Male Infants Born to Older Pregnant Women Are Affected by Maternal Physique at the Beginning of the Pregnancy through Birth until 18 Months of Age.

We have previously reported a positive correlation between the body mass index (BMI) of mothers and the Kaup index of infants at birth and that this correlation may have a stronger influence on the male infants, born to older mothers. Therefore, in this study, we aimed to clarify the correlation between maternal age and gender of the infants as maternal BMI and the Kaup index of infants from birth till 18 mo of age. This study was conducted from April 2010 to September 2011 in Japan. Public health nurses and registered dietitians interviewed the mothers individually under anonymous conditions, and they transferred the required information from the maternity passbook at the 18-mo health checkup. In male infants, significant positive correlations were demonstrated between maternal BMI at the beginning of pregnancy and the Kaup index of infants in mothers older than 35 y when the infants were at birth, at 4 mo and at 18 mo old. In female infants, there was no correlation between maternal BMI and the Kaup index of infants at birth. However, significant positive correlations were demonstrated between maternal BMI and the Kaup index of infants in mothers in their 20s and older than 35 y old when the infants were 4 and 18 mo old. Women who plan on pregnancy and medical professionals need to know that the maternal physique at the beginning of pregnancy affects the physique of the infants at birth and at 4 mo and 18 mo old by gender of the infants.

Genome Editing for Cardiovascular Diseases-A Brief Review for Cardiologists.

The recent technical advances in genome engineering have accelerated our understanding of the molecular mechanisms of human diseases and are leading to increased clinical applications of gene-targeting therapies. The field of cardiovascular medicine, rich in knowledge of molecular level disease mechanisms, is particularly well positioned to receive significant benefits from this technology. Specifically, a new generation of genome editing tools capable of introducing targeted sequence modifications at high frequencies initiated by induced DNA double-strand breaks has been developed. Of note is the RNA-guided genome editing system, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), which has provided researchers and clinicians a malleable gene-targeting platform with high specificity. Recent reports have robustly demonstrated proof-of-concept in using CRISPR-Cas9 based gene therapy for treating common cardiovascular diseases and are testaments that the new genome editing technology holds promise for treating patients with cardiovascular ailments in the clinic in the near future. In light of this trend, a basic understanding of genome editing technology is becoming more relevant to clinical cardiologists. To this end, a concise explanation of terms and the biological basis of genome editing, on-going research, and clinical trials highly relevant to clinical application are presented. In conclusion, the aim of this short review is to introduce clinicians to the core concepts of current genome editing technology.

Application of active learning modalities to achieve medical genetics competencies and their learning outcome assessments.

The steadily falling costs of genome sequencing, coupled with the growing number of genetic tests with proven clinical validity, have made the use of genetic testing more common in clinical practice. This development has necessitated nongeneticist physicians, especially primary care physicians, to become more responsible for assessing genetic risks for their patients. Providing undergraduate medical students a solid foundation in genomic medicine, therefore, has become all the more important to ensure the readiness of future physicians in applying genomic medicine to their patient care. In order to further enhance the effectiveness of instructing practical skills in medical genetics, the emphasis of active learning modules in genetics curriculum at medical schools has increased in recent years. This is because of the general acceptance of a better efficacy of active learner-centered pedagogy over passive lecturer-centered pedagogy. However, an objective standard to evaluate students' skill levels in genomic medicine achieved by active learning is currently missing. Recently, entrustable professional activities (EPAs) in genomic medicine have been proposed as a framework for developing physician competencies in genomic medicine. EPAs in genomic medicine provide a convenient guideline for not only developing genomic medicine curriculum but also assessing students' competency levels in practicing genomic medicine. In this review, the efficacy of different types of active learning modules reported for medical genetics curricula is discussed using EPAs in genomic medicine as a common evaluation standard for modules' learning outcomes. The utility of the EPAs in genomic medicine for designing active learning modules in undergraduate medical genetics curricula is also discussed.

High Maternal Age and Low Pre-Pregnancy Body Mass Index Correlate with Lower Birth Weight of Male Infants.

In Japan, the percentage of leanness has been increasing in young women, and the percentage of low birth weight infants (< 2,500 g) has increased. Moreover, the average age of primiparas rose 3.5 years during the last 30 years. The purpose of this study was to clarify the relationship between maternal age and the influence of maternal pre-pregnancy physique on the neonatal physique of infants. Questionnaires were issued to the participants and collected when they submitted their gestational notifications at their local ward office in Kyoto Prefecture. After delivery, we obtained information on the course of the pregnancy and the neonatal physique of the infants from the participant's maternal passbooks. A total of 454 mothers (age 20 ≥) were analyzed: 161 young mothers (aged 20 to 29 years), 185 mothers (aged 30 to 34 years), and 108 older mothers (age ≥ 35). Overall, the mean rate of leanness (pre-pregnancy BMI < 18.5) was 23.8%. We found that birth weight was significantly lower in female infants, born to lean young mothers, compared to non-lean young mothers, whereas no significant difference was detected in other mothers (age ≥ 30), irrespective of pre-pregnancy BMI. By contrast, male infants, born to older lean mothers (age ≥ 35), showed significantly lower birth weight. Thus, maternal pre-pregnancy BMI exerts differential effects on the fetal growth (neonatal physique), depending on the maternal age and the sex of infants. We need to improve BMI in pre-pregnancy women, especially those in the twenties and 35 years old or over.

Transcriptome Dynamics and Potential Roles of Sox6 in the Postnatal Heart.

The postnatal heart undergoes highly coordinated developmental processes culminating in the complex physiologic properties of the adult heart. The molecular mechanisms of postnatal heart development remain largely unexplored despite their important clinical implications. To gain an integrated view of the dynamic changes in gene expression during postnatal heart development at the organ level, time-series transcriptome analyses of the postnatal hearts of neonatal through adult mice (P1, P7, P14, P30, and P60) were performed using a newly developed bioinformatics pipeline. We identified functional gene clusters by principal component analysis with self-organizing map clustering which revealed organized, discrete gene expression patterns corresponding to biological functions associated with the neonatal, juvenile and adult stages of postnatal heart development. Using weighted gene co-expression network analysis with bootstrap inference for each of these functional gene clusters, highly robust hub genes were identified which likely play key roles in regulating expression of co-expressed, functionally linked genes. Additionally, motivated by the role of the transcription factor Sox6 in the functional maturation of skeletal muscle, the role of Sox6 in the postnatal maturation of cardiac muscle was investigated. Differentially expressed transcriptome analyses between Sox6 knockout (KO) and control hearts uncovered significant upregulation of genes involved in cell proliferation at postnatal day 7 (P7) in the Sox6 KO heart. This result was validated by detecting mitotically active cells in the P7 Sox6 KO heart. The current report provides a framework for the complex molecular processes of postnatal heart development, thus enabling systematic dissection of the developmental regression observed in the stressed and failing adult heart.

Maternal Body Mass Index Correlates with the Neonatal Physique of Male Infants.

Recently, in Japan, the percentage of leanness has risen in young women, and the average birth weight has decreased. An increase in the risk of low birth weight has been reported in lean expectant mothers. In this study, we aimed to clarify the relationship between mother's physique at the beginning of pregnancy and the infant's physique, by focusing on sex differences. The participants were 3,722 mothers who attended health checkups for 18-month-old infants in an urban Japanese city. The participants were limited to those with full-term births, thereby excluding the influence of gestational length. A total of 1,287 mothers, with 621 boys and 666 girls, were analyzed. Public health professionals interviewed the mothers, and transferred the required information from their maternity passbooks. We examined the physical characteristics of the mothers and their infants. Partial correlation analysis, adjusted by gestational length and the mother's age at delivery, was applied to study the association between the mother's BMI and the infant's physique at birth. In the primipara group, only the boys showed significant positive correlation between the mother's BMI and the birth weight (P = 0.025) and the Kaup index (P = 0.035). In the pluripara group, only the boys showed significant positive correlation between the mother's BMI and the head circumference (P = 0.035). Thus, mother's physique may have a stronger influence on the physique of male infants, compared to female infants. The growth-promoting effect of the mother's physique is more apparent in the infants born to the pluripara.

Expression of Sox genes in tooth development.

Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

Genome-wide analysis of transcription factor-binding sites in skeletal muscle cells using ChIP-seq.

Transcriptional regulation of gene expression constitutes a fundamental mechanism of many developmental processes. Therefore, identification and characterization of binding sites of transcription factors are important for uncovering the mechanisms of a particular developmental process. Here, we describe detailed procedures for genome-wide analysis of binding sites of a transcription factor involved in the fiber-type differentiation of skeletal muscle. By conducting ChIP-seq followed by a series of downstream analyses, in-depth information on binding sites of transcription factors can be obtained in a genome-wide manner.

Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells.

BACKGROUND: A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. METHODS: Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. RESULTS: We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. CONCLUSIONS: Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy.

Genome-wide mapping of Sox6 binding sites in skeletal muscle reveals both direct and indirect regulation of muscle terminal differentiation by Sox6.

BACKGROUND: Sox6 is a multi-faceted transcription factor involved in the terminal differentiation of many different cell types in vertebrates. It has been suggested that in mice as well as in zebrafish Sox6 plays a role in the terminal differentiation of skeletal muscle by suppressing transcription of slow fiber specific genes. In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes in mouse fetal myotubes and generated muscle-specific Sox6 knockout (KO) mice to determine the Sox6 null muscle phenotype in adult mice. RESULTS: We have identified 1,066 Sox6 binding sites using mouse fetal myotubes. The Sox6 binding sites were found to be associated with slow fiber-specific, cardiac, and embryonic isoform genes that are expressed in the sarcomere as well as transcription factor genes known to play roles in muscle development. The concurrently performed RNA polymerase II (Pol II) ChIP-seq analysis revealed that 84% of the Sox6 peak-associated genes exhibited little to no binding of Pol II, suggesting that the majority of the Sox6 target genes are transcriptionally inactive. These results indicate that Sox6 directly regulates terminal differentiation of muscle by affecting the expression of sarcomere protein genes as well as indirectly through influencing the expression of transcription factors relevant to muscle development. Gene expression profiling of Sox6 KO skeletal and cardiac muscle revealed a significant increase in the expression of the genes associated with Sox6 binding. In the absence of the Sox6 gene, there was dramatic upregulation of slow fiber-specific, cardiac, and embryonic isoform gene expression in Sox6 KO skeletal muscle and fetal isoform gene expression in Sox6 KO cardiac muscle, thus confirming the role Sox6 plays as a transcriptional suppressor in muscle development. CONCLUSIONS: Our present data indicate that during development, Sox6 functions as a transcriptional suppressor of fiber type-specific and developmental isoform genes to promote functional specification of muscle which is critical for optimum muscle performance and health.

Sox6, jack of all trades: a versatile regulatory protein in vertebrate development.

Approximately 20,000 genes are encoded in our genome, one tenth of which are thought to be transcription factors. Considering the complexity and variety of cell types generated during development, many transcription factors likely play multiple roles. Uncovering the versatile roles of Sox6 in vertebrate development sheds some light on how an organism efficiently utilizes the limited resources of transcription factors. The structure of the Sox6 gene itself may dictate its functional versatility. First, Sox6 contains no known regulatory domains; instead, it utilizes various cofactors. Second, Sox6 has a long 3'-UTR that contains multiple microRNA targets, thus its protein level is duly adjusted by cell type-specific microRNAs. Just combining these two characteristics alone makes Sox6 extremely versatile. To date, Sox6 has been reported to regulate differentiation of tissues of mesoderm, ectoderm, and endoderm origins, making Sox6 a truly multifaceted transcription factor.

Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Sox6 is required for normal fiber type differentiation of fetal skeletal muscle in mice.

Sox6, a member of the Sox family of transcription factors, is highly expressed in skeletal muscle. Despite its abundant expression, the role of Sox6 in muscle development is not well understood. We hypothesize that, in fetal muscle, Sox6 functions as a repressor of slow fiber type-specific genes. In the wild-type mouse, differentiation of fast and slow fibers becomes apparent during late fetal stages (after approximately embryonic day 16). However, in the Sox6 null-p(100H) mutant mouse, all fetal muscle fibers maintain slow fiber characteristics, as evidenced by expression of the slow myosin heavy chain MyHC-beta. Knockdown of Sox6 expression in wild-type myotubes results in a significant increase in MyHC-beta expression, supporting our hypothesis. Analysis of the MyHC-beta promoter revealed a Sox consensus sequence that likely functions as a negative cis-regulatory element. Together, our results suggest that Sox6 plays a critical role in the fiber type differentiation of fetal skeletal muscle.

Sox6 directly silences epsilon globin expression in definitive erythropoiesis.

Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG) DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p100H, epsilony globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic) cells define a 36-base pair region of the epsilony proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that Sox6 acts as a repressor by directly binding to the epsilony promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of epsilony in p100H homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of epsilony globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of epsilony globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia.

Slow and fast fiber isoform gene expression is systematically altered in skeletal muscle of the Sox6 mutant, p100H.

We have previously demonstrated that p100H mutant mice, which lack a functional Sox6 gene, exhibit skeletal and cardiac muscle degeneration and develop cardiac conduction abnormalities soon after birth. To understand the role of Sox6 in skeletal muscle development, we identified muscle-specific genes differentially expressed between wild-type and p100H mutant skeletal muscles and investigated their temporal expression in the mutant muscle. We found that, in the mutant skeletal muscle, slow fiber and cardiac isoform genes are expressed at significantly higher levels, whereas fast fiber isoform genes are expressed at significantly lower levels than wild-type. Onset of this aberrant fiber type-specific gene expression in the mutant coincides with the beginning of the secondary myotube formation, at embryonic day 15-16 in mice. Together with our earlier report, demonstrating early postnatal muscle defects in the Sox6 null-p100H mutant, the present results suggest that Sox6 likely plays an important role in muscle development.

Characterization of two transgene insertional mutations at pirouette, a mouse deafness locus.

The mouse mutant 'pirouette' (pi) exhibits profound hearing loss and vestibular defects due to inheritance of a recessive mutation on chromosome 5. Dysfunction has been correlated with defects during maturation of sensory cells in the inner ear. As an initial step in characterizing pirouette at the genetic level, we have localized the candidate interval to a small region on central chromosome 5 by analysis of a congenic strain of pirouette mice. This region exhibits conserved synteny with human chromosome 4 and suggests that pirouette may be a genetic model of the human nonsyndromic deafness disorder DFNB25, which has been localized to 4p15.3-q12. In addition to the original spontaneous pirouette strain, we have identified and characterized 2 additional mouse strains with allelic mutations at the same locus. Analysis of the morphology in each of the 3 pirouette alleles indicated very similar early postnatal alterations in maturation of stereocilia and suggests that the gene affected in pirouette normally plays a role in building or maintaining these structures that are critical for sensory mechanotransduction.

Sox6 regulation of cardiac myocyte development.

A mouse mutation (p100H/p100H) has been identified that is associated with cardioskeletal myopathy, heart block, delayed growth and early postnatal death. The gene that is disrupted in this mutation encodes the transcription factor Sox6. P19CL6 cells were used as an in vitro cardiomyocyte differentiation system and revealed that Sox6 is expressed exclusively when the cells are committed to differentiate to beating cardiac myocytes. We used the yeast two-hybrid system to identify the Prtb (Proline-rich transcript of the brain) protein as a Sox6 interactor, and subsequently confirmed the interaction by co-immunoprecipitation. Prtb expression in P19CL6 cells increased with differentiation to beating cardiomyocytes. Using the P19CL6 cells stably transfected with noggin, an antagonist of BMP (Bone Morphogenic Protein), we found that BMP expression is required for Sox6 expression in cardiomyocyte differentiation. Surprisingly, the expression of the alpha1c-subunit gene of the L-type Ca2+ channel decreased in P19CL6 cells as they differentiated to beating cardiac cells. Ectopic expression of Sox6 or Prtb alone in P19CL6 cells caused down-regulation of L-type Ca2+ alpha1c expression, but when Sox6 and Prtb were co-transfected to the cells, L-type Ca2+ alpha1c remained at basal levels. A similar relationship of Sox6 and L-type Ca2+ alpha1c expression was seen in vivo (comparing wild-type and p(100H)/p(100H) mutant mice). Thus, Sox6 is within the BMP pathway in cardiac differentiation, interacts with Prtb and may play a critical role in the regulation of a cardiac L-type Ca2+ channel.

Nonneuronal expression of the GABA(A) beta3 subunit gene is required for normal palate development in mice.

Cleft palate is one of the most common birth defects in humans, in which both genetic and environmental factors are involved. In mice, loss of the GABA(A) receptor beta3 subunit gene (Gabrb3) or the targeted mutagenesis of the GABA synthetic enzyme (Gad1) leads to cleft palate. These observations indicate that a GABAergic system is important in normal palate development. To determine what cell types, neuronal or nonneuronal, are critical for GABA signaling in palate development, we used the neuron-specific enolase promoter to express the beta3 subunit in Gabrb3 mutant mice. Expression of this construct was able to rescue the neurological phenotype, but not the cleft palate phenotype. Combined with the previous observation demonstrating that ubiquitous expression of the beta3 subunit rescued the cleft palate phenotype, a nonneuronal GABAergic system is implicated in palate development. Using immunohistochemistry, we detected GABA in the developing palate, initially in the nasal aspect of palatal epithelium of the vertical shelves; later in the medial edge epithelium of the horizontally oriented palatal shelves and in the epithelial seam during fusion. Based on these observations, we propose that GABA, synthesized by the palatal epithelium, acts as a signaling molecule during orientation and fusion of the palate shelves.