LAUSR

Dear Editor, Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a rare Mendelian disorder characterized by genetic and phenotypic heterogeneity. Apart from failure of sexual maturation and pubertal development (i.e., the normosmic form of idiopathic hypogonadotropic hypogonadism (nIHH) or the anosmic form of Kallmann syndrome (KS)), the disorder is also characterized by various non-reproductive features, including unilateral renal agenesis (URA). Interestingly, family members of affected patients usually display milder forms of IGD or its non-reproductive features, such as isolated anosmia and URA, highlighting the variable expressivity of the disorder [1, 2]. We have previously reported the prepubertal diagnosis of KS in a male patient with URA, who carried a mutation in the gene of anosmin 1-ANOS1, previously known as kallmann-1 (KAL1) [3], while other non-reproductive IGD features have been utilized for an early KS diagnosis [4]. With this letter, we would like to share our experience in examining the role of URA as an early prepubertal marker for IGD. Nine prepubertal normosmic male patients were clinically evaluated and screened for mutations in 14 IGD genes. Genomic DNA was obtained from peripheral blood samples by standard phenol-chloroform extraction. Exonic and proximal intronic (≤ 15 bp from splice sites) DNA sequences of the following genes were amplified by PCR and determined by direct sequencing: ANOS1 (OMIM 308700),GNRH1 (OMIM 152760), GNRHR (OMIM 138850), KISS1R (OMIM 604161), KISS1 (OMIM 603286), CHD7 (OMIM 608892), NMSF (OMIM 608137), FGF8 (OMIM 600483), FGFR1 (OMIM 136350), PROK2 (OMIM 607002), PROKR2 (OMIM 607212), HS6ST1 (OMIM 604846), TAC3 (OMIM 162330), and TACR3 (OMIM 162332). To detect exonic deletions/duplications across the entire coding region of ANOS1, gene dosage analysis was performed using the SALSA MLPA kit P132 Kallmann-1 (MRC Holland). Informed consent was obtained. A summary of the clinical characteristics is shown in Table 1. Importantly, none of the children evaluated was anosmic, though signs of GnRH development disruption, such as cryptorchidism, as well as other non-reproductive features were seen in family members. A rare sequence variant (RSV) was defined as a variant (a) affecting splice junctions within 10 bp of the coding sequence or a protein-altering/protein-truncating non-synonymous variant and (b) present in < 1% minor allele frequency (MAF) in the Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA, USA (URL: http://evs.gs.washington.edu/ EVS./) [February 2016], 1000 Genomes Project [http://www. 1000genomes.org/home] [5] and the non-Finnish European population of the Exome Aggregation Consortium (ExAC), Cambridge, MA, USA (URL: http://exac.broadinstitute.org) [February 2016]. Neither RSVs in any of the 14 genes nor intergenic dosage alterations in KAL1 were detected. The lack of a genetic association in this prepubertal normosmic cohort could be attributed to (i) the clinical presentation of our patients: even though nIHH cases with URA have been previously reported [6], the majority of studies have linked URA to KS and ANOS1 genomic alterations; (ii) a missed genetic alternation in the rest of the 37 known IGD genes not screened here; or (iii) an independent genetic cause of URA: since nephrogenesis is controlled by genes enhancing or inhibiting precursor cell * M. I. Stamou [email protected]