A woman with recurrent "infections" since birth--a new mevalonate kinase mutation.

A tired 32-year-old woman complaining of tiredness was referred for work-up of a possible immune deficiency. She had a history of recurrent infections since birth, which usually responded to antibiotics within a few days. Her mother, a nurse, had reported that early charts had disappeared. Munchausen's by proxy was suspected for years. Careful anamnesis indicated possible recurrent fever. Serum IgD levels were high, which led us to suspect Hyper IgD Syndrome. Sequencing of the mevalonate kinase gene revealed 2 mutations, leading to amino acid substitutions: one already described (V3771) and R40W: never reported before. Mevalonate kinase activity was very low in the patient's peripheral blood cells. We used the "Poly Phen" prediction program successfully. Our experiments confirmed the diagnosis of mevalonate kinase deficiency. We used steroids to abort recurrent crises.

[Preimplantation genetic diagnosis (PGD): the Erasme Hospital experience]. / Le diagnostic génétique préimplantatoire (DPI): l'expérience de i'Hôpital Erasme.

The clinical activity of the preimplantation genetic diagnosis (PGD) at Erasme Hospital was carried out since September 1999 for a 47,XYY patient. Up to 31 December 2007, 79 PGD cycles were carried out (45 couples) for either chromosomal structural abnormalities (robertsonian and reciprocal translocations, pericentric inversion, deletion) (n = 41), chromosomal numerical abnormalities (47,XXY, 47,XYY, 45,X/46,XX) (n = 10), aneuploidy screening for recurrent miscarriages or multiple in vitro fertilization failures (n = 10), autosomal recessive diseases (cystic fibrosis and sickle cell anaemia) (n = 12) or X-linked disorders (n = 6). A total of 475 embryos were biopsied for genetic analysis. Unaffected embryos were transferred in 58 cycles, resulting in 22 pregnancies, including fifteen clinical pregnancies. Up to now, 9 babies were born and 3 pregnancies are still ongoing. After a learning curve, our current PGD efficiency shows a total pregnancy rate per transfer of 60.0% and an implantation rate of 28.6%. Each PGD result was confirmed by prenatal or postnatal diagnosis. Our data demonstrate that PGD is a valid technique to allow couples at high risk of transmitting a genetic abnormality to increase their chances of a healthy pregnancy, but considering its complexity, patients must be counselled and selected rigorously.

Prevalence of germline mutations in the TSH receptor gene as a cause of juvenile thyrotoxicosis.

AIM: To ascertain the prevalence of germline mutations in the TSH receptor gene as a cause of juvenile thyrotoxicosis (JT) in non-autoimmune patients. TSH receptor gene mutations are not seen in autoimmune-active patients. METHODS: In a nationwide study on JT, 123 patients were re-examined 10 y (range 4 to 21 y) after diagnosis. Two patients with toxic adenoma were excluded. In 25 patients, no TPO, TG or TSH-R antibodies were found. In 17 patients, DNA material was available for TSH receptor gene analysis. The entire TSH receptor gene was sequenced in five patients. TSH receptor "hot spots" for mutations in exon 9 and 10 were sequenced in the remaining 12 patients. RESULTS: A TSH receptor gene germline mutation was identified in only one patient of a total number of 121 patients with JT, of which 17 patients were presumed to have non-autoimmune JT by the lack of thyroid autoantibodies. CONCLUSION: In Denmark the prevalence of germline mutations in the TSH receptor gene is one in 121 patients with JT (0.8%; 95% CI: 0.02-4.6%) and one in 17 patients with presumed non-autoimmune JT (6%; 95% CI: 5.88% (0.15-28.69)).

Predictive factors for pancreatic cancer in patients with chronic pancreatitis in association with K-ras gene mutation.

BACKGROUND AND STUDY AIMS: Chronic pancreatitis is considered to be a predisposing factor for pancreatic ductal adenocarcinoma (PAC). The purpose of this study was to examine the prognostic value of a finding of mutated (K- ras) gene in predicting the development of PAC in patients with chronic pancreatitis. PATIENTS AND METHODS: The pancreatic duct brushings of 146 patients with chronic pancreatitis were examined in order to identify K- ras gene mutations. A total of 112 patients were followed up (median duration 42 months) using clinical evaluation, serum CA19 - 9 levels, and imaging studies. RESULTS: One or more K- ras mutations were found in 57 of the 146 patients with chronic pancreatitis (39 %). Patients harboring K- ras mutations had a higher incidence of persistent alcohol consumption ( P = 0.041) and of prior rupture of the main pancreatic duct ( P = 0.040). A finding of nuclear atypia in brushing cytology was also more common in patients with K- ras mutation ( P = 0.048). Out of the 112 patients who were followed up, PAC occurred in four of the 44 patients who had a K- ras mutation, but in none of the 68 patients with the wild genotype ( P = 0.022). PAC occurred in three of the 25 patients who did not have pancreatic calcifications ( P = 0.034) and in four of the 54 patients who had demonstrated exocrine insufficiency, but in none of the 58 patients with preserved exocrine function ( P = 0.051). Using stepwise logistic regression, the absence of calcifications, the presence of exocrine insufficiency, and the presence of K- ras mutation were identified as independent predictive factors for cancer development in all patients with chronic pancreatitis. CONCLUSIONS: K- ras gene mutations occur in chronic pancreatitis and are associated with evolution towards PAC. The absence of pancreatic calcifications and the presence of exocrine insufficiency were identified as additional predictive factors for the development of PAC.

Oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in the Japanese population.

OBJECTIVE: Constitutively activating mutations of the thyrotropin receptor (TSHR) have been found in the majority of autonomously functioning thyroid nodules (AFTNs) in European patients. The reported frequency of these mutations varies among reports but amounts to 50-80%. To date, only one such mutation responsible for AFTNs has been identified in the Japanese population and the pathogenic role of such mutations in Japanese AFTNs has been questioned. In the present study, we evaluated the frequency of activating mutations in the TSHR and G(alpha)s in 10 Japanese AFTNs. DESIGN: Genomic DNA was extracted from fresh frozen tissue. The TSHR and the almost entire sequence of the gene coding for the alpha subunit of Gs have been amplified and sequenced. RESULTS: In sequence analysis, four mutations in the TSHR (T632A, I486M, M453T and L512R) were found. To complete our analysis, we searched mutations in the gene coding for the alpha subunit of Gs, in the samples negative for TSHR mutations. In one case a mutation (R201H) affecting GTPase activity was found. CONCLUSIONS: If we focus on the solitary nodules, we obtain the same mutation proportion as in European patients (70%). The absence of TSHR and G(alpha)s mutations in a significant proportion of autonomous adenomas in multinodular goiters suggests that other causes may also play a role in the genesis of these lesions.

[Medical genetics service]. / Le Service de Génétique Médicale.

Created in 1987, the department of medical genetics finds its origins in molecular endocrinology research which had developed from the seventies at the Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) of the Faculty of Medicine. After its fusion with the Center of Human Genetics of the ULB, in 1992, the department is composed of three units: the lab of molecular genetics and oncology, the lab of cytogenetics and a clinical genetics unit. One thousand consultations of genetic counseling and more than 15,000 molecular or cytogenetic diagnostic procedures are performed annually. The development of the clinical activities was paralleled by a very active research activity, resulting in a series of "firsts". Amongst the main results are: the identification of the first mutations responsible for congenital hypothyroidism; the molecular cloning of the TSH receptor and of a series of "orphan" G protein-coupled receptors; the identification of a novel neuropeptide, nociceptin, by the first example of "reverse pharmacology"; the identification of olfactory receptors on the sperm of mammals, including man; the identification in molecular terms of the mechanisms responsible for acquired and hereditary hyperthyroidisms; the identification of the chemokine receptor CCR5 as the major coreceptor of HIV-1, and of the prevalent mutation of CCR5 conferring resistance to HIV to about 1% of the European population.

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.

[Prevention of hemoglobinopathies in Brussels: a necessity?]. / Prévention des hémoglobinopathies à Bruxelles: une nécessité?

Haemoglobinopathies are the most frequent genetic diseases in the world. The estimated frequency of carriers in the world is of 200 millions while there is about 300.000 births per year of major forms of haemoglobinopathies. The neonatal screening of haemoglobinopathies performed at the Hospital Erasme on around 80% of births in Brussels since 1994 has demonstrated that the frequency of carriers of an abnormal haemoglobin is around 1.5% while more than 1/2.000 newborns has a major haemoglobinopathy. A strategy must be adopted to manage the haemoglobinopathies in Brussels.

Mutation analysis of the Epac--Rap1 signaling pathway in cold thyroid follicular adenomas.

OBJECTIVE: The cyclic AMP (cAMP) cascade is the main regulatory pathway in thyrocytes. Whilst activating mutations in the TSH receptor or in the Gs alpha-subunit, which increase cAMP levels, have been shown to be responsible for 80% of the autonomous adenomas, no such mutations have been observed in the other types of thyroid tumors, suggesting that other mechanisms exist. The discovery of Epac ('exchange nucleotide protein directly activated by cAMP'), a novel cAMP-binding protein, which is strongly expressed in the thyroid, raised the possibility of a role for this protein in the generation of the unexplained cold thyroid follicular adenomas. Thus, we investigated whether activating mutations in either Epac or Rap (the downstream target of Epac) could be responsible for the generation of these thyroid nodules. DESIGN: Epac and Rap1 (Rap1A and Rap1B) cDNAs were sequenced in 10 patients. The sequencing of the cDNAs was realized on both strands in the cold nodule and the juxtanodular tissue of each patient. RESULTS: No mutations in either Epac or Rap1 cDNAs were found. For five patients, a polymorphism in Epac at codon 332 (Gly--Ser) was observed. CONCLUSIONS: In this report, we show that the cAMP--Epac--Rap1 signaling pathway in the thyroid gland does not play a major role in the generation of cold thyroid follicular adenomas, since no mutations in either Epac or Rap1 could be observed in the 10 nodules studied.

Autosomal dominant transmission of congenital thyroid hypoplasia due to loss-of-function mutation of PAX8.

Congenital hypothyroidism (CH) is a relatively frequent and potentially severe disease. It is classically subdivided into: 1) thyroid dysgenesis (TD), a defect in the organogenesis of the gland leading to hypoplastic, ectopic, or absent thyroid gland; or 2) thyroid dyshormonogenesis, a defect in one of the biochemical mechanisms responsible for thyroid hormone synthesis. Most cases of TD are sporadic, although familial occurrences have occasionally been described. Recently, several genes have been implicated in a small proportion of TD, but, in the majority of the cases, the etiology remains unknown. PAX8 is a transcription factor involved in thyroid development. So far, three loss-of-function mutations of PAX8 have been described, two in sporadic cases and one in familial thyroid hypoplasia. Here, we describe a novel mutation of PAX8 causing autosomal dominant transmission of CH with thyroid hypoplasia. The mutation consists of the substitution of a tyrosine for cysteine 57 in the paired domain of PAX8. When tested in cotransfection experiments with a thyroid peroxidasse promoter construct, the mutant allele was unable to exert its normal transactivation effect on transcription. Our results give further evidence that, contrary to the situation in knockout mice, haplo-insufficiency of PAX8 is a cause of CH in humans.

Hyperfunctioning malignant thyroid nodule in an 11-year-old girl: pathologic and molecular studies.

We identified a papillary carcinoma in an 11-year-old girl with a hyperfunctioning thyroid nodule. A met453thr mutation in TSHR was found in the nodule but not in normal thyroid tissue or in leukocytes. This case documents that this activating mutation is associated with neoplasia.

A quantitative study of peripheral blood stem cell contamination in diffuse large-cell non-Hodgkin's lymphoma: one-half of patients significantly mobilize malignant cells.

Autologous transplantation using peripheral blood stem cells (PBSCs) collected after chemotherapy, followed by growth factor administration (ASCT), is increasingly used in the treatment of non-Hodgkin's lymphoma (NHL). However, quantitative data regarding contaminating malignant cells in the harvests are still scarce. We prospectively investigated 37 diffuse large-cell lymphomas (DLCLs) in complete remission (CR) that were treated according to multicentric protocols at our centre. DNA was extracted from the diagnostic lymph node. The complementarity-determining region (CDR) III was sequenced and a patient-specific oligomer synthesized. Contamination was evaluated semiquantitatively by polymerase chain reaction (PCR) and was confirmed by a limiting dilution analysis. PBSCs collected at regeneration after administration of granulocyte colony-stimulating factor (G-CSF), steady-state bone marrow (BM) and peripheral blood samples at CR were compared. DNA was available in 37 patients, from which 22 rearrangements could be sequenced. Patients (n = 15) who had both the required follow-up samples and a suitable clonal marker were investigated. In two cases, the patient-specific PCR assay set up at diagnosis later gave false-negative results in samples in which clonal DNA was still detectable by other sets of primers. PBSC contamination was highly variable: 7 out of 15 patients showed a PBSC/BM ratio of NHL cells greater than 1 log, whereas 8 out of 15 patients showed no difference and could vary from one apheresis to another. Eight ASCTs were performed, five of which used highly contaminated PBSCs: four patients relapsed early, three with disseminated lymphoma. Thus, 50% of DLCLs in CR seem to mobilize significantly malignant cells at regeneration under G-CSF. Considering the higher numbers of cells reinfused, this translates into a much higher number of lymphoma cells reinfused when compared with autologous bone marrow transplantation (ABMT). However, their clonogenic potential remains unknown and, despite concerning observations in certain cases, it is still unclear whether this has an impact upon the outcome of ASCT.

Congenital central isolated hypothyroidism caused by a homozygous mutation in the TSH-beta subunit gene.

We report a Belgian girl born in 1983 with isolated thyrotropin (TSH) deficiency. Hypothyroidism without goiter was diagnosed at the age of 2 months, with extremely low total thyroxine (T4) at 0.3 microg/dL (4 nmol/L; N[normal]: 5.6-11.4 microg/dL). Basal TSH, only moderately elevated at 14.8 mU/L (N: 0-5.3; competitive radioimmunoassay, RIA), increased to 18.2 mU/L after thyrotropin-releasing hormone (TRH) stimulation, whereas prolactin increased normally. At age 15 years, after withdrawal of levothyroxine (LT4) therapy for 6 weeks, TRH stimulation slightly increased serum TSH using two immunometric assays, from less than 0.03 to 0.07 and from 0.2 to 0.3 (a monoclonal and polyclonal antibody), and from 1.9 to 4.1 mU/L using a polyclonal TSH antibody and iodinated recombinant TSH. Sequencing of the TSH-beta subunit gene revealed a homozygous single nucleotide deletion in codon 105 producing a frame shift that results in a truncated TSH-beta with nonhomologous 9 carboxyterminal amino acids and a loss of the 5 terminal residues. This mutation was previously reported in one Brazilian and two German families. The abnormal, and presumably biologically inactive, TSH can be detected in serum using appropriate antibodies. Its relatively small amount in serum is due to either reduced secretion or rapid degradation. The occurrence of the same mutation in three families of different ethnic origin suggests that this mutation may be prevalent in the population. Common ancestry or de novo mutations in a hot spot cannot be excluded. Finally, we must be aware that neonatal screening of congenital hypothyroidism based on blood spot TSH measurement will not detect this rare but severe genetic defect.

Gene rearrangement and Chernobyl related thyroid cancers.

The increase in thyroid carcinoma post-Chernobyl has been largely confined to a specific subtype of papillary carcinoma (solid/follicular). This subtype is observed predominantly in children under 10 in unirradiated populations, but maintains a high frequency in those aged 10-15 from those areas exposed to fallout from the Chernobyl accident. The aim of this study was to link morphology with molecular biology. We examined 106 papillary carcinomas from children under the age of 15 at operation. All were examined for rearrangements of the RET oncogene by reverse transcription polymerase chain reaction (RT-PCR); a subset of these cases were also examined for mutations of the three ras oncogenes, exon 10 of the thyroid stimulating hormone receptor, associated more usually with a follicular rather than papillary morphology, and exons 5, 6, 7 and 8 of the p53 gene, commonly involved in undifferentiated thyroid carcinoma. Rearrangements of the REToncogene were found in 44% of papillary carcinomas in which we studied fresh material; none of the tumours examined showed mutation in any of the other genes. The two rearrangements resulting from inversion of part of chromosome 10 (PTC1 and PTC3) accounted for the majority of RET rearrangements identified, with PTC1 being associated with papillary carcinomas of the classic and diffuse sclerosing variants and PTC3 with the solid/follicular variant.

The hypothyroidism in an inbred kindred with congenital thyroid hormone and glucocorticoid deficiency is due to a mutation producing a truncated thyrotropin receptor.

Growth and function of the thyroid and adrenal glands are maintained and controlled by thyrotropin (TSH) and adrenocorticotrophic hormone (ACTH), respectively. The action of these trophic hormones requires the presence of functional TSH and ACTH receptors. We describe a large inbred Bedouin kindred in which profound congenital hypothyroidism and hypoadrenocortisolism occurred alone or together in eight family members belonging to four nuclear families. The high serum TSH and ACTH levels in the presence of normal or hypoplastic thyroid glands and low glucocorticoid, but not mineralocorticoid concentrations, are characteristic of resistance to TSH and ACTH. Linkage analysis, using specific polymorphic markers, excluded the involvement of the ACTH receptor but not thyrotropin receptor (TSHR). A novel point mutation was identified in exon 10 of the TSHR that replaces the normal cytosine in nucleotide 2024 with a thymidine. As a result the normal arginine in codon 609 (CGA) is replaced with a stop codon (TGA). This mutation produces a truncated TSHR lacking the third intracellular and extracellular loops, the sixth and seventh transmembrane segments, and the intracytoplasmic tail. The presence of hypothyroidism did not affect the timing, severity, and manner of clinical manifestation of hypoadrenocortisolism.

A germline mutation of the thyrotropin receptor gene associated with thyrotoxicosis and mitral valve prolapse in a Chinese family.

Activating mutations of the TSH receptor (TSH-R) have been reported to result in toxic adenomas, multinodular goiters, sporadic neonatal hyperthyroidism, and familial autosomal dominant nonautoimmune hyperthyroidism. To date, all descriptions of such mutations, whether somatic or genomic, have been confined to the Caucasian population. We describe a Chinese family in whom a germline proline to serine substitution in position 639 resulted in familial thyrotoxicosis. This constitutively activating mutation has been previously described in a hyperfunctioning thyroid nodule. The three children in this family developed thyrotoxicosis during childhood; their father was diagnosed as thyrotoxic at the age of 38 yr. Two of the children and the father had mitral valve prolapse (MVP) associated with mitral regurgitation. There was a close temporal relationship between the onset of thyrotoxicosis and the diagnosis of mitral valvular disease in these patients. An increased prevalence of MVP has been reported in Graves' disease and chronic lymphocytic thyroiditis, but the pathophysiological mechanisms linking MVP and autoimmune thyroid disease are still not understood. This is the first report of an association between activating TSH-R mutations and MVP. We postulate that TSH-R activation may increase the clinical expression of MVP in genetically predisposed individuals.

Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?

Intensification using peripheral blood stem cells collected after chemotherapy followed by growth factors is being increasingly investigated as an alternative to conventional chemotherapy for mantle cell non-Hodgkin lymphoma. We investigated 14 grades III-IV, t(11;14)-positive cases for contamination of PBSC collected after a polychemotherapy regimen followed by G-CSF. Patients were first treated with a polychemotherapy regimen. There were four CR, seven PR, two refractory and one early death. Seven patients have been transplanted, in whom PBSC were mobilized, using either cyclophosphamide/VP16 or Dexa-BEAM followed by G-CSF. For all patients, whether actually autografted or not, PB cells were tested at the time of regeneration on G-CSF after the first polychemotherapy or after the mobilizing regimen. PCR evaluation of contamination was performed first by a semi-quantitative approach, using serial dilutions of initial DNA, then confirmed using a limiting-dilution analysis. Two patients were not informative (one early death and one without an available molecular marker). PB cells collected at regeneration contained at least one log more lymphoma cells than steady-state blood or marrow, apart from in two cases. Moreover, where a mobilizing treatment diminished tumor burden in the patient, at the same time it increased PB contamination in most cases. We conclude that advanced mantle cell NHL appears to be largely resistant to significant in vivo purging by conventional chemotherapy. Where treatment brings benefits by reducing tumor load, it may at the same time negate it by mobilizing malignant cells into the collections used to intensify. Although the clonogenic potential of this massive infiltration is unknown (only gene marking studies could provide a definitive answer regarding the source of relapses), strategies aimed at reducing the level of contamination in the graft should be considered when designing future protocols.

Long-term follow-Up of an infant with thyrotoxicosis due to germline mutation of the TSH receptor gene (Met453Thr).

UNLABELLED: A 18-year clinical follow-up period in a male patient with a germline TSH-R gene mutation (Met453Thr) is described. Nonautoimmune thyrotoxicosis was diagnosed at the age of 7 months. The patient had exophthalmus, failure to thrive, advanced bone age and no goiter. Long-term antithyroid drug treatment (ATD) was necessary during childhood. At the age of 7 years he developed a goiter. Subtotal thyroidectomy was performed at the age of 9 years, followed by repeated ablative radiotherapy at the age of 9.5-13 years due to a toxic multinodular goiter. After 13 years ATD could be discontinued and the patient was euthyroid until 16 years of age, where L-thyroxine substitution had to be started. The exophthalmus diminished, and had disappeared at the age of 18 years, when CT scan of the orbit was performed. CONCLUSION: TSH-R mutation must be considered in early nonautoimmune thyrotoxicosis. A very aggressive treatment strategy is necessary.