SF1 and spleen development: new heterozygous mutation, literature review and consequences for NR5A1-mutated patient's management.

Steroidogenic factor 1 (encoded by SF1/NR5A1) is a transcription factor with multiple target genes involved in the development and function of multiple steroidogenic and non-steroidogenic tissues. NR5A1 mutations lead to several phenotypes, including sex reversal, spermatogenesis failure, premature ovarian failure and adrenocortical insufficiency. The implication of NR5A1 mutations in spleen development anomalies was recently highlighted. We provide new evidence of this involvement, describing a novel heterozygous non-sense NR5A1 mutation in a 46,XY-DSD with polysplenia female proband and her father, who had hypospadias and asplenia.

Endocrine and molecular investigations in a cohort of 25 adolescent males with prominent/persistent pubertal gynecomastia.

Pubertal gynecomastia is a common condition observed in up to 65% of adolescent males. It is usually idiopathic and tends to regress within 1-2 years. In this descriptive cross-sectional study, we investigated 25 adolescent males with prominent (>B3) and/or persistent (>2 years) pubertal gynecomastia (P/PPG) to determine whether a hormonal/genetic defect might underline this condition. Endocrine investigation revealed the absence of hormonal disturbance for 18 boys (72%). Three patients presented Klinefelter syndrome and three a partial androgen insensitivity syndrome (PAIS) as a result of p.Ala646Asp and p.Ala45Gly mutations of the androgen receptor gene. The last patient showed a 17α-hydroxylase/17,20-lyase deficiency as a result of a compound heterozygous mutation of the CYP17A1 gene leading to p.Pro35Thr(P35T) and p.Arg239Stop(R239X) in the P450c17 protein. Enzymatic activity was analyzed: the mutant protein bearing the premature stop codon R239X showed a complete loss of 17α-hydroxylase and 17,20-lyase activity. The mutant P35T seemed to retain 15-20% of 17α-hydroxylase and about 8-10% of 17,20-lyase activity. This work demonstrates that P/PPG had an endocrine/genetic cause in 28% of our cases. PAIS may be expressed only by isolated gynecomastia as well as by 17α-hydroxylase/17,20-lyase deficiency. Isolated P/PPG is not always a 'physiological' condition and should thus be investigated through adequate endocrine and genetic investigations, even though larger studies are needed to better determine the real prevalence of genetic defects in such patients.

Is Serum Serotonin Involved in the Bone Loss of Young Females with Anorexia Nervosa?

Recent experimental data suggest that circulating serotonin interacts with bone metabolism, although this is less clear in humans. This study investigated whether serum serotonin interferes with bone metabolism in young women with anorexia nervosa (AN), a clinical model of energy deprivation. Serum serotonin, markers of bone turnover [osteocalcin (OC), procollagen type I N-terminal propeptide (PINP), type I-C telopeptide breakdown products (CTX)], leptin, soluble leptin receptor (sOB-R), and insulin-like growth factor-1 (IGF-1) and its binding protein (IGFBP-3) were assessed. Whole body, spine, hip, and radius areal bone mineral density BMD (aBMD) were assessed by dual-energy X-ray absorptiometry in 21 patients with AN and 19 age-matched controls. Serum serotonin, leptin, IGF-1, IGFBP-3, OC, PINP, and aBMD at all sites, radius excepted, were significantly reduced in AN whereas CTX and sOB-R were increased compared with controls. Serum serotonin levels were positively correlated with weight, body mass index, whole body fat mass, leptin, and IGF-1, and negatively with CTX for the entire population. Low serum serotonin levels are observed in patients with AN. Although no direct link between low serum serotonin levels and bone mass was identified in these patients, the negative relationship between serotonin and markers of bone resorption found in all population nevertheless suggests the implication of serotonin in bone metabolism. Impact of low serum serotonin on bone in AN warrants further studies.

Evidence of a link between resting energy expenditure and bone remodelling, glucose homeostasis and adipokine variations in adolescent girls with anorexia nervosa.

UNLABELLED: Low bone mass is a consequence of anorexia nervosa (AN). This study assessed the effects of energy deficiency on various bone and hormonal parameters. The interrelationships between energy deficiency and bone remodelling, glucose homeostasis and adipokines underscore the importance of preventing energy deficiency to limit demineralisation and hormonal alterations in AN patients. INTRODUCTION: Low areal bone mineral density (aBMD) is a well-known consequence of AN. However, the impact of reduced energy expenditure on bone metabolism is unknown. This study assessed the effects of energy deficiency on bone remodelling and its potential interactions with glucose homeostasis and adipose tissue-derived hormones in AN, a clinical model for reduced energy expenditure. METHODS: Fifty women with AN and 50 age-matched controls (mean age 18.1 ± 2.7 and 18.0 ± 2.1 years, respectively) were enrolled. aBMD was determined with DXA. Resting energy expenditure (REEm), a marker of energy status, was indirectly assessed by calorimetry. Bone turnover markers, undercarboxylated osteocalcin (ucOC), parameters of glucose homeostasis, adipokines and growth factors were concomitantly evaluated. RESULTS: AN patients presented low aBMD at all bone sites. REEm, bone formation markers, ucOC, glucose, insulin, HOMA-IR, leptin and IGF-1 were significantly reduced, whereas the bone resorption marker, leptin receptor (sOB-R) and adiponectin were elevated in AN compared with CON. In AN patients, REEm was positively correlated with weight, BMI, whole body (WB) fat mass, WB fat-free soft tissue, markers of bone formation, glucose, insulin, HOMA-IR, leptin and IGF-1 and negatively correlated with the bone resorption marker and sOB-R. Biological parameters, aBMD excepted, appeared more affected by the weight variation in the last 6 months than by the disease duration. CONCLUSIONS: The strong interrelationships between REEm and bone remodelling, glucose homeostasis and adipokines underscore the importance of preventing energy deficiency to limit short- and long-term bone demineralisation and hormonal alterations in AN patients.

Polymorphisms of MAMLD1 gene in hypospadias.

PURPOSE: Mastermind-like domain containing 1 (MAMLD1) is a causative gene for the fetal development of male external genitalia. Almost 10% of patients with both severe and non-severe hypospadias exhibit mutations of MAMLD1. The aim of this work was to determine whether polymorphisms of MAMLD1 are a genetic risk factor for hypospadias. MATERIAL AND METHODS: This study included 150 hypospadias with a range of severities and 150 controls. Direct sequencing of the MAMLD1 coding exons and their flanking splice sites was performed. In silico secondary and tertiary structure prediction and accessibility of changed amino acids were evaluated using JPred, Netsurf and PHYRE software. Functional studies of the transactivation of haplotypes on Hes3 promoter were performed in vitro using cDNAs of missense variants of MAMLD1. RESULTS: The p.P286S polymorphism was identified in 17/150 patients and 12/150 controls (11.3% vs. 8.0%, p = 0.32). The p.N589S polymorphism was identified in 22/150 patients and 12/150 controls (14.6% vs. 8.0%, p = 0.068). The double polymorphism (S-S haplotype) was present in 16/150 patients and 6/150 controls (10.6% vs. 4.0%, p = 0.044, OR = 2.87, CI from 1.09 to 7.55). The association of polymorphisms consistently revealed a modification in the structure prediction or amino acid accessibility in all three in silico models. The P286S, N589S and P286S + N589S proteins did not exhibit reduced transactivating activity on Hes3 promoter. CONCLUSION: Polymorphisms of MAMLD1 gene are frequent in patients with hypospadias. Although no change in transactivation was noted on Hes3 promoter, the in silico studies and the significantly increased incidence of the S-S haplotype in hypospadiac patients raise the hypothesis of a particular susceptibility conferred by these variants.

Isolated 'idiopathic' micropenis: hidden genetic defects?

Micropenis is defined as a stretched penile length of less than 2-2.5SD for age. Aetiologies include hypogonadotropic hypogonadism, testicular dysgenesis, defects in testosterone synthesis, androgen resistance [5α-reductase (5αR) deficiency or partial androgen insensitivity] and other rare causes like growth hormone GH deficiency. Often, the cause remains unknown. The aim of this study was to determine whether isolated micropenis with normal plasma testosterone could hide a molecular defect in the androgen pathway. Twenty-six boys with isolated micropenis were included in this study. All of them had 46,XY karyotype, normal luteinizing hormone and follicle-stimulating hormone and a normal plasma testosterone response to human chorionic gonadotropin testing. Androgen receptor (AR), 5αR and steroidogenic factor 1 (SF1) genes were sequenced. A mutation in the AR gene was found in two patients, and a new mutation in the SF1 gene was found in one patient who was the only one to have a low level of inhibin B (InhB). This is the first report of isolated micropenis as a revealing symptom of AR and SF1 mutations. Anti-Mullerian hormone and InhB should thus be evaluated in patients with isolated micropenis, even when plasma testosterone is in the normal range. Detection of gene mutations is helpful for diagnosis, treatment and genetic counselling for probands.

In peripubertal girls, artistic gymnastics improves areal bone mineral density and femoral bone geometry without affecting serum OPG/RANKL levels.

UNLABELLED: Peripubertal artistic gymnasts display elevated areal bone mineral density at various bone sites, despite delayed menarche and a high frequency of menstrual disorders, factors that may compromise bone health. The concomitant improvement in femoral bone geometry and strength suggested that this type of physical activity might have favourable clinical impact. INTRODUCTION: The purpose of this study is to evaluate the effect of artistic gymnastics (GYM) on areal bone mineral density (aBMD), femoral bone geometry and bone markers and its relationship with the osteoprotegerin (OPG)/rank-ligand (RANKL) system in peripubertal girls. METHODS: Forty-six girls (age 10-17.2 years) were recruited for this study: 23 elite athletes in the GYM group (training 12-30 h/week, age at start of training 5.3 years) and 23 age-matched (± 6 months; leisure physical activity ≤ 3 h/week) controls (CON). The aBMD at whole body, total proximal femur, lumbar spine, mid-radius and skull was determined using dual-X-ray absorptiometry. Hip structural analysis (HSA software) was applied at the femur to evaluate cross-sectional area (CSA, cm(2)), cross-sectional moment of inertia (CSMI, cm(4)), and the section modulus (Z, cm(3)) and buckling ratio at neck, intertrochanteric region and shaft. Markers of bone turnover and OPG/RANKL levels were also analysed. RESULTS: GYM had higher (5.5-16.4%) non-adjusted aBMD and adjusted aBMD for age, fat-free soft tissue and fat mass at all bone sites, skull excepted and the difference increased with age. In the three femoral regions adjusted for body weight and height, CSA (12.5-18%), CSMI (14-18%), Z (15.5-18.6%) and mean cortical thickness (13.6-21%) were higher in GYM than CON, while the buckling ratio (21-27.1%) was lower. Bone markers decreased with age in both groups and GYM presented higher values than CON only in the postmenarchal period. A similar increase in RANKL with age without OPG variation was observed for both groups. CONCLUSION: GYM is associated not only with an increase in aBMD but also an improvement in bone geometry associated with an increase in bone remodelling. These adaptations seem to be independent of the OPG/RANKL system.

Hypospadias: interactions between environment and genetics.

Hypospadias is one of the most common congenital malformations. It is considered to be a mild form of the 46,XY disorders of sex development (DSD), but its precise etiology remains to be elucidated. Compromised androgen synthesis or effects can cause this frequent malformation, although the mutational analyses of the genes involved in androgen actions have identified abnormalities in only a very small portion of patients. The overwhelming majority of cases remain unexplained and hypospadias may be a highly heterogeneous condition subject to multiple genetic and environmental factors. We here review the recent advances in this field and discuss the potential interactions between the environment and genetics.

The IVS1-2A>G mutation in the SRD5A2 gene predominates in Cypriot patients with 5α reductase deficiency.

BACKGROUND: 5α steroid reductase deficiency (5αSRD) is an autosomal recessive enzymatic deficiency and mutations in the 5α steroid reductase type 2 gene (SRD5A2) result in male pseudohermaphrodism caused by decreased dihydrotestosterone (DHT) synthesis. AIM: To identify the specific mutations of the SRD5A2 gene in Cypriot patients with 5αSRD. SUBJECTS AND METHODS: Five unrelated patients with 46,XY karyotype were examined. Four of them were born with ambiguous genitalia and 1 patient, who was raised as girl, presented with primary amenorrhea. The hCG test was informative (elevated testosterone/DHT) of 5αSRD in 3 out of 4 subjects. Sequencing of the SRD5A2 gene was completed for all patients. Genomic DNA was also isolated from a total of 204 healthy unrelated Cypriot subjects. Screening for the IVS1-2A>G mutation was performed by using direct sequencing and restriction enzyme analysis. RESULTS: The IVS1-2A>G was identified in homozygosity in 3 patients and in a compound heterozygote state in the other 2 patients, in combination with p.P181L and p.R171S in exon 3, respectively. The carrier frequency in the Cypriot population for the IVS1-2A>G mutation was estimated to be 0.98% or 2 in 204. CONCLUSIONS: The same IVS1-2A>G mutation in the SRD5A2 gene seems to characterize all Cypriot patients with 5αSRD diagnosed so far. Furthermore this relatively rare genetic defect, which has only been reported previously in a single case in the Eastern Mediterranean region, is very likely to be the result of a founder effect.

Undervirilization in XY newborns may hide a 5α-reductase deficiency: report of three new SRD5A2 gene mutations.

The observation of ambiguous genitalia in the newborn signals a medical, surgical and psychological emergency. The most crucial decision will be the choice of sex assignment. Rapid and precise diagnosis is thus essential. In XY newborns with normal/high plasma testosterone (T), partial androgen insensitivity syndrome (PAIS) is usually the first diagnosis evoked, which implies an androgen receptor (AR) defect. The diagnosis of steroid-5-alpha-reductase deficiency is rarely considered by the paediatrician. We report three new SRD5A2 gene mutations in four newborns from France, Morocco and Turkey. The newborns presented with ambiguous genitalia and normal plasma T values and the initial diagnosis\PAIS. In all four cases, normal sequences of the complete AR gene excluded this diagnosis and raised the hypothesis of 5α-reductase deficiency. The entire coding region (5 exons) of the SRD5A2 gene was assessed by PCR and direct sequencing analysis. For patient 1, we identified a new homozygous 2bp deletion in exon 1 (c.122_123delAG). Patient 2 had a known homozygous mutation, p.G115D, in exon 2. New compound heterozygous mutations in exon 4 (p.A215V) and exon 5 (p.X255Q) were found in patient 3. Patient 4 presented a new substitution in exon 1 (p.S14R) associated with a known polymorphism (p.V89L). Our data confirm our previous experience and clearly demonstrate that a 5-α reductase defect should be considered in all XY newborns with ambiguous genitalia and normal plasma T secretion, whatever their geographic area or ethnic group; moreover, this defect was not linked to specific phenotype. Early molecular diagnosis is indispensable for the crucial decision of the newborn's sex of rearing.

Phenotype, hormonal profile and genotype of subjects with partial androgen insensitivity syndrome: report of a family with four adult males and one child with disorder of sexual differentiation.

There is little information on the molecular basis of intrafamilial and inter-familial phenotypic heterogeneity with the same androgen receptor (AR) mutation in patients with partial androgen insensitivity syndrome. A genetic analysis was performed in a large kindred with ambiguous genitalia and the genotype-phenotype correlations were analysed. The index case was brought for sex assignment. Family history revealed four other affected members who had hypospadias and varying degrees of virilisation. All the affected males had hemizygous mutations in the third exon of the AR gene (A596T). One was also found to have a heterozygous mutation in the fourth exon of the 5 alpha reductase type 2 gene (G196S). This affected male with double mutations was better virilised compared with the other affected members with a single mutation. The degree of virilisation correlated with serum testosterone levels. Gynaecomastia was not present in any of these subjects. It is concluded that the subject with dual gene defects also had higher levels of testosterone and pubertal virilsation. Testosterone levels possibly govern the degree of pubertal virilisation in subjects with A596T gene defects. It is not clear whether the better pubertal virilsation and higher testosterone are in any way causally related to the SRD5A2 gene defect.

Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2.

BACKGROUND: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF). RESULTS: We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient. CONCLUSIONS: Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.

[Juvenile granulosa-cell tumor: clinical and molecular expression]. / Tumeurs juvéniles de la granulosa: expression clinique et moléculaire.

Ovarian sex cord-stromal tumors are rare tumors that originate from the nongerminal cells of ovary. Two decades ago, the identification of juvenile granulosa-cell tumors (GCT), as a specific entity inside this group, allowed a better treatment of these tumors in children. However, little data have been reported on the natural course of the disease and reliable prognostic factors have not been yet defined. We here review the clinical and genetics aspects of granulosa tumors, based on a series of 40 children. This national collaborative study involved the French Society of Children Cancer and eight clinical departments of pediatric endocrinology. We found that early diagnosis of a tumor, revealed by clinical signs of hyperoestrogeny, is an important prognostic factor. The pathophysiology of these tumors is still debatable and several cellular- and molecular-abnormal signals could be implicated in their development. The role of growth factors and oncogenes through the signaling pathway of MAP kinase is still discussed. According to our data, FSH signaling-transduction pathway, such as a constitutionally activated Galphas, could also be implicated in the induction of granulosa cell proliferation and seems to modulate the invasiveness of the tumor. Last, we have described a low-expression pattern or an extinction of an ovarian-determination gene, FOXL2, which is related to a worse prognosis of this tumor.

Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm.

Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

Searching for somatic mutations in McCune-Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples.

BACKGROUND: Activating mutations of the Gsalpha gene (GNAS), which encodes for the alpha-subunit of the stimulatory G protein, have been identified in patients with McCune-Albright syndrome (MAS). Accuracy and sensitivity in the molecular diagnosis of MAS is mandatory for optimal therapeutic strategy and adapted follow-up, especially for incomplete clinical forms of MAS. To date, the highly sensitive nested PCR method with intermediary digestion by a restriction enzyme at the mutation site is one of the most widely used techniques. This study evaluated a new diagnostic method using a peptidic nucleic acid (PNA) and compared it with the nested PCR method. MATERIAL AND METHODS: One hundred and forty-eight DNA samples from eighty-eight patients presenting clinical symptoms compatible with MAS were included. The DNA samples were mainly obtained from peripheral blood, ovarian tissue or cyst liquid, and bone lesions. The nested PCR method required 4 days. PNA clamping required 1.5 days and utilized the higher thermal stability and specificity of PNA-DNA coupling to inhibit PCR product formation. Direct sequencing was subsequently performed in all cases. RESULTS: The sensitivity of mutation detection was 54% (n = 80) for nested PCR and 46.6% (n = 69) for PNA (P > 0.05). The 11 cases where PNA failed to detect the mutation were mainly incomplete and atypical clinical forms of MAS (n = 10/11). The cost per sample was 50 Euros for PNA clamping versus 136 Euros for nested PCR. CONCLUSION: PNA clamping is a rapid, reliable, and economical method to diagnose MAS. It should be the first-line diagnostic method, although negative results, especially for incomplete clinical forms of MAS, should be confirmed by nested PCR.

[The stakes in preventing opportunistic MAI infections in AIDS. From an interview with Dr. Patrick Philibert]. / Les enjeux d'une prophylaxie des infections opportunistes à MAC du SIDA. D'après une interview du Dr Patrick Phlibert.

Development of HIV infection is littered with many pathological episodes of which the physical and psychological repercussions on the patient are full of consequences. In this context, full co-operation between the hospital services and general practitioners is fundamental, from the moment of the announcement of seropositivity, to place under anti-retroviral treatment and to follow the pathological events. Opportunistic infections that threaten the life of the patient are severe because of the fall in the level of CD4 lymphocytes. Nowadays it is important to start prophylactic treatments and to make the patient understand the fight to follow these treatments that alone can help to maintain his autonomy and quality of life. The quality of the relationship established between the practitioner and patient is here very important. At the start of prophylactic treatment for several opportunistic infections in AIDS, such as pneumocystosis or toxoplasmosis, it is essential to have available an effective therapeutic strategy for infection by Mycobacterium avium complex (MAC). In effect, these serious infections following after the development of AIDS, graviously threaten the life of patients and their probability to survive. Today, practitioners have available rifabutine (Ansatipine) which is an efficacious treatment for MAC infections. It is sure that many patients will find benefit.

Diet-induced thermogenesis in HIV infection.

OBJECTIVE: To assess whether postprandial dietary thermogenesis contributes to weight loss during HIV infection. METHODS: The thermogenic response to a test meal (15 kcal/kg) was evaluated with indirect calorimetry in 16 HIV-infected patients in a stable condition and compared with a control group. Patients were compared according to AIDS (n = 8) or non-AIDS (n = 8) status and to body weight loss (WL; n = 9) or no loss (NL; n = 7). Indirect calorimetry was performed after fasting 6 h and during 5 h after the test meal. RESULTS: Maximum value of energy expenditure was reached later in the WL group than in the control and NL group (200 versus 30 min, respectively). Energy expenditure returned to the initial value 300 min after the test meal (last measurement) in the control group but remained elevated in the patient group. Energy expenditure after food intake was more elevated in HIV-infected patients than in controls, especially in patients with detectable clinical change in their nutritional status (0.96 versus 0.72 kcal/kg body weight). CONCLUSION: Both kinetics and quantitative aspect of dietary thermogenesis are modified during HIV infection and the different variations are dependent on the extent of body weight loss.