Identification of SLC20A1 and SLC15A4 among other genes as potential risk factors for combined pituitary hormone deficiency.

PURPOSE: Combined pituitary hormone deficiency (CPHD) is characterized by a malformed or underdeveloped pituitary gland resulting in an impaired pituitary hormone secretion. Several transcription factors have been described in its etiology, but defects in known genes account for only a small proportion of cases. METHODS: To identify novel genetic causes for congenital hypopituitarism, we performed exome-sequencing studies on 10 patients with CPHD and their unaffected parents. Two candidate genes were sequenced in further 200 patients. Genotype data of known hypopituitary genes are reviewed. RESULTS: We discovered 51 likely damaging variants in 38 genes; 12 of the 51 variants represent de novo events (24%); 11 of the 38 genes (29%) were present in the E12.5/E14.5 pituitary transcriptome. Targeted sequencing of two candidate genes, SLC20A1 and SLC15A4, of the solute carrier membrane transport protein family in 200 additional patients demonstrated two further variants predicted as damaging. We also found combinations of de novo (SLC20A1/SLC15A4) and transmitted variants (GLI2/LHX3) in the same individuals, leading to the full-blown CPHD phenotype. CONCLUSION: These data expand the pituitary target genes repertoire for diagnostics and further functional studies. Exome sequencing has identified a combination of rare variants in different genes that might explain incomplete penetrance in CPHD.

Investigation of SHANK3 in schizophrenia.

The postsynaptic scaffolding protein SHANK3 is essential for the normal function of glutamatergic synapses in the brain. Emerging evidence suggests that impaired plasticity of glutamatergic synapses contributes to the pathology of schizophrenia (SCZ). To investigate whether variants in the SHANK3 gene contribute to the etiology of SCZ, we sequenced SHANK3 in 500 affected individuals (cohort C1). In total, we identified 48 variants and compared them to European controls from the 1000 Genomes Project and the Exome Variant Server. Five variants showed significant differences in frequencies between patients and controls. We were able to follow three of them up in an independent cohort (C2) comprising 993 SCZ patients and 932 German controls. We could not confirm an association for three of these variants (rs140201628, rs1557620, and rs61729471). Two rare variants with predicted functional relevance were identified in further SCZ individuals of cohort C1: c.3032G>T (p.G1011V) and c.*27C>T. The latter variant was found in one additional SCZ individual and the p.G1011V variant was identified in two additional SCZ individuals from cohort C2. The p.G1011V variant was the most interesting variant in our study; together with previous studies this variant has been identified in 4 out of 1,524 SCZ patients and in 4 out of 2,147 individuals with autism spectrum disorder (ASD), but not in 2468 European Sanger-sequenced controls. Therefore, we consider this variant a promising candidate variant for follow-up studies in larger samples and functional investigations. © 2017 Wiley Periodicals, Inc.

Coding and non-coding variants in the SHOX2 gene in patients with early-onset atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia with a strong genetic component. Molecular pathways involving the homeodomain transcription factor Shox2 control the development and function of the cardiac conduction system in mouse and zebrafish. Here we report the analysis of human SHOX2 as a potential susceptibility gene for early-onset AF. To identify causal variants and define the underlying mechanisms, results from 378 patients with early-onset AF before the age of 60 years were analyzed and compared to 1870 controls or reference datasets. We identified two missense mutations (p.G81E, p.H283Q), that were predicted as damaging. Transactivation studies using SHOX2 targets and phenotypic rescue experiments in zebrafish demonstrated that the p.H283Q mutation severely affects SHOX2 pacemaker function. We also demonstrate an association between a 3'UTR variant c.*28T>C of SHOX2 and AF (p = 0.00515). Patients carrying this variant present significantly longer PR intervals. Mechanistically, this variant creates a functional binding site for hsa-miR-92b-5p. Circulating hsa-miR-92b-5p plasma levels were significantly altered in AF patients carrying the 3'UTR variant (p = 0.0095). Finally, we demonstrate significantly reduced SHOX2 expression levels in right atrial appendages of AF patients compared to patients with sinus rhythm. Together, these results suggest a genetic contribution of SHOX2 in early-onset AF.

Identification of novel genes including NAV2 associated with isolated tall stature.

Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211 cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism.

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.

Evidence That Non-Syndromic Familial Tall Stature Has an Oligogenic Origin Including Ciliary Genes.

Human growth is a complex trait. A considerable number of gene defects have been shown to cause short stature, but there are only few examples of genetic causes of non-syndromic tall stature. Besides rare variants with large effects and common risk alleles with small effect size, oligogenic effects may contribute to this phenotype. Exome sequencing was carried out in a tall male (height 3.5 SDS) and his parents. Filtered damaging variants with high CADD scores were validated by Sanger sequencing in the trio and three other affected and one unaffected family members. Network analysis was carried out to assess links between the candidate genes, and the transcriptome of murine growth plate was analyzed by microarray as well as RNA Seq. Heterozygous gene variants in CEP104, CROCC, NEK1, TOM1L2, and TSTD2 predicted as damaging were found to be shared between the four tall family members. Three of the five genes (CEP104, CROCC, and NEK1) belong to the ciliary gene family. All genes are expressed in mouse growth plate. Pathway and network analyses indicated close functional connections. Together, these data expand the spectrum of genes with a role in linear growth and tall stature phenotypes.

Identification of Transient Receptor Potential Channel 4-Associated Protein as a Novel Candidate Gene Causing Congenital Primary Hypothyroidism.

BACKGROUND: Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. METHODS: To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (TRPC4AP), was sequenced in 179 further patients with TD. Expression of TRPC4AP in human thyroid was investigated using RT-PCR. Trpc4ap- functional analysis was performed in Xenopus laevis using Morpholino (MO) antisense oligomers. RESULTS: WES identified a likely damaging mutation in TRPC4AP leading to a de novo stop codon p.Q552*. Targeted sequencing of TRPC4AP demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that TRPC4AP is expressed in human thyroid gland tissue. Using Xenopus laevis, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in Trpc4ap MO knockdowns compared to controls and by 41% in "Clustered Regularly Interspaced Short Palindromic Repeats"/Cas9-mediated gene knockout experiments. DISCUSSION: A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by IKBKG gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. CONCLUSION: TRPC4AP was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.

Evaluation of the bioaccumulation of octocrylene after dietary and aqueous exposure.

Octocrylene is used as UV filter in personal care products with a high production volume and can be detected in surface water and biota. It is liquid at ambient temperature, highly lipophilic, has a high adsorption capacity to organic material and is considered as persistent in the environment. The very low water solubility complicates the evaluation of potential long-term effects in aquatic toxicity testing, since effect thresholds are often above the water solubility limit. Thus, the evaluation of the bioaccumulation potential becomes highly relevant for the assessment of long-term environmental effects. However, even the determination of the water solubility limit for a substance with such difficult properties is challenging. The following experiments are described, and results compared to available environmental monitoring data: A bioconcentration study with aqueous exposure (BCF) in zebrafish and a biomagnification study with dietary exposure (BMF) in rainbow trout, as well as supporting experiments to evaluate the water solubility. The growth and lipid corrected BCF determined by aqueous exposure was 858 L kg-1 while the corrected BMF was 0.0335. The model-based estimation of the BCF from BMF (152-1182 L kg-1) is in good agreement with the measured BCF value. Environmental monitoring data provide only limited information on the bioaccumulation potential of octocrylene, as only few investigations were made in biota and water in parallel and concentrations of octocrylene vary by several orders of magnitude during seasons. Based on the determined fish BCF data, we conclude that OCR is not bioaccumulative according to the criteria as laid down by ECHA, 2017. Furthermore, the low BMF value indicates no accumulation along the food chain.

Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation.

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing SHOX2 gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended SHOX2 gene analysis in patients with distinct arrhythmias, we investigated SHOX2 as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated in vivo and in vitro, together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. In vitro reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced Bmp4 target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the SHOX2 gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving SHOX2. Moreover, our data highlight the importance of functional investigations of rare variants.

Functional missense and splicing variants in the retinoic acid catabolizing enzyme CYP26C1 in idiopathic short stature.

Height is a complex quantitative trait with a high heritability. Short stature is diagnosed when height is significantly below the average of the general population for that person's age and sex. We have recently found that the retinoic acid degrading enzyme CYP26C1 modifies SHOX deficiency phenotypes toward more severe clinical manifestations. Here, we asked whether damaging variants in CYP26C1 alone could lead to short stature. We performed exome and Sanger sequencing to analyze 856 individuals with short stature where SHOX deficiency was previously excluded. Three different damaging missense variants and one splicing variant were identified in six independent individuals; the functional significance of the identified variants was tested in vitro or in vivo using zebrafish as a model. The genetic and functional data reported here indicate that CYP26C1 represents a novel gene underlying growth disorders and that damaging variants in the absence of SHOX variants can lead to short stature.

Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency.

Mutations in the homeobox gene SHOX cause SHOX deficiency, a condition with clinical manifestations ranging from short stature without dysmorphic signs to severe mesomelic skeletal dysplasia. In rare cases, individuals with SHOX deficiency are asymptomatic. To elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with SHOX deficiency. The variant p.Phe508Cys of the retinoic acid catabolizing enzyme CYP26C1 co-segregated with the SHOX variant p.Val161Ala in the affected individuals, while the SHOX mutant alone was present in asymptomatic individuals. Two further cases with SHOX deficiency and damaging CYP26C1 variants were identified in a cohort of 68 individuals with LWD The identified CYP26C1 variants affected its catabolic activity, leading to an increased level of retinoic acid. High levels of retinoic acid significantly decrease SHOX expression in human primary chondrocytes and zebrafish embryos. Individual morpholino knockdown of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. Together, our findings describe CYP26C1 as the first genetic modifier for SHOX deficiency.

The definition of the Y chromosome growth-control gene (GCY) critical region: relevance of terminal and interstitial deletions.

Deletions of the long arm of the Y chromosome are often associated with short stature and infertility. Initial mapping assignments to narrow down the localization of the Y chromosome growth-control gene, GCY, were based on molecular analysis of patients carrying a 46,XYq- karyotype as defined by classic karyotyping. Two non-overlapping critical regions for GCY were defined. To determine whether one or both of these regions contained GCY, adult patients with monocentric Yq- chromosomes were reinvestigated in detail. Fluorescence in situ hybridization (FISH) analysis showed that all patients previously defined as having a pure 46,XYq- karyotype were in fact mosaics, with cells containing an idic(Y) or ring(Y) chromosome in association with 45,X cells. This excluded patients with terminal deletions as reliable subjects of study to locate the stature gene on Yq. In contrast, detailed molecular deletion analysis of patients who had interstitial deletions of the long arm of the Y chromosome pointed unequivocally to a single region next to the centromere that had a possible impact on growth. No functional gene has been identified in this region.

Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation.

Using microarrays, we identified de novo copy number variations in the SHANK2 synaptic scaffolding gene in two unrelated individuals with autism-spectrum disorder (ASD) and mental retardation. DNA sequencing of SHANK2 in 396 individuals with ASD, 184 individuals with mental retardation and 659 unaffected individuals (controls) revealed additional variants that were specific to ASD and mental retardation cases, including a de novo nonsense mutation and seven rare inherited changes. Our findings further link common genes between ASD and intellectual disability.

BNP is a transcriptional target of the short stature homeobox gene SHOX.

Short stature due to SHOX deficiency represents a common congenital form of growth failure and is involved in the aetiology of 'idiopathic' short stature and the growth deficits and skeletal anomalies in Leri-Weill, Langer and Turner syndromes. Although much is known on the clinical and molecular aspects of SHOX haploinsufficiency, the integration of SHOX in the signalling pathways regulating bone growth is currently not defined. Here we identify NPPB encoding the natriuretic peptide, BNP, a well-known cardiac and natriuretic peptide hormone, as a transcriptional target of SHOX. The ability of SHOX to transactivate the NPPB endogenous promoter was demonstrated in luciferase reporter assays using serial deletions of the NPPB promotor region. Binding of SHOX to the NPPB promoter was also demonstrated in vivo by chromatin fixation and immunoprecipitation. We also demonstrate the lack of promoter activation in two SHOX mutants from patients with Leri-Weill syndrome. In addition, immunohistochemical analysis of human growth plate sections showed for the first time a co-expression of BNP and SHOX in late proliferative and hypertrophic chondrocytes. Together these data strongly suggest that BNP represents a direct target of SHOX.

Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients.

Defects in long-range regulatory elements have recently emerged as previously underestimated factors in the genesis of human congenital disorders. Léri-Weill dyschondrosteosis is a dominant skeletal malformation syndrome caused by mutations in the short stature homeobox gene SHOX. We have analysed four families with Léri-Weill dyschondrosteosis with deletions in the pseudoautosomal region but still with an intact SHOX coding region. Using fluorescence in situ hybridization and single nucleotide polymorphism studies, we identified an interval of approximately 200 kb that was deleted in all tested affected family members but retained in the unaffected members and in 100 control individuals. Comparative genomic analysis of this interval revealed eight highly conserved non-genic elements between 48 and 215 kb downstream of the SHOX gene. As mice do not have a Shox gene, we analysed the enhancer potential in chicken embryos using a green fluorescent protein reporter construct driven by the beta-globin promoter, by in ovo electroporation of the limb bud. We observed cis-regulatory activity in three of the eight non-genic elements in the developing limbs arguing for an extensive control region of this gene. These findings are consistent with the idea that the deleted region in the affected families contains several distinct elements that regulate Shox expression in the developing limb. Furthermore, the deletion of these elements in humans generates a phenotype apparently undistinguishable to those patients identified with mutations in the SHOX coding region and, for the first time, demonstrates the potential of an in vivo assay in chicken to monitor putative enhancer activity in relation to human disease.

Interchromosomal segmental duplications of the pericentromeric region on the human Y chromosome.

Basic medical research critically depends on the finished human genome sequence. Two types of gaps are known to exist in the human genome: those associated with heterochromatic sequences and those embedded within euchromatin. We identified and analyzed a euchromatic island within the pericentromeric repeats of the human Y chromosome. This 450-kb island, although not recalcitrant to subcloning and present in 100 tested males from different ethnic origins, was not detected and is not contained within the published Y chromosomal sequence. The entire 450-kb interval is almost completely duplicated and consists predominantly of interchromosomal rather than intrachromosomal duplication events that are usually prevalent on the Y chromosome. We defined the modular structure of this interval and detected a total of 128 underlying pairwise alignments (>/=90% and >/=1 kb in length) to various autosomal pericentromeric and ancestral pericentromeric regions. We also analyzed the putative gene content of this region by a combination of in silico gene prediction and paralogy analysis. We can show that even in this exceptionally duplicated region of the Y chromosome, eight putative genes with open reading frames reside, including fusion transcripts formed by the splicing of exons from two different duplication modules as well as members of the homeobox gene family DUX.

Molecular and evolutionary analysis of the growth-controlling region on the human Y chromosome.

The human Y chromosome has been predicted to harbour a locus termed GCY, affecting height in males. GCY has been positioned by deletion mapping to the pericentromeric region on the long arm of the Y chromosome. As the relevant gene has not been identified yet, we have carried out exon amplification and isolated nine different exon trap clones within the critical region. Gene prediction programs have proposed 17 different gene models and standard BLASTN searches with the genomic sequence detected significant homologies to six known genes/pseudogenes or expressed sequence tags. Large-scale cDNA library screening and reverse transcription of polyA(+) RNAs, however, could not demonstrate unequivocally the existence of a novel transcriptional unit. All potential transcriptional units are embedded in subintervals of the GCY critical region that have been transposed to the human Y during different stages of primate evolution. These results challenge our present view on the Y-chromosomal stature locus GCY, proposing the existence of an unusual gene with an extremely confined spatial and/or temporal expression pattern, albeit the structural impact of the nearby pericentromeric heterochromatin should not be excluded.