Background

Noninvasive prenatal screening (NIPS) by next-generation sequencing of cell-free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21 in high risk pregnancies. NIPS can identify fetal genomic microdeletions; however, sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus who tested positive for atypical 22q deletion via maternal plasma cfDNA testing.

Methods

We performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. Fluorescence in situ hybridization and chromosomal microarray diagnostic genetic tests were done postnatally.

Conclusion

NIPS detected a 22q microdeletion that, upon diagnostic workup, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.

Background/objective

The non-invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non-invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM).

Methods

Maternal plasma was obtained from a pregnancy affected by a 4.2-Mb fetal microdeletion and three normal controls. Plasma DNA was subjected to capture an 8-Mb sequence spanning the breakpoint region and sequence. Data were analyzed with our published method, MINK, and a new method called GCREM.

Results

The 8-Mb capture segment was divided into either 38 or 76 non-overlapping regions of 200 and 100 Kb, respectively. At 200 Kb resolution, using GCREM (but not MINK), we obtained significant adjusted p-values for all 20 regions overlapping the deleted sequence, and non-significant p-values for all 18 reference regions. At 100 Kb resolution, GCREM identified significant adjusted p-values for all but one 100-Kb region located inside the deleted region.

Conclusion

Targeted sequencing and GCREM analysis may enable cost effective detection of fetal microdeletions and microduplications at high resolution.

Objective

To examine gonadal protective properties of granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF) in female mice treated with high-dose alkylating chemotherapy.

Design

Experimental laboratory animal study.

Setting

Tertiary care academic hospital and research institute.

Animal(s)

Six- and 8-week-old C57Bl/6 female mice.

Intervention(s)

Adult female mice were treated with [1] cyclophosphamide and busulfan (CTx), [2] CTx + G-CSF/SCF, [3] CTx + G-CSF, or [4] normal saline and dimethyl sulfoxide (DMSO; vehicle control).

Main outcome measure(s)

Follicle counts, microvessel density, cellular response to DNA damage, and litter production.

Result(s)

G-CSF ± SCF increased microvessel density and decreased follicle loss in CTx-treated female mice compared with CTx-only treated female mice. Mice administered CTx alone exhibited premature ovarian insufficiency, with only 28% of mice producing two litters. However, 100% of mice receiving CTx with G-CSF + SCF, and 80% of mice receiving CTx + G-CSF alone produced at least three litters and 20% of mice in each group produced five litters.

Conclusion(s)

Treatment of mice with G-CSF decreases chemotherapy-induced ovarian follicle loss and extends time to premature ovarian insufficiency in female mice. Further studies are needed to validate these preclinical results in humans and compare efficacy with the established GnRH analogue treatments.

Minimally Invasive Karyotyping (MINK) was communicated in 2009 as a novel method for the non-invasive detection of fetal copy number anomalies in maternal plasma DNA. The original manuscript illustrated the potential of MINK using a model system in which fragmented genomic DNA obtained from a trisomy 21 male individual was mixed with that of his karyotypically normal mother at dilutions representing fetal fractions found in maternal plasma. Although it has been previously shown that MINK is able to non-invasively detect fetal microdeletions, its utility for aneuploidy detection in maternal plasma has not previously been demonstrated. The current study illustrates the ability of MINK to detect common aneuploidy in early gestation, compares its performance to other published third party methods (and related software packages) for prenatal aneuploidy detection and evaluates the performance of these methods across a range of sequencing read inputs. Plasma samples were obtained from 416 pregnant women between gestational weeks 8.1 and 34.4. Shotgun DNA sequencing was performed and data analyzed using MINK RAPIDR and WISECONDOR. MINK performed with greater accuracy than RAPIDR and WISECONDOR, correctly identifying 60 out of 61 true trisomy cases, and reporting only one false positive in 355 normal pregnancies. Significantly, MINK achieved accurate detection of trisomy 21 using just 2 million aligned input reads, whereas WISECONDOR required 6 million reads and RAPIDR did not achieve complete accuracy at any read input tested. In conclusion, we demonstrate that MINK provides an analysis pipeline for the detection of fetal aneuploidy in samples of maternal plasma DNA.