Use of cell-free non-invasive prenatal testing in pregnancies affected by placental mosaicism.

OBJECTIVE: To evaluate cell-free non-invasive prenatal testing (cfNIPT) in pregnancies affected by mosaicism. METHOD: We assessed paired cfNIPT and chorionic villus sample (CVS) results from the same pregnancies in a case series of mosaicism detected in Central and North Denmark Regions from April 2014 to September 2018. Indications for the clinically obtained CVS, pregnancy markers and outcome were retrieved from The Danish Fetal Medicine Database. RESULTS: Mosaicisms in CVS involved common aneuploidy, n = 14; sex chromosomal aneuploidies, n = 14; rare autosomal trisomies (RATs), n = 16 and copy number variants (CNVs) >5Mb, n = 9. Overall, 24/53 (45.3%; CI 95%: 31.8%-59.4%) of cases with mosaicism were detected by cfNIPT; highest for RATs (56%) and lowest for CNVs (22%). CfNIPT more commonly detected high-level than low-level mosaic cases (p = 0.000). CfNIPT detected 7/16 (43.8%; CI 95%: 21%-69%) clinically significant mosaic cases, either true fetal mosaicism or confined placental mosaicisms with adverse pregnancy outcome. There was a trend toward a higher risk for adverse outcome in pregnancies where mosaicism was detected by cfNIPT compared to pregnancies where mosaicism was not detected by cfNIPT (p = 0.31). CONCLUSION: CfNIPT has a low detection rate of mosaicism, including pregnancies with clinically significant mosaicism. However, abnormal cfNIPT results may be a predictor of adverse pregnancy outcomes.

A decade of change - lessons learned from prenatal diagnostics in Central Denmark region in 2008-2018.

INTRODUCTION: In 2011, it was decided to implement chromosomal microarray in prenatal testing in the Central Denmark Region, mainly due to the expected higher diagnostic yield. Chromosomal microarray was introduced gradually for an increasing number of pregnancies and without a transition period where both karyotyping and chromosomal microarray were performed: first malformations (2011), then large nuchal translucency (2013), then high risk at combined first trimester risk screening (2016) and finally for all indications (2018). This retrospective study summarizes 11 years of using chromosomal microarray in invasive prenatal testing and presents the effect on diagnostic yield and turnaround time. Furthermore, the concerns when introducing chromosomal microarray are presented and discussed. MATERIAL AND METHODS: Registry data from the Danish Fetal Medicine Database, the regional fetal medicine database, the Danish Cytogenetic Central Register and the local laboratory database at Department of Clinical Genetics were all combined, and a cohort of 147 158 singleton pregnancies with at least one ultrasound examination was established RESULTS: Of the 147 158 pregnancies, invasive sampling was performed (chorionic villi or amniocytes) in 8456, corresponding to an overall invasive rate of 5.8%. Between 2016 and 2018, 3.4% (95% confidence interval [CI] 2.8-4.2%; n = 86) of the invasive samples (n = 2533) had a disease causing copy number variant and 5.3% (95% CI 4.4-6.2%; n = 133) had trisomies and other aneuploidies. The turnaround time more than halved from 14 days to an average of 5.5 days for chorionic villus sampling. CONCLUSIONS: Chromosomal microarray identified 5.3% trisomies and 3.4% copy number variants, thereby increased the diagnostic yield by more than 64% compared with karyotype only and it also more than halved the turnaround time. Some preliminary concerns proved real, eg prenatal counseling complexity, but these have been resolved over time in a clinical path with expert consultations.

National data on the early clinical use of non-invasive prenatal testing in public and private healthcare in Denmark 2013-2017.

INTRODUCTION: In Denmark, non-invasive prenatal testing (NIPT) has been used since 2013. We aimed to evaluate the early clinical use of NIPT in Danish public and private healthcare settings before NIPT became an integrated part of the national guidelines on prenatal screening and diagnosis in 2017. MATERIAL AND METHODS: NIPT data were collected between March 2013 and June 2017 from national public registries and private providers. Results from follow-up samples (chorionic villi, amniotic fluid, postnatal blood or fetal tissue) were included from The Danish Cytogenetics Central Registry and indications and outcome from The Danish Fetal Medicine Database. RESULTS: A total of 3936 NIPT results were included in the study from public hospitals (n = 3463, 88.0%) and private clinics (n = 473, 12.0%). The total number of prenatal tests was 19 713 during the study period: 20% were NIPT analyses (n = 3936) and 80% invasive procedures (n = 15 777). Twenty-five percent of NIPTs in the private clinics were performed before gestational week 11+0 , whereas NIPT in public settings was used only after combined first trimester screening (P < .001). Regardless of indication, the national public sensitivity was 96.9% (95% CI 82.0%-99.8%) for trisomy 21, 100% (95% CI 46.3%-100%) for trisomy 18, 100% (95% CI 5.5%-100%) for trisomy 13, and 87.0% (95% CI 74.5%-92.4%) for any fetal chromosomal aberration. Forty-seven true-positive NIPT results included cases of common aneuplodies (trisomy 21, n = 31; trisomy 18, n = 5; and trisomy 13, n = 1), sex chromosomal aberrations (n = 7) and atypical chromosomal aberrations (n = 3). One false-negative NIPT result occurred (trisomy 21). Of 47 cases, 21 (45%) cases with a true-positive NIPT result resulted in live births by choice; 11 of these children had Down and 4 had Edwards syndrome. CONCLUSIONS: The total number of NIPT analyses was low compared with the number of invasive procedures in the implementation period. In contrast to the generally high termination rate after a positive result following invasive testing in Denmark, a high proportion of true-positive NIPT results from the public setting resulted in live births. NIPT may be an important risk-free alternative to invasive testing for a minority of women in the public setting who wish to use prenatal genetic testing for information only and not for reproductive decision-making.

Fetal Heart Defects and Measures of Cerebral Size.

OBJECTIVES: To estimate the association between fetal congenital heart defects (CHDs) and measures of brain size throughout pregnancy, from the end of the first trimester to birth. STUDY DESIGN: The cohort consisted of all fetuses scanned in Western Denmark in 2012 and 2013. Anthropometric measures in fetuses with isolated CHDs diagnosed within 12 months after birth were compared with those in the fetuses without CHDs. Z-scores standardized to gestational age were calculated for first trimester biparietal diameter, second trimester head circumference, fetal weight, birthweight, head circumference, and placental weight. RESULTS: We obtained data from 63 349 pregnancies and identified 295 fetuses with isolated CHDs (major n = 145; minor n = 150). The first trimester mean biparietal diameter Z-scores were not different between those with and those without CHDs. The head circumference mean Z-score difference was -0.13 (95% CI, -0.24 to -0.01; P = .03) in the second trimester and -0.22 (95% CI, -0.35 to -0.09; P < .001) at birth. Fetuses with univentricular physiology or tetralogy of Fallot showed the most pronounced compromise in cerebral size. CONCLUSIONS: Our results suggest that the brain alterations inducing an increased risk of impaired neurodevelopment in children with CHDs begin during pregnancy. Although fetuses with univentricular physiology or tetralogy of Fallot exhibited the most pronounced compromise in cerebral size, we recommend neurodevelopmental follow-up for all children with CHDs.

Ocular albinism with infertility and late-onset sensorineural hearing loss.

Ocular albinism type 1 (OA1) is caused by mutations in the GPR143 gene located at Xp22.2. The manifestations, which are due to hypopigmentation, are confined to the eyes and optic pathway. OA1 associated with late-onset sensorineural hearing loss was previously reported in a single family and hypothesized to be caused by a contiguous gene deletion syndrome involving GPR143 and the adjacent gene, TBL1X. Here, we report on a family with OA1, infertility, late-onset sensorineural hearing loss, and a small interstitial Xp microdeletion including the GPR143, TBL1X, and SHROOM2 genes. In addition, we re-examined a patient previously described with OA1, infertility and a similar Xp deletion with audiologic follow-up showing a late-onset sensorineural hearing loss. Our results raise an intriguing question about the possibility for TBL1X (absence) involvement in this type of hearing loss. However, our study cannot claim a causative relationship and more convincing evidence is needed before the hypothesis can be accepted that TBL1X could be involved in late-onset sensorineural hearing loss and that ocular albinism with late-onset sensorineural hearing loss can present itself as a contiguous gene deletion/microdeletion syndrome. The finding of infertility in all affected male patients demonstrates that this deletion, including the SHROOM2 gene, may be a potentially causative X-linked genetic factor of male infertility.

On the road to replacing invasive testing with cell-based NIPT: Five clinical cases with aneuploidies, microduplication, unbalanced structural rearrangement, or mosaicism.

OBJECTIVE: Trophoblastic fetal cells harvested from maternal blood have the capacity to be used for copy number analyses in a cell-based non-invasive prenatal test (cbNIPT). Potentially, this will result in increased resolution for detection of subchromosomal aberrations due to high quality DNA not intermixed with maternal DNA. We present 5 selected clinical cases from first trimester pregnancies where cbNIPT was used to demonstrate a wide range of clinically relevant aberrations. METHOD: Blood samples were collected from high risk pregnancies in gestational week 12 + 1 to 12 + 5. Fetal trophoblast cells were enriched and stained using fetal cell specific antibodies. The enriched cell fraction was scanned, and fetal cells were picked using a capillary-based cell picking instrument. Subsequently, whole genome amplification (WGA) was performed on fetal cells, and the DNA was analyzed blindly by array comparative genomic hybridization (aCGH). RESULTS: We present 5 cases where non-invasive cell-based prenatal test results are compared with aCGH results on chorionic villus samples (CVS), demonstrating aneuploidies including mosaicism, unbalanced translocations, subchromosomal deletions, or duplications. CONCLUSION: Aneuploidy and subchromosomal aberrations can be detected using fetal cells harvested from maternal blood. The method has the future potential of being offered as a cell-based NIPT with large high genomic resolution.

Comprehensive Evaluation of TFF3 Promoter Hypomethylation and Molecular Biomarker Potential for Prostate Cancer Diagnosis and Prognosis.

Overdiagnosis and overtreatment of clinically insignificant tumors remains a major problem in prostate cancer (PC) due to suboptimal diagnostic and prognostic tools. Thus, novel biomarkers are urgently needed. In this study, we investigated the biomarker potential of Trefoil factor 3 (TFF3) promoter methylation and RNA expression levels for PC. Initially, by quantitative methylation specific PCR (qMSP) analysis of a large radical prostatectomy (RP) cohort (n = 292), we found that the TFF3 promoter was significantly hypomethylated in PC compared to non-malignant (NM) prostate tissue samples (p < 0.001) with an AUC (area under the curve) of 0.908 by receiver operating characteristics (ROC) curve analysis. Moreover, significant TFF3 promoter hypomethylation (p ≤ 0.010) as well as overexpression (p < 0.001) was found in PC samples from another large independent patient sample set (498 PC vs. 67 NM) analyzed by Illumina 450K DNA methylation arrays and/or RNA sequencing. TFF3 promoter methylation and transcriptional expression levels were inversely correlated, suggesting that epigenetic mechanisms contribute to the regulation of gene activity. Furthermore, low TFF3 expression was significantly associated with high ERG, ETS transcription factor (ERG) expression (p < 0.001), as well as with high Gleason score (p < 0.001), advanced pathological T-stage (p < 0.001), and prostate-specific antigen (PSA) recurrence after RP (p = 0.013; univariate Cox regression analysis). There were no significant associations between TFF3 promoter methylation levels, ERG status, or PSA recurrence in these RP cohorts. In conclusion, our results demonstrated diagnostic biomarker potential of TFF3 promoter hypomethylation for PC as well as prognostic biomarker potential of TFF3 RNA expression. To the best of our knowledge, this is the most comprehensive study of TFF3 promoter methylation and transcriptional expression in PC to date.

17q12 deletion and duplication syndrome in Denmark-A clinical cohort of 38 patients and review of the literature.

17q12 deletions and duplications are two distinct, recurrent chromosomal aberrations usually diagnosed by chromosomal microarray analysis (CMA). The aberrations encompass the genes, HNF1B, LHX1, and ACACA, among others. We here describe a large national cohort of 12 phenotyped patients with 17q12 deletions and 26 phenotyped patients with 17q12 duplications. The total cohort includes 19 index patients and 19 family members. We also reviewed the literature in order to further improve the basis for the counseling. We emphasize that renal disease, learning disability, behavioral abnormalities, epilepsy, autism, schizophrenia, structural brain abnormalities, facial dysmorphism, and joint laxity are features seen in both the 17q12 deletion syndrome and the reciprocal 17q12 duplication syndrome; and we extend the list of features seen in both patient categories to include strabismus, esophageal defects, and duodenal atresia. Delayed language development, learning disability, kidney involvement, and eye dysmorphism and strabismus were the most consistently shared features among patients with 17q12 deletion. Patients with 17q12 duplications were characterized by an extremely wide phenotypic spectrum, including a variable degree of learning disabilities, delayed language development, delayed motor milestones, and a broad range of psychiatric and neurological features. This patient group also included adults achieving an academic degree. Assessing index patients and non-index patients separately, our observations illustrate that an overall milder disease burden is seen, in particular in patients with 17q12 duplications who are ascertained on the duplication rather than the phenotype. This evidence may be useful in prenatal counseling. © 2016 Wiley Periodicals, Inc.

Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women.

OBJECTIVE: Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. METHODS: We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. RESULTS: An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X). CONCLUSION: Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.

Absolute immature platelet count may predict imminent platelet recovery in thrombocytopenic children following chemotherapy.

BACKGROUND: Immature platelets are the youngest circulating platelets and they reflect the rate of thrombopoiesis. The immature platelet fraction (IPF) or the absolute immature platelet count (AIPC) may predict platelet count recovery following chemotherapy-induced thrombocytopenia in pediatric patients and help guiding prophylactic platelet transfusion therapy. PROCEDURE: To test IPF and AIPC as predictors of platelet recovery, 19 children with platelet nadirs of less than 20 × 10(9)/L after chemotherapy were prospectively enrolled. IPF, platelet count, and C-reactive protein (CRP) were analyzed in 416 paired samples from 37 patients with malignancies to test if IPF-levels were CRP dependent. RESULTS: A significant increase of 0.6 × 10(9)/L in AIPC was seen between 1 and 2 days prior to platelet count recovery. No predictive day-to-day differences were found for IPF. Platelet count recovery did not occur significantly earlier for patients with a peak IPF > 10% than for those patients with a peak IPF < 10%. IPF and AIPC showed no correlation to CRP. AIPC was in contrast to IPF not influenced by platelet transfusions. CONCLUSION: AIPC increased significantly between 24 and 48 hours before platelet recovery whereas IPF showed no significant increase during the same time period. AIPC may be a better indicator than IPF for predicting platelet recovery after chemotherapy in pediatric patients.

Prenatal diagnosis: array comparative genomic hybridization in fetuses with abnormal sonographic findings.

OBJECTIVE: To evaluate the clinical value of a high-resolution whole-genome array method for examination of genomic imbalances in prenatal samples (46 amniotic fluid, 17 chorionic villus, and 26 products of conception) from fetuses with abnormal ultrasound, in a clinical setting where more than 90% of pregnant women receive first-trimester combined screening and a second-trimester anomaly scan. DESIGN: Cross-sectional study. SETTING: Fetal medicine units (national healthcare system) in Central and North Denmark Regions from March 2009 to April 2012. SAMPLES: Eighty-nine samples obtained at 11.5-35.0 (mean 19.3) gestational weeks, either during ongoing pregnancy or after termination. METHODS: DNA was extracted directly from amniotic fluid cells and chorionic villus samples, or from cultured cells, and examined with 80-kb resolution oligonucleotide array-based comparative genomic hybridization (aCGH). MAIN OUTCOME MEASURES: Clinically significant copy number variations identified by aCGH. RESULTS: We detected clinically significant copy number variations in 11 fetuses (12%, confidence interval 6.0-19%) with structural malformations. Three fetuses (3.4%) had uncertain clinical significant variations and incidental findings. CONCLUSIONS: aCGH is a valuable diagnostic tool when fetal malformations are detected. More affected fetuses may be diagnosed at an earlier gestational age providing better possibilities for postnatal treatment and allowing for women to decide earlier on termination of pregnancy. When a normal result has reduced the risk of significant chromosomal aberration, aCGH may facilitate parental decision-making on whether to continue the pregnancy.

Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture.

The T(-13910) variant located in the enhancer element of the lactase (LCT) gene correlates perfectly with lactase persistence (LP) in Eurasian populations whereas the variant is almost nonexistent among Sub-Saharan African populations, showing high prevalence of LP. Here, we report identification of two new mutations among Saudis, also known for the high prevalence of LP. We confirmed the absence of the European T(-13910) and established two new mutations found as a compound allele: T/G(-13915) within the -13910 enhancer region and a synonymous SNP in the exon 17 of the MCM6 gene T/C(-3712), -3712 bp from the LCT gene. The compound allele is driven to a high prevalence among Middle East population(s). Our functional analyses in vitro showed that both SNPs of the compound allele, located 10 kb apart, are required for the enhancer effect, most probably mediated through the binding of the hepatic nuclear factor 1 alpha (HNF1 alpha). High selection coefficient (s) approximately 0.04 for LP phenotype was found for both T(-13910) and the compound allele. The European T(-13910) and the earlier identified East African G(-13907) LP allele share the same ancestral background and most likely the same history, probably related to the same cattle domestication event. In contrast, the compound Arab allele shows a different, highly divergent ancestral haplotype, suggesting that these two major global LP alleles have arisen independently, the latter perhaps in response to camel milk consumption. These results support the convergent evolution of the LP in diverse populations, most probably reflecting different histories of adaptation to milk culture.

Trefoil factor family peptides in human saliva and cyclical cervical mucus. Method evaluation and results on healthy individuals.

BACKGROUND: Trefoil peptides are 7-12 kDa molecules, se-creted by a variety of mucin-producing epithelial cells from different tissues and believed to be essential for protection and maintenance of gastrointestinal mucosa. Data on concentrations of trefoil peptides in secretions are limited. METHODS: We validated in-house ELISA assays, developed for measurement of trefoil peptide concentrations (TFF1, TFF2 and TFF3) in serum, for use with saliva and cervical mucus. Saliva from healthy individuals (n=30), and cervical mucus as well as blood collected three times during the menstrual cycle from healthy women (n=18) were analyzed. RESULTS: Recovery of all trefoil peptides in the initial supernatants of saliva and (cervical mucus) were 86 and (92)% or more. Recovery of exogenously added trefoil peptides was 93 and (95)% or more. Western blotting showed that antibodies used in the TFF3-ELISA assay recognised one molecule of the same size as TFF3 in both saliva and cervical mucus. Median concentrations of TFF1, TFF2 and TFF3 in saliva and (cervical mucus) were 2.7 (2.7), 0.08 (0.58) and 14 (430) nmol/g protein, with a significant decrease in concentrations in cervical mucus after ovulation. Serum concentrations resembled previously measured values in blood donors and showed no cyclic change. CONCLUSIONS: Previously established ELISA assays can be employed for measurement of trefoil peptides in saliva and cervical mucus. TFF3 was the predominant trefoil peptide in both saliva and cervical mucus, and TFF3 in cervical mucus represents the highest concentration measured in a biological fluid to date.

[Haemoglobin A1c measurements when to use and when not].

Haemoglobin A1c (HbA1c) reflects the glycaemic status of the latest 2-3 month and is used in both diagnosing and monitoring diabetes. Different circumstances may lead to spurious HbA1c results as summarised in this review. HbA1c is susceptible to changes in erythrocyte turnover (e.g. anaemia) regardless of measurement method, and to analytical interference (e.g. haemoglobin variants) depending on the method. The laboratory may detect and warn of suspected analytical interference. However, if the clinical presentation and glycaemic measures are incoherent, spurious HbA1c should be suspected and fasting glucose should be measured.

Promoter hypomethylation and upregulation of trefoil factors in prostate cancer.

Trefoil factors, mucin-associated peptides, are overexpressed in prostate cancer (PC). We hypothesized that promoter methylation contributes to the regulation of trefoil factors (TFF1, TFF2 and TFF3) in human prostate cells. Here we show hypomethylation of promoter regions of TFF1 and TFF3 in PC cell lines with significant TFF expression as compared to benign immortalized prostate cell lines and PC cell lines not expressing trefoil factor. The most striking difference was observed for CpG sites located close to the AUG start codon overlapping several putative binding sites for cellular transcription factors. TFF2 was hypermethylated and had no or very low expression in all prostate cell lines investigated. Treatment of methylated cell lines with 5-aza-2'-deoxycytidine restored TFF expression in cell lines not expressing TFF and increased expression significantly in low-expressing cell lines. In clinical samples, methylation of the promoter/enhancer regions of TFF1 and TFF3 was significantly lower in PC compared to benign prostatic hyperplasia. The present study shows an inverse relation between promoter methylation and expression of trefoil factors. Preliminary analysis on clinical samples suggests that this regulatory mechanism is responsible for the increased levels of TFF1 and TFF3 observed in PC. The overexpression and promoter hypomethylation of trefoil factors may serve as biomarkers in PC.

Placental mosaicism in the era of chromosomal microarrays.

OBJECTIVE: Placental mosaicism for a subset of a chromosome, a structural chromosomal aberration, is thought to be a very rare finding in chorionic villus samples. Here, we present clinical and laboratory data on five cases with such mosaicism for structural chromosomal aberrations. METHODS: During a period of 6 months, chromosomal microarray was carried out on DNA extracted from 100 uncultured chorion villous samples from high-risk pregnancies. RESULTS: In five of 100 consecutively collected samples (5/100), mosaicism for a structural chromosomal aberration was detected. The mosaic aberration was subsequently detected in fetal tissue in three of the five cases. CONCLUSION: Chromosomal microarray can detect placental mosaicism for structural chromosomal aberrations. This kind of mosaicism may be more frequent than previously anticipated, and the fetal involvement seems difficult to predict. These findings highlight the complexity of mosaicism for structural chromosomal aberrations in prenatal samples in the chromosomal microarray era.

Trefoil factors in human milk.

We measured concentrations of the gastrointestinal protective peptides Trefoil factors in human milk. By the use of in-house ELISA we detected high amounts of TFF3, less TFF1 and virtually no TFF2 in human breast milk obtained from 46 mothers with infants born extremely preterm (24-27 wk gestation), preterm (28-37 wk gestation), and full term (38-42 wk gestation). Samples were collected during the first, second, third to fourth weeks and more than 4 wks postpartum. Median (range) TFF1 [TFF3] concentrations in human milk were 320 (30-34000) [1500 (150-27,000)] pmol/L in wk 1, 120 (30-720) [310 (50-7100)] pmol/L in wk 2, 70 (20-670) [120 (20-650)] pmol/L in wks 3 to 4, and 60 (30-2500) [80 (20-540)] pmol/L in >4 wks after delivery. The lowest concentrations of TFF1 and TFF3 were found later than 2 wks after birth. In conclusion, TFF was present in term and preterm human milk with rapidly declining concentrations during the first weeks post partum. The clinical significance of TFF present in human milk remains to be explored, both regarding development of the fetal gut and protection against necrotizing enterocolitis.

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.

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents.

Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2-3h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.