Subtelomeric FISH analysis in 76 patients with syndromic developmental delay/intellectual disability

Background Intellectual disability affects approximately 1 to 3% of the general population. The etiology is still poorly understood and it is estimated that one-half of the cases are due to genetic factors. Cryptic subtelomeric aberrations have been found in roughly 5 to 7% of all cases. Methods We performed a subtelomeric FISH analysis on 76 unrelated children with normal standard karyotype ascertained by developmental delay or intellectual disability, associated with congenital malformations, and/or facial dysmorphisms. Results Ten cryptic chromosomal anomalies have been identified in the whole cohort (13,16%), 8 in the group of patients characterized by developmental delay or intellectual disability associated with congenital malformations and facial dysmorphisms, 2 in patients with developmental delay or intellectual disability and facial dysmorphisms only. Conclusion We demonstrate that a careful clinical examination is a very useful tool for pre-selection of patients for genomic analysis, clearly enhancing the chromosomal anomaly detection rate. Clinical features of most of these patients are consistent with the corresponding emerging chromosome phenotypes, pointing out these new clinical syndromes associated with specific genomic imbalances.


Background
Developmental delay (DD) and intellectual disability (ID) represent common conditions affecting 1 to 3% of the general population and it has been estimated that onehalf of the cases are due to genetic factors [1][2][3][4]. With a prevalence of 5 to 16% and 1 to 2% of cases respectively, trisomy 21 and Fragile X syndrome are the most common genetic causes of ID [5].
Routine cytogenetic analysis detects chromosomal aberrations involving at least 3-5 megabases (Mb) of DNA, in concordance with the technique resolution power. Fluo-rescent in situ hybridisation (FISH) overcomes this limitation allowing to investigate specific loci or subtelomeric regions for cryptic aberrations that are responsible of roughly 5 to 7% of all DD/ID cases [6,7]. These anomalies represent one of the most common causes of idiopathic DD/ID [8][9][10][11], being identified in 6.3% to 10.2% of moderate to severe DD/ID and in a significantly lower rate, less than 1%, in mildly retarded patients [12]. Major malformations and/or dysmorphisms, pre and post-natal growth retardation, and/or positive family history can be observed in the majority of patients with moderate to severe DD/ID related to criptic chromosomal imbalances [9,[13][14][15]. As a matter of fact, clinical pre-selection of DD/ ID patients improves the detection rate. It has been suggested for this purpose the use of specific check-list, such as the five item of De Vries et al. [16]. In recent years the study of genotype-phenotype correlations of these anomalies has allowed the definition of new emerging chromosomal phenotypes [15,17,18]. The recent introduction of the technology of array comparative genomic hybridization (CGH), that allows the detection of submicroscopic copy number variations in the whole genome, represents the next step forward in this effort.
We performed subtelomeric FISH analysis in 76 unrelated children affected by various degree of DD/ID, congenital malformations (CM) and facial dysmorphisms (FD), with normal standard karyotype. Ten subtelomeric anomalies have been identified (13.16%), underlying the role of cryptic subtelomeric anomalies in the pathogenesis of complex clinical presentation associated with DD/ID.

Patients
Seventy-six patients, aged from 3 days to 14 years, recruited at the Department of Pediatrics, University of Torino, were enrolled in the study. In order to better define the clinical features correlated with chromosomal subtelomeric imbalances, we have divided the cohort in three subgroups: 32/76 patients (42,1%) with DD/ID associated with CM and striking FD, 18/76 patients (23,68%) with DD/ID associated with FD, 26/76 patients (34,21%) with DD/ID associated with CM and not relevant FD.

Methods
Routine cytogenetic analysis at 400-550 bands level was performed in all patients and it did not detect any imbalance. Chromosome preparations from peripheral blood cells were used for FISH analysis. The Chromoprobe-T kit with telomeric specific clones was used according to the supplier's instructions (Cytocell, UK) with minor modifications. When a criptic subtelomeric rearrangements was identified by FISH, prometaphase chromosomes were re-analysed in order to rule -out if the rearrangement could have been detected in retrospect.

Patient 1
A 4 year-old girl was evaluated for DD and seizures. She was born at 37 weeks of gestation, after an uneventful dizygotic pregnancy, with a birth weight of 2.45 kg (3 rd percentile), length 48 cm (10 th percentile), head circumference 32 cm (3 rd percentile). APGAR scores were 7 and 8 at the first and fifth minute respectively. DD was evident in the first two years of life: she sat alone at 13 months and she stood at 27 months. Partial seizures with focal irritative complexes at EEG were diagnosed at 12 months of age. When she was evaluated frontal bossing, convergent strabismus, straight eyebrows, deep-set eyes and low-set ears were noted. She was not able to walk and language was limited to few words. Subtelomeric FISH analysis detected a de novo terminal deletion of chromosome 1p (46,XX.ish.del (1p)(pVYS218C-)).

Patient 2
A 16 month-old girl was evaluated for DD and seizures. She was born at 37 weeks of gestation by spontaneous delivery after an uneventful pregnancy. Neonatal weight was 2.49 kg (25 th percentile), length 47.8 cm (25 th percentile), head circumference 32.2 cm (3 rd percentile). APGAR scores were 9 at the first and fifth minute. At birth, a ventricular septal defect (VSD) and a bicuspid aortic valve were detected by ecochardiogram. She was able to sit alone at 7 months, but indipendent walking was still not achieved at 16 months. At 10 months she presented generalized seizures characterized by gaze, circumoral cyanosis, masticatory movements. Right temporal spike and wave activity, associated with irritative multifocal anomalies were recorded on EEG; even if valproic acid therapy Facial aspects and specific malformations in patients with criptic subtelomeric anomalies

Patient 6
A 1 year-old boy was referred for DD and CM. Family history revealed a maternal uncle affected by ID and progressive chronic renal failure who died at 16 years of age for acute pneumonia. The child was born by spontaneous delivery at 36 weeks of gestation after a pregnancy complicated by IUGR, oligohydramnios, mild hypertension and gestational diabetes. Birth weight was 2.13 kg (< 3 rd percentile), head circumference 30 cm (3 rd percentile), length not reported. He presented a complex cranial vault anomaly, characterized by brachicephaly and anterior plagiochephaly, associated with bilateral renal hypoplasia. When he was evaluated at 12 months of age, weight was 5 kg (<< 3 rd percentile), length 56 cm (<< 3 rd percentile), head circumference 44 cm (3 rd percentile); bilateral ptosis, micrognathia with hypoplastic teeth and low-set ears were evident. Cerebral MRI revealed a hypoplastic corpus callosum. Surgical correction of cranial vault anomaly was performed, with good aesthetic and neurologic outcome. At 3 years of age, he developed chronic renal failure; kidney biopsy was consistent with membranous glomerulonephritis and he underwent kidney transplantation at the age of 4. At follow up at 4 years, weight was 9.5 kg (<< 3 rd percentile), length 85 cm (<< 3 rd percentile). When he was revaluated at 8 years of age, a good recovery of auxometric parameters was evident, being his weight 25 kg (50 th percentile) and his height 121 cm (50 th percentile). Moreover he was attending a normal school program, with a backup teacher. Subtelomeric FISH analysis revealed an unbalanced maternally derived translocation with a partial trisomy of chromosome 6p and partial monosomy of chromosome 1q (46, XY, ish der(6) t(6;1)(p22.3;q44)mat). The balanced chromosomal anomaly identified in the mother was also present in a phenotypically normal maternal uncle.

Patient 7
A 3 day-old boy was evaluated for hypotonia, CM and FD.
He was born at 36 weeks of gestation after a pregnancy complicated by IUGR. A previous miscarriage was reported. Birth weight was 1.8 kg (< 3 rd percentile), length and head circumference were not reported. At birth, facial dysmorphisms, microcephaly, macrotia, hypospadia, bilateral cryptorchidism, anal atresia and club feet were noted. Chorioretinal coloboma, neurological bladder, VSD, intestinal malrotation with right diaphragmatic hernia complicated the phenotype. Multiple skeletal abnormalities including fusion of the 10 th and 11 th ribs, sacral agenesis, congenital bilateral hip dislocation and luxation of the right rotula were observed at X-ray. At follow-up at 2 years of age severe DD was evident, being the child not able to sit nor to speak. Weight was 9.5 kg (< 3 rd percentile), length 70 cm (<< 3 rd percentile), head circumference 42 cm (<< 3 rd percentile). Subtelomeric FISH analysis revealed a maternally derived unbalanced translocation, resulting in a partial deletion of chromosome 7q and partial duplication of chromosome 12q (46, XY. ish der(7) t(7;12)(q34;q24.32) mat).

Patient 8
A 7 month-old girl was evaluated for CM and FD at birth. Ultrasound detection of cystic hygroma in the first trimester led to perform karyotype on chorionic villi, resulting in a normal female karyotype. She was born at term, with a birth weight of 3.17 kg (25 th percentile), length 48 cm (25 th percentile), head circumference 33.5 cm (25 th percentile). APGAR scores were 8 and 9 at the first and fifth minute, respectively. At birth, right microtia, hypertelorism, epichantal folds, micrognathia and pterigium colli were noted. Acoustic oto-emission screening test was normal. Posterior embriotoxon was observed on ophtalmological examination. At 7 months of age, weight was 6.8 kg (3 rd percentile), length 64 (25 th percentile), head circumference 42 cm (25 th percentile). Mild motor DD associated with persistent neonatal phenotype, suggested the subtelomeric analysis, which revealed a de novo, chromosomal anomaly consistent with a partial monosomy of the short arm of chromosome 6 and a partial duplication of the long arm of the same chromosome (46, XX, ish der(6)(ptel-;qtel++) de novo). The long term follow-up at 5 years revealed a normal cognitive and motor development and auxometric parameters within normal limits (50 th percentile).

Patient 9
A six day-old female was evaluated for hypotonia, CM and FD. Family history was remarkable for a first trimester spontaneous miscarriage. She was born at 41 weeks of gestation by caesarean section after a pregnancy complicated by IUGR. Birth weight was 2.2 kg (< 3 rd percentile), length 46.5 cm (3 rd percentile) and head circumference 31.5 cm (< 3 rd percentile); APGAR scores were 6 and 8 at the first and fifth minute, respectively. Severe hypotonia, flat facies, down-slanting palpebral fissures, high-arched palate, low-set ears and II, III, IV toes syndactyly were observed, associated with ASD and VSD. Tracheostomy was placed in the first month of life to prevent recurrent apnea and severe desaturation, and a feeding tube was positioned up to the first year of life. Cerebral MRI performed at 1 year of age revealed fronto-temporal cerebral atrophy. Follow-up at 2 years of life revealed severe DD, characterized by absent speech, poor head control and ineffective deglutition reflex. At 3 years of age, head circumference was 44.5 cm (<< 3 rd percentile), length 95 cm (50 th percentile), weight 11 kg (3 rd percentile). Language and independent walking were absent. Subtelomeric FISH analysis revealed a de novo unbalanced translocation with partial distal monosomy of chromosome 5p and partial distal trisomy of chromosome 10q (46, XX, ish der(5)t(5pter;10qter) de novo).

Patient 10
A 7 year-old boy was evaluated for mild ID and FD. Family history was unremarkable for ID and CM. He was born at term after an uneventful pregnancy, with birth weight of 3.8 kg (75 th percentile), length 51 cm (50 th percentile), head circumference 36.5 cm (97 th percentile). APGAR scores were 9 and 10 at the first and fifth minute, respectively. Independent walking was reached at 24 months of age and language was completely absent at the age of 36 months. When he was evaluated at 7 years of age, a mild cognitive impairment was observed. Weight was 21 kg (50 th percentile), length 114 cm (50 th percentile) and head circumference 55 cm (> 97 th percentile). Facial dysmorphisms including prominent forehead, deep-set eyes, thick superior lip and prominent inferior lip first suggested the clinical diagnosis of α-talassemia mental retardation X linked syndrome (ATRX-syndrome; OMIM Number 30032). Cerebral MRI, EEG, visual evoked potentials (VEP), electroretinogram (ERG) and metabolic workup were normal. Subtelomeric FISH analysis revealed a de novo translocation, consisting with a partial monosomy of 13q and partial duplication of 1p (46, XY, ish t(1;13)(p32.2;q31.1) de novo).

Discussion
Chromosomal subtelomeric anomalies are a relevant cause of DD/ID and birth defects [14,20,21]. Here we present the results of a subtelomeric analysis performed on 76 paediatric patients, aged from 3 days to 14 years, presenting with a complex clinical phenotype associated with DD/ID. Ten cryptic chromosomal anomalies have been identified in the cohort, with a detection rate of 13.16%: 3 de novo deletions, 4 unbalanced translocations of parental origin, and 3 de novo unbalanced translocations. Interestingly we have observed relevant differences in the detection rate among the 3 groups identified on clinical features. In particular, the detection rate was 25% in the first group of patients (DD/ID, CM and FD), percentage reduced to 11,11% in the second one (DD/ID and FD), while no anomalies were identified in the third group (DD/ID and CM). This gradient in the detection rate reflects the probability that a chromosomal imbalance is responsible for complex developmental disturbances and leads to relevant facial dysmorphisms.
Some frequent cryptic telomeric anomalies are characterized by a specific phenotype and are emerging as recognizable subtelomeric syndromes, such as 1p terminal deletion (Patients 1-2). The phenotype is characterized by neurodevelopmental disability and a recognizable pattern of malformation associated with specific facial dysmorphisms, straight eyebrows, deep set eyes and frontal bossing that may direct towards the correct clinical diagnosis [10,17,18,22,23]. Also terminal 9q deletion (Patient 3) is a new emerging recognizable phenotype, described in about 30 patients [24], being characterized by severe ID, hypotonia, brachycephaly or microcephaly, flat face with hypertelorism, synophrys, anteverted nares, a distinctive mouth with macroglossia and a thickened lower lip, and congenital hearth defects. Moreover, sleep disturbances, autistic features and obesity due to food seeking behaviour have been reported in those patients [24][25][26]. Agenesis of corpus callosum associated with diaphragmatic hernia characterized Patient 4, suggesting the diagnosis of Donnai-Barrow syndrome (OMIM 222448). Subsequently an unbalanced t(9;16)(9qter;16q24.3qter)pat with partial monosomy 9q34-qter and partial trisomy 16q24-qter has been identified suggesting the hypothesis that 9q terminal deletion can cause Donnai-Barrow syndrome [19]. Patient 5 carried an unbalanced paternally derived der(20) t(16;20)(q24;q13.3), with partial 16q trisomy and 20q monosomy. Trisomy 16q24 is a clinical recognizable phenotype characterized by periorbital oedema in the neonatal period, generalized hypotonia, failure to thrive, severe DD/ID and a distinctive facies with a high forehead and bitemporal narrowing, associated with a variable genital and anal abnormalities [27,28]. This patient presented basopenienal hypospadia, supporting the hypothesis that one or more genes involved in uro-genital development are located in this chromosomal region [29,30]. Deletion of 20q13 is a very rare anomaly and it is associated with severe malformations of the limbs, short neck, flat occiput, midfacial dysmorphism, and failure to thrive [31][32][33]. Patient 6 carried a maternally derived unbalanced translocation, consisting with 6p trisomy and 1q monosomy. He presented a complex craniosynostosis, renal hypoplasia, hystologically characterized by membranous glomerulonephritis, leading to progressive renal failure. Interestingly, Pierpoint et al described a patient carrying a 6p terminal trisomy, who presented a striking overlapping facial phenotype associated with progressive renal failure [34]. These observations allow the hypothesis that a gene locus for syndromic renal failure maps to 6p22.3-pter. Moreover, we propose 6p terminal deletion as a new chromosomal syndrome, characterized by a peculiar facial appearance, renal failure and DD/ID. Interestingly the patient we described reached a good cognitive development and school performance after the surgical correction of the craniosynostosis, the kidney transplantation and an intensive rehabilitation program. Chromosome 1q terminal deletion [16,[35][36][37] is emerging as a new specific chromosomal phenotype characterized by DD/ID, pre-postnatal growth retardation, microcephaly, seizures, bow shaped eyebrows, hand -foot anomalies and midline defects, including corpus callosum hypoplasia [36,38], and may probably contribute to the pathogenesis of the complex phenotype of this patient. The clinical presentation of Patient 7 confirms the role of genes mapping to terminal region of 7q in caudal regression pathogenesis [39]. Moreover, the peculiar facial traits, such as microcephaly, hypertelorism, epicanthus, coloboma/cataract, blepharoptosis/phimosis, broad nasal bridge with bulbous nasal tip, ear anomalies, short neck, and abnormal genitalia could be considered a specific phenotype for 7q34-qter deletion, as argued by Lukusa et al. [40]. Partial terminal 12q duplication is a rare event described in about 35 cases with mild phenotypical effects [41,42]. In Patient 8 facial dysmorphisms, characterized by hypertelorism and a striking external ear malformation, short neck associated with early motor developmental delay led to the clinical suspicion of 6p terminal deletion subsequently confirmed by subtelomeric FISH analysis. She is now 6 years old and she displays normal cognitive development at periodic clinical evaluation. This patient confirms that criptic chromosomal aberrations do not always result in DD/ID, suggesting caution in the definition of prognosis, particularly in prenatal genetic counselling. More extensive reports about the size of subtelomeric imbalances in normally neurodeveloped patients are needed in order to investigate which subtelomeric loci are implicated in syndromic DD/ID [43]. In Patient 9, IUGR, severe hypotonia associated with flat facies, hypertelorism, down-slanting palpebral fissures, and toes syndactyly first suggested us the clinical hypoth-esis of Smith-Lemli-Opitz syndrome (OMIM 270400). Subtelomeric FISH analysis revealed a partial monosomy 5p associated with trisomy 10q, already described in a single patient with an overlapping phenotype, characterized by hands and feet anomalies and facial dysmorphisms [44]. Patient 10 carried a partial monosomy 13q31.1 and partial trisomy 1p32.2. Eight patients with pure monosomy 13q33 have been previously reported, delineating a well-recognized syndrome, characterized by growth and psychomotor retardation, microcephaly and deficiency of coagulation factors VII and X [44,45]. Partial duplication of chromosome 1p has been described in 10 patients: major characteristics are abnormal genitalia in males, congenital heart defects, craniofacial and hand anomalies [46].

Conclusion
Our report illustrates the relevance of careful dysmorphologic evaluation of patients with DD/ID variably associated with CM and/or FD as an useful tool for pre-selection for genomic analysis. This is particularly noteworthy in respect of the recent availability in the clinical setting of new technologies, such as array genomic hybridization, that allow whole-genome screening for sub-microscopic genomic anomalies, not previously detectable by conventional methods. Literature reports of patients affected by subtelomeric imbalances are very helpful in increasing clinicians awareness of subtle chromosomal phenotypes, and in revealing genomic candidate region for specific developmental defect. Paediatricians and clinical geneticists might improve their expertise in correlating new specific patterns of congenital anomalies with specific chromosomal imbalances, in order to optimize, both in terms of time and cost, the yield of chromosomal studies.