Genetics of congenital hypogonadotropic hypogonadism in Denmark

Genetics of congenital hypogonadotropic hypogonadism in Denmark

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 ...

257KB Sizes 5 Downloads 31 Views

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

EJMG2924_proof ■ 17 April 2014 ■ 1/4

European Journal of Medical Genetics xxx (2014) 1e4

Contents lists available at ScienceDirect

European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg

Clinical research

Genetics of congenital hypogonadotropic hypogonadism in Denmark Q9

Johanna Tommiska a, b, *, Johanna Känsäkoski a, b, Peter Christiansen c, Niels Jørgensen c, Jacob G. Lawaetz c, Anders Juul c, Taneli Raivio a, b a

Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland Children’s Hospital, Helsinki University Central Hospital (HUCH), Helsinki, Finland c Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 December 2013 Accepted 3 April 2014 Available online xxx

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 (G97D, 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. Ó 2014 Published by Elsevier Masson SAS.

Keywords: CHARGE syndrome Congenital hypogonadotropic hypogonadism Kallmann syndrome Puberty

1. Introduction Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder characterized by incomplete or absent puberty caused by the lack or deficient number of hypothalamic gonadotropinreleasing hormone (GnRH) neurons, disturbed secretion or action of GnRH, or both [Seminara et al., 1998]. When HH presents with deficient sense of smell (anosmia), the condition is termed Kallmann syndrome (KS). X-linked, autosomal dominant, and autosomal recessive, as well as di- and oligogenic inheritance of CHH have all been described [Falardeau et al., 2008; Pitteloud et al., 2007a; Sykiotis et al., 2010], and several genes have been connected with the disorder, including KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, GNRHR, GNRH1, KISS1R, KISS1, TAC3, and TACR3 [Bianco and Kaiser, 2009; Brioude et al., 2010]. In the majority of cases, however, the molecular genetic cause remains unresolved, implying the existence of additional genes underlying the

* Corresponding author. Institute of Biomedicine/Physiology, University of Helsinki, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), FI-00014 University of Helsinki, Helsinki, Finland. Tel.: þ358 9 19125286; fax: þ358 9 19125302. E-mail address: [email protected]fi (J. Tommiska).

condition [Martin et al., 2011]. We have previously described the phenotypic and genotypic features of Finnish patients with CHH [Laitinen et al., 2011a; Laitinen et al., 2012a]. They had mutations in FGFR1, KAL1, CHD7, or GNRHR, whereas biallelic mutations in KISS1, KISS1R, TAC3, TACR3, PROK2, PROKR2 or GNRH1 genes were not found [Laitinen et al., 2011a; Laitinen et al., 2012a]. Herein, we describe genotypic and phenotypic features of CHH in another Nordic country, as a relatively large series of Danish patients was examined.

2. Patient data All patients with hypogonadotropic hypogonadism in our tertiary referral center (pediatric endocrinology and andrology) were identified through our register. Thus, patients registered with an ICD10 code corresponding to IHH (DE23.0D) were included. The diagnosis was validated by careful evaluation of the patient record files. All clinical (medical history, phenotypic description, auxological data) and biochemical data were reordered, and results from DEXA and brain MRI included when available, as well as the sense of smell, if reported. The data on sense of smell are based on patient records; tested or self-reported. DNA was collected from our

http://dx.doi.org/10.1016/j.ejmg.2014.04.002 1769-7212/Ó 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Tommiska J, et al., Genetics of congenital hypogonadotropic hypogonadism in Denmark, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.04.002

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

EJMG2924_proof ■ 17 April 2014 ■ 2/4

2

J. Tommiska et al. / European Journal of Medical Genetics xxx (2014) 1e4

biobank and available in 42 male patients with CHH (our unit primarily diagnoses and treats male patients).

4. Ethical issues Patients gave their informed consent, and the study was approved by the regional ethical committee (KF01328087) and the Danish Data Protection Agency (2006-41-7251).

3. Methods DNA from peripheral blood lymphocytes was purified using the QuickGene-810 Nucleic Acid Isolation System (Fujifilm, Life Science-Products, Tokyo, Japan), by means of the QIAmp 96 DNA Blood kit (Qiagen, Inc., Chatsworth, CA, USA), or Nucleo Spin 96 Blood kit (MachereyeNagel, Select Science Ltd, Corston, UK). Fortyone patients with congenital HH were screened for mutations in KAL1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R. In addition, CHD7 was screened for mutations in two patients with hearing loss. The GNRHR mutations Q106R and R262Q as well as the TAC3 M1L mutation were also screened in 95 healthy prepubertal girls from The COPENHAGEN Puberty Study [Aksglaede et al., 2009; Tommiska et al., 2011]. The coding exons and exon-intron boundaries of the genes were PCR-amplified from the genomic DNA of the patients. PCR products were purified with ExoSAP-IT treatment (Amersham Biosciences, Piscataway, NJ, USA), and bi-directionally sequenced using the ABI BigDyeTerminator Cycle Sequencing Kit (v3.1) and ABI Prism 3730xl DNA Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned and read with SequencherÒ 4.9 software (Gene Codes Corporation, AnnArbor, MI, USA). All primer sequences and PCR conditions are available upon request. The effects of identified missense mutations were predicted with the web version of PolyPhen-2 software (v.2.2.2). The effect on splicing of the identified synonymous change in FGFR1 was predicted with the online bioinformatics tool Human Splicing Finder (v. 2.4.1).

5. Results and discussion The patients enrolled to this study were from a single tertiary referral center, in which the patients with CHH are followed up from birth to senescence. This system is probably beneficial for the patients, as the potentially vulnerable transition period from the pediatric to adult health care system can be avoided [Laitinen et al., 2012b]. Similar to our earlier results in Finnish patients [Laitinen et al., 2011a], a relatively high proportion of Danish males with CHH had a history of microphallus (23/41, 56%), a phenotypic finding consistent with profound neonatal congenital gonadotropin deficiency. Thirteen patients were reported to have a normal sense of smell (patient’s own statement or tested), and 16 were hyposmic/anosmic; for 12 patients this was not reported at all. Because of the incomplete data on olfaction, KAL1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R were analyzed in all patients regardless of the reported sense of smell. CHD7 was screened in two patients with hearing loss. Twelve (29%) of the 41 Danish patients with CHH were detected as having a conclusive mutation in FGFR1, KAL1, GNRHR, or CHD7. The phenotypes of these patients are summarized in Table 1. Five patients (12%) had a mutation in FGFR1 (c.290G>A (p.G97D), c.625C>T (p.R209C), c.1535C>T (p.A512V), c.1936C>T (p.R646W), and a synonymous change c.1614C>T, (p.I538I), predicted to affect splicing). They all had cryptorchidism and/or

Table 1 Clinical features of the Danish HH patients with FGFR1, KAL1, GNRHR, and CHD7 mutations. Proband Family history

1 2 3

4 5 6 7

Anosmic father No Brother with cleft lip and palate, tooth agenesis No

Gene

FGFR1 FGFR1 FGFR1

Mutation

c.625C>T (p.R209C) c.1936C>T (p.R646W) c.1535C>T (p.A512V)

History of

Olfaction

Micro- Cryptor- Puberty penis chidism

SelfClinical reported examination

Yes Yes Yes

c.1614C>T Yes (p.I538I, affects splicingb) Yes Two anosmic sons FGFR1 TAC3 c.290G>A (p.G97D)d c.1A>T (p.M1L) No KAL1 c.309C>A þ c.310delT (p.S103R þ p.C105VfsX13) Brother with KS KAL1 c.154_157duplAGGT (p.C53X)

Yes Yes Yes

FGFR1

8

No

KAL1

del ex5-8

9 10

No No

KAL1 GNRHR

11

No

CHD7

12

No

CHD7

c.769C>T (p.R257X)e c.785G>A/c.785G>A (p.R262Q/p.R262Q)f c.7832_7841del (p.K2611MfsX25) c.2443-2A>C

Yes

No

No No No

H

No

N

Partialc

A

Associated phenotypes

A H

Cleft lip or palate, tooth agenesis Cleft lip or palate; normal MRI

No

A

Normal MRI

Yes

No

A

Yes

No

Hypoplastic olfactory groove and sulci in MRI; olfactory bulbs not visible Hearing loss, rheumatoid arthritis, normal MRI

Yes

A

Cleft lip and palate, esophagotracheal fistula, hearing loss Syndactyly, hearing loss

Yes

Yes

No A Delayed; N reversal; LOH No

Yes

Yes

No

N

Forty-one Danish patients with congenital hypogonadotropic hypogonadism (CHH) were screened for mutations in KAL1, FGFR1, FGF8, PROK2, PROKR2, GNRHR, TAC3, TACR3, and KISS1R. In addition, CHD7 was screened for mutations in two patients with hearing loss. In 12 patients, a molecular genetic cause for CHH was found. Mutations were found in KAL1, FGFR1, GNRHR, and CHD7. KS, Kallmann syndrome; LOH, late-onset hypogonadism; A, anosmia; H, hyposmia; N, normosmia; MRI, magnetic resonance imaging; US, ultrasound. a Q10 Testicular volume less than 4 mL. b Predicted by Human Splicing Finder. c Testicular volume 6 mL at the age of 32. d Mutation first reported in Dode et al. [2003]. e Mutation previously reported in Hardelin et al. [1993] and Bhagavath et al. [2007]. f Case previously reported in Tommiska et al. [2013].

Please cite this article in press as: Tommiska J, et al., Genetics of congenital hypogonadotropic hypogonadism in Denmark, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.04.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

EJMG2924_proof ■ 17 April 2014 ■ 3/4

J. Tommiska et al. / European Journal of Medical Genetics xxx (2014) 1e4

micropenis, and two also had cleft lip/palate. All the missense mutations were predicted by PolyPhen2 to be “possibly damaging” with score 1.000 (sensitivity: 0.00; specificity: 1.00). None of them was reported in the dbSNP database nor in the Exome Variant Server (EVS) database. The c.290G>A (p.G97D) mutation is a known mutation in KS [Dodé et al., 2003], but to the best our knowledge, none of the other mutations has been previously reported in CHH patients. However, a c.626G>A (p.R209H) mutation, affecting the same codon as the c.625C>T (p.R209C) mutation, was detected in a Finnish patient with KS. Interestingly, neither of these two patients had any additional phenotypic features other than HH and anosmia [Laitinen et al., 2011a]. Three of the 4 patients having mutations in KAL1 had a mutation creating a premature stop codon; c.309C>A þ c.310delT (p.S103R þ p.C105VfsX13), c.154_157duplAGGT (p.C53X), and c.769C>T (p.R257X) [Bhagavath et al., 2007; Hardelin et al., 1993]. The fourth patient had a deletion of exons 5 to 8 as these exons could not be amplified from the patient’s genomic DNA while the control samples amplified at the same time did produce the correct amplicons. Degradation of the DNA sample was ruled out because the other exons of KAL1 could also be amplified from the same DNA after unsuccessful amplification of exons 5e8. One patient with CHH had a homozygous c.785G>A (p.R262Q) mutation in GNRHR, coding for GnRH receptor. He had recovered from HH before the age of 32, was without Rx for 30 yrs, and at the age of 60 yrs, presented with signs of late-onset hypogonadism [Tommiska et al., 2013]. Three heterozygous c.785G>A (p.R262Q) mutations and one heterozygous c.317A>G (p.Q106R) mutation in GNRHR were also found in our series of Danish patients. Both mutations have been implicated in autosomal recessive normosmic CHH, and have been shown to partially impair receptor function [de Roux et al., 1997]. The lack of more patients with biallelic GNRHR mutations is a bit surprising, as GNRHR mutations are the most common known genetic cause for normosmic CHH. It is not likely, however, that we would have missed the second mutation in those heterozygous patients, as three of them were reported anosmic. In addition, the carrier frequency for both of these mutations in the Danish population was 1/95, as determined in the controls from The COPENHAGEN Puberty Study, and similar carrier frequencies have been reported in other populations as well [Laitinen et al., 2011b; Vaaralahti et al., 2011]. As mutations in CHD7, the gene initially implicated in CHARGE syndrome, may also underlie CHH [Kim et al., 2008], especially when CHARGE-like phenotypic features are present [Bergman et al., 2012], we also screened two patients for CHD7 mutations, and both of them were indeed found to have CHD7 mutations. The patient with a frameshift mutation, c.7832_7841del (p.K2611MfsX25), was born with an esophagotracheal fistula and a bilateral cleft lip and palate. He also had anosmia, deafness of the left ear, and partial hearing loss on the right ear. The other one had severe HH (micropenis, cryptorchidism), syndactyly, and hearing loss. He had a mutation in the conserved donor splicesite, c.2443-2A>C. Husu et al. [2013] very recently reported the phenotypic features of 18 Danish CHARGE patients with verified CHD7 mutations and found an extensive variability in clinical presentation. CHD7 mutations are not common in patients with CHH, but they can be found even in patients with reversal of HH [Laitinen et al., 2012a]. It is thus important to screen CHH patients with CHARGE syndrome eassociated features for CHD7 mutations, so that relevant genetic counseling can be given to the affected family. One KS patient had a PROK2 mutation c.163delA (p.I55fsX1) previously identified both in autosomal recessive KS and normosmic CHH [Leroy et al., 2008; Pitteloud et al., 2007b], but in a

3

heterozygous state. Although another KS patient heterozygous for the same mutation has been described [Pitteloud et al., 2007b], the mutation is not likely to be a monoallelic cause of the disease phenotype as it has also been found in a healthy sibling [Abreu et al., 2008]. Similarly, one KS patient with a mutation in FGFR1 (c.290G>A (p.G97D)) also had a heterozygous variant in TAC3, c.1A>T, which changes the start codon ATG to TTG, coding for leucine, possibly resulting in the deletion of the first three amino acids of the polypeptide (the next methionine in the amino acid sequence is found in the fourth amino acid position). TAC3 mutations were first implicated in autosomal recessive normosmic CHH [Topaloglu et al., 2009]. However, our patient is anosmic and has two anosmic sons (Table 1), so it is most likely that the FGFR1 mutation alone explains the patient’s phenotype. We conclude that KAL1, FGFR1, GNRHR, and CHD7 mutations were found in this representative patient series from the largest tertiary center in Denmark; the situation very similar to that recently described in Finland [Laitinen et al., 2011a; Laitinen et al., 2012a], although genetically the Finns differ from the Danes and other Northern Europeans [Beekman et al., 2013]. Interestingly, no FGF8 mutations or biallelic mutations in PROK2, PROKR2, TAC3, TAC3R, or KISS1R, were found in either population. This may be due to different mutation frequencies in different populations, which has already been documented for PROKR2 mutations [Sarfati et al., 2013]. Mutations in PROKR2 and PROK2 have also been reported to be frequently involved in oligogenicity [Sykiotis et al., 2010]; thus the lack of them partly explains the lack of oligogenicity in the Finnish and Danish patients. Finally, the CHD7 mutations found in two patients with hearing loss further emphasize the genetic and clinical overlap of CHH and CHARGE syndrome.

6. Study limitations We chose to focus on genes that are the most relevant in a clinical setting and thus did not screen all of the genes that have recently been connected with CHH, such as GNRH1, KISS1, HS6ST1, SEMA3A, and SOX10. Mutations in GNRH1 and KISS1 in CHH patients are extremely rare (less than five families with homozygous mutations reported in the world [Bouligand et al., 2009; Chan et al., 2009; Topaloglu et al., 2012]). As these two rare recessive genes were not screened, it is not impossible that they would contribute to CHH more in the Danish population than in other populations, although we do not see recessive inheritance (affected siblings) in the families. In addition, the role of mutations in genes such as HS6ST1 [Tornberg et al., 2011] and SEMA3A [Hanchate et al., 2012; Känsäkoski et al., 2014; Young et al., 2012] in the pathogenesis of CHH is still unclear, and even if a mutation or variant in one of these genes was found, its contribution would be impossible to evaluate and would thus not be of much value to the patient or to the treating physician. Screening of SOX10 is reasonable when the patient has hearing loss and/or other specific phenotypic features [Pingault et al., 2013; Vaaralahti et al., in press], but both our patients with hearing loss were already found to have mutations in CHD7. It is also possible that oligogenicity would have been observed if all the genes had been screened, as especially SEMA3A mutations have been found in patients with KAL1 and FGFR1 mutations [Hanchate et al., 2012; Känsäkoski et al., 2014]. We did not perform functional assays on the novel FGFR1 mutations found in this study. However, FGFR1 is already a wellestablished CHH gene so there is no great reason to doubt the involvement of these rare mutations in the disorder. The in vitro assays commonly used to study the impact of FGFR1 missense

Please cite this article in press as: Tommiska J, et al., Genetics of congenital hypogonadotropic hypogonadism in Denmark, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.04.002

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

EJMG2924_proof ■ 17 April 2014 ■ 4/4

4

J. Tommiska et al. / European Journal of Medical Genetics xxx (2014) 1e4

mutations also do not seem to help in predicting the phenotype of the patient. Complete phenotyping information on all the patients was unfortunately not available, especially on additional non-reproductive features such as renal agenesis, tested olfaction, or olfactory MRI data. For mutation positive patients, all the information available is given in Table 1. It is of note that because not all the information was available (for example, sense of smell), we screened the same genes in all of the patients, except for CHD7, which was sequenced only in patients with hearing loss. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgments We thank Ms Lea Puhakka for skillful technical assistance. This Q1,2 work was supported by the Academy of Finland, the Finnish Q3

Foundation for Pediatric Research, the Emil Aaltonen Foundation,

Q4,5 the Sigrid Juselius Foundation, the Danish Medical Research Q6,7 Council, the Research fund of the Capital Region, and the Lundbeck

Foundation. References Abreu AP, Trarbach EB, De Castro M, Costa EMF, Versiani B, Baptista MTM, et al. Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome. J Clin Endocrinol Metab 2008;93:4113e8. Aksglaede L, Sørensen K, Petersen JH, Skakkebaek NE, Juul A. Recent decline in age at breast development: the Copenhagen Puberty Study. Pediatrics 2009;123: e932e9. Beekman M, Blanché H, Perola M, Hervonen A, Bezrukov V, Sikora E, et al. Genomewide linkage analysis for human longevity: genetics of Healthy Aging Study. Aging Cell 2013;12:184e93. Bergman JE, de Ronde W, Jongmans MC, Wolffenbuttel BH, Drop SL, Hermus A, et al. The results of CHD7 analysis in clinically well-characterized patients with Kallmann syndrome. J Clin Endocrinol Metab 2012;97:E858e62. Bhagavath B, Xu N, Ozata M, Rosenfield RL, Bick DP, Sherins RJ, et al. KAL1 mutations are not a common cause of idiopathic hypogonadotrophic hypogonadism in humans. Mol Hum Reprod 2007;13:165e70. Bianco SD, Kaiser UB. The genetic and molecular basis of idiopathic hypogonadotropic hypogonadism. Nat Rev Endocrinol 2009;5:569e76. Bouligand J, Ghervan C, Tello JA, Brailly-Tabard S, Salenave S, Chanson P, et al. Isolated familial hypogonadotropic hypogonadism and a GNRH1 mutation. N Engl J Med 2009;360:2742e8. Brioude F, Bouligand J, Trabado S, Francou B, Salenave S, Kamenicky P, et al. Nonsyndromic congenital hypogonadotropic hypogonadism: clinical presentation and genotype-phenotype relationships. Eur J Endocrinol 2010;162:835e51. Chan Y-M, De Guillebon A, Lang-Muritano M, Plummer L, Cerrato F, Tsiaras S, et al. GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci U S A 2009;106:11703e8. de Roux N, Young J, Misrahi M, Genet R, Chanson P, Schaison G, et al. A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor. N Engl J Med 1997;337:1597e602. Dodé C, Levilliers J, Dupont JM, De Paepe A, Le Dû N, Soussi-Yanicostas N, et al. Lossof-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet 2003;33:463e5. Falardeau J, Chung WC, Beenken A, Raivio T, Plummer L, Sidis Y, et al. Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice. J Clin Invest 2008;118:2822e31. Hanchate NK, Giacobini P, Lhuillier P, Parkash J, Espy C, Fouveaut C, et al. SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome. PLoS Genet 2012;8:e1002896. Hardelin JP, Levilliers J, Blanchard S, Carel JC, Leutenegger M, Pinard-Bertelletto JP, et al. Heterogeneity in the mutations responsible for X chromosome-linked Kallmann syndrome. Hum Mol Genet 1993;2:373e7. Husu E, Hove HD, Farholt S, Bille M, Tranebjærg L, Vogel I, et al. Phenotype in 18 Danish subjects with genetically verified CHARGE syndrome. Clin Genet 2013;83:125e34. Kim HG, Kurth I, Lan F, Meliciani I, Wenzel W, Eom SH, et al. Mutations in CHD7, encoding a chromatin-remodeling protein, cause idiopathic hypogonadotropic hypogonadism and Kallmann syndrome. Am J Hum Genet 2008;83:511e9.

Känsäkoski J, Fagerholm R, Laitinen EM, Vaaralahti K, Hackman P, Pitteloud N, et al. Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism. Pediatr Res 2014; Feb 12. http://dx.doi.org/ 10.1038/pr.2014.23 [Epub ahead of print]. Laitinen E-M, Vaaralahti K, Tommiska J, Eklund E, Tervaniemi M, Valanne L, et al. Incidence, phenotypic features and molecular genetics of Kallmann syndrome in Finland. Orphanet J Rare Dis 2011a;6:41. Laitinen EM, Tommiska J, Virtanen HE, Oehlandt H, Koivu R, Vaaralahti K, et al. Isolated cryptorchidism: no evidence for involvement of genes underlying isolated hypogonadotropic hypogonadism. Mol Cell Endocrinol 2011b;341:35e 8. Laitinen E-M, Tommiska J, Sane T, Vaaralahti K, Toppari J, Raivio T. Reversible congenital hypogonadotropic hypogonadism in patients with CHD7, FGFR1 or GNRHR mutations. PLoS ONE 2012a;7:e39450. http://dx.doi.org/10.1371/ journal.pone.0039450. Laitinen EM, Hero M, Vaaralahti K, Tommiska J, Raivio T. Bone mineral density, body composition and bone turnover in patients with congenital hypogonadotropic hypogonadism. Int J Androl 2012b;35:534e40. Leroy C, Fouveaut C, Leclercq S, Jacquemont S, Boullay HD, Lespinasse J, et al. Biallelic mutations in the prokineticin-2 gene in two sporadic cases of Kallmann syndrome. Eur J Hum Genet 2008;16:865e8. Martin C, Balasubramanian R, Dwyer AA, Au MG, Sidis Y, Kaiser UB, et al. The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations. Endocr Rev 2011;32:225e46. Pingault V, Bodereau V, Baral V, Marcos S, Watanabe Y, Chaoui A, et al. Loss-offunction mutations in SOX10 cause Kallmann syndrome with deafness. Am J Hum Genet 2013;92:707e24. Pitteloud N, Quinton R, Pearce S, Raivio T, Acierno J, Dwyer A, et al. Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism. J Clin Invest 2007a;117:457e63. Pitteloud N, Zhang C, Pignatelli D, Li JD, Raivio T, Cole LW, et al. Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci USA 2007b;104:17447e52. Sarfati J, Fouveaut C, Leroy C, Jeanpierre M, Hardelin JP, Dodé C. Greater prevalence of PROKR2 mutations in Kallmann syndrome patients from the Maghreb than in European patients. Eur J Endocrinol 2013;169:805e9. Seminara SB, Hayes FJ, Crowley Jr WF. Gonadotropin-releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallmann’s syndrome): pathophysiological and genetic considerations. Endocr Rev 1998;19:521e39. Sykiotis GP, Plummer L, Hughes VA, Au M, Durrani S, Nayak-Young S, et al. Oligogenic basis of isolated gonadotropin-releasing hormone deficiency. Proc Natl Acad Sci USA 2010;107:15140e4. Tommiska J, Sørensen K, Aksglaede L, Koivu R, Puhakka L, Juul A, et al. LIN28B, LIN28A, KISS1, and KISS1R in idiopathic central precocious puberty. BMC Res Notes 2011;4:363. Tommiska J, Jørgensen N, Christiansen P, Juul A, Raivio T. A homozygous R262Q mutation in the gonadotropin-releasing hormone receptor presenting as reversal of hypogonadotropic hypogonadism and late-onset hypogonadism. Clin Endocrinol 2013;78:316e7. Topaloglu AK, Reimann F, Guclu M, Yalin AS, Kotan LD, Porter KM, et al. TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat Genet 2009;41: 354e8. Topaloglu AK, Tello JA, Kotan LD, Ozbek MMN, Yilmaz MB, Erdogan S, et al. Inactivating KISS1 mutation and hypogonadotropic hypogonadism. N Engl J Med 2012;366:629e35. Tornberg J, Sykiotis GP, Keefe K, Plummer L, Hoang X, Hall JE, et al. Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism. Proc Natl Acad Sci U S A 2011;108:11524e9. Vaaralahti K, Wehkalampi K, Tommiska J, Laitinen EM, Dunkel L, Raivio T. The role of gene defects underlying isolated hypogonadotropic hypogonadism in patients with constitutional delay of growth and puberty. Fertil Steril 2011;95:2756e8. Vaaralahti K, Tommiska J, Tillmann V, Liivak N, Känsäkoski J, Laitinen E-M, et al. Novel phenotypes in Kallmann syndrome: de novo SOX10 nonsense mutation in a patient with Kallmann syndrome and hearing loss. Pediatr Res 2014 [in press]. Q8 Young J, Metay C, Bouligand J, Tou B, Francou B, Maione L, et al. SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development. Hum Reprod 2012;27:1460e5.

Web resources PolyPhen-2 software: http://genetics.bwh.harvard.edu/pph2/. Human Splicing Finder online bioinformatics tool: http://www.umd.be/HSF/. dbSNP database: http://www.ncbi.nlm.nih.gov/SNP/. Exome Variant Server database: http://evs.gs.washington.edu/EVS.

Please cite this article in press as: Tommiska J, et al., Genetics of congenital hypogonadotropic hypogonadism in Denmark, European Journal of Medical Genetics (2014), http://dx.doi.org/10.1016/j.ejmg.2014.04.002

65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128