The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3Ј-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs Cold Spring Harbor Laboratory Press on May 10, 2018 -Published by genome.cshlp.org Downloaded from annotated in the FANTOM-2 annotation. We have also produced 547,149 5Ј end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, 5Ј-end clusters identify regions that are potential promoters for 8637 known genes and 5Ј-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.[Supplemental material available online at www.genome.org.]One of the primary goals of genome sequencing projects is to identify the genome sequences that are transcribed into functional mRNAs, so that full-length cDNAs can be isolated to allow further downstream biology, and functional and structural genomics. The limitations of a priori genome annotation dictate that the transcriptome needs to be identified experimentally via cDNA cloning and sequencing. Although expressed sequence tags (ESTs) (Adams et al. 1991(Adams et al. , 1995Hillier et al. 1996;Marra et al. 1999;Kargul et al. 2001) and ORESTES (Camargo et al. 2001) have been extremely valuable for new gene discovery, these approaches have not allowed highthroughput recovering of full-length cDNA clones nor definition of protein sequence derived from actual cDNA clones. To overcome such problems, we undertook from the year 1995, a strategic project aimed at the comprehensive collection of at least one full-length cDNA derived from each mouse gene, a strategy that is recently becoming useful in similar projects to collect full-length gene collections (Stapleton et al. 2002;Strausberg et al. 2002).Because of the limited processivity of reverse transcriptase and other limitations, standard cDNA libraries generally contain a majority of truncated transcripts. The introductio...

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor ( MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98 , another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or nonsyndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.

The anterior and intermediate lobes of the pituitary gland are formed from Rathke's pouch. FGF, BMP and WNT signals emanating from the ventral diencephalon influence pouch growth and development. In order to examine the role of canonical WNT signaling during pituitary development we examined the pituitary expression of the TCF/LEF family of transcription factors, which mediate WNT signaling through the binding of beta-catenin. We report here the expression of several members of this family during pituitary development and the functional role of one member, TCF4 (TCF7L2), in the induction of the pituitary primordium. TCF4 is expressed in the ventral diencephalon early in pituitary development, rostral to a domain of BMP and FGF expression. Tcf4 deficient mice express Fgf10 and Bmp4; however, the Bmp and Fgf expression domains are expanded rostrally. As a result, additional pituitary progenitor cells are recruited into Rathke's pouch in Tcf4 mutants. Mutants also exhibit an expansion of the Six6 expression domain within Rathke's pouch, which may increase the number of proliferating pouch cells, resulting in a greatly enlarged anterior pituitary gland. This suggests that TCF4 negatively regulates pituitary growth through two mechanisms. The first mechanism is to restrict the domains of BMP and FGF signaling in the ventral diencephalon, and the second mechanism is the restriction of Six6 within Rathke's pouch. Thus, TCF4 is necessary both intrinsically and extrinsically to Rathke's pouch to ensure the proper growth of the pituitary gland.

The 'paired'-like homeodomain transcription factor Prop1 is essential for the expansion of the pituitary primordia and for the differentiation and/or function of the hormone-producing cells of the anterior pituitary gland. Prop1 expression is normally extinguished before transcription of most differentiation markers is initiated. We report that constitutive expression of Prop1 interferes with anterior pituitary cell differentiation and increases the susceptibility for pituitary tumors. The terminal differentiation of pituitary gonadotropes is delayed, resulting in transient hypogonadism and a delay in the onset of puberty. Thyrotrope differentiation occurs normally, but thyrotrope function is impaired resulting in mild hypothyroidism. Aged mice exhibit defects consistent with misregulation of pituitary cell proliferation, including adenomatous hyperplasia with the formation of Rathke's cleft cysts and tumors. Thus, silencing Prop1 is important for normal pituitary development and function. These data suggest that gain-of-function mutations in PROP1 could contribute to the most common human pituitary endocrinopathies and tumors.