Johanna Känsäkoski

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.

A missense mutation in MKRN3 in a Danish girl with central precocious puberty and her brother with early puberty

Background:Idiopathic central precocious puberty (ICPP) results from the premature reactivation of the hypothalamic–pituitary–gonadal axis leading to development of secondary sexual characteristics prior to 8 y in girls or 9 y in boys. Since the initial discovery of mutations in the maternally imprinted MKRN3 gene in 2013, several case reports have described mutations in this gene in ICPP patients from different populations, highlighting the importance of MKRN3 as a regulator of pubertal onset.Methods:We screened 29 Danish girls with ICPP for mutations in MKRN3. Expression of MKRN3 in human hypothalamic complementary DNA (cDNA) was investigated by PCR.Results:One paternally inherited rare variant, c.1034G>A (p.Arg345His), was identified in one girl with ICPP and in her brother with early puberty. The variant is predicted to be deleterious by three different in silico prediction programs. Expression of MKRN3 was confirmed in adult human hypothalamus.Conclusion:Our results are in line with previous studies in which paternally inherited MKRN3 mutations have been found both in males and in females with ICPP or early puberty. Our report further expands the set of MKRN3 mutations identified in ICPP patients across diverse populations, thus supporting the major regulatory function of MKRN3 in pubertal onset.

Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.

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.

Congenital hypogonadotropic hypogonadism, functional hypogonadotropism or constitutional delay of growth and puberty? An analysis of a large patient series from a single tertiary center

STUDY QUESTION What diagnoses underlie delayed puberty (DP) and predict its outcome? SUMMARY ANSWER A multitude of different diagnoses underlie DP, and in boys a history of cryptorchidism, small testicular size and slow growth velocity (GV) predict its clinical course. WHAT IS KNOWN ALREADY DP is caused by a variety of underlying etiologies. Hormonal markers can be used in the differential diagnosis of DP but none of them have shown complete diagnostic accuracy. STUDY DESIGN, SIZE, DURATION Medical records of 589 patients evaluated for DP in a single tertiary care center between 2004 and 2014 were retrospectively reviewed. PARTICIPANTS/MATERIALS, SETTING, METHODS Clinical and biochemical data of 174 boys and 70 girls who fulfilled the criteria of DP were included in the analyses. We characterized the frequencies of underlying conditions and evaluated the predictive efficacy of selected clinical and hormonal markers. MAIN RESULTS AND THE ROLE OF CHANCE Thirty etiologies that underlie DP were identified. No markers of clinical value could be identified in the girls, whereas a history of cryptorchidism in the boys was associated with an increase in the risk of permanent hypogonadism (odds ratio 17.2 (95% CI; 3.4-85.4, P < 0.001)). The conditions that cause functional hypogonadotropic hypogonadism were more frequent in boys with a GV below 3 cm/yr than in those growing faster (19% vs 4%, P < 0.05). In this series, the most effective markers to discriminate the prepubertal boys with constitutional delay of growth and puberty (CDGP) from those with congenital hypogonadotropic hypogonadism (CHH) were testicular volume (cut-off 1.1 ml with a sensitivity of 100% and a specificity of 91%), GnRH-induced maximal LH (cut-off 4.3 IU/L; 100%, 75%) and basal inhibin B (INHB) level (cut-off 61 ng/L; 90%, 83%). LIMITATIONS, REASONS FOR CAUTION The main limitation of the study is the retrospective design. WIDER IMPLICATIONS OF THE FINDINGS Prior cryptorchidism and slow GV are two important clinical cues that may help clinicians to predict the clinical course of DP in boys, whereas markers of similar value could not be identified in girls. In prepubertal boys, testicular size appeared as effective as INHB and GnRH-induced LH levels in the differential diagnosis between CHH and CDGP. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Academy of Finland (268356), Foundation for Pediatric Research (7495), Sigrid Juselius Foundation (2613) and the Finnish Medical Foundation (011115). The authors have no competing interests to report. TRIAL REGISTRATION NUMBER Not applicable.

Genetics of congenital hypogonadotropic hypogonadism in Denmark

Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder characterized by incomplete/absent puberty caused by deficiency or defective action of gonadotropin-releasing hormone (GnRH). The phenotypic features of patients with CHH vary from genital hypoplasia and absent puberty to reversal of HH later in life. We examined the genetics and clinical features of CHH in Denmark. Forty-one male patients were screened for mutations in KAL1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R. CHD7 was screened in two patients with hearing loss. In 12 patients, a molecular genetic cause for CHH was found. Four patients had mutations in KAL1 (C105VfsX13, C53X, ex5-8del, R257X), and five in FGFR1 (G97S, R209C, A512V, R646W, and c.1614C>T, (p.I538I), predicted to affect splicing). All 9 had severe HH (cryptorchidism and/or micropenis), and 2 had cleft lip/palate. One patient with a previously reported homozygous R262Q mutation in GNRHR displayed fascinating temporal variation in his phenotype. Two patients with hearing loss had CHD7 mutations (c.7832_7841del (p.K2611MfsX25) and c.2443-2A>C), confirming that CHH patients with CHARGE syndrome-associated features should be screened for mutations in CHD7.

Two missense mutations in KCNQ1 cause pituitary hormone deficiency and maternally inherited gingival fibromatosis

Familial growth hormone deficiency provides an opportunity to identify new genetic causes of short stature. Here we combine linkage analysis with whole-genome resequencing in patients with growth hormone deficiency and maternally inherited gingival fibromatosis. We report that patients from three unrelated families harbor either of two missense mutations, c.347G>T p.(Arg116Leu) or c.1106C>T p.(Pro369Leu), in KCNQ1, a gene previously implicated in the long QT interval syndrome. Kcnq1 is expressed in hypothalamic GHRH neurons and pituitary somatotropes. Co-expressing KCNQ1 with the KCNE2 β-subunit shows that both KCNQ1 mutants increase current levels in patch clamp analyses and are associated with reduced pituitary hormone secretion from AtT-20 cells. In conclusion, our results reveal a role for the KCNQ1 potassium channel in the regulation of human growth, and show that growth hormone deficiency associated with maternally inherited gingival fibromatosis is an allelic disorder with cardiac arrhythmia syndromes caused by KCNQ1 mutations.Growth retardation is most commonly caused by genetic defects in the growth hormone pathway. Here, in families with growth retardation and gingival fibromatosis, the authors identify mutations in the potassium channel gene KCNQ1 that cause electrophysiological aberrations and altered ACTH secretion in vitro.

Childhood growth in boys with congenital hypogonadotropic hypogonadism

Background:We describe childhood growth patterns in a series of well-characterized patients with congenital hypogonadotropic hypogonadism (CHH) with special emphasis on genotype–phenotype correlation.Methods:We retrospectively evaluated the growth charts of 36 males with CHH (27 from Finland and 9 from Denmark). Fifteen patients (42%) had representative growth measurements during the first year of life. Genetically verified diagnosis of CHH was made in 15 (42%) patients (KAL1, FGFR1, GNRHR, or PROK2).Results:We found a deceleration of growth rate during early childhood. The mean (SD) length standard deviation score (SDS) at birth (0.2 (1.6) SDS) decreased significantly during the first 3 (to −0.9 (1.2) SDS) and 6 mo of life (to −0.7 (1.3) SDS). At the average age of 3 y, mean height SDS (−0.2 (1.3) SDS) did not differ from mid-parental target height (MPH). Mean height SDS reached its nadir (−1.7 (1.4) SDS) at an average age of 15.8 (0.8) years reflecting pubertal failure. Final heights did not differ from MPH. No clear genotype-growth associations emerged.Conclusion:Moderate postnatal length deflection is a novel feature of CHH and may reflect early androgen deficiency. Childhood growth patterns are not of clinical value in targeting molecular genetic diagnosis of CHH.

Circulating makorin ring-finger protein-3 (MKRN3) levels in healthy men and in men with hypogonadotropic hypogonadism.

Abstract The initiation of puberty is dependent on loosening of the influence of inhibitory factors acting on the GnRH neurons, which control the hypothalamic-pituitary-gonadal (HPG) axis activity. One of these factors was identified recently, as loss-of-function mutations in the maternally imprinted MKRN3 gene were implicated in central precocious puberty (CPP).(1) MKRN3 encodes Makorin RING-finger protein 3, a putative E3 ubiquitin ligase belonging to the Makorin family of zinc-finger proteins. Recently, circulating MKRN3 was measured in humans for the first time.(2) MKRN3 levels declined prior to clinical onset of puberty and correlated negatively with gonadotropins in prepubertal girls, lending support to the view that a decline in MKRN3 expression precedes the onset of puberty, and circulating MKRN3 reflects this phenomenon. To date it is not known whether circulating MKRN3 in males could serve as a marker of HPG axis integrity. This article is protected by copyright. All rights reserved.