De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals where it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologs. Using RNA-sequencing, we show how 5' splice site usage is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 bp region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Towards improved genetic diagnosis of human differences of sex development.

Despite being collectively among the most frequent congenital developmental conditions worldwide, differences of sex development (DSD) lack recognition and research funding. As a result, what constitutes optimal management remains uncertain. Identification of the individual conditions under the DSD umbrella is challenging and molecular genetic diagnosis is frequently not achieved, which has psychosocial and health-related repercussions for patients and their families. New genomic approaches have the potential to resolve this impasse through better detection of protein-coding variants and ascertainment of under-recognized aetiology, such as mosaic, structural, non-coding or epigenetic variants. Ultimately, it is hoped that better outcomes data, improved understanding of the molecular causes and greater public awareness will bring an end to the stigma often associated with DSD.

Beyond the exome: What's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order, and emerging technologies, such as optical genome mapping and long-read DNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to research consortia focused on elucidating the underlying cause of rare unsolved genetic disorders.

Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis.

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting.

nanotatoR: a tool for enhanced annotation of genomic structural variants.

BACKGROUND: Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity. RESULTS: We developed an R-based package, nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external (DGV; DECIPHER; Bionano Genomics BNDB) and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patient's phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted (e.g. from an RNA-Seq dataset, allowing the user to assess the effects of SVs on the transcriptome). Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern (INDELs, inversions, translocations, de novo, etc.). nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoR's annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM. CONCLUSIONS: The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting.

Paucity and Disparity of Publicly Available Sex-Disaggregated Data for the COVID-19 Epidemic Hamper Evidence-Based Decision-Making.

COVID-19 has joined the long list of sexually dimorphic human disorders. Higher lethality in men, evident in the first reports from China, was confirmed in the subsequent Italian outbreak. Newspapers and scientific journals commented on this finding and the preexisting conditions, biological processes, and behavioral differences that may underlie it. However, little appeared to be released about sex differences in severity of disease, comorbidities, rate of recovery, length of hospital stay, or number of tests performed. Systematic analysis of official websites for 20 countries and 6 US states revealed a wide disparity in sex-disaggregated data made available to the public and scholars. Only a handful reported cases by sex. None of the other characteristics, including deaths, were stratified by sex at the time. Beyond suboptimal sex disaggregation, we found a paucity of usable raw data sets and a generalized lack of standardization of captured data, making comparisons difficult. A second round of data capture in April found more complete, but even more disparate, information. Our analysis revealed a wide range of sex ratios among confirmed cases. In countries where a male bias was initially reported, the proportion of women dramatically increased in 3 weeks. Analysis also revealed a complex pattern of sex ratio variation with age. Accurate, peer-reviewed, analysis of harmonized, sex-disaggregated data for characteristics of epidemics, such as availability of testing, suspected source of infection, or comorbidities, will be critical to understand where the observed disparities come from and to generate evidence-based recommendations for decision-making by governments.

Objective documentation of hypospadias anatomy with three-dimensional scanning.

INTRODUCTION: The absence of a standardized classification of hypospadias hinders understanding of the anatomic differences among patients and the evaluation of outcomes following surgical repair. In working towards a standardized, objective method of recording patients' hypospadias anatomy, we describe our initial experience using a non-invasive three-dimensional scanner. MATERIAL AND METHODS: An Artec3D Space Spider scanner was used to obtain 3D scans in 29 patients undergoing hypospadias repair. Measurements of the urethral plate width, urethral plate length, glans width, penile shaft length, and penile shaft width were made by 2 pediatric urology attendings and 1 pediatric urology fellow. Measurements were compared and inter-rater reliability was calculated. RESULTS: A total of 435 measurements were made on 29 successfully generated 3D scans, ranging from distal to proximal hypospadias. The inter-rater reliability of measurements from the generated 3D models shown good inter-rater reliability of urethral plate width (ICC0.87 [95%CI:0.76,0.93]), penile shaft length (ICC0.87 [95%CI:0.70,0.94]) and glans width (ICC0.83 [95%CI:0.68,0.92]), excellent inter-rater reliability of urethral plate length (ICC0.96) and moderate inter-rater reliability of penile shaft width (ICC0.69 [95%CI:0.44,0.84]). DISCUSSION: There was a high degree of reliability of measurements made across multiple users. Calculation of the ratio of the urethral plate length/total penile shaft length objectively defined the initial position of the urethral meatus. When compared to the 3-dimensional volume of the glans, a more proximally positioned urethral meatus was associated with a lower glans volume. CONCLUSION: 3D scanning offers a rapid, reproducible, and non-invasive method of documenting hypospadias anatomy. The ability to evaluate three dimensional features (i.e. glans volume) offers an exciting opportunities for robust investigation of hypospadias outcomes and further understanding of the relationship between a patient's genotype and phenotype.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Membrane ß-catenin and adherens junctions in early gonadal patterning.

BACKGROUND: Mechanisms involved in early patterning of the mammalian gonad as it develops from a bipotential state into a testis or an ovary are as yet not well understood. Sex-specific vascularization is essential in this process, but more specific mechanisms required to, for example, establish interstitial vs. cord compartments in the testis or ovigerous cords in the ovary have not been reported. Adherens junctions (AJs) are known for their roles in morphogenesis; we, therefore, examined expression of AJ components including ß-catenin, p120 catenin, and cadherins for possible involvement in sex-specific patterning of the gonad. RESULTS: We show that, at the time of early gonadal sex differentiation, membrane-associated ß-catenin and p120 catenin colocalize with cell-specific cadherins in both sex-nonspecific and sex-specific patterns. These expression patterns are consistent with an influence of AJs in overall patterning of the testis vs. ovary through known AJ mechanisms of cell-cell adhesion, cell sorting, and boundary formation. CONCLUSIONS: Together these complex and dynamic patterns of AJ component expression precisely mirror patterning of tissues during gonadogenesis and raise the possibility that AJs are essential effectors of patterning within the developing testis and ovary.

Genome-wide neonatal epigenetic changes associated with maternal exposure to the COVID-19 pandemic.

BACKGROUND: During gestation, stressors to the fetus, including viral exposure or maternal psychological distress, can fundamentally alter the neonatal epigenome, and may be associated with long-term impaired developmental outcomes. The impact of in utero exposure to the COVID-19 pandemic on the newborn epigenome has yet to be described. METHODS: This study aimed to determine whether there are unique epigenetic signatures in newborns who experienced otherwise healthy pregnancies that occurred during the COVID-19 pandemic (Project RESCUE). The pre-pandemic control and pandemic cohorts (Project RESCUE) included in this study are part of a prospective observational and longitudinal cohort study that evaluates the impact of elevated prenatal maternal stress during the COVID-19 pandemic on early childhood neurodevelopment. Using buccal swabs collected at birth, differential DNA methylation analysis was performed using the Infinium MethylationEPIC arrays and linear regression analysis. Pathway analysis and gene ontology enrichment were performed on resultant gene lists. RESULTS: Widespread differential methylation was found between neonates exposed in utero to the pandemic and pre-pandemic neonates. In contrast, there were no apparent epigenetic differences associated with maternal COVID-19 infection during pregnancy. Differential methylation was observed among genomic sites that underpin important neurological pathways that have been previously reported in the literature to be differentially methylated because of prenatal stress, such as NR3C1. CONCLUSIONS: The present study reveals potential associations between exposure to the COVID-19 pandemic during pregnancy and subsequent changes in the newborn epigenome. While this finding warrants further investigation, it is a point that should be considered in any study assessing newborn DNA methylation studies obtained during this period, even in otherwise healthy pregnancies.

Beyond the exome: what's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order and emerging technologies, such as optical genome mapping and long-read DNA or RNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to a consortium such as GREGoR, which is focused on elucidating the underlying cause of rare unsolved genetic disorders.

Newborn Screening Protocols and Positive Predictive Value for Congenital Adrenal Hyperplasia Vary across the United States.

Newborn screening for congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency is mandated throughout the US. Filter paper blood specimens are assayed for 17-hydroxyprogesterone (17OHP). Prematurity, low birth weight, or critical illness cause falsely elevated results. The purpose of this report is to highlight differences in protocols among US state laboratories. We circulated a survey to state laboratory directors requesting qualitative and quantitative information about individual screening programs. Qualitative and quantitative information provided by 17 state programs were available for analysis. Disease prevalence ranged from 1:9941 to 1:28,661 live births. Four state laboratories mandated a second screen regardless of the initial screening results; most others did so for infants in intensive care units. All but one program utilized birthweight cut-points, but cutoffs varied widely: 17OHP values of 25 to 75 ng/mL for birthweights >2250-2500 g. The positive predictive values for normal birthweight infants varied from 0.7% to 50%, with the highest predictive values based in two of the states with a mandatory second screen. Data were unavailable for negative predictive values. These data imply differences in sensitivity and specificity in CAH screening in the US. Standardization of newborn screening protocols could improve the positive predictive value.

New technologies to uncover the molecular basis of disorders of sex development.

The elegant developmental biology experiments conducted in the 1940s by French physiologist Alfred Jost demonstrated that the sexual phenotype of a mammalian embryo depended whether the embryonic gonad develops into a testis or not. In humans, anomalies in the processes that regulate development of chromosomal, gonadal or anatomic sex result in a spectrum of conditions termed Disorders/Differences of Sex Development (DSD). Each of these conditions is rare, and understanding of their genetic etiology is still incomplete. Historically, DSD diagnoses have been difficult to establish due to the lack of standardization of anatomical and endocrine phenotyping procedures as well as genetic testing. Yet, a definitive diagnosis is critical for optimal management of the medical and psychosocial challenges associated with DSD conditions. The advent in the clinical realm of next-generation sequencing methods, with constantly decreasing price and turnaround time, has revolutionized the diagnostic process. Here we review the successes and limitations of the genetic methods currently available for DSD diagnosis, including Sanger sequencing, karyotyping, exome sequencing and chromosomal microarrays. While exome sequencing provides higher diagnostic rates, many patients still remain undiagnosed. Newer approaches, such as whole-genome sequencing and whole-genome mapping, along with gene expression studies, have the potential to identify novel DSD-causing genes and significantly increase total diagnostic yield, hopefully shortening the patient's journey to an accurate diagnosis and enhancing health-related quality-of-life outcomes for patients and families.

Psychosocial Screening in Disorders/Differences of Sex Development: Psychometric Evaluation of the Psychosocial Assessment Tool.

BACKGROUND/AIMS: Utilization of a psychosocial screener to identify families affected by a disorder/difference of sex development (DSD) and at risk for adjustment challenges may facilitate efficient use of team resources to optimize care. The Psychosocial Assessment Tool (PAT) has been used in other pediatric conditions. The current study explored the reliability and validity of the PAT (modified for use within the DSD population; PAT-DSD). METHODS: Participants were 197 families enrolled in the DSD-Translational Research Network (DSD-TRN) who completed a PAT-DSD during a DSD clinic visit. Psychosocial data were extracted from the DSD-TRN clinical registry. Internal reliability of the PAT-DSD was tested using the Kuder-Richardson-20 coefficient. Validity was examined by exploring the correlation of the PAT-DSD with other measures of caregiver distress and child emotional-behavioral functioning. RESULTS: One-third of families demonstrated psychosocial risk (27.9% "Targeted" and 6.1% "Clinical" level of risk). Internal reliability of the PAT-DSD Total score was high (α = 0.86); 4 of 8 subscales met acceptable internal reliability. A priori predicted relationships between the PAT-DSD and other psychosocial measures were supported. The PAT-DSD Total score related to measures of caregiver distress (r = 0.40, p < 0.001) and to both caregiver-reported and patient self-reported behavioral problems (r = 0.61, p < 0.00; r = 0.37, p < 0.05). CONCLUSIONS: This study provides evidence for the reliability and validity of the PAT-DSD. Given variability in the internal reliability across subscales, this measure is best used to screen for overall family risk, rather than to assess specific psychosocial concerns.

Identification of novel candidate genes for 46,XY disorders of sex development (DSD) using a C57BL/6J-Y POS mouse model.

BACKGROUND: Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. METHODS: To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (Y POS ) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-Y B6 and undervirilized B6-Y POS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-Y POS males). RESULTS: We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-Y POS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. CONCLUSION: The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation.

Genetics of Disorders of Sex Development: The DSD-TRN Experience.

Although many next-generation sequencing platforms are being created around the world, implementation is facing multiple hurdles. A strong hurdle to the full adherence of clinical teams to the Disorders of Sex Development Translational Research Network (DSD-TRN) guidelines for standardization of reporting and practice is the current lack of integration of the standardized clinical forms into the various electronic medical records at different sites. Time allocated to research is also limited. In spite of these hurdles, genetic information for half the enrolled patients is already available in the DSD-TRN registry, and early results demonstrate the value of such an infrastructure.

Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis.

BACKGROUND: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations. METHODS: To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene. RESULTS: We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45-250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing. CONCLUSIONS: We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome.

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Abnormal venous and arterial patterning in Chordin mutants.

Classic dye injection methods yielded amazingly detailed images of normal and pathological development of the cardiovascular system. However, because these methods rely on the beating heart of diffuse the dyes, the vessels visualized have been limited to the arterial tree, and our knowledge of vein development is lagging. In order to solve this problem, we injected pigmented methylsalicylate resins in mouse embryos after they were fixed and made transparent. This new technique allowed us to image the venous system and prompted the discovery of multiple venous anomalies in Chord-/- mutant mice. Genetic inactivation of Chordin, an inhibitor of the Bone Morphogenetic Protein signaling pathway, results in neural crest defects affecting heart and neck organs, as seen in DiGeorge syndrome patients. Injection into the descending aorta of Chrd-/- mutants demonstrated how a very severe early phenotype of the aortic arches develops into persistent truncus arteriosus. In addition, injection into the atrium revealed several patterning defects of the anterior cardinal veins and their tributaries, including absence of segments, looping and midline defects. The signals that govern the development of the individual cephalic veins are unknown, but our results show that the Bone Morphogenetic Protein pathway is necessary for the process.

Differential perturbation of the Fgf/Erk1/2 and Shh pathways in the C57BL/6N and SWV embryonic limb buds after mid-gestational cadmium chloride administration.

The differential susceptibility of inbred mouse strains to teratogen-induced malformations can serve as a model to assess the molecular pathogenesis of dysmorphology. Using such a model, the teratogenic effect of cadmium chloride (CdCl(2)), which results in limb reduction deformities in the C57BL/6N mouse strain, but not in the SWV strain, was found to correlate with reduction of the expression domains of Fgf8/4 (fibroblast growth factor-8 and -4) in the apical ectodermal ridge (AER) and Shh (sonic hedgehog) in the posterior mesenchyme, as well as reduction of MAPK/Erk1/2 (the mitogen-activated protein kinase/extracellular regulated kinase 1/2) phosphorylation (pErk1/2) in the mesenchyme throughout the limb bud. The pattern of pErk1/2 reduction did not consistently reflect the pattern of Fgf8/4 reduction suggesting that CdCl(2) might affect pErk1/2 through an Fgf-independent pathway. Other potential downstream mediators of the Fgf pathway including Mkp3 and Fgf10 as well as pMek (phosphorylated MAPK/Erk1/2 kinase) were not different in limb buds between the two strains at the studied time points. The effect of CdCl(2) on skeletogenesis was traced in time to the early stages of pre-chondrogenic condensation as determined by the Sox9 expression domain. The data of the present study indicate that a differential strain response to CdCl(2)-induced forelimb digital loss may be due to a polymorphic interference with the Fgf/Shh positive feedback loop and Erk1/2 phosphorylation.