Novel mutations in LHX3 are associated with hypopituitarism and sensorineural hearing loss

Homozygous loss-of-function mutations in the transcription factor LHX3 have been associated with hypopituitarism with structural anterior pituitary defects and cervical abnormalities with or without restricted neck rotation. We report two novel recessive mutations in LHX3 in four patients from two unrelated pedigrees. Clinical evaluation revealed that all four patients exhibit varying degrees of bilateral sensorineural hearing loss, which has not been previously reported in association with LHX3 mutations, in addition to hypopituitarism including adrenocorticotropic hormone deficiency and an unusual skin and skeletal phenotype in one family. Furthermore, re-evaluation of three patients previously described with LHX3 mutations showed they also exhibit varying degrees of bilateral sensorineural hearing loss. We have investigated a possible role for LHX3 in inner ear development in humans using in situ hybridization of human embryonic and fetal tissue. LHX3 is expressed in defined regions of the sensory epithelium of the developing inner ear in a pattern overlapping that of SOX2, which precedes the onset of LHX3 expression and is known to be required for inner ear and pituitary development in both mice and humans. Moreover, we show that SOX2 is capable of binding to and activating transcription of the LHX3 proximal promoter in vitro. This study therefore extends the phenotypic spectrum associated with LHX3 mutations to encompass variable sensorineural hearing loss and suggests a possible interaction between LHX3 and SOX2 likely to be important for development of both the inner ear and the anterior pituitary in human embryonic development

Increased Transactivation Associated with SOX3 Polyalanine Tract Deletion in a Patient with Hypopituitarism

Backgound and Aims: Correct gene dosage of SOX3 is critical for the development of the hypothalamo-pituitary axis. Both overdosage of SOX3, as a result of gene duplication, and loss of function resulting from expansion of the first polyalanine (PA) tract are associated with variable degrees of hypopituitarism, with or without mental retardation. The aim of this study was to further investigate the contribution of SOX3 in the etiology of hypopituitarism and the mechanisms involved in the phenotypic variability. Methods: We screened 154 patients with congenital hypopituitarism and an undescended posterior pituitary for mutations in SOX3 and variability in the length of the first PA tract. In addition, 300 patients with variable septooptic dysplasia were screened for variability of the PA tract. Results: We report a novel 18-base pair deletion (p.A243_A248del6, del6PA) in a female patient with hypopituitarism resulting in a 2-fold increase in transcriptional activation in vitro, compared with wild-type SOX3. We also identified a previously reported seven-alanine expansion (p.A240_A241ins7, +7PA) in two male siblings with isolated GH deficiency and a distinct phenotype, in addition to the nonsynonymous variant p.R5Q in an unrelated individual; this appears to have no functional effect on the protein. In contrast to +7PA, del6PA maintained its ability to repress beta-catenin mediated transcription in vitro. Conclusion: This is the first study to report that PA tract deletions associated with hypopituitarism have functional consequences in vitro, possibly due to increased activation of SOX3 target genes. In addition, we have expanded the phenotypic spectrum associated with PA tract expansion (+7PA) mutations to include panhypopituitarism or isolated GH deficiency, with or without mental retardation. (J Clin Endocrinol Metab 96: E685-E690, 2011)Medical Research Council (MRC) United KingdomEuropean Society for Pediatric Endocrinolog

<i>POMC</i> intron2 exon3 genomic sequence, P300 ChIP assay, real-time PCR analysis, and real-time <i>POMC</i> PCR.

<p>(A) <i>POMC</i> intron2 exon3 genomic sequence with annotated ChIP primer localization and P300 binding site. (B) P300 ChIP assay was performed at three independent occasions within human peripheral blood cells. <i>POMC</i> fragment was amplified with two different primer pairs (fragment 1 and 2). Confirmation of a published P300 binding site within the insulin gene promoter in β-TC3 cells was used as a positive control <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002543#pgen.1002543-Chakrabarti1" target="_blank">[34]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002543#pgen.1002543.s005" target="_blank">Figure S5</a>). (C) Real time PCR analysis (PCR fragment 2) of the P300 ChIP results of 4 obese patients with a hypermethylation variant and 6 individuals with hypomethylated intron 2-exon 3 intersection at <i>POMC</i> CpG island 2. (D) Real-time <i>POMC</i> PCR (<i>POMC</i> exon 2–3 and <i>POMC</i> exon3) of cDNA extracted from PBC of hypomethylated normal weight individuals (n = 20), hypermethylated obese patients (n = 20), and obese individuals without the hypermethylated variant (n = 20) reveal reduced POMC gene expression in the hypermethylated samples.</p

<i>POMC</i> CpG methylation pattern of CpG island 1 and 2.

<p>Analysis example of the DNA methylation pattern of human peripheral blood cells (PBC) from a normal weight individual after sequencing of 10 different clones of the PCR amplification product. Filled cycles represent methylated CpGs, open cycles non-methylated CpG positions, which are numbered according to their relative position to the start of the next exon. Alu element positions and P300 binding site are marked.</p