Developmental delay (DD) is a condition wherein developmental milestones and learning skills do not occur at the expected age range for patients under 5 years of age. Areas used for evaluating developmental stages are gross and fine motor skills, speech and language skills, cognition, and personal-social development. Intellectual disability (ID) is characterized by limited or insufficient development of mental abilities, including intellectual functioning impairments, such as learning and cause-effect relationship [1]. Intellectual disability cases are often diagnosed in the early school-age period. The incidence of DD is 1.0–3.0% in the general population, whereas that of ID is approximately 2.7% among early school-age children [2]. Some cases have DD or ID as the only finding and are called isolated cases. Conversely, cases accompanied by facial dysmorphism, autism spectrum disorder (ASD), epilepsy and congenital anomalies, are called syndromic DD/ID [3]. Recent studies have shown that biological signaling pathways causing DD/ID, ASD, and epilepsy phenotypes are common. Additionally, the relationship between signaling pathways involved in early brain development, synaptic plasticity, and neuronal migration and the formation of these phenotypes has been demonstrated [4].
Isolated and syndromic DD/ID cases show extreme genetic heterogeneity. Genetic etiology can be detected in approximately 40.0% of the cases, whereas chromosomal abnormalities are observed in 25.0% [5,6]. Conventional cytogenetic testing can be used for detecting ≥5 Mb chromosome abnormalities. Moreover, specific chromosomal abnormalities can be investigated using fluorescence
The study included 139 patients diagnosed with isolated or syndromic DD/ID (78 females, 62 males) at the Department of Pediatric Neurology, Giresun University, Giresun, Turkey; Department of Medical Genetics, Karadeniz Technical University, Trabzon, Turkey; Department of Medical Genetics, Erzurum City Hospital, Erzurum, Turkey and Department of Medical Genetics, Marmara University, Istanbul, Turkey. All patients were evaluated a by medical geneticist for dysmorphologic phenotyping. Patients with abnormal metabolic and thyroid function test results, brain tumor, brain infection, and signs of hypoxic ischemic encephalopathy, were excluded from the study. All cases were evaluated using prenatal history, family history, anthropometric measurements, detailed dysmorphological examination, hearing examination, eye examination and cardiac analysis (echocardiography). Electroencephalogram (EEG) and brain magnetic resonance imaging (MRI) tests were performed in cases where it was deemed necessary. For genetic analysis, blood samples were obtained from all patients whose parents provided written informed consent.
All experimental procedures were conducted in accordance with the principles of the Declaration of Helsinki, and informed written consent was obtained from patients or their guardians. This was a retrospective clinical study approved by Erzurum Research and Training Hospital Ethics Committee, Erzurum, Turkey [Approval #2020/23-219].
All patients first underwent standard karyotyping using the G-banding technique. At least 20 metaphases were analyzed at 450–500 band resolution for each patient. Chromosomal abnormalities were reported according to the recommendations of the International System for Human Cytogenetic Nomenclature 2016 [12].
For aCGH analysis, genomic DNA was isolated from peripheral blood leukocytes using Siam® DNA Mini Kit (Qiagen GmbH, Hilden, Germany). Affymetrix CytoScan Optima 315K arrays (Thermo Fisher Scientific, Waltham, MA, USA) were used according to the manufacturer's instructions for detecting CNVs. The aCGH results were evaluated using Chromosome Analysis Suite version 3.1.0 (Thermo Fisher Scientific). Technical specifications of the aCGH platform are available on the manufacturer's website (
The study included 139 cases (77 females, 62 males) who met the patient selection criteria. The mean age was 6.3 ± 5.1 (range 1.0–25.0). Data analysis revealed 38 different CNVs in 35 cases. The average size of CNVs was 7.01 ± 11.38 Mb (range 0.215–50.379 Mb). Of the 38 CNVs, 19 were gains and 19 were losses. Additionally, 73.6% (28/38) of all CNVs were
In this study, 19 cases with pathogenic CNVs (13.6%,
Of the 35 cases detected with CNVs, 16 had microcephaly, 15 had epilepsy, three had ASD, 14 had facial dysmorphism, 10 had short stature, two had congenital heart defect, and 10 had structural brain anomaly. The demographic and clinical findings and detailed neurological findings of the patients are summarized in Tables 1 and 2, respectively.
Clinical and genetic features of the patients.
1 | M-4 | DD, hypotonia, short stature, microcephaly, micrognathia, small mouth, proximally placed thumb, fifth finger clinodactyly, broad forehead, strabismus, uplanting palpebral fissures, scoliosis | 46,XY | 1721.32q21.33 (47,346,528–48,900,875)×3 | 1554 |
2 | M-6 | DD, epilepsy, uncal dysplasia | 46,XY | Xp11.23 (48,888,996–49,401,262)×2 |
512 |
3 | M-1 | DD, IUGR, short stature failure to thrive, microcephaly, round face, low-set ears, epicanthus, hypotonia, cat-like cry | 46,XY | 5p15.33p15.2 (113,576–14,739,104)×1 | 14,625 |
4 | M-6 | DD, VSD, curly eyelashes, thin upper lip, prominent methopic suture, synophrys, triangular face, large ears, epilepsy | 46,XY | 8q24.21q24.3 (130,459,411–140,444,375)×1 | 9985 |
5 | F-5 | DD, epilepsy | 46.XX.der(8) |
8p24.3p23.1 (158,048–10,161,482)×1 |
10,003 |
6 | F-3 | DD, short stature, failure to thrive, hypotonia, large ears, depressed nasal bridge, thin upper lip, epilepsy | 46,XX | 2q12.2q12.3 (106,925,594–188,257,773)×3 | 50,379 |
7 | F-3 | DD, bifid thumb, microcephaly, strabismus, broad nasal tip, depressed nasal bridge, telecanthus, short neck, low-set ears, epilepsy | 46,XX,dup(4) |
4q28.2q35.1 (137,877,879–188,257,773)×3 | 50,379 |
8 | F-1 | DD, microcephaly, short stature, IUGR, prominent glabella, short philtrum, strasbismus, hypertelorism, epicanthus, epilepsy | 46,XX | 4p16.3 (68,345–1,881,435)×1 | 1800 |
9 | F-4 | DD, short stature, micrognathia, low-set ears, hyperterlorism, short philtrum, hypocalcemia | 46,XX | 23q11.21 (18,894,820–20,311,733)×1 | 1416 |
10 | M-1 | DD, microcephaly, hypertonicity, epilepsy | 46,XY,der(3) |
3p26.3p26.1 (61,891–5,528,884)×1 |
5467 |
11 | F-1 | DD, hypotonia, iris coloboma | 46,XX | 15q13.1q13.3 (29,013,163–32,915,723)×1 | 3900 |
12 | F-4 | DD, epilepsy, ataxia, broad nasal tip | 46,XX | 6q21q23.31 (114,502,807–121,158,975)×1 | 6656 |
13 | F-1 | DD, hypotonia, brachycephaly, long eyelashes, small philtrum, telecanthus, pectus excavatum | 46,XX | Xp22.2 (11,279,310–12,016,067)×4 | 737 |
14 | M-10 | DD, epilepsy | 46,XY | 9q13q21.11 (68,240,211–70,984,588)×1 | 2744 |
15 | M-3 | DD, sensorineural hearing loss, ptosis, microcephaly | 46,XY | 9p24.3 (204,193–500,584)×3 | 296 |
16 | F-10 | DD, ASD, microcephaly, hypertonicity, self mutilation, optic atrophy, EEG abnormality | 46,XX | 16p12.2 (21,601,714–21,816,543)×1 | 215 |
17 | F-8 | DD, epilepsy, hypertonicity, hydrocephaly, obesity, short stature | 46,XX | 3p12 (44,626,845–45,983,652)×1 | 1357 |
18 | F-10 | DD, webbed neck, epilepsy, tall stature | 46,X,der(X) | Xp22.2p21.3 (14,036,105026,666,672)×3 | 126 |
19 | M-6 | DD, ADHD, VSD, epilepsy, hypotonia, microcephaly | 46,XY | Yp11.32q11.223 (118,546–25,415,912)×2 | 25,287 |
20 | F-7 | DD, pachygyria, lissencephaly, microcephaly, hypertonicity, epilepsy | 46,XX | 8q24.23 (137,278,410–138,539,014)×3 | 1261 |
21 | M-4 | DD, microcephaly, epilepsy, hypertonicity, macrodontia, optic atrophy, limb contractures | 46,XY | 16p13.11p12.3 (16,295,900–16,873,547)×1 | 578 |
22 | F-3 | DD, microcephaly | 46,XX | 14q32.33 (106,505,480–107,285,437)×1 | 780 |
23 | F-2 | DD, ASD, microcephaly, epilepsy, cone dystrophy | 46,XX | 8p1.21p11.1 (42,908,376–43,822,214)×3 | 914 |
24 | F-1 | DD, microcephaly, short stature, failure to thrive, prominent metopic suture, synophrys, asymetric head shape, triangular and asymetric face, telecanthus, epicanthal folds, down-slanting palpebral fissures, microphthalmia of the left eye, anteverted nares, smooth and tented philtrum, microretrognathia, low-set ears, auricular pits, high-arched palate, thin upper lip and hypotonia | 46,XX,der(16) |
16q121q23.5 (52,459,169–82,285,105)×3 | 29,800 |
25 | F-2 | DD, microcephaly, short stature, low-set ears, convex nasal ridge | 46,XX | 3p14.2 (60,681,991–61,207,077)×1 | 520 |
26 | F-14 | ID, obesity, behavioral problems | 46,XX | 8p21.3 (21,157,621–22,987,837)×3 | 1800 |
27 | M-12 | ID, impaired social interactions | 46,XY | 15q13.3 (31,999,631–32,914,239)×3 | 446 |
28 | M-3 | DD, epilepsy | 46,XY | 16p13.1 (14,866,283–16,391,910)×1 | 1500 |
29 | F-7 | ID, ASD, short stature, hand stereotypies | 46,XX | 14q32.2q32.33 (97,377,993–107,282,437)×3 | 9904 |
30 | F-4 | DD, epilepsy | 46,XX | 20p13 (2,911,855–4,931,592)×3 | 2020 |
31 | M-14 | ID, IUGR, hypotonia, microcephaly, short stature, low-set ears, small mouth, prominent forehead, hypertelorism | 46,XY | 19p13.3 (2,572,666–4,192,224)×3 | 1619 |
32 | M-25 | ID, diabetes mellitus, renal cysts, obesity, stereotyped movements | 46,XY | 15q11.2q13.1 (23,164,31–28,530,182)×3 | 5365 |
33 | F-4 | DD, epilepsy, microcephaly, micrognathia | 46,XX | 4q34.2q34.3 (177,322,096–180,306,130)×3 | 2984 |
34 | M-14 | DD, synophrys, thin upper lip, short fingernails | 46,XY,der(10) |
10p15.3p15.1 (135,608–6,054,675)×1 | 5919 |
35 | M-12 | DD/ID, microcephaly, cerebral atrophy, synophrys, flat philtrum, 2-3-4-5 toe syndactyly | 46,XY | 2q31.1q31.3 (170,694,601–182,623,003)×1 | 11,900 |
1 | M-4 | 25 | pathogenic | – | ||
2 | M-6 | >30 |
maternal | pathogenic | chromosome Xp11.23-11.22 duplication syndrome | |
3 | M-1 | 10 | pathogenic | chromosome 5p deletion syndrome (Cri-du-Chat syndrome) | ||
4 | M-6 | >30 | pathogenic | – | ||
5 | F-5 | >30 |
paternal balanced reciprocal translocation | pathogenic | – | |
6 | F-3 | 3 | VUS, likely pathogenic | – | ||
7 | F-3 | >30 | VUS, likely pathogenic | – | ||
8 | F-1 | 27 | pathogenic | chromosome 4p16.3 deletion syndrome (Wolf-Hirschhorn syndrome) | ||
9 | F-4 | >30 | maternal | pathogenic | chromosome 11q11.2 deletion syndrome (DiGeorge syndrome) | |
10 | M-1 | 13 |
paternal inv(3)(p25q25) | pathogenic | 3p syndrome |
|
11 | F-1 | 12 | pathogenic | chromosome 15q13.3 deletion syndrome | ||
12 | F-4 | 18 | pathogenic | – | ||
13 | F-1 | 3 | VUS, no sub-classification | – | ||
14 | M-10 | 17 | – | benign | – | |
15 | M-3 | 1 | benign | – | ||
16 | F-10 | 3 | benign | – | ||
17 | F-8 | 4 | VUS, no subclassification | – | ||
18 | F-10 | >30 | pathogenic | – | ||
19 | M-6 | >30 | VUS, likely benign | – | ||
20 | F-7 | 0 | – | maternal | benign | – |
21 | M-4 | 2 | VUS, no subclassification | – | ||
22 | F-3 | 0 | – | paternal | benign | – |
23 | F-2 | 4 | – | maternal | benign | – |
24 | F-1 | 211 | pathogenic | – | ||
25 | F-2 | 1 | VUS, likely pathogenic | – | ||
26 | F-14 | >30 | VUS, likely pathogenic | – | ||
27 | M-12 | 2 | paternal | pathogenic | chromosome 15q13.3 duplication syndrome | |
28 | M-3 | 12 | pathogenic | – | ||
29 | F-7 | >30 | pathogenic | – | ||
30 | F-4 | 29 | VUS, no subclassification | – | ||
31 | M-14 | >30 | pathogenic | – | ||
32 | M-25 | 19 | pathogenic | chromosome 15q11-q13 duplication syndrome | ||
33 | F-4 | 4 | VUS, likely pathogenic | – | ||
34 | M-14 | 19 | pathogenic | – | ||
35 | M-12 | >30 | pathogenic | – |
#: patient number; aCGH: array-based comparative genomic hybridization; M: male; F: female; DD: developmental delay; IUGR: intrauterine growth retardation; VSD: ventricular septal defect; VUS: uncertain clinical significance; DD/ID: developmental delay/intellectual disability; EEG: electroencephalogram; ASD: autism spectrum disorder; ADHD: attention deficit hyperactivity disorder.
Detailed neurological findings of the patients.
1 | M-4 | – | normal | normal | – |
2 | M-6 | focal temporal lobe epilepsy started at the age of 5 months | left temporal discharges | left temporal uncal dysplasia | seizures controlled with the use of multi anti epileptic drugs |
3 | M-1 | – | slowing of background activity | midbrain and pontine hypoplasia with enlargement of lateral ventricles | – |
4 | M-6 | absence of seizures at the of 3 years | generalized SWDs maximally located at the post regions triggered with hyperventilation | normal | seizures controlled with the use of multi anti epileptic drugs |
5 | F-5 | head drop seizures started at the age of 3 years | SWDs located on bilateral central regions | normal | seizures controlled with the use of multi anti epileptic drugs |
6 | F-3 | focal motor seizures started at the age of 5 months | SWDs located on cetro-temporal regions | normal | seizures controlled with the use of multi anti epileptic drugs |
7 | F-3 | focal motor seizures started at the age of 3 months | SWDs located on frontotemporal discharge | normal | seizures controlled with the use of multi anti epileptic drugs |
8 | F-1 | focal motor seizures started at the age of 5 months | multifocal epileptic discharges with normal background activity | normal | seizures controlled with the use of multi anti epileptic drugs |
9 | F-4 | – | normal | normal | – |
10 | M-1 | focal motor seizures started at the age of 7 months | SWDs located on frontotemporal discharge | normal | seizures controlled with the use of multi anti epileptic drugs |
11 | F-1 | – | normal | normal | – |
12 | F-4 | myoclonic asthatic seizures started at the age of 3 years | 3.0–3.5 hz generalized SWDs | normal | seizures controlled with the use of multi anti epileptic drugs |
13 | F-1 | – | difuse slowing of the background activity without epileptic activity | cerebral and white matter atrophy | – |
14 | M-10 | migratuar clonic seizures started as newborn | hypsarrhythmia | cerebral and white matter atrophy with enlargement of lateral ventricles | seizures controlled with the use of multi anti epileptic drugs |
15 | M-3 | – | normal | normal | – |
16 | F-10 | – | difuse slowing of the background activity without epileptic activity | cerebral and white matter atrophy with enlargement of lateral ventricles | – |
17 | F-8 | focal hypomotor seizures started at the age of 5 months | SWDs located on temporoparietal and occipital regions | cerebral and white matter atrophy with enlargment of lateral ventricle and hydrocephalus | seizures controlled with the use of multi anti epileptic drugs |
18 | F-10 | absence seizures started at the age of 4 years | 3.0–3.5 hz generalized SWDs | normal | seizures controlled with the use of multi anti epileptic drugs |
19 | M-6 | secondary generalized seizures and status epilepticus started at the age of 6 months | multifocal epileptic discharges with normal background activity | normal | seizures controlled with the use of multi anti epileptic drugs |
20 | F-7 | multiple types of seizures started at the age of 18 months | multifocal epileptic discharges with slowing of background activity | Type 1 tip1 pachygyria, lis-sencephaly, nodular heterotropy | seizures were resistant to anti epileptic therapy |
21 | M-4 | infantile spasm seizures started at the age of 4 months | hypsarrythmia | bilateral gliosis on the occipital regions | seizures were controlled with ACTH therapy |
22 | F-3 | – | normal | normal | – |
23 | F-2 | infantile spasm seizures started at the age of 4 months | multifocal epileptic discharges with slowing of background activity | bilateral gliosis on the occipital regions and enlargement of lateral ventricles | seizures were resistant to anti epileptic therapy |
24 | F-1 | – | normal | normal | – |
25 | F-2 | – | normal | normal | – |
26 | F-14 | – | normal | normal | – |
27 | M-12 | – | normal | normal | – |
28 | M-3 | febrile seizures started at the age of 12 months and restarted at the age of 2 years | normal | normal | seizures were controlled with the use of a single anti epileptic drug |
29 | F-7 | – | normal | normal | – |
30 | F-4 | myoclonic seizures started at the age of 18 months | generalized polyspike waves | normal | seizures were controlled with the use of multi anti epileptic drug |
31 | M-14 | – | normal | normal | – |
32 | M-25 | – | normal | normal | – |
33 | F-4 | generalized polyspike waves | normal | seizures were controlled with ACTH therapy | |
34 | M-14 | – | normal | normal | – |
35 | M-12 | – | normal | cerebral atrophy | – |
#: patient number; M: male; F: female; EEG: electroencephalogram; MRI: magnetic resonance imaging; SWDs: sleep-wake disturbances, ACTH: adrenocorticotropic hormone.
The aCGH analysis revealed pathogenic CNVs showing clinical features in 19 (13.6%) of the total 139 cases. The findings of karyotype analysis were normal in 29 (
Array-based comparative genomic hybridization is recommended as the first-tier test in unexplained DD/ID cases as it can detect submicroscopic deletions and duplications below 5.0 Mb that cannot be detected by conventional karyotype analysis [11]. The widespread use of aCGH technology in recent years has resulted in increased diagnostic rates for DD/ID cases and identification of new microdeletion/microduplication syndromes.
The diagnosis rates vary between 5.1–35.0% in the literature [14,15]. The variability in diagnosis rates may be related to differences in criteria for patient selection, resolution of the aCGH platform used, and classification of detected CNVs. With the use of aCGH as a first-tier test in DD/ID cases, the frequency of VUS variants also increases in addition to the increase in diagnosis rates, making it difficult to demonstrate the genotype-phenotype correlation. CNVs associated with recurrent/well-defined syndromes, inherited CNVs from parents with a similar phenotype, and CNVs containing defined morbid genes in the OMIM database were identified as pathogenic, whereas polymorphic CNVs frequently seen in population databases were considered benign [16]. However, clinical interpretations of unique non recurrent CNVs are not always easy. The low number of these CNV cases in the literature, the unclear dosage sensitivity status of the genes, and the difference in penetrance, make interpretation difficult. In this study, pathogenic CNVs were detected in 19 cases according to the ACMG criteria, of which eight were cases of recurrent microdeletion/microduplication syndrome: 22q11.21 deletion (DiGeorge) syndrome in one, 5p deletion (Cri-Du-Chat) syndrome in one, 4p16.3 deletion (Wolf-Hirschhorn) syndrome in one, Xp11.23-p11.22 duplication syndrome in one, 3q26 microduplication syndrome and 3p deletion syndrome in one, 15q13.3 deletion syndrome in one, 15q13.3 duplication syndrome in one, and 15q11-q13 duplication syndrome in one. Additionally, rare pathogenic CNVs were detected in 11 cases: 2q31.1 q31.3 deletion in one, 10p15.3p15.1 deletion in one, 19p13.3 duplication in one, 14q32.2q32.33 duplication in one, 16p13.11 deletion in one, 16q12.1q23.3 duplication in one, Xp22.2p21.3 duplication in one, 6q21q22.31 deletion in one, 8p23.3p23.1 deletion and 9p24.3p23 duplication in one, 8q24.21q24.3 deletion in one, and 17q21.32q 21.33 duplication in one. All five CNVs that were considered likely pathogenic have been previously reported in at least one case with DD/ID in the literature and contain morbid genes. The CNVs detected in all four cases in the VUS, no subclassification group were previously reported as VUS in cases diagnosed with DD/ID in the DECIPHER database. Cases of frequent CNVs in the general population were grouped as benign. According to the two-hit model proposed by Girirajan
Of the 26 pathogenic/likely pathogenic CNVs, 13 were gains and 13 were losses. Additionally, 88.4% (23/26) of these CNVs were alterations larger than 1.0 Mb. It has been reported in the literature that microdeletion syndromes are more frequently observed, and microduplication syndromes are overlooked owing to their mild phenotype [18,19]. The more frequent detection of microduplication syndromes in this study can be attributed to the inclusion of cases with a mild phenotype. The interpretation of pathogenicity of microduplications is more difficult due to incomplete penetrance and unclear triplosensitivity status of the genes it involves. Microduplications that are not found in the general population, larger than 1.0 Mb,
Some cases of pathogenic CNVs with novel clinical and radiological findings are rarely described in the literature. Patient 1, a 4-year-old male with DD, short stature, microcephaly and scoliosis findings, was diagnosed with a
The widespread use of aCGH analysis in DD/ID cases increases the diagnostic rate. However, karyotype analysis must also be considered in each case for evaluating the cases for balanced translocations, inversions, and low-level mosaicisms that cannot be detected by the aCGH method. Determination of the location, size, and involved genes of the chromosomal abnormality using aCGH is important in terms of genotype-phenotype correlations. Additionally, a clear presentation of the chromosomal abnormality is critical for prognosis, clinical follow-up, and rehabilitation program planning. In terms of the family of the index case, it becomes possible to present prenatal diagnosis and pre-implantation genetic diagnosis options by screening other family members for chromosomal anomalies and explaining the risk of recurrence in subsequent pregnancies to the family [29].
The first limitation of this study is the small number of patients. The small sample size may have resulted in the low detection rate of frequently observed microdeletion/microduplication cases. The second limitation of the study was that
In conclusion, the use of aCGH analysis as a first-tier test in DD/ID cases contributes significantly to the diagnosis rates and enables the detection of rare microdeletion/microduplication syndromes. The clear determination of genetic etiology contributes to the literature in terms of genotype-phenotype correlation.