Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with β-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic β-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in β-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM.

OBJECTIVE

To precisely classify the various forms of TD, and then to screen for mutations in transcription factor genes active in thyroid development.

METHODS

Patients underwent ultrasound, thyroid scan, and serum thyroglobulin measurement to accurately diagnose the form of TD. DNA was extracted from peripheral leukocytes. The PAX8, and NKX2.5 genes were evaluated in all patients, and TSH receptor (TSHR) gene in those with hypoplasia.

RESULTS

In 27 nonconsanguineous patients with TD, 13 were diagnosed with ectopia, 11 with hypoplasia, and 3 with athyreosis. No mutations were detected in any of the genes studied.

CONCLUSIONS

Sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.

The distinction between breast and müllerian carcinomas from each other and from tumors with a similar cytokeratin profile can be difficult. We tested the usefulness of 2 new markers, NY-BR-1 and PAX8, by staining a variety of breast and gynecologic carcinomas, along with tumors of pancreas, bile ducts, stomach, and gastroesophageal junction. NY-BR-1 expression (ie, H score >10) was seen in 58.4% of breast carcinomas (111/190), 5.6% of müllerian carcinomas (8/142), 7% of pancreatic tumors (1/15), 0% of cholangiocarcinomas (0/22), 0% of gastric tumors (0/36), and 0% of gastroesophageal carcinomas (0/25). All 188 breast carcinomas were negative for PAX8. PAX8 expression was seen in 72.4% of müllerian tumors (105/145). All pancreatic tumors (n = 15), cholangiocarcinomas (n = 23), and gastric (n = 35) and gastroesophageal junction (n = 25) carcinomas were negative for PAX8. Addition of NY-BR-1 and PAX8 in a panel would be useful in distinguishing breast cancer, gynecologic tumors, and tumors of the upper gastrointestinal tract.

BACKGROUND

PAX8, a member of the paired-box family of genes, is expressed in many tumors of Müllerian origin. However, it is unclear whether PAX8 is a useful marker in diagnosing endocervical glandular lesions because of limited data.

OBJECTIVE

To study the expression of PAX8 in endocervical glandular lesions.

METHODS

We first studied a cohort of 29 cervical cone biopsies, followed by a second cohort of 17 cases of endocervical adenocarcinoma and 20 cases of uterine endometrioid adenocarcinoma.

RESULTS

In the first cohort, we found that PAX8 was expressed in 23 of 23 (100%) benign endocervical glandular epithelium, 15 of 16 (94%) adenocarcinoma in situ, and 21 of 26 (81%) invasive endocervical adenocarcinoma specimens. In the second cohort, endocervical adenocarcinomas were positive for PAX8 in 14 of 17 (82%), strongly and diffusely positive for p16 in 14 of 17 (82%), positive for carcinoembryonic antigen in 12 of 17 (71%), positive for vimentin in 2 of 17 (12%), and positive for estrogen receptor in 7 of 17 cases (41%). Uterine endometrioid cancer was positive for PAX8 in 20 of 20 (100%), weakly and/or patchy positive for p16 in 17 of 20 (85%), positive for carcinoembryonic antigen in 2 of 20 (10%), positive for vimentin in 19 of 20 (95%), and positive for estrogen receptor in 20 of 20 cases (100%).

CONCLUSIONS

PAX8 is expressed in the majority of benign, premalignant, and malignant endocervical glandular lesions. The usefulness of PAX8 in differentiating endocervical from endometrial lesions is limited.

Mesonephric remnants, usually located deep in the lateral cervical wall, may become hyperplastic resulting in a florid proliferation. These can be misinterpreted as malignant and confused with endocervical adenocarcinomas. Recent data have shown that PAX2 is diffusely expressed in mesonephric remnants and hyperplasias. PAX8 is a related transcription protein that is expressed in tissues of müllerian and wolffian origin. In this study, we have investigated the utility of an immunohistochemical panel comprising of PAX8, estrogen receptor (ER), and p16 in the differential diagnosis between mesonephric proliferations and cervical adenocarcinomas. A database search was conducted for cases of mesonephric remnants/hyperplasia/carcinoma of cervix and invasive cervical adenocarcinomas. Immunohistochemical stains for PAX8, ER, and p16 were performed using the avidin-biotin peroxidase technique on the most representative tissue. The search yielded 28 cases of mesonephric proliferations of cervix (15 mesonephric remnants, 12 mesonephric hyperplasias, and 1 mesonephric adenocarcinoma) and 16 cases of cervical adenocarcinomas (15 usual type and 1 adenoma malignum). Immunohistochemically, all the mesonephric proliferations, regardless of being benign or malignant, displayed a consistent staining pattern-diffusely and strongly positive for PAX8, negative for ER, and patchy cytoplasmic staining for p16. The usual type cervical adenocarcinomas exhibited a variable staining pattern with PAX8 and ER but all were strongly and diffusely positive for p16. The case of adenoma malignum was PAX8 positive, ER negative, and showed weak and patchy staining with p16. Our study suggests that a panel of immunohistochemical stains composed of PAX8, p16, and ER is useful in the distinction between mesonephric proliferations and cervical adenocarcinomas.

The present study aimed to investigate the clinical significance of paired-box 8 (PAX8) in primary epithelial ovarian cancer (PEOC). Using immunohistochemical (IHC) staining, the expression of PAX8 in 60 patients with PEOC, 20 patients with ovarian benign lesions and 10 patients with metastatic ovarian cancer (MOC), was examined based on the clinicopathological profiles of the patients. The correlation between PAX8 expression and the clinicopathological parameters or prognosis of patients was statistically analyzed. PAX8 was revealed to be highly expressed in PEOC, but not in MOC, as indicated by IHC staining. The rate of positivity of PAX8 in PEOC was 92% (57/60) with no significant difference of PAX8 expression found between the various pathological types of PEOC (P=0.871). The rate of positivity of PAX8 in ovarian benign tumors was 85%, demonstrating no significant difference in comparison with that of PEOC (P=0.761). PAX8 staining and statistical analysis revealed that the higher the grade of PEOC, the less the cancer cell had differentiated (P=0.033) and the more the cancer had advanced according to International Federation of Gynaecological Oncologists (FIGO) staging (P=0.003). Survival rate statistics showed that PEOC patients with higher PAX8 expression exhibited a shorter postoperative survival rate (P=0.009). PAX8 was specifically expressed in PEOC, and its expression level was associated with the degree of cancer cell differentiation, FIGO stage, and survival rate, indicating that PAX8 is a potential marker for the diagnosis of PEOC.

OBJECTIVE

To study the frequency of mutations in the Pax8 gene in a cohort of patients with congenital hypothyroidism (CH) in South West Germany.

METHODS

A cohort of 95 patients with CH (60 females, 35 males), identified in our newborn screening program, was analyzed for mutations in Pax8 by single-stranded conformational polymorphism (SSCP) and DNA sequencing.

RESULTS

SSCP analysis and direct sequencing of exon 3 of a female patient with a hypoplastic thyroid gland revealed two heterozygous mutations in Pax8 resulting in a transition of T to C (codon 34) and G to A (codon 35), replacing isoleucine by threonine and valine by isoleucine. Using allele-specific PCR we could demonstrate that both mutations are located on the same allele. Furthermore, a polymorphism was documented in 24 patients with thyroid hypoplasia in intron 6 at nucleotide +51 (CC, GG, CG). Comparison of the polymorphisms between hypothyroid patients and controls revealed no significant differences suggesting that this polymorphism does not play a role in the pathogenesis of hypothyroidism. No further mutations or polymorphisms were found in the cohort.

CONCLUSIONS

These findings confirm the contribution of mutations in the Pax8 gene to the etiology of thyroid dysgenesis with a variable penetrance, but also demonstrate the rare overall incidence in CH.

Background Due to its very low prevalence, little is known about the genetic characteristics of pediatric follicular thyroid cancer (FTC). Methods We investigated genetic alterations in tumor tissues from 15 patients aged < 20 years (median: 14.3 years; range: 2.4-19.0 years) using multifaceted approaches. Whole-exome sequencing, targeted next-generation sequencing using a cancer gene panel, and Sanger sequencing of the major exons of the H/K/N-RAS and DICER1 genes and the promoter region of the TERT gene, were performed. Normal tissues or blood of patients with DICER1- or PTEN-positive tumors were evaluated to determine whether the variant is germline. Results The median tumor size was 3.1 cm (range: 0.6-6.4 cm). Four patients exhibited angioinvasion, and one extensive capsular invasion; none showed evidence of disease over a median of 8.1 years. Eight patients (53.3%) had DICER1 variants, including four with DICER1 syndrome (three patients were <10 years of age). One patient had a germline PTEN frameshift variant with the diagnosis of PTEN hamartoma tumor syndrome. One patient had a PAX8/PPARγ rearrangement, and two patients have no genetic driver alteration other than multiple loss of heterozygosity with or without copy number alterations in their tumors. Nodular hyperplasia and follicular adenoma (FA) coexisted in DICER1 variant-positive FTCs more frequently than variant-negative FTCs (p=0.026). All DICER1 variant-positive FTCs had a somatic missense variant at metal binding sites (six at codon p.E1813 and two at codon p.D1709) within the RNase IIIb domain; seven had other missense, nonsense, or frameshift variants in the DICER1 gene. Six coexisting FAs of two patients with DICER1 syndrome (three of each) had additional somatic variants at metal binding sites within the RNase IIIb domain (codon p.E1705, p.D1709, p.D1810 or p.E1813), different from each other and from the indexed FTC tumor. Conclusions Pediatric FTCs have distinct genomic alterations and pathogenesis compared to adults, particularly those characterized by DICER1 variants. The DICER1 variant should be considered in pediatric FTCs, especially in cases < 10 years of age. In all DICER1 variant-positive FTCs and FAs, recurrent hotspot variants were found at metal binding sites within the RNase IIIb domain, suggesting they impact tumorigenesis.

Primary thyroid teratomas are exceedingly rare. Mature and immature variants recapitulate their gonadal counterparts (predilection for infants/children, triphasic germ layer differentiation, and favorable outcome). On the other hand, the so-called malignant teratomas affect predominantly adults and elderly, are highly aggressive, and, according to a few published cases, harbor DICER1 mutations. We describe three highly aggressive sporadic malignant teratoid thyroid tumors in 2 females (17 and 45 years) and one male (17 years). Histology showed triphasic neoplasms composed of solid nests of small primitive monomorphic cells embedded in a cellular stroma with primitive immature rhabdomyosarcoma-like (2) or pleomorphic sarcoma-like (1) phenotype. The third component was represented by TTF1+/PAX8+ primitive teratoid epithelial tubules reminiscent of primitive thyroid follicles and/or Wilms tumor, admixed with scattered respiratory- or enteric-type tubules, neuroepithelial rosettes, and fetal-type squamoid nests. Foci of cartilage were seen in two cases, but none contained mature organoid adult-type tissue or skin adnexa. SALL4 was expressed in the small cell (2) and stromal (1) component. Other germ cell markers were negative. Molecular testing revealed a known "hotspot" pathogenic DICER1 mutation in two cases. In addition, case 1 had a missense TP53 variant. This type of thyroid malignancy is distinct from genuine teratomas. The immunoprofile suggests primitive thyroid- or branchial cleft-like differentiation. Given that "blastoma" is a well-accepted terminology in the spectrum of DICER1-associated malignancies, the term "thyroblastoma" might be more convenient for these malignant teratoid tumors of the thyroid gland. Relationship of thyroblastoma to the DICER1 syndrome remains to be addressed.

Keywords: DICER1; Germ cell tumor; Head and neck; Malignant teratoma; Rhabdomyosarcoma; Teratocarcinosarcoma; Thyroblastoma; Thyroid.

Thyroid diseases constitute a heterogeneous collection of abnormalities associated with mutations in genes responsible for the development of thyroid: thyroid transcription factor-1 (TTF-1), thyroid transcriptions factor-2 (TTF-2) and PAX8, or in one of the genes coding for the proteins involved in thyroid hormone biosynthesis such as thyroglobulin (TG), thyroperoxidase (TPO), hydrogen peroxide-generating system (DUOX2), sodium/iodide symporter (NIS), pendrin (PDS), TSH and TSH receptor (TSHr). Congenital hypothyroidism occurs with a prevalence of 1 in 4000 newborns. Patients with this syndrome can be divided into two groups: nongoitrous (dysem/bryogenesis) or goitrous (dyshormonogenesis) congenital hypothyroidism. The dysembryogenesis group, which accounts for 85% of the cases, results from ectopy, agenesis and hypoplasia. In a minority of these patients, the congenital hypothyroidism is associated with mutations in TTF-1, TTF-2, PAX-8, TSH or TSHr genes. The presence of congenital goiter (15% of the cases) has been linked to mutations in the NIS, TG, TPO, DUOX2 or PDS genes. The congenital hypothyroidism with dyshormonogenesis is transmitted as an autosomal recessive trait. Somatic mutations of the TSHr have been identified in hyperfunctioning thyroid adenomas. Another established thyroid disease is the resistance to thyroid hormone (RTH). It is a syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the thyroid hormone receptor beta (TRbeta) gene. Mutant TRbetas interfere with the function of the wild-type receptor by a dominant negative mechanism. In conclusion, the identification of mutations in the thyroid expression genes has provided important insights into structure-function relationships. The thyroid constitutes an excellent model for the molecular study of genetic diseases.

Di-(2-ethylhexyl) phthalate (DEHP) has the potential to disrupt the thyroid endocrine system, but the underlying mechanism is unknown. In this study, zebrafish (Danio rerio) embryos were exposed to different concentrations of DEHP (0, 40, 100, 200, 400 μg/L) from 2 to 168 hours post fertilization (hpf). Thyroid hormones (THs) levels and transcriptional profiling of key genes related to hypothalamus-pituitary-thyroid (HPT) axis were examined. The result of whole-body thyroxine (T4) and triiodothyronine (T3) indicated that the thyroid hormone homeostasis was disrupted by DEHP in the zebrafish larvae. After exposure to DEHP, the mRNA expressions of thyroid stimulating hormone (tshβ) and corticotrophin releasing hormone (crh) genes were increased in a concentration dependent manner, respectively. The expression level of genes involved in thyroid development (nkx2.1 and pax8) and thyroid synthesis (sodium/iodide symporter, nis, thyroglobulin, tg) were also measured. The transcripts of nkx2.1 and tg were significantly increased after DEHP exposure, while those of nis and pax8 had no significant change. Down-regulation of uridinediphosphate-glucuronosyl-transferase (ugt1ab) and up-regulation of thyronine deiodinase (dio2) might change the THs levels. In addition, the transcript of transthyretin (ttr) was up-regulated, while the mRNA levels of thyroid hormone receptors (trα and trβ) remained unchanged. All the results demonstrated that exposure to DEHP altered the whole-body thyroid hormones in the zebrafish larvae and changed the expression profiling of key genes related to HPT axis, proving that DEHP induced the thyroid endocrine toxicity and potentially affected the synthesis, regulation and action of thyroid hormones.

A subset of thyroid carcinomas expresses an oncogenic paired box 8 (PAX8) and peroxisome proliferator activated receptor γ (PPARγ) fusion protein (PPFP). The PPARγ/PPFP ligand pioglitazone is highly therapeutic in a transgenic mouse model of PPFP thyroid carcinoma, but whether pioglitazone is therapeutic in patients with PPFP thyroid carcinoma is unknown.

Tumor blocks from 40 patients with progressive thyroid cancer despite standard-of-care therapy were screened for PPFP, and the tumor from only one patient (2.5%) was positive. The patient had a 6.0-cm acetabular soft tissue metastasis from Hürthle cell carcinoma that caused severe pain on weight bearing and had a serum thyroglobulin level of 1974 ng/mL. After 24 weeks of therapy with pioglitazone, the metastatic lesion was 3.9 cm, the thyroglobulin level was 49.4 ng/mL, and the patient was pain-free. Thirteen months after discontinuation of pioglitazone, the metastatic lesion was 3.6 cm, the thyroglobulin level was 4.7 ng/mL, and the patient remained pain-free.

Pioglitazone may be therapeutic in patients with PPFP thyroid cancer. However, thyroid cancers that are progressive despite standard-of-care therapy appear to only rarely express PPFP.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease with an increasing global burden. It is hypothesized that fibroblasts have a number of functions that may affect the development and progression of IPF. However, the present understanding of cellular and molecular mechanisms associated with fibroblasts in IPF remains limited. The present study aimed to identify the dysregulated genes in IPF fibroblasts, elucidate their functions and explore potential microRNA (miRNA)‑mRNA interactions. mRNA and miRNA expression profiles were obtained from IPF fibroblasts and normal lung fibroblasts using a next‑generation sequencing platform, and bioinformatic analyses were performed in a step‑wise manner. A total of 42 dysregulated genes (>2 fold‑change of expression) were identified, of which 5 were verified in the Gene Expression Omnibus (GEO) database analysis, including the upregulation of neurotrimin (NTM), paired box 8 (PAX8) and mesoderm development LRP chaperone, and the downregulation of ITPR interacting domain containing 2 and Inka box actin regulator 2 (INKA2). Previous data indicated that PAX8 and INKA2 serve roles in cell growth, proliferation and survival. Gene Ontology analysis indicated that the most significant function of these 42 dysregulated genes was associated with the composition and function of the extracellular matrix (ECM). A total of 60 dysregulated miRNAs were also identified, and 1,908 targets were predicted by the miRmap database. The integrated analysis of mRNA and miRNA expression data, combined with GEO verification, finally identified Homo sapiens (hsa)‑miR‑1254‑INKA2 and hsa‑miR‑766‑3p‑INKA2 as the potential miRNA‑mRNA interactions in IPF fibroblasts. In summary, the results of the present study suggest that dysregulation of PAX8, hsa‑miR‑1254‑INKA2 and hsa‑miR‑766‑3p‑INKA2 may promote the proliferation and survival of IPF fibroblasts. In the functional analysis of the dysregulated genes, a marked association between fibroblasts and the ECM was identified. These data improve the current understanding of fibroblasts as key cells in the pathogenesis of IPF. As a screening study using bioinformatics approaches, the results of the present study require additional validation.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

Keywords: MRKHS; MRKHS, Müllerian duct; Mayer-Rokitansky-Küster-Hauser syndrome; Müllerian aplasia; PAX8; Wölffian duct.

OBJECTIVE

Paediatric follicular thyroid carcinomas are uncommon, and their clinicopathological features and molecular profiles are still unknown. In the present study, we aimed to investigate the clinicopathological aspects of a large series of follicular thyroid carcinomas (FTCs) in paediatric patients and to analyse the point mutations in codons 12, 13 and 61 of NRAS, HRAS and KRAS genes and the rearrangements of PAX8-PPARG.

RESULTS

A total of 41 paediatric FTCs less than 21 years of age were enrolled into the present study. We used direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) to detect RAS mutations and PAX8-PPARG fusions, respectively. The paediatric FTCs were 6:1 in a female to male ratio, with a mean tumour size of 52.7 mm. Distant metastasis was found in one case at the time of presentation. During a median follow-up time of 69 months, two cases had lung metastasis and all patients were alive. Histologically, all cases were minimally invasive FTCs and varied in growth patterns: microfollicular (39%), follicular (14.6%), solid/trabecular (6%), oncocytic (4.9%) and mixed patterns (26.8%). The mean Ki67 index was 5.7% and it was not statistically different among the growth patterns. NRAS mutations were found in five cases (12.2%) and associated significantly with small tumour size (P = 0.014). PAX8-PPARG fusion was not detected in our series.

CONCLUSIONS

Paediatric FTCs are indolent in clinical course in spite of their large tumour size and have a distinct genetic background. RAS mutations and PAX8-PPARG fusions may not play major roles in the tumorigenesis of paediatric FTCs.

Zebrafish is a good model for studying vertebrate development because of the availability of powerful genetic tools. We are interested in the study of the craniofacial skeletal structure of the zebrafish. For this purpose, we performed a gene trap screen and identified a Gal4 gene trap line, SAGFF(LF)134A. We then analyzed the expression pattern of SAGFF(LF)134A;Tg(UAS:GFP) and found that green fluorescent protein (GFP) was expressed not only in craniofacial skeletal elements but also in the vascular system, as well as in the nervous system. In craniofacial skeletal elements, strong GFP expression was detected not only in chondrocytes but also in the perichondrium. In the vascular system, GFP was expressed in endothelium-associated cells. In the spinal cord, strong GFP expression was found in the floor plate, and later in the dorsal radial glia located on the midline. Taking advantage of this transgenic line, which drives Gal4 expression in specific tissues, we crossed SAGFF(LF)134A with several UAS reporter lines. In particular, time-lapse imaging of photoconverted floor-plate cells of SAGFF(LF)134A;Tg(UAS:KikGR) revealed that the floor-plate cells changed their shape within 36 h from cuboidal/trapezoidal to wine glass shaped. Moreover, we identified a novel mode of association between axons and glia. The putative paths for the commissural axons, including pax8-positive CoBL interneurons, were identified as small openings in the basal endfoot of each floor plate. Our results indicate that the transgenic line would be useful for studying the morphogenesis of less-well-characterized tissues of interest, including the perichondrium, dorsal midline radial glia, late-stage floor plate, and vascular endothelium-associated cells.

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBötzinger Complex (preBötC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBötC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBötC neurons express κ-opioid receptors in addition to μ-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBötC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBötC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBötC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development.

Although typically arranged in solid alveolar fashion, chromophobe renal cell carcinoma (RCC) may also show several other architectural growth patterns. We include in this series 8 chromophobe RCC cases with prominent papillary growth, a pattern very rarely reported or only mentioned as a feature of chromophobe RCC, which is lacking wider recognition The differential diagnosis of such cases significantly varies from the typical chromophobe RCC with its usual morphology, particularly its distinction from papillary RCC and other relevant and clinically important entities. Of 972 chromophobe RCCs in our files, we identified 8 chromophobe RCCs with papillary growth. We performed immunohistochemistry and array Comparative Genomic Hybridisation (aCGH) to investigate for possible chromosomal aberrations. Patients were 3 males and 5 females with age ranging from 30 to 84 years (mean 57.5, median 60 years). Tumor size was variable and ranged from 2 to 14 cm (mean 7.5, median 6.6 cm). Follow-up was available for 7 of 8 patients, ranging from 1 to 61 months (mean 20.1, median 12 months). Six patients were alive with no signs of aggressive behavior, and one died of the disease. Histologically, all cases were composed of dual cell population consisting of variable proportions of leaf-like cells with pale cytoplasm and eosinophilic cells. The extent of papillary component ranged from 15 to 100% of the tumor volume (mean 51%, median 50%). Sarcomatoid differentiation was identified only in the case with fatal outcome. Immunohistochemically, all tumors were positive for CK7, CD117 and Hale's Colloidal Iron. PAX8 was positive in 5 of 8 cases, TFE3 was focally positive 3 of 8 tumors, and Cathepsin K was focally positive in 2 of 8 tumors. All cases were negative for vimentin, AMACR and HMB45. Fumarate hydratase staining was retained in all tested cases. The proliferative activity was low (up to 1% in 7, up to 5% in one case). Three cases were successfully analyzed by aCGH and all showed a variable copy number variation profile with multiple chromosomal gains and losses. CONCLUSIONS: Chromophobe RCC demonstrating papillary architecture is an exceptionally rare carcinoma. The diagnosis can be challenging, although the cytologic features are consistent with the classic chromophobe RCC. Given the prognostic and therapeutic implications of accurately diagnosis other RCCs with papillary architecture (i.e., Xp11.2 translocation RCC, FH-deficient RCC), it is crucial to differentiate these cases from chromophobe RCC with papillary architecture. Based on this limited series, the presence of papillary architecture does not appear to have negative prognostic impact. However, its wider recognition may allow in depth studies on additional examples of this rare morphologic variant.

Thyroid transcription factor-1 (TTF-1) is required for maximal expression of thyrotropin receptor (TSHR) in the thyroid. Extrathyroidal TSHR expression is detectable in normal orbital adipose tissues, with increased levels found in orbital tissues from patients with Graves' ophthalmopathy (GO), and in orbital preadipocyte cultures following differentiation. In order to determine whether TTF-1 might be involved in orbital TSHR expression, we used quantitative real-time polymerase chain reaction (PCR) to assess relative expression of this and other thyroid-associated transcription factors (TTF-2 and Pax-8) in GO orbital tissue specimens (n = 28) and cultures (n = 3), and in normal orbital tissues (n = 19) and cultures (n = 3). We detected TTF-1 and TTF-2 mRNA in GO and normal orbital tissue samples, with no difference in levels noted between the tissues. In the GO orbital cultures, TTF-1 mRNA was higher in differentiated than in control (undifferentiated) cultures (p < 0.05), while TTF-2 was unchanged. In the normal cultures, neither TTF-1 nor TTF-2 mRNA levels increased in differentiated cultures. Pax8 was undetectable in all orbital tissues and cell cultures. The presence of mRNA encoding TTF-1 in orbital tissues and cultures suggest that this transcription factor may play an important role in extrathyroidal, as it does in thyroidal, TSHR expression.

OBJECTIVE

The aim of this study is to evaluate the detection of paired box gene 8 (PAX8) and p53 with immunohistochemistry in pelvic washing cell block sections.

METHODS

A total of 92 cases were used in this study, which were assigned to three groups according to the cytopathology files. The first group with positive cytology including endometrial and ovarian carcinomas comprised 32 cases. The second group with suspicious cytology for endometrial or ovarian carcinomas consisted of 29 cases. The third group with negative cytology (regarded as mesothelial cells) included 31 cases. The pelvic washing cell blocks underwent immunohistochemistry to detect PAX8 and p53 expression.

RESULTS

Immunoreactivity for PAX8 was found in 75% (24/32) of the cases in the group with positive cytology, in 6.9% (2/29) of the cases with suspicious cytology, and in none of the 31 cases with negative cytology (sensitivity: 75%; specificity: 100%; p<0.05). p53 expression was detected in 37.5% (12/32) of the cases in the first group, in 3.4% (1/29) of the cases in the second group, and in none of the cases in the third group (sensitivity: 37.5%; specificity: 100%; p<0.05). Moreover, the combined expression of PAX8 and p53 showed the same result as the single expression of p53 in the three groups.

CONCLUSIONS

The detection of PAX8 and p53 is beneficial in recognizing metastatic carcinomas in pelvic washings, especially in cases with suspicious cytology, which additionally supports the Müllerian origin of these carcinomas.

Pax genes encode evolutionarily conserved transcription factors that play critical roles in embryonic development and organogenesis. Pax proteins are subdivided into four subfamilies: group I (Pax1and 9), II (Pax2, 5, and 8), III (Pax3 and 7), and IV (Pax4 and 6), based on the presence of a paired domain, an octapeptide motif and part or all of the homeodomain. Studies of the evolution of this gene family are incomplete. Nevertheless, it is known that each family evolved via duplication from four corresponding ancestral genes. Pax gene functions have been shown to be conserved within subgroups. It remains unclear, however, whether any (early) conserved function is shared between subgroups. To investigate conserved functions between subfamily II and III, we replaced an allele of Pax3 with a Pax8-coding sequence via gene targeting in the mouse. Homozygote Pax3(Pax8/Pax8) embryos display phenotypes indistinguishable from Pax3-deficient mutant embryos, with neural tube closure defects, a deficit in neural crest cells in the trunk, and skeletal muscle defects including absence of long-range migratory myogenic progenitors and impaired somite development. Interestingly, despite Pax8 expression in the neural tube in a domain ventral to that of Pax3, Pax8 cannot replace Pax3 function in the dorsal neural tube. Altogether, our results demonstrate that expression of Pax8 fails to compensate for Pax3 deficiency, demonstrating the absence of functional compensation between one subfamily of Pax genes and another in the mouse embryo. Our result suggests that Pax3/7 and Pax2/5/8 functions evolved independently after duplication of the ancestral progenitor Pax genes.

Congenital hypothyroidism (CH) may cause severe and irreversible neurologic and developmental abnormalities when not recognized early. Many millions of newborns have now been screened and many thousands of patients with CH have been identified. Approximately 80%-85% have defects of thyroid gland development, while 15%-20% have congenital errors of thyroid hormone biosynthesis. An entire population screened for CH over a long period of time, was studied in the present report, using a population-based approach. In particular, two CH phenotypes, both presenting with in situ thyroid gland (patients with either goiter or with thyroid gland volume ranging from normal to hypoplasic) were analyzed. Mutations were searched in some of the most likely candidate genes: thyroperoxidase (TPO) in patients with CH goiter, Pax8 and thyrotropin receptor (TSHR) in the other group. In the former group (n = 8), four TPO gene mutations were identified in three patients. One patient was a compound heterozygous. In two cases an already described mutation (1277(insGGCC)) was present; in two other cases mutations not previously described (1996(G->>T) and 2295(G->>A)), which induced aminoacid variations with a Glu ->> Stop and Val ->> Ile changes, respectively, were identified. In all patients mutations were inherited from one of the parents. In the case of the compound heterozygous patient, one mutation was inherited from the mother (1277(insGGCC)) and the other from the father (1996(G->>T), Glu ->> Stop). In the latter group (n = 8), a patient with a 16-base pair C(T)(13)CC deletion in TSHR gene intron 8, 42-bp distal to exon/intron 8 splice junction, was identified. No mutation was identified in Pax8 gene.