Kirsi Vaaralahti

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.

Reversible congenital hypogonadotropic hypogonadism in patients with CHD7, FGFR1 or GNRHR mutations.

Background Congenital hypogonadotropic hypogonadism (HH) is a rare cause for delayed or absent puberty. These patients may recover from HH spontaneously in adulthood. To date, it is not possible to predict who will undergo HH reversal later in life. Herein we investigated whether Finnish patients with reversal of congenital hypogonadotropic hypogonadism (HH) have common phenotypic or genotypic features. Methods and Findings Thirty-two male HH patients with anosmia/hyposmia (Kallmann Syndrome, KS; n = 26) or normal sense of smell (nHH; n = 6) were enrolled (age range, 18–61 yrs). The patients were clinically examined, and reversal of HH was assessed after treatment withdrawal. KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, KISS1R, KISS1, TAC3, TACR3, and LHβ were screened for mutations. Six HH patients (2 KS, 4 nHH) were verified to have reversal of HH. In the majority of cases, reversal occurred early in adulthood (median age, 23 yrs; range, 21–39 yrs). All had spontaneous testicular growth while on testosterone replacement therapy (TRT). One nHH subject was restarted on TRT due to a decline in serum T. Two reversal variants had a same GNRHR mutation (R262Q), which was accompanied by another GNRHR mutation (R139H or del309F). In addition, both of the KS patients had a mutation in CHD7 (p.Q51X) or FGFR1 (c.91+2T>A). Conclusions Considerable proportion of patients with HH (8% of KS probands) may recover in early adulthood. Spontaneous testicular enlargement during TRT was highly suggestive for reversal of HH. Those with the GNRHR mutation R262Q accompanied by another GNRHR mutation may be prone to reversal, although even patients with a truncating mutation in CHD7 or a splice-site mutation in FGFR1 can recover. We recommend that all adolescents and young adults with congenital HH should be informed on the possibility of reversal.

The role of gene defects underlying isolated hypogonadotropic hypogonadism in patients with constitutional delay of growth and puberty

Variation in FGFR1, GNRHR, TAC3, and TACR3 was evaluated in 146 Finnish subjects with constitutional delay of growth and puberty. Although one male subject carried a previously undescribed heterozygous deletion (Phe309del) in GNRHR, which segregated with delayed puberty in his family, mutations in the coding regions of FGFR1, GNRHR, TAC3, and TACR3 are not likely to underlie common constitutional delay of growth and puberty.

Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism

Background:Congenital hypogonadotropic hypogonadism (HH), a rare disorder characterized by absent, partial, or delayed puberty, can be caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (GnRH) neurons. SEMA3A was recently implicated in the etiology of the disorder, and Sema7A-deficient mice have a reduced number of GnRH neurons in their brains.Methods:SEMA3A and SEMA7A were screened by Sanger sequencing in altogether 50 Finnish HH patients (34 with Kallmann syndrome (KS; HH with hyposmia/anosmia) and 16 with normosmic HH (nHH)). In 20 patients, mutation(s) had already been found in genes known to be implicated in congenital HH.Results:Three heterozygous variants (c.458A>G (p.Asn153Ser), c.1253A>G (p.Asn418Ser), and c.1303G>A (p.Val435Ile)) were found in SEMA3A in three KS patients, two of which also had a mutation in FGFR1. Two rare heterozygous variants (c.442C>T (p.Arg148Trp) and c.1421G>A (p.Arg474Gln)) in SEMA7A were found in one male nHH patient with a previously identified KISS1R nonsense variant and one male KS patient with a previously identified mutation in KAL1, respectively.Conclusion:Our results suggest that heterozygous missense variants in SEMA3A and SEMA7A may modify the phenotype of KS but most likely are not alone sufficient to cause the disorder.

Bone mineral density, body composition and bone turnover in patients with congenital hypogonadotropic hypogonadism

Patients with congenital hypogonadotropic hypogonadism (HH) may have reduced peak bone mass in early adulthood, and increased risk for osteoporosis despite long-term hormonal replacement therapy (HRT). To investigate the relationship between HRT history and measures of bone health in patients with HH, we recruited 33 subjects (24 men, nine women; mean age 39.8 years, range: 24.0-69.1) with congenital HH (Kallmann syndrome or normosmic HH). They underwent clinical examination, were interviewed and medical charts were reviewed. Twenty-six subjects underwent dual-energy X-ray absorptiometry for evaluation of BMD of lumbar spine, hip, femoral neck and whole body; body composition and vertebral morphology were evaluated in 22 and 23 subjects, respectively. Circulating PINP, ICTP and sex hormone levels were measured. HRT history clearly associated to bone health: BMDs of lumbar spine, femoral neck, hip and whole body were lower in subjects (n = 9) who had had long (≥5 years) treatment pauses or low dose testosterone (T) treatment as compared to subjects without such history (n = 17; all p-values < 0.05). In addition, fat mass and body mass index (BMI) were significantly higher in men with deficient treatment history (median fat mass: 37.5 vs. 23.1%, p = 0.005; BMI: 32.6 vs. 25.2 kg/m(2), p < 0.05). Serum PINP correlated with ICTP (r(s) = 0.61; p < 0.005) in men, but these markers correlated neither with circulating T, nor with serum estradiol levels in women. In conclusion, patients with congenital HH require life-long follow-up to avoid inadequate HRT, long treatment pauses and further morbidity.

Circulating antimüllerian hormone levels in boys decline during early puberty and correlate with inhibin B

OBJECTIVE To investigate peripheral levels of inhibin B and antimüllerian hormone (AMH) in boys during peripuberty and in patients with congenital hypogonadotropic hypogonadism (HH). DESIGN Randomized, placebo-controlled trial (peripubertal boys); and cross-sectional clinical study (males with HH). SETTING University central hospital. PATIENT(S) Twenty-eight peripubertal boys with idiopathic short stature (ISS), 19 males with Kallmann syndrome. INTERVENTION(S) Letrozole (2.5 mg/day) or placebo in boys with ISS for 2 years. MAIN OUTCOME MEASURE(S) Longitudinal follow-up observation of serum AMH and its relationship with inhibin B during early puberty and the influence of high (letrozole-treated boys) and low (males with HH) gonadotropin exposure on circulating AMH. RESULT(S) In boys with ISS receiving placebo, the decrease in AMH levels and the increase in inhibin B levels were correlated. The serum AMH level had already declined before a clinically significant increase in testis volume or serum testosterone occurred. Letrozole did not appear to modulate the decline in AMH. The AMH levels were lower in boys and young adults with Kallmann syndrome and prepubertal testes (mean: 20.9 ± 4.7 ng/mL, n = 6) as compared with prepubertal ISS boys (102.3 ± 11.9 ng/mL). CONCLUSION(S) The gonadotropin-mediated early pubertal increase in inhibin B is tightly coupled to decrease in AMH levels and may reflect androgen-mediated differentiation of Sertoli cells. Profound gonadotropin deficiency is associated with low AMH levels, suggesting impaired development of the Sertoli cell population.

Decrease in circulating fibroblast growth factor 21 after an oral fat load is related to postprandial triglyceride-rich lipoproteins and liver fat

BACKGROUND Elevated levels of circulating fibroblast growth factor 21 (FGF21) are commonly encountered in type 2 diabetes, dyslipidemia, and non-alcoholic fatty liver disease, all of which share exaggerated postprandial lipemia as a common proatherogenic feature. How FGF21 responds to an oral fat load in man is unknown. METHODS We measured liver fat contents and subcutaneous and visceral fat volumes in 47 healthy subjects, who also underwent an oral fat load with measurements of plasma FGF21 and free fatty acid (FFA). Triglyceride (TG), apolipoprotein B-48 (apoB-48), and apoB-100 concentrations were measured in triglyceride-rich lipoprotein (TRL) fractions. RESULTS When compared with fasting levels, the concentration of FGF21 decreased significantly at 4 h (P < 0.05) and tended to return to fasting levels at 8 h after an oral fat load. Fasting and postprandial FGF21 correlated significantly with liver fat as well as with TRLs in the chylomicron and especially in very low-density lipoprotein 1 (VLDL1) and VLDL2 fractions representing remnant particles, but not with FFA. Subjects with increased liver fat (>5%, n = 12) showed impaired suppression of FGF21 at 4 h (P < 0.05) and at 8 h (P=0.01) and demonstrated higher postprandial TG area under the curve in plasma and TRL fractions (P ≤ 0.032) compared with those with normal liver fat (≤ 5%, n = 35). CONCLUSIONS We observed a significant decrease of FGF21 concentration after an oral fat load. Fasting and postprandial FGF21 levels were closely related to large VLDL and remnants, but not to plasma FFA. Our pilot findings suggest that the postprandial accumulation of TRL remnants and liver fat may modulate postprandial FGF21 levels.

De novo SOX10 nonsense mutation in a patient with Kallmann syndrome and hearing loss

K syndrome (KS; Mendelian Inheritance in Man (MIM)# 147950), a combination of congenital hypogonadotropic hypogonadism (MIM# 146110) and decreased/ absent sense of smell, results from disturbed migration of gonadotropin-releasing hormone neurons along the olfactory sensory neurons from the olfactory placode to the hypothalamus. The reproductive phenotype may vary from severe hypogonadism to reversal of hypogonadotropism later in life. Patients may also have additional nonreproductive features, which include cleft lip/palate, dental agenesis, syndactyly, limb anomalies, renal agenesis, scoliosis, mirror movements, and deafness (1). Very recently, Pingault et al. described a high prevalence of SOX10 mutations in patients with KS and deafness (2). SOX10 is a transcription factor involved in many cellular and developmental processes, especially in the development and differentiation of the neural crest (3). Mutations in SOX10 are known to cause Waardenburg syndrome (WS), which is a rare developmental disorder characterized by pigmentation abnormalities and deafness (specifically type 4C, which combines Waardenburg and Hirschsprung disease MIM# 613266; its neurological variant PCWH MIM#609136; and also WS type 2E MIM# 611584) (4–6). Involvement of SOX10 mutations in KS was suspected when a high frequency of olfactory bulb agenesis was found among patients with WS. Indeed, seven novel loss-of-function mutations (four missense mutations, one frameshift mutation, one splice-site mutation, and one mutation located in the initiation codon) in SOX10 were found among altogether 103 patients with KS (2). Notably, six of these seven mutation carriers had a hearing impairment suggesting that SOX10 mutations should be screened among patients with KS and deafness (2). We further investigated the role of SOX10 in KS by screening 20 patients with KS with special interest in four patients with hearing impairment. The clinical features of 18 Finnish male subjects with KS have been reported in Laitinen et al. (7). In addition, one female patient with KS with a history of spontaneous puberty followed by secondary amenorrhea, congenital anosmia, absent olfactory bulbs in the magnetic resonance imaging, and low estradiol (E2) in the setting of normal gonadotropin levels, thus representing mild gonadotropin-releasing hormone deficiency, was included. Three of the male subjects had hearing impairment. One had bilateral hearing impairment and other CHARGE syndrome (coloboma, heart defects, choanal atresia, retarded growth and development, genital abnormalities, and ear anomalies; MIM# 214800) associated features (cleft lip and palate, unilateral microphthalmia and coloboma, left facial nerve palsy, cup-shaped ears, upper body muscular atrophy, and hypoplastic semicircular canals), the other had bilateral sensorineural hearing loss, and the third had unilateral hearing loss. In addition, one patient had CHARGE syndrome-associated features without hearing impairment (cup-shaped ears and upper body muscular atrophy). The fourth patient with KS and hearing loss was from Estonia. His phenotypic features are described below. None of the subjects has been found to carry mutations in the seven genes known to underlie KS (KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11), and no intragenic deletions were found in CHD7 or FGFR1 multiplex ligationdependent probe amplification assays (MRC-Holland, Amsterdam, the Netherlands) as described in Laitinen et al. (7). This study was performed with appropriate permissions from the Ethics Committee (E7) of the Helsinki University Central Hospital and from each university hospital in Finland, and from the Ethics Review Committee on Human Research of the University of Tartu. All subjects obtained and signed the written consent. Genomic DNA from peripheral blood leukocytes of the subjects was extracted, and the coding exons and exon-intron boundaries of SOX10 (NM_006941.3) were PCR amplified and screened by direct sequencing. Primer sequences and PCR conditions are available on request. Mutation was confirmed from a second PCR product. One male patient with KS and hearing loss carried a heterozygous nonsense mutation c.184G>T (p.Glu62X) in the first coding exon of SOX10. This mutation was confirmed as de novo (absence of the mutation was verified by sequencing in both parents), and it was not present in 90 control individuals or reported in relevant databases (dbSNP:http://www.ncbi. nlm.nih.gov/projects/SNP/, 1000 Genomes Project:http:// www.1000genomes.org/, NHLBI Exome Variant Server: http:// evs.gs.washington.edu/EVS/, Leiden Open Variation Database: http://grenada.lumc.nl/LOVD2/WS/home.php?select_ db1⁄4SOX10). The Glu62X mutation most likely activates the De novo SOX10 nonsense mutation in a patient with Kallmann syndrome and hearing loss

PROKR2 mutations in autosomal recessive Kallmann syndrome

OBJECTIVE To investigate the inheritance pattern of two missense PROKR2 changes within a single family. DESIGN This is a descriptive study. SETTING Tertiary referral center. PATIENT(S) The proband and his brother, both with congenital hypogonadotropic hypogonadism and anosmia (Kallmann syndrome). INTERVENTION(S) Clinical and biochemical evaluation of Kallmann syndrome. Sequence analysis of the coding exons and exon-intron boundaries of KAL1, FGFR1, FGF8, PROK2, and PROKR2 from polymerase chain reaction (PCR)-amplified genomic DNA. Recombinant human FSH treatment of the proband. MAIN OUTCOME MEASURE(S) Phenotypic and genotypic features, and inhibin B response to recombinant human FSH. RESULT(S) The proband and his brother were homozygous for two variants in PROKR2; a novel mutation c.701G>A (p.G234D), and a polymorphism c.802C>T (p.R268C). Recombinant human FSH therapy of the proband increased serum inhibin B from <16 to 136 ng/L. The heterozygous parents were fertile and had six children. CONCLUSION(S) These findings are consistent with recessive mode of inheritance. PROKR2 signaling does not directly affect Sertoli cell function.

Isolated cryptorchidism: no evidence for involvement of genes underlying isolated hypogonadotropic hypogonadism.

Mutations in FGFR1, GNRHR, PROK2, PROKR2, TAC3, or TACR3 underlie isolated hypogonadotropic hypogonadism (IHH) with clinically variable phenotypes, and, by causing incomplete intrauterine activation of the hypothalamic-pituitary-gonadal axis, may lead to cryptorchidism. To investigate the role of defects in these genes in the etiology of isolated cryptorchidism, we screened coding exons and exon-intron boundaries of these genes in 54 boys or men from 46 families with a history of cryptorchidism. Control subjects (200) included 120 males. None of the patients carried mutation(s) in FGFR1, PROK2, PROKR2, TAC3 or TACR3. Two of the 46 index subjects with unilateral cryptorchidism were heterozygous carriers of a single GNRHR mutation (Q106R or R262Q), also present in male controls with a similar frequency (3/120; p=0.62). No homozygous or compound heterozygous GNRHR mutations were found. In conclusion, cryptorchidism is not commonly caused by defects in genes involved in IHH.