An Improved Method to Extract DNA from 1 ml of Uncultured Amniotic Fluid from Patients at Less than 16 Weeks’ Gestation

<div><p>The aim of this study was to develop an improved technique for DNA extraction from 1 ml of uncultured AF from patients with a gestational age less than 16 weeks and to allow the use of array-CGH without DNA amplification. The DNA extraction protocol was tested in a series of 90 samples including 41 of uncultured AF at less than 16 weeks of gestation. Statistical analyses were performed using linear regression. To evaluate the sensitivity and the specificity of array-CGH on 1 ml of uncultured AF, five samples with an abnormal karyotype (three with aneuploidy, two with structural abnormalities) and five with a normal karyotype were studied. This protocol was reproducible and we were able to show a great improvement with higher yield of DNA obtained from all patients, including those with a gestational age less than 16 weeks (p = 0.003). All chromosomal abnormalities were detected and characterized by array-CGH and normal samples showed normal profiles. This new DNA extraction protocol associated with array-CGH analysis could be used in prenatal testing even when gestational age is less than 16 weeks, especially in cases with abnormal ultrasound findings.</p> </div

Prospective study of amniotic fluid (AF) by array-CGH.

<p>AMA, advanced maternal age; AU,abnormal ultrasound.</p

Summary of karyotype, array-CGH results, indication and DNA isolated from 1 ml of uncultured amniotic fluid at less than 16 weeks’ gestation.

<p>AMA, Advanced maternal age; IUGR, intrauterine growth retardation; AF, amniotic fluid; R, risk evaluation after triple test screening’.</p

Genome views of five number and structural abnormalities characterized by array-CGH, extracted from 1 ml of uncultured amniotic fluid from patients at less than 16 weeks of gestation.

<p>(cases 1 to 5).</p

Retrospective study of amniotic fluid (AF) by array-CGH.

<p>AF cffDNA, amniotic fluid supernatant cell-free fetal DNA.</p

Genome views of 12 chromosomal abnormalities characterized by array-CGH, extracted from 1 ml of uncultured amniotic fluid from patients at more than 16 weeks of gestation.

<p>(cases 11 to 22).</p

Summary of DNA yield, purity (260/280 and 260/230 reading) extract by this optimized protocol of the 90 uncultured amniotic fluid.

<p>Summary of DNA yield, purity (260/280 and 260/230 reading) extract by this optimized protocol of the 90 uncultured amniotic fluid.</p

Results of multiple linear regression analysis performed to assess the independent relationship between DNA level, the study (ours vs. Bi et al. 2008) and gestational age.

<p>The multiple linear regression analysis model adjusted for gestational age (R² = 0.42).</p

Comparison of DNA yield and gestational age with Bi et al. [2008] study.

<p>Comparison of DNA yield and gestational age with Bi et al. [2008] study.</p

Table1_Combining globally search for a regular expression and print matching lines with bibliographic monitoring of genomic database improves diagnosis.DOCX

Introduction: Exome sequencing has a diagnostic yield ranging from 25% to 70% in rare diseases and regularly implicates genes in novel disorders. Retrospective data reanalysis has demonstrated strong efficacy in improving diagnosis, but poses organizational difficulties for clinical laboratories.Patients and methods: We applied a reanalysis strategy based on intensive prospective bibliographic monitoring along with direct application of the GREP command-line tool (to “globally search for a regular expression and print matching lines”) in a large ES database. For 18 months, we submitted the same five keywords of interest [(intellectual disability, (neuro)developmental delay, and (neuro)developmental disorder)] to PubMed on a daily basis to identify recently published novel disease–gene associations or new phenotypes in genes already implicated in human pathology. We used the Linux GREP tool and an in-house script to collect all variants of these genes from our 5,459 exome database.Results: After GREP queries and variant filtration, we identified 128 genes of interest and collected 56 candidate variants from 53 individuals. We confirmed causal diagnosis for 19/128 genes (15%) in 21 individuals and identified variants of unknown significance for 19/128 genes (15%) in 23 individuals. Altogether, GREP queries for only 128 genes over a period of 18 months permitted a causal diagnosis to be established in 21/2875 undiagnosed affected probands (0.7%).Conclusion: The GREP query strategy is efficient and less tedious than complete periodic reanalysis. It is an interesting reanalysis strategy to improve diagnosis.</p