Taneli Raivio

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling.

Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone (GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome (IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releasing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embryonic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2 that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize to produce a more severe phenotype in IHH families than either alone. This genetic model could account for some phenotypic heterogeneity seen in GnRH deficiency.

Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone (GnRH) deficiency in the human presents either as normosmic idiopathic hypogonadotropic hypogonadism (nIHH) or with anosmia [Kallmann syndrome (KS)]. To date, several loci have been identified to cause these disorders, but only 30% of cases exhibit mutations in known genes. Recently, murine studies have demonstrated a critical role of the prokineticin pathway in olfactory bulb morphogenesis and GnRH secretion. Therefore, we hypothesize that mutations in prokineticin 2 (PROK2) underlie some cases of KS in humans and that animals deficient in Prok2 would be hypogonadotropic. One hundred IHH probands (50 nIHH and 50 KS) with no known mutations were examined for mutations in the PROK2 gene. Mutant PROK2s were examined in functional studies, and the reproductive phenotype of the Prok2−/− mice was also investigated. Two brothers with KS and their sister with nIHH harbored a homozygous deletion in the PROK2 gene (p.[I55fsX1]+[I55fsX1]). Another asymptomatic brother was heterozygous for the deletion, whereas both parents (deceased) had normal reproductive histories. The identified deletion results in a truncated PROK2 protein of 27 amino acids (rather than 81 in its mature form) that lacks bioactivity. In addition, Prok2−/− mice with olfactory bulb defects exhibited disrupted GnRH neuron migration, resulting in a dramatic decrease in GnRH neuron population in the hypothalamus as well as hypogonadotropic hypogonadism. Homozygous loss-of-function PROK2 mutations cause both KS and nIHH.

Comparison of solution-based exome capture methods for next generation sequencing

BackgroundTechniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison.ResultsWe developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays.ConclusionsLibraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons.

Expert consensus document: European Consensus Statement on congenital hypogonadotropic hypogonadism—pathogenesis, diagnosis and treatment

Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder caused by the deficient production, secretion or action of gonadotropin-releasing hormone (GnRH), which is the master hormone regulating the reproductive axis. CHH is clinically and genetically heterogeneous, with >25 different causal genes identified to date. Clinically, the disorder is characterized by an absence of puberty and infertility. The association of CHH with a defective sense of smell (anosmia or hyposmia), which is found in ∼50% of patients with CHH is termed Kallmann syndrome and results from incomplete embryonic migration of GnRH-synthesizing neurons. CHH can be challenging to diagnose, particularly when attempting to differentiate it from constitutional delay of puberty. A timely diagnosis and treatment to induce puberty can be beneficial for sexual, bone and metabolic health, and might help minimize some of the psychological effects of CHH. In most cases, fertility can be induced using specialized treatment regimens and several predictors of outcome have been identified. Patients typically require lifelong treatment, yet ∼10–20% of patients exhibit a spontaneous recovery of reproductive function. This Consensus Statement summarizes approaches for the diagnosis and treatment of CHH and discusses important unanswered questions in the field.

Fecal calprotectin remains high during glucocorticoid therapy in children with inflammatory bowel disease

Objective. Fecal calprotectin is a promising marker for the assessment of gastrointestinal inflammation. Fecal calprotectin levels were followed-up in children with inflammatory bowel disease (IBD) who were introduced to glucocorticoid therapy. The aim of this study was to assess whether the changes in fecal calprotectin levels reflect therapeutic responses. Material and methods. Fecal calprotectin was measured by enzyme immunoassay in 57 children (mean age 9.8 years, range 0.9–18 years) who underwent colonoscopies (IBD n=31, non-IBD disease n=13, normal n=13) and followed-up in 15 children (mean age 13 years, range 3.6–18 years) who were introduced to glucocorticoid therapy because of active IBD at 0, 2, and 4 weeks and at 4-week intervals until one month after discontinuation of the therapy. Results. Fecal calprotectin was <100 µg/g in 70% of the children with normal findings on colonoscopy or a non-IBD disease. Fecal calprotectin was >100 µg/g in all but one child with active IBD and in 13/15 of those children who were introduced to glucocorticoids by the clinicians. Fecal calprotectin values decreased within 4 weeks in line with clinical improvement in 7 children and normalized in 4/15 children during the follow-up. Fecal calprotectin increased in 5/8 of the non-steroid-dependent children after discontinuation of glucocorticoids. Conclusions. Fecal calprotectin is a sensitive marker for chronic colitis. In active disease treated with glucocorticoids, fecal calprotectin levels declined in line with the clinical improvement but seldom fell within the normal range, which suggests ongoing inflammation in a clinically silent disease. The measurement of fecal calprotectin may provide new tools for the assessment of the level of gut inflammation in children with chronic colitis in the follow-up of clinical responses.

Genetic Overlap in Kallmann Syndrome, Combined Pituitary Hormone Deficiency, and Septo-Optic Dysplasia

CONTEXT Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.

Are Adolescent Boys with Klinefelter Syndrome Androgen Deficient? A Longitudinal Study of Finnish 47,XXY Boys

Testosterone (T)-substitution therapy is widely used in adult patients with Klinefelter syndrome (KS) to prevent symptoms and sequels of androgen deficiency, but it is currently unknown if adolescent boys with KS benefit from early T therapy. To evaluate the optimal age to start T substitution, we searched for signs of androgen deficiency in pubertal boys with KS. 14 nonmosaic 47,XXY boys, aged 10–13.9 y, were followed up for 4–37 mo with staging of puberty and frequent reproductive hormone measurements. Furthermore, indices reflecting androgen action (serum SHBG, leptin, and prostate-specific antigen (PSA) levels) were studied. Both onset and progression of puberty according to Tanner stages were normal in boys with KS. Consistently, serum T concentrations increased as expected and remained normal throughout follow-up. Changes in the indices of androgen action (decreases in serum SHBG and leptin, and increase in serum PSA concentrations) occurred normally, except that average leptin levels were higher in the boys with KS (KS boys 11.8 ± 7.0 μg/L; controls 7.6 ± 4.7 μg/L; p = 0.033). Despite normal T concentrations, the KS boys displayed from the age of 13 y elevated serum FSH and LH levels, and exaggerated gonadotropin responses to gonadotropin-releasing hormone. These data do not demonstrate an unequivocal androgen deficiency in adolescent boys with KS that would necessitate androgen supplementation therapy during early puberty.

Incidence, Phenotypic Features and Molecular Genetics of Kallmann Syndrome in Finland

BackgroundKallmann syndrome (KS), comprised of congenital hypogonadotropic hypogonadism (HH) and anosmia, is a clinically and genetically heterogeneous disorder. Its exact incidence is currently unknown, and a mutation in one of the identified KS genes has only been found in ~30% of the patients.MethodsHerein, we investigated epidemiological, clinical, and genetic features of KS in Finland.ResultsThe minimal incidence estimate of KS in Finland was 1:48 000, with clear difference between males (1:30 000) and females (1:125 000) (p = 0.02). The reproductive phenotype of 30 probands (25 men; 5 women) ranged from severe HH to partial puberty. Comprehensive mutation analysis of all 7 known KS genes (KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, and WDR11) in these 30 well-phenotyped probands revealed mutations in KAL1 (3 men) and FGFR1 (all 5 women vs. 4/25 men), but not in other genes.ConclusionsOur results suggest that Finnish KS men harbor mutations in gene(s) yet-to-be discovered with sex-dependent penetrance of the disease phenotype. In addition, some KS patients without CHD7 mutations display CHARGE-syndrome associated phenotypic features (e.g. ear or eye anomalies), possibly implying that, in addition to CHD7, there may be other genes associated with phenotypes ranging from KS to CHARGE.

Liver Fat But Not Other Adiposity Measures Influence Circulating FGF21 Levels in Healthy Young Adult Twins

CONTEXT Emerging as an important metabolic regulator, fibroblast growth factor 21 (FGF21) has gained considerable interest in diabetes and obesity research. The circulating FGF21 concentration is fairly low in normal individuals, but elevated hormone levels may associate with obesity. The determining factors of FGF21 levels in humans are not clear. OBJECTIVE Our objective was to study the influence of genetic and acquired components to serum FGF21 variability in healthy young adult twins. DESIGN AND PARTICIPANTS Fasting serum FGF21, lipids, body fat, and oral glucose tolerance test were investigated in 46 monozygotic (MZ) and 75 dizygotic twin pairs aged 22.8-33.1 yr. Subcutaneous, intraabdominal, and liver fat content were measured by magnetic resonance imaging/spectroscopy in a subsample of 24 MZ pairs. RESULTS Genetic factors contributed moderately (heritability 40%) to circulating serum FGF21 levels. Subjects with high FGF21 concentrations (≥ 250 pg/ml, n = 30) had higher fasting triglycerides, insulin, homeostasis model assessment index, and area under the curve glucose and lower high-density lipoprotein cholesterol but similar measures of overall adiposity (body mass index, body fat percent) than subjects with lower FGF21 (<100 pg/ml, n = 148). Importantly, in the MZ subsample, higher liver fat but not sc or intraabdominal fat content was found in subjects with high FGF21. Furthermore, in analyses controlling for genetic/familial effects in twin pairs, within-pair differences in liver fat (MZ) and triglycerides (dizygotic pairs) were the major acquired factors that correlated with differences in FGF21 concentrations. CONCLUSIONS Genetic factors influence serum FGF21 levels. Of the acquired components, high liver fat and triglycerides rather than overall adiposity associate with high FGF21 levels.