Mowat-Wilson Syndrome: Case Report and Review of ZEB2 Gene Variant Types, Protein Defects and Molecular Interactions.

Mowat-Wilson syndrome (MWS) is a rare genetic neurodevelopmental congenital disorder associated with various defects of the zinc finger E-box binding homeobox 2 (ZEB2) gene. The ZEB2 gene is autosomal dominant and encodes six protein domains including the SMAD-binding protein, which functions as a transcriptional corepressor involved in the conversion of neuroepithelial cells in early brain development and as a mediator of trophoblast differentiation. This review summarizes reported ZEB2 gene variants, their types, and frequencies among the 10 exons of ZEB2. Additionally, we summarized their corresponding encoded protein defects including the most common variant, c.2083 C>T in exon 8, which directly impacts the homeodomain (HD) protein domain. This single defect was found in 11% of the 298 reported patients with MWS. This review demonstrates that exon 8 encodes at least three of the six protein domains and accounts for 66% (198/298) of the variants identified. More than 90% of the defects were due to nonsense or frameshift changes. We show examples of protein modeling changes that occurred as a result of ZEB2 gene defects. We also report a novel pathogenic variant in exon 8 in a 5-year-old female proband with MWS. This review further explores other genes predicted to be interacting with the ZEB2 gene and their predicted gene-gene molecular interactions with protein binding effects on embryonic multi-system development such as craniofacial, spine, brain, kidney, cardiovascular, and hematopoiesis.

Chromosomal Microarray Study in Prader-Willi Syndrome.

A high-resolution chromosome microarray analysis was performed on 154 consecutive individuals enrolled in the DESTINY PWS clinical trial for Prader-Willi syndrome (PWS). Of these 154 PWS individuals, 87 (56.5%) showed the typical 15q11-q13 deletion subtypes, 62 (40.3%) showed non-deletion maternal disomy 15 and five individuals (3.2%) had separate unexpected microarray findings. For example, one PWS male had Klinefelter syndrome with segmental isodisomy identified in both chromosomes 15 and X. Thirty-five (40.2%) of 87 individuals showed typical larger 15q11-q13 Type I deletion and 52 individuals (59.8%) showed typical smaller Type II deletion. Twenty-four (38.7%) of 62 PWS individuals showed microarray patterns indicating either maternal heterodisomy 15 subclass or a rare non-deletion (epimutation) imprinting center defect. Segmental isodisomy 15 was seen in 34 PWS subjects (54.8%) with 15q26.3, 15q14 and 15q26.1 bands most commonly involved and total isodisomy 15 seen in four individuals (6.5%). In summary, we report on PWS participants consecutively enrolled internationally in a single clinical trial with high-resolution chromosome microarray analysis to determine and describe an unbiased estimate of the frequencies and types of genetic defects and address potential at-risk genetic disorders in those with maternal disomy 15 subclasses in the largest PWS cohort studied to date.

Connective Tissue Disorders and Fragile X Molecular Status in Females: A Case Series and Review.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disabilities and the second most common cause after Down syndrome. FXS is an X-linked disorder due to a full mutation of the CGG triplet repeat of the FMR1 gene which codes for a protein that is crucial in synaptogenesis and maintaining functions of extracellular matrix-related proteins, key for the development of normal neuronal and connective tissue including collagen. In addition to neuropsychiatric and behavioral problems, individuals with FXS show physical features suggestive of a connective tissue disorder including loose skin and joint laxity, flat feet, hernias and mitral valve prolapse. Disturbed collagen leads to hypermobility, hyperextensible skin and tissue fragility with musculoskeletal, cardiovascular, immune and other organ involvement as seen in hereditary disorders of connective tissue including Ehlers−Danlos syndrome. Recently, FMR1 premutation repeat expansion or carrier status has been reported in individuals with connective tissue disorder-related symptoms. We examined a cohort of females with features of a connective tissue disorder presenting for genetic services using next-generation sequencing (NGS) of a connective tissue disorder gene panel consisting of approximately 75 genes. In those females with normal NGS testing for connective tissue disorders, the FMR1 gene was then analyzed using CGG repeat expansion studies. Three of thirty-nine females were found to have gray zone or intermediate alleles at a 1:13 ratio which was significantly higher (p < 0.05) when compared with newborn females representing the general population at a 1:66 ratio. This association of connective tissue involvement in females with intermediate or gray zone alleles reported for the first time will require more studies on how the size variation may impact FMR1 gene function and protein directly or in relationship with other susceptibility genes involved in connective tissue disorders.

Clinical genetics evaluation and testing of connective tissue disorders: a cross-sectional study.

BACKGROUND: Heritable connective tissue disorders (HCTDs) consist of heterogeneous syndromes. The diagnosis of HCTDs is aided by genomic biotechnologies (e.g., next-generation sequencing panels) facilitating the discovery of novel variants causing disease. METHODS: Detailed clinical exam data and CLIA-approved genetic testing results from next generation sequencing of 74 genes known to play a role in HCTDs were manually reviewed and analyzed in one hundred consecutive, unrelated patients with phenotypic features indicative of a HCTD referred over a 3.5-year period (2016-2020) to a specialized academic genetics clinic. The prevalence of symptoms was evaluated in the context of genetic variants. We also determined if symptoms among different organ systems were related and performed latent class analysis to identify distinct groups of patients based on symptomatology. RESULTS: In the cohort of 100 consecutive, unrelated individuals there were four pathogenic, six likely pathogenic and 35 classified potentially pathogenic variants of unknown clinical significance. Patients with potentially pathogenic variants exhibited similar symptom profiles when compared to patients with pathogenic/likely pathogenic variants in the same genes. Although results did not meet a multiple testing corrected threshold, patients with connective tissue symptoms had suggestive evidence of increased odds of having skin (odds ratio 2.18, 95% confidence interval 1.12 to 4.24) and eye symptoms (odds ratio 1.89, 95% confidence interval 0.98 to 3.66) requiring further studies. The best performing latent class analysis results were identified when dividing the dataset into three distinct groups based on age, gender and presence or absence of symptoms in the skeletal, connective tissue, nervous, gastrointestinal and cardiovascular systems. These distinct classes of patients included individuals with: (1) minimal skeletal symptoms, (2) more skeletal but fewer connective tissue, nervous or gastrointestinal symptoms and (3) more nervous system symptoms. CONCLUSIONS: We used novel approaches to characterize phenotype-genotype relationships, including pinpointing potentially pathogenic variants, and detecting unique symptom profiles in patients with features of HCTDs. This study may guide future diagnosis and disease/organ system monitoring with continued improvement and surveillance by clinicians for patients and their families.

Next-generation sequencing and analysis of consecutive patients referred for connective tissue disorders.

Heritable connective tissue disorders (HCTDs) consist of a wide array of genetic disorders such as Ehlers-Danlos syndrome, Marfan syndrome, and osteogenesis imperfecta. The diagnosis relies on clinical presentation and family history to guide genetic testing with next-generation sequencing (NGS) for identification of gene variants in HCTDs. NGS was performed on a cohort of 100 consecutive, unrelated patients referred for a connective tissue disorder at Fulgent Genetics, an accredited commercial laboratory. One hundred seventeen gene variants were found in 76 patients with 10 recognized pathogenic or likely pathogenic variants seen in nine patients. The remaining variants were grouped as unknown clinical significance with 36 meeting three out of four pathogenicity criteria, or potentially pathogenic, as defined in our study in 33 patients. They were judged as potentially pathogenic for clinical care and management with disease surveillance based on the specific gene and phenotypic presentation. Gene variants in collagen-related proteins were the most frequent with ZNF469 and ADAMTSL2 variants most often identified. Joint hypermobility was the most frequent clinical finding. Variants were found in 76% of patients who had distinct clinical features of a HCTD. The data were stratified to provide insight into frequency and types of variants, their classification, and clinical manifestations.

Central adrenal insufficiency screening with morning plasma cortisol and ACTH levels in Prader-Willi syndrome.

OBJECTIVES: Prader-Willi syndrome (PWS) is a complex genetic disorder with severe hypotonia, failure to thrive, childhood obesity, hypogonadism/hypogenitalism and learning/behavioral problems with endocrine-related growth and other hormone deficiencies. The prevalence of central adrenal insufficiency (CAI) using dynamic testing ranges from rare to 60%. We compared routine morning plasma cortisol (MPC) and ACTH levels in large cohorts of PWS and control children to address CAI. METHODS: Retrospective analysis of MPC and ACTH levels was undertaken in 128 PWS growth hormone (GH)-treated children under medical care before considering dynamic testing for CAI and 128 non-syndromic control children with short stature evaluated for GH deficiency. RESULTS: The average MPC level in PWS was 9.7 ± 3.7 µg/dL with no difference in age, gender or PWS genetic subtype and 13.4 ± 5.7 µg/dL in the control group. MPC levels were significantly lower (p < 0.05) in PWS but in the normal range. The morning plasma ACTH level in the PWS group was 22.1 ± 8.0 pg/mL with one individual having an initial low plasma ACTH level (8 pg/mL), but normal upon repeat. CONCLUSIONS: MPC levels in PWS are normal and comparable with control children, without evidence or increased risk of CAI. Lower but normal MPC levels were seen in PWS and suggestive of reduced local regeneration of cortisol from cortisone in adipose tissue by the GH-IGF-I system. Hence, MPC measures alone or in combination with ACTH should be considered for initial screening for CAI in PWS but prior to dynamic testing.

A Streamlined Approach to Prader-Willi and Angelman Syndrome Molecular Diagnostics.

Establishing or ruling out a molecular diagnosis of Prader-Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.

Genomic, Clinical, and Behavioral Characterization of 15q11.2 BP1-BP2 Deletion (Burnside-Butler) Syndrome in Five Families.

The 15q11.2 BP1-BP2 deletion (Burnside-Butler) syndrome is emerging as the most common cytogenetic finding in patients with neurodevelopmental or autism spectrum disorders (ASD) presenting for microarray genetic testing. Clinical findings in Burnside-Butler syndrome include developmental and motor delays, congenital abnormalities, learning and behavioral problems, and abnormal brain findings. To better define symptom presentation, we performed comprehensive cognitive and behavioral testing, collected medical and family histories, and conducted clinical genetic evaluations. The 15q11.2 BP1-BP2 region includes the TUBGCP5, CYFIP1, NIPA1, and NIPA2 genes. To determine if additional genomic variation outside of the 15q11.2 region influences expression of symptoms in Burnside-Butler syndrome, whole-exome sequencing was performed on the parents and affected children for the first time in five families with at least one parent and child with the 15q1l.2 BP1-BP2 deletion. In total, there were 453 genes with possibly damaging variants identified across all of the affected children. Of these, 99 genes had exclusively de novo variants and 107 had variants inherited exclusively from the parent without the deletion. There were three genes (APBB1, GOLGA2, and MEOX1) with de novo variants that encode proteins evidenced to interact with CYFIP1. In addition, one other gene of interest (FAT3) had variants inherited from the parent without the deletion and encoded a protein interacting with CYFIP1. The affected individuals commonly displayed a neurodevelopmental phenotype including ASD, speech delay, abnormal reflexes, and coordination issues along with craniofacial findings and orthopedic-related connective tissue problems. Of the 453 genes with variants, 35 were associated with ASD. On average, each affected child had variants in 6 distinct ASD-associated genes (x¯ = 6.33, sd = 3.01). In addition, 32 genes with variants were included on clinical testing panels from Clinical Laboratory Improvement Amendments (CLIA) approved and accredited commercial laboratories reflecting other observed phenotypes. Notably, the dataset analyzed in this study was small and reported results will require validation in larger samples as well as functional follow-up. Regardless, we anticipate that results from our study will inform future research into the genetic factors influencing diverse symptoms in patients with Burnside-Butler syndrome, an emerging disorder with a neurodevelopmental behavioral phenotype.

ADAMTSL2 gene variant in patients with features of autosomal dominant connective tissue disorders.

Ehlers-Danlos syndrome (EDS) consists of a heterogeneous group of genetically inherited connective tissue disorders. A family with three affected members over two generations with features of Dermatosparaxic EDS (dEDS) autosomal dominant transmission was reported by Desai et al. and having a heterozygous nonsynonymous missense variant of ADAMTSL2 (c.1261G > A; p. Gly421Ser). Variation in this gene is also reported to cause autosomal recessive geleophysic dysplasia. We report five unrelated patients with the Gly421Ser variant identified from a large series of patients presenting with features of connective tissue disorders, each with a positive family history consistent with autosomal dominant transmission. Clinical features of a connective tissue disorder included generalized joint hypermobility and pain with fragility of internal and external tissues including of skin, dura, and arteries. Overall, our analyses including bioinformatics, protein modeling, and gene-protein interactions with the cases described would add evidence for the Gly421Ser variant in ADAMTSL2 as causative for variable expressivity of autosomal dominant connective tissue disorders.

Relationship Between Body Habitus and Aggression Subtypes Among Healthy Young Adults from the American Midwest.

This study examined associations between body habitus and functions of aggression, in a sample of 474 college students from the Midwestern region of the United States (age range = 18-25y; 73% Caucasian). Two instruments of aggression, the Reactive-Proactive Aggression Questionnaire from Dodge & Coie 1987 (DC) and Raine et al. 2006 (RPQ) were given as self-assessments. Body habitus measures standardized by age and gender specific weight and height were collected. Subjects considered to have a large body habitus in our study had both weight and height measures above the 75th percentile. Large body habitus was positively correlated with both proactive and reactive functions of aggression among adult males but not females; however, regression analyses indicated that body habitus was most strongly and robustly associated with proactive aggression. Findings suggest that even in a healthy homogeneous population, large body size in males is associated with aggression, particularly proactive aggression including bullying rather than retaliatory aggression. The presence of a large body physique may reinforce aggressive behavioral traits acquired through life experiences and activities evoking physical dominance. Alternatively, the relationship may reflect neurological processes related to size influenced by genetic factors and hormones leading to antisocial behaviors requiring future research on the role of genes for aggression.

Analysis of the Prader-Willi syndrome imprinting center using droplet digital PCR and next-generation whole-exome sequencing.

BACKGROUND: Detailed analysis of imprinting center (IC) defects in individuals with Prader-Willi syndrome (PWS) is not readily available beyond chromosomal microarray (MA) analysis, and such testing is important for a more accurate diagnosis and recurrence risks. This is the first feasibility study of newly developed droplet digital polymerase chain reaction (ddPCR) examining DNA copy number differences in the PWS IC region of those with IC defects. METHODS: The study cohort included 17 individuals without 15q11-q13 deletions or maternal disomy but with IC defects as determined by genotype analysis showing biparental inheritance. Seven sets of parents and two healthy, unrelated controls were also analyzed. RESULTS: Copy number differences were distinguished by comparing the number of positive droplets detected by IC probes to those from a chromosome 15 reference probe, GABRß3. The ddPCR findings were compared to results from other methods including MA, and whole-exome sequencing (WES) with 100% concordance. The study also estimated the frequency of IC microdeletions and identified gene variants by WES that may impact phenotypes including CPT2 and NTRK1 genes. CONCLUSION: Droplet digital polymerase chain reaction is a cost-effective method that can be used to confirm the presence of microdeletions in PWS with impact on genetic counseling and recurrence risks for families.

Molecular genetic classification in Prader-Willi syndrome: a multisite cohort study.

BACKGROUND: Prader-Willi syndrome (PWS) is due to errors in genomic imprinting. PWS is recognised as the most common known genetic cause of life-threatening obesity. This report summarises the frequency and further characterises the PWS molecular classes and maternal age effects. METHODS: High-resolution microarrays, comprehensive chromosome 15 genotyping and methylation-specific multiplex ligation probe amplification were used to describe and further characterise molecular classes of maternal disomy 15 (UPD15) considering maternal age. RESULTS: We summarised genetic data from 510 individuals with PWS and 303 (60%) had the 15q11-q13 deletion; 185 (36%) with UPD15 and 22 (4%) with imprinting defects. We further characterised UPD15 findings into subclasses based on the presence (size, location) or absence of loss of heterozygosity (LOH). Additionally, significantly older mothers (mean age=32.5 years vs 27.7 years) were found in the UPD15 group (n=145) compared with the deletion subtype (n=200). CONCLUSIONS: We report on molecular classes in PWS using advanced genomic technology in the largest cohort to date. LOH patterns in UPD15 may impact the risk of having a second genetic condition if the mother carries a recessive mutant allele in the isodisomic region on chromosome 15. The risk of UPD15 may also increase with maternal age.

Sex-Dimorphic Interactions of MAOA Genotype and Child Maltreatment Predispose College Students to Polysubstance Use.

Polysubstance use (PSU) is highly prevalent among college students. Recent evidence indicates that PSU is based on gene x environment (G×E) interactions, yet the specific biosocial factors underlying this problem remain elusive. We recently reported that lifetime use of tobacco and cannabis in college students is influenced by the interaction of the X-linked MAOA (monoamine oxidase A) gene and child maltreatment. Building on these premises, here we evaluated whether the same G×E interaction may also predict PSU in this population. Students of a large Midwestern university (n = 470; 50.9% females) took part in a computer survey for substance use, as well as childhood trauma exposure, using the Child Trauma Questionnaire (CTQ). DNA was extracted from their saliva samples and genotyped for MAOA variable-number of tandem repeat (VNTR) variants. Findings indicated that the highest number of substances were used by male students harboring low-activity MAOA alleles with a history of childhood emotional abuse. In contrast, female homozygous high-activity MAOA carriers with a history of emotional and physical abuse reported consumption of the greatest number of substances. Our results indicate that PSU among college students is influenced by the interaction of MAOA and child maltreatment in a sex-specific fashion. Further studies are warranted to understand the mechanisms of sex differences in the biosocial interplays underlying PSU in this at-risk group.

Preliminary observations of mitochondrial dysfunction in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a complex multisystem disorder because of errors in genomic imprinting with severe hypotonia, decreased muscle mass, poor suckling, feeding problems and failure to thrive during infancy, growth and other hormone deficiency, childhood-onset hyperphagia, and subsequent obesity. Decreased energy expenditure in PWS is thought to contribute to reduced muscle mass and physical activity but may also relate to cellular metabolism and disturbances in mitochondrial function. We established fibroblast cell lines from six children and adults with PWS and six healthy controls for mitochondrial assays. We used Agilent Seahorse XF extracellular flux technology to determine real-time measurements of several metabolic parameters including cellular substrate utilization, Adenosine Triphosphate (ATP)-linked respiration, and mitochondrial capacity in living cells. Decreased mitochondrial function was observed in the PWS patients compared to the healthy controls with significant differences in basal respiration, maximal respiratory capacity, and ATP-linked respiration. These results suggest disturbed mitochondrial bioenergetics in PWS although the low number of studied subjects will require a larger subject population before a general consensus can be reached to identify if mitochondrial dysfunction is a contributing factor in PWS.

A descriptive study on selected growth parameters and growth hormone receptor gene in healthy young adults from the American Midwest.

CONTEXT: The first study of growth hormone receptor (GHR) genotypes in healthy young adults in the United States attending a Midwestern university and impact on selected growth parameters. OBJECTIVE: To describe the frequency of GHR genotypes in a sample of healthy young adults from the United States attending a university in the Midwest and analyze the relationship between GHR genotypes and selected growth parameters. DESIGN: Saliva was collected from 459 healthy young adults (237 females, 222 males; age range = 18-25 y) and DNA isolated for genotyping of GHR alleles (fl/fl, fl/d3, or d3/d3). Selected growth parameters were collected and GHR genotype data examined for previously reported associations (e.g., height, weight or bone mass density) or novel findings (e.g., % body water and index finger length). RESULTS: We found 219 participants (48%) homozygous for fl/fl, 203 (44%), heterozygous fl/d3 and 37 (8%) homozygous d3/d3. The distribution of GHR genotypes in our participants was consistent with previous reports of non-US populations. Several anthropometric measures differed by sex. The distribution of GHR genotypes did not significantly differ by sex, weight, or other anthropometric measures. However, the fl/d3 genotype was more common among African-Americans. CONCLUSIONS: Our study of growth and anthropometric parameters in relationship to GHR genotypes found no association with height, weight, right index finger length, BMI, bone mass density, % body fat or % body water in healthy young adults. We did identify sex differences with increased body fat, decreased bone density, body water and index finger length in females.

Three siblings with Prader-Willi syndrome caused by imprinting center microdeletions and review.

Prader-Willi syndrome (PWS) is a complex genetic imprinting disorder characterized by childhood obesity, short stature, hypogonadism/hypogenitalism, hypotonia, cognitive impairment, and behavioral problems. Usually PWS occurs sporadically due to the loss of paternally expressed genes on chromosome 15 with the majority of individuals having the 15q11-q13 region deleted. Examples of familial PWS have been reported but rarely. To date 13 families have been reported with more than one child with PWS and without a 15q11-q13 deletion secondary to a chromosome 15 translocation, inversion, or uniparental maternal disomy 15. Ten of those 13 families were shown to carry microdeletions in the PWS imprinting center. The microdeletions were found to be of paternal origin in nine of the ten cases in which family studies were carried out. Using a variety of techniques, the microdeletions were identified in regions within the complex SNRPN gene locus encompassing the PWS imprinting center. Here, we report the clinical and genetic findings in three adult siblings with PWS caused by a microdeletion in the chromosome 15 imprinting center inherited from an unaffected father that controls the activity of genes in the 15q11-q13 region and summarize the 13 reported cases in the literature.

Interactive roles of fibroblast growth factor 2 and neurotrophin 3 in the sequence of migration, process outgrowth, and axonal differentiation of mouse cochlear ganglion cells.

A growth factor may have different actions depending on developmental stage. We investigated this phenomenon in the interactions of fibroblast growth factor 2 (FGF2) and neurotrophins on cochlear ganglion (CG) development. The portions of the otocyst fated to form the CG and cochlear epithelium were cocultured at embryonic day 11 (E11). Cultures were divided into groups fed with defined medium, with or without FGF2 and neurotrophin supplements, alone or in combination, for 7 days. We measured the number of migrating neuroblasts and distances migrated, neurite outgrowth, and axonlike processes. We used immunohistochemistry to locate neurotrophin 3 (NT3) and its high-affinity receptor (TrkC) in the auditory system, along with FGF2 and its R1 receptor, at comparable developmental stages in vitro and in situ from E11 until birth (P1) in the precursors of hair cells, support cells, and CG cells. Potential sites for interaction were localized to the nucleus, perikaryal cytoplasm, and cell surfaces, including processes and growth cones. Time-lapse imaging and quantitative measures support the hypothesis that FGF2 alone or combined with neurotrophins promotes migration and neurite outgrowth. Synergism or antagonism between NT3 and other factors suggest interactions at the receptor level. Formation of axons, endings, and synaptic vesicle protein 2 were increased by interactions of NT3 and FGF2. Similar experiments with a mutant overexpressor for FGF2 suggest that endogenous FGF2 supports migration and neurite outgrowth of CG neuroblasts as well as proliferation, leading to accelerated development. The findings suggest interactive and sequential roles for FGF2 and NT3.

Site-specific interactions of neurotrophin-3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus.

Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes.

Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Where is the spike generator of the cochlear nerve? Voltage-gated sodium channels in the mouse cochlea.

The origin of the action potential in the cochlea has been a long-standing puzzle. Because voltage-dependent Na+ (Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the type I and type II ganglion cells. In most type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.