Beyond MEN1, when to think about MEN4? Retrospective study on 5600 patients in the French population & literature review.

CONTEXT: Germline CDKN1B variants predispose patients to multiple endocrine neoplasia type 4 (MEN4), a rare MEN1-like syndrome, with <100 reported cases since its discovery in 2006. Although CDKN1B mutations are frequently suggested to explain cases of genetically-negative MEN1, the prevalence and phenotype of MEN4 patients is poorly known, and genetic counseling is unclear. OBJECTIVE: To evaluate the prevalence of MEN4 in MEN1-suspected patients and characterize the phenotype of MEN4 patients. DESIGN: Retrospective observational nationwide study. Narrative review of literature and variant class reassessment. PATIENTS: We included all adult patients with class 3/4/5 CDKN1B variants identified by the laboratories from the French TENGEN network between 2015 and 2022 through germline genetic testing for MEN1 suspicion. After class reassessment, we compared the phenotype of symptomatic patients with class 4/5 CDKN1B variants, i.e. with genetically-confirmed MEN4 diagnosis, in our series and in literature with 66 matched MEN1 patients from the UMD-MEN1 database. RESULTS: From 5600 MEN1-suspected patients analyzed, four patients with class 4/5 CDKN1B variant were found (0.07%). They presented with multiple duodenal NET, PHPT and adrenal nodule, isolated PHPT, PHPT and pNET. We listed 29 patients with CDKN1B class 4/5 variants from literature. Compared to matched MEN1 patients, MEN4 patients presented lower NET incidence and older age at PHPT diagnosis. CONCLUSION: The prevalence of MEN4 is low. PHPT and PA represent the main associated lesions, NETs are rare. Our results suggest a milder and later phenotype than in MEN1. Our observations will help to improve genetic counseling and management of MEN4 families.

An unusual ectopic thyroid tissue location & review of literature.

Ectopic thyroid is well-known pathology for several decades. Many locations have been described. Nowadays, only three cases of skull base location have been described in literature. In our case, we reported a single case of a 39-year-old male presenting with a supra-sellar and suprachiasmatic location. He presented no clinical symptoms, no endocrine alteration on biology and no skull base invasion which make this case unique compared to actual literature. Such diagnosis should always be an exclusion diagnosis as secondary neoplastic should be first ruled out.

Pituitary adenoma & nuclear medicine: Recent outcomes and ongoing developments.

In order to explore pituitary adenoma (PA), magnetic resonance imaging (MRI) remains the cornerstone. However, there are some limitations and MRI can be non-conclusive. The development of additional imaging modalities like nuclear medicine explorations may help to confirm PA diagnosis, guide management and follow up. Nuclear medicine uses radiopharmaceuticals for imaging with single photon emission computed tomography (SPECT), or positron emission tomography (PET), coupled to CT scan. Radiopharmaceuticals products target specific cellular elements which allow to explore several biological pathways. Nuclear medicine may also be used for therapeutic purposes and recent developments of approach based on Peptide Receptor Radionuclide Therapy (PRRT) for treatment of aggressive PA and pituitary carcinoma will be reviewed. Several radiotracers have been studied in the context of PA, and the aim of this paper is to discuss their respective performances and clinical interest.

Gamma Knife surgery for recurrent or persistent Cushing disease: long-term results and evaluation of biological effective dose in a series of 26 patients.

INTRODUCTION: Here we report long-term results after stereotactic radiosurgery (SRS) with Gamma Knife (GKRS) for Cushing disease. We further evaluated the potential role of the biological effective dose (BED) in the cure of this disease. METHODS: A retrospective review of a prospectively collected database (n = 26) was undertaken at Lille University Hospital, France. The mean follow-up period was 66 months (median 80, range 19–108). The mean marginal prescribed dose was 28.5 Gy (median 27.5, range 24–35) and the mean BED was 208.5 Gy2.47 (median 228.1, range 160–248). We divided patients with endocrine remission into a high BED group (160–228 Gy2.47, n = 6) and a low BED group (228–248 Gy2.47, n = 12). RESULTS: Eighteen (69.2%) patients had endocrine remission in the absence of any pharmacological therapy after a mean of 36 months (median 24, range 6–98). The actuarial probability of endocrine remission was 59% at 3 years and 77.6% at 7 years, which remained stable up to 10 years. There was a tendency to a higher overall probability of biological remission associated with higher BED values (77% versus 66% at last follow-up), although this did not reach statistical significance. Of note, the numbers of patients reflecting this actuarial probability at 12, 24, 36, 51 and 96 months were 21, 15, 11, 7 and 3, respectively. Tumour control was achieved in all cases (mean decrease in size for patients experiencing one was 29.4%, range 0–100%). Seven patients developed new pituitary insufficiency after GKRS. CONCLUSONS: Gamma Knife radiosurgery offers high rates of tumour control and endocrine remission on a long-term basis for ACTH-secreting pituitary adenomas. In this small series, higher BED values appeared to be associated with better endocrine remission rates. Owing to the limited sample size, such results should be validated in a larger cohort.

Pegvisomant treatment in acromegaly in clinical practice: Final results of the French ACROSTUDY (312 patients).

OBJECTIVE: We report the final analysis of the French ACROSTUDY, using data revised and enriched since the 2013 interim analysis. Our objective was to validate the use of pegvisomant (PEGV) in the treatment of acromegaly and to determine efficacy and safety. PATIENTS AND METHODS: Patients with acromegaly treated with PEGV and followed up for at least 5 years were included. Eighty-eight investigators from 62 clinical centers in France included patients from April 2007 to April 2014. PEGV dose and administration frequency were determined by the physicians, based on their clinical evaluation and local habits. No additional examinations beyond those performed in normal follow-up were required. Minimum recommended follow-up included check-ups at treatment initiation, 6 months, 12 months and then annually. RESULTS: In total, 312 patients were enrolled. Mean age was 46.1±14.3 years at introduction of PEGV. Median PEGV treatment duration was 6.3 years and median follow-up was 5.6 years. Median dose at initiation was 10mg/day. The percentages of patients with IGF-1 ≤ ULN (upper limit of normal) were 10% (n=300) at baseline, 54% at 6 months (n=278), and 61.7% (n=253) at 2 years, then stabilizing at 64.4% (n=180) at 5 years. Mean PEGV dose was 17.4±11.7mg in patients with controlled disease versus 21.1±17.3mg in those without control at 5 years. At 5 years, 21.8% of patients (54/248) were receiving >30mg PEGV per day. In patients with at least one pituitary imaging procedure during the 5-year follow-up (n=292), the most recent image showed stable tumor volume in 212 subjects (72.6%), increased volume in 13 (4.5%), and decreased volume in 30 (10.3%). No PEGV treatments were permanently discontinued due to transaminase elevation. There were no cases of liver failure. CONCLUSION: The French ACROSTUDY showed normalization of IGF-1 levels in 64.4% of a real-life cohort of patients, mostly with uncontrolled disease despite multiple prior therapies. Long-term follow-up showed a sustained effectiveness and good long-term safety.

Pegvisomant in combination or pegvisomant alone after failure of somatostatin analogs in acromegaly patients: an observational French ACROSTUDY cohort study.

OBJECTIVE: After surgery, when somatostatin analogs (SAs) do not normalise IGF-I, pegvisomant (PEG) is indicated. Our aim was to define the medical reasons for the treatment of patients with PEG as monotherapy (M) or combined with SA, either as primary bitherapy, PB (PEG is secondarily introduced after SA) or as secondary bitherapy, SB (SAs secondarily introduced after PEG). METHODS: We retrospectively analysed French data from ACROSTUDY. RESULTS: 167, 88 and 57 patients were treated with M, PB or SB, respectively, during a median time of 80, 42 and 70 months. The median PEG dose was respectively 15, 10 and 20 mg. Before PEG, the mean IGF-I level did not differ between M and PB but the proportion of patients with suprasellar tumour extension was higher in PB group (67.5% vs. 44.4%, P = 0.022). SB regimen was used preferentially in patients with tumour increase and IGF-I level difficult to normalise under PEG. In both secondary regimens, the decrease of the frequency of PEG's injections, compared to monotherapy was confirmed. However, the mean weekly dose of PEG between M and PB remained the same. CONCLUSIONS: The medical rationale for continuing SAs rather than switching to PEG alone in patients who do not normalise IGF-I under SAs was a tumour concern with suprasellar extension and tumour shrinkage under SA. A potential explanation for introducing SA in association with PEG appears to be a tumour enlargement and difficulties to normalise IGF-I levels under PEG given alone. In both regimens, the prospect of lowering PEG injection frequency favoured the choice.

Gamma Knife radiosurgery for acromegaly: Evaluating the role of the biological effective dose associated with endocrine remission in a series of 42 consecutive cases.

INTRODUCTION: Stereotactic radiosurgery (SRS) is a valuable treatment option for persistent and/or recurrent acromegaly secondary to growth hormone (GH) secreting pituitary adenoma (PA). Here, we assess the role of biological effective dose (BED) received by PA treated with SRS in relation with endocrine remission. METHODS: Forty-two patients (minimum 6 months follow-up) were included. Mean marginal dose was 27.7 (median 28, 20-35), and mean BED received by tumour was 193.1 Gy2.47 (median 199.7, 64.1-237.1). Based on the median values, we divided the patients in high tumour BED group (H-BEDtm, 199.7-237.1 Gy2.47, n = 12) and low BED one (L- BEDtm, 64.1-199.7 Gy2.47 , n = 10). The two groups did not differ by pretherapeutic IGF-1 levels (p = .1) or by the prescribed dose (p = .6). RESULTS: Mean follow-up period was 62.5 months (median 60.5, 9-127). Probability of IGF-1 normalization was 65% at 3 years and 72.4% at 4 years, remaining stable until last follow-up. Twenty-two (52.4%) patients had complete endocrine remission in absence of any Somatostatin analogues. Actuarial rates were 33% at 3 years and 57.4% at 7 years, further remaining stable during follow-up course. In univariate analysis, only statistically significant parameter was pretherapeutic serum IGF-1 and IGF-1 index (p = .01). Five patients (5/26, 19.3%) without previous hypopituitarism developed new pituitary insufficiency. H-BEDtm was associated with higher rates of endocrine remission compared with L-BEDtm, with actuarial probability of 70.2% versus 48.2% at 9 years, although this did not reach statistical significance (p > .05). CONCLUSION: Our study confirms that SRS by Gamma Knife is safe and effective for GH-secreting PA. Pretherapeutic serum levels of IGF-1 were only statistically significant parameter for endocrine remission.

Clinical lessons learned in constitutional hypopituitarism from two decades of experience in a large international cohort.

CONTEXT: The international GENHYPOPIT network collects phenotypical data and screens genetic causes of non-acquired hypopituitarism. AIMS: To describe main phenotype patterns and their evolution through life. DESIGN: Patients were screened according to their phenotype for coding sequence variations in 8 genes: HESX1, LHX3, LHX4, PROP1, POU1F1, TBX19, OTX2 and PROKR2. RESULTS: Among 1213 patients (1143 index cases), the age of diagnosis of hypopituitarism was congenital (24%), in childhood (28%), at puberty (32%), in adulthood (7.2%) or not available (8.8%). Noteworthy, pituitary hormonal deficiencies kept on evolving during adulthood in 49 of patients. Growth Hormone deficiency (GHD) affected 85.8% of patients and was often the first diagnosed deficiency. AdrenoCorticoTropic Hormone deficiency rarely preceded GHD, but usually followed it by over 10 years. Pituitary Magnetic Resonance Imaging (MRI) abnormalities were common (79.7%), with 39.4% pituitary stalk interruption syndrome (PSIS). The most frequently associated extrapituitary malformations were ophthalmological abnormalities (16.1%). Prevalence of identified mutations was 7.3% of index cases (84/1143) and 29.5% in familial cases (n = 146). Genetic analysis in 449 patients without extrapituitary phenotype revealed 36 PROP1, 2 POU1F1 and 17 TBX19 mutations. CONCLUSION: This large international cohort highlights atypical phenotypic presentation of constitutional hypopituitarism, such as post pubertal presentation or adult progression of hormonal deficiencies. These results justify long-term follow-up, and the need for systematic evaluation of associated abnormalities. Genetic defects were rarely identified, mainly PROP1 mutations in pure endocrine phenotypes.

[The mouth of patients with acromegaly]. / La bouche de l'acromégale.

Orofacial changes are frequent in acromegaly. Their evolution is slowly progressive. The lips (everted and thickened), the mandibular morphology (prognathism), the tongue (macroglossia), the soft palate and the uvula (increased and thickened), the parodontis (gingival hyperplasia, paradontitis), the teeth (increased interdental spaces, hypercementosis, increased dental mobility, multiple tooth loss) are concerned. Functional consequences are significant (obstructive sleep apnea syndrome, malocclusion, pain of the oral maxillofacial area, decrease of the quality of life). They are rarely noticed as the first symptoms of the disease and rarely responsible for the diagnosis of acromegaly because of a progressive development over a long period of time, and because of the low prevalence of the disease which can be unknown by dentists and dental surgeons. When patients are cured or well-controlled, abnormalities of soft tissues improve but are not always completely reversible and bone enlargement remain unchanged. If any corrective surgical procedures are to be performed, this should be carried out only after normalization of GH and IGF I levels.

Changes in the management and comorbidities of acromegaly over three decades: the French Acromegaly Registry.

CONTEXT: Acromegaly is a rare disease associated with chronic multisystem complications. National registries have been created in several countries. DESIGN: The French Registry contains data on acromegaly epidemiology, management and comorbidities recorded over more than three decades, retrospectively until 1999 and prospectively from 1999 to 2012. RESULTS: Data could be analyzed for 999 of the 1034 patients included in the registry (46% males). Disease control, defined as IGF-I normalization (adjusted for age and sex), was achieved in 75% of patients at the last follow-up visit. Half the patients with uncontrolled disease had IGF-I levels below 1.5 times the upper limit of normal (ULN). The proportion of patients with surgically cured disease did not change markedly over time, whereas the proportion of patients with uncontrolled disease fell and the proportion of patients with medically controlled disease rose. Cardiovascular, metabolic, respiratory and rheumatologic comorbidities and their outcomes were recorded for most patients, and no noteworthy overall deterioration was noted over time. Cancer occurred in 10% of patients, for a standardized incidence ratio of 1.34 (95% CI: 0.94-1.87) in men and 1.24 (0.77-1.73) in women. Forty-one patients died during follow-up, for a standardized mortality ratio of 1.05 (0.70-1.42). Most deaths were due to cancer. CONCLUSIONS: The majority of patients with acromegaly now have successful disease control thanks to the multistep management. The incidence of comorbidities following diagnosis of acromegaly is very low. Life expectancy is now close to that of the general population, probably owing to better management of the GH/IGF-I excess and comorbidities.

Post-surgical management of non-functioning pituitary adenoma.

Post-surgical surveillance of non-functioning pituitary adenoma (NFPA) is based on magnetic resonance imaging (MRI) at 3 or 6 months then 1 year. When there is no adenomatous residue, annual surveillance is recommended for 5 years and then at 7, 10 and 15 years. In case of residue or doubtful MRI, prolonged annual surveillance monitors any progression. Reintervention is indicated if complete residue resection is feasible, or for symptomatic optic pathway compression, to create a safety margin between the tumor and the optic pathways ahead of complementary radiation therapy (RT), or in case of post-RT progression. In case of residue, unless the tumor displays elevated growth potential, it is usually recommended to postpone RT until progression is manifest, as efficacy is comparable whether treatment is immediate or postponed. The efficacy of the various RT techniques in terms of tumor volume control is likewise comparable. RT-induced hypopituitarism is frequent, whatever the technique. The choice thus depends basically on residue characteristics: size, delineation, and proximity to neighboring radiation-sensitive structures. Reduced rates of vascular complications and secondary brain tumor can be hoped for with one-dose or hypofractionated stereotactic RT, but there has been insufficient follow-up to provide evidence. Somatostatin analogs and dopaminergic agonists have yet to demonstrate sufficient efficacy. Temozolomide is an option in aggressive NFPA resistant to surgery and RT.

Management of clinically non-functioning pituitary adenoma.

Clinically NFPA is currently the preferred term for designing all the pituitary adenomas which are not hormonally active (in other words, not associated with clinical syndromes such as amenorrhea-galactorrhea in the context of prolactinomas, acromegaly, Cushing's disease or hyperthyroidism secondary to TSH-secreting adenomas). They account for 15-30% of pituitary adenomas. Diagnosis is usually made either in the context of mass effect due to a macroadenoma or, increasingly, fortuitously during imaging performed for some unrelated purpose; the latter case is known as pituitary incidentaloma. Surgery is indisputably indicated in case of tumoral syndrome, but other aspects of NFPA (hormonal work-up, follow-up, and especially postoperative follow-up, management of remnant or recurrence, the special case of incidentaloma, or apoplexy) remain controversial. The French Endocrinology Society (SFE) therefore set up an expert working group of endocrinologists, neurosurgeons, ophthalmologists, neuroradiologists, pathologists and biologists to draw up guidelines, at the 2012 SFE Congress in Toulouse, France. The present article presents the guidelines suggested by this group of French-speaking experts.

[Biomarkers in endocrinology]. / Biomarqueurs en endocrinologie.

TSH assay is the best parameter of the thyroid function. For adults, the normal interval of TSH concentrations range from 0.4 to 4 mUI/L. At the first trimester of pregnancy, TSH levels must be <2.5 mUI/L. Normal TSH levels increase with aging and obesity. The biological diagnosis relies on the identification of excessive secretion of the metanephrines which are more sensitive and specific than those of catecholamines. The concentrations of the free plasmatic metanephrines reflect the ongoing production of tumor. Plasma methoxytyramine is a novel biomarker of metastatic pheochromocytomas and paragangliomas. Serum IGF1 is a reliable measure of integrated GH concentrations in patients with acromegaly. Accurate assessment of IGF1 concentrations requires age and sex-matched control values. IGF1 is a sensitive tool for the diagnosis of acromegaly and efficacy of therapies. Serum AMH assay is more sensitive, more specific and more reproducible that counting of ovarian follicles by ultrasound. AMH level above 5 ng/mL (35 pmol/L) could be chosen as one of the diagnostic criteria for the polycystic ovary syndrome. In early or "incipiens" ovarian failure, the decrease in serum AMH is far ahead of the increase in FSH. Thyroglobulin (TG) and calcitonin (CT) are the sensitive and specific markers of respectively well-differentiated thyroid cancers of follicular origin and of the medullary thyroid cancers. The same tumour marker assay should be used to monitor a given patient. Chromogranin A (CgA) is a highly efficient biomarker for diagnosis and follow-up of various endocrine tumours. Despite the lack of international standardisation, some CgA assays are reliable.

A new prognostic clinicopathological classification of pituitary adenomas: a multicentric case-control study of 410 patients with 8 years post-operative follow-up.

Pituitary adenomas are currently classified by histological, immunocytochemical and numerous ultrastructural characteristics lacking unequivocal prognostic correlations. We investigated the prognostic value of a new clinicopathological classification with grades based on invasion and proliferation. This retrospective multicentric case-control study comprised 410 patients who had surgery for a pituitary tumour with long-term follow-up. Using pituitary magnetic resonance imaging for diagnosis of cavernous or sphenoid sinus invasion, immunocytochemistry, markers of the cell cycle (Ki-67, mitoses) and p53, tumours were classified according to size (micro, macro and giant), type (PRL, GH, FSH/LH, ACTH and TSH) and grade (grade 1a: non-invasive, 1b: non-invasive and proliferative, 2a: invasive, 2b: invasive and proliferative, and 3: metastatic). The association between patient status at 8-year follow-up and age, sex, and classification was evaluated by two multivariate analyses assessing disease- or recurrence/progression-free status. At 8 years after surgery, 195 patients were disease-free (controls) and 215 patients were not (cases). In 125 of the cases the tumours had recurred or progressed. Analyses of disease-free and recurrence/progression-free status revealed the significant prognostic value (p < 0.001; p < 0.05) of age, tumour type, and grade across all tumour types and for each tumour type. Invasive and proliferative tumours (grade 2b) had a poor prognosis with an increased probability of tumour persistence or progression of 25- or 12-fold, respectively, as compared to non-invasive tumours (grade 1a). This new, easy to use clinicopathological classification of pituitary endocrine tumours has demonstrated its prognostic worth by strongly predicting the probability of post-operative complete remission or tumour progression and so could help clinicians choose the best post-operative therapy.

Growth hormone replacement therapy in patients with traumatic brain injury.

In patients with severe traumatic brain injury (TBI), a growth hormone deficiency (GHD) is frequent and may contribute to the cognitive sequelae and reduction in quality of life (QoL). Recent studies have suggested that GH replacement therapy (GHRT) can improve processing speed and memory. The aim of the study was to analyze the efficacy of GHRT on cognition, activities of daily living (ADL), and QoL and the factors that predicted and contributed to these effects. We included patients at least 1 year after their TBI and assessed pituitary functions (with stimulation tests), cognition (attention, memory, and executive function), participation in ADL and QoL. GHD was treated for at least 1 year in 23 patients, who were compared with 27 non-treated patients. Other deficiencies were also treated. Measurements were performed at baseline and 1 year later. An analysis of variance of the factors group and session (p ≤ 0.05) showed that most cognitive parameters had improved at 1 year (evidencing a session effect). A stronger effect of GHRT (i.e. a group x session interaction) was found for Rey Osterrieth complex figure recall and 2/6 domains in the QoL questionnaire ("personal" and "functional"). Trends (p ≤ 0.07) were also found for spatial orientation and immediate recall in the verbal memory test. Greatest improvements were associated with lower performance before treatment. The magnitude of the improvements in ADL and QoL was moderately correlated with the improvement in cognition. In conclusion, replacement therapy can improve cognition and QoL in patients with TBI who have GHD, especially in those with severe disabilities.

Genetic analysis in young patients with sporadic pituitary macroadenomas: besides AIP don't forget MEN1 genetic analysis.

CONTEXT: Germline mutations in the aryl hydrocarbon receptor interacting protein gene (AIP) have been identified in young patients (age ≤30 years old) with sporadic pituitary macroadenomas. Otherwise, there are few data concerning the prevalence of multiple endocrine neoplasia type 1 (MEN1) mutations in such a population. OBJECTIVE: We assessed the prevalence of both AIP and MEN1 genetic abnormalities (mutations and large gene deletions) in young patients (age ≤30 years old) diagnosed with sporadic and isolated macroadenoma, without hypercalcemia and/or MEN1-associated lesions. DESIGN: The entire coding sequences of AIP and MEN1 were screened for mutations. In cases of negative sequencing screening, multiplex ligation-dependent probe amplification was performed for the detection of large genetic deletions. PATIENTS AND SETTINGS: One hundred and seventy-four patients from endocrinology departments of 15 French University Hospital Centers were eligible for this study. RESULTS: Twenty-one out of 174 (12%) patients had AIP (n=15, 8.6%) or MEN1 (n=6, 3.4%) mutations. In pediatric patients (age ≤18 years old), AIP/MEN1 mutation frequency reached nearly 22% (n=10/46). AIPmut and MEN1mut were identified in 8/79 (10.1%) and 1/79 (1.2%) somatotropinoma patients respectively; they each accounted for 4/74 (5.4%) prolactinoma (PRL) patients with mutations. Half of those patients (n=3/6) with gigantism displayed mutations in AIP. Interestingly, 4/12 (33%) patients with non-secreting adenomas bore either AIP or MEN1 mutations, whereas none of the eight corticotroph adenomas or the single thyrotropinoma case had mutations. No large gene deletions were observed in sequencing-negative patients. CONCLUSION: Mutations in MEN1 can be of significance in young patients with sporadic isolated pituitary macroadenomas, particularly PRL, and together with AIP, we suggest genetic analysis of MEN1 in such a population.

Hyperandrogenism in adolescent girls.

When the symptoms of hyperandrogenism are mild in an adolescent girl, it is difficult to establish whether they are related to the physiological androgenization of puberty or to an incipient disease. The most frequent etiology is the polycystic ovary syndrome (PCOS). Its diagnosis is defined in adult women by precise criteria which need to be adapted to the particular case of the adolescent girl. Nonclassic adrenal hyperplasia is the main differential diagnosis to PCOS. Although much less frequent, other etiologies of peripubertal hyperandrogenism such as adrenal or ovarian tumors or Cushing's disease must be considered by analyzing the rapidity of installation and intensity of hyperandrogenic symptoms and by a systematic hormonal work-up. In addition to etiologic measures, the treatment consists of administration of an anti-androgen, mainly cyproterone acetate. In the case of PCOS, the patient needs to be educated regarding the metabolic risks.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Cyclin-dependent kinase inhibitor 1B (CDKN1B) gene variants in AIP mutation-negative familial isolated pituitary adenoma kindreds.

Familial isolated pituitary adenoma (FIPA) occurs in families and is unrelated to multiple endocrine neoplasia type 1 and Carney complex. Mutations in AIP account only for 15-25% of FIPA families. CDKN1B mutations cause MEN4 in which affected patients can suffer from pituitary adenomas. With this study, we wanted to assess whether mutations in CDKN1B occur among a large cohort of AIP mutation-negative FIPA kindreds. Eighty-eight AIP mutation-negative FIPA families were studied and 124 affected subjects underwent sequencing of CDKN1B. Functional analysis of putative CDKN1B mutations was performed using in silico and in vitro approaches. Germline CDKN1B analysis revealed two nucleotide changes: c.286A>C (p.K96Q) and c.356T>C (p.I119T). In vitro, the K96Q change decreased p27 affinity for Grb2 but did not segregate with pituitary adenoma in the FIPA kindred. The I119T substitution occurred in a female patient with acromegaly. p27(I119T) shows an abnormal migration pattern by SDS-PAGE. Three variants (p.S56T, p.T142T, and c.605+36C>T) are likely nonpathogenic because In vitro effects were not seen. In conclusion, two patients had germline sequence changes in CDKN1B, which led to functional alterations in the encoded p27 proteins in vitro. Such rare CDKN1B variants may contribute to the development of pituitary adenomas, but their low incidence and lack of clear segregation with affected patients make CDKN1B sequencing unlikely to be of use in routine genetic investigation of FIPA kindreds. However, further characterization of the role of CDKN1B in pituitary tumorigenesis in these and other cases could help clarify the clinicopathological profile of MEN4.