Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and results in mental retardation if untreated. Eighty-five percent of CH cases are due to disruptions in thyroid organogenesis and are mostly sporadic, but about 2% of thyroid dysgenesis is familial, indicating the involvement of genetic factors in the aetiology of the disease. In this study, we aimed to investigate the Mendelian (single-gene) causes of non-syndromic and non-goitrous congenital hypothyroidism (CHNG) in consanguineous or multi-case families. Here we report the results of the second part (n=105) of our large cohort (n=244), representing the largest such cohort in the literature, and interpret the overall results of the whole cohort. Additionally, 50 sporadic cases with thyroid dysgenesis and 400 unaffected control subjects were included in the study. In familial cases, first, we performed potential linkage analysis of four known genes causing CHNG (TSHR, PAX8, TSHB, and NKX2-5) using microsatellite markers and then examined the presence of mutations in these genes by direct sequencing. In addition, in silico analyses of the predicted structural effects of TSHR mutations were performed and related to the mutation specific disease phenotype. We detected eight new TSHR mutations and a PAX8 mutation but no mutations in TSHB and NKX2-5. None of the biallelic TSHR mutations detected in familial cases were present in the cohort of 50 sporadic cases. Genotype/phenotype relationships were established between TSHR mutations and resulting clinical presentations. Here we conclude that TSHR mutations are the main detectable cause of autosomal recessively inherited thyroid dysgenesis. We also outline a new genetic testing strategy for the investigation of suspected autosomal recessive non-goitrous CH.

Genomic imprinting disorders often exhibit delayed neurobehavioral development, suggesting this unique mechanism of epigenetic regulation plays a role in mental and neurological health. While major errors in imprinting have been linked to adverse health outcomes, there has been little research conducted on how moderate variability in imprinted gene expression within a population contributes to differences in neurobehavioral outcomes, particularly at birth. Here, we profiled the expression of 108 known and putative imprinted genes in human placenta samples from 615 infants assessed by the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). Data reduction identified 10 genes (DLX5, DHCR24, VTRNA2-1, PHLDA2, NPAP1, FAM50B, GNAS-AS1, PAX8-AS1, SHANK2, and COPG2IT1) whose expression could distinguish between newborn neurobehavioral profiles derived from the NNNS. Clustering infants based on the expression pattern of these genes identified 2 groups of infants characterized by reduced quality of movement, increased signs of asymmetrical and non-optimal reflexes, and increased odds of demonstrating increased signs of physiologic stress and abstinence. Overall, these results suggest that common variation in placental imprinted gene expression is linked to suboptimal performance on scales of neurological functioning as well as with increased signs of physiologic stress, highlighting the central importance of the control of expression of these genes in the placenta for neurobehavioral development.

BACKGROUND

Screening of the known candidate genes involved in thyroid organogenesis has revealed mutations in a small subset of patients with congenital hypothyroidism due to thyroid dysgenesis (TD).

OBJECTIVE

We studied a girl with TD who had mutations in two transcription factors involved in thyroid development.

RESULTS

Sequencing analysis of candidate genes involved in thyroid gland development revealed a new paternally inherited heterozygous mutation in the NKX2.5 gene (S265R) and a new maternally inherited heterozygous mutation in the PAX8 promoter region (-456C>T). Both parents and a brother, who was also heterozygous for both mutations, were phenotypically normal. Immunofluorescence microscopy showed a correct nuclear localization of both wild-type (WT) and mutant NKX2.5 proteins. EMSA demonstrated that the mutant NKX2.5 binds to the NKE_2, DIO2, TG, and TPO promoter elements equally well as the WT protein. However, the mutant NKX2.5 protein showed a 30-40% reduced transactivation of the thyroglobulin and the thyroid peroxidase promoters and a dominant-negative effect of the mutant NKX2.5. EMSA studies of the WT and mutant PAX8 promoter sequences incubated with nuclear extracts from PCCL3 cells exhibited a loss of protein binding capacity of the mutant promoter. In addition, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro. Thus, this promoter mutation is expected to lead to reduced PAX8 expression.

CONCLUSIONS

We identified new heterozygous mutations in both NKX2.5 and PAX8 genes of a girl with TD. Both defects might contribute to the phenotype.

PAX8 is a useful immunohistochemical marker for the diagnosis of gynecologic tract malignancies. Several studies have described PAX8 expression in a wide variety of epithelial neoplasms, including ovarian and endometrial carcinomas. The goal of this study was to evaluate PAX8 expression in various types of uterine adenocarcinomas and mesonephric proliferations. Ninety-four cases of uterine adenocarcinomas (52 endometrial endometrioid carcinomas, 21 endometrial serous carcinomas, and 21 human papillomavirus-related endocervical carcinomas), 11 cases of benign mesonephric proliferations (remnants/hyperplasia), and normal endometrial and endocervical glandular epithelium in 58 cases were studied. Immunohistochemical staining was performed with the rabbit polyclonal anti-PAX8 antibody. All adenocarcinoma groups demonstrated a high frequency of PAX8 expression but with relatively high variability in the extent of staining among different subtypes. Both serous carcinomas and endometrioid carcinomas were positive in most cases (95% and 96%, respectively), but serous carcinomas displayed a significantly higher level of expression (immunohistochemical composite scores based on combined extent and intensity of expression) compared with endometrioid carcinomas (mean immunohistochemical composite scores: 8.3 vs. 5.3, respectively; P<0.006). Endocervical adenocarcinomas also had a high frequency of PAX8 expression (86% of cases), but the level of expression was significantly less than that of endometrial adenocarcinomas (mean immunohistochemical composite scores: 2.9 vs. 5.3-8.3, respectively; P<0.004). Among benign glandular epithelia, normal endocervical glands exhibited a significantly lower level of expression compared with either normal endometrial glands or benign mesonephric proliferations (mean immunohistochemical composite scores: 2.6 vs. 6.6-11.2, respectively; P<0.0006). We conclude that PAX8 is expressed in the vast majority of uterine adenocarcinomas, including those of both endometrial and endocervical origin, and that the level of expression based on combined extent and intensity is highest in endometrial serous carcinoma and lowest in endocervical adenocarcinoma. However, the high prevalence of PAX8 expression in the various types of uterine adenocarcinomas precludes use of this marker for distinguishing these tumors. In extrauterine sites, PAX8 can serve as a useful marker for adenocarcinomas of uterine origin (also positive in the majority of ovarian carcinomas), being most sensitive for identification of endometrial adenocarcinomas (both serous and endometrioid). The sensitivity for identifying metastatic endocervical adenocarcinomas is likely less and dependent on the degree to which the significantly lower extent of expression in these tumors is maintained in metastatic sites.

BACKGROUND

The three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap) of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis.

RESULTS

We have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter.

CONCLUSIONS

The early nephrogenic transcription factors play an important role in nephrogenesis, but have no pronephros induction potential upon overexpression in animal caps. They activate transcriptional cascades that partially reflect the gene activation initiated by activin A and retinoic acid. Significantly, HNF1B activates the lhx1 promoter directly, thus extending the known activin A regulation of the lhx1 gene via an activin A responsive element.

Whole-organ decellularization technology has emerged as a new alternative for the fabrication of bioartificial lungs. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are potentially useful for recellularization since they can be directed to express phenotypic marker genes of lung epithelial cells. Normal pulmonary development takes place in a low oxygen environment ranging from 1 to 5%. By contrast, in vitro ESC and iPSC differentiation protocols are usually carried out at room-air oxygen tension. Here, we sought to determine the role played by oxygen tension on the derivation of Nkx2.1+ lung/thyroid progenitor cells from mouse ESC and iPSC. A step-wise differentiation protocol was used to generate Nkx2.1+ lung/thyroid progenitors under 20% and 5% oxygen tension. On day 12, gene expression analysis revealed that Nkx2.1 and Foxa2 (endodermal and early lung epithelial cell marker) were significantly upregulated at 5% oxygen tension in ESC and iPSC differentiated cultures compared to 20% oxygen conditions. In addition, quantification of Foxa2+Nkx2.1+Pax8- cells corresponding to the lung field, with exclusion of the potential thyroid fate identified by Pax8 expression, confirmed that the low physiologic oxygen tension exerted a significant positive effect on early pulmonary differentiation of ESC and iPSC. In conclusion, we found that 5% oxygen tension enhanced the derivation of lung progenitors from mouse ESC and iPSC compared to 20% room-air oxygen tension.

BACKGROUND

Dyshormonogenesis due to genetic defect in thyroglobulin (Tg) synthesis and secretion can lead to congenital hypothyroidism.

OBJECTIVE

The aim of the study was to analyze the TG gene for the presence of mutations and to study the underlying mechanisms leading to dyshormonogenesis.

METHODS

Two siblings aged 25 and 31 yr presented with recurrent goitrous hypothyroidism with undetectable serum Tg. The older sibling was diagnosed with follicular variant of papillary thyroid carcinoma (FVPTC) at age 21 and metastatic FVPTC 8 yr later.

METHODS

The entire coding region of TG gene was sequenced. BRAF, RAS, and P53 mutations or PAX8/PPAR-gamma rearrangement were screened in the FVPTC. Tg expression was studied by immunohistochemistry.

RESULTS

Biallelic c.6725G>A (p.R2223H) and c.6396C>T (p.S2113L) sequence variations were detected in both patients and monoallelic variations in their family members. The c.6396C>T (p.S2113L) sequence variation was found in 14% of 100 population controls, whereas c.6725G>A variation was not present in the controls. Two previously reported polymorphisms (c.2200T>G and c.3082A>G) were present in all the family members. Strong cytoplasmic immunostaining of Tg was observed in the hyperplastic thyroid epithelial cells and weak or no staining in the follicular lumen. Cytoplasmic staining was localized in the endoplasmic reticulum. Reduced staining was found in the FVPTC. Neither RAS, BRAF, or P53 gene mutation nor a PAX8/PPAR-gamma rearrangement was detected in the tumor tissue.

CONCLUSIONS

Biallelic c.6725G>A (p.R2223H) mutation causes Tg retention in the endoplasmic reticulum, resulting in dyshormonogenesis. Prolonged TSH stimulation may promote malignant transformation and development of thyroid cancer. The c.6396C>T (p.S2113L) is a novel polymorphism.

OBJECTIVE

Dedifferentiated endometrial carcinoma (DDEC) is defined by the presence of an undifferentiated carcinoma together with an endometrioid carcinoma. Inactivation of SMARCA4 (BRG1) and inactivation of SMARCB1 (INI1) were recently described as potential mechanisms underlying the histological dedifferentiation. The aim of this study was to characterize the immunophenotypic features of DDECs, particularly in cases with prototypical histological and molecular features (BRG1/INI1 deficiency).

RESULTS

We evaluated PAX8, oestrogen receptor (ER) and p53 immunostaining in the endometrioid and the undifferentiated components of 20 BRG1/INI1-deficient DDECs and 15 BRG1/INI1-intact DDECs, and compared the results with those of 23 grade 3 endometrioid carcinomas. The differentiated endometrioid component was positive for PAX8 and/or ER in 19 of 20 BRG1/INI1-deficient DDECs, whereas the corresponding undifferentiated component of all 20 tumours showed a complete absence of PAX8 and ER staining. All except one of the BRG1/INI1-deficient tumours showed a wild-type p53 staining pattern. PAX8 and ER expression in the undifferentiated component was absent in 67% and 80% of BRG1/INI1-intact DDECs, respectively, whereas 47% of the BRG1/INI1-intact DDECs showed a mutated p53 staining pattern. In comparison, absent PAX8 expression and absent ER expression were each observed in the more solid area of 48% and 48% of grade 3 endometrioid carcinomas.

CONCLUSIONS

The consistent absence of PAX8 and ER expression in molecularly defined (BRG1/INI1-deficient) DDECs suggests that the loss of PAX8 and ER expression is a fundamental feature of dedifferentiation. The frequent findings of a mutated p53 staining pattern in BRG1/INI1-intact DDECs indicate that BRG1/INI1-intact DDECs may be biologically different from BRG1/INI1-deficient tumours.

OBJECTIVE

Von Hippel-Lindau protein (VHL) provides the degradation of hypoxia-inducible factor (HIF). Tetracycline-induced, Pax8-rtTA-based knockout of VHL (VHL-KO) affects all renal tubules and periportal hepatocytes and leads to sustained upregulation of HIF. Here, we study the phenotype of VHL-KO in both organs, the time course of changes, and long-term morpho-functional outcome.

METHODS

Mice with doxycycline-induced VHL-KO and controls (CON) were followed for up to 9 months. Systemic and tissue parameters were evaluated using clinical chemistry, histology, immunohistochemistry, RT-PCR and in situ hybridisation.

RESULTS

At day 3 following VHL-KO, substantial abundance of HIF-1α and -2α was detected in the nuclei of hepatocytes and renal tubular epithelia. Hypoxia, induced by bleeding anaemia, did not further augment HIF signal. Erythropoietin mRNA was detectable in hepatocytes but not in the kidney. Vascular endothelial growth factor mRNA was upregulated in kidney but not in liver. At day 7 following VHL-KO, the renal capillary density was enhanced, reaching its maximum at day 14. Blood haemoglobin increased constantly up to day 28 (23.3 vs. 15.8 g dL(-1) , VHL-KO vs. CON). Thereafter, it was kept within the normal range by weekly blood collections. Pathological changes were absent from kidney and liver 9 months after VHL-KO.

CONCLUSIONS

Inducible, Pax8-rtTA-based deletion of VHL leads to organ-specific expression of epithelial HIF and erythropoietin in liver and kidney without causing pathological changes. Uniform, maximal and sustained HIF activation along the renal tubule may serve to study the potential benefits of hypoxia adaptation in experimental renal injury.

In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP(+) neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP(+) cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP(+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP(+) cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons.

Recent advances in molecular diagnostics have led to significant insights into the genetic basis of thyroid tumorigenesis. Among the mutations commonly seen in thyroid cancers, the vast majority are associated with the mitogen-activated protein kinase pathway. B-Raf proto-oncogene (BRAF) mutations are the most common mutations observed in papillary thyroid cancers (PTCs), followed by RET/PTC rearrangements and RAS mutations, while follicular thyroid cancers are more likely to harbor RAS mutations or PAX8/peroxisome proliferator-activated receptor γ (PPARγ) rearrangements. Beyond these more common mutations, alterations in the telomerase reverse transcriptase (TERT) promoter have recently been associated with clinicopathologic features, disease prognosis, and tumorigenesis in thyroid cancer. While the mutations underlying thyroid tumorigenesis are well known, the frequency of these mutations is strongly associated with geography, with clear differences reported between Asian and Western countries. Of particular interest is the prevalence of BRAF mutations, with Korean patients exhibiting the highest rate of BRAF-associated thyroid cancers in the world. Here, we review the prevalence of each of the most common mutations in Asian and Western countries, and identify the characteristics of well-differentiated thyroid cancer in Asians.

BACKGROUND

Terminal differentiation of the human thyroid is characterized by the onset of follicle formation and thyroid hormone synthesis at 11 gestational weeks (GW).

OBJECTIVE

This study aimed to investigate the ontogeny of thyroglobulin (Tg), thyroid peroxidase (TPO), sodium/iodide symporter (NIS), pendrin (PDS), dual oxidase 2 (DUOX2), thyroid-stimulating hormone receptor (TSHR), and thyroid transcription factor 1 (TITF1), forkhead box E1 (FOXE1), and paired box gene 8 (PAX8) in the developing human thyroid.

METHODS

Thyroid tissues from human embryos and fetuses (7-33 GW; n = 45) were analyzed by quantitative PCR to monitor mRNA expression for each gene and by immunohistochemistry to determine the cellular distribution of TITF1, TSHR, Tg, TPO, NIS, and the onset of T4 production. A broken line regression model was fitted for each gene to compare the loglinear increase in expression before and after the onset of T4 synthesis.

RESULTS

TITF1, FOXE1, PAX8, TSHR, and DUOX2 were stably expressed from 7 to 33 GW. Tg, TPO, and PDS expression was detectable as early as 7 GW and was correlated with gestational age (all, P < 0.01), and the slope of the regression line was significantly different before and after the onset of T4 synthesis at 11 GW (all, P < 0.01). NIS expression appeared last and showed the highest fit by the broken line regression model of all genes (correlation age P < 0.0001, broken line regression P < 0.0001). Immunohistochemical studies detected TITF1, TSHR, and Tg in unpolarized thyrocytes before follicle formation. T(4) and NIS labeling were only found in developing follicles from 11 GW on.

CONCLUSIONS

These results imply a key role of NIS for the onset of human thyroid function.

The transcriptional regulation of the human sodium/iodide symporter (NIS) gene in normal and transformed thyroid cells is a crucial issue in attempting to restore iodide uptake and use radioiodine as a therapeutic treatment of thyroid cancer. Previous investigations have shown that the multifunctional protein apurinic apyrimidinic endonuclease/redox factor 1 (APE/Ref-1) plays an important role in regulation of thyroid-specific gene transcription. In this study, we investigated the effects of APE/Ref-1 on human NIS promoter activity. Cotransfection experiments performed in nonthyroid HeLa cells demonstrated that APE/Ref-1 exerts both PAX8-dependent and PAX8-independent effects. In fact, in the absence of PAX8, overexpression of APE/Ref-1 enhanced NIS promoter activity 2-fold. When the expression plasmid of APE/Ref-1 was transfected together with an expression plasmid for PAX8, a strong cooperative effect was detected with an increase of NIS promoter activity 9-fold over control. The PAX8-independent effect of APE/Ref-1 was specific for the NIS promoter, resulting not present for the promoter of the thyroperoxidase (TPO) gene. It was, at least in part, due to the up-regulation of the transcriptional activity of the ubiquitous factor early growth response-1 (Egr-1). In the thyroid tumor cell lines TPC-1 and B-CPAP, APE/Ref-1 was not effective by itself, and it also failed to increase PAX8 stimulation on NIS promoter activity. These data demonstrate a role for APE/Ref-1 protein in the transcriptional regulation of NIS gene expression by itself and in cooperation with PAX8. However, restoring the PAX8-APE/Ref-1 expression in tumor cells may not be sufficient to obtain adequate levels of NIS gene expression.

Routine morphologic cytology is the basis for any kind of (integrated) molecular FNA diagnostics. The rule out (gene expression classifier) approach requires confirmation by independent studies, whereas the rule in approach (detection of BRAF, NRAS, HRAS, and KRAS and PAX8/PPARG- and RET/PTC rearrangements) has been investigated by several groups with overall reproducible results. Moreover, molecular screening for point mutations and rearrangements is feasible in routine air-dried FNA smears, offering several advantages over obtaining additional fresh FNA material. The current panel of somatic mutations (rule in approach) for indeterminate FNAs clarifies only a subgroup of indeterminate FNAs. Therefore, further markers are urgently needed that can reliably identify the malignant, but mutation negative and especially the many benign nodules, among the indeterminate FNAs. miRNA markers and the targeted next generation sequencing (NGS) technology do have the potential to identify those nodules that are mutation negative by current approaches.

BACKGROUND

Although thyroid dysgenesis is the most common cause of congenital hypothyroidism (CH), its molecular basis remains largely elusive. Indeed, in only a minority of cases with thyroid dysgenesis (2%-3%) was it possible to identify an underlying genetic defect. The objective of this study was to screen the PAX8 gene and the PAX2 gene in a family with six cases of CH spanning three generations and presenting urogenital malformations. Herein, we report a case series and in vitro characterization of the PAX8 gene mutation.

METHODS

Investigations were conducted at a tertiary care referral center. The index case was diagnosed to have congenital hypothyroidism at 7 months of age when he presented with severe impairment of suckling, constipation, and poor development. Treatment with levothyroxine corrected the symptoms and was associated with catch-up growth. His progeny, including two sons, one daughter, and two granddaughters, were affected by CH, and three of them received the diagnosis at neonatal screening. Ultrasound demonstrated normally located thyroid glands with reduced volumes. Five of the six affected family members, including the index case, had urogenital malformations, including incomplete horseshoe kidney, undescended testicles, hydrocele, and ureterocele. Strabismus was found in three out of six affected patients. No other somatic malformations were found.

RESULTS

Direct sequencing of the PAX8 gene revealed a new heterozygous mutation (c.74C>> G) in all affected individuals. This mutation leads to substitution of proline with arginine at codon 25 (P25R). Fluorescence microscopy showed that P25R is normally located in the nucleus. In transient transfection studies, this mutation causes reduced transcriptional activation ability when using a luciferase reporter construct under the control of a thyroglobulin promoter. This diminished transactivation ability is due to loss of DNA binding capability as shown in electrophoresis mobility shift assay. The sequencing analysis of the PAX2 gene was normal.

CONCLUSIONS

We conclude that this novel PAX8 mutation is responsible for a severe form of dominantly inherited CH. The mutation seems to be associated with abnormalities of the urogenital tract.

BACKGROUND

Individuals carrying a heterozygous inactivating PAX8 mutation are affected by congenital hypothyroidism (CH), although heterozygous Pax8 knockout mice are not. It has remained unclear whether CH in PAX8 mutation carriers is caused by haploinsufficiency or a dominant negative mechanism.

OBJECTIVE

To report clinical and molecular findings of four novel PAX8 mutations, including one early-truncating frameshift mutation.

METHODS

Four probands were CH patients. Two had family history of congenital or childhood hypothyroidism. Three probands were diagnosed in the frame of newborn screening for CH, while one had a negative result in screening but was diagnosed subsequently. Three had thyroid hypoplasia and one had a slightly small thyroid with low echogenicity. For these probands and their family members, we sequenced PAX8 using a standard PCR-based method. Pathogenicity of identified mutations was verified in vitro.

RESULTS

We found four novel heterozygous PAX8 mutations in the four probands: L16P, F20S, D46SfsX24, and R133Q. Family studies showed four additional mutation carriers, who were confirmed to have high serum TSH levels. Expression experiments revealed that three mutations (L16P, F20S, and R133Q) had defects in target DNA binding, while D46fs had protein instability that was rescued by the proteasome inhibitor MG132. All four mutations had reduced transactivation on the thyroglobulin promoter, supporting that they were inactivating mutations.

CONCLUSIONS

D46fs is the first PAX8 mutation with confirmed protein instability. Our clinical and in vitro findings together suggest that pure PAX8 haploinsufficiency can cause CH in humans.

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the roles of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish and humans. Although Pax2 and the related gene, Pax8, are essential for early intermediate mesoderm specification and are found in the epithelial lineage arising from that mesoderm, how these proteins regulate cell lineage restriction and gene expression patterns has remained obscure. Our recent data, suggests that Pax proteins help establish chromatin domains within cell lineages by providing the locus and tissue specificity for epigenetic imprinting complexes that modify histones. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. Given the prevalence of Pax2 expression in kidney development and in kidney disease, we now need to address the effects of epigenetics on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.

The transcription factor Pax8 plays an important role in the expression of the differentiated phenotype of thyroid follicular cells. It has recently been shown that Pax8 is necessary for thyroglobulin (Tg) gene expression in the fully differentiated rat thyroid cell line PC. We have used the PC model system to investigate the role of Pax8 as a mediator of TSH regulation of Tg gene expression. We have demonstrated that Pax8 expression, as well as Tg expression, is severely reduced in cells grown in the absence of hormones and serum. The re-addition of TSH or forskolin to the culture medium is able to restore to wild-type levels the expression of both Pax8 and Tg. We have determined that the action of TSH/forskolin on Pax8 is at the transcriptional level. However, the re-expression of Pax8 can be observed several hours before that of Tg, suggesting that either another factor is needed or that Pax8 itself must be post-translationally modified by a newly synthesized protein to become active. To distinguish between these two possibilities we have stably transfected into PC cells an exogenous Pax8 that is expressed independently of TSH. Our results indicate that in these cells the Tg promoter is still dependent on TSH despite the constitutive presence of Pax8. Furthermore, we also show that in this condition Tg gene transcription requires de novo protein synthesis. In conclusion, TSH regulates the expression of Pax8 at a transcriptional level and also regulates the activity of Pax8 by controlling the expression of one or more as yet unknown factors.

OBJECTIVE

To compare mutation analysis of cytology specimens and (99m)Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm.

METHODS

Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and (99m)Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of (99m)Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method.

RESULTS

In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. (99m)Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative (99m)Tc-MIBI scan reliably excluded malignancy.

CONCLUSIONS

In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of (99m)Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material.

BACKGROUND

Hemangioblastomas account for up to 2.5% of all intracranial tumors. They may occur sporadically or as a part of the multisystem genetic syndrome of Von Hippel-Lindau syndrome (VHL). Patients with VHL are also at an increased risk of developing clear cell renal cell carcinoma (ccRCC). Distinguishing hemangioblastomas from metastatic ccRCC to the central nervous system (CNS) can be challenging at times when based solely on hematoxylin and eosin-stained sections. We propose an immunohistochemistry (IHC) panel of combination of PAX2, PAX8, and inhibin A as a helpful approach in distinguishing the 2 lesions.

METHODS

Archival tissues from 20 hemangioblastomas and 16 ccRCCs metastatic to the CNS were retrieved from our surgical pathology files (2001 to 2010). IHC for PAX2, PAX8, and inhibin A was performed on routine or tissue microarray sections using standard IHC protocol. The intensity of nuclear staining was evaluated for each marker and was assigned an incremental 0, 1+, 2+, and 3+ score. The extent of staining was categorized as focal (<25%), multifocal (25% to 75%), or diffuse (>75%).

RESULTS

(1) Hemangioblastoma: The Von Hippel-Lindau syndrome was diagnosed in 4 of 16 (25%) patients, 2 of whom developed multiple hemangioblastomas. All 20 (100%) hemangioblastomas were positive for inhibin A (cytoplasmic). The staining intensity was moderate or strong (2+ or 3+) in 19 cases (95%), all of which were multifocal or diffuse in extent. Nuclear PAX2 staining was present in 1 of 19 evaluable lesions (5%), whereas PAX8 staining was not present in any of the 20 examined lesions. (2) Metastatic ccRCC to the CNS: Fourteen of 16 (88%) examined ccRCCs were positive for PAX2, whereas 15 of 16 (94%) lesions showed PAX8 staining. None of 16 (0%) examined ccRCCs were positive for inhibin A.

CONCLUSIONS

We propose the use of the combination of PAX2, PAX8, and inhibin A as a helpful ancillary IHC panel to resolve the differential diagnosis of hemangioblastoma versus metastatic ccRCC. The immunoprofile of PAX2(+) or PAX8(+) and inhibin A(-) supports the diagnosis of metastatic ccRCC with a sensitivity of 94%, specificity of 100%, and positive predictive value of 100%. The PAX2(-), PAX8(-), and inhibin A(+) profile supports the diagnosis of hemangioblastoma with a sensitivity of 95%, specificity of 100%, and positive predictive value of 100%.

Papillary and follicular carcinomas, commonly referred to as follicular cell-derived differentiated thyroid carcinomas (DTC), account for 90% of all thyroid carcinomas. The prognosis of DTC is generally good, depending on the biologic behavior of the tumor and on the appropriate initial treatment which includes total thyroidectomy and ablation by radioiodine-131. However, a considerable number of patients, approximately 30%, as shown after 30 years of follow-up, have recurrent disease. It is thus of utmost importance to evaluate the prognostic factors, as derived from retrospective studies, and identify high risk patients. Age of more than 45 years or less than 25 years is a particularly strong independent prognostic factor; on the contrary gender is a poor prognostic factor. Histological type of the cancer especially tall cancer cells and columnar cancer cells, as well as increased vascular invasion of the tumor, lymph-node and distant metastases, are all considered as risk factors that can lead to poor prognosis. Combined prognostic factors have been used to form scoring systems (SS) such as AGES, MACIS, AMES, EORTC and TNM for a more precise description of high or low risk patients. However, prognostic significance of the SS is limited, since they do not take into consideration the clinical status or the treatment procedure during the course of the disease. Molecular factors such as rearrangements of genes RET/PTC, RAS mutations and fusion of, paired box and 8/peroxisome proliferator-activated receptor gamma (PAX8/PPARgamma) are also involved in thyroid cancer prognosis, while some others: human Pituitary- Tumor Transforming Gene (e.g. MIB-1, hPTTG) have been reported as additional prognostic factors. In this review we describe the risk and the prognostic factors of DTC as related to management and the outcome of DTC.

BACKGROUND

Paired box gene 8 (PAX8), a member of the thyroid-specific transcription factors, has recently been investigated as a useful marker of thyroid epithelial neoplasms. To the authors' knowledge, its utility in the immunohistochemistry of papillary thyroid carcinoma (PTC) has not been well studied to date. The goal of the current study was to compare the immunohistochemical expression of PAX8 with HBME-1 and cytokeratin 19 (CK19) expression on cell block preparations of PTC and benign thyroid nodules (BTN).

METHODS

Thirty-two cases of PTC and 20 cases of BTN diagnosed on fine-needle aspiration were included. The cell blocks were immunohistochemically stained for PAX8, HBME-1, and CK19.

RESULTS

PAX8 demonstrated nuclear positivity in >90% of cases with PTC (31 of 32 cases; 96.9%) and BTN (20 of 20 cases; 100%), with a high sensitivity (0.97), but low specificity (0). HBME-1 and CK19 were found to have high sensitivity (0.78 and 0.75, respectively) and specificity (0.95 for both) for PTC. Statistically significant differences in staining intensity and distribution were noted for the PAX8 antibody (negative [1 of 32 cases] to weakly positive [20 of 32 cases] in 65.6% of PAX8-positive PTCs, and intermediate to strong in 100% of BTNs).

CONCLUSIONS

In contrast to PAX8, HBME-1 and CK19 were found to have a high degree of both sensitivity and specificity for PTC. The results of the current study demonstrate that strong PAX8 staining appears to be typical of BTNs, whereas negative to weakly positive staining is noted in PTC. PAX8, HMBE-1 and CK19 may all play a role, particularly as a panel; however, the interpretation of PAX8 should consider the intensity and distribution of staining.

The LIM homeodomain transcription factor Isl1 was investigated in mouse thyroid organogenesis. All progenitor cells of the midline thyroid diverticulum and lateral primordia (ultimobranchial bodies) expressed Isl1. This pattern persisted until the growing anlagen fused at embryonic day (E) 13.5. In Isl1 null mutants thyroid progenitors expressing Nkx2.1 and Pax8 were readily specified in the anterior endoderm but the size of the thyroid rudiment was reduced. In late development, only immature C-cells expressed Isl1. In the adult gland the number of Isl1+ cells was small compared with cells expressing calcitonin. Analysis of microarray profiles indicated a higher level of Isl1 expression in medullary thyroid carcinomas than in tumors derived from follicular cells. Together, these findings suggest that Isl1 may be a novel regulator of thyroid development before terminal differentiation of the endocrine cell types. Isl1 is an embryonic C-cell precursor marker that may be relevant also in cancer developed from the mature C-cell.

Recently, reports using immunohistochemistry and a polyclonal antibody directed against the N-terminal region of PAX8 describe PAX8 expression in malignant lymphomas. As the N-terminal regions of PAX family members, including the B-cell transcription factor PAX5, have high sequence homology, we investigated PAX8 positivity in malignant lymphomas. Comparative sequence analysis between the N- and C-terminal regions of PAX8 and PAX5 proteins confirmed homologies of 70% and 39%, respectively. We then compared the results using N-terminal (high homology) and C-terminal (lower homology) anti-PAX8 antibodies to assess PAX8 expression in reactive tissues, diffuse large B-cell lymphoma and classical Hodgkin lymphoma, using routine immunohistochemical methods. Expression of PAX8 was also assessed in diffuse large B-cell lymphoma and classical Hodgkin lymphoma cell lines using real-time qRT-PCR methods. Our results show that reactive and neoplastic B-cells are positive for PAX8 using the N-terminal antibody, but negative for PAX8 when the C-terminal antibody was used. PAX8 mRNA levels were not detected in any of the B-cell lymphoma cell lines studied. These results indicate that benign and malignant B-cells do not express PAX8. We conclude that positivity for PAX8 reported by others in B-cell lymphomas is likely due to cross-reactivity between the N-terminal regions of PAX8 and PAX5, due to the high sequence homology of these two regions.