Identification of microdeletion and microduplication syndromes by chromosomal microarray in patients with intellectual disability with dysmorphism
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.241346
Source of Support: None, Conflict of Interest: None
Keywords: Chromosomal microarray, chromosomal microdeletion, DiGeorge syndrome, FISH, Kleefestra syndrome, Prader–Willi syndrome, SNP array
Chromosome aberrations have long been recognized as the most common cause of developmental disabilities and mental retardation (MR). Any technological advances in the cytogenetic screening translate into an increased chance of a positive diagnosis for patients with mental retardation and their families. The conventional karyotyping is useful in identifying many syndromes. Despite a constant improvement in the chromosomal culture and banding techniques, however, subtle structural rearrangements of chromosomes which are less than one band (5–10 Mb) remain invisible (as in microdeletion or microduplication syndromes, and cryptic translocations). Fluorescent in situ hybridization (FISH) provides a link between the two techniques: conventional banding cytogenetics and molecular genetics. The development of array comparative genomic hybridization (array CGH) has also enabled the simultaneous testing of multiple loci to asssess for copy number differences., In this study, we describe our experience on the stepwise detection of microdeletion and microduplication syndromes and the application of chromosomal microarray analysis (CMA; single-nucleotide polymorphism-based array CGH).
The patients on follow-up in the genetic clinic of the institute for the past 3 years with suspected chromosomal microdeletion and/or microduplication syndromes were included in this retrospective analysis. The cases with known chromosomal monosomies and trisomies such as Down and Turner syndromes were excluded. Conditions such as 22q11 deletion syndrome [DiGeorge/velocardiofacial syndrome (DGS)], Prader–Willi syndrome (PWS), and Williams–Beuren syndrome (WBS) were also excluded from final analysis.
A flow chart given in [Figure 1] represents the usual protocol followed in the genetics clinic for the diagnosis of cases with dysmorphism with mental retardation or malformations. The references and databases used for the clinical diagnosis of these patients included Smith's Book on Malformations, Oxford Medical Databases, Brigg's Book on Drugs, and Online Mendelian Inheritance in Man (OMIM).,,,
CMA was performed in 18 selected cases with significant dysmorphism with or without mental retardation after excluding common causes by radiological, biochemical, and molecular analyses. CMA testing was done using Illumina HumanCytoSNP-12 array which is specifically used for the detection of chromosomal abnormalities such as deletions and duplications. The CMA abnormalities detected were checked on databases such as Decipher, PubMed, and OMIM for assessing their possible clinical significance. Prenatal diagnosis was done in one family by the CMA and multiplex ligation probe amplification (MLPA) analysis.
A total of 65 patients with suspected chromosomal syndromes were shortlisted. One patient with partial trisomy of chromosome 13 was excluded from the final analysis. In suspected PWS, FISH and methylation testing confirmed six cases to be really PWS. Five cases of DGS and one case of WBS were also detected by FISH analysis with specific probes. These cases were excluded from final analysis. Finally, 18 cases were tested by CMA for possible unidentified chromosomal deletion or duplication syndromes.
The details of 9 of 18 patients tested by CMA are listed in [Table 1]. One additional case with DGS (case 4), one case each of a terminal and an interstitial deletion (cases 2 and 5), and two cases each with terminal and interstitial duplications (cases 1, 3, 6, and 7) were identified after CMA. Finally, there was one case of a maternal or paternal uniparental disomy of 15q13.3–15q14 (case 8). One case showed clinically significant deletion on one chromosome and duplication on another chromosome (case 9). Prenatal diagnosis was done in the family of case 5 on DNA extracted from chorionic villus sample, and the fetus was found to be normal. Three interesting cases are described in detail below. Case 13, a 4-year old male child who had infantile spasms, microcephaly, and gastro-esophageal reflux disease, showed chimerism on CMA testing—Chi 46, XY/46, XX. Five of 18 cases (cases 10, 11, 12, 17 and 18) did not show any significant clinical abnormality on CMA testing. The detected variants were compared with the variations in Database of Genomic Variants (DGV) and the local population-specific common variants.
Case 14, a 15-month old male patient presented to us for the first time at 4 months of age with respiratory infection (pneumonia) along with developmental delay. He was the first-born child to non-consanguineous parents with no family history of any congenital malformations or early deaths. No other child in the family presented with a developmental delay or mental retardation in the family. Antenatally, his mother conceived spontaneously after 2 years of married life, with the mother's and the father's age being 22 and 26 years, respectively. The mother underwent regular monitoring during the antenatal period. There was no history of drug intake, gestational diabetes, hypothyroidism, or hypertension. The child was born full-term, through a normal delivery, with average birth weight, with perinatal evidence of decreased spontaneous motor activity. The baby was top fed with formula feed.
The examination showed dysmorphic features in the form of coarse facies, depressed nasal bridge, synophrys, long philtrum, thick ear lobules, seborrhea, and low set posterior placed ears [Figure 2]. He also had neonatal teeth, a short neck, short stubby fingers, and few overlapping toes. Systemic examination revealed axial and appendicular hypotonia and ejection systolic murmur of grade 3 along with bilateral crepitations. Anthropometric parameters suggested significant growth retardation with microcephaly. On developmental assessment, the developmental age corresponded to only 1–2 months and he continued to lag behind significantly compared with his peers. On follow-up visits, the child had recurrent episodes of lower respiratory tract infections (LRTIs) for which he required multiple hospitalizations and treatment with intravenous antibiotics. The first admission was at 4 months of age for lower respiratory tract infection (LRTI) for 5 days, that was treated with antibiotics, and the baseline syndrome evaluation was done along with other supportive care. His thyroid function tests were normal, and two-dimensional (2D) echocardiography showed an 8-mm ostium secundum atrial septal defect. The ultrasonography and infantogram did not reveal any abnormalities. The second admission was at 7 months of age with fever for 2 months, along with symptoms of LRTI. He was evaluated for determining the causes of chronic fever along with recurrent respiratory infections. He had one gastric lavage for acid-fast bacilli (AFB) positive although there was no family history of active tuberculosis (TB) in the family. He was started on anti-tuberculous drugs and his fever responded well. He was admitted again for the third time at the age of 9½ months for a short respiratory illness signifying the presence of a fresh LRTI. He was treated with antibiotics and supportive care. Due to the presence of recurrent LRTI, he was also evaluated for an immunodeficiency state but had normal immunoglobulin (Ig) G/IgM/IgA levels as well as normal age-appropriate total blood counts, differential counts, and lymphocyte counts on flow cytometry. He was discharged after 5 days when he became better symptomatically.
A CMA was done, which revealed 2-Mb deletion at 9q34.3. He also carried a duplication of 7.7 Mb at cytoband 3p26.3p26.1. The child's features were fitting with 9q34.3 deletion spectrum or Kleefestra syndrome. [Figure 3] depicts the 9q34.3 deletion, identified on microarray analysis. He was again admitted for the fourth time at 13 months of age for similar complaints for which he was evaluated further with a high-resolution computed tomography to look for any bronchiectatic changes in the lung and to look for any other treatable causes. However, the entire work-up for his recurrent LRTI was negative, and therefore, the infection was thought to be related to his primary illness, that had led to hypotonia and difficulty in proper feeding.
Case 15, a 3-year old male child, presented with global developmental delay with the developmental age being 1 year. He was the first-born child to non-consanguineous parents. He was born at term in the hospital. There was no family history of a developmental delay, mental retardation, congenital malformation or any early death of a sibling. Antenatally, he was conceived by spontaneous conception and there was no history of any drug intake or any febrile illness during pregnancy. His weight at birth was 2.75 kg, and he had a normal perinatal transition. On examination, the child had microcephaly, with a normal weight and height. He had subtle dysmorphic features such as a narrow forehead, low set ears, retrognathia, upturned nostrils, deep-seated eyes, and strabismus. On detailed developmental assessment, he had significant developmental delay encompassing all the sectors tested. A cognitive or intelligence quotient (IQ) assessment was done, and he was found to be having an subnormal IQ (74%) but a normal hearing and vision. As a part of the work up of the syndrome, an ultrasonography for kidneys, a two-dimensional (2D) echocardiography, X-rays of the spine, and thyroid function tests were done, which were normal. Magnetic resonance imaging of brain showed temporal atrophy along with malrotated hippocampus.
The child was further evaluated with microarray and showed a 9p24 duplication of size 35.3 Mb. This child also carried a homozygous deletion of 8.5 Mb at cytoband Yq11.221q11.23 encompassing approximately 31 genes, and a duplication of 360 kb at cytoband 16q23.1 and a duplication of 460 kb at cytoband 17p11.2.
Case 16, the index case, a 5-year old female child, presented with seizures, severe mental retardation, microcephaly, a prominent metopic ridge, repeated mouthing and facial abnormality, with a right preauricular tag. X-rays of the spine did not reveal any vertebral anomalies, and the screening for metabolic disorders was normal. Since the mother was pregnant, and a clinical suspicion of chromosomal disorder was considered, CMA was done. This revealed duplication of the cytoband 16p13.3-p13.2, which also encompassed the cyclic adenosine monophosphate-response element-binding protein (CREB) binding protein [CREBBP] gene, the deletion of which is responsible for Rubinstein–Taybi syndrome. Since the mother had a history of abortions, her karyotype was done and it showed 46, XX, t(15:16)(q11.2;p13.3) karyotype. [Figure 4] depicts the translocation. Genetic counseling was done for this patient.
Microdeletion syndromes are genetic syndromes that are associated with small chromosomal deletions that are beyond the resolution power of the conventional banding karyotyping analysis. Recognition of the phenotype almost always relies on detecting and combining minor dysmorphic stigmata characteristic of the syndrome. Analysis of patients with intellectual disability and dysmorphism should be done in a stepwise manner. Detection of whole chromosome trisomies and monosomies, as well as detection of gross chromosomal rearrangements may be done by banding and molecular cytogenetic techniques. Until a few years back, the high-resolution G-banding karyotyping and the FISH studies using probes targeted to the subtelomeres and/or known microdeletion loci were considered the gold standard for detecting cytogenetic aberrations.
However, such aberrations, especially when present in low mosaic levels, might easily be missed when only performing targeted molecular techniques. Targeted FISH, multiplex ligation-dependent probe amplification (MLPA), and chromosome-based comparative genomic hybridization (cCGH) or CMA could be the next steps. Array-based CGH has a much greater sensitivity than high-resolution karyotyping and can target more loci than FISH in a cost-effective manner. Furthermore, it is more successful than FISH at detecting small chromosomal deletions or duplications., The degree of resolution provided by different array platforms of different companies (Illumina/Affymetrix) can be variable.
At present, CMA is available at only a few centers and is expensive; here we suggest a stepwise, clinical diagnosis-based analysis scheme [Figure 1] and [Figure 5]. [Figure 1] delineates the suggested approach to a case presenting in the clinic with facial dysmorphism with malformations with or without developmental delay/mental retardation. Known microdeletion syndromes such as PWS, WBS, and DGS can be identified in most cases by the FISH analysis. In patients with cardiac defects and subtle dysmorphic features, the search for associated abnormalities can help in picking up cases of DGS or WBS. PWS should be kept in the differential diagnosis of a child with infantile hypotonia or suspected genetic obesity., The regions identified to be of clinical significance on CMA are listed in [Table 1] and revealed patients with microdeletion and duplication syndromes found in this study. Case 1 had a 38.9 Mb duplication on chromosome 10, consistent with a 10p duplication syndrome. Case 7 had a 10q26.13 duplication, which was with clinical significance. The article by Weise et al., describes in detail the different chromosome regions where abnormalities found are associated with certain microdeletion or duplication syndromes. A case of DGS was also identified after CMA, There is also a DGS2 region on 10q22-q23. DGS is one of the commoner syndromes associated with cardiac defects.,, Case 5 with dysmorphism and a cardiac defect had the 9.4 Mb deletion on chromosome 7p encompassing the Saethre–Chotzen syndrome (SCS) region. Patients with SCS have a short stature, facial dysmorphism, craniosynostosis, and cardiac defects., We identified one patient with chimerism, who presented with microcephaly and seizures. Chimeras are usually described in post-transplantation cases. Further studies are needed to clarify the physical effects of chimerism in humans.
Constitutional deletions of the distal long arm of chromosome 9 (MIM 610253) encompassing the EHMT1 gene, or loss-of-function mutations in EHMT1, result in a clinically recognizable syndrome that is characterized by suggestive craniofacial features, hypotonia, childhood obesity, microcephaly, and substantial speech delay and mental retardation., The child in this study—case 14—also had duplication of 7.7 Mb at cytoband 3p26.3p26.1, but this was considered clinically less relevant, as the overall features were fitting into the Kleefestra syndrome due to presence of the 9q34.3 deletion. In a recent report, the predominant problem with patients with the Kleefestra syndrome was found to be muscular hypotonia and its consequences. Another child also showed a large duplication (35.3 Mb) on chromosome 9 at the 9p24 region (case 15) and presented with microcephaly and developmental delay. In general, patients with chromosomal duplication syndromes have milder phenotypes, and a case manifestating with a chromosomal deletion tends to have severe phenotypes and more intellectual disability. The size and position of the deletion or duplication can also determine the specific phenotypes.
Balanced translocation carriers have an increased risk of early first-trimester abortions and also are at a greater risk of having children with abnormal chromosomal complement. This was seen in the family of case 16. The child had the chromosome16p13.3 duplication syndrome. Chromosome 16p13.3 duplications are reported to cause intellectual disability, facial dysmorphism, developmental defects of the heart, a posterior cleft palate, duplicated thumbs, hypoplastic genitalia, abnormal eyes including microcorneae, and behavioral problems. Our child had trigonocephaly, a right preauricular tag, and presented with seizures.
Thus, the diagnosis of the cause of intellectual disability or behavioral disorders can be challenging for the neurologist, pediatrician, or geneticist. The common disorders include the Down syndrome, Fragile X syndrome, and Rett syndrome.,,, The availability of newer techniques such as triplet primed polymerase chain reaction (for fragile X syndrome), MLPA (for certain disorders such as Rett syndrome), CMA, and next-generation sequencing (NGS) has enabled better characterization of disorders with intellectual disability with or without seizures or behavioral abnormalities. The CMA can help in the determination of underlying defects in many cases of developmental delay, intellectual disability, and autistic spectrum disorder., The disadvantages include an inability to identify balanced chromosomal rearrangements, inversions, insertions, and point mutations, and some cases of polyploidy. However, CMA can sometimes give a clue to the regions of loss of heterozygosity, which can enable further diagnostic testing, especially in case of consanguineous families. Further testing is by DNA analysis, which can be either in the form of specific gene testing, if this can be identified by revisiting the clinical phenotype, or in the form of NGS for a panel of disorders encompassing multiple genes.
[Figure 5] depicts the interpretation of copy number variants (CNV) found on CMA testing. Further assessment after the identification of the abnormality on CMA is the testing in parents for carrier of balanced translocation by the routine chromosomal analysis, which could have led to the phenotype occurring in the child. Alternatively, if there are multiple CNVs found on CMA testing in the child, the CMA test in the parents may be warranted to find out which CNV could be benign (also found in normal parents) and which is most likely to be pathogenic or the cause of the phenotype in the child.
The present write-up summarizes the use of CMA in cases with intellectual disability after a thorough clinical evaluation and routine diagnostic testing. This ensures that a maximum positivity is obtained from the CMA results and a prenatal diagnosis can be planned accordingly in positive cases with pathogenic or likely-pathogenic chromosomal deletions or duplications. In general, the diagnosis of microdeletion and microduplication disorders presents a clinical challenge to the clinician or the dysmorphologist. The variability of the phenotype brings counseling issues, and a detailed knowledge of the genes involved, the databases, and parental testing can help in better characterization of these disorders.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The authors would like to thank Sandor Proteomics for help in evaluation of selected patients. They also thank Dr. Thomas Liehr for critical appraisal of the initial draft of this article.
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Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]