Disruptive CHD8 mutations define a subtype of autism early in development.

Autism spectrum disorder (ASD) is a heterogeneous disease in which efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in postmitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation.

Toward 3D facial analysis for recognizing Mendelian causes of autism spectrum disorder.

Recognizing Mendelian causes is crucial in molecular diagnostics and counseling for patients with autism spectrum disorder (ASD). We explored facial dysmorphism and facial asymmetry in relation to genetic causes in ASD patients and studied the potential of objective facial phenotyping in discriminating between Mendelian and multifactorial ASD. In a cohort of 152 ASD patients, 3D facial images were used to calculate three metrics: a computational dysmorphism score, a computational asymmetry score, and an expert dysmorphism score. High scores for each of the three metrics were associated with Mendelian causes of ASD. The computational dysmorphism score showed a significant correlation with the average expert dysmorphism score. However, in some patients, different dysmorphism aspects were captured making the metrics potentially complementary. The computational dysmorphism and asymmetry scores both enhanced the individual expert dysmorphism scores in differentiating Mendelian from non-Mendelian cases. Furthermore, the computational asymmetry score enhanced the average expert opinion in predicting a Mendelian cause. By design, our study does not allow to draw conclusions on the actual point-of-care use of 3D facial analysis. Nevertheless, 3D morphometric analysis is promising for developing clinical dysmorphology applications in diagnostics and training.

Taking the risk. A systematic review of ethical reasons and moral arguments in the clinical use of polygenic risk scores.

Debates about the prospective clinical use of polygenic risk scores (PRS) have grown considerably in the last years. The potential benefits of PRS to improve patient care at individual and population levels have been extensively underlined. Nonetheless, the use of PRS in clinical contexts presents a number of unresolved ethical challenges and consequent normative gaps that hinder their optimal implementation. Here, we conducted a systematic review of reasons of the normative literature discussing ethical issues and moral arguments related to the use of PRS for the prevention and treatment of common complex diseases. In total, we have included and analyzed 34 records, spanning from 2013 to 2023. The findings have been organized in three major themes: in the first theme, we consider the potential harms of PRS to individuals and their kin. In the theme "Threats to health equity," we consider ethical concerns of social relevance, with a focus on justice issues. Finally, the theme "Towards best practices" collects a series of research priorities and provisional recommendations to be considered for an optimal clinical translation of PRS. We conclude that the use of PRS in clinical care reinvigorates old debates in matters of health justice; however, open questions, regarding best practices in clinical counseling, suggest that the ethical considerations applicable in monogenic settings will not be sufficient to face PRS emerging challenges.

3D facial phenotyping by biometric sibling matching used in contemporary genomic methodologies.

The analysis of contemporary genomic data typically operates on one-dimensional phenotypic measurements (e.g. standing height). Here we report on a data-driven, family-informed strategy to facial phenotyping that searches for biologically relevant traits and reduces multivariate 3D facial shape variability into amendable univariate measurements, while preserving its structurally complex nature. We performed a biometric identification of siblings in a sample of 424 children, defining 1,048 sib-shared facial traits. Subsequent quantification and analyses in an independent European cohort (n = 8,246) demonstrated significant heritability for a subset of traits (0.17-0.53) and highlighted 218 genome-wide significant loci (38 also study-wide) associated with facial variation shared by siblings. These loci showed preferential enrichment for active chromatin marks in cranial neural crest cells and embryonic craniofacial tissues and several regions harbor putative craniofacial genes, thereby enhancing our knowledge on the genetic architecture of normal-range facial variation.

Exploring attitudes and experiences with reproductive genetic carrier screening among couples seeking medically assisted reproduction: a longitudinal survey study.

PURPOSE: This study aimed to assess the attitudes and experiences of subfertile couples applying for medically assisted reproduction (MAR) using their own gametes towards reproductive genetic carrier screening (RGCS) for monogenic conditions. METHODS: A prospective survey study was conducted where subfertile couples were recruited from the fertility centre of a university hospital in Flanders, Belgium. Participants were offered RGCS free of charge and completed self-administered questionnaires at three different time points. RESULTS: The study sample consisted of 26 couples. Most participants had no children, did not consider themselves as religious, and had some form of higher education. Overall, attitudes towards RGCS were mostly positive and the intention to participate in RGCS was high. Anxiety scores were only elevated and clinically relevant for a limited number of participants. A large proportion of participants would consider preventive reproductive options like prenatal diagnosis or in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) combined with pre-implantation genetic testing for monogenic conditions (PGT-M) in the event of an increased likelihood of conceiving a child with a hereditary condition. Participants were satisfied with their decision to undergo RGCS, and the majority would recommend RGCS to other couples. CONCLUSION: Our study findings suggest that subfertile couples applying for MAR using their own gametes find RGCS acceptable and have a positive attitude towards it. This study provides valuable insights into the perspectives of these couples, highlighting the need for appropriate counseling and timely information provision.

NCKAP1 Disruptive Variants Lead to a Neurodevelopmental Disorder with Core Features of Autism.

NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development.

Refining nosology by modelling variation among facial phenotypes: the RASopathies.

BACKGROUND: In clinical genetics, establishing an accurate nosology requires analysis of variations in both aetiology and the resulting phenotypes. At the phenotypic level, recognising typical facial gestalts has long supported clinical and molecular diagnosis; however, the objective analysis of facial phenotypic variation remains underdeveloped. In this work, we propose exploratory strategies for assessing facial phenotypic variation within and among clinical and molecular disease entities and deploy these techniques on cross-sectional samples of four RASopathies: Costello syndrome (CS), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC) and neurofibromatosis type 1 (NF1). METHODS: From three-dimensional dense surface scans, we model the typical phenotypes of the four RASopathies as average 'facial signatures' and assess individual variation in terms of direction (what parts of the face are affected and in what ways) and severity of the facial effects. We also derive a metric of phenotypic agreement between the syndromes and a metric of differences in severity along similar phenotypes. RESULTS: CFC shows a relatively consistent facial phenotype in terms of both direction and severity that is similar to CS and NS, consistent with the known difficulty in discriminating CFC from NS based on the face. CS shows a consistent directional phenotype that varies in severity. Although NF1 is highly variable, on average, it shows a similar phenotype to CS. CONCLUSIONS: We established an approach that can be used in the future to quantify variations in facial phenotypes between and within clinical and molecular diagnoses to objectively define and support clinical nosologies.

De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder.

PURPOSE: KLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20. METHODS: Patients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed. RESULTS: We studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type ß-propeller domain of the KLHL20 protein, which shapes the substrate binding surface. CONCLUSION: Our findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity.

Reasons affecting the uptake of reproductive genetic carrier screening among nonpregnant reproductive-aged women in Flanders (Belgium).

Reproductive genetic carrier screening (RGCS) allows to identify couples who have an increased likelihood of conceiving a child affected with an autosomal recessive or X-linked monogenic condition. Multiple studies have reported on a wide and fragmented set of reasons to accept or decline RGCS. Only a few studies have been performed to assess the uptake of RGCS. Nonpregnant women visiting their gynecologist were invited to complete a questionnaire assessing perceived susceptibility, the acceptability of offering RGCS, attitudes, the intention to participate in RGCS, reasons to accept or decline RGCS, and sociodemographic characteristics. Women who showed the intention to have RGCS were asked to consider a free RGCS offer. Most women (n = 127) were between 25 and 34 years old (60%), in a relationship (91%), and wanted to have children (65%). Study participants had positive attitudes towards RGCS and the intention to consider RGCS in the future. Reasons to accept RGCS were being able to share genetic information with children or relatives (n = 104/127, 82%), to prevent the birth of a child affected with a hereditary condition (n = 103/127, 81%), and/or to know the chance of conceiving a child with a hereditary condition (n = 102/127, 80%). Reasons for declining RGCS were the possible concerns that could arise when receiving test results (n = 27/127, 21%), having no family history of hereditary disorders (n = 19/127, 15%), and not wanting to take action based on test results (n = 13/127, 10%). Among test intenders that met the inclusion criteria, 53% decided to participate in RGCS together with their male reproductive partner. More in-depth research on the decision-making process behind the choice to accept or decline an RGCS offer would be highly valuable to make sure couples are making informed reproductive choices.

8p21.3 deletions are rare causes of non-syndromic autism spectrum disorder.

A de novo 0.95 Mb 8p21.3 deletion had been identified in an individual with non-syndromic autism spectrum disorder (ASD) through high-resolution copy number variant analysis. Subsequent screening of in-house and publicly available databases resulted in the identification of six additional individuals with 8p21.3 deletions. Through case-based reasoning, we conclude that 8p21.3 deletions are rare causes of non-syndromic neurodevelopmental and neuropsychiatric disorders. Based on literature data, we highlight six genes within the region of minimal overlap as potential ASD genes or genes for neuropsychiatric disorders: DMTN, EGR3, FGF17, LGI3, PHYHIP, and PPP3CC.

NFIB Haploinsufficiency Is Associated with Intellectual Disability and Macrocephaly.

The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.

NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons.

BACKGROUND: NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/ß/γ. Previous studies on cultured cells show that the short NRXN1ß primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. METHODS: In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. RESULTS: NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. CONCLUSIONS: Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

Biallelic MADD variants cause a phenotypic spectrum ranging from developmental delay to a multisystem disorder.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.

The clinical relevance of intragenic NRXN1 deletions.

BACKGROUND: Intragenic NRXN1 deletions are susceptibility variants for neurodevelopmental disorders; however, their clinical interpretation is often unclear. Therefore, a literature study and an analysis of 43 previously unpublished deletions are provided. METHODS: The literature cohort covered 629 heterozygous NRXN1 deletions: 148 in controls, 341 in probands and 140 in carrier relatives, and was used for clinical hypothesis testing. Exact breakpoint determination was performed for 43 in-house deletions. RESULTS: The prevalence of exonic NRXN1 deletions in controls was ~1/3000 as compared with ~1/800 in patients with neurodevelopmental/neuropsychiatric disorders. The differential distribution of deletions across the gene between controls and probands allowed to distinguish distinct areas within the gene. Exon 6-24 deletions appeared only twice in over 100000 control individuals, had an estimated penetrance for neurodevelopmental disorders of 32.43%, a de novo rate of 50% and segregated mainly with intellectual disability (ID) and schizophrenia. In contrast, exon 1-5 deletions appeared in 20 control individuals, had an estimated penetrance of 12.59%, a de novo rate of 32.5% and were reported with a broad range of neurodevelopmental phenotypes. Exact breakpoint determination revealed six recurrent intron 5 deletions. CONCLUSION: Exon 6-24 deletions have a high penetrance and are mainly associated with ID and schizophrenia. In contrast, the actual contribution of exon 1-5 deletions to a neurodevelopmental/neuropsychiatric disorder in an individual patient and family remains very difficult to assess. To enhance the clinical interpretation, this study provides practical considerations for counselling and an interactive table for comparing a deletion of interest with the available literature data.

Compound heterozygous loss-of-function mutations in KIF20A are associated with a novel lethal congenital cardiomyopathy in two siblings.

Congenital or neonatal cardiomyopathies are commonly associated with a poor prognosis and have multiple etiologies. In two siblings, a male and female, we identified an undescribed type of lethal congenital restrictive cardiomyopathy affecting the right ventricle. We hypothesized a novel autosomal recessive condition. To identify the cause, we performed genetic, in vitro and in vivo studies. Genome-wide SNP typing and parametric linkage analysis was done in a recessive model to identify candidate regions. Exome sequencing analysis was done in unaffected and affected siblings. In the linkage regions, we selected candidate genes that harbor two rare variants with predicted functional effects in the patients and for which the unaffected sibling is either heterozygous or homozygous reference. We identified two compound heterozygous variants in KIF20A; a maternal missense variant (c.544C>T: p.R182W) and a paternal frameshift mutation (c.1905delT: p.S635Tfs*15). Functional studies confirmed that the R182W mutation creates an ATPase defective form of KIF20A which is not able to support efficient transport of Aurora B as part of the chromosomal passenger complex. Due to this, Aurora B remains trapped on chromatin in dividing cells and fails to translocate to the spindle midzone during cytokinesis. Translational blocking of KIF20A in a zebrafish model resulted in a cardiomyopathy phenotype. We identified a novel autosomal recessive congenital restrictive cardiomyopathy, caused by a near complete loss-of-function of KIF20A. This finding further illustrates the relationship of cytokinesis and congenital cardiomyopathy.

Frequency and Management of Craniofacial Syndromes.

BACKGROUND: Craniofacial syndromes occur in approximately 1 in 5600 to 100,000 infants, often resulting in significant morbidity. Due to the heterogeneity of this patient population, no clear consensus consists on optimal treatment modalities and timing. The aim of this study was to analyze the craniofacial syndrome population that were treated at the University Hospital Leuven. METHODS: A retrospective analysis of patients with a clinical diagnosis of a craniofacial syndrome was performed. Inclusion criteria were patients with a clinical diagnosis of a craniofacial syndrome and that received treatment between "2000-2005" and "2010-2015." Patients with nonsyndromic conditions were excluded. Data regarding patient characteristics, treatment modalities, and treatment outcomes were analyzed. RESULTS: After matching the inclusion criteria, 98 eligible patients, affected by 40 different syndromes were included. In the period of "2000 to 2005," 48 patients were treated, as compared to 50 patients in the period of "2010 to 2015." A statistically significant decrease over time is seen for cleft surgery and orthodontic treatment (P = 0.0017 and P = 0.0015, respectively). No statistically significant differences were found concerning the age at which treatment was received (P = 0.42). Significant associations between treatment modalities were found for orthognathic surgery and distraction osteogenesis (P < 0.0001), orthognathic surgery and orthodontic treatment (P < 0.0001), and between orthodontic treatment and distraction osteogenesis (P = 0.03311). CONCLUSION: A decline in cleft reconstruction surgery and orthodontic treatment for patients with craniofacial syndromes was seen over time. A significant association was found between distraction osteogenesis and orthognathic surgery, possibly due to higher reintervention rates for patients treated at a young age.

Maternal copy-number variations in the DMD gene as secondary findings in noninvasive prenatal screening.

PURPOSE: Noninvasive prenatal screening (NIPS) using genome sequencing also reveals maternal copy-number variations (CNVs). Those CNVs can be clinically actionable or harmful to the fetus if inherited. CNVs in the DMD gene potentially causing dystrophinopathies are among the most commonly observed maternal CNVs. We present our experience with maternal DMD gene CNVs detected by NIPS. METHODS: We analyzed the data of maternal CNVs detected in the DMD gene revealed by NIPS. RESULTS: Of 26,123 NIPS analyses, 16 maternal CNVs in the DMD gene were detected (1/1632 pregnant women). Variant classification regarding pathogenicity and phenotypic severity was based on public databases, segregation analysis in the family, and prediction of the effect on the reading frame. Ten CNVs were classified as pathogenic, four as benign, and two remained unclassified. CONCLUSION: NIPS leverages CNV screening in the general population of pregnant women. We implemented a strategy for the interpretation and the return of maternal CNVs in the DMD gene detected by NIPS.

Noise-robust assessment of SNP array based CNV calls through local noise estimation of log R ratios.

Arrays based on single nucleotide polymorphisms (SNPs) have been successful for the large scale discovery of copy number variants (CNVs). However, current CNV calling algorithms still have limitations in detecting CNVs with high specificity and sensitivity, especially in case of small (<100 kb) CNVs. Therefore, this study presents a simple statistical analysis to evaluate CNV calls from SNP arrays in order to improve the noise-robustness of existing CNV calling algorithms. The proposed approach estimates local noise of log R ratios and returns the probability that a certain observation is different from this log R ratio noise level. This probability can be triggered at different thresholds to tailor specificity and/or sensitivity in a flexible way. Moreover, a comparison based on qPCR experiments showed that the proposed noise-robust CNV calls outperformed original ones for multiple threshold values.

The East Flanders Prospective Twin Survey (EFPTS): 55 Years Later.

The East Flanders Prospective Twin Survey (EFPTS) is a registry of multiple births in the province of East Flanders, Belgium. Since its start in 1964, over 10,000 twin-pairs have been registered. EFPTS has several unique features: it is population-based and prospective, with the possibility of long-term follow-up; the twins (and higher order multiple births) are recruited at birth; basic perinatal data are recorded; chorion type and zygosity are established; since 1969, placental biopsies have been taken and frozen at -20°C for future research. Since its origin, the EFPTS has included placental data and allows differentiation of three subtypes of monozygotic twins based on the time of the initial zygotic division: the dichorionic-diamniotic pairs (early, with splitting before the fourth day after fertilization), the monochorionic-diamniotic pairs (intermediate, splitting between the fourth- and the seventh-day postfertilization) and the monochorionic-monoamniotic pairs (late, splitting after the eighth day postfertilization). Studies can be initiated taking into account primary biases, those originating 'in utero'. Such studies could throw new light on the consequences of early embryological events and the gene-environment interactions as far as periconceptional and intrauterine environment are concerned.