Laurence Duprez

Familial congenital hypothyroidism due to inactivating mutation of the thyrotropin receptor causing profound hypoplasia of the thyroid gland.

Thyroid gland agenesis is the most common cause of congenital hypothyroidism and is usually sporadic. We investigated a brother and sister from consanguineous parents, ascertained through systematic newborn screening, and initially diagnosed with thyroid agenesis. Careful cervical ultrasonography in both patients revealed a very hypoplastic thyroid gland. By direct sequencing of the thyrotropin receptor gene, we identified the substitution of threonine in place of a highly conserved alanine at position 553, in the fourth predicted transmembrane domain. The mutation was found homozygous in the affected siblings, and heterozygous in both parents and two unaffected siblings. Functional analysis in transfected COS-7 cells showed that it resulted in extremely low expression at the cell surface as compared with the wild-type receptor, in spite of an apparently normal intracellular synthesis. The small amount of mutated receptor expressed at the surface of transfected cells bound thyrotropin with normal affinity and responded in terms of cAMP production, but the in vivo significance of these data from overexpressed receptor in transfected cells is unclear. Of note, blood thyroglobulin was unexpectedly elevated in the patients at the time of diagnosis, a finding that might prove useful in refining etiologies of congenital hypothyroidism.

TSH receptor and disease

The TSH, LH/CG and FSH receptors belong to a subfamily of G protein-coupled receptors. As such, their primary structures deduced from the sequence of the corresponding cDNA (Parmentieret al., 1989; Nagayama et al., 1989; Libertet al., 1989; Minegishiet al., 1990; 1991), predict the existence of seven segments with hydropathy compatible with a transmembrane location. The glycoprotein hormone receptor subfamily (TSH, LH/CG, FSH) share characteristics that distinguish them from the other G protein-coupled receptors. They contain a signal peptide (20 amino acids for the TSH receptor) and they have a long extracellular aminoterminal domain (398 aminoacids for the TSH receptor) comprising the loose repetition of a motif of 25 residues rich in leucine (Parmentieret al., 1989; McFarlandet al., 1989). Similar leucine-rich motifs are also found in a number of widely different proteins (Roth, 1991), in which they are believed to confer the ability to interact with other proteins. The threedimensional structure of one such protein, the ribonuclease inhibitor, has been determined (Kobe & Deisenhofer, 1993), and provides a basis for the modelling of other leucine-rich protein segments. Site-directed mutagenesis studies involving chimeric receptors have clearly shown that the binding specificity and the effector properties of the glycoprotein hormone receptors are encoded in separate domains of the proteins (Xie et al., 1990; Braunet al., 1991; Nagayamaet al., 1991; Vassart & Dumont, 1992); the extracellular domain mediates the binding specificities and the ‘serpentine’ portion with the seven transmembrane segments displays the effector properties triggering G protein activation. This duality is reflected at the genomic level where a single exon encodes the serpentine portion of the receptors and many exons (nine for the TSH receptor) encode the extracellular domain (Gross et al., 1991). When aligned, the three glycoprotein hormone receptors show stronger conservation in the serpentine domain (approximately 70% similarity) than in the extracellular domain (approximately 40% similarity). A peculiarity of the TSH receptor with no counterpart in the FSH or LH/CG receptors is a 50-residue insert upstream from the hinge between the aminoterminal extracellular portion and the first transmembrane segment. A first model for the three-dimensional structure of the aminoterminal extracellular segment of the thyrotrophin receptor has been recently proposed (Kajavaet al., 1995) (Fig. 1). It is based on the known structure of the ribonuclease inhibitor (Kobe & Deisenhofer, 1993).

Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Cell lines are crucial to elucidate mechanisms of tumorigenesis and serve as tools for cancer treatment screenings. Therefore, careful validation of whether these models have conserved properties of in vivo tumors is highly important. Thyrocyte-derived tumors are very interesting for cancer biology studies because from one cell type, at least five histologically characterized different benign and malignant tumor types can arise. To investigate whether thyroid tumor-derived cell lines are representative in vitro models, characteristics of eight of those cell lines were investigated with microarrays, differentiation markers, and karyotyping. Our results indicate that these cell lines derived from differentiated and undifferentiated tumor types have evolved in vitro into similar phenotypes with gene expression profiles the closest to in vivo undifferentiated tumors. Accordingly, the absence of expression of most thyrocyte-specific genes, the nonresponsiveness to thyrotropin, as well as their large number of chromosomal abnormalities, suggest that these cell lines have acquired characteristics of fully dedifferentiated cells. They represent the outcome of an adaptation and evolution in vitro, which questions the reliability of these cell lines as models for differentiated tumors. However, they may represent useful models for undifferentiated cancers, and by their comparison with differentiated cells, can help to define the genes involved in the differentiation/dedifferentiation process. The use of any cell line as a model for a cancer therefore requires prior careful and thorough validation for the investigated property.

Isodisomy of chromosome 6 in a newborn with methylmalonic acidemia and agenesis of pancreatic beta cells causing diabetes mellitus.

Isodisomy (ID) is a genetic anomaly defined as the inheritance of two copies of the same genetic material from one parent. ID in an offspring is a rare cause of recessive genetic diseases via inheritance of two copies of a mutated gene from one carrier parent. We studied a newborn female with a mut(o) of methylmalonic acidemia and complete absence of insulin-producing beta cells in otherwise normal-appearing pancreatic islets, causing insulin-dependent diabetes mellitus. The patient died 2 wk after birth. Serotyping of the HLA antigens, DNA typing of HLA-B and HLA class II loci, study of polymorphic DNA markers of chromosome 6, and cytogenetic analysis demonstrated paternal ID, involving at least a 25-centiMorgan portion of the chromosome pair that encompasses the MHC. ID probably caused methylmalonic acidemia by duplication of a mutated allele of the corresponding gene on the chromosome 6 inherited from the father. It is also very likely that ID was etiologically related to the agenesis of beta cells and consequent insulin-dependent diabetes mellitus in our patient. We thus speculate on the existence of a gene on chromosome 6 involved in beta cell differentiation.

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age.

An excess of chromosome 1 breakpoints in male infertility.

In a search for potential infertility loci, which might be revealed by clustering of chromosomal breakpoints, we compiled 464 infertile males with a balanced rearrangement from Mendelian Cytogenetics Network database (MCNdb) and compared their karyotypes with those of a Danish nation-wide cohort. We excluded Robertsonian translocations, rearrangements involving sex chromosomes and common variants. We identified 10 autosomal bands, five of which were on chromosome 1, with a large excess of breakpoints in the infertility group. Some of these could potentially harbour a male-specific infertility locus. However, a general excess of breakpoints almost everywhere on chromosome 1 was observed among the infertile males: 26.5 versus 14.5% in the cohort. This excess was observed both for translocation and inversion carriers, especially pericentric inversions, both for published and unpublished cases, and was significantly associated with azoospermia. The largest number of breakpoints was reported in 1q21; FISH mapping of four of these breakpoints revealed that they did not involve the same region at the molecular level. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility.

A Familial Case of Congenital Hypothyroidism Caused by a Homozygous Mutation of the Thyrotropin Receptor Gene

Most of the time congenital hypothyroidism appears as a sporadic disease. In addition to the rare defects in hormonosynthesis associated with goiters, the causes of congenital hypothyroidism include agenesis and ectopy of the thyroid gland. The study of some familial cases has allowed the identification of a few genes responsible for congenital hypothyroidism. We report here a familial case of congenital hypothyroidism, transmitted as a recessive trait, and caused by a homozygous mutation in the thyrotropin receptor (TSH-R). The initial diagnosis of thyroid agenesis, based on the absence of tracer uptake on scintiscan, was incorrect, because ultrasound examination identified severely hypoplastic thyroid tissue in the cervical region.

A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly

Primary microcephaly (microcephalia vera) is a developmental abnormality resulting in a small brain, with mental retardation. It is usually transmitted as an autosomal recessive trait, and six loci have been reported to date. We analyzed a translocation breakpoint previously reported in a patient with apparently sporadic primary microcephaly, at 1q31, where locus MCPH5 maps. The patient was lost to follow-up, and we sampled a maternal aunt who carried the familial translocation. FISH analyses showed that the insert of BAC clone RP11-32D17 spanned the breakpoint. The breakpoint was further located within a fragment of this insert corresponding to intron 17 of the ASPM gene, resulting in a predicted transcript truncated of more than half of its coding sequence. It is very likely that the proband carried a second ASPM mutation in trans, but he was not available for sampling and hence we could not confirm this hypothesis. Our observation adds to the mutation spectrum of ASPM in primary microcephaly, and is to our knowledge the second example of a constitutional, reciprocal translocation responsible for a bona fide autosomal recessive phenotype.

Constitutively active receptors as a disease-causing mechanism

Membrane receptors have appeared early in evolution as the means for the unicellular organism to sense its environment. With the emergence of social cellular life in multicellular organisms, membrane receptors have acquired the additional functions of sensing the presence of similar cells (as in the aggregation phenomenon of Dictyostelium discoideum) (Klein et al., 1988) or the presence of the mate (Saccharomyces cerevisiae) (Cross et al., 1988), and to detect endocrine signals emitted by cells in distant tissues. As the latter function is central to homeostasis and regulation of cell growth, the downstream regulatory cascades under receptor control are the subject of intense research with implications in virtually all fields of biomedical science. The impact of the analysis of tyrosine kinase-activated cascades on our understanding of carcinogenesis is but one example of such an advance.

Truncation of the CNS-expressed JNK3 in a patient with a severe developmental epileptic encephalopathy

We have investigated the breakpoints in a male child with pharmacoresistant epileptic encephalopathy and a de novo balanced translocation t(Y;4)(q11.2;q21). By fluorescence in situ hybridisation, we have identified genomic clones from both chromosome 4 and chromosome Y that span the breakpoints. Precise mapping of the chromosome 4 breakpoint indicated that the c-Jun N-terminal kinase 3 (JNK3) gene is disrupted in the patient. This gene is predominantly expressed in the central nervous system, and it plays an established role in both neuronal differentiation and apoptosis. Expression studies in the patient lymphoblastoid cell line show that the truncated JNK3 protein is expressed, i.e. the disrupted transcript is not immediately subject to nonsense-mediated mRNA decay, as is often the case for truncated mRNAs or those harbouring premature termination codons. Over-expression studies with the mutant protein in various cell lines, including neural cells, indicate that both its solubility and cellular localisation differ from that of the wild-type JNK3. It is plausible, therefore, that the presence of the truncated JNK3 disrupts normal JNK3 signal transduction in neuronal cells. JNK3 is one of the downstream effectors of the GTPase-regulated MAP kinase cascade, several members of which have been implicated in cognitive function. In addition, two known JNK3-interacting proteins, β-arrestin 2 and JIP3, play established roles in neurite outgrowth and neurological development. These interactions are likely affected by a truncated JNK3 protein, and thereby provide an explanation for the link between alterations in MAP kinase signal transduction and brain disorders.