Mark D. Pertile

Rapid, high throughput prenatal detection of aneuploidy using a novel quantitative method (MLPA)

Prenatal diagnosis of syndromes caused by chromosomal abnormality is a long established part of obstetric care in developed countries. In this area, there have been recent significant advances in the identification of high risk pregnancies using sophisticated serum analyte and ultrasound screening methods.1,2 For follow up diagnostic testing, karyotyping has provided the gold standard. This technology has remained essentially unchanged over 30 years, as no new technology has yet proven superior in terms of being able to detect such a wide range of abnormalities with the necessary precision. Nevertheless, molecular tests, such as fluorescent in situ hybridisation (FISH) 3 and short tandem repeat analysis,4 are now in common practice for the diagnosis of specific abnormalities. These adjunctive tests importantly decrease turnaround times from 1–2 weeks to 1–2 days. We assessed the performance of multiplex ligation dependent probe amplification (MLPA) as an alternative method for the detection of aneuploidy, which is by far the most common prenatal chromosome abnormality. This novel technique5 detects sequence dosage differences in a semi-quantitative manner and has many potential applications in diagnostic molecular genetics and cytogenetics. For example, a recent report describes its use for detection of large genomic deletions and duplications in the BRCA1 gene.6 This technology appears to have significant advantages in that it is extremely versatile in its applications, flexible in its target loci, highly automated, suitable for high throughput testing, efficient, and cost effective. Its application for aneuploidy detection has not been reported in a clinical setting. In order to assess the precision and robustness of the test, we conducted a prospective blind trial on 492 consecutive amniocentesis samples referred to our cytogenetics laboratory. ### Study design This study was designed to blind test amniocentesis samples prospectively. The samples were collected over a 4 month period as they were referred …

Pathogenic aberrations revealed exclusively by single nucleotide polymorphism (SNP) genotyping data in 5000 samples tested by molecular karyotyping

Background Several recent studies have demonstrated the use of single nucleotide polymorphism (SNP) arrays for the investigation of intellectual disability, developmental delay, autism or congenital abnormalities. In addition to LogR ‘copy number’ data, these arrays provide SNP genotyping data for gene level autozygosity mapping, estimating low levels of mosaicism, assessing long continuous stretches of homozygosity (LCSH), detection of uniparental disomy, and ‘autozygous’ regions. However, there remains little specific information on the clinical utility of this genotyping data. Methods Molecular karyotyping, using SNP array, was performed on 5000 clinical samples. Results Clinically significant ‘LogR neutral’ genotyping abnormalities were detected in 0.5% of cases. Among these were a single case of chimerism, 12 cases with low level chromosome mosaicism, and 11 cases with an LCSH associated with uniparental disomy. In addition, the genotyping data revealed several LCSH associated with clinically relevant ‘recessive type’ genetic defects. Conclusions These results demonstrate the utility of SNP genotyping data for detection of clinically significant abnormalities, including chimerism/mosaicism and recessive Mendelian disorders associated with autozygosity. The incidence of clinically significant low level mosaicism inferred from these cases suggests that this has hitherto been underestimated and chromosome mosaicism frequently occurs in the absence of indicative clinical features. The growing appreciation among clinicians and demand for SNP genotyping data poses significant challenges for the interpretation of LCSH, especially where there is no detailed phenotypic description to direct laboratory analysis. Finally, reporting of unexpected or hidden consanguinity revealed by SNP array analysis raises potential ethical and legal issues.

High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy.

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low‐resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation‐Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy‐number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty‐two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost‐effective, high‐throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.

Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity

The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother’s tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDHbr) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDHbr cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDHbr cells than CMSC. Finally, flow sorted ALDHbr cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments.

Prospective ranking of the sonographic markers for aneuploidy: Data of 2143 prenatal cytogenetic diagnoses referred for abnormalities on ultrasound

Objective: To design a scheme to rank sonographic anomalies as indicators of aneuploidy and record the distribution of data from 2143 prenatal amniotic fluid/chorionic villous sample diagnoses referred for karyotyping because of fetal anomalies detected with ultrasound.

Rare autosomal trisomies, revealed by maternal plasma DNA sequencing, suggest increased risk of feto-placental disease

Maternal plasma cell-free DNA sequencing reveals rare autosomal trisomies that are associated with perinatal complications. A complete look at fetal chromosomes Genetic analysis of fetal DNA in maternal blood is becoming increasingly common, but the standard clinical tests typically consider only the chromosomes that are most frequently found to be aneuploid: 13, 18, 21, X, and Y. Pertile et al. analyzed patient data from two clinical laboratories and discovered that this approach may be ignoring valuable information. In both cohorts, the authors found a number of rare autosomal trisomies that are not reported on routine testing and showed that these are associated with an increased risk of pregnancy complications, indicating their potential relevance for clinical care. Whole-genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta, fetus, or pregnant woman. Current bioinformatics algorithms typically only report on chromosomes 21, 18, 13, X, and Y; sequencing results from other chromosomes may be masked. We hypothesized that by systematically analyzing WGS data from all chromosomes, we could identify rare autosomal trisomies (RATs) to improve understanding of feto-placental biology. We analyzed two independent cohorts from clinical laboratories, both of which used a similar quality control parameter, normalized chromosome denominator quality. The entire data set included 89,817 samples. Samples flagged for analysis and classified as abnormal were 328 of 72,932 (0.45%) and 71 of 16,885 (0.42%) in cohorts 1 and 2, respectively. Clinical outcome data were available for 57 of 71 (80%) of abnormal cases in cohort 2. Visual analysis of WGS data demonstrated RATs, copy number variants, and extensive genome-wide imbalances. Trisomies 7, 15, 16, and 22 were the most frequently observed RATs in both cohorts. Cytogenetic or pregnancy outcome data were available in 52 of 60 (87%) of cases with RATs in cohort 2. Cases with RATs detected were associated with miscarriage, true fetal mosaicism, and confirmed or suspected uniparental disomy. Comparing the trisomic fraction with the fetal fraction allowed estimation of possible mosaicism. Analysis and reporting of aneuploidies in all chromosomes can clarify cases in which cfDNA findings on selected “target” chromosomes (21, 18, and 13) are discordant with the fetal karyotype and may identify pregnancies at risk of miscarriage and other complications.

Questionable pathogenicity of FOXG1 duplication

Chromosome microarray analysis has revolutionised the diagnosis of patients with neurocognitive impairment and has resulted in the association of many phenotypes with copy number changes in particular genes.

Trisomy 13 mosaicism at prenatal diagnosis: dilemmas in interpretation

We describe six cases of trisomy 13 mosaicism detected at prenatal diagnosis. Most level I and level II trisomy 13 mosaicism detected at prenatal diagnosis is pseudomosaicism or confined placental mosaicism. Rarely, low‐level mosaicism at chorionic villus sampling or amniocentesis reflects a true fetal mosaicism. In this case, a normal phenotype is a possible, but not a certain, outcome. Genetic counselling is not straightforward.

Maternal mosaicism for a large segmental duplication of 18q as a secondary finding following non‐invasive prenatal testing and implications for test accuracy

Non-invasive prenatal testing (NIPT) for fetal aneuploidies by massively parallel sequencing has transformed the approach to prenatal care. The presence of circulating fetal cell-free DNA (cfDNA) in maternal plasma, which is derived primarily from the placenta, is the basis of this test. Studies comparing NIPT to serum or combined screening have shown that NIPT outperforms these screening options in trisomy detection and has significantly lower false positive rates. Biological causes of false positive NIPT results may include maternal mosaicism (primarily sex chromosome mosaicism), confined placental mosaicism, co-twin demise, maternal malignancy and maternal copy number variation. Here, we present a case of a nonstandard NIPT result involving a large and pathogenic partial duplication of chromosome 18q first indicated by NIPT. Subsequent prenatal diagnosis confirmed the presence of the duplication in the fetus, which was inherited from the phenotypically normal mother who carried the same chromosomal imbalance, but in mosaic form. A review of the NIPT data indicates that maternal mosaicism for this large segmental imbalance is sufficient to cause a false positive NIPT result in subsequent pregnancies, in the absence of the imbalance being transmitted to the fetus. Therefore, maternal mosaicism for large segmental copy number abnormalities may be yet another rare cause of false positive NIPT results, which further highlights the need for confirmatory invasive testing whenever an abnormal NIPT result is returned. The patient was a 38-year-old G3P2 woman who underwent first trimester combined screening. Risks for trisomy 18 and trisomy 21 were calculated at 1:13,100 and 1:1,690, respectively. No further investigations for Down syndrome or trisomy 18 were recommended on the basis of these low risk results. A fetal anomaly ultrasound scan at 20weeks of gestation identified an isolated aberrant right subclavian artery. The sonologist counselled a low risk for chromosome abnormality but informed the patient of the option of NIPT and amniocentesis. The patient was not referred for formal genetic counselling and pursued NIPT independently. Maternal venous blood was collected for NIPT at 20 + 6weeks of gestation, and testing was performed by Natera Panorama test, which is a targeted, multiplexed, single nucleotide polymorphism (SNP)-based NIPT assay. The NIPT returned an unexpected finding with no result for chromosome 18 because of an ‘atypical finding outside the current scope of the test’. The atypical finding was a suspicion of a partial duplication of chromosome 18. To help direct follow-up testing, a request was made for genomic or cytogenetic breakpoint information but was declined on the basis that the information being sought was outside the current scope of the test. Genetic counselling was arranged, and the patient elected amniocentesis at 22weeks of gestation, at which time a small ventricular septal defect on ultrasound was also noted. As no positional information for the suspected duplication was available, a rapid interphase fluorescence in situ hybridization test, microarray analysis and a conventional karyotype were requested. Fluorescence in situ hybridization testing for chromosomes 13, 18, 21, X and Y using a Vysis AneuVysion probe set (Abbott Molecular) was consistent with a male fetus with two copies of the chromosome 18 centromere. Chromosome microarray on DNA extracted from uncultured amniotic fluid cells using an Affymetrix CytoScan 750K array (Affymetrix, Santa Clara, CA) revealed a 16.1Mb duplication from the chromosome 18 long arm, region q12.1 to q21.1 (Figure 1), with molecular karyotype arr[hg19] 18q12.1q21.1(29,524,180-45,659,732)x3. Conventional G-banded chromosome analysis on cultured amniotic fluid

Monosomy 21 Seen in Live Born Is Unlikely to Represent True Monosomy 21: A Case Report and Review of the Literature

We report a case of a neonate who was shown with routine chromosome analysis on peripheral blood lymphocytes to have full monosomy 21. Further investigation on fibroblast cells using conventional chromosome and FISH analysis revealed two additional mosaic cell lines; one is containing a ring chromosome 21 and the other a double ring chromosome 21. In addition, chromosome microarray analysis (CMA) on fibroblasts showed a mosaic duplication of chromosome region 21q11.2q22.13 with approximately 45% of cells showing three copies of the proximal long arm segment, consistent with the presence of a mosaic ring chromosome 21 with ring instability. The CMA also showed complete monosomy for an 8.8 Mb terminal segment (21q22.13q22.3). Whilst this patient had a provisional clinical diagnosis of trisomy 21, the patient also had phenotypic features consistent with monosomy 21, such as prominent epicanthic folds, broad nasal bridge, anteverted nares, simple ears, and bilateral overlapping fifth fingers, features which can also be present in individuals with Down syndrome. The patient died at 4.5 months of age. This case highlights the need for additional studies using multiple tissue types and molecular testing methodologies in patients provisionally diagnosed with monosomy 21, in particular if detected in the neonatal period.