Novel FGFR1 and KISS1R Mutations in Chinese Kallmann Syndrome Males with Cleft Lip/Palate

Kallmann syndrome (KS) is characterized by isolated hypogonadotropic hypogonadism (IHH) with anosmia and is sometimes associated with cleft lip/palate (CLP). In order to describe the clinical features, genetic etiology, and treatment outcome of KS males with CLP, we performed genetic screening for 15 known causal IHH genes (KAL1, FGFR1, NELF, FGF8, CHD7, WDR11, SEMA3A, KISS1R, KISS1, PROKR2, PROK2, TAC3, TACR3, GNRH1, and GNRHR) in four KS with CLP patients and six IHH patients without CLP. Two novel heterozygous missense mutations in FGFR1, (NM_001174066): c.776G>A (p.G259E) and (NM_001174066): c.358C>T (p.R120C), were identified in a 23-year-old KS male with cleft lip and an 18-year-old KS patient with cleft lip and palate, dental agenesis, and high arched palate, respectively. These two mutations were not presented in their healthy parents and 200 normal controls. One novel heterozygous missense mutation in KISS1R, (NM_032551): c.587C>A (p.P196H), was identified in an 18-year-old KS male with cleft lip and dental agenesis who developed sperm after being treated with gonadotropin. This mutation was also presented in his healthy father and grandfather. These results have implications for the diagnosis, genetic counseling, and treatment of KS and CLP males with mutations in FGFR1 gene.

Cleft lip/palate (CLP) occurs in 1 in 500-2,500 live births, and 30% of CLP cases are part of a syndrome with additional congenital anomalies. The remaining 70% of cases are isolated [5]. CLP and other facial and oral developmental defects, such as dental agenesis and high arched palate, have been described in KS patients. However, the genetic causes and treatment outcome of KS with CLP patients have not been well described.
This study aimed to perform clinical evaluation and observe the treatment outcome of four Chinese men with KS and CLP and explore their causative mutations.
Each patient's family history of pubertal development and associated nonreproductive phenotypes was obtained. Detailed physical examinations were performed, including testicular volume and penis length measurements using a Prader orchidometer and vernier caliper, respectively. Pubic hair and genitalia were evaluated according to Tanner pubertal stages. Olfaction was tested by the Alcohol Sniff Test [6]. In men able to ejaculate, a semen sample was analyzed using the World Health Organization (WHO) normal values based on the WHO 2010 reference limits [7]. The olfactory system was examined by brain magnetic resonance imaging (MRI). Renal structure and cryptorchidism were assessed by ultrasound. Patients were treated with human chorionic gonadotropin (hCG) and followed up for at least three years after their initial diagnosis.
The study protocol was approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from each participant after a full explanation of the purpose and nature of all study procedures.

DNA Extraction and Next-Generation Sequencing.
Each participant's genomic DNA was extracted from whole blood using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Fifteen known causal genes with the highest mutation frequency in IHH patients (KAL1, FGFR1, PROKR2, PROK2, NELF, FGF8, CHD7, WDR11, SEMA3A, KISS1R, KISS1, TAC3, TACR3, GNRH1, GNRHR) were selected, and a custom semiconductor targeted resequencing panel was designed as follows. Primers of 319 overlapping amplicons covering approximately 30 kb of coding sequence and flanking regulatory regions of each target gene were automatically designed with the Ion AmpliSeq Ready-to-Use custom designer platform (https:// www.ampliseq.com/protected/dashboard.action). The primers (Life Technologies, Carlsbad, California, USA) which are able to perform ultrahigh-multiplex PCR reaction in one tube parallelly overlap 98.52% of the targeted region (30884 bp) by 319 amplicons. Ion Torrent adapter-ligated libraries were generated according to the Ion Ampliseq Library Kit 2.0 (Life Technologies, Carlsbad, CA, USA) protocol within about 5 hours. Briefly, 20 ng gDNA for every sample was quantitated by Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) for multiplex PCR amplification with the 2X primer pools. Then, the resulting amplicons were ligated to barcodes and Ion Torrent adapters (Life Technologies). Subsequently, we purified the libraries with AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified the libraries by Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Then, multiplexed barcoded libraries were enriched by clonal amplification using emulsion PCR on an Ion OneTouch 2 system (Life Technologies, CA, USA) according to the manufacturer's protocol. The emulsion PCR was performed as follows: the enzyme was activated at 99 ∘ C for 2 min. DNA was denatured at 99 ∘ C for 15 seconds, and annealing and extension were performed at 60 ∘ C for 4 min for 17 cycles. Massively parallel semiconductor sequencing was performed on Ion 318 Chips using the Ion PGM Sequencing 200 v2 Kit (Life Technologies, CA, USA) and a Personal Genome Machine Sequencer (Ion Torrent) according to the manufacturer's instructions.

Bioinformatic Analysis.
Data outputs obtained by semiconductor sequencing were processed using Torrent Suite version 3.6.2 (Life Technologies, Carlsbad, CA, USA) to generate sequence reads, trim adapter sequences, remove poor signal-profile reads, align to the hg19 human reference genome, analyze coverage, and call variants. Detected variants were then annotated with Ion Reporter (https://ionreporter.lifetechnologies.com/ir) and filtered using dbSNP137, Hapmap, and 1000-Genome Project. Functional prediction and conservation analysis were performed using ANNOVAR.

Sanger Sequencing Validation.
Only the novel variants that had not been previously described in dbSNP137, 1000-Genome Project, and were predicted to cause nonsynonymous coding changes were sequenced by Sanger sequencing to validate whether these mutations detected by PGM are true mutations. Briefly, PCR amplification of the DNA region across the variants in the patients who carried the variants was performed using Taq Hot Start Version Kit (Takara Bio, Otsu, Shiga, Japan) and specific primer (see Supplementary Table 1 Table 1) to detect any possible false positive errors. In order to determine whether the true mutations were presented in the 200 normal controls, we performed PCR amplification of the DNA region across the true mutations using Taq Hot Start Version Kit (Takara Bio, Otsu, Shiga, Japan) and specific primer (Supplementary Table 1) in 200 normal controls samples. Then, Sanger sequencing was performed in the PCR products with the specified primers. Similarly, we sequenced the DNA regions across the true mutations by Sanger sequencing in the family members of each patient who harbored true mutation to find weather these mutations were presented in the other family members. The technically  Table 3).
We excluded variants presented in dbSNP137 or the general population with a minor allele frequency greater than 5% according to the 1000-Genome Project, intronic variants, and synonymous exonic variants, leaving 21 remaining variants (Supplementary Table 4). These mutations were predicted to cause nonsynonymous coding changes in seven genes, with an average of three variants per patient (range 0-5 variants).

Sanger Sequencing Validation.
We performed Sanger sequencing to rule out false positives and confirmed that 3 of the 21 variants (Table 2)  high arched palate, respectively. These two mutations were not presented in their parents and other heathy family members. One heterozygous missense mutation in KISS1R which was reported previously, (NM 032551): c.587C>A (p.P196H) (Figure 2), was identified in an 18-year-old KS male with cleft lip and dental agenesis. However, it was presented in the healthy father and grandfather of this patient. We did not identify these three mutations in any of the 200 control subjects. Furthermore, we did not identify any mutations in the six IHH/KS patients without CLP.

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Fibroblast growth factor receptor 1 (FGFR1) encodes one of four FGFRs, which are tyrosine kinase family cell surface receptors. They function in the olfactory system and GnRH ontogeny was consistent with a role in the KS patient phenotype [14]. FGFR1 mutated in as many as 10% of all KS cases and patients harboring FGFR1 mutations exhibited a broad spectrum of associated nonreproductive phenotypes, including CLP and dental agenesis [14]. It has been reported that KS linked to FGFR1 with or without CLP is transmitted as an autosomal dominant disease [12,14]. We identified two FGFR1 mutations in two of the four KS patients with CLP. The parents of the two patients had a normal phenotype and no other family members were affected by this disease (Supplementary Figure 1). Both patients exhibited anosmia and bilateral absence of the olfactory bulbs and tracts by MRI (Figure 3), consistent with complete KS. One patient also had dental agenesis and a high arched palate. This phenotype heterogeneity of KS caused by FGFR1 mutations in our patients is consistent with previous report [4]. To our knowledge, this is the first report of an FGFR1 mutation in KS male with a high arched palate. Besides, the c.776G>A mutation was not presented in the normal parents of case 1 and the c.358C>T mutation was not presented in the normal parents and old sister of patient 3 (data not shown), suggesting that these mutations are novel de novo mutations. Our data supports that KS with CLP linked to FGFR1 is transmitted as an autosomal dominant disorder which has been reported previously [14].
The KISS1R gene encodes the G protein-coupled receptor GPR54, which regulates GnRH secretion. Studies have shown that KISS1R loss-of-function mutations contribute to nIHH by affecting GnRH secretion, but not olfactory bulb development or GnRH neuronal migration [15,16]. Relatively few KISS1R mutations have been reported in IHH patients to date, and all reported mutations are compound heterozygous or homozygous mutations [17][18][19][20][21][22]. Here, we identified one heterozygous KISS1R mutation in a KS patient with IHH, anosmia, lack of bilateral olfactory bulbs (Figure 3), cleft lip, and dental agenesis. This mutation was also presented in the normal father and grandfather of this patient (Supplementary Figure 2). Interestingly, this mutation has been previously reported in a girl with central precocious puberty [23]. This evidence suggested that this mutation may not be a pathogenic mutation in KS patient though functional prediction indicates the c.587C>A mutation in KISS1R causing damage to the KISS1R. However, in vitro functional studies are needed to confirm the consequence of this mutation. Besides, we did not detect any mutations in the other known causal KS genes, including KAL1, FGFR1, NELF, FGF8, CHD7, WDR11, SEMA3A, PROKR2, and PROK2.
In the present study, we observed that the testicular size and length of penis did increase in the cases 1, 2, and 3. These can be explained as results of the normalized serum testosterone levels in these three patients after being treated with hCG. The serum testosterone levels of case 4 remained in low level, so no obvious increase of the testicular size and phallus length was observed. Furthermore, the hCG treatment succeeded in inducing spermatogenesis in the KISS1R mutation patient. However, it failed to result in sperm in the ejaculation of the two FGFR1 mutation patients (Table 3). Our data showed that the heterogeneity response of testis to the hCG treatment existed. Recent studies may cast new light on the mechanism of this heterogeneity. It has reported that KAL1 gene, which plays a key role in the GnRH neuron migration by regulating FGFR1 signaling activation, is expressed in the marsupial gonad and participates in gametogenesis directly within the gonads themselves [24]. More recently, some authors reported that Fgfr1 was expressed in the entire population of undifferentiated spermatogonia in mammalian testes and FGF8 signal positively regulated the numbers of BioMed Research International 7   BioMed Research International undifferentiated spermatogonia through FGFR1 [25]. Based on these data we predict that mutations in the FGFR1 may affect the spermatogenesis outcome in the testes of the KS patients after being treated with gonadotropin. However, more studies should be done in the future to prove the association with this phenotype.