar a mutação e/ou marcadores informativos nas famílias com criança previamente afetada. O DNA fetal pode ser obtido pela biópsia da vilosidade coriônica ou amniocentese. O diagnóstico genético pré-implantação tem surgido como alternativa que dispensa a interrupção da gestação. Essa técnica, que envolve fertilização in vitro e teste genético do embrião. vem sendo realizada para genodermatoses em poucos centros de referência. A ultra-sonografia é exame não invasivo, mas tem uso limitado no diagnóstico pré-natal de genodermatoses. A ultrasonografia tridimensional geralmente estabelece o diagnóstico tardiamente na gestação, e há apenas relatos anedóticos de diagnóstico pré-natal de genodermatoses usando esse método.

Prenatal diagnostic testing is indicated for some severe genodermatoses, such as recessive dystrophic epidermolysis bullosa and junctional epidermolysis bullosa. Fetal skin biopsy was introduced in 1980, but it cannot be performed before 15th gestational week. Fetal DNA analysis is a precise method and can be performed earlier in pregnancy. However, the molecular basis of the genodermatoses must be known and it is essential to determine the gene mutations and/or informative markers in the families with a previously affected child. Fetal DNA can be obtained by chorionic villus sampling or amniocentesis. Preimplantation genetic diagnosis is an alternative approach obviating the need for termination of pregnancy. It involves in vitro fertilization and genetic testing of embryos. However, this technique has been performed for genodermatoses in only a few reference centers. Ultrasonography is a non-invasive test, but has a limited use in prenatal diagnosis of genodermatoses. Tridimensional ultrasonography usually establishes diagnosis late in pregnancy and there are only anecdotal reports of prenatal diagnosis of genodermatoses using this method.

Fetal obstructive uropathy: is urine sampling useful for prenatal counselling?

OBJECTIVES: To evaluate whether fetal urinary sodium and chloride provide clinically useful information in addition to ultrasound in bilateral obstructive uropathy. METHODS: Sonographic features and urinary concentrations of sodium and chloride were evaluated in fetuses with bilateral obstructive uropathy. After a minimum of 12 months of postnatal follow-up, cases that developed increased serum creatinine (greater than 50 micromol/L) were compared with those that did not. RESULTS: Of the cases studied, 16/35 died perinatally, all showing anamnios and markedly elevated urinary electrolytes. Of the survivors, ten maintained normal postnatal serum creatinine, whereas nine did not. The frequency of reduced amniotic fluid/olygohydramnios was higher in cases that developed increased serum creatinine (four out of nine) than in those that did not (nil). Sodium above the 95th percentile was 100% specific and 44% sensitive to predict an increased serum creatinine during early infancy, while chloride above the 95th percentile was 70% specific and 56% sensitive. All seven cases in which urinary sodium was elevated and/or amniotic fluid volume was reduced developed renal failure. CONCLUSION: Urine sampling slightly improved renal function prediction, but this must be balanced against its fetal risks.

Determinação ultra-sonográfica do sexo fetal pela medida dos ângulos do apêndice genital / Sonographic determination of fetal gender by measurement of the angles of the genital tubercle

OBJETIVO: avaliar a acurácia da ultra-sonografia para predição do sexo fetal entre a 11ª e a 13ª semana e 6 dias por meio da medida dos ângulos anterior e posterior do apêndice genital. MÉTODOS: os ângulos anterior e posterior do apêndice genital foram medidos em corte sagital de 455 fetos entre a 11ª e a 13ª semana e 6 dias. A probabilidade de predição correta do sexo fetal (confirmado após o nascimento) foi categorizada de acordo com a medida dos ângulos, idade gestacional e comprimento crânio-caudal. Os pontos de corte de melhor acurácia para predizer o sexo fetal foram obtidos por meio de curva ROC. A variação interobservador foi avaliada pelo método de Bland-Altman. RESULTADOS: a taxa de predição correta do sexo fetal se elevou com a idade gestacional e comprimento crânio-nádegas. Utilizando o ponto de corte de 42 graus para o ângulo anterior (curva ROC), a predição correta do sexo fetal ocorreu em 72 por cento dos fetos da 11ª à 11ª semana e 6 dias, 86 por cento da 12ª à 12ª semana e 6 dias e 88 por cento da 13ª à 13ª semana e 6 dias. As taxas de predição obtidas com o ângulo posterior (ponto de corte de 24 graus) foram, respectivamente: 70, 87 e 87 por cento. A avaliação da variação interobservador revelou diferença média entre medidas pareadas de 15,7 e 9 graus para as medidas dos ângulos posterior e anterior, respectivamente. CONCLUSAO: a acurácia da medida dos ângulos do apêndice genital na predição do sexo fetal foi elevada a partir da 12ª semana de gestação mas ainda insuficiente para sua aplicação em gestações com risco de doenças graves ligadas ao cromossomo X.

Association between prenatal sonographic findings and post-natal outcomes in 30 cases of isolated spina bifida aperta.

This study presents 30 cases of fetal isolated spina bifida aperta (SBA) to identify prenatal ultrasound findings that could predict the prognosis. Comparisons between surviving patients who had normal (group 1) and abnormal (group 2) post-natal neurological outcomes were made for three different prenatal signs, that is, site of vertebral lesion, presence and degree of ventriculomegaly and presence of talipes. The site of the lesion was the most significant outcome predictor, as high spinal dysraphisms were observed in 2 patients (2/7-28.6%) in group 1 and in 15 patients (15/19-79.0%) in group 2 (p = 0.03). The presence of fetal ventriculomegaly was associated with impaired post-natal neurological development, as it occurred in 4 patients (4/7-57.1%) in group 1 and in 18 patients (18/19-94.7%) in group 2 (p = 0.04). The presence of talipes did not significantly differ between the two groups. Patients with abnormal intellectual outcome (8/26-30.8%) had significantly greater (p = 0.018) lateral ventricle/hemisphere ratios (mean = 0.74, standard deviation = 0.13) than those with normal intellectual development (mean = 0.54, standard deviation = 0.18). Mean post-natal follow-up was at 23 months (standard deviation = 15 months).

Rastreamento antenatal da Síndrome de Down utilizando parâmetros ultra-sonográficos / Screening for fetal Down Syndrome using ultrasonographic parameters

Objetivo: avaliar o valor de parâmetros ultra-sonográficos para o diagnóstico da síndrome de Down fetal (T21), com a finalidade de permitir sua aplicação na prática clínica rotineira. Métodos: estudo do tipo coorte transversal prospectivo, utilizando múltiplos parâmetros ultra-sonográficos para predição da T21. Foram avaliados 1662 exames ultra-sonográficos no estudo de coorte e outros 289 exames como amostra diferencial para testar a curva de normalidade, de outubro de 1993 a novembro de 2000. A análise estatística se baseou no cálculo das variabilidades intra e interoperadores, na construção das curvas de normalidade dos parâmetros e provas de validação destas, nos cálculos de sensibilidade, especificidade, risco relativo, razão de probabilidade e dos valores preditivos pós-teste. Resultados: entre os 1662 casos do estudo, ocorreram 22 casos (1,32 por cento) de fetos com T21. As curvas de normalidades foram construídas para os parâmetros espessura nucal, relação fêmur/pé e comprimento dos ossos próprios do nariz, sendo que a pelve renal obedeceu a distribuição semiquantitativa e o nível de corte estabelecido foi de 4,0 mm. A partir do cálculo da razão de probabilidade e da incidência de T21 nas diversas idades maternas, construiu-se tabela para risco pós-teste dos parâmetros ultra-sonográficos estudados. Conclusões: foram estabelecidas as curvas de normalidade e os índices para cálculo de risco populacional da síndrome de Down fetal utilizando diferentes idades maternas e os fatores multiplicadores por nós propostos. Não foi possível estabelecer curva de normalidade para o parâmetro medida da pelve renal, pois seus resultados tiveram comportamento semiquantitativo, tendo sido, portanto, analisados por faixa de normalidade

Resultados obstetricos e perinatais em gestantes adolescentes atendidas em pre-natal especializado / Perinatal and obstetric results in pregnant adolescents cared at specialized prenatal service

Analisou-se retrospectivamente 100 gestantes adolescentes submetidas a uma assitencia pre-natal diferenciada em grupo multidisciplinar no Hospital das Clinicas da FMUSP, durante os anos de 1992 a 1994, e com o parto realizado na propria instituicao. Os indices avaliados foram: a frequencia dos diversos tipos de parto, suas indicacoes e complicacoes, a distribuicao do peso e do Apgar dos recem-nascidos, a incidencia de CIUR, prematuridade e de asfixia neonatal, alem de outras complicacoes neonatais. Percebeu-se uma incidencia aumentada de parto forcipe e cesarea, em detrimento do parto normal. O parto pelvico tambem teve uma taxa elevada, correlacionando-se com a asfixia neonatal dos recem-nascidos. A incidencia de baixo peso ao nascer foi de 14,85 por cento, a de CIUR de 12,8 por cento e a de prematuridade de 11,88 por cento, todos um pouco acima das medias da populacao geral, mas ainda diminuidas em relacao a algumas cifras referidas em adolescentes...

Malformaçöes fetal do trato urinário / Fetal urinary tract anomalies

No presente artigo, os autores fazem uma atualizaçäo sobre as malformaçöes renais e do trato urinário. Säo abordadas as anormalidades proprias do rim, assim como, as alteraçöes obstrutivas das vias excretoras, com enfase nos aspectos ultra-sonográficos e o estudo da funçäo renal do feto. Finalmente, säo comentados aspectos prognósticos dessas malformaçöes

Os efeitos do exercício na gravidez / Exercises effects during pregnancy

Através de reviséo da literatura os autores fazem consideraçöes sobre a fisiologia e as consequências do exercício físico na gestaçäo com enfase na conduta prática durante o pré-natal. Säo esclarecidos normas e conselhos para uma boa orientaçäo da gestante segundo o grau e tipo de atividade física que esta deseja desenvolver durante os meses do pré-natal. Alguns conceitos e repercussöes fisiológicas de exercício sobre o binomio mäe-feto säo também considerados