Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Cranial neural crest cells on the move: their roles in craniofacial development.

The craniofacial region is assembled through the active migration of cells and the rearrangement and sculpting of facial prominences and pharyngeal arches, which consequently make it particularly susceptible to a large number of birth defects. Genetic, molecular, and cellular processes must be temporally and spatially regulated to culminate in the three-dimension structures of the face. The starting constituent for the majority of skeletal and connective tissues in the face is a pluripotent population of cells, the cranial neural crest cells (NCCs). In this review we discuss the newest scientific findings in the development of the craniofacial complex as related to NCCs. Furthermore, we present recent findings on NCC diseases called neurocristopathies and, in doing so, provide clinicians with new tools for understanding a growing number of craniofacial genetic disorders.

Craniofacial ciliopathies: A new classification for craniofacial disorders.

Craniofacial anomalies are some of the most variable and common defects affecting the population. Herein, we examine a group of craniofacial disorders that are the result of defects in primary cilia; ubiquitous, microtubule-based organelles that transduce molecular signals and facilitate the interactions between the cell and its environment. Based on the frequent appearance of craniofacial phenotypes in diseases born from defective primary cilia (ciliopathies) we propose a new class of craniofacial disorders referred to as craniofacial ciliopathies. We explore the most frequent phenotypes associated with ciliopathic conditions and the ciliary gene mutations responsible for craniofacial defects. Finally, we propose that some non-classified disorders may now be classified as craniofacial ciliopathies.

Holoprosencephaly-Polydactyly syndrome: in search of an etiology.

Holoprosencephaly-Polydactyly (HPS) or Pseudotrisomy 13 syndrome are names conferred to clinically categorize patients whose phenotype is congruent with Trisomy 13 in the context of a normal karyotype. The literature suggests that this entity may be secondary to submicroscopic deletions in holoprosencephaly (HPE) genes; however, a limited number of investigations have been undertaken to evaluate this hypothesis. To test this hypothesis we studied a patient with HPE, polydactyly, and craniofacial dysmorphologies consistent with the diagnosis of Trisomy 13 whose karyotype was normal. We performed mutational analysis in the four main HPE causing genes (SHH, SIX3, TGIF, and ZIC2) and GLI3, a gene associated with polydactyly as well as fluorescent in situ hybridization (FISH) to search for microdeletions in these genes and two candidate HPE genes (DISP1 and FOXA2). No mutations or deletions were detected. A whole genome approach utilizing array Comparative Genomic Hybridization (aCGH) to screen for copy number abnormalities was then taken. No loss or gain of DNA was noted. Although a single case, our results suggest that coding mutations in these HPE genes and copy number anomalies may not be causative in this disorder. Instead, HPS likely involves mutations in other genes integral in embryonic development of the forebrain, face and limbs. Our systematic analysis sets the framework to study other affected children and delineate the molecular etiology of this disorder.

Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence.

The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.

Cocaine body packing in pregnancy.

Ingesting multiple packets of drugs ("body packing") is a well-described method of smuggling. Although older reports suggested that body packers were mostly young men, the demographics of this group may be changing because children, older patients, and pregnant women may be involved. Pregnant patients represent a challenge in management, particularly in the event of package rupture. Modification of standard management protocols, which were developed for nonpregnant body packers, may be necessary to address the anatomic and physiologic changes of pregnancy. We report the case of a pregnant cocaine body packer who required a perimortem cesarean section after the rupture of a cocaine packet. The care of the pregnant body packer is discussed.

Molecular interactions coordinating the development of the forebrain and face.

From an architectural point of view, the forebrain acts as a framework upon which the middle and upper face develops and grows. In addition to serving a structural role, we present evidence that the forebrain is a source of signals that shape the facial skeleton. In this study, we inhibited Sonic hedgehog (Shh) signaling from the neuroectoderm then examined the molecular changes and the skeletal alterations resulting from the treatment. One of the first changes we noted was that the dorsoventral polarity of the forebrain was disturbed, which manifested as a loss of Shh in the ventral telencephalon, a reduction in expression of the ventral markers Nkx2.1 and Dlx2, and a concomitant expansion of the dorsal marker Pax6. In addition to changes in the forebrain neuroectoderm, we observed altered gene expression patterns in the facial ectoderm. For example, Shh was not induced in the frontonasal ectoderm, and Ptc and Gli1 were reduced in both the ectoderm and adjacent mesenchyme. As a consequence, a signaling center in the frontonasal prominence was disrupted and the prominence failed to undergo proximodistal and mediolateral expansion. After 15 days of development, the upper beaks of the treated embryos were truncated, and the skeletal elements were located in more medial and proximal locations in relation to the skeletal elements of the lower jaw elements. These data indicate that a role of Shh in the forebrain is to regulate Shh expression in the face, and that together, these Shh domains mediate patterning within the frontonasal prominence and proximodistal outgrowth of the middle and upper face.

New insights into craniofacial morphogenesis.

No region of our anatomy more powerfully conveys our emotions nor elicits more profound reactions when disease or genetic disorders disfigure it than the face. Recent progress has been made towards defining the tissue interactions and molecular mechanisms that control craniofacial morphogenesis. Some insights have come from genetic manipulations and others from tissue recombinations and biochemical approaches, which have revealed the molecular underpinnings of facial morphogenesis. Changes in craniofacial architecture also lie at the heart of evolutionary adaptation, as new studies in fish and fowl attest. Together, these findings reveal much about molecular and tissue interactions behind craniofacial development.

Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes.

One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway.

Anatomical studies of the fibroblast growth factor-10 mutant, Sonic Hedge Hog mutant and androgen receptor mutant mouse genital tubercle.

OBJECTIVES: Congenital genital abnormalities have a diverse spectrum from hypospadias to cloacal anomalies. The molecular events in the normal and abnormal development of the genital tubercle (GT) are still obscure. Genetically engineered mice with specific gene deletions that affect genital anatomy are a useful tool to better understand the etiology of genital abnormalities. In this study, we compared the genital tubercle anatomy of the androgen receptor (AR) deficient, fibroblastic growth factor (FGF)-10 deficient and Sonic HedgeHog (Shh) deficient mutant male mice to that of the wild type male and female mouse. MATERIALS AND METHODS: The lower pelvis of the androgen receptor deficient, FGF-10 deficient, Shh deficient mutant male and wild type male and female mouse at different gestational days (E13-21) and post natal ages (1 day-1 week) were studied. GTs were imaged, serially sectioned and stained immunohistochemically with antibodies raised against E-Cadherin, Cytokeratin 7, 10 and 14. Serial sections of the GTs were selected and three-dimensional computerized images were created to better elucidate the anatomy. RESULTS: AR deficient mutant male mouse revealed a distinctive GT anatomy, different from both sexes. The corporal bodies and glans remained hypoplastic whereas the urethral spongiosa was more developed than the wild type female counterpart. This finding is consistent with the AR mutant mouse being a unique morphologic phenotype distinct from the normal male and female. FGF-10 deficient mutant male mouse revealed normal corporal bodies with failure of the urethral plate to fuse ventrally consistent with hypospadias. The Shh deficient mutant mouse demonstrated complete agenesis of GT outgrowth and a persistent cloaca. CONCLUSION: Animal models bred by gene knockout technology or natural occurring mutants contribute to the basic understanding of normal and abnormal GT development. The anatomy of the these three mutant mice confirms the importance of the androgen receptor, FGF-10 and Shh in genital development.