Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.

DISP1 deficiency: Monoallelic and biallelic variants cause a spectrum of midline craniofacial malformations.

PURPOSE: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog, a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with division transporter dispatched-1 (DISP1) variants. METHODS: This study was based on the identification of at least 1 pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available. RESULTS: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor, and congenital nasal pyriform aperture stenosis. These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (ie, oligogenic inheritance). CONCLUSION: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of Sonic hedgehog. We also demonstrated that the very rare solitary median maxillary central incisor and congenital nasal pyriform aperture stenosis combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant.

A statistical shape model for estimating missing soft tissues of the face in a black South African population.

PURPOSE: Facial disfigurement may affect the quality of life of many patients. Facial prostheses are often used as an adjuvant to surgical intervention and may sometimes be the only viable treatment option. Traditional methods for designing soft-tissue facial prostheses are time-consuming and subjective, while existing digital techniques are based on mirroring of contralateral features of the patient, or the use of existing feature templates/models that may not be readily available. We aim to support the objective and semi-automated design of facial prostheses with primary application to midline or bilateral defect restoration where no contralateral features are present. Specifically, we developed and validated a statistical shape model (SSM) for estimating the shape of missing facial soft tissue segments, from any intact parts of the face. MATERIALS AND METHODS: An SSM of 3D facial variations was built from meshes extracted from computed tomography and cone beam computed tomography images of a black South African sample (n = 235) without facial disfigurement. Various types of facial defects were simulated, and the missing parts were estimated automatically by a weighted fit of each mesh to the SSM. The estimated regions were compared to the original regions using color maps and root-mean-square (RMS) distances. RESULTS: Root mean square errors (RMSE) for defect estimations of one orbit, partial nose, cheek, and lip were all below 1.71 mm. Errors for the full nose, bi-orbital defects, as well as small and large composite defects were between 2.10 and 2.58 mm. Statistically significant associations of age and type of defect with RMSE were observed, but not with sex or imaging modality. CONCLUSION: This method can support the objective and semi-automated design of facial prostheses, specifically for defects in the midline, crossing the midline or bilateral defects, by facilitating time-consuming and skill-dependent aspects of prosthesis design.

Congenital Athymia: Genetic Etiologies, Clinical Manifestations, Diagnosis, and Treatment.

Congenital athymia is an ultra-rare disease characterized by the absence of a functioning thymus. It is associated with several genetic and syndromic disorders including FOXN1 deficiency, 22q11.2 deletion, CHARGE Syndrome (Coloboma, Heart defects, Atresia of the nasal choanae, Retardation of growth and development, Genitourinary anomalies, and Ear anomalies), and Complete DiGeorge Syndrome. Congenital athymia can result from defects in genes that impact thymic organ development such as FOXN1 and PAX1 or from genes that are involved in development of the entire midline region, such as TBX1 within the 22q11.2 region, CHD7, and FOXI3. Patients with congenital athymia have profound immunodeficiency, increased susceptibility to infections, and frequently, autologous graft-versus-host disease (GVHD). Athymic patients often present with absent T cells but normal numbers of B cells and Natural Killer cells (T-B+NK+), similar to a phenotype of severe combined immunodeficiency (SCID); these patients may require additional steps to confirm the diagnosis if no known genetic cause of athymia is identified. However, distinguishing athymia from SCID is crucial, as treatments differ for these conditions. Cultured thymus tissue is being investigated as a treatment for congenital athymia. Here, we review what is known about the epidemiology, underlying etiologies, clinical manifestations, and treatments for congenital athymia.

Mesenchymal Wnt signaling promotes formation of sternum and thoracic body wall.

Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine-an O-acetyl transferase mediating Wnt ligand acylation-with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wls(f/f);Dermo1(Cre/+) embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wls(f/f);Dermo1(Cre/+) embryos, we tested whether Wnt/ß-catenin signaling was operative in developing the embryonic ventral body wall using Axin2(LacZ) and BatGal reporter mice. While Wnt/ß-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wls(f/f);Dermo1(Cre/+) thoracic body wall partially due to anomalous ß-catenin independent Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/ß-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wls(f/f);Dermo1(Cre/+) embryos supporting a role for Wnt/ß-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt ligands from the developing ventral body wall mesenchyme plays a critical role in fusion of the sternum and closure of the secondary body wall. Thus, impaired Wls activity in the ventral body wall mesenchyme is a mechanism underlying ectopia cordis and unfused sternum.

Trio-based whole exome sequencing in patients with ectopic posterior pituitary.

Introduction: Ectopic posterior pituitary (EPP) is a rare congenital abnormality, sometimes associated with other midline defects, such as pituitary stalk interruption syndrome (PSIS), in which thin or absent pituitary stalk and anterior pituitary hypoplasia are combined to EPP. Most cases are sporadic, with few reports of familial cases, and many congenital hypopituitarism (CH) cases remain unsolved. Objective: To search for candidate genes associated with this condition, we performed trio-based whole-exome sequencing (WES) on patients with EPP, including two familial cases. Methods: This study included subjects with EPP and PSIS diagnosed by a simple MRI protocol (FAST1.2). We performed two distinct analyses in the trio-based WES. We looked for previously described genes associated with pituitary development. Next, we investigated the whole exome for variants inherited in a pattern consistent with a monogenic etiology. Results: Ten families were evaluated; eight were composed of a child with EPP and healthy parents, one has two affected siblings, and one family has a son and mother with EPP. When analyzing the previously described candidate variants associated with pituitary development, we found variants in GLI2 and FGFR1 in three families. We also found six other variants of interest in three patients: KMT2A, GALR3, RTN4R, SEMA3A, NIPBL, and DSCAML1. Conclusion: The analysis allowed us to find previously reported and not reported GLI2 variants, all inherited from healthy parents, which reinforces the incomplete penetrance pattern of GLI2 variants in the development of EPP and draws attention to possible future functional studies of those variants that have a recurrent expression in CH. We also found novel FGFR1 and SEMA3A variants that suggest an oligogenic mechanism in PSIS and EPP, as seen in patients with hypogonadotropic hypogonadism. We report the first case of a patient with Wiedemann-Steiner syndrome and PSIS, suggesting that the KMT2A gene may be related to pituitary development. Furthermore, the trios' analysis allowed us to find five other variants of interest. Future investigations may clarify the roles of these variants in the etiology of EPP and PSIS.

Whole Exome Sequencing in Patients With Ectopic Posterior Pituitary.

Context: Ectopic posterior pituitary (EPP), a condition in which the posterior pituitary gland is displaced due to defective neuronal migration, is frequently associated with hypopituitarism. Genetic variants play a role, but many cases remain unexplained. Objective: A large EPP cohort was studied to explore the importance of genetic variants and how they correlate with clinical findings. Methods: Whole exome sequencing was performed on a discovery sample of 27 cases to identify rare variants. The variants that met the criteria for rarity and biological relevance, or that were previously associated with EPP (ROBO1 and HESX1), were then resequenced in the 27 cases plus a replication sample of 51 cases. Results: We identified 16 different variants in 12 genes in 15 of the 78 cases (19.2%). Complete anterior pituitary deficiency was twice as common in cases with variants of interest compared to cases without variants (9/15 [60%] vs 19/63 [30.1%], respectively; Z test, P = 0.06). Breech presentation was more frequent in the variant positive group (5/15 vs 1/63; Z test, P = 0.003). Four cases had variants in ROBO1 and 1 in HESX1, genes previously associated with EPP. The ROBO1 p.S18* variant has not been reported previously; ROBO1 p.Q1227H has not been associated with EPP previously. Conclusion: EPP cases with variants of interest identified in this study were more likely to present with severe clinical disease. Several variants were identified in genes not previously associated with EPP. Our findings confirm that EPP is a multigenic disorder. Future studies are needed to identify additional genes.

Neuropathology of Septo-optic Dysplasia: A Report of 4 Autopsy Cases.

Septo-optic dysplasia (SOD) is defined by the presence of 2 or more features in a diagnostic triad: (1) optic nerve hypoplasia, (2) pituitary dysfunction, and (3) midline forebrain anomalies. SOD arises due to diverse pathogenetic mechanisms including acquired and genetic factors, and it shows considerable clinical and phenotypic variability. Our knowledge of SOD is incomplete in part because of a paucity of published neuropathology data, so we reviewed the autopsy neuropathology of 4 SOD patients. All patients met SOD criteria according to the triad. Additional neuropathologic findings included malformations involving non-forebrain structures and possible secondary phenomena. Autopsies demonstrate that SOD patients often have additional neuropathologic findings beyond the triad and we feel that use of the term SOD-complex appropriately underscores this diversity and its likely clinical impact. This study suggests that autopsies enhance our understanding of SOD and may be an asset in performing needed clinical and phenotypic correlation studies.

Pituitary stalk interruption syndrome.

Pituitary stalk interruption syndrome (PSIS) is a distinct developmental defect of the pituitary gland identified by magnetic resonance imaging and characterized by a thin, interrupted, attenuated or absent pituitary stalk, hypoplasia or aplasia of the adenohypophysis, and an ectopic posterior pituitary. The precise etiology of PSIS still remains elusive or incompletely confirmed in most cases. Adverse perinatal events, including breech delivery and hypoxia, were initially proposed as the underlying mechanism affecting the hypothalamic-pituitary axis. Nevertheless, recent findings have uncovered a wide variety of PSIS-associated molecular defects in genes involved in pituitary development, holoprosencephaly (HPE), neural development, and other important cellular processes such as cilia function. The application of whole exome sequencing (WES) in relatively large cohorts has identified an expanded pool of potential candidate genes, mostly related to the Wnt, Notch, and sonic hedgehog signaling pathways that regulate pituitary growth and development during embryogenesis. Importantly, WES has revealed coexisting pathogenic variants in a significant number of patients; therefore, pointing to a multigenic origin and inheritance pattern of PSIS. The disorder is characterized by inter- and intrafamilial variability and incomplete or variable penetrance. Overall, PSIS is currently viewed as a mild form of an expanded HPE spectrum. The wide and complex clinical manifestations include evolving pituitary hormone deficiencies (with variable timing of onset and progression) and extrapituitary malformations. Severe and life-threatening symptomatology is observed in a subset of patients with complete pituitary hormone deficiency during the neonatal period. Nevertheless, most patients are referred later in childhood for growth retardation. Prompt and appropriate hormone substitution therapy constitutes the cornerstone of treatment. Further studies are needed to uncover the etiopathogenesis of PSIS.

Hypogonadotropic hypogonadism and pituitary hypoplasia as recurrent features in Ulnar-Mammary syndrome.

Ulnar-mammary syndrome (UMS) is characterized by ulnar defects, and nipple or apocrine gland hypoplasia, caused by TBX3 haploinsufficiency. Signs of hypogonadism were repeatedly reported, but the mechanisms remain elusive. We aim to assess the origin of hypogonadism in two families with UMS. UMS was suspected in two unrelated probands referred to an academic center with delayed puberty because of the evident ulnar ray and breast defects in their parents. Clinical, biochemical and genetic investigations proved the existence of congenital normosmic IHH (nIHH) associated with pituitary hypoplasia in the two probands who were heterozygous for novel TBX3 pathogenic variants. The mutations co-segregated with delayed puberty, midline defects (nose, teeth and tongue anomalies) and other variable features of UMS in the two families (absent axillary hairs and nipple hypoplasia, asymmetrical features including unilateral ulnar or renal abnormalities). The combined analysis of these findings and of the previous UMS reports showed delayed puberty and other signs of hypogonadism in 79 and 37% of UMS males, respectively. Proband 1 was followed up to adulthood with persistence of nIHH. In conclusion, UMS should be suspected in patients with delayed puberty and midline defects, including pituitary hypoplasia, in the presence of mild cues for TBX3 mutation, even in the absence of limb malformations. In addition, TBX3 should be included among candidate genes for congenital nIHH.

Mid1/Mid2 expression in craniofacial development and a literature review of X-linked opitz syndrome.

BACKGROUND: Opitz syndrome (OS) is a genetic disorder that affects mainly the development of midline structures, including the craniofacial region, embryonic heart, and urogenital system. The manifestations of X-linked OS are believed to be results of a malfunctioned gene, MID1, whose product has been shown to have ubiquitin E3 ligase activity and regulate the turnover of microtubular protein phosphatase 2Ac. MID2, a homolog of MID1, shares high structural and functional similarities with MID1. Identification of a missense mutation in MID2 in an Indian family causing overlapping phenotypes with OS provided the first evidence that MID2 might be involved in similar pathogenesis. METHODS: The clinic features and the genetic findings of all reported X-linked OS were collectively summarized in this research. Real-time RT-PCR and in situ hybridization were used in the expression studies of Mid1/Mid2 in mouse embryos. RESULTS: Up-to-date, 88 different mutations have been identified in MID1 and most mutations occurred on the conserved amino acids of MID1 and MID2. Expression studies using real-time RT-PCR implicated a tendency of a mutually repressive expression pattern between Mid1 and Mid2 in mouse embryos. Further investigations using in situ hybridization revealed strong expressions of Mid1 and Mid2 in the epithelium of approaching facial prominences and downregulated expressions after fusion in mouse embryos. CONCLUSIONS: Our results support the hypothesis of functional redundancy of Mid1/Mid2 and their potential roles in regulating tissue remodelling in early development.

Complex rearrangement of the exon 6 genomic region among Opitz G/BBB Syndrome MID1 alterations.

Opitz G/BBB Syndrome (OS) is a multiple congenital anomaly disorder characterized by developmental defects of midline structures. The most relevant clinical signs are ocular hypertelorism, hypospadias, cleft lip and palate, laryngo-tracheo-esophageal abnormalities, imperforate anus, and cardiac defects. Developmental delay, intellectual disability and brain abnormalities are also present. The X-linked form of this disorder is caused by mutations in the MID1 gene coding for a member of the tripartite motif family of E3 ubiquitin ligases. Here, we describe 12 novel patients that carry MID1 mutations emphasizing that laryngo-tracheo-esophageal defects are very common in OS patients and, together with hypertelorism and hypospadias, are the most frequent findings among the full spectrum of OS clinical manifestations. Besides missense and nonsense mutations, small insertions and deletions scattered along the entire length of the gene, we found that a consistent number of MID1 alterations are represented by the deletion of single coding exons. Deep characterization of one of these deletions reveals, for the first time within the MID1 gene, a complex rearrangement composed of two deletions, an inversion and a small insertion that may suggest the involvement of concurrent non-homologous mechanisms in the generation of the observed structural variant.

Prevalence of olfactory and other developmental anomalies in patients with central hypogonadotropic hypogonadism.

INTRODUCTION: Hypogonadotropic hypogonadism (HH) is a heterogeneous disease caused by mutations in several genes. Based on the presence of hyposmia/anosmia it is distinguished into Kallmann syndrome (KS) and isolated HH. The prevalence of other developmental anomalies is not well established. METHODS: We studied 36 patients with HH (31 males, 5 females, mean age 41.5), 9 with familial and 27 with sporadic HH (33 congenital, 3 adult-onset), by physical examination, smell test (BSIT Sensonics), audiometry, renal ultrasound, and magnetic resonance imaging of the olfactory structures. RESULTS: Based on the smell test, patients were classified as normosmic (n = 21, 58.3%) and hypo/anosmic (n = 15, 41.6%). Hypoplasia/agenesis of olfactory bulbs was found in 40% of patients (10/25; 75% hypo/anosmic, 7.6% normosmic, p < 0.01, Fisher's test). Remarkably, olfactory structures were normal in two anosmic patients, while one normosmic patient presented a unilateral hypoplastic bulb. Fourteen of 33 patients (42.4%) presented neurosensorial hearing loss of various degrees (28.5% hypo/anosmic, 52.6% normosmic, p = NS). Renal ultrasound revealed 27.7% of cases with renal anomalies (26.6% hypo/anosmic, 28.5% normosmic, p = NS). At least one midline defect was found in 50% of the patients (53.3% hypo/anosmic, 47.6% normosmic, p = NS), including abnormal palate, dental anomalies, pectus excavatum, bimanual synkinesis, iris coloboma, and absent nasal cartilage. Anamnestically 4/31 patients reported cryptorchidism (25% hypo/anosmic, 5.2% normosmic, p = NS). CONCLUSION: Hypo/anosmia is significantly related to anatomical anomalies of the olfactory bulbs/tracts but the prevalence of other developmental anomalies, especially midline defects and neurosensorial hearing loss, is high both in HH and KS and independent of the presence of anosmia/hyposmia. From the clinical standpoint KS and normosmic HH should be considered as the same complex, developmental disease.