Methods
This retrospective study was based on national records from the Israeli
Ministry of Health (MOH). We searched the electronic database of the MOH
for CMA tests performed from January 2016 to March 2018 for the
indication of short fetal long bones, below the 5thpercentile for gestational age. All testing was financed by the MOH
after approval by a clinical geneticist.
The study was approved by the Institutional Review Board for Human
Subjects (September 6, 2016, registration number – MOH2016).
Clinical data were obtained from the Israeli national database and from
patients’ medical records. Detailed clinical information including
maternal age, maternal chronic illness, familial background of genetic
conditions, obstetrical history of recurrent spontaneous abortions,
elevated nuchal translucency, biochemical screening results, gestational
age at diagnosis, lowest bone length percentile recorded during the
pregnancy, and the presence of additional sonographic findings were
retrieved.
CMA findings were reviewed independently by two authors (R.S.H. and
I.M.) and grouped independently into four categories:
- Normal (including benign and variants of unknown significance - likely
benign categories),
- Pathogenic (P)/likely pathogenic (LP) variants,
- Microdeletion/duplication with low penetrance, and
- Variants of unknown clinical significance (VUS).
The categorization was based on laboratory reports, as well as on new
information gained from the medical literature and from the authors’
experience.
For VUS, only cases with deletions ≤1 Mb and duplications ≥2 Mb were
included. These variants are reported by the lab, according to the
guidelines determined by Israeli Society of Medical Geneticists.
Microarray results were also categorized into ”karyotype-detectable”
(i.e., copy number variants of at least 10 MB) or not
“karyotype-detectable” in order to assess the incremental yield of CMA
over karyotype.
Two cohorts were used to assess the background risk.
- A large local cohort of 5,541
cases with normal prenatal ultrasounds in a large, hospital-based
clinical laboratory.13 The detection rate for this
cohort was 1.4% (78 cases).
- A second cohort of 10,614 cases was extrapolated from a meta-analysis
by Srebniak et al.14 We calculated the background
risk by adding the risk for submicroscopic chromosomal abnormalities
to the risk for karyotype detectable chromosomal
abnormalities.15 This risk was 1:384, based on the
average maternal age of our cohort. The detection rate for this cohort
was 1.1%.
Various platforms were used by 12 laboratories to perform CMA. Most
medical center laboratories in Israel use the CytoScan 750K array, which
is composed of 550,000 nonpolymorphic copy number variant probes and
more than 200,000 single-nucleotide polymorphism probes, with an average
resolution of 100 Kb.16 One laboratory uses Infinium
OmniExpress-24 v1.2 BeadChip, which includes 713,599 genome-wide markers
at an average spacing of 4,080 bases and has a targeting minor allele
frequency of 5%,17 and one laboratory performs
microarray analysis using a Cytochip ISCA 8360K format,
BlueGnome.18 Two additional centers (one previously
working with the BlueGnome platform and one using BlueGnome and then
Illumina) switched to Affymetrix in 2017. One laboratory uses GenetiSure
Unrestricted CGH + SNP (43180K) P/N G5976A Agilent. Genomic coordinates
were evaluated in accordance with genome build GRCh37/hg19 in all
laboratories. All analyses performed in the laboratories met the
standards and guidelines of the American College of Genetics and
Genomics for constitutional cytogenomic microarray analysis, including
postnatal and prenatal applications,13, 15 adopted by
the recommendations of the Israeli Medical Genetics
Association.16