Prevalence and phenotypic characterization of MC4R variants in a large pediatric cohort.

OBJECTIVE: We aimed to determine the prevalence of melanocortin-4 receptor (MC4R) variants in a large German cohort of children with obesity in a pediatric outpatient clinic and to ascertain whether there is a specific phenotype associated with loss-of-function variants as previously reported. STUDY DESIGN: Eight hundred and ninety-nine patients from our pediatric obesity clinic were screened for MC4R variants by DNA sequencing after PCR amplification. Retrospective statistical analysis of anthropometric and metabolic characteristics was performed, comparing patients with and without MC4R variants across the entire cohort (n=586) as well as in case-control analysis using patients with common sequence MC4R individually matched for age, sex and body mass index standard deviation score (SDS) (n=11 case-control pairs). RESULTS: We identified heterozygous variants within the coding region of the MC4R gene in n=22 (2.45%) patients. Fourteen (1.56%) had a variant that impaired receptor function. One new frameshift (p.F152Sfs), an yet unpublished nonsense mutation (p.Q156X) and one nonsynonymous variation (p.V65E) described in the Mouse Genome Database were detected. Across the whole cohort, at all ages, mean height SDS in subjects with impaired receptor function was higher than in patients with common sequence MC4R. In matched individuals, this trend persisted (8 of the 11 pairs) within the case-control setting. No differences were found regarding metabolic characteristics. CONCLUSIONS: The observed prevalence of mutations causing impaired receptor function in this large cohort is comparable to other pediatric cohorts. MC4R deficiency tends to lead to a taller stature, confirming previous clinical reports. The association of MC4R mutations with a distinct phenotype concerning metabolic characteristics remains questionable.

Loss of enteroendocrine cells in autoimmune-polyendocrine-candidiasis-ectodermal-dystrophy (APECED) syndrome with gastrointestinal dysfunction.

BACKGROUND: Enteroendocrine (EE) cells are necessary for the regulation of gastrointestinal function. The lack of intestinal enteroendocrine cells in enteroendocrine cell dysgenesis causes severe malabsorptive diarrhea. Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) is often accompanied by gastrointestinal (GI) symptoms. AIMS: We hypothesized that an autoimmune attack against the cells of the GI-associated diffuse endocrine system may be a specific feature of GI dysfunction in APECED disorders. METHODS: Biopsies were obtained during routine diagnostic endoscopy from 35 pediatric patients with gastrointestinal symptoms as well as from five healthy controls; biopsies were immunostained for chromogranin A and serotonin. Four patients were classified as APECED syndrome on molecular and clinical grounds. RESULTS: Immunohistological analysis of biopsies along the GI tract (stomach, duodenum, colon) immunostained with chromogranin A and serotonin revealed a widespread reduction or complete loss of EE cells in all four patients with APECED syndrome suffering from severe diarrhea, vomiting, malabsorption, or constipation. In contrast, EE cells were present in pediatric patients with similar gastrointestinal symptoms caused by inflammatory bowel disease, celiac disease, lymphocytic colitis, and autoimmune disorders without endocrinopathy or graft vs. host disease of the gut. CONCLUSIONS: The reduction of EE cells is a specific and important early event in the pathogenesis of APECED with GI dysfunction. We propose a diagnostic algorithm integrating clinics, genetics and immunohistology.

Osteopetrosis due to homozygous chloride channel ClCN7 mutation mimicking metabolic disease with haematological and neurological impairment.

UNLABELLED: We report on the fatal clinical course of a 3 year old male Turkish patient suffering from osteopetrosis caused by a homozygous mutation in the chloride channel gene ClCN7 with developing pancytopenia and severe neurological impairment. Hepatosplenomegaly due to extramedullary hematopoesis, severe transfusion-dependent anemia and growth failure initially suggested metabolic or oncologic disorder. Particular haematological parameters like tear drop cells basophilic punctation of the polymorphonuclear cells in the absence of haemolysis caused the diagnostic X-ray investigations of the skull and vertebral column. Raised serum creatinkinase-BB isoenzyme and genetic testing were in line with the diagnose of osteopetrosis at an age of 2(1/2) years. CONCLUSION: Osteopetrosis is a rare but considerable differential diagnose for unclarified change in haematopoetic cell lines combined with severe neurological symptoms mimicking metabolic or haematological disease. Because of this rare disease a consensus protocol for diagnostics, treatment and follow up of patients suffering from osteopetrosis is recently worked out from the European Group of Blood and Marrow Transplantation (EBMT) and the European Society for Immundeficiencies (ESID) to build up a central registry for this disease (available by ansgar.schulz@uniklinik-ulm.de).

Novel homozygous AIRE mutation in a German patient with severe APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder typically presenting with chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal failure variably accompanied by other symptoms. APECED is caused by a mutation in the autoimmune regulator gene (AIRE). Today over 60 different mutations are known world-wide, most of them localized in exons 2, 8, and 10. We report here a German girl with rheumatoid factor positive arthritis, chronic mucocutaneous candidiasis, autoimmune hepatitis, chronic diarrhea, vitiligo, hypothyroidism, hypoparathyroidism, and adrenal failure who is homozygous for a novel mutation at the end of exon 3 of the AIRE gene (c.462G>A), within the conserved splice donor sequence. This mutation probably introduces a frameshift after amino acid 154 (p.Pro154fs) by skipping exon 4. In addition, we analyzed five other family members out of three generations for the AIRE gene mutation and for polymorphisms in the cytotoxic T lymphocyte antigen 4 (CTLA4) gene region and lymphoid protein tyrosine phosphatase (PTPN22) gene, which are associated with the occurrence of sporadic autoimmune Addison's disease, type 1 diabetes mellitus, and generalized vitiligo.

Heterogeneity in disease severity in a family with a novel G68V GCK activating mutation causing persistent hyperinsulinaemic hypoglycaemia of infancy.

BACKGROUND/AIM: Glucokinase (GCK)-activating mutations cause persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI). GCK-PHHI patients have regulated insulin secretion and can usually be treated with diazoxide. The six reported cases suggest that the severity of the mutation predicts the clinical phenotype. The aim of this study was to relate genotype to phenotype [clinical phenotype, glucose-stimulated insulin release (GSIR) and GCK functional analysis] in a large pedigree with eight affected individuals. METHODS: The genes encoding B-cell GCK and the K(ATP) channel subunits (ABCC8 and KCNJ11) were sequenced to identify mutations for functional analysis. Genetic variants influencing B-cell function were genotyped in affected individuals. Islet secretory capacity was determined by oral glucose tolerance test RESULTS: A novel GCK mutation (G68V) co-segregating with hypoglycaemia was identified in eight family members. Kinetic analysis revealed that G68V-GCK activity is ~16 times more than wild-type-GCK with an increased affinity for glucose [concentration at half maximal activation (S(0.5)) 1.94 +/- 0.16 vs. 7.43 +/- 0.12, mutant vs. wild type, mean +/- sem]. Mathematical modelling predicted a threshold for GSIR of 1.9 mmol/l in the mutant. Oral glucose tolerance tests showed regulated insulin secretion. The severity of hypoglycaemia and related symptoms in affected subjects were heterogeneous. Clinical presentations were asymptomatic (n = 1), extreme hunger (n = 3), seizures (n = 2) and loss of consciousness (n = 2); 7/8 were managed with diet but the proband was treated with diazoxide and octreotide. Phenotypic modification by a second mutation in the K(ATP) channel genes (ABCC8, KCNJ11) or by common genetic variants in KCNJ11, GCK and TCF7L2 was excluded. CONCLUSION: The novel activating GCK mutation G68V is associated with variable phenotypic severity, supporting modification of GSIR by genetic and/or environmental factors.

Stem cell transplantation in children with infantile osteopetrosis is associated with a high incidence of VOD, which could be prevented with defibrotide.

Malignant infantile osteopetrosis (MIOP) is a rare hereditary disorder of osteoclast function, which can be reversed by hematopoietic stem cell transplantation (SCT). We observed a high incidence of hepatic veno-occlusive disease (VOD) in transplanted patients and explored the prevention of this complication by using defibrotide (DF) as a prophylaxis. Twenty children with MIOP were consecutively transplanted in our center between 1996 and 2005. Eleven of these patients were transplanted between 1996 and 2001 and experienced an overall incidence of VOD of 63.6% (7/11). VOD was severe in three patients and one patient succumbed to VOD-related multi-organ failure. Owing to this very high incidence of VOD, DF prophylaxis was initiated in nine patients consecutively transplanted between 2001 and 2005. In this group, only one patient (11.1%) was diagnosed with moderate VOD. We report here a very high risk in patients with MIOP to develop VOD after transplantation. Prophylactic DF was implemented in our current transplant protocol and reduced the VOD rate significantly in this high-risk population.

Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs.

One of the key end points for understanding the molecular basis of embryogenesis is the analysis of spatiotemporal patterns of gene expression. Methodical limitations due to low mRNA levels often prevent a tissue-specific resolution. In this study, we developed an improved laser microdissection technique and RT-PCR that allows marker gene detection in small tissue areas from sections of formalin-fixed paraffin-embedded Xenopus embryos. Tissue pieces were isolated by laser microbeam microdissection and captured by laser pressure catapulting. Neither laser treatment nor conventional histological or immunochemical staining impaired subsequent RNA analysis. Transcripts of tissue-specific marker genes such as endodermin (endoderm), epidermal cytokeratin (epidermal ectoderm), N-CAM (neural tube), myoD (somites), and sonic hedgehog (floor plate) were amplified by nested RT-PCR analysis from small areas of single sections.

Diagnosis of papillary thyroid carcinoma is facilitated by using an RT-PCR approach on laser-microdissected archival material to detect RET oncogene activation.

OBJECTIVE: The purpose of this study was to investigate the value of the expression of the RET oncogene (rearranged during transfection) in papillary thyroid carcinomas (PTC) and its variants in the differential diagnosis of thyroid neoplasias. According to the literature RET oncogene activation by chromosomal rearrangements has been exclusively implicated in PTCs. METHODS: To establish the incidence of RET activation in PTCs we used 5- to 10-microm sections from archival paraffin blocks. Either parts of the tissue slices were manually dissected or a few distinct cells were microdissected by laser-mediated manipulation with the Robot-MicroBeam system. RNA was extracted from paraffin-embedded thyroid tumors and the corresponding normal tissue. RT and nested PCR were performed using primers for RET/PTC1, PTC2 and PTC3, or for RET exons 12 and 13. PCR products were resolved by gel electrophoresis. RESULTS: We detected RET transcription in approximately 85% of the PTCs including follicular variants and in isolated cells of the same tissues, but not in nonmalignant thyroid tissue. CONCLUSIONS: Our method may serve as an additional diagnostic tool to characterize ambiguous neoplasias and to identify especially nonpapillary, i.e. follicular tumors, as papillary carcinomas. Additionally, this study has demonstrated that expressed genes can be analyzed from routine histopathological tissue slides or pooled single cells. Large retrospective studies can also be performed with this method.

Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine.

The UV-laser microbeam has been established as a valuable tool in a wide area of molecular biology as well as in medical research and applications. This system allows to cut or fuse microscopically small specimen. An important application of the cutting laser is laser microbeam microdissection (LMM) combined with laser pressure catapulting (LPC), which allows to procure single cells or small homogeneous cell areas for subsequent molecular analysis in an entirely "non-contact" manner. With LMM minute tissue areas, single cells or chromosomes are microdissected and separated from their surroundings. Subsequently, LPC ejects the dissectates directly into the cap of a sample tube without any mechanical contact. This enables the rapid procurement of homogeneous specimen from less than one up to several hundreds of micrometers in diameter without encroachment of the adjacent region. The mRNA information of the selected specimen as well as of the remaining probe are well preserved, as demonstrated with laser isolated samples from a routinely prepared tissue section of a differentiated colorectal adenocarcinoma. Reverse transcription of specific mRNA coding for cytoplasmic beta-actin and subsequent hemi-nested PCR amplification was not impaired. Any kind of tissue, as well as single cells from different sources and even subcellular structures can be captured using this laser method. Wherever homogeneous samples are required to analyze cell or chromosome-specific genetic alterations such as in cancer research or prenatal diagnosis this unique and rapid laser micropreparation method will become a key technology of great value.

Identification of expressed genes by laser-mediated manipulation of single cells.

We describe a rapid noncontact method for the capture of single cells or small tissue areas of any size or shape directly within the cap of a common microfuge tube. Prior to the laser-mediated transfer, the specimen is isolated by laser microbeam microdissection, forming a clear-cut gap around the selected area. Laser treatment does not impair subsequent RNA analysis. We have used this method to isolate a single cell from archival colon adenocarcinoma, and were able to detect point mutations within codon 12 of c-Ki-ras2 mRNA after nested RT-PCR analysis.

The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain.

Sexual differentiation of the brain is thought to be regulated by hormonal signals from the developing male gonad. However, more-recent experimental and clinical data throw some doubt on the general validity of the "classical" steroid hypothesis and suggest that additional intervening factors or mechanisms need to be considered. In particular, it is now envisaged that neurons are capable of acquiring sex-specific properties independently of their hormonal environment. Here we show that two Y-chromosomal genes involved in sex determination of the gonad, SRY and ZFY, are transcribed in hypothalamus, and frontal and temporal cortex of the adult male human brain. These genes are candidates for male-specific transcriptional regulators that could confer upon human brain cells the potential for hormone-independent realization and maintenance of genetic sex.

Visualizing the expression of a human growth hormone (hGH) transgene in the liver: intrahepatic regional and intracellular differences of expression are associated with morphological alterations and hepatocellular proliferation.

Growth hormone acts directly on liver cells; it binds to its receptor and induces a multitude of intracellular events leading, for example, to the production of insulin-like growth factor-1 (IGF-1). While much is known about the biochemical side of these events, their structural correlates are less well examined. Here, we examined livers of transgenic mice (TM) expressing human GH, in an attempt to correlate at the cellular level the site of GH gene expression with effects on morphology and mitotic behavior of liver cells within the hepatic architecture. Using in situ hybridization histochemistry we observed a striking expression pattern of the hGH gene in hepatocytes near the periportal spaces. In the same regions, the hGH protein, but no IGF-1 immunoreactive product, was detected using immunohistochemical methods. In the sections of TM livers, 6.8-31.9% of cells were hGH-immunoreactive. However, the cellular hGH staining pattern was not homogeneously distributed in the immunoreactive cells. Two main patterns became obvious. In the majority of the immunoreactive cells a cytoplasmic stain was present. These cells exhibited normal liver cell features and were not enlarged (type I). In the other group (type II), the staining was stronger and concentrated, sometimes punctuate, and often confined to cytoplasmic compartments which were in a perinuclear position. The latter staining pattern was generally seen in morphologically altered cells, which were enlarged and possessed intranuclear inclusions and invaginations. In the the periportal regions, mitotically active hepatocytes were evident, but these cells, as judged from immunocytochemistry, apparently did not express the transgene. In conclusion, different staining patterns for hGH may indicate different levels of transgene expression, which could be associated with difficulties in the cells with regard processing and/or secreting the hormone. In addition to the endocrine actions implied by the high hGH levels in the peripheral circulation of these TM, intracrine actions are also suggested (type II staining pattern), but para- and autocrine loops are possible as well (type I staining pattern). Whether IGF-1 is involved, and the mechanism underlying hepatocyte cell proliferation, remain to be examined.

The neural cell adhesion molecule (NCAM) provides clues to the development of testicular Leydig cells.

As previously shown, Leydig cells in culture dramatically up-regulate the expression of the neural cell adhesion molecule (NCAM) gene and express alternatively spliced forms. Because the family of NCAM adhesion molecules is known to be involved in cell migration and differentiation, we examined the potential involvement of NCAMs in Leydig cell differentiation in the developing testis. We detected NCAM-immunoreactive cells in the rat and mouse at embryonic day (ED) 13-14 in epithelia of mesonephric tubules and cell clusters between the mesonephros and testis. At about ED 17-18, strongly NCAM-immunoreactive cells were demonstrated extending from the mesonephros/mesonephric tubules into the region occupied by the forming rete testis and spreading into the testis itself. Within the testis, interstitial fetal Leydig cells, identified with an antiserum directed against P450-side-chain cleavage enzyme, were also NCAM immunoreactive, although to a lesser degree, and in part expressed NCAM-associated polysialic acid. In situ hybridization histochemistry demonstrated the presence of NCAM mRNA, mainly in the cell population corresponding to the strongly immunoreactive cells. NCAM forms with molecular weights of 140 and 180 kDa, the latter polysialylated, predominate in immunoblots of fetal testes. Because testicular development occurs along the mesonephros, from which precursor cells are thought to migrate into the developing gonad and then differentiate into the various testicular cell types, our results may suggest that the expression of NCAMs and NCAM modification are associated with cells that appear to migrate into the developing testis. Although it remains to be proven whether these cells could be precursor cells of Leydig cells, this assumption is supported by the fact that within the developing testis, NCAMs and NCAM modifications are expressed during differentiation of testicular P450-side-chain cleavage enzyme-positive Leydig cells. Thus, the adhesion molecule NCAM and its variants and modifications are expressed in the developing testis and may be of functional, "morphogenic" importance. Because Leydig cells in vitro and during fetal development express NCAMs, these molecules may prove to be a suitable specific marker for the study of Leydig cell development and differentiation.

Transcription of the Y chromosomal gene, Sry, in adult mouse brain.

The Y chromosomal gene Sry encodes a putative transcription factor which appears to serve as a master switch initiating testicular development. Here we show that this gene is transcribed in hypothalamus, midbrain, and testis of adult male but not adult female mice. In contrast to its circular transcripts in adult testis, those in brain are linear and may be translated. We propose that Sry exerts a role in the regulation of sex differentiation of the mammalian nervous system.

Expression and alternative splicing of the neural cell adhesion molecule NCAM in human granulosa cells during luteinization.

Freshly aspirated human granulosa cells from pre-ovulatory follicles and granulosa cells luteinized in culture possess the neural cell adhesion molecule (NCAM) of approximate molecular mass of 140,000 and NCAM mRNA as confirmed by S1-nuclease protection assays and RT-PCR. Moreover, in the process of luteinization the NCAM isoform pattern is modified. Isoforms containing an insert of 10 amino acids (termed VASE) in the extracellular domain of NCAM were supplemented by alternatively spliced isoforms without this insert. NCAM immunoreactivity, at light and electron microscope levels, was associated with the cell membrane of most granulosa cells which formed clusters. During time in culture an increasing subpopulation of granulosa cells, devoid of NCAM immunoreactivity, spread out and formed monolayers. This differential expression and the alternative splicing of NCAM during luteinization of granulosa cells raise the possibility that NCAM could be involved in folliculogenesis and the formation of the corpus luteum in the human.