Orofacial clefts are the most common craniofacial human birth defect, affecting the lip only (CL), both the lip and the palate (CLP) or the palate alone (CP). CL and CLP can occasionally occur within the same pedigree. These defects are considered to arise from perturbations of a common pathway that underlies the growth or fusion of the maxillary, lateral and medial nasal processes, which form the upper lip and the primary palate.1 CP is considered to be a distinct developmental entity, which arises from a defective growth, elevation or fusion of the palatal shelves. The palatal shelves emerge from the maxillary processes to form the secondary palate, which separates the nasal and oral cavities.1, 2 Defective palatal shelf development can be evoked by genetic variants, environmental factors or a combination thereof.3 These hazards can be intrinsic to the palatal shelves, or can affect bones in the vicinity of the palatal shelves with a secondary effect on palatal development through mechanical hindrance (developmental deformation sequence). For example, palatal elevation depends on mandibular elongation.4 As the mandible elongates, the tongue is drawn forward and the palatal shelves are able to elevate and orientate horizontally. Therefore, the cleft palate can arise from primary defects of mandibular outgrowth, or from problems that are external to the mandibular skeleton but secondarily restrict its growth (eg, intrauterine constraint due to oligohydramnion).4 This triad of mandibular hypoplasia, glossoptosis and cleft secondary palate is referred to as Pierre–Robin sequence (PRS). Isolated PRS was found to be associated with variants in non-coding elements upstream of the SOX9 gene,5 whereas common genetic causes for syndromic PRS include Stickler syndrome (COL2A1, COL11A1, COL11A2), Campomelic Dysplasia (SOX9), trisomy 18 and 22q11 microdeletion syndrome.

Most clefts are isolated (70% of CL or CLP and 50% of CP), sporadic and likely have a multifactorial cause. Orofacial clefts, associated with developmental delay, dysmorphic features or other major congenital anomalies, are defined syndromic. These mostly have a single genetic cause, either chromosomal or monogenic. Genetic causes of syndromic and nonsyndromic CL, CLP and CP were recently reviewed.6, 7 Next to overt cleft lip or palate, the spectrum of orofacial clefts covers subclinical phenotypes, including microform clefts of the lip, defects of the musculus orbicularis oris,8 bifid uvula, submucous CP and velopharyngeal insufficiency. These subphenotypes may complicate genetic counseling and should be considered in genetic study design.

Palatal defects and PRS in association with schwannoma of the vestibular nerve have been recently described in patients with deletions of 22q12.9 Here we report on a further seven patients with a submicroscopic deletion involving chromosome 22q12, and presenting with multiple congenital anomalies, including a heterogeneous spectrum of palatal defects, vestibular schwannomas and corpus callosum agenesis (Table 1, Figure 1).

Table 1 Clinical and molecular data associated with 22q12.1 deletions in our patient cohort
Figure 1
figure 1

Facial dysmorphic features in patients with 22q12 deletions. (a) Patient 1 presented with a cleft soft palate and hypoplastic terminal phalanges. (b) Patient 3 aged 17 years. (c) Patient 5 at 5 years of age. (d) Patient 6 at the age of 6 years and 6 months. (e) Patient 7 at the age of 17 with postoperative facial nerve paralysis and mild dysmorphic features.

Case reports

Patient 1 – Decipher 287905

This 2.5-year-old boy was the second child of healthy, unrelated parents. Family history was unremarkable with regard to congenital anomalies or developmental delay. He was born at term after an uneventful pregnancy. His birth weight was 3.835 kg, his length was 52 cm and his head circumference was 36.5 cm (all at the 75th centile). At birth, he presented with facial dysmorphic features, including a large anterior fontanel, hypertelorism and a depressed nasal bridge. He had retrognathia and a hypoplastic soft palate with a cleft. The terminal phalanges and the nails of the toes were hypoplastic, and the nipples were widely spaced. He had feeding problems, requiring tube feeding. He was hypotonic, with uncontrolled, irregular movements. No epileptic activity was recorded on EEG monitoring. Brain MRI revealed the presence of corpus callosum hypoplasia. Eye exam and cardiac ultrasound were normal. No other organ malformations were noted. At 7 months of age, transtympanic drains were inserted to treat for serous otitis media with bilateral conductive hearing loss. Surgery for cleft palate was performed at the age of 15 months. At 2.5 years he weighs 13.3 kg (25–50th centile), his length is 90 cm (10–25th centile), and his head circumference is 50.7 cm (50–75th centile). He presented with strabismus en thoracic kyphosis. Moderate motor delay was noted. The patient was able to sit and walk, independently, at 10 months and at 2 years, respectively. At 2.5 years his receptive and productive language skills are in accord with those of a 15-month-old (Figure 1a).

A 22q11q12.2 deletion was detected in this boy by means of comparative genome hybridization (CGH) using a 180 k oligo array platform (OGT CytoSure Syndrome Plus array, OGT Oxford, UK), performed according to the manufacturer’s instructions. The deleted region spans maximally 4.3 Mb (25 689 977_30 038 041del) with the distal break point affecting the NF2 gene. This deletion was not found in the parents using the same array platform.

Patient 2

This 7-year-old girl was born as the first child from healthy, nonconsanguineous parents, after a pregnancy which was complicated by hyperemesis and polyhydramnios. Her birth weight and length (52 cm, 75th centile) were normal. She presented with a large anterior fontanel, and a posterior cleft of the soft palate, which was operated twice. She had an atrial septal defect, which was closed percutaneously in infancy. A right pelvic ectopic kidney was detected on abdominal ultrasound. At the age of 2, she underwent ophthalmic surgery for bilateral lateral rectus muscle palsy, causing strabismus. She was susceptible to recurrent respiratory infections. Brain MRI at 6 months revealed a thin corpus callosum with mild ventriculomegaly. No seizures were noted. Her motor development was severely delayed: head control, sitting without support and walking independently were obtained from the age of 1 year, 1.5 year and 3 years, respectively. She has an affable, friendly character, and uses simple words and three-word-sentences. She has had intensive physical and speech therapy. At the age of 7 years, she was unable to read or write.

At 7 years of age, biometry was normal, with a relatively large head (52 cm, 50–75th centile), in comparison with height (at 10th centile). Clinical evaluation revealed facial dysmorphism: wide forehead with depressed supraorbital ridge, short downslanting palpebral fissures, arched eyebrows, a prominent wide nasal bridge, wide alae nasi, malar hypoplasia and low-set, posteriorly rotated ears. She showed an open mouth appearance with a high arched palate and full lips. She had multiple teeth decay and retrognathia, requiring orthodontic treatment. She presented with a narrow thorax and long, slender fingers and toes. Thus far, no hearing loss was found (Figure 1b). Follow-up by means of brain and spine imaging was planned.

High-resolution genome-wide DNA copy-number analysis, using platform CytoScan HD Array (Affymetrix, Santa Clara, CA, USA), was performed according to the manufacturer’s protocol, and revealed a 3.58-Mb deletion in 22q12.1q12.2 (27 266 032_30 848 647del), comprising NF2. Data were evaluated with the Affymetrix Chromosome Analysis Suite software. Parental analysis by means of FISH analysis showed that the deletion occurred de novo.

Patient 3 – Decipher 256661

This 18-year-old girl was the second child of healthy, unrelated parents. Familial medical history was unremarkable. She was born at term. Her birth weight and length were 2885 g (25th centile) and 46 cm (10th centile), respectively. During the first year, her development was delayed and at 9 months she had a serious apnea event. Clinical evaluation revealed a unilateral choanal atresia, which was corrected surgically. Facial dysmorphic features included short palpebral fissures, hypertelorism, a prominent nose and maxilla, a receding chin and small ears. She presented with nasal speech and a high palate without overt clefting. She has had a late eruption of teeth and strabismus. Her fingers and toes were long. She had four café au lait spots. Ultrasound of the heart and kidneys was normal. She has been susceptible to infections and had suffered from meningitis at several occasions. She walked independently at two and a half year of age. Although her hearing was normal, her speech development was delayed. She had been diagnosed with mild intellectual disability and high functioning autism. At age 17, she presented with hearing impairment, mainly on the right side. Brain MRI revealed a large vestibular schwannoma on the right side and a smaller tumor on the left (Figure 1c). One year later, a 12-mm schwannoma emerged from the right hypoglossal nerve and progression of both vestibular schwannomas was noticed. MRI imaging of the spine at the age of 18 years, revealed 1–2 mm schwannomas in the roots of the second and third cervical nerve, left and right, respectively. In addition, a schwannoma, sized 4 mm, was detected in the cauda equina at the level of lumbar 2.

Genetic evaluation with chromosome analysis, subtelomeric FISH screening and mutation analysis of CHD7 were normal. Because of the combined picture of NF2-related symptoms with facial dysmorphism and developmental delay a chromosomal disorder was suspected. Array CGH, using the Agilent 244k platform (Agilent Technologies, Santa Clara, CA, USA), was performed according the manufacturer’s instructions, and revealed a 3.59-Mb deletion in 22q12.1q12.2 (26 727 554_30 313 733del), encompassing the NF2 gene. The deletion occurred de novo.

Patient 4 – Decipher 290734

This boy was born from healthy, nonconsanguineous parents, after an uneventful pregnancy. At birth, he presented with facial dysmorphic features, a broad hallux, tapered fingers and laryngomalacia. No palatal cleft nor velopharyngeal insufficiency were noted. Birth weight was normal. At the age of 2 years, biometry was normal, with a relatively short stature (84 cm, 9th centile), compared with his weight (13.7 kg, 75th centile) and head size 51.2 cm (75th centile). His development was globally delayed: he sat and walked independently since the age of 8 months and 18 months, respectively. At 2 years of age, he had a limited vocabulary, saying a handful of simple words. He showed copious drooling. His hearing was not impaired. Imaging of the brain has not been performed thus far.

Molecular karyotyping, using the Affy Cytoscan 750 k SNP genotyping array (Affymetrix), was performed according to the manufacturer’s instructions. It revealed a 3.4-Mb deletion on 22q12.1q12.2, ranging from 26 980 074 to 30 016 888, hereby presumably affecting the promoter region and first exons of the NF2 gene. Parental analysis, using the same array platform, showed that the deletion occurred de novo in the patient.

Patient 5 – Decipher 269448

This girl was born from healthy, unrelated parents with facial dysmorphic features, including epicanthic folds, a webbed neck and narrow ear canals. Birth weight was normal (3.997 kg). At birth, she presented with sublingual cysts, which resolved spontaneously. No overt cleft palate was noted. This girl developed a bilateral conductive hearing loss, which was attributed to bilateral glue ear. Hearing aids, as well as speech and language therapy, were introduced to improve speech, as she presented with nasal speech and speech delay, performing at the level of a 3-year old at the age of 5. She showed significant improvement with speech therapy, but today she still misses off ends of words and still has a nasal speech. Aged 5, normal motor and social development was observed. Weight and length were at the 25th and 75th centile, respectively. Head size was 49.5 cm (3rd centile). She was hypotonic and hypermobile, and presented with a 1-cm leg length discrepancy and a postural scoliosis. No brain imaging has been performed.

Molecular karyotyping was performed by means of the BlueGnome ISCA 8 × 60 k OligoArray v2.0 and analyzed with Bluefuse Multi software (BlueGnome Ltd, Cambridge, UK). A 2.17 Mb deletion on 22q12.1 was detected, ranging from 26 857 719 to 29 029 963. The deletion occurred de novo.

Patient 6 – Decipher 274296

At birth, this boy presented with neonatal vocal cord paralysis and arthrogryposis. In addition, he featured large dysplastic ears, an ogival palate and severe velopharyngeal insufficiency. He suffered from feeding problems and required a gastrostomy for enteral nutrition. Biometric parameters were all at the 50th centile. At 5 years of age, vocal language was totally absent, but he could communicate using pictograms and signs, and he understood simple orders (Figure 1e). Brain MRI revealed large ventricles and global cortical atrophy. His mother is healthy. Paternal data could not be retrieved.

A 22q12.2 deletion was detected by means of array CGH, using the Agilent 60 K array platform (SurePrint G3 Human CGH Microarray 8 × 60 K) and Agilent CytoGenomics software (Agilent Technologies), according to the manufacturer’s instructions, and was confirmed by FISH analysis. The deletion spans 0.93 Mb (27 375 542_28 307 008del), and comprises MN1, but not the NF2 gene. The deletion was not found in the mother by means of FISH analysis. The father was not available for further testing.

Patient 7 – Decipher 758

This girl was the third child of healthy, nonconsanguineous parents. Family history was negative with regard to malformations or mental handicap. She was born at term with birth weight 4 kg (90th centile), length 52 cm (75th centile) and head circumference of 35.5 cm (75th centile). She had feeding problems, with nasal regurgitation. Mild pulmonary valve stenosis and peripheral pulmonary stenosis were observed, which did not require any intervention. She had a cleft uvula. At the age of 3 years, she had surgery for strabismus. She had developed amblyopia, with residual vision of 1/10 at the left eye. She had a high myopia of – 5 and – 8 Diopter.

At the age of 4 years 8 months, she showed a normal growth. Her weight was 16.7 kg (at the 25th centile), her length was 104 cm (at the 25th centile) and head circumference was 52.5 cm (at the 90th centile). Facial features were not remarkable, with mild hypertelorism and smooth philtrum. She had long fingers and toes, with shorter 5th toes. The uvula was bifid. Speech was hypernasal due to velopharyngeal insufficiency, for which a pharyngoplasty was performed at age 5.

She walked at the age of 18 months, but especially her language development was delayed. At the age of 4 years and 9 months her total, verbal and performance IQ were 83, 85 and 84, respectively (Wechsler scales). She followed special education for children with mild learning difficulties. In addition, she was followed by a children’s psychiatrist because of hyperactivity. Brain MRI at 3 years of age was normal. When re-examined at the age of 14 years, she experienced no medical problems. Biometry was normal.

A tentative diagnosis of 22q11 deletion syndrome could not be confirmed by means of fluorescence in situ hybridization. This patient was described previously in a set of 60 patients with idiopathic syndromic heart defects, which were screened for submicroscopic anomalies by means of array CGH with an estimated resolution of 1Mb.10 A de novo microdeletion was detected on chromosome 22q12.2. The deletion break points were further delineated using a 180k oligo array platform, performed as described above (OGT CytoSure Syndrome Plus array). The deletion spans 1.15 Mb (29 281 852_30 429 113del) and comprises the NF2 gene. As haploinsufficiency of NF2 predisposes to neurofibromatosis type 2, brain MRI was repeated at 14 years of age, and revealed the presence of bilateral acoustic schwannomata, before clinical manifestation. A spine MRI was normal. Rapid tumor growth with progressive perceptive hearing loss at the age of 17 years necessitated for a resection of the left-sided acoustic schwannoma, which was complicated by a postoperative facial nerve paralysis (Figure 1d).


We report on seven patients with submicroscopic deletions within the 22q12.1q12.2 region. Informed consent was obtained from all patients or their legal representatives to share clinical data and pictures. Molecular and clinical details are summarized in Table 1, and were submitted to DECIPHER, a public database for chromosomal variants ( Deletion sizes range from 1.15 to 4.3 Mb. All deletion break points are unique, advocating non-homologous end-joining as the underlying pathogenic mechanism (Figure 2). Five out of seven patients present with palatal malformation or dysfunction, ranging from a high arched palate to overt cleft palate. Overt clefting of the secondary palate is observed in two patients. Mandibular hypoplasia is associated in three patients. Other recurrent features include schwannoma of the auditory vestibular nerve and agenesis of the corpus callosum.

Figure 2
figure 2

Overview of 22q12 deletions in our patient cohort, complemented with four previously reported cases9, 11, 12 and one Decipher patient (Decipher 4110) with a complex chromosomal rearrangement on chromosome 22, including a de novo 2 Mb deletion of 22q12.2 and a typical duplication of the diGeorge region on 22q11. Deletions associated with overt cleft palate are depicted in deep purple, those associated with minor palatal defects (high arched palate or velopharyngeal insufficiency) in light purple, and those without palatal malformations in white. All genome coordinates are according to NCBI human genome build 37 (hg19, February 2009). A full color version of this figure is available at the European Journal of Human Genetics journal online.

Palatal defects, including PRS, have been described in three patients harboring a 22q12.1q12.2 deletion9, 11, 12 and in one Decipher patient (Decipher 4110)9 with a complex chromosomal rearrangement on chromosome 22, including a de novo 2 Mb deletion on 22q12.2 and a typical duplication of the diGeorge region on 22q11. Clinical and molecular details are provided in Table 2 and Figure 2. Review of the literature for 22q12.2 deletions reveals a further three patients with vestibular schwannoma but without palatal defects,13, 14 and one additional Decipher patient (Decipher 999)9 with poor clinical data (Supplementary Table 1). Combining our data with those provided in literature, we discern 22q12.2 deletions in six patients with an overt cleft secondary palate (Figure 2), and in three with minor palatal defects, including velopharyngeal insufficiency, high arched palate and cleft uvula. The overlapping region for overt CP covers 1.47 Mb (chr22: 27 411 505 - 28 885 620) and contains three genes: MN1, PITPNB and TTC28, of which MN1 has been suggested as a candidate for CP and PRS before.9 MN1, an oncogene in murine hematopoiesis, is considered a candidate gene for sporadic meningioma15 and is involved in human myeloid leukemia.16, 17, 18 Mn1 is shown to be crucial in the development of membranous bones in the murine skull. In homozygous Mn1-knockout mice, cranial bone development is severely affected with decreased mineralization, hypoplasia or agenesis of several bones. In addition, palatal shelf elevation, particular in the middle and posterior regions, is impaired. This results in cleft secondary palate, leading to death of Mn1 null mice shortly after birth. Heterozygous Mn1 ± mice present an intermediate phenotype, including hypoplasia of the maxillary and palatal shelves and cleft secondary palate with incomplete penetrance.19 The role for MN1 in cleft palate development is further supported by the finding of an increased allele frequency (although not significant) at SNP rs5752638 of MN1 in patients with submucous cleft palate compared to control individuals.20 Moreover, Mn1 acts as a transcriptional activator of Tbx22 during palatal development in mice.21 Variants in the human TBX22 gene have been associated with X-linked cleft palate and ankyloglossia22, 23 and with isolated CP.24 Taken together, we assume that haploinsufficiency of MN1 contributes to the association of cleft palate in humans with 22q12.2 deletions, either resulting from primary defects in palatal growth or from secondary effects of other craniofacial abnormalities (eg, micrognathia). However, consistent with the mouse model, we observe incomplete penetrance for palatal defects. Patients 3, 5 and 6 present with retrognathia, a high arched palate or velopharyngeal insufficiency, but no overt clefting. Moreover, patient 7 features cleft uvula, but the detected 22q12 deletion is located beyond MN1. We assume that haploinsufficiency of other genes within the 22q12 region may modify MN1 expression or may independently affect the genesis of palatal defects.

Table 2 Clinical and molecular data previously described in patients with 22q12.1 deletions

Patients with loss of NF2 function through nucleotide changes within or deletions comprising NF2, a tumor suppressor gene on 22q12.2, are predisposed to Neurofibromatosis type 2 (OMIM: 101000,25). Constitutional NF2 mutations in combination with a somatic second hit of NF2 may lead to the development of schwannoma of the vestibular nerve with progressive hearing loss or tinnitus. In addition, other tumors, including schwannomas of other cranial nerves, peripheral nerve schwannoma, meningioma and ependymoma and non-neoplastic features such as cataracts, retinal hamartomas, epiretinal membranes, meningoangiomatosis and axonal polyneuropathy may manifest. Truncating NF2 variants are considered to cause a more severe phenotype, with younger age of onset, higher proportion of meningiomas and spinal tumors and higher number of peripheral nerve tumors, compared with those with an in-frame NF2 deletion26 or with a large deletion that comprises NF2 completely.13, 27 This hypothesis was challenged recently by the report of a severe NF2-related phenotype in a patient with a large 22q12 deletion, including NF2.9 Within our series of 22q12 deletions, two out of three patients with complete loss of NF2 presented with schwannomas of the vestibulocochlear nerve, one of which was identified before clinical manifestation. The NF2 phenotype in these two patients is considered severe, given the age at tumor onset, and the presence of peripheral nerve manifestations and spinal tumors in patient 3. The deletions in patient 1 and 4 only partially affect the NF2 gene. These chromosomal imbalances include the promoter region of NF2 and therefore we speculate that the tumor risk in these patients will be similar to those lacking a complete allele of NF2. Long-term follow-up, including brain and spine MRI, will permit more accurate delineation of the severity of the phenotype in all patients harboring a complete or partial germline loss of the NF2 gene. Haploinsufficiency of neighboring genes within 22q12 may alleviate or aggravate the patients’ phenotype. Co-deletion of CHEK2, a DNA repair and tumor suppressor gene on 22q12, in combination with NF2 may provoke a higher incidence of aggressive meningioma, as enforced Chk2 knockdown in human meningioma cells decreases DNA repair and increases chromosomal instability.28 To support this hypothesis, regular follow-up by clinical neurologic examinations, audiometry, ophthalmologic examination and imaging of brain and spine by MRI, is recommended in all patients harboring a 22q12.2 deletion, comprising NF2. The deletions detected within our cohort do not comprise SMARCB1 nor LZTR1. Constitutional loss-of-function variants in either of these two tumor suppressor genes located on 22q11 were shown to predispose to multiple schwannomatosis. The schwannomas in these individuals show a three-stage four-hit sequence with somatic loss of one copy of 22q together with inactivating somatic variants of NF2 on the remaining chromosome 22.29, 30

Agenesis or hypoplasia of the corpus callosum is associated with 22q12 deletions in two of the patients reported here, and in two previously described patients.11, 12 This region does not contain any known genes involved in corpus callosum development. The association of corpus callosum agenesis, PRS and facial dysmorphism fits the diagnosis of the Toriello–Carey syndrome, a rare presumable autosomal recessive entity with unknown genetic etiology (TCS; OMIM: 217980).31 Interestingly, 22q12 deletions were detected before in three patients with clinical diagnosis of TCS,12, 32 suggesting clinical resemblance between the 22q12 deletion syndrome and TCS. However, in our case series, the facial phenotype does not correspond to the TCS facial gestalt, which includes short palpebral fissures, hypertelorism, a small nose and full cheeks. Nevertheless, chromosomal analysis by means of array CGH is advisory in all patients with a clinical phenotype of TCS.


We further delineate a novel contiguous gene deletion syndrome on chromosome 22q12, featuring NF2-related schwannoma of the vestibular nerve and palatal defects with or without mandibular hypoplasia. We propose that the patients with a microdeletion comprising NF2 should be followed by a multidisciplinary NF2 team in addition to the follow-up for cleft palate and intellectual disability. Combining our results with those provided in the literature,9, 11, 12, 13, 14 eight patients are recorded with palatal defects in association with a 22q12 deletion, involving the MN1 gene. These observations, together with an increased MN1 SNP frequency in patients with submucous cleft palate,20 with the mouse expression data and the finding of craniofacial malformations including cleft palate in a Mn1-knockout mouse model further suggest that this gene contributes to the cleft palate observed in the reported patients.