Pubertal delay is defined as the lack of occurrence of secondary sexual characteristics at an age that is at least two standard deviations later than the population mean, as defined by Marshall and Tanner (1969, 1970). This means the absence of thelarche, that is, the appearance of the breast bud, at the age of 13 years in girls or of testicular volume increase, that is, >3 mL, at the age of 14 years in boys (Palmert and Dunkel, 2012). Amongst the genetic disorders underlying pubertal delay, hypogonadotropic (or central) hypogonadism (HH) explains 3.2% of the cases in girls and 13.3% in boys (Jonsdottir-Lewis et al., 2021). Hypogonadotropic hypogonadism may occur isolated or in association with anosmia or hyposmia, known as Kallmann syndrome (OMIM #308700) (Dwyer et al., 2023). The advent of high-throughput sequencing techniques has led to the understanding of the genetic basis of hypogonadotropic hypogonadism in patients with complex syndromes, such as CHARGE syndrome (OMIM #214800) due to CHD7 variants, Waardenburg syndrome (OMIM #613266) associated with SOX10 mutations, TUBB3 syndrome (OMIM #600638) due to E410K mutation in TUBB3 (Dentici et al., 2020), and Pallister–Hall syndrome (OMIM #146510) caused by mutations in GLI3 (Grinspon, 2022). In many of these syndromes, the reproductive phenotype remains underdiagnosed, with the clinical focus driven to the complex symptomatology. Recently, it has been suggested that hypogonadotropic hypogonadism may overlap with Witteveen–Kolk syndrome (WITKOS, OMIM #613406) (Liu and Mapow, 2020; Schnoll et al., 2023).

WITKOS is associated with heterozygous loss-of-function (LOF) variants in switch-independent 3 transcription regulatory family member A (SIN3A) (OMIM *607776) or 15q24 microdeletions encompassing SIN3A. This autosomal dominant disorder is characterized by a broad spectrum of neurological and physical phenotypes. Various degrees of intellectual disability, neurodevelopmental disorders, behavioral problems, and neurological symptoms have been well-established in the early descriptions of the syndrome (Witteveen et al., 2016). Typical facial dysmorphisms have been consistently reported. Additionally, abnormalities in growth, skeletal structure, hearing, visual disorders, gastrointestinal function, and ectodermal features have also been documented in patients with WITKOS (Balasubramanian et al., 2021). Early or late signs of congenital hypogonadotropic hypogonadism, such as micropenis, cryptorchidism, or pubertal delay, have only been reported in patients with 15q24 microdeletions (Liu and Mapow, 2020). However, whether hypogonadotropic hypogonadism is due to haploinsufficiency of SIN3A or any of the other eight genes located in the shortest region of overlap (∼260 kb) of 15q24 could not be ascertained (Magoulas and El-Hattab, 2012; Liu and Mapow, 2020). Our aim was to report the case of a girl with WITKOS and congenital hypogonadotropic hypogonadism carrying a pathogenic heterozygous frameshift variant in SIN3A and, furthermore, to conduct a literature review to find evidence supporting the hypothesis that hypogonadotropic hypogonadism could be an additional feature within the spectrum of WITKOS.

The propositus was a 6-year-old girl referred to us for short stature. She was born at 34 weeks by cesarean section due to oligohydramnios and intrauterine growth retardation (IUGR) and small for gestational age [1,510 g, −1.76 standard deviation score (SDS) for Argentine population (Lejarraga et al., 2009)]. She required mechanical ventilation due to respiratory distress syndrome and nutritional recovery after an episode of hypoglycemia in the neonatal period. During follow-up, she required attention for anemia and thrombocytopenia, recurrent bronchiolitis complicated with pneumonia, and febrile seizures. She was the second daughter of healthy non-consanguineous parents, with two siblings and four healthy paternal siblings. There was no family history of developmental delay. Her developmental milestones were delayed with multiple dyslalias and deficit in motor coordination. Based on neurological examinations, she was diagnosed with intellectual disability and began a speech therapy and school integration program. She also showed a behavioral disorder that was characterized as an attention-deficit hyperactivity disorder (ADHD).

At first visit, she presented with microcephaly (47.5 cm, −3.00 SDS), long face with a high anterior hairline, broad forehead, epicanthal folds, strabismus, wide medial eyebrows, wide nasal base with prominent nasal bridge, retrognathia, short and smooth philtrum, ears with overfolded helix and large lobes, hands with thin digits and bilateral radial deviation of the second digits, clinodactyly of the fifth finger, and joint laxity. Further assessment revealed sensorineural hearing impairment and a bicuspid aortic valve.

At the age of 6 years 10 months, her height was 109.2 cm (−1.62 SDS), below mid-parental height (164.6 cm, 0.60 SDS), and her weight was 15.4 kg (−2.54 SDS). At 7 years 8 months, her growth velocity was 4.2 cm/year (third centile), and her height SDS was impaired (−2.10 SDS; 110.6 cm). Routine blood tests (RBC, WBC, glucose, BUN, creatinine, SGOT, SGPT, cholesterol, triglycerides, and celiac antibodies) showed normal results. TSH, fT4, IGF1, IGFBP3, cortisol, prolactin, and insulin were within normal ranges. With a diagnosis of small for gestational age without catch-up growth, she was treated with recombinant growth hormone at 0.33 mg/kg/week with a good response: between 8 years 7 months and 11 years 8 months, the height velocity ranged between 5.4 and 7.4 cm/year (Figure 1). At the age of 14 years (Figure 2), she showed the absence of thelarche (Tanner stage M1) and pubarche (PH1); her height was 154 cm (−0.66 SDS); her growth velocity was prepubertal at 4.6 cm/year; and her bone age was delayed (12 years 6 months). She was therefore evaluated for delayed puberty.

To evaluate gonadotropin function, a GnRH IV infusion test was performed as previously published (Grinspon et al., 2010). In brief, the patient was subjected to a GnRH IV infusion test (100 μg GnRH, Luteoliberina; Elea SACIFyA, Buenos Aires, Argentina; 0.83 μg/min for 120 min) using the AVI 270 infusion pump (AVI Inc., 3M Healthcare, St. Paul, MN). Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in the serum at 0–120 min and estradiol at baseline in the serum sample. All hormones were measured using validated assays, as previously published (Bergadá et al., 2006). Serum estradiol was undetectable, and gonadotropins were low after the GnRH infusion test (basal LH 0.2 IU/L, peak 0.7 IU/L; basal FSH 0.1 IU/L, peak 0.6 IU/L), leading to the diagnosis of hypogonadotropic hypogonadism. Pelvic ultrasound showed a prepubertal normal uterus, and the ovaries were 1.0 and 0.9 cm³. Magnetic resonance imaging (MRI) showed bilateral agenesis of the olfactory bulbs (Figure 2), and anosmia was detected by olfactometry. Altogether, a clinical diagnosis of Kallmann syndrome was reached. Hormone replacement treatment was started gradually with normal evolution. During follow-up, severe scoliosis, aggressive behavior, and ulcerative colitis were found. The final height was 158 cm (−0.4 SDS).

The patient's medical history summary is shown in Figure 3.

The karyotype was 46,XX. To investigate a potential genetic etiology for syndromic hypogonadotropic hypogonadism, a next-generation sequencing (NGS) approach was followed.

In this work, we report the genetic diagnosis of a novel heterozygous frameshift pathogenic variant in SIN3A in a girl with clinical features of both WITKOS and Kallmann syndrome (hypogonadotropic hypogonadism with anosmia/hyposmia). No other potential candidate variants in the genes associated with Kallmann syndrome were found, and no clinically relevant CNVs were predicted from the WES data.

SIN3A is part of a core histone deacetylase complex, involved in transcriptional silencing mediated through interactions with repressors and corepressors. SIN3–HDAC–MECP2 corepressor complexes play an important role during various phases of embryonic development, such as cell cycle events and cell proliferation (Grzenda et al., 2009). Interestingly, MECP2 has been suggested to participate in the epigenetic control of human pubertal timing (Canton et al., 2023). On the other hand, Witteveen et al. (2016) demonstrated that SIN3A is expressed throughout the brain in developing mice, with higher levels in the subventricular zone, rostral migratory stream, and olfactory bulb. In vivo functional knockdown of Sin3a leads to reduced cortical neurogenesis, altered neuronal identity, and aberrant corticocortical projections. In humans, haploinsufficiency of SIN3A leads to a broad range of neurodevelopmental disorders, explained by reduced cortical neurogenesis (Witteveen et al., 2016). Altogether, the evidence from patients with WITKOS and animal model studies has allowed to suggest that alterations in cortical expansion would be a direct consequence of SIN3A haploinsufficiency, leading to a broad range of neurodevelopmental disorders. An impaired expression in the structures originating the olfactory bulb and GnRH neurons can explain the reproductive and olfactory phenotypes observed in our patient and another case recently reported with WITKOS and Kallmann syndrome (Schnoll et al., 2023).

Pubertal delay, cryptorchidism, and micropenis, which can be due to hypogonadotropic hypogonadism, have been reported in patients with WITKOS due to 15q24 microdeletions (Cushman et al., 2005; Magoulas and El-Hattab, 2012; Schnoll et al., 2023). The shortest region of overlap contains eight disease-related genes: COX5A, CYP11A1, EDC3, MAN2C1, MPI, SEMA7A, SIN3A, and STRA6. Of them, only SEMA7A plays a role in GnRH neuron migration in mice (Messina et al., 2011). However, which of the eight deleted genes present in the shortest region of overlap is responsible for the reproductive phenotype in patients with WITKOS has not been determined. Here, we describe the first female patient with pubertal delay due to Kallmann syndrome with WITKOS and a loss-of-function variant in SIN3A. The analysis of the other contiguous seven genes did not reveal any clinically relevant variant. Additionally, no clinically significant variants were found in the 96 candidate genes that we selected, according to the literature evidence linking them with hypogonadotropic hypogonadism. Interestingly, defects in SIN3A have recently been associated with syndromic congenital hypogonadotropic hypogonadism in two male patients with WITKOS, one of them with a 550-kb deletion at 15q24.1 and another with a nonsense rare variant in the N-terminal region of SIN3A (Schnoll et al., 2023). Altogether, these data indicate that SIN3A haploinsufficiency could underlie the reproductive phenotype in patients with overlapping phenotypes of WITKOS and Kallmann syndrome.

Like in many other complex syndromic disorders with neurodevelopmental and behavior issues, the neurologic symptoms and intellectual disability predominantly capture medical attention and drive patient management, interfering with a wider clinical assessment, which may explain the probable underdiagnosis of pubertal developmental disorders in patients with WITKOS. Indeed, cryptorchidism and micropenis can anticipate pubertal delay in male populations with hypogonadotropic hypogonadism, but may go undiagnosed if not specifically sought. In the female population, the situation is even more difficult given that there are no early clinical signs of hypogonadism before the age of puberty (Palmert and Dunkel, 2012). In the present case, the absence of thelarche and the endocrine compromise of the GnRH axis only at the age of 14 years drove the genetic assessment in search for the etiology of hypogonadotropic hypogonadism. The unexpected result of the WES study and the consequent reverse phenotyping allowed to reach the diagnosis of WITKOS and to explain the short stature, which was the reason for referral to the pediatric endocrinologist, together with the neurodevelopmental phenotype and multiple dysmorphic features. An early etiologic diagnosis is important for the establishment of a multidisciplinary management from an early age, for instance, therapy for neurodevelopmental delay; pharmacological treatment for psychiatric problems and epilepsy; and interventions for cardiac, digestive, and ophthalmologic problems. From the endocrinological standpoint, the surveillance of growth and pubertal onset impact the patient’s quality of life deriving from social adjustment related to the acquisition of secondary sexual characteristics, adult height, and sexual maturity in the less severe cases. Finally, further mechanistic studies will be necessary to elucidate the role of SIN3A in the regulation of the hypothalamic–pituitary–gonadal axis.