A 5.8 Mb interstitial deletion on chromosome Xq21.1 in a boy with intellectual disability, cleft palate, hearing impairment and combined growth hormone deficiency

Cytogenetically detectable chromosomal deletions involving chromosome X are rare in males due to the incompatibility with life of nullisomy for large portions of this chromosome. In particular, deletions at Xq21 are poorly described in the literature and only a few cases are reported of male patients with complex phenotypes including neurosensorial deafness, choroideremia, intellectual disability and dysmorphisms [1]. About 50 % of the carrier females display almost or completely skewed X inactivation, silencing the abnormal X resulting in a relatively benign phenotype [2]. The development of high resolution genomic arrays has made it possible to increase the imbalance detection rate with the identification of cryptic small rearrangements on the X chromosome, some of these containing only a few genes or even just a single gene [3, 4]. However, despite the improvement of the analysis resolution, rearrangements at Xq21 are still rarely reported.

Here we describe a male patient with an interstitial deletion of the Xq21.1 chromosome, determined through array comparative genome hybridization (aCGH). We sought to explain the clinical outcome, according to genotype-phenotype correlation, focusing on the genes possibly involved in the clinical phenotype including intellectual impairment, hearing loss, cleft-palate and short stature.

The patient was born at the 41st week of gestation by caesarean section due to fetal macrosomia. The birth weight was 4,530 g (>97° percentile), birth length 54 cm (90–97° percentile), and head circumference 40 cm (>97° percentile). Apgar scores were 9 and 10 at 1 and 5 minutes, respectively. The propositus was the only child of non-consanguineous Italian parents. The father was 36 and the mother was 28 years old. At birth, the patient showed cleft palate (plastic surgery of the hard palate was performed at the 5th month of life), hip dysplasia (corrected with retractor), right blepharoptosis (surgically corrected at 7 years of age) and dysmorphic face. At the fourth month of age, the parents noted the presence of severe head and limb hypotonia and a delayed psychomotor development. At 18 months, bilateral sensorineural hearing loss was diagnosed and a bilateral hearing aid was implanted.

The standard cytogenetic analysis revealed a normal male karyotype (46,XY). The patient proved negative for chromosome 22.11.2 deletion syndrome and fragile-X syndrome analysis. The family history was negative for intellectual disability.

Neurological examinations were performed, including a brain MRI at the age of 1 year with evidence of perinatal outcome suffering of the white matter, thinning of the corpus callosum and mild ventricular asymmetry. At 8 years of age, the brain MRI was repeated and showed the presence of mild ectasia of the perivascular spaces, soft signal hyperintensity of the periventricular white matter in the occipital region bilaterally as incomplete myelination.

At 3 years of age, because of short stature, a growth hormone (GH) stimulation test with arginine (GH peak: 5 ng/ml; n.v. > 10 ng/ml) and with glucagon (GH peak: 2.1 ng/ml; n.v. > 10 ng/ml) were performed and led to a diagnosis of complete growth hormone deficiency (GHD). GH replacement therapy was therefore started at the age of 4 years and stopped at the age of 9 years, for poor growth response. The patient did not return for further medical examination until the age of 15 years, when he was reassessed at our center for growth arrest associated with suspected hypogonadism (gonadal volume 2 cc bilaterally). His height was 151 cm (−2.23 SDS), BMI 26.97 kg/m² (1.47 SDS). The gonadotropin evaluation tests performed with luteinizing-hormone-releasing hormone (LHRH) revealed deficit of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with a LH peak of 4.1 mU/ml (prepubertal value) and FSH peak of 7.3 mU/ml (prepubertal value). The stimulation test with growth-hormone-releasing hormone (GHRH) and arginine confirmed the severe GHD (GH peak: 0.1 ng/ml; n.v. > 19 ng/ml). Insulin-like growth factor (IGF-1) was below the normal range (<25.0 ng/ml; −7.81 SDS). Adrenal, thyroid, liver and kidney function showed normal range values. The ECG and the cardiological examination were normal. A delayed bone age of 2 years compared with chronological age was detected. A pituitary MRI was also performed and showed a normal morphology of the adenohypophysis, smaller than normal for the age without significant alterations in signal intensity before, during and after the administration of contrast medium. The patient therefore restarted GH replacement therapy.

For the psychomotor retardation with hyperactivity, the child attends psychomotor and speech therapy regularly and he is currently being treated with Levomepromazine (75 mg/day) and Promazine (50 gtt/day). The boy has a poor ability to tolerate frustration and there have been some episodes of verbal aggression against its peers. A neuropsychiatric test (Wisc IV test) showed an intellectual disability with an IQ < 70.

Concerning the morphological aspect (Fig. 1), the patient presents gynoid habitus, abdominal fat, pseudogynecomastia (presence of only adipose tissue), hypoplastic genitalia with microtestes (volume of 2 ml), generalized hypotonia, genus valgus, pes planus, short toes, café au lait spot on the left shoulder and in the right lumbar region. Facial features (Fig. 2) are characterized by: macrocephaly, broad forehead, prominent nose root and coarse nasal pyramid, anteverted nares, white tuft of hair on the top of his head (Fig. 3).

The mother of the boy presents some atypical characteristics, such as coarse facial features, prominent frontal bossing, big hands and progressive hearing impairment. She showed normal height and good intellectual development.

At the age of 18 months, the patient was tested for the presence of causative mutations in the gene encoding connexin 26 (GJB2), as he manifested bilateral neurosensorial hearing loss. No mutation associated with hearing impairment was identified in this gene.

The array-CGH analysis, performed at the age of 13 years, revealed the presence of an interstitial deletion at Xq21.1 (77.456.818x1,77.489.632-83.255.802x0,83.287.869x1), of approximately 5.8 Mb (Fig. 4). The same deletion was present at the heterozygous state in the mother.

A paternal inherited deletion on chromosome 22q11.2 of about 105 kb (18.894.864x2, 18.905.139-19.010.478x1, 1.190.35352x2) was also identified, included within the 3 Mb deletion of the DiGeorge syndrome. However, it is likely a benign CNV as it is present in the Database of Genomic Variants (http://dgv.tcag.ca/dgv).

We report a maternally inherited deletion of approximately 5.8 Mb at Xq21.1, in a male subject with developmental delay, dysmorphic facial features, cleft palate, intellectual disability, short stature and hearing loss. The deleted region includes 14 OMIM genes: CYSLTR1, ZCCHC5, LPAR4, P2RY10, GPR174, ITM2A, TBX22, BRWD3, HMGN5, SH3BGRL, HMGN5, SH3BGRL, POU3F4, CYLC1 (Fig. 4 and Table 1). Among these, TBX22, BRWD3 and POU3F4 well explain some of the clinical features of this patient.

Mutations in the TBX22 gene are a well established cause of X-linked cleft palate with ankyloglossia as well as contributing to the prevalence of isolated cleft palate [5]. More rarely, other craniofacial anomalies including cleft lip and hypodontia have also been related to TBX22 variants [6]. The phenotypic spectrum of subjects bearing TBX22 mutations can vary, even within the same family, from asymptomatic females to males or females with a bifid uvula, a cleft of the soft palate, or a complete cleft of the hard and soft secondary palate, along with ankyloglossia [7].

Genetic hearing loss has an extremely varied etiology, with a plethora of genes involved in the autosomal and X-linked forms with overlapping phenotypes [8]. Among the main genetic defects, mutations within connexin 26 (GJB2) and connexin 30 (GJB6) are usually associated with severe to profound sensorineural deafness. Our patient had been screened for the presence of mutations in GJB2 before performing array-CGH, and was negative. The deafness causative gene is undoubtedly POU3F4, which causes the X-linked neurosensorial deafness DFN3. This defect is associated with either POU3F4 point mutations or small deletions in a region located at 900 Kb upstream of the gene and disturbing a regulatory element [9]. Male patients show a well characterized phenotype with both stapes fixation and progressive mixed hearing loss [10, 11]. Hearing loss was also observed in the mother of our patient as well as in about 40 % of the POU3F4 mutation carrier females. Carrier females usually exhibit a postlingual onset of the hearing impairment that progresses over time [12] with a variable expressivity attributable to variations in the degree of skewing of X inactivation.

Loss of function mutations affecting BRWD3 are associated with a phenotype including mild to moderate intellectual disability, macrocephaly, dysmorphic facial features, skeletal signs and behavioral disturbance [13, 14]. Among these alterations, a partial deletion encompassing 74 Kb and including the 30 last exons of the BRWD3 gene was reported in a male with high forehead, deep-set eyes, hypertelorism, short palpebral fissures, anteverted nares, downturned corners of the mouth, pointed chin, and skeletal anomalies. This phenotype is similar to that of the patient presented here: macrocephaly, dysmorphic facial features including prominent forehead and abnormal ears, behavioral disturbance, skeletal symptoms like pes planus, and cubitus valgus. These findings strongly suggest that the lack of BRWD3 causes intellectual disability, macrocephaly and possibly the skeletal symptoms (pes planus and cubitus valgus) observed in our patient. It has been demonstrated that BRWD3 plays a crucial role in ubiquitination, as part of the ubiquitin/proteasome system. In Drosophila, BRWD3 belongs to the CUL4-ROC1-DDB1 E3 ligase complex in which it acts as a CULLIN (CUL)4-associated factor that mediates light-dependent binding of CRY (Cryptochrome, a circadian photoreceptor) to the complex, inducing the ubiquitination of dCRY and its light-induced degradation [15]. The ubiquitin-proteasome system plays a crucial role in brain development and is a critical regulator of the synaptic plasticity and long-term memory formation [16]. Intriguingly, in humans mutations of the CUL4B, which encode a ubiquitin 3 ligase subunit cause an X-linked syndrome characterized by intellectual impairment, macrocephaly, central obesity, hypogonadism, pes cavus and tremor [17], a phenotype that largely overlaps with that observed in patients carrying BRWD3 mutations/deletions, including the present case. As both BRWD3 and CUL4B are part of the same complex it is likely that alterations of BRWD3 influence the ubiquitin-proteasome system similarly to other intellectual disability syndromes, whose prototype is represented by the Angelman Syndrome which is caused by mutations of the ubiquitin ligase-encoding UBE3A gene (MIM 105830).

The search in the Decipher and ISCA databases for similar size deletions at Xq21.1 led to the identification of six deletions in males with syndromic intellectual disability and developmental delay (DECIPHER 287069,253735, 257570,292180 and ISCA nsv533989, nsv530231, Fig. 4) and in one patient with no further phenotype details (DECIPHER 283100, Fig. 4) but none of these exactly corresponds to that of our patient. However, among those observed in female patients, there are two deletions (DECIPHER 262726 and 265126, Fig. 4) that completely cover that of our patient. The milder associated phenotype includes broad nasal tip, skeletal defects, speech delay, and cleft palate, which are also features of the present patient.

Other remarkable clinical signs of our patient are short stature and multiple pituitary hormone deficiency. The presence of mutations in PROP1, the most common genetic cause of combined pituitary hormone deficiency (CPHD) associated with short stature [18], was excluded. It is interesting that the deletion partially overlaps with regions duplicated in patients with multiple congenital anomalies, developmental delay, GH deficiency and short stature [19–21]. This suggests that Xq21.1 might contain either a protein coding gene or a regulatory element involved in pituitary hormone secretion. Within the 5.8 Mb region there is no evidence for a protein coding gene directly involved in pituitary functioning. However, ITM2A, included in the deletion, might be related to the severe short stature as it encodes an integral transmembrane protein involved in early cartilage development [22]. It has been suggested that the expression of ITM2A influences the chondrogenic differentiation potential of mesenchymal stem cells in vitro [23]. It might be hypothesized that the absence of ITM2A greatly influences the cartilage development with a possible impact on postnatal growth.

In conclusion, a detailed comparison of the clinical characteristics and the function of the genes included in the Xq21.1 delete region confirm the causative role of this rearrangement in our patient. Moreover, the presence of a gene involved in cartilage differentiation suggests that ITM2A might be responsible for growth impairment.

Written informed consent was obtained from the mother of the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.