Kallmann syndrome in a patient with Weiss–Kruszka syndrome and a de novo deletion in 9q31.2

Patients with deletions on chromosome 9q31.2 may exhibit delayed puberty, craniofacial phenotype including cleft lip/palate, and olfactory bulb hypoplasia. We report a patient with congenital HH with anosmia (Kallmann syndrome, KS) and a de novo 2.38 Mb heterozygous deletion in 9q31.2. The deletion breakpoints (determined with whole-genome linked-read sequencing) were in the FKTN gene (9:108,331,353) and in a non-coding area (9:110,707,332) (hg19). The deletion encompassed six protein-coding genes (FKTN, ZNF462, TAL2, TMEM38B, RAD23B, and KLF4). ZNF462 haploinsufficiency was consistent with the patient’s Weiss–Kruszka syndrome (craniofacial phenotype, developmental delay, and sensorineural hearing loss), but did not explain his KS. In further analyses, he did not carry rare sequence variants in 32 known KS genes in whole-exome sequencing and displayed no aberrant splicing of 15 KS genes that were expressed in peripheral blood leukocyte transcriptome. The deletion was 1.8 Mb upstream of a KS candidate gene locus (PALM2AKAP2) but did not suppress its expression. In conclusion, this is the first report of a patient with Weiss–Kruszka syndrome and KS. We suggest that patients carrying a microdeletion in 9q31.2 should be evaluated for the presence of KS and KS-related features.


Introduction
Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease that prevents pubertal development and elicits infertility due to deficient secretion or action of gonadotropin-releasing hormone (GnRH) (1). CHH is called normosmic (nCHH) if the patient has a normal sense of smell, and Kallmann syndrome (KS) if the patient has an absent or deficient ability to smell (1). Both nCHH and KS may manifest with accompanying anomalies, such as bone abnormalities, hearing impairment and ear abnormalities, cleft lip or palate, and anophthalmia and/or coloboma (1). These diseases present wide phenotypic and genetic heterogeneity, as over 60 genes underlying nCHH and KS have been identified to date (1,2). However, several nCHH and KS genes remain to be discovered, since the currently known genes account for only half of all cases (1). nCHH and KS might be inherited in a Mendelian or oligogenic fashion depending on the causative gene; large cohort studies suggest that a significant proportion of the cases are oligogenic (1). reminiscent of KS. However, no clear link between the 9q31.2 deletions and complete KS currently exists. The closest connection comes from disruption or pathogenic variants in the closely located PALM2AKAP2 locus. Indeed, PALM2 and AKAP2, which can form fusion transcripts, constitute potential KS disease genes, since a female with KS and Graves' disease carried a missense mutation, which was predicted to be deleterious, in PALM2 (16), and a male with KS and bone anomalies carried a balanced chromosomal translocation that disrupted AKAP2 expression (17).
To investigate the putative relationship between the proband's 9q31.2 deletion and KS, we first defined the exact deletion breakpoints with linked-read whole genome sequencing. Next, we investigated if the deletion was associated with decreased expression of PALM2AKAP2 and screened this gene in a set of Finnish KS patients. Since these investigations did not support the connection between PALM2AKAP2 and KS, we performed whole exome and RNA sequencing in the proband and his family members to exclude defects in genes implicated in KS and to reveal potential new candidate genes.
Our results, together with the CHH and KS-related phenotypes in previous patients with 9q31.2 deletions, suggest that microdeletions in this chromosomal region underlie KS.

Subjects
We investigated a Finnish family whose son (the proband) had been assessed for delayed development and subsequently for the absence of puberty. He was the second child of healthy nonconsanguineous parents (Figure 1), and he had a healthy sister, who had normal timing of puberty since she reached menarche at the age of 12 years. The mother's menarche age had been 11 years, and the father had no reported history of delayed puberty. Following an uneventful pregnancy, the proband was born at gestation week H42+1. His birth weight was 3240 grams and length 50 cm. He received 4, 6, and 9 Apgar points at the ages of 1, 5, and 10 minutes, respectively. He was noted to have a muscular ventricular septal defect that closed spontaneously. His testes were normally descended. Before going to school, he was diagnosed with attention deficit disorder and mild developmental delay. He had distinctive facial features including ptosis, flat nasal tip, low set ears, and mild bilateral sensorineural hearing loss with normal semicircular canals in MRI. At the age of 6.5 years, he underwent comparative genetic hybridization (Agilent 44K), in which a 2.38 Mb deletion in the 9q31.2 area was detected, yet the precise breakpoints remained undetermined. The deletion was de novo, as his parents tested negative for the presence of the deletion.
At the age of 13.5 years, the proband was referred for the evaluation of delayed puberty and was noted to have a hypoplastic scrotum and testes. He had selfreported anosmia, which had been well appreciated in the family, and absent olfactory bulbs in the MRI. He had no synkinesia, hand or foot deformities, pigmentation defects or missing teeth, nor he reported problems with balance. Abdominal ultrasound revealed two kidneys. His circulating reproductive hormone levels were low (LH 0.1 IU/L, FSH 0.2 IU/L, testosterone 0.7 nM, and inhibin B level was very low, 14 ng/l). At the age of 14 years and Inc., San Diego, CA, USA) using S4 flow cell and XP workflow. Read length for the pairedend run was 2x151. The reads were aligned to the GRCh37 (hg19) reference genome.

Investigation of PALM2AKAP2 expression with RT-qPCR
The RNA extracted from the proband and his parents were converted into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions in a regular thermocycler with 1 µg of total RNA. The synthesized cDNAs were used as templates to assess the mRNA expression with a ready-to-use qPCR mix (Solis Biodyne) in a quantitative PCR machine (Roche LC480 II). The expression levels of PALM2AKAP2 were normalized to GAPDH. The relative expression levels were standardized against a healthy unrelated control sample, which was given an arbitrary value of 1.0. The PCR conditions and primers are available on request.

RNA sequencing
The quality and integrity of the RNA was assessed with high sensitivity D5000 Screen

RNA-sequencing data analysis
Raw adaptor-trimmed FASTQ files were subjected to quality assessment with the FASTQC tool (Simon Andrews, Babraham Bioinformatics). Based on the overall data quality and the individual reads, additional trimming was unnecessary. The sequences were aligned to the GRCh37 human genome version with the STAR package (30).
Splicing events of known KS genes were visually verified with the Sashimi plot feature of the MISO framework (31) in the IGV browser from Broad Institute (32).

Statistics and the probability estimate for the occurrence of a 9q31.2 microdeletion and KS
The relative expression values of PALM2AKAP2 in the PBLs of the proband (n=4) and his parents (n=4) were compared with one way ANOVA, and followed by Tukey's HSD posthoc analysis. P-value <0.05 was accepted to indicate statistical significance. To evaluate the role of the 9q31.2 microdeletion in our proband, we utilized the CNV data obtained

Ethics
Informed consents were obtained from all patients, and in the case of a minor/children, also a parent or guardian gave the consent. The study was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa and was conducted in accordance with the Declaration of Helsinki.

Results
We investigated the genetic and phenotypic features of a patient with KS and Weiss-  (Fig. 2). Instead, the deletion disrupted FKTN, which contains 10 exons, at 9:108,331,353 in the first intron, and no other known gene at 9:110,707,332. The deletion encompassed six protein-coding genes (FKTN, TAL2, TMEM38B, ZNF462, RAD23B, and KLF4), none of which has been implicated in KS.

PALM2AKAP2 expression in peripheral blood leukocytes
Given that PALM2AKAP2 has been implicated in KS and our proband's deletion laid approximately 1.8 Mb upstream of the PALM2AKAP2 locus (Fig. 2), we investigated whether the expression of PALM2AKAP2 was altered in peripheral blood leukocyte RNA.
However, the proband's PALM2AKAP2 transcript was expressed at a higher level than in the father (Figure 3), indicating that the deletion does not suppress PALM2AKAP2 expression. fulfilled the criteria of CHH (1). The ~35 kb region shared by our proband and Xu's patient (11), contained only a ribosomal pseudogene RNA5SP293 (Fig. 2). This region also contains binding sites for ZNF143 (a transcription factor which interacts with a pubertyrelated factor, LIN28B, in neuroblastoma cells (50)), JUND (JunD binds to GnRHR activator protein-1 (AP-1) site in mouse gonadotrope-derived αT3-1 cells (51), and is expressed in the mouse olfactory bulb (52)), GATA2 and GATA3 (crucial for neurogenesis and expressed in the olfactory bulbs (53)); and CEBPB (Cebpb in rats is expressed in the olfactory bulbs and olfactory ensheathing cells (54), and is known to be an upstream transcriptional regulator of Gnrh in mice (55)). However, the mechanism by which a loss of transcription binding site(s) alone would cause a disease is difficult to decipher, especially as the closest KS candidate gene locus (PALM2AKAP2) is relatively far away, 1.8 Mb, downstream from the 3' breakpoint of the deletion. This locus in our patient with KS was, however, spared and expressed PALM2AKAP2 efficiently (Fig. 3). Moreover, screening a set of Finnish KS patients revealed no pathogenic variants in PALM2AKAP2. Of note, the cases displayed in Fig. 2 showed wide phenotypic variability and we were able to verify KS-related features in only a proportion of them (see Supplementary Table 2        ***Patient's deletion (del(9)(q31.1q33.1)) was inherited from her unaffected mother with an inverted insertion (18;9)(q12.2;q33.1q31.1) ****Reported age within or at the borderline of the normal limit (± 2SD; 8 years -12 years) for Tanner breast stage M2

Rare variants of uncertain significance detected in WES
The proband harbored two maternally inherited, rare heterozygous variants of uncertain significance detected in WES (Supplementary Table 1