Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Genetic diagnosis of congenital hypopituitarism in Turkish patients by a target gene panel: novel pathogenic variants in GHRHR, GLI2, LHX4 and POU1F1 genes

ABSTRACT Objective: Congenital hypopituitarism (CH) is a rare disease characterized by one or more hormone deficiencies of the pituitary gland. To date, many genes have been associated with CH. In this study, we identified the allelic variant spectrum of 11 causative genes in Turkish patients with CH. Materials and methods: This study included 47 patients [21 girls (44.6%) and 26 boys (55.4%)] from 45 families. To identify the genetic etiology, we screened 11 candidate genes associated with CH using next-generation sequencing. To confirm and detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. Results: We identified 12 possible pathogenic variants in GHRHR, GH1, GLI2, PROP-1, POU1F1, and LHX4 in 11 patients (23.4%), of which six were novel variants: two in GHRHR, two in POU1F1, one in GLI2, and one in LHX4. In all patients, these variants were most frequently found in GLI2, followed by PROP-1 and GHRHR. Conclusion: Genetic causes were determined in only 23.4% of all patients with CH and 63% of molecularly diagnosed patients (7/11) from consanguineous families. Despite advances in genetics, we were unable to identify the genetic etiology of most patients with CH, suggesting the effect of unknown genes or environmental factors. More genetic studies are necessary to understand the etiology of CH.

Genetic diagnosis of congenital hypopituitarism in Turkish patients by a target gene panel: novel pathogenic variants in GHRHR, GLI2, LHX4 and POU1F1 genes.

Objective: Congenital hypopituitarism (CH) is a rare disease characterized by one or more hormone deficiencies of the pituitary gland. To date, many genes have been associated with CH. In this study, we identified the allelic variant spectrum of 11 causative genes in Turkish patients with CH. Materials and methods: This study included 47 patients [21 girls (44.6%) and 26 boys (55.4%)] from 45 families. To identify the genetic etiology, we screened 11 candidate genes associated with CH using next-generation sequencing. To confirm and detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. Results: We identified 12 possible pathogenic variants in GHRHR, GH1, GLI2, PROP-1, POU1F1, and LHX4 in 11 patients (23.4%), of which six were novel variants: two in GHRHR, two in POU1F1, one in GLI2, and one in LHX4. In all patients, these variants were most frequently found in GLI2, followed by PROP-1 and GHRHR. Conclusion: Genetic causes were determined in only 23.4% of all patients with CH and 63% of molecularly diagnosed patients (7/11) from consanguineous families. Despite advances in genetics, we were unable to identify the genetic etiology of most patients with CH, suggesting the effect of unknown genes or environmental factors. More genetic studies are necessary to understand the etiology of CH.

Association of birth type and LHX4 gene polymorphism with reproductive hormones, growth hormone, and prolactin in Awassi ewes.

BACKGROUND: LIM homeobox transcription factor 4 (LHX4) is a promising candidate gene for mammalian reproductive traits. LHX4 polymorphism has previously been associated with phenotypic traits in goats and cattle. However, there have been no LHX4 gene polymorphisms identified in Awassi sheep. Therefore, this study investigated the effects of the LHX4 polymorphism on reproductive hormones, growth hormones, and prolactin in Awassi ewes. METHODS AND RESULTS: A total of 232 ewes between the ages of 3 and 4 years were selected for this study (123 single-progeny ewes and 109 twin-producing ewes). Serum was collected to measure reproductive hormones, growth hormone, and prolactin using ELISA kits made by ELK Biotechnology. Genomic DNA was extracted from sheep blood, genotyped, and sequenced to confirm variations in LHX4 (exon 1, 207 bp). Genotyping revealed three genotypes in 207 bp: AA, AG, and GG. Sequence analysis detected a novel mutation in exon 1: 160 A > G. Statistically, the 160 A > G SNP was significantly associated with the phenotypic traits. Ewes carrying AA genotypes had higher estrogen, progesterone, follicle-stimulating hormones/luteinizing hormones, and growth hormone, and lower prolactin levels (65.63 ± 3.84) (pg/mL), (6.67 ± 0.38) (ng/mL), (22.34 ± 1.27) (ng/mL)/(23.89 ± 2.13) (ng/mL), (1.30 ± 0.05) (ng/mL), and (13.16 ± 0.75) (pg/mL), respectively, compared to AG and GG genotypes in the fourth month of twin-pregnant ewes compared to single-pregnant ewes. CONCLUSION: This study suggests that the 160 A > G SNP negatively affects the Awassi sheep's hormone levels. It provides valuable insight into the sheep LHX4 gene, which could be an effective marker in marker-assisted selection.

Novel gross deletion at the LHX4 gene locus in a child with growth hormone deficiency.

OBJECTIVE: To identify and characterize a novel deletion at the LHX4 gene locus in a proband with growth hormone deficiency (GHD). METHODS: Long range polymerase chain reaction (PCR) amplification was used to confirm the suspected deletion and to identify the rough locations of the end points. Sanger sequencing was carried out to identify the exact end points of the deletion. RESULTS: Suspected deletion was confirmed via long range PCR amplification. Sanger sequencing identified the end points of the deletion within three nucleotide repeat sequences ("CTT"). The total length of the deleted segment was 12 127 base pairs and it includes complete exon 5 and exon 6 of the LHX4 gene. Therefore the homeodomain motif coded by exons 4 and 5, might be affected. CONCLUSION: We have identified a novel deletion that spans exon 5 and exon 6 of the LHX4 gene that could have occurred via microhomology mediated non-recurrent rearrangement. The deletion characterized does not appear to have been reported before. To our knowledge this novel deletion is the first identified LHX4 variant from Sri Lanka and it explains the phenotype of the proband characterized by growth hormone deficiency, hypoplastic anterior pituitary and subsequent deficiency of thyroid stimulating hormone and adrenocorticotropic hormone (ACTH).

Clinical lessons learned in constitutional hypopituitarism from two decades of experience in a large international cohort.

CONTEXT: The international GENHYPOPIT network collects phenotypical data and screens genetic causes of non-acquired hypopituitarism. AIMS: To describe main phenotype patterns and their evolution through life. DESIGN: Patients were screened according to their phenotype for coding sequence variations in 8 genes: HESX1, LHX3, LHX4, PROP1, POU1F1, TBX19, OTX2 and PROKR2. RESULTS: Among 1213 patients (1143 index cases), the age of diagnosis of hypopituitarism was congenital (24%), in childhood (28%), at puberty (32%), in adulthood (7.2%) or not available (8.8%). Noteworthy, pituitary hormonal deficiencies kept on evolving during adulthood in 49 of patients. Growth Hormone deficiency (GHD) affected 85.8% of patients and was often the first diagnosed deficiency. AdrenoCorticoTropic Hormone deficiency rarely preceded GHD, but usually followed it by over 10 years. Pituitary Magnetic Resonance Imaging (MRI) abnormalities were common (79.7%), with 39.4% pituitary stalk interruption syndrome (PSIS). The most frequently associated extrapituitary malformations were ophthalmological abnormalities (16.1%). Prevalence of identified mutations was 7.3% of index cases (84/1143) and 29.5% in familial cases (n = 146). Genetic analysis in 449 patients without extrapituitary phenotype revealed 36 PROP1, 2 POU1F1 and 17 TBX19 mutations. CONCLUSION: This large international cohort highlights atypical phenotypic presentation of constitutional hypopituitarism, such as post pubertal presentation or adult progression of hormonal deficiencies. These results justify long-term follow-up, and the need for systematic evaluation of associated abnormalities. Genetic defects were rarely identified, mainly PROP1 mutations in pure endocrine phenotypes.

[Thyroid disruptor p, p'-DDE inhibited the expression of LHX4 and DIS3L protein in Nthy-ori-3-1 cells].

Objective: To observe the changes of LHX4 and DIS3L mRNA and protein expression in Nthy-ori-3-1 cells after the treatment of thyroid disruptor p, p'-DDE. Methods: Nthy-ori-3-1 cells in logarithmic growth phase were treated with 0, 0.5, 1.0, 2.0 and 5.0 µg/ml p, p'-DDE solution. The growth state and morphology of the cells were observed by microscope. The mRNA levels of LHX4 and DIS3L were detected by real-time fluorescent quantitative PCR, and the protein expression levels of LHX4 and DIS3L were detected by Western blot. Results: when the concentrations of p, p'-DDE were 0, 0.5, 1.0 and 2.0 µg/ml, Nthy-ori-3-1 cells grew normally. There were 33 differential genes in 2.0 µg/ml group, among which 13 genes were down regulated and 20 genes were up-regulated. Compared with the control group, the protein expression levels of LHX4 and DIS3L in 1.0 and 2.0 µg/ml groups were significantly decreased (P<0.05) , and the relative expression levels of LHX4 and DIS3L protein mRNA in 1.0 µg/ml group were significantly decreased (P<0.05) . Conclusion: p, p'-DDE can affect the protein expression of LHX4 and dis3l in nthy-ori-3-1 cells.

Genetic analysis of adult Slovenian patients with combined pituitary hormone deficiency.

PURPOSE: Among genetic causes of combined pituitary hormone deficiency (CPHD), mutations of genes coding for transcription factors involved in pituitary development have been implicated. Congenital CPHD is a rare disease; therefore, it is important to expand the knowledge about incidence and regional distribution of specific mutations. The aim of this paper is to report results of genetic analyses of adult Slovenian patients with CPHD. METHODS: Twenty-three adult Slovenian patients with early childhood onset CPHD were included in the study. Blood samples were collected through the GENHYPOPIT network to assess possible mutations of six genes (PROP1/HESX1/LHX4/LHX3/POU1F1) involved in the pituitary development following an established algorithm. RESULTS: In seven out of 23 patients (30%) a specific mutation in genes encoding pituitary transcription factors was discovered. In five patients, two different mutations of the PROP1 gene (c.150delA and c.301-302delAG) were identified. One patient was heterozygous for a missense variant in the LHX4 gene. Additionally, one patient was positive for a mutation in the gene coding for prokineticin receptor-2. CONCLUSIONS: Our study confirms that the two most common mutations of the PROP1 gene globally are also the most frequent mutations in the cohort of adult Slovenian patients with CHPD. Other mutations of pituitary transcription factor genes are extremely rare.

Interstitial duplication of chromosome region 1q25.1q25.3: report of a patient with mild cognitive deficits, tall stature and facial dysmorphisms.

Isolated interstitial duplications of chromosome band 1q25 are apparently very rare; no patients with detailed molecular and clinical characterization of duplications restricted to this region have been published to date. We report on a 9-year-old girl with mild cognitive deficits, tall stature, macrocephaly and discrete dysmorphic features in whom a de novo interstitial 7.5 Mb duplication of 1q25.1q25.3 was detected by SNP array analysis (arr[hg19] 1q25.1q25.3(173,925,505-181,381,242)x3 dn). The duplicated region was inversely inserted into chromosome band 1q42.2: 46,XX,der(1)(pter→q42.2::q25.3→q25.1::q42.2→qter). Overexpression of one or several of the 87 genes in the duplicated interval was presumably the major causative factor for the clinical manifestations. Reports of additional patients with overlapping duplications will be needed to establish detailed karyotype-phenotype correlations and to gain a better understanding of the underlying pathomechanisms.

Impact of mineralocorticoid receptor polymorphisms on urinary electrolyte excretion with and without diuretic drugs.

AIM: Polymorphisms in the mineralocorticoid receptor may affect urinary sodium and potassium excretion. We investigated polymorphisms in the MR gene in relation to urinary electrolyte excretion in two separate studies. PATIENTS & METHODS: The genotype-phenotype association was studied in healthy volunteers after single doses of bumetanide, furosemide, torsemide, hydrochlorothiazide, triamterene and after NaCl restriction. RESULTS: High potassium excretion under all conditions except torsemide, and high NaCl excretion after bumetanide and furosemide were associated with the A allele of the intron-3 polymorphism (rs3857080). This polymorphism explained 5-10% of the functional variation and in vitro, rs3857080 affected DNA binding of the transcription factor LHX4. CONCLUSION: rs3857080 may be a promising new candidate for research in cardiac and renal disorders and on antialdosteronergic drugs like spironolactone.

Lhx3 and Lhx4 suppress Kolmer-Agduhr interneuron characteristics within zebrafish axial motoneurons.

A central problem in development is how fates of closely related cells are segregated. Lineally related motoneurons (MNs) and interneurons (INs) express many genes in common yet acquire distinct fates. For example, in mouse and chick Lhx3 plays a pivotal role in the development of both cell classes. Here, we utilize the ability to recognize individual zebrafish neurons to examine the roles of Lhx3 and its paralog Lhx4 in the development of MNs and ventral INs. We show that Lhx3 and Lhx4 are expressed by post-mitotic axial MNs derived from the MN progenitor (pMN) domain, p2 domain progenitors and by several types of INs derived from pMN and p2 domains. In the absence of Lhx3 and Lhx4, early-developing primary MNs (PMNs) adopt a hybrid fate, with morphological and molecular features of both PMNs and pMN-derived Kolmer-Agduhr' (KA') INs. In addition, we show that Lhx3 and Lhx4 distinguish the fates of two pMN-derived INs. Finally, we demonstrate that Lhx3 and Lhx4 are necessary for the formation of late-developing V2a and V2b INs. In conjunction with our previous work, these data reveal that distinct transcription factor families are deployed in post-mitotic MNs to unequivocally assign MN fate and suppress the development of alternative pMN-derived IN fates.

Linkage disequilibrium and haplotype distribution of the bovine LHX4 gene in relation to growth.

LIM homeobox transcription factor 4 (LHX4) is a LIM homeodomain transcription factor involved in pituitary gland and nervous system development. The aim of this study was to examine the association of the LHX4 polymorphisms with growth traits in beef cattle breed. A total of 7 single nucleotide polymorphyisms (SNPs) have been identified in the coding region and noncoding region of the bovine LHX4 by sequencing pooled DNA samples (Pool-Seq) and PCR-single strand conformation polymorphism (PCR-SSCP) methods. The linkage disequilibrium was assessed in 871 individuals representing four main cattle breeds from China. The SNPs 2-5 and 7-8 were found to be in complete linkage disequilibrium, respectively. The result of haplotype analysis of 13 SNPs showed that 31 haplotypes were found in four Chinese cattle breeds, and 20 genotypes were only found in Nanyang cattle. The statistical analyses indicated that the SNP1-5, and 6 are associated with the body weight at 18, and 6 months of age in Nanyang cattle population (P<0.05), but no significant associations between their twenty combined genotypes. Our results provide evidence that some polymorphisms in LHX4 are associated with growth traits at certain ages, and may be used as candidates for marker-assisted selection and management in cattle.

Genetic variants and effects on milk traits of the caprine paired-like homeodomain transcription factor 2 (PITX2) gene in dairy goats.

The paired-like homeodomain transcription factor 2 (PITX2) gene plays a critical role in cell proliferation, differentiation, hematopoiesis and organogenesis. This gene regulates several genes' expressions in the Wnt/beta-catenin and POU1F1 pathways, thereby probably affecting milk performance. The goal of this study was to characterize the genetic variants of the PITX2 gene and test their associations with milk traits in dairy goats. Herein, four novel single nucleotide polymorphisms (SNPs), AC_000163:g.18117T>C, g.18161C>G, g.18322C>A and g.18353T>C, within the caprine PITX2 gene, were found in two famous Chinese dairy goat breeds. These SNPs mapping at Cys28Arg, Pro42Pro, IVS1+79C>A and IVS1+110T>C, were genotyped by the MvaI, SmaI, MspI and RsaI aCRS-RFLP or PCR-RFLP methods, respectively. Accordingly, two main haplotypes (CGCT and CGCC) were identified among the specimens. Association testing revealed that the SmaI and RsaI polymorphisms were significantly associated with the milk fat content, milk lactose content and milk density (P<0.05 or P<0.01) in the Guanzhong (GZ) dairy goats, respectively. At the same time, the RsaI locus was also found to significantly link to the second lactation milk yield, milk fat content, milk lactose content, milk density and milk total solid content (P<0.05 or P<0.01) in the Xinong Saanen (XNSN) dairy goats, respectively. These results indicated that the caprine PITX2 gene had the significant effects on milk traits. Hence, the RsaI and SmaI loci could be regarded as two DNA markers for selecting superior milk performance in dairy goats. These preliminary findings not only would extend the spectrum of genetic variation of the goat PITX2 gene, but also would contribute to implementing marker-assisted selection (MAS) in breeding and genetics in dairy goats.

A critical functional missense mutation (H173R) in the bovine PROP1 gene significantly affects growth traits in cattle.

The PROP1 protein, encoded by the prophet of Pit-1 (PROP1) gene, exhibits both DNA-binding and transcriptional activation abilities. Its expression leads to the ontogenesis of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), and pituitary hormone. The missense mutation H173R in PROP1 may result in deficiencies of GH, PRL, TSH, and Pit-1, thereby affecting growth traits. The objective of this study was to characterize the H173R mutation within the PROP1 gene and examine its associations with growth traits in cattle. Accordingly, the H173R mutation was genotyped in 1207 cows belonging to five Chinese native breeds. Three genotypes were identified among the specimens, with genotype AA being the major one. Consequently, the "G" allele was the minor allele. Association testing revealed that the H173R mutation was significantly associated with body weight, average daily weight gain and physical parameters in the analyzed breeds. Interestingly, the cows with genotype AG and/or AA had superior growth traits compared with those expressing the GG genotype, in all tested breeds. These findings revealed that the "A" allele had positive effects on growth traits, which was consistent with the increasing binding ability and enhanced activation capacity associated with the bovine isoform PROP1-173H, representing the "A" allele. Therefore, the H173R mutation can be considered as a DNA marker for selecting individuals with superior growth traits, thereby contributing to research on breeding and genetics in the beef industry.

Molecular and Clinical Findings in Patients with LHX4 and OTX2 Mutations.

The pituitary gland produces hormones that play important roles in both the development and homeostasis of the body. Ontogeny of the anterior and posterior pituitary is orchestrated by inputs from neighboring tissues, cellular signaling molecules and transcription factors. Disruption of expression or function of these factors has been implicated in the etiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1F1, LHX3, LHX4, OTX2, SOX2, SOX3 and GLI2. This review focuses on summarizing most recent mutations in LHX4 and OTX2 responsible for pituitary hormone deficiency. In both genetic defects of LHX4 and OTX2, there is high variability in clinical manifestations even in the same family. In addition, there is no clear phenotype-genotype correlation. These findings indicate that the other genetic and/or environmental factors influence the phenotype. In addition, the variability might reflect a plasticity during pituitary development and maintenance. Over the past two decades, a genetic basis for pituitary hormone deficiency and the mechanism of pituitary development have been clarified. It should be kept in mind that this review is not comprehensive, and defects of other transcriptional factors have been described in patients with CPHD. Furthermore, the causes in many patients with CPHD have not yet been determined. Therefore, continuing efforts for the clarification of the etiology are necessary.

Cat eye syndrome and growth hormone deficiency with pituitary anomalies: a case report and review of the literature.

Cat eye syndrome is a rare congenital disease characterized by the existence of a supernumerary chromosome derived from chromosome 22, with a variable phenotype comprising anal atresia, coloboma of the iris and preauricular tags or pits. We report a girl with cat eye syndrome, presenting short stature, with growth hormone deficiency due to posterior pituitary ectopia. Short stature is a common feature of this syndrome, and the association with a structural pituitary anomaly has been described, however growth hormone deficiency and the underlying mechanisms are rarely reported. A review on short stature and growth hormone deficiency in cat eye syndrome is conducted.

Congenital pituitary hormone deficiencies: role of LHX3/LHX4 genes.

LHX3 and LHX4 are LIM domain transcription factors involved in the early steps of pituitary organogenesis. They are necessary for the proper differentiation of Rathke's pouch that gives rise to the anterior pituitary lobe. Mutations of these transcription factors are involved in congenital hypopituitarism: to date, nine mutations of LHX3 have been reported, responsible for variable pituitary hormone deficiencies and extrapituitary manifestations, including limited neck rotation. By contrast, only five LHX4 mutations have been reported, responsible for variable hormone deficiencies, and pituitary/intracranial abnormalities. Future investigations will aim to better understand human pituitary organogenesis and to shed light on the interspecies differences in the roles of these transcription factors.

Hypothalamic-pituitary magnetic resonance imaging in growth hormone deficiency.

The accurate analysis of the hypothalamic-pituitary area is essential in the diagnosis of endocrine-related diseases. High-quality magnetic resonance imaging represents the examination modality of choice in the evaluation of hypothalamic-pituitary morphology. Indeed, the advent of molecular biology and neuroimaging techniques has led to significant progress in the understanding of the pathogenesis of disorders affecting the pituitary gland, specifically by demonstrating a clear phenotype-genotype relationship. Animal studies, along with the correlation of a particular genetic profile to certain endocrine and magnetic resonance imaging phenotypes in humans, have yielded great insights into pituitary development. Today, there is convincing evidence to support the hypothesis that marked magnetic resonance imaging differences in pituitary morphology indicate a variety of disorders that affect anterior pituitary gland organogenesis and function with a variety of diverse prognoses.