Fetal Genotype Research Articles

Haplotype-based noninvasive prenatal diagnosis of methylmalonic acidemia and the discovery of a recurrent pathogenic haplotype associated with c.609G>A.

Early diagnosis and intervention are crucial for the prognosis of methylmalonic acidemia (MMA). However, research focused on early prenatal diagnosis of MMA is limited. A 161.89kb capture panel was designed for selectively enriching highly heterozygous SNPs. Fetal genotypes were inferred using relative haplotype dosage (RHDO) and Bayes factor, followed by invasive prenatal diagnosis (IPD) for validation. A core pathogenic haplotype associated with c.609G>A was identified based on the frequency differences between pathogenic and normal haplotypes. We recruited 41 pregnancies at risk of MMA with a median gestational age of 8+2 weeks. The assay success rate of NIPD-MMA for maternal variants was 92.7% (38/41), and after incorporating the paternal result, the overall assay success rate reached 100% (41/41). All NIPD results were concordant with IPD. Notably, a core haplotype (hap_2), comprising 28 SNPs, demonstrates significant enrichment within pathogenic haplotypes bearing the c.609G>A variation. On average, c.609G>A carriers had 22.38 heterozygous loci within these 28 SNPs. NIPD-MMA presents a viable choice for early, accurate, and safe prenatal diagnosis. Furthermore, the discovery of the recurrent core pathogenic haplotype provides a novel approach for haplotype phasing and has the potential for realizing proband-independent NIPD in the future.

Fetal RHD Screening in RH1 Negative Pregnant Women: Experience in Switzerland.

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

Noninvasive screening of fetal RHD genotype in Chinese pregnant women with serologic RhD-negative phenotype.

Noninvasive fetal RHD genotyping has been provided to nonimmunized RhD-negative pregnant women to guide anti-D prophylaxis. Among the Chinese, more than 30% of the RhD-negative phenotype is associated with variant RHD alleles, which would limit the accuracy of fetal RHD status prediction; thus, more targeting and proper programs need to be developed. Fluorescence quantitative polymerase chain reaction PCR (qPCR) or Sanger sequencing on all RHD exons was used to detect maternal RHD genotypes. For pregnant women with RHD*01N.01 or RHD*01N.03 alleles, the presence of RHD exons 5 and 10 in cell-free DNA was determined by qPCR. For pregnant women with the RHD(1227G>A) allele, high-throughput sequencing on exon 9 of the RHD gene and RHCE gene was used to predict fetal RhD phenotype. Among 65 cases of Chinese pregnant women with the serologic RhD-negative phenotype, three major genotypes were identified: RHD*01N.01/RHD*01N.01 (61.5%), RHD*01N.01/RHD(1227G>A) or RHD*01N.03/RHD(1227G>A) (20%), and RHD*01N.01/RHD*01N.03 (13.8%), along with three cases of minor genotypes (4.6%). For 43 pregnant women with the RHD*01N.01 or RHD*01N.03 alleles, qPCR on maternal cell-free DNA yielded a 98.5% (42/43) accuracy rate and 100% successful prediction rate. High-throughput sequencing was successfully used to predict fetal RhD phenotypes for 13 pregnant women with RHD(1227G>A). On the basis of maternal RHD genotyping, fetal genotyping through qPCR or high-throughput sequencing can improve the accuracy and success rate of prenatal fetal RhD phenotype prediction among Chinese pregnant women. It plays a potential role in guiding anti-D prophylaxis and pregnancy management in Chinese pregnant women.

Bringing precision medicine to the management of pregnancy in women with glucokinase-MODY: a study of diagnostic accuracy and feasibility of non-invasive prenatal testing.

In pregnancies where the mother has glucokinase-MODY (GCK-MODY), fetal growth is determined by fetal genotype. When the fetus inherits a maternal pathogenic GCK variant, normal fetal growth is anticipated, and insulin treatment of maternal hyperglycaemia is not recommended. At present, fetal genotype is estimated from measurement of fetal abdominal circumference on ultrasound. Non-invasive prenatal testing of fetal GCK genotype (NIPT-GCK) using cell-free DNA in maternal blood has recently been developed. We aimed to compare the diagnostic accuracy of NIPT-GCK with that of ultrasound, and determine the feasibility of using NIPT-GCK to guide pregnancy management. We studied an international cohort of pregnant women with hyperglycaemia due to GCK-MODY. We compared the diagnostic accuracy of NIPT-GCK with that of measurement of fetal abdominal circumference at 28 weeks' gestation (n=38) using a directly genotyped offspring sample as the reference standard. In a feasibility study, we assessed the time to result given to clinicians in 43 consecutive pregnancies affected by GCK-MODY between July 2019 and September 2021. In terms of diagnostic accuracy, NIPT-GCK was more sensitive and specific than ultrasound in predicting fetal genotype (sensitivity 100% and specificity 96% for NIPT-GCK vs sensitivity 53% and specificity 61% for fetal abdominal circumference 75th percentile). In terms of feasibility, a valid NIPT-GCK fetal genotype (≥95% probability) was reported in all 38 pregnancies with an amenable variant and repeated samples when needed. The median time to report was 5 weeks (IQR 3-8 weeks). For the 25 samples received before 20 weeks' gestation, results were reported at a median gestational age of 20 weeks (IQR 18-24), with 23/25 (92%) reported before 28 weeks. Non-invasive prenatal testing of fetal genotype in GCK-MODY pregnancies is highly accurate and is capable of providing a result before the last trimester for most patients. This means that non-invasive prenatal testing of fetal genotype is the optimal approach to management of GCK-MODY pregnancies.

Validation of a non-invasive prenatal test for fetal RhD, C, c, E, K and Fya antigens

We developed and validated a next generation sequencing-(NGS) based NIPT assay using quantitative counting template (QCT) technology to detect RhD, C, c, E, K (Kell), and Fya (Duffy) fetal antigen genotypes from maternal blood samples in the ethnically diverse U.S. population. Quantitative counting template (QCT) technology is utilized to enable quantification and detection of paternally derived fetal antigen alleles in cell-free DNA with high sensitivity and specificity. In an analytical validation, fetal antigen status was determined for 1061 preclinical samples with a sensitivity of 100% (95% CI 99–100%) and specificity of 100% (95% CI 99–100%). Independent analysis of two duplicate plasma samples was conducted for 1683 clinical samples, demonstrating precision of 99.9%. Importantly, in clinical practice the no-results rate was 0% for 711 RhD-negative non-alloimmunized pregnant people and 0.1% for 769 alloimmunized pregnancies. In a clinical validation, NIPT results were 100% concordant with corresponding neonatal antigen genotype/serology for 23 RhD-negative pregnant individuals and 93 antigen evaluations in 30 alloimmunized pregnancies. Overall, this NGS-based fetal antigen NIPT assay had high performance that was comparable to invasive diagnostic assays in a validation study of a diverse U.S. population as early as 10 weeks of gestation, without the need for a sample from the biological partner. These results suggest that NGS-based fetal antigen NIPT may identify more fetuses at risk for hemolytic disease than current clinical practice, which relies on paternal genotyping and invasive diagnostics and therefore is limited by adherence rates and incorrect results due to non-paternity. Clinical adoption of NIPT for the detection of fetal antigens for both alloimmunized and RhD-negative non-alloimmunized pregnant individuals may streamline care and reduce unnecessary treatment, monitoring, and patient anxiety.

Spectrum of congenital anomalies of the kidney and urinary tract (CAKUT) including renal parenchymal malformations during fetal life and the implementation of prenatal exome sequencing (WES)

Objectives and backgroundCongenital malformations of the kidney and urinary tract (CAKUT) have a prevalence of 4–60 in 10,000 livebirths and constitute for 40–50% of all end stage pediatric kidney disease. CAKUT can have a genetic background due to monogenetic inherited disease, such as PKD or ciliopathies. They can also be found in combination with extra-renal findings as part of a syndrome. Upon detection of genitourinary malformations during the fetal anomaly scan the question arises if further genetic testing is required. The purpose of this study was to determine the phenotypic presentation of CAKUT cases and the results of exome analysis (WES).MethodsThis is a retrospective analysis of 63 fetal cases with a diagnosis of CAKUT or DSD at a single center between August 2018 and December 2022.ResultsA total of 63 cases (5.6%) out of 1123 matched CAKUT phenotypes including renal parenchyma malformations. In 15 out of 63 WES analysis a pathogenic variant was detected (23.8%). In fetuses with isolated CAKUT the rate of detecting a pathogenic variant on exome sequencing was five out of 44 (11.4%). Ten out of 19 fetuses (52.6%) that displayed extra-renal findings in combination with CAKUT were diagnosed with a pathogenic variant.ConclusionsWES provides an increase in diagnosing pathogenic variants in cases of prenatally detected CAKUT. Especially in fetuses with extra-renal malformations, WES facilitates a gain in information on the fetal genotype to enhance prenatal counselling and management.

Determination of Rh type and gender using circulating cell-free fetal DNA in early pregnancy of Rh negative women in turkey.

Choosing the right clinical approach for early and reliable diagnosis/screening is becoming more important day by day. The aim of the study was to determine the early RhD type with cff-DNA obtained from maternal plasma, especially in the light of recent developments. In this way, it is aimed to apply Rh Ig only to mothers who are determined to have RhD (+) fetuses and to prevent unnecessary further tests that may possess a risk for RhD (-) fetuses. Prediction of fetal gender and RH genotype was performed by using RT-qPCR method. With simultaneous amplification of sequences of SRY, DYS14 and RH genes (exon 7 and exon 10). Fetal gender and RhD were determined in 30 RHD (-) pregnant women with cfDNA. As a result of genotyping, the gender of 67% (20/30) fetuses was determined as male; the gender of 33% (10/30) fetuses was determined as female in a sample group of 30 pregnancies. It was determined that the DYS14 100% (20/20) gene was more sensitive than the SRY 97% (18/20) gene in gender determination after examining prenatal and postnatal results. As a result of the analysis, the presence of 17% (5/30) RhD (-) fetuses and 83% (25/30) RhD (+) fetuses were determined which is 100% compatible with postnatal results. Detecting fetal RhD gene in maternal plasma made an important contribution to its use in non-invasive prenatal screening. This study shows that unnecessary intervention and cost can be avoided with successful genotyping analysis performed with RT-qPCR.

Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

Terminating Routine Cord Blood RhD Typing of the Newborns to Guide Postnatal Anti-D Immunoglobulin Prophylaxis Based on the Results of Fetal RHD Genotyping

Introduction: Targeted routine antenatal prophylaxis with anti-D immunoglobulin (Ig) only to RhD-negative pregnant women who carry RhD-positive fetuses (determined by fetal RHD genotyping) has reduced D-alloimmunization significantly when administered in addition to postnatal prophylaxis. Achieving high analysis sensitivity and few false-negative fetal RHD results will make RhD typing of the newborn redundant. Postnatal prophylaxis can then be given based on the result of fetal RHD genotyping. Terminating routine RhD typing of the newborns in cord blood will streamline maternity care. Accordingly, we compared the results of fetal RHD genotyping with RhD typing of the newborns. Methods: Fetal RHD genotyping was performed, and antenatal anti-D Ig was administered at gestational week 24 and 28, respectively. Data for 2017–2020 are reported. Results: Ten laboratories reported 18,536 fetal RHD genotypings, and 16,378 RhD typing results of newborns. We found 46 false-positive (0.28%) and seven false-negative (0.04%) results. Sensitivity of the assays was 99.93%, while specificity was 99.24%. Conclusion: Few false-negative results support the good analysis quality of fetal RHD genotyping. Routine cord blood RhD typing will therefore be discontinued nationwide and postnatal anti-D Ig will now be given based on the result of fetal RHD genotyping.

A gene-based association test of interactions for maternal-fetal genotypes identifies genes associated with nonsyndromic congenital heart defects.

The risk of congenital heart defects (CHDs) may be influenced by maternal genes, fetal genes, and their interactions. Existing methods commonly test the effects of maternal and fetal variants one-at-a-time and may have reduced statistical power to detect genetic variants with low minor allele frequencies. In this article, we propose a gene-based association test of interactions for maternal-fetal genotypes (GATI-MFG) using a case-mother and control-mother design. GATI-MFG can integrate the effects of multiple variants within a gene or genomic region and evaluate the joint effect of maternal and fetal genotypes while allowing for their interactions. In simulation studies, GATI-MFG had improved statistical power over alternative methods, such as the single-variant test and functional data analysis (FDA) under various disease scenarios. We further applied GATI-MFG to a two-phase genome-wide association study of CHDs for the testing of both common variants and rare variants using 947 CHD case mother-infant pairs and 1306 control mother-infant pairs from the National Birth Defects Prevention Study (NBDPS). After Bonferroni adjustment for 23,035 genes, two genes on chromosome 17, TMEM107 (p = 1.64e-06) and CTC1 (p = 2.0e-06), were identified for significant association with CHD in common variants analysis. Gene TMEM107 regulates ciliogenesis and ciliary protein composition and was found to be associated with heterotaxy. Gene CTC1 plays an essential role in protecting telomeres from degradation, which was suggested to be associated with cardiogenesis. Overall, GATI-MFG outperformed the single-variant test and FDA in the simulations, and the results of application to NBDPS samples are consistent with existing literature supporting the association of TMEM107 and CTC1 with CHDs.

Effect of the Fetal THRB Genotype on the Placenta.

Pregnant women with mutations in the thyroid hormone receptor beta (THRB) gene expose their fetuses to high TH levels shown to be detrimental to a normal fetus (NlFe) but not to an affected (AfFe) fetus. However, no information is available about differences in placental TH regulators. To investigate whether there are differences in placentas associated with a NlFe compared to an AfFe, we had the unique opportunity to study placentas from two pregnancies of the same woman with THRB mutation G307D. One placenta supported a NlFe while the other an AfFe. Sections of placentas were collected and frozen at -80°C after term delivery of NlFe and AfFe. Two placentas from healthy women of similar gestational age were also obtained. The fetal origin of the placental tissues was established by gDNA quantitation of genes on the X and Y chromosomes and THRB gene. Expression and enzymatic activity of deiodinases 2 and 3 were measured. The following genes expression was also quantitated: MCT10, MCT8, LAT1, LAT2, THRB, THRA. The placenta carrying the AfFe exhibited a significant reduction of deiodinase 2 and 3 activities as well as the expression of the thyroid hormone transporters MCT10, LAT1 and LAT2 and the THRA. Here, we present the first study of the effect of fetal THRB genotype on the placenta. Though limited by virtue of the rarity of THRB mutations and sample availability, we show that the fetal THRB genotype influences the levels of thyroid hormone regulators in the placenta.

Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

Internationale richtlijnen voor het gebruik van Rhesus D immunoglobuline: een vergelijkend onderzoek en de plaats van foetale Rhesus D genotypering

International guidelines for the use of Rhesus D immunoglobulin: a comparative study and the contribution of fetal Rhesus genotyping The introduction of immunoglobulin D (Anti-D, Rhogamâ) has been a major advancement in the prevention of Rhesus D alloimmunisation. However, the required dose and timing of administration remain unclear. Fetal Rhesus genotyping, to administer Anti-D more selectively, is not yet available as part of standard care for Rhesus negative pregnant women in Belgium. We performed a comparative literature search, using the most recent international guidelines regarding the use of Anti-D and fetal Rhesus genotyping. There is international consensus that Anti-D should be administered prophylactically between 28-34 weeks of gestation and especially postpartum. The recommended dose and number of administrations vary strongly in different countries. There is no international consensus regarding the necessity to administer Anti-D nor the dose of Anti-D to be administered in the event of possible sensitisation during pregnancy. The results of our guideline study suggest that several possible causes of sensitisation require a lower dose of Anti-D than the currently available dose in Belgium (1500 IU). One could consider introducing a lower dose in Belgium in order to use Anti-D more efficiently. Nonetheless, further research concerning the ideal dose per indication and the ideal timing of administration is indicated. Fetal Rhesus D genotyping has a sensitivity of 99.9% and specificity of 99.2% when performed after 11 weeks of gestation. Given the high sensitivity and specificity of fetal Rhesus genotyping, a standard implementation of this test in Belgium certainly seems worth considering.

Integration of targeted sequencing and pseudo-tetraploid genotyping into clinically assisted decision support for β-thalassemia invasive prenatal diagnosis.

The high prevalence of β-thalassemia indicates the severe medical burden in Guangxi province in China. Millions of thousands of prenatal women with healthy or thalassemia-carrying fetuses received an unnecessary prenatal diagnosis. We designed a prospective single-center proof-of-concept study to evaluate the utility of a noninvasive prenatal screening method in the stratification of beta-thalassemia patients before invasive procedures. Next-generation and optimized pseudo-tetraploid genotyping-based methods were utilized in preceding invasive diagnosis stratification to predict the mater-fetus genotype combinations in cell-free DNA, which is from maternal peripheral blood. Populational linkage disequilibrium information with additional neighboring loci to infer the possible fetal genotype. The concordance of the pseudo-tetraploid genotyping with the gold standard invasive molecular diagnosis was used to evaluate the effectiveness of this method. 127 β-thalassemia carrier parents were consecutively recruited. The total genotype concordance rate is 95.71%. The Kappa value was 0.8248 for genotype combinations and 0.9118 for individual alleles. This study offers a new approach to picking out the health or carrier fetus before invasive procedures. It provides valuable novel insight into patient stratification management on β-thalassemia prenatal diagnosis.

Maternal and Infant Outcomes in GCK-MODY Complicated by Pregnancy.

Challenges exist in the management of Glucokinase-maturity-onset diabetes of the young (GCK-MODY), especially during pregnancy. This work aimed to evaluate the prevalence of congenital anomaly in newborns from GCK-MODY mothers, and the relationship between fetus genotype and the risk of congenital malformation as well as other adverse pregnancy outcomes. Electronic databases including PubMed, EMBASE, and Cochrane database last updated July 16, 2022, were searched. We included observational studies conducted in GCK-MODY complicated by pregnancy, and reporting at least one pregnancy outcome. We extracted data in duplicate, and the risk of bias was evaluated by the Newcastle-Ottawa Quality Assessment Scale (NOS). All statistical analysis was performed by Cochrane Review Manager. Eight studies were selected in the meta-analysis. Five were of high quality and 3 were of medium quality evaluated by NOS. A total of 257 GCK-MODY mothers and 499 offspring were enrolled. Among them, 370 offspring were divided into 2 groups: GCK-affected offspring (GCK+, n = 238) and GCK-unaffected offspring (GCK-, n = 132). The percentage of congenital malformations in GCK pregnant women's offspring was 2.4%. The risk of congenital malformations was similar between the GCK+ and GCK- group (odds ratio = 0.56; 95% CI, 0.07-4.51; I2 = 0%; P = .59). The risk of macrosomia/large for gestational age, neonatal hypoglycemia, and combined adverse neonatal outcome was significantly lower in offspring with the GCK mutation compared with non-GCK mutation carriers. The percentage of congenital malformations was 2.4% in GCK-MODY pregnant women's offspring, and newborns with the GCK mutation have lower birth complication than non-GCK mutation carriers.

A theoretical base for non-invasive prenatal paternity testing

There is an increasing demand for prenatal paternity testing in the forensic applications, which identify biological fathers before the birth of children. Currently, one of the most effective and safe Non-Invasive Prenatal Paternity Testing (NIPPT) methods is high-throughput Next-Generation Sequencing (NGS)-based SNP genotyping of cell-free DNA in maternal peripheral blood. To the best of our knowledge, nearly all methods being used in such applications are based on traditional postnatal paternity tests and/or statistical models of conventional polymorphism sites. These methods have shown unsatisfactory performance due to the uncertainty of fetal genotype. In this study, we propose a cutting-edge methodology called the Prenatal paternity Test Analysis System (PTAS) for cell-free fetal DNA-based NIPPT using NGS-based SNP genotyping. With the implementation of our proposed PTAS methodology, 63 out of 64 early-pregnancy (i.e., less than seven weeks) samples can be precisely identified to determine paternity, except for one sample that does not meet quality control requirements. Although the fetal fraction of the non-identified sample is extremely low (0.51%), its paternity can still be detected by our proposed PTAS methodology through unique molecular identifier tagging. Paternity of the total 313 samples for mid-to-late pregnancy (i.e., more than seven weeks) can be accurately identified. Extensive experiments indicate that our methodology makes a significant breakthrough in the NIPPT theory and will bring substantial benefits to forensic applications.

Maternal and fetal origins of offspring blood pressure: statistical analysis using genetic correlation and genetic risk score-based Mendelian randomization.

Epidemiological studies demonstrated that adverse in utero environment was associated with increased risk of offspring high blood pressure, by using birthweight as the proxy of maternal intrauterine exposure; however, the nature of such association remains less understood. With maternal/fetal-specific summary statistics of birthweight (n = 297 356 for own birthweight and n = 210 248 for offspring birthweight) and summary statistics of blood pressure [i.e. systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (PP)] (n = 757 601), we evaluated the genetic correlation between fetal-specific birthweight and blood pressure using cross-trait linkage disequilibrium score regression, and next detected pleiotropic genes for them with a pleiotropy mapping method called mixture-adjusted intersect-union pleiotropy test. Furthermore, we conducted a genetic risk score (GRS)-based Mendelian randomization analysis in parent-offspring pairs (n = 6031) of the UK Biobank cohort, to assess the causal relation between maternal-specific GRS and blood pressure conditioning on fetal genotypes. We found fetal-specific birthweight had a negative genetic correlation with DBP (ρ^g = -0.174, P = 1.68 × 10-10), SBP (ρ^g = -0.198, P = 8.09 × 10-12), and PP (ρ^g = -0.152, P = 6.04 × 10-8), and detected 143, 137 and 135 pleiotropic genes shared between fetal-specific birthweight and PP, SBP and DBP, respectively. These genes often exhibited opposite genetic effects, and were more likely to be differentially expressed in pancreas, liver, heart, brain, whole blood and muscle skeletal tissues. A causal negative association of maternal-specific birthweight was identified with SBP (P = 2.20 × 10-2) and PP (P = 7.67 × 10-3) but not DBP (P = 0.396) in mother-offspring pairs, after accounting for the influence of fetal-specific GRS; and the two significant relations were robust against the horizontal pleiotropy of instruments and the confounding influence of gestational duration and preterm birth. However, these causal associations could not be detected in father-offspring pairs. This study revealed common genetic components underlying birthweight and blood pressure, and provided important insight into aetiology and early prevention of high blood pressure.

Prenatal screening service for fetal RHD genotyping to guide prophylaxis: the two-year experience of the Friuli Venezia Giulia region in Italy.

Fetal RHD genotyping of cell-free fetal DNA (cff-DNA) from RhD-negative pregnant women can be used to guide anti-D prophylaxis: the knowledge of fetal RhD type can direct and restrict the use of prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus. Since November 2019 in the region of Friuli Venezia Giulia (Italy) a prenatal screening service has been offered to RhD-negative women at 22-24 weeks of gestation. The cff-DNA is extracted from a simple peripheral maternal blood sample to analyze the fetal RHD gene: the results are interpreted as RHD-positive fetus, RHD-negative fetus, or Inconclusive. The service is shared with all regional hospitals and tests are provided free of charge by the National Health System. Overall, 142 RhD-negative pregnant women were recruited in nearly 2 years. Fetal RHD genotyping was negative in 53 pregnancies and positive in 89 pregnancies. Thus, unnecessary treatment of pregnant women and exposure to a scarce plasma-derived medicinal product was avoided, by the use of a single blood sample, in 37.8% of cases, representing 100% of the RhD-negative women carrying a RhD-negative fetus in our cohort. The first Italian region-wide screening service for fetal RHD genotyping has been implemented for 2 years, despite the COVID-19 pandemic, in order to obtain the predicted fetal RhD phenotype before the 28th week of gestation, during which prenatal prophylaxis is usually administered. Giving prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus reduces the overall use of anti-D immunoglobulin, which is becoming an ever more limited resource. The high sensitivity of the procedure provides evidence that the implementation of a diagnostic test in a reference laboratory guarantees the quality of the results, the concordance of reports and the sustainability of costs, representing an excellent guide to targeted use of prophylaxis.

The power of digital PCR in fetal blood group genotyping: a review

: There is a clinical need to determine the fetal blood group status for pregnant women who present with clinically significant red cell antibodies to optimize perinatal care. Non-invasive prenatal testing (NIPT) to assess the fetal blood group status is problematic for blood groups that arise from single nucleotide variants (SNVs) because the cell-free DNA from the maternal allele is present in greater abundance than the fetal allele, resulting in competition, especially in traditional real-time quantitative polymerase chain reaction (qPCR) platforms. This review discusses the power for digital (d)PCR to overcome this limitation. The dPCR is based on first diluting and compartmentalizing individual DNA molecules into individual PCR mixes, so that the competing (e.g., maternal) molecules are separated from the target (fetal) DNA molecule prior to amplification. In comparison to qPCR platforms, dPCR is amenable to the quantitation of a positive signal from an individual DNA molecule, providing the ability to calculate the fractional abundance of the fetal sequence of interest. Comparison studies, though small in number, show that dPCR is more precise and sensitive. Clinical feasibility studies to date show that dPCR for fetal Kell antigen, Rhc, RhE and Duffy antigens have high accuracy. The key limitation of the dPCR approach is that separate assays need to be established and validated for each blood group antigen. Despite that limitation, the power of dPCR for fetal blood group genotyping provides an added diagnostic tool in the management of women with red cell antibodies.

Using Crisper in the Treatment of Sickle Cell Disease

Individuals with sickle cell disease (SCD) experience many complications and health problems due to their mutated hemoglobin gene; however, these problems could be alleviated by using CRISPR gene editing to alter which hemoglobin variants hematopoietic stem progenitor cells produce. The aim of this review is to determine if it is safe and effective to treat patients with SCD with CRISPR. The findings of “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β Thalassemia” found an increase in fetal hemoglobin expression in patients after transplanting hematopoietic stem progenitor cells with edits to the BCL11A transcription factor. “Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia” also raised fetal hemoglobin by editing hematopoietic stem progenitor cells within their β-globin loci to artificially apply a hereditary persistence of fetal hemoglobin deletional genotype to the cells. Researchers in “CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells” raised wild-type hemoglobin expression in hematopoietic stem progenitor cells affected with SCD by editing the β-globin loci and “fixing” the point mutation that causes SCD. Target deletion efficiency ranged from 18%-80%, and every study used either SpCas9 or SaCas9. Each study found no punitive off-target effects, and they also succeeded in raising hematopoietic stem progenitor cells levels of their targeted hemoglobin. These results point to using CRISPR as both a safe and effective way to treat SCD.