Introduction Kallmann syndrome (KS) combines hypogonadotropic hypogonadism and anosmia or hyposmia, i.e., a deficiency of the sense of smell . Anosmia/hyposmia is related to the absence or hypoplasia of the olfactory bulbs and tracts . Hypogonadism is due to deficiency in gonadotropin-releasing hormone  and probably results from a failure of embryonic migration of gonadotropin-releasing hormone-synthesizing neurons . These cells normally migrate from the olfactory epithelium to the forebrain along the olfactory nerve pathway . In some KS patients other developmental anomalies can be present, which include renal agenesis, cleft lip and/or palate, selective tooth agenesis, and bimanual synkinesis . This is a genetically heterogeneous disease, which affects 1:8000 males and approximately five times less females. Two different genes have so far been identified. Loss-of-function mutations in KAL1 (NCBI GeneID: 3730) [7–9] and FGFR1 (NCBI GeneID: 2260)  account for the X-chromosome linked form and an autosomal dominant form of the disease, respectively. KAL1 encodes anosmin-1, a locally restricted glycoprotein of embryonic extracellular matrices , which is likely to be involved in fibroblast growth factor-signaling [6,12]. Nearly 80% of the KS patients, however, do not carry a mutation in either of these genes . Because the common infertility in affected individuals and, most importantly, the incomplete penetrance of the disease impede linkage analysis, the positional cloning strategies that have been taken to find causative genes were based on the analysis of rare KS individuals who carry chromosomal rearrangements detectable by cytogenetics techniques [7,8,10]. Here, we used a direct candidate gene approach and identified two novel genes underlying the disease. Results/Discussion We first considered GPR73L1/PROKR2 (NCBI GeneID: 128674), encoding the prokineticin receptor-2 (PROKR2) [13–15], a most relevant candidate because olfactory bulbs do not develop normally in mutant mice lacking this G protein-coupled transmembrane receptor, and these mice also have a severe atrophia of the reproductive system related to the absence of gonadotropin-releasing hormone-synthesizing neurons in the hypothalamus . We thus sequenced the two coding exons of PROKR2 and flanking splice sites in 192 unrelated individuals (144 males and 48 females) affected by KS, including 38 familial cases. Ten different mutations (one frameshift and nine missense mutations) were detected in 14 patients (four familial and ten apparently sporadic cases) in the heterozygous (ten cases), homozygous (two cases), or compound heterozygous (two cases) state (Figure S1, Table 1, and Figure 1). Conservation of the mutated amino acid residues in bovine, murine, and rat orthologous sequences (Figure S2) argues in favor of a deleterious effect for all the missense mutations. However, two of these mutations, p.R268C and p.V331M, as well as a mutation (c.253C>T, p.R85C) affecting the same residue as the p.R85H mutation found in two KS cases and another missense mutation (c.1004C>G, p.T335M) not found in the cohort of KS patients, were detected, once each, in 500 alleles from ethnically matched (Caucasian) control individuals. No other nonsynonymous variant was found in the controls. In the absence of functional testing, one cannot be sure that each missense mutation found in KS individuals is causative of the disease. Nevertheless, together with the KS-like phenotype of Prokr2 knockout mice, the fact that the overall proportion of PROKR2 alleles carrying nonsynonymous mutations is significantly higher in KS patients (18 out of 384 alleles) than in controls (four out of 500 alleles; chi-square value = 13.5, p A (p.R85H) and c.518T>G (p.L173R), were also found in the homozygous state in one patient each. (2.4 MB TIF) Click here for additional data file. Figure S2 Alignment of PROKR2 and PROK2 Amino Acid Sequences in Man, Cow, Mouse, and Rat (CLUSTALW) The missense mutations found in Kallmann syndrome patients are indicated by arrowheads. In the PROK2 sequence, the additional peptide encoded by exon 3 (alternative splicing) is underlined, and the N-terminal AVITGA motif that is critical for the bioactivity of the protein is highlighted in yellow. (91 KB PDF) Click here for additional data file. Figure S3 DNA Sequence Electrophoretograms from the Kallmann Syndrome Patient Carrying Missense Mutations in PROKR2 and KAL1, and Interspecies Comparison of the Amino Acid Sequence of KAL1 (Anosmin-1) around the Mutated Residue Control electrophoretograms are shown on the top. The mutations in PROKR2 and KAL1 are indicated by vertical arrows on the patient's electrophoretograms (bottom). Alignment of the KAL1 amino acid sequences from man, cow, chicken, zebrafish (kal1.1 and kal1.2), Caenorhabditis elegans, and Drosophila melanogaster shows the conservation of the mutated residue (Ser396) in vertebrates and invertebrates (either serine or threonine), whereas most of the surrounding residues are more variable. (943 KB TIF) Click here for additional data file.