Pregnancy in Women With Monogenic Diabetes due to Pathogenic Variants of the Glucokinase Gene: Lessons and Challenges

Heterozygous loss-of-function variants of the glucokinase (GCK) gene are responsible for a subtype of maturity-onset diabetes of the young (MODY). GCK-MODY is characterized by a mild hyperglycemia, mainly due to a higher blood glucose threshold for insulin secretion, and an up-regulated glucose counterregulation. GCK-MODY patients are asymptomatic, are not exposed to diabetes long-term complications, and do not require treatment. The diagnosis of GCK-MODY is made on the discovery of hyperglycemia by systematic screening, or by family screening. The situation is peculiar in GCK-MODY women during pregnancy for three reasons: 1. the degree of maternal hyperglycemia is sufficient to induce pregnancy adverse outcomes, as in pregestational or gestational diabetes; 2. the probability that a fetus inherits the maternal mutation is 50% and; 3. fetal insulin secretion is a major stimulus of fetal growth. Consequently, when the fetus has not inherited the maternal mutation, maternal hyperglycemia will trigger increased fetal insulin secretion and growth, with a high risk of macrosomia. By contrast, when the fetus has inherited the maternal mutation, its insulin secretion is set at the same threshold as the mother’s, and no fetal growth excess will occur. Thus, treatment of maternal hyperglycemia is necessary only in the former situation, and will lead to a risk of fetal growth restriction in the latter. It has been recommended that the management of diabetes in GCK-MODY pregnant women should be guided by assessment of fetal growth by serial ultrasounds, and institution of insulin therapy when the abdominal circumference is ≥ 75th percentile, considered as a surrogate for the fetal genotype. This strategy has not been validated in women with in GCK-MODY. Recently, the feasibility of non-invasive fetal genotyping has been demonstrated, that will improve the care of these women. Several challenges persist, including the identification of women with GCK-MODY before or early in pregnancy, and the modalities of insulin therapy. Yet, retrospective observational studies have shown that fetal genotype, not maternal treatment with insulin, is the main determinant of fetal growth and of the risk of macrosomia. Thus, further studies are needed to specify the management of GCK-MODY pregnant women during pregnancy.

Maternal Choline Supplementation Modulates Placental Markers of Inflammation, Angiogenesis, and Apoptosis in a Mouse Model of Placental Insufficiency

Dlx3 (distal-less homeobox 3) haploinsufficiency in mice has been shown to result in restricted fetal growth and placental defects. We previously showed that maternal choline supplementation (4X versus 1X choline) in the Dlx3+/− mouse increased fetal and placental growth in mid-gestation. The current study sought to test the hypothesis that prenatal choline would modulate indicators of placenta function and development. Pregnant Dlx3+/− mice consuming 1X (control), 2X, or 4X choline from conception were sacrificed at embryonic (E) days E10.5, E12.5, E15.5, and E18.5, and placentas and embryos were harvested. Data were analyzed separately for each gestational day controlling for litter size, fetal genotype (except for models including only +/− pups), and fetal sex (except when data were stratified by this variable). 4X choline tended to increase (p < 0.1) placental labyrinth size at E10.5 and decrease (p < 0.05) placental apoptosis at E12.5. Choline supplementation decreased (p < 0.05) expression of pro-angiogenic genes Eng (E10.5, E12.5, and E15.5), and Vegf (E12.5, E15.5); and pro-inflammatory genes Il1b (at E15.5 and 18.5), Tnfα (at E12.5) and Nfκb (at E15.5) in a fetal sex-dependent manner. These findings provide support for a modulatory effect of maternal choline supplementation on biomarkers of placental function and development in a mouse model of placental insufficiency.

Maternal and fetal genetic effects on birth weight and their relevance to cardio-metabolic risk factors

Birth weight (BW) variation is influenced by fetal and maternal genetic and non-genetic factors, and has been reproducibly associated with future cardio-metabolic health outcomes. These associations have been proposed to reflect the lifelong consequences of an adverse intrauterine environment. In earlier work, we demonstrated that much of the negative correlation between BW and adult cardio-metabolic traits could instead be attributable to shared genetic effects. However, that work and other previous studies did not systematically distinguish the direct effects of an individual’s own genotype on BW and subsequent disease risk from indirect effects of their mother’s correlated genotype, mediated by the intrauterine environment. Here, we describe expanded genome-wide association analyses of own BW (n=321,223) and offspring BW (n=230,069 mothers), which identified 278 independent association signals influencing BW (214 novel). We used structural equation modelling to decompose the contributions of direct fetal and indirect maternal genetic influences on BW, implicating fetal- and maternal-specific mechanisms. We used Mendelian randomization to explore the causal relationships between factors influencing BW through fetal or maternal routes, for example, glycemic traits and blood pressure. Direct fetal genotype effects dominate the shared genetic contribution to the association between lower BW and higher type 2 diabetes risk, whereas the relationship between lower BW and higher later blood pressure (BP) is driven by a combination of indirect maternal and direct fetal genetic effects: indirect effects of maternal BP-raising genotypes act to reduce offspring BW, but only direct fetal genotype effects (once inherited) increase the offspring’s later BP. Instrumental variable analysis using maternal BW-lowering genotypes to proxy for an adverse intrauterine environment provided no evidence that it causally raises offspring BP. In successfully separating fetal from maternal genetic effects, this work represents an important advance in genetic studies of perinatal outcomes, and shows that the association between lower BW and higher adult BP is attributable to genetic effects, and not to intrauterine programming.

The Immunosignature of Mother/Fetus Couples in Gestational Diabetes Mellitus: Role of HLA-G 14 bp ins/del and PAPP-A A/C Polymorphisms in the Uterine Inflammatory Milieu

We enrolled 151 healthy mother/newborn couples and 26 with gestational diabetes mellitus (GDM). HLA-G and PAPP-A plasma levels were measured by ELISA at first and second trimesters, at delivery, and in cord blood. HLA-G 14 bp ins/del and PAPP-A A/C polymorphisms were genotyped. HLA-G del/del and PAPP-A C/C genotypes were more frequent among GDM mothers than controls. We observed a genetic epistasis between the two polymorphisms: the HLA-G del/del and PAPP-A C/C combination was carried by 8% of GDM mothers and 1.3% of controls (OR = 9.5, 95% CI = 0.8–109, p=0.07). GDM mothers showed increased sHLA-G levels compared to controls (p=0.004), and those carrying the HLA-G del/del genotype produced more sHLA-G at the second trimester and at delivery (p=0.014). A genetic pressure by fetal genotype on maternal sHLA-G production was observed in GDM mothers with heterozygous HLA-G del/ins newborns (p=0.02). Babies born to GDM mothers showed higher sHLA-G concentrations compared to those born to healthy mothers, and those carrying HLA-G del/del showed the highest sHLA-G levels (p=0.013). PAPP-A amounts significantly increased along pregnancy (p<0.001), but the median levels at the first and second trimesters were significantly lower in GDM (p=0.03). Our findings first suggest an involvement of HLA-G and PAPP-A gene-protein interaction in GDM and highlight a possible contribution of the fetus in balancing maternal inflammation.

Effects of fetal genotype and sex on developmental response to maternal malnutrition

The present study aimed to determine whether developmental patterns, adiposity level and fatty-acid composition of fetuses exposed to maternal malnutrition are driven by their sex or their genotype, or both, as these may modulate the adaptive response to the intrauterine environment independently of the maternal genotype. We used a single maternal genotype (purebred Iberian (IB) sows), which was inseminated with heterospermic semen (obtained by mixing semen from Iberian and Large White (LW) boars), to obtain four different subsets of fetuses (male and female, purebred (IB × IB) and crossbred (IB × LW)) in Iberian purebred sows. Analysis of fetal phenotypes indicated a better adaptive response of the female offspring, which was modulated by their genotype. When faced with prenatal undernutrition, females prioritised the growth of vital organs (brain, liver, lungs, kidneys and intestine) at the expense of bone and muscle. Moreover, the analysis of fat composition showed a higher availability of essential fatty acids in the female sex than in their male counterparts and also in the Iberian genotype than in crossbred fetuses. These results are of high translational value for understanding ethnic differences in prenatal programming of postnatal health and disease status, and show evidence that prenatal development and metabolic traits are primarily determined by fetal sex and strongly modulated by fetal genotype.

Towards the Development of a Noninvasive Prenatal Test for Beta-Thalassemia: Utilization of Probe Capture Enrichment and Next Generation Sequencing

β-thalassemia causes significant morbidity and mortality worldwide. Currently, the diagnosis can be made prenatally for couples at risk using invasive procedures such as chorionic villus sampling and amniocentesis. Noninvasive prenatal testing (NIPT) on maternal blood samples would enable earlier fetal diagnosis and eliminate risks associated with these procedures. The discovery of cell-free fetal DNA (cfFDNA) in maternal plasma and advances in next generation sequencing (NGS) have made NIPT a clinical reality for aneuploidies. Diagnosing autosomal recessive (AR) disorders is more challenging as it requires determination of both the paternally and maternally inherited alleles and can be particularly difficult when both parents carry the same mutation. Previous studies used methods to identify the paternally inherited allele in maternal plasma through the detection of a DNA sequence variant present in the father and absent in the mother. Our method expands the target region beyond the β-globin gene into highly polymorphic regions to increase the likelihood of identifying unique paternal sequences. Unlike the detection of the paternal allele, there is no direct qualitative approach for determining the maternally inherited allele. Our solution is an indirect quantitative method that compares the ratio of allelic sequence reads to infer which maternal allele was inherited by the fetus. We have developed a novel NGS assay which utilizes probe capture enrichment, a method employing thousands of short overlapping oligonucleotide probes complementary to a target sequence region. This design makes it uniquely applicable to short, fragmented DNA, such as cell-free DNA (~150 base pairs). Our probe assay targets a contiguous 900 bp region which spans a portion of the β-globin gene (part of exon 2 as well as the entirety of IVS-1 and exon 1) and extends 579 bp into the highly polymorphic 5' UTR region to identify linked sequence variations. Additionally, the assay targets 451 single nucleotide polymorphisms (SNPs) throughout the genome. SNPs that are "informative" (i.e. an allele present in the fetus and absent in the mother) are used to calculate the fraction of cfDNA from the fetus. We have evaluated our assay's performance in a set of experiments designed to simulate the challenging aspects of cfFDNA: very low quantity and short fragment size. The assay's sensitivity was tested by preparing libraries containing amounts of DNA ranging from 50 to 0.05 ngs. At DNA amounts as low as 5 ng (5-fold lower than that expected in plasma), 100% coverage was achieved for the targeted β-globin region and SNPs. The probe/NGS system successfully captured and sequenced DNA fragments as short as 35 bps and as little as 0.5 ng of DNA with >95% coverage. To test the ability of the probe/NGS system to resolve mixtures, DNA samples from patients with known β-globin mutations were combined in ratios ranging from 2.5:97.5 to 20:80 in 25 ng total DNA to mimic the maternal/fetal cfDNA mixture. Our assay was able to detect minority heterozygous mutant alleles at proportions as low as 1.25% and 0.3 ng of DNA. In a mixture designed to simulate a case with a shared parental mutation (CD41/42(-TTCT)), we identified a linked SNP (rs713040) allele, which was within 250 bp of the mutation and unique to the minor fraction. We used this SNP to distinguish the CD41/42 (-TTCT) mutation contributed by the minor fraction. Based on 6 mixtures, we observed 24.3% (110/451) of SNPs to be informative, allowing for a precise estimate of the minor fraction. We estimated the minor fraction to be 11.88% and 5.98% compared to the expected 10% and 5%, respectively. To show proof of concept for inferring the minor ("fetal") genotype when the major ("maternal") genotype was heterozygous mutant (IVS1-5 (G>C)), the estimated minor fraction (10.4% based on 104 informative SNPs) was used to calculate the expected allelic ratios of the 3 different possible minor ("fetal") genotypes. The minor genotype was correctly inferred as wt/wt based on the observed mixture ratio (42.56/57.41 mut/wt) compared to the expected (44.93/55.07). These data show that our probe capture/NGS system can overcome the challenges implicit in the analysis of cfFDNA for NIPT: low DNA amount (<5 ng) and short fragments (<150 bp). We expect that our approach using the fetal fraction estimate will allow us to successfully infer the fetal genotype when applied to maternal plasma. Disclosures Erlich: Allen and Overy, Law Office: Consultancy.

Clinical Potential of Effective Noninvasive Exclusion of KEL1-Positive Fetuses in KEL1-Negative Pregnant Women

Background: The clinical importance of assessing the fetal KEL genotype is to exclude ‘K'-positive fetuses (genotype KEL1/KEL2) in ‘K'-alloimmunized pregnant women (genotype KEL2/KEL2). Noninvasive assessment of the fetal KEL genotype is not yet available in the Czech Republic. Objective: The aim of this study was to assess the fetal KEL1/KEL2 genotype from cell-free fetal DNA in the plasma of KEL2/KEL2 pregnant women. Methods: The fetal genotype was assessed by minisequencing (a dilution series including control samples). A total of 138 pregnant women (between the 8th and 23rd gestational week) were tested by minisequencing. The fetal genotype was further verified by analysis of a buccal swab from the newborn. Results: Minisequencing proved to be a reliable method. In 2.2% (3/138) of the examined women, plasma sample testing failed; 94.8% (128/135) had the KEL2/KEL2 genotype, and a total of 3.1% of fetuses (4/128) had the KEL1/KEL2 genotype. Sensitivity and specificity reached 100% (p < 0.0001). Conclusion: Minisequencing is a reliable method for the assessment of the fetal KEL1 allele from the plasma of KEL2/KEL2 pregnant women.