Case Report: Aggressive neural crest tumors in a child with familial von Hippel Lindau syndrome associated with a germline VHL mutation (c.414A>G) and a novel KIF1B gene mutation.

Introduction: Von Hippel Lindau (VHL) syndrome is caused by an autosomal dominant hereditary or sporadic germline mutation of the VHL gene with more than five hundred pathogenic mutations identified. Pheochromocytomas and rarely paragangliomas occur in 10-50% of patients with VHL syndrome usually around 30 years of age and exceptionally before the age of 10. Case presentation: We diagnosed a 9-year-old girl of normal appearance and severe refractory hypertension, with a norepinephrine-secreting pheochromocytoma related to VHL syndrome due to a known familial germline heterozygous mutation of VHL gene (c.414A>G), also present in three members of her family. At age 13, a pelvic tumor and a left adrenal pheochromocytoma that showed to be multi-metastatic to both lungs were discovered in the patient leading to left adrenalectomy and pelvic tumor resection. In addition to the germline VHL gene mutation, blood analysis using Next Generation Sequencing identified a novel heterozygous germline mutation of the KIF1B gene (c.3331_3332del; p.Asn1111Glnfs*21), which is only present in the girl and not the other family members. The patient is currently under steroid substitution therapy and leads a normal life. Discussion: This family is notable by the early age of onset of multiple neural crest tumors associated with a high propensity for malignancy and metastatic spread. Most reports in the literature associated the VHL mutation with a later onset in adulthood and a benign course, which contrast with our findings and question the role of this mutation in the phenotype expressed in this kindred. Also, the presence of concomitant mutations in two susceptibility genes for neural crest tumors poses the question of their respective roles in the development of tumors in this family. Our familial case description illustrates the potential for systematic use of targeted Next Generation Sequencing with multi-gene panels in patients with neural crest tumors to confirm the role of known susceptibility genes as well as identifying new ones, but also to contribute to comprehensive databases on gene variants and their phenotypic counterparts in this specific area of medicine.

Congenital hydrocephalus: new Mendelian mutations and evidence for oligogenic inheritance.

BACKGROUND: Congenital hydrocephalus is characterized by ventriculomegaly, defined as a dilatation of cerebral ventricles, and thought to be due to impaired cerebrospinal fluid (CSF) homeostasis. Primary congenital hydrocephalus is a subset of cases with prenatal onset and absence of another primary cause, e.g., brain hemorrhage. Published series report a Mendelian cause in only a minority of cases. In this study, we analyzed exome data of PCH patients in search of novel causal genes and addressed the possibility of an underlying oligogenic mode of inheritance for PCH. MATERIALS AND METHODS: We sequenced the exome in 28 unrelated probands with PCH, 12 of whom from families with at least two affected siblings and 9 of whom consanguineous, thereby increasing the contribution of genetic causes. Patient exome data were first analyzed for rare (MAF < 0.005) transmitted or de novo variants. Population stratification of unrelated PCH patients and controls was determined by principle component analysis, and outliers identified using Mahalanobis distance 5% as cutoff. Patient and control exome data for genes biologically related to cilia (SYScilia database) were analyzed by mutation burden test. RESULTS: In 18% of probands, we identify a causal (pathogenic or likely pathogenic) variant of a known hydrocephalus gene, including genes for postnatal, syndromic hydrocephalus, not previously reported in isolated PCH. In a further 11%, we identify mutations in novel candidate genes. Through mutation burden tests, we demonstrate a significant burden of genetic variants in genes coding for proteins of the primary cilium in PCH patients compared to controls. CONCLUSION: Our study confirms the low contribution of Mendelian mutations in PCH and reports PCH as a phenotypic presentation of some known genes known for syndromic, postnatal hydrocephalus. Furthermore, this study identifies novel Mendelian candidate genes, and provides evidence for oligogenic inheritance implicating primary cilia in PCH.

Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing.

STUDY QUESTION: Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER: Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY: Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION: This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5-10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE: The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS: Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation.

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.

Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations.

OBJECTIVE: To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. METHODS: We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. RESULTS: Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. CONCLUSION: Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations.

Outcome of publicly funded nationwide first-tier noninvasive prenatal screening.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation. METHODS: The performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered. RESULTS: Trisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%. CONCLUSION: Expanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.

Comparison of the mutation spectrum and association with pre and post treatment lipid measures of children with heterozygous familial hypercholesterolaemia (FH) from eight European countries.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is commonly caused by mutations in the LDLR, APOB or PCSK9 genes, with untreated mean low density lipoprotein-cholesterol (LDL-C) concentrations being elevated in APOB mutation carriers, even higher in LDLR mutation and highest in those with a PCSK9 mutation. Here we examine this in children with FH from Norway, UK, The Netherlands, Belgium, Czech Republic, Austria, Portugal and Greece. METHODS: Differences in characteristics and pre- and post-treatment lipid concentrations in those with different molecular causes were compared by standard statistical tests. RESULTS: Data were obtained from 2866 children, of whom 2531 (88%) carried a reported LDLR/APOB/PCSK9 variant. In all countries, the most common cause of FH was an LDLR mutation (79% of children, 297 different), but the prevalence of the APOB p.(Arg3527Gln) mutation varied significantly (ranging from 0% in Greece to 39% in Czech Republic, p < 2.2 × 10-16). The prevalence of a family history of premature CHD was significantly higher in children with an LDLR vs APOB mutation (16% vs 7% p=0.0005). Compared to the LDLR mutation group, mean (±SD) concentrations of pre-treatment LDL-C were significantly lower in those with an APOB mutation (n = 2260 vs n = 264, 4.96 (1.08)mmol/l vs 5.88 (1.41)mmol/l, p < 2.2 × 10-16) and lowest in those with a PCSK9 mutation (n = 7, 4.71 (1.22)mmol/l). CONCLUSIONS: The most common cause of FH in children from eight European countries was an LDLR mutation, with the prevalence of the APOB p.(Arg3527Gln) mutation varying significantly across countries. In children, LDLR-FH is associated with higher concentrations of LDL-C and family history of CHD compared to those with APOB-FH.

Heterogeneous colonic content: A prenatal sonographic manifestation of lysinuric protein intolerance.

We report a fetus with heterogeneous colonic content, an isolated sonographic prenatal sign of lysinuric protein intolerance, a very rare metabolic disease. Familial genetic enquiries confirmed heterozygote mutation in the implicated gene in parents. The prenatal diagnosis led to neonatal dietary adaptation and avoided acute complications.

Prenatally detected copy number variants in a national cohort: A postnatal follow-up study.

OBJECTIVE: Belgian genetic centers established a database containing data on all chromosomal microarrays performed in a prenatal context. A study was initiated to evaluate postnatal development in children diagnosed prenatally with a non-benign copy number variant (CNV). METHODS: All children diagnosed with a prenatally detected non-benign CNV in a Belgian genetic center between May 2013 and February 2015 were included in the patient population. The control population consisted of children who had undergone an invasive procedure during pregnancy, with no or only benign CNVs. Child development was evaluated at 36 months using three (3) questionnaires: Ages and Stages Questionnaire Third edition, Ages and Stages Questionnaire Social-Emotional Second Edition and a general questionnaire. RESULTS: A significant difference in communication and personal-social development was detected between children with a reported susceptibility CNV and both children with an unreported susceptibility CNV and the control population. The outcome of children with a particular CNV is discussed in a case-by-case manner. CONCLUSION: Our postnatal follow-up project of children with a prenatally detected non-benign CNV is the first nationwide project of its kind. A higher number of cases for each CNV category is however needed to confirm our findings.

Tumor sequencing is useful to refine the analysis of germline variants in unexplained high-risk breast cancer families.

BACKGROUND: Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. METHODS: Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. RESULTS: Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. CONCLUSION: Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.

A Belgian consensus strategy to identify familial hypercholesterolaemia in the coronary care unit and its subsequent cascade screening and treatment: BEL-FaHST (The BELgium Familial Hypercholesterolaemia STrategy).

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is an autosomal dominant lipoprotein disorder characterized by significant elevation of low-density lipoprotein cholesterol (LDL-C) and markedly increased risk of premature cardiovascular disease (CVD). Because of the very high coronary artery disease risk associated with this condition, the prevalence of FH among patients admitted for CVD outmatches many times the prevalence in the general population. Awareness of this disease is crucial for recognizing FH in the aftermath of a hospitalization of a patient with CVD, and also represents a unique opportunity to identify relatives of the index patient, who are unaware they have FH. This article aims to describe a feasible strategy to facilitate the detection and management of FH among patients hospitalized for CVD. METHODS: A multidisciplinary national panel of lipidologists, cardiologists, endocrinologists and cardio-geneticists developed a three-step diagnostic algorithm, each step including three key aspects of diagnosis, treatment and family care. RESULTS: A sequence of tasks was generated, starting with the process of suspecting FH amongst affected patients admitted for CVD, treating them to LDL-C target, finally culminating in extensive cascade-screening for FH in their family. Conceptually, the pathway is broken down into 3 phases to provide the treating physicians with a time-efficient chain of priorities. CONCLUSIONS: We emphasize the need for optimal collaboration between the various actors, starting with a "vigilant doctor" who actively develops the capability or framework to recognize potential FH patients, continuing with an "FH specialist", and finally involving the patient himself as "FH ambassador" to approach his/her family and facilitate cascade screening and subsequent treatment of relatives.

The Belgian MicroArray Prenatal (BEMAPRE) database: A systematic nationwide repository of fetal genomic aberrations.

OBJECTIVE: With the replacement of karyotyping by chromosomal microarray (CMA) in invasive prenatal diagnosis, new challenges have arisen. By building a national database, we standardize the classification and reporting of prenatally detected copy number variants (CNVs) across Belgian genetic centers. This database, which will link genetic and ultrasound findings with postnatal development, forms a unique resource to investigate the pathogenicity of variants of uncertain significance and to refine the phenotypic spectrum of pathogenic and susceptibility CNVs. METHODS: The Belgian MicroArray Prenatal (BEMAPRE) consortium is a collaboration of all genetic centers in Belgium. We collected data from all invasive prenatal procedures performed between May 2013 and July 2016. RESULTS: In this three-year period, 13 266 prenatal CMAs were performed. By national agreement, a limited number of susceptibility CNVs and no variants of uncertain significance were reported. Added values for using CMA versus conventional karyotyping were 1.8% in the general invasive population and 2.7% in cases with an ultrasound anomaly. Of the reported CNVs, 31.5% would have remained undetected with non-invasive prenatal test as the first-tier test. CONCLUSION: The establishment of a national database for prenatal CNV data allows for a uniform reporting policy and the investigation of the prenatal and postnatal genotype-phenotype correlation.

Reevaluation of ATR signaling in primary resting chronic lymphocytic leukemia cells: evidence for pro-survival or pro-apoptotic function.

ATM, primarily activated by DNA double-strand breaks, and ATR, activated by single-stranded DNA, are master regulators of the cellular response to DNA damage. In primary chronic lymphocytic leukemia (CLL) cells, ATR signaling is considered to be switched off due to ATR downregulation. Here, we hypothesized that ATR, though expressed at low protein level, could play a role in primary resting CLL cells after genotoxic stress. By investigating the response of CLL cells to UV-C irradiation, a prototypical activator of ATR, we could detect phosphorylation of ATR at Thr-1989, a marker for ATR activation, and also observed that selective ATR inhibitors markedly decreased UV-C-induced phosphorylation of ATR targets, including H2AX and p53. Similar results were obtained with the purine analogs fludarabine and cladribine that were also shown to activate ATR and induce ATR-dependent phosphorylation of H2AX and p53. In addition, ATR inhibition was found to sensitize primary CLL cells to UV-C by decreasing DNA repair synthesis. Conversely, ATR inhibition rescued CLL cells against purine analogs by reducing expression of the pro-apoptotic genes PUMA and BAX. Collectively, our study indicates that ATR signaling can be activated in resting CLL cells and play a pro-survival or pro-apoptotic role, depending on the genotoxic context.

Routine use of gene panel testing in hereditary breast cancer should be performed with caution.

Breast cancer is the most frequent cancer occurring in women. Ten percent of these cancers are considered hereditary. Among them, 30% are attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. Other genes of lower penetrance are also known, explaining together up to 40% of the hereditary risk of breast cancer. New techniques, such as next-generation sequencing, allow the simultaneous analysis of multiple genes in a cost-effective way. As a logical consequence, gene panel testing is entering clinical practice with the promise of personalized care. We however advocate that gene panel testing is not ready for non-specialist clinical use, as it generates many variants of unknown significance and includes more genes than are presently considered clinically useful. We hereby review the data for each gene that can change the risk management of patients carrying a pathogenic variant.

A familial heterozygous null mutation of MET in autism spectrum disorder.

Autism spectrum disorder (ASD) results from interactions of genetic and environmental factors. The MET proto-oncogene has been identified as a candidate gene for autism susceptibility, and is implicated in neurodevelopment and social brain circuitry. Here, we describe the first case of a familial mutation of MET, consisting of an interstitial genomic deletion removing exons 12 through 15, causing a frameshift and premature stop codon, with evidence of nonsense-mediated mRNA decay. On the other allele, patients carried the C allele of the MET promoter rs1858830 polymorphism, known to decrease MET expression and previously associated with autism susceptibility. The heterozygous mutation was associated with autism in one patient, and language and social impairment in a sibling. Our observations delineate the phenotypic spectrum associated with a clearly defined, very likely complete loss of function mutation of MET. Incomplete penetrance in this family was consistent with MET as a partial susceptibility gene for ASD. Implication of MET in normal and pathological brain development opens new perspectives for understanding the pathophysiology of autism and for eventual therapeutical clues.

Implementation of genomic arrays in prenatal diagnosis: the Belgian approach to meet the challenges.

After their successful introduction in postnatal testing, genome-wide arrays are now rapidly replacing conventional karyotyping in prenatal diagnostics. While previous studies have demonstrated the advantages of this method, we are confronted with difficulties regarding the technology and the ethical dilemmas inherent to genomic arrays. These include indication for testing, array design, interpretation of variants and how to deal with variants of unknown significance and incidental findings. The experiences with these issues reported in the literature are most often from single centres. Here, we report on a national consensus approach how microarray is implemented in all genetic centres in Belgium. These recommendations are subjected to constant re-evaluation based on our growing experience and can serve as a useful tool for those involved in prenatal diagnosis.

A family with a novel TSH receptor activating germline mutation (p.Ala485Val).

Autosomal dominant nonautoimmune hyperthyroidism (ADNAH) is caused by gain of function mutations in the TSH receptor (TSHr) gene and characterized by toxic thyroid hyperplasia with a variable age of onset in the absence of thyroid antibodies and clinical symptoms of autoimmune thyroid disease in at least two generations. We report here a Turkish family with a novel TSHr gene mutation with distinct features all consistent with ADNAH. Thyroid function tests of the proband were as follows: free T3: 13.1 pg/ml (N: 1.8-4.6); free T4: 5.1 ng/dl (N: 0.9-1.7); TSH: 0.01 microIU/ml (N: 0.2-4.2); and TSH receptor antibody: 2 IU/ml (N: 0-10). A heterozygous missense mutation in exon 10 of the TSHr gene (c.1454C>T) resulting in the substitution of valine for alanine at codon 485 (p.Ala485Val) was found in the father and his son and daughter. This mutation had arisen de novo in the father. Functional studies of the novel TSHr germline mutation demonstrated a higher constitutive activation of adenyl cyclase than wild type without any effect on phospholipase C activity. In conclusion, our data indicate that gain of function germline mutations in the TSHr gene should be investigated in families with members suffering from thyrotoxicosis and progressive growth of goiter, but without clinical and biochemical evidence of autoimmune thyroid disease. In addition, patients harboring the same mutation of the TSHr gene may show wide phenotypic variability with respect to the age at onset, and severity of hyperthyroidism and thyroid growth.

Identification of the first germline mutation in the extracellular domain of the follitropin receptor responsible for spontaneous ovarian hyperstimulation syndrome.

The receptors for follitropin (FSHR), thyrotropin (TSHR), and lutropin/chorionic gonadotropin (LHCGR) are the members of the glycoprotein hormone (GPH) receptors (GPHR) family. They present a bipartite structure with a large extracellular amino-terminal domain (ECD), responsible for high-affinity hormone binding, and a carboxyl-terminal serpentine region, implicated in transduction of the activation signal. Spontaneous ovarian hyperstimulation syndrome (sOHSS) is a rare genetic condition in which human chorionic gonadotropin (hCG) promiscuously stimulates the FSHR during the first trimester of pregnancy. Surprisingly, germline FSHR mutations responsible for the disease have so far been found only in the transmembrane helices of the serpentine region of the FSHR, outside the hormone binding domain. When tested functionally, all mutants were abnormally sensitive to both hCG and thyrotropin (TSH) while displaying constitutive activity. This loss of ligand specificity was attributed to the lowering of an intramolecular barrier of activation rather than to an increase of binding affinity. Here we report the first germline mutation responsible for sOHSS (c.383C>A, p.Ser128Tyr), located in the ECD of the FSHR. Contrary to the mutations described previously, the p.Ser128Tyr FSHR mutant displayed increase in affinity and sensitivity toward hCG and did not show any constitutive activity, nor promiscuous activation by TSH. Thus, sOHSS can be achieved from different molecular mechanisms involving each functional domains of the FSHR. Based on the structure of the FSHR/FSH complex and site-directed mutagenesis studies, we provide robust molecular models for the GPH/GPHR complexes and we propose a molecular explanation to the binding characteristics of the p.Ser128Tyr mutant.

Understanding ovarian hyperstimulation syndrome.

The ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation treatments. Severe forms are characterized by a massive ovarian enlargement with the formation of multiple ovarian cysts associated with extravascular fluid shifts resulting in the development of ascites, pleural and/or pericardial effusion. The pathophysiology of the syndrome has not been completely elucidated yet. The vascular fluid leakage is thought to result from an increased capillary permeability of mesothelial surfaces under the action of one or several vasoactive ovarian factor(s) produced by the multiple corpora lutea. The paper focuses on the recent identification of mutations in the FSH receptor gene that display an increased sensitivity to hCG and are responsible for the development of spontaneous OHSS occurring during pregnancy. These findings have shed light for the first time on the molecular basis of the pathophysiology of the spontaneous form of the syndrome. As spontaneous and iatrogenic OHSS share similar pathophysiological sequences including massive recruitment and growth of ovarian follicles, extensive luteinization provoked by hCG, and oversecretion of vasogenic molecules by the corpora lutea, they have also opened new research perspectives for the understanding of the much more frequent iatrogenic OHSS.

Evaluation of a new generation of plastic evacuated blood-collection tubes in clinical chemistry, therapeutic drug monitoring, hormone and trace metal analysis.

Polyethylene terephthalate (PET) tubes have several advantages over glass tubes: they are unbreakable, lighter and more easily disposed of. Despite a steady increase in their use and an expansion of the range of available tubes, few studies validating their use have been published in the literature. This paper describes the various studies that have been performed to compare VENOJECT glass, VENOSAFE PET and VENOSAFE PET/heparin tubes for the assay of a panel of analytes in routine clinical chemistry, immunochemistry, hormone and tumor marker analysis and trace metal determination. These studies demonstrate that VENOSAFE PET tubes are a suitable alternative to glass tubes.