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Background The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. Methods Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly. Key results Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. Conclusions & inferences Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.

Background: The short stature homeodomain transcription factors SHOX and SHOX2 play key roles in limb formation. To gain more insight into genes regulated by Shox2 during limb development, we analyzed expression profiles of WT and Shox2−/− mouse embryonic limbs and identified the T-Box transcription factor Tbx4 as a potential downstream target. Tbx4 is known to exert essential functions in skeletal and muscular hindlimb development. In humans, haploinsufficiency of TBX4 causes small patella syndrome, a skeletal dysplasia characterized by anomalies of the knee, pelvis, and foot. Results: Here, we demonstrate an inhibitory regulatory effect of Shox2 on Tbx4 specifically in the forelimbs. We also show that Tbx4 activates Shox2 expression in fore- and hindlimbs, suggesting Shox2 as a feedback modulator of Tbx4. Using EMSA studies, we find that Tbx4/TBX4 is able to bind to distinct T-box binding sites within the mouse and human Shox2/SHOX2 promoter. Conclusions: Our data identifies Tbx4 as a novel transcriptional activator of Shox2 during murine fore- and hindlimb development. Tbx4 is also regulated by Shox2 specifically in the forelimb bud possibly via a feedback mechanism. These data extend our understanding of the role and regulation of Tbx4 and Shox2 in limb development and limb associated diseases. Developmental Dynamics 243:629–639, 2014. © 2013 Wiley Periodicals, Inc.

Mutations in the SRGAP3 gene residing on chromosome 3p25 have previously been associated with intellectual disability. Genome-wide association studies have also revealed SRGAP3, together with genes from the same cellular network, as risk genes for schizophrenia. SRGAP3 regulates cytoskeletal dynamics through the RHO protein RAC1. RHO proteins are known to be involved in cytoskeletal reorganization during brain development to control processes such as synaptic plasticity. To elucidate the importance of SRGAP3 in brain development, we generated Srgap3-knockout mice. Ten percent of these mice developed a hydrocephalus and died before adulthood. Surviving mice showed various neuroanatomical changes, including enlarged lateral ventricles, white matter tracts, and dendritic spines together with molecular changes, including an increased basal activity of RAC1. Srgap3(-/-) mice additionally exhibited a complex behavioral phenotype. Behavioral studies revealed an impaired spontaneous alternation and social behavior, while long-term memory was unchanged. The animals also had tics. Lower locomotor activity was observed in male Srgap3(-/-) only. Srgap3(-/-) mice showed increased methylphenidate stimulation in males and an impaired prepulse inhibition in females. Together, the results show neurodevelopmental aberration in Srgap3(-/-) mice, with many of the observed phenotypes matching several schizophrenia-related intermediate phenotypes. Mutations of SRGAP3 may thus contribute to various neurodevelopmental disorders.

A translocation chromosome in a woman with the karyotype 46,X,der(X)t(X;Y)(p22.3; q11.2) was investigated by FISH and STS analysis with molecular probes derived from the sex chromosomes. Due to the partial deletion of the short arm pseudoautosomal region (PAR1) from DXYS14 to DXYS147 in the translocation chromosome, the proband is hemizygous for the gene responsible for growth control (SS) located in this region, yet does not show growth retardation. Molecular analysis of the Yq arm of the translocation chromosome revealed the presence of markers DYS273 to DYS246 harboring the hypothesized growth control gene critical region (GCY) on Yq, thereby placing the deletion breakpoint between markers DYS11 and DYS273. These results suggest that the Y-specific growth gene GCY on Yq compensates for the missing growth gene SS on Xp22.3.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) mediates a multitude of central nervous functions by activating 5-HT receptor subtypes. A dysfunction of serotonergic neurotransmission is considered to play a major role in the pathophysiology of complex neuropsychiatric disorders. In our study, a mutation screen of the serotonin receptor gene HTR3B was carried out to explore a putative contribution to the etiology of bipolar affective disorder (BPAD) and schizophrenia (SZ). Screening of 49 patients suffering from BPAD, 78 patients with SZ and 62 control individuals revealed eleven sequence variations including a 3 bp deletion within the 5′UTR (5′ untranslated region), four exonic and five intronic SNPs as well as a point mutation in the 3′UTR of HTR3B. Four of these sequence variations have not been described previously. Statistical computation rated most variants as probably non-disease related polymorphisms. However, IVS6 + 31C > T, IVS6 + 40C > A, and 1386T > C were solely detected in bipolar affective patients and in none of the controls. Interestingly, we observed a significant underrepresentation of the 3 bp deletion −100_−102delAAG in an extended sample of 162 bipolar affected patients compared to controls (allele-wise: 8% vs. 15%, P = 0.006, OR = 0.49, 95% CI: 0.3–0.82; genotype-wise: 15,5% vs. 29,0%, P = 0.005, OR = 0.45, 95% CI: 0.26–0.77). We suggest that this deletion may influence translational efficiency, thereby possibly affecting the development of bipolar affective disease. © 2004 Wiley-Liss, Inc.

The Turner syndrome (TS) is a complex disorder associated with almost invariant short stature and gonadal dysgenesis, as well as a variety of other major organ malformations. Recently, a homeobox-containing gene entitled short-stature homeobox-containing gene (SHOX), was isolated from a minimal short stature gene interval from the pseudoautosomal region of Xp (and Yp). Together with the demonstrable escape of SHOX from X-inactivation, this suggested SHOX to be a strong candidate gene for the short stature component of TS, and as SHOX haploinsufficiency appears to be the molecular basis of a mesomelic short statured skeletal dysplasia (Leri-Weill syndrome), this suggested that SHOX protein expression levels may confer a dosage effect on human stature. However, in this communication we report a normal statured female with gonadal dysgenesis, due to the inheritance of a recombinant duplication-deletion X-chromosome. The karyotype of the proband was 46,X,rec(X)dup-(Xp)inv(X)(p11.22q21.2)mat and fluorescent in situ hybridization of her metaphases with a SHOX cosmid confirmed the proband to be trisomic for SHOX. This communication suggests the relationship between levels of SHOX expression and human stature to be more complex than envisaged previously. The presence of normal stature in our patient rather than tall stature is likely to represent the natural variation seen in patients with transcription factor disorders.

Here we report an 8-year-old male patient who had mesomelic shortening of forearms and legs, brachytelephalangia and ichthyotic skin lesions. Chromosomal analysis showed an X;Y translocation involving the short arm of the X chromosome (Xp). Fluorescence in situ hybridization (FISH) and molecular studies localized the breakpoints on Xp22.3 in the immediate vicinity of the KAL gene demonstrating deletions of steroid sulfatase (STS), arylsulfatase E (ARSE), and short stature homeo box (SHOX) genes. It was suspected that the patient was suffering from chondrodysplasia punctata because of a loss of the arylsulfatase E (ARSE) gene. However, no stippled epiphyses were to be seen in the neonatal radiograph. Interestingly, this patient is the first case with a proven loss of the ARSE gene without chondrodysplasia punctata, assuming that chondrodysplasia punctata is not an obligatory sign of ARSE gene loss. Brachytelephalangia was the only result of ARSE gene deletion in this case. The patient's mother also had dwarfism and showed Madelung deformity of the forearms. She was detected as a carrier of the same aberrant X chromosome. The male patient did not show Madelung deformity, demonstrating that Lerri-Weill syndrome phenotype may be still incomplete in children with SHOX gene deletion. The wide clinical spectrum in the male and the Leri-Weill phenotype in his mother are the results of both a deletion involving several sulfatase genes in Xp22.3 and the SHOX gene located in the pseudoautosomal region. Nevertheless, there is no explanation for the absence of chondrodysplasia punctata despite the total loss of the ARSE gene. Further studies are necessary to investigate genotype/phenotype correlation in cases with translocations or microdeletions on Xp22.3, including the ARSE and the SHOX gene loci.

We report the results of detailed molecular-cytogenetic studies of two isodicentric Y [idic(Y)] chromosomes identified in patients with complex mosaic karyotypes. We used fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) to determine the structure and genetic content of the abnormal chromosomes. In the first patient, classical cytogenetics and FISH analysis with Y chromosome-specific probes showed in peripheral blood lymphocytes a karyotype with 4 cell lines: 45,X[128]/46,X,+idic(Y)(p11.32)[65]/47,XY,+idic(Y)(p11.32)[2]/47,X,+2idic(Y)(p11.32)[1]. No Y chromosome material was found in the removed gonads. For precise characterization of the Yp breakpoint, FISH and fiberFISH analysis, using a telomeric probe and a panel of cosmid probes from the pseudoautosomal region PAR1, was performed. The results showed that the breakpoint maps ∼1,000 Kb from Ypter. The second idic(Y) chromosome was found in a boy with mild mental retardation, craniofacial anomalies, and the karyotype in lymphocytes 47,X,+idic(Y)(q11.23),+i(Y)(p10)[77]/46,X,+i(Y)(p10)[23]. To our knowledge, such an association has not been previously described. FISH and PCR analysis indicated the presence of at least two copies of the SRY gene in all analyzed cells. Using 17 PCR primers, the Yq breakpoint was shown to map between sY123 (DYS214) and sY121 (DYS212) loci in interval 5O in AZFb region. Possible mechanisms of formation of abnormal Y chromosomes and karyotype-phenotype correlations are discussed. © 2001 Wiley-Liss, Inc.

We report on a three-month-old boy with a 46,XY,der(Y)t(Y;7)(p11.32;p15.3) karyotype and growth deficiency, postnatal microcephaly with large fontanels, wide sagittal and metopic sutures, hypertelorism, choanal stenosis, micrognathia, bilateral cryptorchidism, hypospadias, abnormal fingers and toes, and severe developmental delay. FISH studies showed partial trisomy 7p resulting from a de novo unbalanced translocation. The application of molecular probes from the TWIST gene region (7p15.3-p21.1) and probes from the pseudoautosomal region (PAR) demonstrated that the 7p15.3-pter fragment was translocated onto Yp with the breakpoint within approximately 20 kb from the Yp telomere. We discuss the possible role of the TWIST gene in abnormal skull development and suggest that trisomy 7p cases with delayed closure of fontanels can be a result of TWIST gene dosage effect.

Twelve patients with different features of Turner syndrome, and with Xp and Yp rearrangements involving the pseudoautosomal region (PAR1) are described. In all patients, FISH analysis showed loss of one copy of the Short Stature Homeobox (SHOX)-containing gene. Ten patients had short stature and one disproportionate (mesomelic) normal stature, while the last one had normal stature. Skeletal abnormalities, including shortened ulna, were detected in nine subjects, and in six of them Madelung deformity was observed. These clinical data indicated a genotype phenotype correlation between haploinsufficiency of SHOX, and short stature and skeletal abnormalities.

We report on a mother and her 5-year old son, both with a terminal deletion of the short arm of the X chromosome. By molecular genetic analysis the breakpoint was located distal to steroid sulfatase gene. The boy manifested, due to nullisomy of this region, short stature (SHOX), chondrodysplasia punctata (ARSE), and mental retardation (putative mental retardation gene MRX 49). Short stature is present in mother and son, but both also had bilateral Madelung deformity, a key finding in the Leri-Weill syndrome. We discuss the phenotype in relationship to hitherto published cases with chromosomal aberrations and contiguous gene syndromes of Xp22.3.

Consistent handedness and language laterality are two of the most striking behavioral and cognitive asymmetries observed in humans. Alterations in the typical pattern of cerebral laterality, termed "anomalous dominance," is observed in left-handers and some patients with verbal learning disabilities. We undertook the study of a genetically distinct group of subjects, XXY males (Klinefelter's syndrome; KS), who demonstrate anomalous dominance in a variety of testing paradigms in order to begin to elucidate the molecular basis of anomalous dominance in this population. KS subjects manifest specific verbal learning disability, evidence of altered functional laterality for phonologic processing, and an increase in left-handedness when measured by skill. It is proposed that an alteration in gene dosage in the pseudoautosomal region (PAR) of the sex chromosomes is the most likely explanation for anomalous dominance in these patients. This is especially intriguing in light of previously described genetic models of cerebral laterality that suggest a contributing locus in the PAR, or adjacent high homology regions of the X chromosome. We have developed an ordered DNA microarray covering the X chromosome PAR at high resolution for hybridization with two-color fluorescently labeled probes. We demonstrate the ability to detect changes in hybridization signal that will facilitate efficient large-scale screening of this region for alterations in gene dosage associated with features of anomalous dominance and other cognitive or behavioral phenotypes.

Hodgkin’s lymphoma (HL) is generally a sporadic disease that may be caused by an infectious agent, possibly Epstein‐ Barr virus (EBV). However, there is consistent evidence of familial clustering in HL (Ferraris et al, 1997), suggesting a role for heritable predisposition in a proportion of patients. Although progress with the identification of an HL-predisposing gene has been hindered by the rarity of multiplepatient HL families, our description of a family in which two sisters with the skeletal dysplasia Leri‐Weill dyschondrosteosis (LWD) both developed HL in adolescence led us to suggest that an HL-predisposing gene might be linked to the LWD gene (Gokhale et al, 1995). Subsequent identification of the LWD gene as the pseudoautosomal short stature homeobox gene SHOX (Shears et al, 1998) led to the hypothesis, based on observations in our HL-LWD family, that an HL-predisposing gene might be located in the major pseudoautosomal region (PAR1) of the X and Y chromosomes (Horowitz & Wiernik, 1999). We have studied further the PAR1 in the HL-LWD family using microsatellite marker analysis and fluorescence in-situ hybridization (FISH). Fluorescent polymerase chain reaction (PCR) primers were used to amplify the pseudoautosomal microsatellite markers DXYS233, DXS6814, DXYS228, DXYS230 and the SHOX CA repeat [located approximately 15 kilobases (kb) upstream of SHOX]. PCR products were analysed using an ABI Prism TM 377 Genetic Analyser and allele sizes calculated using GENOTYPER TM software (Applied Biosystems, Warrington, UK). The microsatellite marker DXYS232 and the 4 basepair (bp) insertion ⁄ deletion polymorphism at the MIC2 locus were amplified using nonfluorescent primers, and PCR products resolved using polyacrylamide gel electrophoresis and silver staining. FISH was performed on metaphase spreads of lymphocytes using a panel of cosmid probes from the Xp ⁄ Yp telomere spanning PAR1. One of these, LLNOYCO3¢M¢34F5, encompasses the SHOX gene. Cosmids were labelled using the Bio-Nick labelling system (Life Technologies). The results of the microsatellite and FISH analysis are summarized in Fig 1A and B. The individuals tested included the probands P1 and P2, both affected with LWD and Hodgkin’s lymphoma, their mother (M), who only had LWD, their unaffected father (F) and their maternal grandmother (GM). Our results confirmed that the siblings with HL-LWD each harboured a maternally inherited microdeletion within PAR1 encompassing SHOX, estimated to be 900 kb, and located between 200 kb and 1100 kb from the Xp telomere. Three genes of potential importance in HL are located within the pseudoautosomal region, including the interleukin 3 receptor a chain (IL3RA), the granulocyte‐ macrophage colony stimulating factor receptor a chain (CSF2RA) and MIC2, which encodes the CD99 cell adhesion protein. These genes were not, however, within the deleted region in the two siblings (Fig 1C). While it is not yet possible to exclude haploinsufficiency due to the deletion of some unknown tumour suppressor gene, an intriguing possibility is that the PAR1 deletion caused a long-range position effect by downregulating expression of one of the above genes. MIC2 is a particularly strong candidate as decreased expression is associated with the development of Hodgkin’s Reed‐Sternberg cells (H-RS cells; Kim et al, 1998) and is downregulated by EBV latent membrane protein 1, which is highly expressed in H-RS cells (Lee et al, 2001). Against this hypothesis is the fact that PAR1 microdeletions of a similar size are the most common defect in LWD, but there is, as yet, no report of an association between LWD and HL. The existence of an HL-predisposing gene in PAR1 (Horwitz & Wiernik, 1999) thus remains speculative, but further studies of genes in this region in familial Hodgkin’s lymphoma should help to resolve this issue.

We have isolated the murine gene encoding the 5−HT3 receptor (5−HT3R), a member of the ligand−gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5−HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use ofexon 9 splice acceptor sites generated two 5−HT3R variants. The 5−HT3R gene, whose structure is closely related to neuronal and muscle AchRα genes, as demonstrated by four common splice junctions, was localised on human chromosome 11