Numbers of prenatal cell-free DNA screens performed: Results of a 2022 CAP exercise.

OBJECTIVE: Determine current analytical methods and number of cell-free (cf) DNA prenatal screening tests performed for common trisomies. METHODS: The College of American Pathologists 2022-B Noninvasive Prenatal Testing exercise was distributed in December 2022 to 93 participants in 22 countries. Supplemental questions included the number of tests performed in a recent month and the proportion of samples originating outside the United States (US). RESULTS: Eighty-three participants from three continents returned results; 74 (89%) were suitable for the analyses. Nine manufacturer/platform combinations were identified, most commonly Illumina/Nextseq (55%). The most common methodology was whole genome sequencing (76%). Annualized cfDNA tests were 2.80 million, with Asian, European and North American participants representing 10.6%, 6.5% and 82.9% of tests, respectively. When restricted to US in-country tests, the annualized rate was 2.18 million, with four of 20 participants testing 79.2%. Among 73 respondents, 63 (86%) were for-profit, eight (11%) were non-profit academic or government supported and the remaining two included hospital-based and private non-profit. Eighteen (25%) supported relevant academic training. CONCLUSION: In 2011, screening for common trisomies was based on serum/ultrasound markers with an estimated 2.96 million US pregnancies screened in 131 laboratories. In 2022, cfDNA-based screening was offered by 20 laboratories testing 2.18 million US pregnancies.

Extending the phenotypes associated with TRIO gene variants in a cohort of 25 patients and review of the literature.

The TRIO gene encodes a rho guanine exchange factor, the function of which is to exchange GDP to GTP, and hence to activate Rho GTPases, and has been described to impact neurodevelopment. Specific genotype-to-phenotype correlations have been established previously describing striking differentiating features seen in variants located in specific domains of the TRIO gene that are associated with opposite effects on RAC1 activity. Currently, 32 cases with a TRIO gene alteration have been published in the medical literature. Here, we report an additional 25, previously unreported individuals who possess heterozygous TRIO variants and we review the literature. In addition, functional studies were performed on the c.4394A > G (N1465S) and c.6244-2A > G TRIO variants to provide evidence for their pathogenicity. Variants reported by the current study include missense variants, truncating nonsense variants, and an intragenic deletion. Clinical features were previously described and included developmental delay, learning difficulties, microcephaly, macrocephaly, seizures, behavioral issues (aggression, stereotypies), skeletal problems including short, tapering fingers and scoliosis, dental problems (overcrowding/delayed eruption), and variable facial features. Here, we report clinical features that have not been described previously, including specific structural brain malformations such as abnormalities of the corpus callosum and ventriculomegaly, additional psychological and dental issues along with a more recognizable facial gestalt linked to the specific domains of the TRIO gene and the effect of the variant upon the function of the encoded protein. This current study further strengthens the genotype-to-phenotype correlation that was previously established and extends the range of phenotypes to include structural brain abnormalities, additional skeletal, dental, and psychiatric issues.

Differential light scattering and the measurement of molecules and nanoparticles: A review.

Within the past few decades, the application of light scattering techniques to a broad range of scientific disciplines has increased significantly, especially in the field of analytical chemistry. The resulting interest in and use of light scattering methods suggests the need for an easily understood introduction and review of material for those new to the method as well as for current users in need of a refresher. In many respects, the theory and its applications may appear so overwhelming for many studying the field for the first time, that they rarely can spend the time just needed to understand the basic measurements and their interpretations. A variety of applications in analytical chemistry especially have resulted in a greater understanding of many of the macromolecular processes themselves from molar mass distributions, to the macromolecular interactions responsible for aggregation processes, to determinations of structure and function. The use of such analytical processes to obtain a better understanding of nanoparticle structure and function has become almost universal.

Prenatal serum screening for Down syndrome and neural tube defects in the United States: Changes in utilization patterns from 2012 to 2020.

OBJECTIVE: To compile current usage of serum-based prenatal screening for Down syndrome in the United States and compare it with results from a similar 2011/2012 survey. SETTING: The College of American Pathologists maternal screening proficiency testing survey includes a supplemental question on the first of three yearly distributions. METHODS: Information regarding tests offered and the monthly number of pregnancies tested for US-based laboratories were reviewed. Results were stratified by size of laboratory, tests offered, and pregnancies tested. Findings were compared to an earlier survey. RESULTS: Fifty-six laboratories reported they will have screened 1,131,336 pregnancies in 2020. Of these, 36% are screened by stand-alone first trimester testing, 48% by stand-alone second trimester testing, and 16% using tests that integrate results from both trimesters. Eighty percent of all serum screens were provided by the five laboratories that performed the most screens (at least 50,000). These five performed similar proportions of first or second trimester screens (42.2% and 41.8%, respectively). Compared to eight years earlier, there are now 54% fewer laboratories. Pregnancies screened using the first trimester, second trimester, and integrated protocols were lower by 27%, 69%, and 72%, respectively. The serum screening activity in the US showed a 62% decrease from 2012 levels. During 2012-2020, the number of cell-free DNA tests increased from negligible to 1,492,332. CONCLUSIONS: Maternal serum screening for common aneuploidies has changed significantly in eight years with fewer laboratories, a shift toward larger laboratories and a 2.5-fold reduction in pregnancies tested, likely due to the introduction of cell-free DNA screening.

Cell-free DNA screening in twin pregnancies: A more accurate and reliable screening tool.

OBJECTIVE: Outcome data from cell-free DNA (cfDNA) screening in twin gestations are limited. This study adds an appreciable number of confirmed outcomes to the literature, and assesses performance of cfDNA screening in twins over a 4.5-year period at one large clinical laboratory. METHOD: Prenatal cytogenetic and SNP microarray results were cross-referenced with cfDNA results for twin pregnancies, yielding 422 matched cases. Using diagnostic results as truth, performance of cfDNA screening in this population was assessed. RESULTS: Of the 422 twin pregnancies with both cfDNA and diagnostic results, 3 specimens failed amniocyte analysis, and 48 samples (11.5%) were nonreportable from the initial cfDNA draw. Analysis of the 371 reportable samples demonstrated a collective sensitivity of 98.7% and specificity of 93.2% for trisomies 21/18/13. Positive predictive values (PPVs) in this study population, which is enriched for aneuploidy, were 78.7%, 84.6%, and 66.7% for trisomy 21, 18, and 13, respectively. CONCLUSION: CfDNA screening in a cohort of twin pregnancies with matched diagnostic results showed superior performance compared to traditional serum biochemical screening in twins. This study adds to a growing body of evidence suggesting that cfDNA is an accurate and reliable screening tool for the major trisomies in twin pregnancies.

Assessment of laboratories offering cell-free (cf) DNA screening for Down syndrome: results of the 2018 College of American Pathology External Educational Exercises.

PURPOSE: Summarize and interpret results from exercises distributed to laboratories offering cell-free (cf) DNA screening for Down syndrome. METHODS: The College of American Pathologists distributed three patient-derived plasma specimens twice in 2018. Sequencing platforms, test methods, results, and responses to supplemental questions were collected. Results were not graded but discrepancies were identified. RESULTS: Sixty-five laboratories from six continents enrolled; six provided no results. The most common methodology was shotgun/genome sequencing (39/56, 70%). Overall, 40% of the gestational or maternal age responses were incorrect but 45% of the errors were corrected by the next distribution. Fetal fractions from 54 responding laboratories generally agreed with the intended response. No genotyping errors occurred (40/40 for trisomy 21 and 226/226 for euploid challenges) but 10 additional tests failed (3.6%). All 213 fetal sex calls were correct. Participants reported their clinical text for a Down syndrome screen positive test; 39% were classified as inadequate or misleading. CONCLUSION: Patient-derived materials are suitable for all enrolled technologies/methodologies, but collecting material is challenging. Suggested clinical text includes the terms "screen positive" and "screen negative." Overall, laboratories performed well. Future efforts will focus on potential manufactured samples, clarifying results reporting and including additional chromosome abnormalities.

Measuring nanoparticles in the size range to 2000 nm.

Measurement of light scattered from suspensions of monodisperse nanoparticles in solution ("turbidity") long has been used to derive their size. Following some means of fractionation, the light (monochromatic) scattered by the particles into a set of distinct angles is collected and a non-linear least squares fit was made to an appropriate theory in order to extract their size. For a wide range of particle structures, where this process becomes very complex and of questionable validity, there is a far simpler interpretive means based upon measurements at extremely small, and often inaccessible, scattering angles. A method is described whereby the required small angle values are derived from measurements made over a range of larger, more readily accessible, angles. Although the basis for the analyses developed is the Rayleigh-Gans approximation, the results presented confirm that the method provides meaningful results up to a size of about 2000 nm. The larger sizes are well beyond the RG limits.

Measuring proteins with greater speed and resolution while reducing sample size.

A multi-angle light scattering (MALS) system, combined with chromatographic separation, directly measures the absolute molar mass, size and concentration of the eluate species. The measurement of these crucial properties in solution is essential in basic macromolecular characterization and all research and production stages of bio-therapeutic products. We developed a new MALS methodology that has overcome the long-standing, stubborn barrier to microliter-scale peak volumes and achieved the highest resolution and signal-to-noise performance of any MALS measurement. The novel design simultaneously facilitates online dynamic light scattering (DLS) measurements. As National Institute of Standards and Technology (NIST) new protein standard reference material (SRM 8671) is becoming the benchmark molecule against which many biomolecular analytical techniques are assessed and evaluated, we present its measurement results as a demonstration of the unique capability of our system to swiftly resolve and measure sharp (20~25 µL full-width-half-maximum) chromatography peaks. Precise measurements of protein mass and size can be accomplished 10 times faster than before with improved resolution. In the meantime the sample amount required for such measurements is reduced commensurately. These abilities will have far-reaching impacts at every stage of the development and production of biologics and bio-therapeutic formulations.

Measurement of special nanoparticle structures by light scattering.

The characterization of nanoparticle size and structure by means of classical light scattering measurements from monodisperse suspensions is examined from both the Rayleigh-Gans (R-G) approximation as well as (for various spherical structures) the exact Lorenz-Mie theory. A means by which the traditional limits of the R-G theory may be extended and simplified is shown by a detailed discussion of the characteristic mean-square radius. This becomes particularly important for irregular particle shapes, where scattering depends on the orientation of such particles with respect to the direction of the incident illumination. A variety of particle structures are addressed, including rods, tubes, ellipsoids, rings, and superellipsoids.

Investigation of NRXN1 deletions: clinical and molecular characterization.

Deletions at 2p16.3 involving exons of NRXN1 are associated with susceptibility for autism and schizophrenia, and similar deletions have been identified in individuals with developmental delay and dysmorphic features. We have identified 34 probands with exonic NRXN1 deletions following referral for clinical microarray-based comparative genomic hybridization. To more firmly establish the full phenotypic spectrum associated with exonic NRXN1 deletions, we report the clinical features of 27 individuals with NRXN1 deletions, who represent 23 of these 34 families. The frequency of exonic NRXN1 deletions among our postnatally diagnosed patients (0.11%) is significantly higher than the frequency among reported controls (0.02%; P = 6.08 × 10(-7) ), supporting a role for these deletions in the development of abnormal phenotypes. Generally, most individuals with NRXN1 exonic deletions have developmental delay (particularly speech), abnormal behaviors, and mild dysmorphic features. In our cohort, autism spectrum disorders were diagnosed in 43% (10/23), and 16% (4/25) had epilepsy. The presence of NRXN1 deletions in normal parents and siblings suggests reduced penetrance and/or variable expressivity, which may be influenced by genetic, environmental, and/or stochastic factors. The pathogenicity of these deletions may also be affected by the location of the deletion within the gene. Counseling should appropriately represent this spectrum of possibilities when discussing recurrence risks or expectations for a child found to have a deletion in NRXN1.

Effect of mild hepatic or renal impairment on maternal serum screening biochemical measures.

BACKGROUND: Integrated maternal serum screening (MSS) is commonly used to screen for fetal trisomies and neural tube defects in early pregnancy. The kidney and liver each play an important role in hormone metabolism, and anecdotal data suggest that MSS biochemical measures may vary with a mother's health status. We examined the correlations between kidney and liver function parameters and MSS markers and the possible association of mild renal or hepatic impairment with MSS measures. METHODS: We completed a prospective cross-sectional study of 257 consecutive women who underwent integrated MSS at a single hospital. Serum analytes (pregnancy associated plasma protein A [PAPP-A], hCG, creatinine [Cr], and alanine aminotransferase [ALT]) were drawn at approximately 12 weeks' gestation, and alpha-fetoprotein and unconjugated estriol were drawn at 16 weeks' gestation. Creatinine clearance was calculated using the Cockcroft-Gault formula. Abnormally elevated serum Cr and ALT were each defined as ≥ 90th percentile among all women. A low creatinine clearance (CrCl) was set at ≤ 10th percentile. RESULTS: Serum hCG, PAPP-A, and alpha-fetoprotein were negatively correlated with CrCl, but not after correction for maternal age, weight, and ethnicity. No association between MSS and serum ALT was observed. The median serum concentrations of both PAPP-A (P = 0.04) and alpha-fetoprotein (P = 0.02) were significantly higher among those whose CrCl was ≤ 10th percentile. At the more extreme concentrations of PAPP-A and alpha-fetoprotein, no significant association with a low CrCl or an elevated serum ALT was seen. CONCLUSIONS: Among a group of apparently healthy pregnant women, mild renal or hepatic impairment had little or no significant correlation with individual MSS markers. Further work should focus on the effect of more severe renal or hepatic dysfunction on MSS measures.

Fragile X testing in obstetrics and gynaecology in Canada.

OBJECTIVE: To provide Canadian family physicians, genetic counsellors, medical geneticists, midwives, and obstetrician-gynaecologists with recommendations regarding screening for fragile X in the obstetrical and gynaecological population. METHODS: Medline, the Cochrane Library, journals, and textbooks were searched for English-language articles, published between 1966 and March 2008, relating to fragile X testing outcomes. Search terms included fragile X, screening, prenatal testing, pregnancy outcome, premutation, trinucleotide repeats, and ovarian failure. All study types were reviewed. Randomized controlled trial results were considered evidence of the highest quality, followed by results of cohort studies. Key individual studies on which the recommendations are based are referenced. Supporting data for each recommendation are summarized with evaluative comments and references. This document represents an abstraction of the information. EVIDENCE: The quality of evidence reported in this document has been described using the criteria outlined in the report of the Canadian Task Force on Preventive Health Care. RECOMMENDATIONS: 1. Any testing for fragile X syndrome must occur only following thorough counselling and with the informed consent of the woman to be tested. (III-A) 2. Fragile X testing is indicated for a woman with a family history of fragile X syndrome, fragile X tremor/ataxia syndrome, or premature ovarian failure (in more than one family member) if the pedigree structure indicates that she is at risk of inheriting the mutated gene. Referral to a medical geneticist for counselling and assessment should be considered in these cases. (II-2A) 3. Fragile X testing is indicated for women who have a personal history of autism or mental retardation/developmental delay of an unknown etiology or who have at least one male relative with these conditions within a three-generation pedigree. (II-2A) 4. Fragile X testing is indicated for women who have reproductive or fertility problems associated with an elevated level of follicle stimulating hormone before the age of 40. (III-A) 5. Prenatal fetal testing via chorionic villus sampling or amniocentesis should be offered to women who are confirmed to be carriers of a premutation or full mutation of the fragile X gene (FMR-1). (II-2A) Pre-implantation genetic diagnosis is available as another reproductive option. (III-A) 6. Population screening for fragile X syndrome for all women in the reproductive age-range is feasible. However, it should be considered only when there is a provincial/regional program that can test and adequately counsel the targeted population about the meaning and implications of the results. (II-2B).

Maternal obesity and the risk of placental vascular disease.

INTRODUCTION: While elevated maternal weight in early pregnancy is associated with a higher rate of preeclampsia, the risk of placental abruption and placental infarction is unknown. METHODS: We evaluated the risk of placental abruption, placental infarction, and preeclampsia in association with maternal weight quintile at approximately 17 weeks' gestation in 386 323 women with a singleton pregnancy who underwent maternal serum screening in Ontario. RESULTS: After adjusting for age, ethnicity, parity, diabetes mellitus, and tobacco use, the odds ratio (OR) for preeclampsia was 4.1 (95% confidence interval [CI] 3.8-4.4) comparing the highest and lowest weight quintiles. Conversely, there was a lower risk of placental abruption or placental infarction, despite further adjustment for preeclampsia, gestational hypertension and drug dependence (OR 0.81, 95% CI 0.75-0.87). CONCLUSION: Higher maternal weight in early pregnancy is associated with a higher risk of preeclampsia and a lower risk of placental abruption or placental infarction, a seeming paradox that requires further elucidation.

Metabolic syndrome features and risk of neural tube defects.

BACKGROUND: Maternal obesity and pre-pregnancy diabetes mellitus, features of the metabolic syndrome (MetSyn), are individual risk factors for neural tube defects (NTD). Whether they, in combination with additional features of MetSyn, alter this risk is not known. We evaluated the risk of NTD in association with maternal features of the MetSyn. METHODS: We used a population-based case-control study design in the province of Ontario, Canada. Cases and controls were derived from women who underwent antenatal maternal screening (MSS) at 15 to 20 weeks' gestation. There were 89 maternal cases with, and 434 controls without, an NTD-affected singleton pregnancy. Maternal features of MetSyn were defined by the presence of pre-pregnancy diabetes mellitus, body weight > or = 90th centile among controls, non-white ethnicity and/or serum highly sensitive C-reactive protein (hsCRP) > or = 75th centile of controls. Since hsCRP naturally increases in pregnancy, analyses were performed with, and without, the inclusion of hsCRP in the model. RESULTS: Mean hsCRP concentrations were exceptionally high among study cases and controls (6.1 and 6.4 mg/L, respectively). When hsCRP was excluded from the model, the adjusted odds ratios for NTD were 1.9 (95% confidence interval 1.1-3.4) in the presence 1 feature of MetSyn, and 6.1 (1.1-32.9) in the presence of 2 or more features. When hsCRP was included, the respective risk estimates were attenuated to 1.6 (0.88-2.8) and 3.1 (1.2-8.3). CONCLUSION: We found about 2-fold and 6-fold higher risk for NTD in the presence 1, and 2 or more features, of the metabolic syndrome, respectively. It is not clear whether this risk is altered by the presence of a high serum hsCRP concentration.