Molecular Genetics of Kallmann Syndrome and Constitutional delay of growth and puberty in Finland

Puberty is governed by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. GnRH neurons originate from the neural crest and from the olfactory placode. From the olfactory placode GnRH neurons migrate to the hypothalamus along the axons of developing olfactory nerves. Defects in GnRH neuron development, migration or in GnRH secretion or action cause congenital hypogonadotropic hypogonadism (HH), which is a rare developmental disorder characterised by delayed or absent puberty. If HH appears with defects in sense of smell, the condition is termed Kallmann syndrome (KS). Clinical and genetic features of KS and congenital HH with normal sense of smell (normosmic HH) are heterogeneous. Only for ~35% of congenital HH patients a molecular genetic diagnosis can be given. The most common cause of delayed puberty is constitutional delay of growth and puberty (CDGP). CDGP is a variant of the normal spectrum of pubertal timing and is characterized by first pubertal signs appearing at an age that is 2.0 standard deviations above the mean age for pubertal onset. The genetic background of CDGP is unknown. The aim of this thesis study was to characterize the molecular genetic features of KS patients in Finland. We also investigated whether mutations in known holoprosencephaly (HPE) or septo-optic dysplasia (SOD) genes could underlie some cases of KS. In addition, we investigated the role of congenital HH genes in CDGP. Thirty-four subjects with KS (6 females, 28 males) were screened for mutation(s) in genes involved in development and/or migration of GnRH neurons and in which mutations are known to cause KS: KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, and SOX10. The effects of FGFR1 missense mutations G48S, R209H, and E670A on receptor function were analysed in vitro. Patients remaining without identified molecular genetic cause in established KS genes were also screened for mutation in SOX2, SHH, SIX3, TGIF1, TDGF1, FOXH1, and GLI2, in which mutations are known to cause HPE or SOD (SOX2). Furthermore, GNRHR, FGFR1, TAC3, and TACR3 were screened in 146 subjects with CDGP (TAC3 and TACR3 in females only). Out of 34 KS patients, 15 got a molecular genetic diagnosis. Nine patients (5 females, 4 males) had an FGFR1 mutation, three males had a KAL1 mutation, one male of Iraqi origin carried a homozygous PROKR2 mutation, one male with CHARGE (coloboma, heart defects, choanal atresia, retarded growth and development, genital abnormalities, and ear anomalies) syndrome associated features had a CHD7 mutation, and one male with KS and deafness carried a de novo SOX10 mutation. FGFR1 missense mutants G48S and E670A displayed impaired mitogen-activated protein kinase signalling in vitro. One male KS patient carried heterozygous missense variants in GLI2 and in SIX3. One male subject with CDGP carried a previously undescribed heterozygous deletion in GNRHR, which segregated with delayed puberty in his family. In conclusion, KS is a male predominant condition. 44% of KS patients received a molecular genetic diagnosis. A clear difference was seen in the distribution of molecular genetic diagnoses in this study and in those reported previously, as the leading molecular genetic cause of KS, mutation in FGFR1, accounted for 26% of the cases, and mutations in PROK2 and PROKR2 were almost completely absent. Also, a significantly higher proportion of women (83%) carried an FGFR1 mutation compared with men (14%). Considerable genotypic and phenotypic overlap is seen between KS, Waardenburg syndrome and CHARGE syndrome. Therefore hearing impairment and/or ear anomalies in KS patient should be considered as an indication for both CHD7 and SOX10 molecular analyses and, in case of identified mutation the possibility of more severely affected future children should be taken under consideration. Mutations in known HPE genes are not a common cause for KS in Finland. Finally, mutations in FGFR1, GNRHR, TAC3 or TACR3 are not a common cause of CDGP.Synnynnäinen hypogonadotrooppinen hypogonadismi (HH) on harvinainen viivästyneen tai puuttuvan murrosikäkehityksen syy. Synnynnäinen HH aiheutuu sukupuolihormonien puutteesta, mikä on seurausta gonadotropiineja vapauttavan hormonin, GnRH:n, vajeesta. Synnynnäistä HH:ta sairastavat potilaat on perinteisesti luokiteltu kahteen ryhmään sen perusteella onko heillä HH:n lisäksi alentunut tai puuttuva hajuaisti (Kallmannin oireyhtymä, KS) vai normaali hajuaisti (normosminen HH, nHH). Synnynnäinen HH on sekä kliinisesti että geneettisesti erittäin heterogeeninen. Vaikka geenejä, joissa mutaatiot aiheuttavat synnynnäistä HH:ta on tähän mennessä tunnistettu jo useita, vain alle puolelle potilaista kyetään antamaan molekyyligeneettinen diagnoosi. Yleisin viivästyneen murrosikäkehityksen syy on konstitutionaalinen viivästynyt murrosikä (constitutional delay of growth and puberty, CDGP). CDGP on normaalin murrosiän ajoittumisen ääripää, ja sille on tyypillistä että ensimmäiset murrosiän merkit ilmaantuvat keskimääräistä myöhäisemmällä iällä. CDGP on ainakin osittain geneettisesti määritelty, mutta toistaiseksi geenit sen taustalla ovat vielä selvittämättä. Tässä väitöskirjatutkimuksessa saatiin uutta tietoa suomalaisten KS-potilaiden geneettisistä piirteistä. Tutkimuksessa selvitettiin myös, aiheuttavatko mutaatiot tunnetuissa holoprosenkefalia (HPE) -geeneissä KS:aa. Lisäksi tutkittiin aiheuttavatko mutaatiot GNRHR-, FGFR1-, TAC3- ja TACR3-geeneissä CDGP:tä. KS-potilaista 44% sai molekyyligeneettisen diagnoosin. Yleisin KS:n syy Suomessa oli mutaatio FGFR1-geenissä (26%), kun taas muualla maailmassa yleiset PROK2- ja PROKR2-mutaatiot puuttuivat miltei kokonaan. Huomattavasti suurempi osa naisista kantoi FGFR1-mutaatiota (83%) verrattuna miehiin (14%). Kolmella miehellä, joilla kaikilla oli vaikea ilmiasu, oli mutaatio KAL1-geenissä. Yhdellä miehellä, jolla oli KS:n lisäksi CHARGE-syndroomalle tyypillisiä piirteitä, oli mutaatio CHD7-geenissä ja yhdellä miehellä, jolla oli KS:n lisäksi kuulovamma, oli mutaatio SOX10-geenissä. Koska KS:n, CHARGE syndrooman ja Waardenburgin syndrooman ilmiasuissa ja genetiikassa on osittain samoja piirteitä, tulisi sekä CHD7- että SOX10-geenit seuloa kaikilta niiltä KS-potilailta, joilla on liitännäisoireena kuulovamma ja/tai korvan epämuodostuma. Mikäli näistä geenistä löytyy mutaatio, tulisi potilaiden tulevien lasten mahdollisesti vakavampi ilmiasu ottaa huomioon ja tarjota perinnöllisyysneuvontaa. Mutaatiot tunnetuissa HPE-geeneissä eivät ole yleinen KS:n syy Suomessa, ainoastaan yhdeltä miespotilaalta löydettiin harvinaiset variantit sekä GLI2- että SIX3-geeneistä. Yhdeltä CDGP potilaalta löydettiin aiemmin kuvaamaton mutaatio GNRHR-geenistä, mutta mutaatiot GNRHR-, FGFR1-, TAC3- ja TACR3-geeneissä eivät ole CDGP:n yleinen syy

Circulating miR-30b levels increase during male puberty

Objective: The role of miRNA as endocrine regulators is emerging, and microRNA mir-30b has been reported to repress Mkrn3. However, the expression of miR-30b during male puberty has not been studied. Design and methods: Circulating relative miR-30b expression was assessed in sera of 26 boys with constitutional delay of growth and puberty (CDGP), treated with low-dose testosterone (T) (n =11) or aromatase inhibitor letrozole (Lz) (n =15) for 6 months and followed up to 12 months (NCT01797718). The associations between the relative expression of miR-30b and hormonal markers of puberty were evaluated. Results: During the 12 months of the study, circulating miR-30b expression increased 2.4 +/- 2.5 (s.D.) fold (P = 0.008) in all boys, but this change did not correlate with corresponding changes in LH, testosterone, inhibin B, FSH, or testicular volume (P = 0.25-0.96). Lz-induced activation of the hypothalamic-pituitary-gonadal (HPG) axis was associated with more variable miR-30b responses at 3 months (P <0.05), whereas those treated with T exhibited significant changes in relative miR-30b levels in the course the study (P <0.01-0.05). Conclusions: Circulating miR-30b expression in boys with CDGP increases in the course of puberty, and appears to be related to the activity of the HPG axis.Peer reviewe

FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time-and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (PPeer reviewe

Kallmann syndrome in a patient with Weiss-Kruszka syndrome and a de novo deletion in 9q31.2

Patients with deletions on chromosome 9q31.2 may exhibit delayed puberty, craniofacial phenotype including cleft lip/palate, and olfactory bulb hypoplasia. We report a patient with congenital HH with anosmia (Kallmann syndrome, KS) and a de novo 2.38 Mb heterozygous deletion in 9q31.2. The deletion breakpoints (determined with wholegenome linked-read sequencing) were in the FKTN gene (9:108,331,353) and in a non-coding area (9:110,707,332) (hg19). The deletion encompassed six protein-coding genes (FKTN, ZNF462, TAL2, TMEM38B, RAD23B, and KLF4). ZNF462 haploinsufficiency was consistent with the patient's Weiss-Kruszka syndrome (craniofacial phenotype, developmental delay, and sensorineural hearing loss), but did not explain his KS. In further analyses, he did not carry rare sequence variants in 32 known KS genes in whole-exome sequencing and displayed no aberrant splicing of 15 KS genes that were expressed in peripheral blood leukocyte transcriptome. The deletion was 1.8 Mb upstream of a KS candidate gene locus (PALM2AKAP2) but did not suppress its expression. In conclusion, this is the first report of a patient with Weiss-Kruszka syndrome and KS. We suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features.Peer reviewe

Circulating Liver-enriched Antimicrobial Peptide-2 Decreases During Male Puberty

Context: Circulating levels of liver-enriched antimicrobial peptide 2 (LEAP2), a ghrelin receptor antagonist, decrease under caloric restriction and increase in obesity. The role of LEAP2 in male puberty, a phase with accelerated energy demand, is unclear. Objective: This work aimed to investigate whether circulating LEAP2 levels are downregulated in boys following the onset of puberty to respond to the energy need required for growth. Methods: We determined circulating LEAP2 levels in 28 boys with constitutional delay of growth and puberty (CDGP) who participated in a randomized controlled trial (NCT017977181, and were treated with letrozole (n = 15) or intramuscular low-dose testosterone (T) (n = 13) for 6 months. Blood sampling and dual-energy x-ray absorptiometry-measured body composition were performed at 0-, 6-, and 12-month visits. Results: Serum LEAP2 levels decreased statistically significantly during pubertal progression (0-6 months: mean decrease -4.3 (10.3) ng/mL, P = .036 and 0-12 months: -3.9 19.31 ng/mL, P = .033). Between 0 and 6 months, the changes in serum LEAP2 levels correlated positively with changes in percentage of body fat (r(s) = 0.48, P = .011), and negatively with growth velocity and estradiol levels (r(s) = -0.43, P = .022, r(s) = -0.55, P = .003, respectively). In the T group only, the changes in serum LEAP2 correlated negatively with changes in T and estradiol levels. Between 0 and 12 months, the change in LEAP2 levels correlated negatively with the change in high-density lipoprotein levels (r(s) = -0.44, P = .022) and positively with the change in insulin (r(s) = 0.50, P = .009) and HOMA-IR (r(s )= 0.51, P = .007) levels. Conclusion: Circulating LEAP2 levels decreased after induction of puberty reciprocally with increased growth rate and energy demand, reflecting the metabolic state of the adolescent. Further, the results suggest that estradiol levels may have a permissive role in downregulating circulating LEAP2 levels.Peer reviewe

GnRH receptor gene mutations in adolescents and young adults presenting with signs of partial gonadotropin deficiency

Biallelic, partial loss-of-function mutations in GNRHR cause a wide spectrum of reproductive phenotypes from constitutional delay of growth and puberty to complete congenital hypogonadotropic hypogonadism. We studied the frequency of GNRHR, FGFR1, TAC3, and TACR3 mutations in nine adolescent and young adult females with clinical cues consistent with partial gonadotropin deficiency (stalled puberty, unexplained secondary amenorrhea), and describe phenotypic features and molecular genetic findings of monozygotic twin brothers with stalled puberty. Two girls out of nine (22%, 95% CI 6-55%) carried biallelic mutations in GNRHR. The girl with compound heterozygous c. 317A>G p.(Gln106Arg) and c. 924_926delCTT p.(Phe309del) GNRHR mutations displayed incomplete puberty and clinical signs of hypoestrogenism. The patient carrying a homozygous c. 785G> A p.(Arg262Gln) mutation presented with signs of hypoestrogenism and unexplained secondary amenorrhea. None of the patients exhibited mutations in FGFR1, TAC3, or TACR3. The twin brothers, compound heterozygous for GNRHR mutations c. 317A> G p.(Gln106Arg) and c. 785G>A p.(Arg262Gln), presented with stalled puberty and were discordant for weight, and the heavier of them had lower testosterone levels. These results suggest that genetic testing of the GNRHR gene should be offered to adolescent females with low-normal gonadotropins and unexplained stalled puberty or menstrual dysfunction. In male patients with partial gonadotropin deficiency, excess adipose tissue may suppress hypothalamic-pituitary-gonadal axis.Peer reviewe

Familial central precocious puberty : two novel MKRN3 mutations

Background Paternally inherited loss-of-function mutations in MKRN3 underlie central precocious puberty (CPP). We describe clinical and genetic features of CPP patients with paternally inherited MKRN3 mutations in two independent families. Methods The single coding exon of MKRN3 was analyzed in three patients with CPP and their family members, followed by segregation analyses. Additionally, we report the patients' responses to GnRH analog treatment. Results A paternally inherited novel heterozygous c.939C>G, p.(Ile313Met) missense mutation affecting the RING finger domain of MKRN3 was found in a Finnish girl with CPP (age at presentation 6 years). Two Polish siblings (a girl presenting with B2 at the age of 4 years and a boy with adult size testes at the age of 9 years) had inherited a novel heterozygous MKRN3 mutation c.1237_1252delGGAGACACATGCTTTT p.(Gly413Thrfs*63) from their father. The girls were treated with GnRH analogs, which exhibited suppression of the hypothalamic-pituitary-gonadal axis. In contrast, the male patient was not treated, yet he reached his target height. Conclusions We describe two novel MKRN3 mutations in three CPP patients. The first long-term data on a boy with CPP due to an MKRN3 mutation questions the role of GnRH analog treatment in augmenting adult height in males with this condition. Impact We describe the genetic cause for central precocious puberty (CPP) in two families. This report adds two novel mutations to the existing literature. One of the mutations, p.(Ile313Met) affects the RING finger domain of MKRN3, which has been shown to be important for repressing the promoter activity of and . MKRN3KISS1TAC3We describe the first long-term observation of a male patient with CPP due to a paternally inherited MKRN3 loss-of-function mutation. Without GnRH analog treatment, he achieved an adult height that was in accordance with his mid-parental target height.Peer reviewe

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

Peer reviewe

Presentation and diagnosis of childhood-onset combined pituitary hormone deficiency : A single center experience from over 30 years

Publisher Copyright: © 2022 The AuthorsBackground: Childhood-onset combined pituitary hormone deficiency (CPHD) has a wide spectrum of etiologies and genetic causes for congenital disease. We aimed to describe the clinical spectrum and genetic etiologies of CPHD in a single tertiary center and estimate the population-level incidence of congenital CPHD. Methods: The retrospective clinical cohort comprised 124 CPHD patients (48 with congenital CPHD) treated at the Helsinki University Hospital (HUH) Children's Hospital between 1985 and 2018. Clinical data were collected from the patient charts. Whole exome sequencing was performed in 21 patients with congenital CPHD of unknown etiology. Findings: The majority (61%;76/124) of the patients had acquired CPHD, most frequently due to craniopharyngiomas and gliomas. The estimated incidence of congenital CPHD was 1/16 000 (95%CI, 1/11 000-1/24 000). The clinical presentation of congenital CPHD in infancy included prolonged/severe neonatal hypoglycaemia, prolonged jaundice, and/or micropenis/bilateral cryptorchidism in 23 (66%) patients; despite these clinical cues, only 76% of them were referred to endocrine investigations during the first year of life. The median delay between the first violation of the growth screening rules and the initiation of GH Rx treatment among all congenital CPHD patients was 2·2 years, interquartile range 1·2–3·7 years. Seven patients harbored pathogenic variants in PROP1, SOX3, TBC1D32, OTX2, and SOX2, and one patient carried a likely pathogenic variant in SHH (c.676G>A, p.(Ala226Thr)). Interpretation: Our study suggests that congenital CPHD can occur in 1/16 000 children, and that patients frequently exhibit neonatal cues of hypopituitarism and early height growth deflection. These results need to be corroborated in future studies and might inform clinical practice. Funding: Päivikki and Sakari Sohlberg Foundation, Biomedicum Helsinki Foundation, and Emil Aaltonen Foundation research grants.Peer reviewe

Circulating Liver-enriched Antimicrobial Peptide-2 Decreases During Male Puberty

Context: Circulating levels of liver-enriched antimicrobial peptide 2 (LEAP2), a ghrelin receptor antagonist, decrease under caloric restriction and increase in obesity. The role of LEAP2 in male puberty, a phase with accelerated energy demand, is unclear.Objective: This work aimed to investigate whether circulating LEAP2 levels are downregulated in boys following the onset of puberty to respond to the energy need required for growth.Methods: We determined circulating LEAP2 levels in 28 boys with constitutional delay of growth and puberty (CDGP) who participated in a randomized controlled trial (NCT01797718), and were treated with letrozole (n = 15) or intramuscular low-dose testosterone (T) (n = 13) for 6 months. Blood sampling and dual-energy x-ray absorptiometry-measured body composition were performed at 0-, 6-, and 12-month visits.Results: Serum LEAP2 levels decreased statistically significantly during pubertal progression (0-6 months: mean decrease -4.3 [10.3] ng/mL, P = .036 and 0-12 months: -3.9 [9.3] ng/mL, P = .033). Between 0 and 6 months, the changes in serum LEAP2 levels correlated positively with changes in percentage of body fat (r s = 0.48, P = .011), and negatively with growth velocity and estradiol levels (r s = -0.43, P = .022, r s = -0.55, P = .003, respectively). In the T group only, the changes in serum LEAP2 correlated negatively with changes in T and estradiol levels. Between 0 and 12 months, the change in LEAP2 levels correlated negatively with the change in high-density lipoprotein levels (r s = -0.44, P = .022) and positively with the change in insulin (r s = 0.50, P = .009) and HOMA-IR (rs = 0.51, P = .007) levels.Conclusion: Circulating LEAP2 levels decreased after induction of puberty reciprocally with increased growth rate and energy demand, reflecting the metabolic state of the adolescent. Further, the results suggest that estradiol levels may have a permissive role in downregulating circulating LEAP2 levels.</p