Derivative chromosomes involving 5p large rearranged segments went unnoticed with the use of conventional cytogenetics
© The Author(s). 2018
Received: 15 January 2018
Accepted: 27 March 2018
Published: 9 May 2018
In countries where comparative genomic hybridization arrays (aCGH) and next generation sequencing are not widely available due to accessibility and economic constraints, conventional 400–500-band karyotyping is the first-line choice for the etiological diagnosis of patients with congenital malformations and intellectual disability. Conventional karyotype analysis can rule out chromosomal alterations greater than 10 Mb. However, some large structural abnormalities, such as derivative chromosomes, may go undetected when the analysis is performed at less than a 550-band resolution and the size and banding pattern of the interchanged segments are similar. Derivatives frequently originate from inter-chromosomal exchanges and sometimes are inherited from a parent who carries a reciprocal translocation.
We present two cases with derivative chromosomes involving a 9.1 Mb 5p deletion/14.8 Mb 10p duplication in the first patient and a 19.9 Mb 5p deletion/ 18.5 Mb 9p duplication in the second patient. These long chromosomal imbalances were ascertained by aCGH but not by conventional cytogenetics. Both patients presented with a deletion of the Cri du chat syndrome region and a duplication of another genomic region. Each patient had a unique clinical picture, and although they presented some features of Cri du chat syndrome, the phenotype did not conclusively point towards this diagnosis, although a chromosomopathy was suspected.
These cases highlight the fundamental role of the clinical suspicion in guiding the approach for the etiological diagnosis of patients. Molecular cytogenetics techniques, such as aCGH, should be considered when the clinician suspects the presence of a chromosomal imbalance in spite of a normal karyotype.
In Mexico and other developing countries, the genetic approach for patients with intellectual disability (ID) and congenital malformations (CM) uses conventional G-banded karyotyping as the first-choice diagnostic test. It is usually performed at a 500-band level that allows the detection of 5–10 Mb abnormalities and results in an etiological diagnosis in approximately 3–10% of patients with a suspected chromosomopathy [1–3]. However, some structurally abnormal chromosomes with rearrangements larger than 5 Mb may go unnoticed by conventional cytogenetics. This may occur with derivative chromosomes, which are unbalanced intra- or inter-chromosomal rearrangements, in which the exchanged segments share a similar size and banding pattern, making them difficult to identify by conventional karyotyping [3, 4].
When a patient presents with a derivative chromosome, phenotypic evaluation and chromosome analysis of the parents are mandatory to rule out the presence of a balanced translocation in one of them . In fact, 70% of derivatives are inherited and this information has a significant impact on genetic counseling [5, 6]. Comparative Genomic Hybridization arrays (aCGH) may uncover this type of abnormalities, because the finding of a combined deletion/duplication in the same patient points towards the presence of a derivative chromosome. Here, we describe the cytogenetic and clinical findings of two patients in whom a clinical phenotype consisting of ID and CM prompted the completion of aCGH despite having a normal 450-band karyotype. The two patients presented here, were ascertained through aCGH during the study of a cohort of 152 patients that presented with ID or CM and a normal conventional karyotype (manuscript in preparation).
Genomic DNA from the two patients and their parents was amplified and labeled using the CGH-labeling kit for oligo arrays (Enzo Life Sciences, New York, USA) and then analyzed with a 60 K oligonucleotide arrays according to the manufacturer’s protocol (Agilent, Santa Clara, USA). The slides were scanned using a microarray scanner with Surescan High Resolution Technology (Agilent, Santa Clara, USA). Image quantification, array quality control and aberration detection were performed using the Agilent Feature Extraction and DNA Analytics software (Agilent, Santa Clara, USA) according to the manufacturer’s instructions. Changes identified in the samples were visualized using the UCSC Genome Browser online tool (http://genome.ucsc.edu) and were compared to the Database of Genomic Variants (http://projects.tcag.ca/variation) to exclude copy number changes considered to be benign variants. The DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources) (https://decipher.sanger.ac.uk/) and ECARUCA (European Cytogeneticists Association Register of Unbalanced Chromosome Aberrations) (http://umcecaruca01.extern.umcn.nl:8080/ecaruca/ecaruca.jsp) databases were used to assist with the genotype-phenotype correlation.
The rearrangements were validated using Fluorescence in situ hybridization (FISH) probes (Sure-FISH, Agilent, Santa Clara, USA). Molecular cytogenetic techniques, and GTG-banded karyotypes were performed on patients and their first-degree relatives to establish the origin (inherited or sporadic) of the chromosomal rearrangement and provide appropriate genetic counseling.
Both patients were males, born to non-consanguineous parents. There was no family history of congenital diseases, intellectual disability, autism, seizures, neurological disorders, metabolic diseases, infertility or recurrent pregnancy loss. Physical examination revealed that both patients had weight, height and head circumference below the 5th percentile. An informed consent letter for each patient was obtained from the parents.
In Fig. 2d, it is evident that the derivative chromosome 5 shows a very similar G-band pattern than the normal chromosome 5, which explains why it was not detected in the conventional karyotype despite the large segment involved in the rearrangement. FISH with the specific probes did not show a balanced chromosomal rearrangement in either parent.
The prevalence of derivative chromosomes is unknown, although the estimated frequency of balanced reciprocal translocations ranges from 1 in 500 to 1 in 1000 in live births [4, 6]. The carriers of a balanced rearrangement may have decreased fertility, miscarriages and children with ID and CM. All of these are consequences of pregnancies with genomic imbalances due to the fertilization with a gamete that received the derivative chromosome during meiotic segregation [8–10].
We describe two patients with derivative chromosomes that despite having large chromosomal imbalances were not identified by conventional karyotyping. Furthermore, even though these rearrangements resulted in a 5p deletion, the patients did not present a phenotype in which the Cri-du-chat syndrome was readily recognized. The first patient’s karyotype is 46,XY,der(5)t(5;10)(p15.2;p13)mat carrying a chromosome 5 derivative that arose from a t(5;10)(p15.2;p13). The derivative chromosome results in a partial monosomy of 5p15.2 → pter, producing haploinsufficiency of the SDHA (5p15.33) and SEMA5A (5p15.31) genes that have been related to psychomotor retardation, microcephaly, pachygyria and microgyria. All these symptoms were present in our patient . The der(5)t(5;10)(p15.2;p13) also results in partial chromosome 10 trisomy 10p13 → pter. This region contains the AKR1C3 gene (10p15.1) that is involved in male gonadal development  although its overexpression is yet to be related to an altered function. Thus, it is possible that the cryptorchidism found in this patient is a phenotype due to the 5p deletion . In addition, the GATA3 gene (10p14) is involved in ear development, and abnormalities at this level have been linked to ear defects . Our patient shares ID, growth delay, microcephaly, low-set ears, downturned corners of mouth, cryptorchidism and abnormal palmar creases with previously reported 5p deletion patients . Patients with the 10p duplication present with ID, microcephaly, low-set ears, hearing impairment, cryptorchidism and abnormal palmar creases [15, 16].
The second patient also has a chromosome 5 derivative with a karyotype 46,XY,der(5)t(5;9)(p14.3;p22.1)dn leading to a partial monosomy of 5p14.3 → pter. This karyotype explains why this patient shared clinical manifestations with Cri du chat syndrome such as microcephaly, round face, epicanthus, telecanthus, downturned corners of mouth and dysplastic pinnae . This derivative also results in partial trisomy of 9p22.1 → pter and the patient presents with clinical manifestations that have been previously reported in patients with a 9p duplication. These include ID, growth delay, microcephaly, hypertelorism, epicanthic folds, low-set ears, transverse fold, short stature, hearing loss, and nail hypoplastic [18, 19].
Genetic counseling for the second family is different because neither parent are carriers. Thus, the theoretical risk of recurrence is zero. However, we cannot rule out the presence of germline mosaicism in one of the parents or a non-paternity situation.
The use of molecular cytogenetics methodology should be mandatory in patients with intellectual disability and multiple congenital malformations
Currently, it is well accepted that conventional karyotyping is an excellent and non-expensive methodology for detecting aneuploidies, low-level mosaicism and large -more than 10 Mb- rearrangements; however, detection of chromosomal structural abnormalities largely depends on the skills and experience of the cytogeneticist and yet, some alterations may go unnoticed . To improve the detection of chromosomal abnormalities, molecular methodologies such as FISH or Multiplex Ligation-dependent Probe Amplification (MLPA) and chromosomal microarrays have been used . Several groups have highlighted the economical and medical convenience of using microarrays as a first-line diagnostic test for detecting constitutional genomic imbalances [26–33]. The high cost of microarrays keeps these methodologies from being the first-line diagnostic tests in some developing countries, forcing a careful selection of patients in whom such studies are performed. Often, the few available microarrays are used in patients in whom a submicroscopic alteration is suspected, overlooking other patients who could also benefit from these types of analyses. Our cases support the benefit of offering microarray analysis to patients with a more severe phenotype such as ID and developmental delay, dysmorphic features or multiple CM, and when the clinical picture does not point toward a specific target region of the genome. A high rate of detection of unbalanced rearrangements using microarray methodology has been observed in patients with these referral indications . It should be noted that microarrays can only detect unbalanced genomic regions, while the chromosomal locations of the duplicated or deleted regions material cannot be defined. In order to do so, the analysis must be complemented with the use of FISH to localize the segments in the karyotype.
The phenotype of the patients with a derivative chromosome are unique, since it combines the clinical features of both the partial deletion and the partial duplication.
Noticeably, only when the clinical geneticist makes a detailed phenotype-genotype correlation, it becomes evident that the patient has clinical manifestations of the specific syndrome, such as Cri du chat syndrome in this study. The typical gestalt is modified by non-classical manifestations resulting from the new genome created by the chromosomal rearrangement, and the clinical diagnosis is not apparent.
Patients presented here are clear examples in which large chromosomal rearrangements go unnoticed by experienced cytogeneticists when using conventional cytogenetics. These cases highlight the importance of performing complementary analyses in patients with developmental delay or ID associated with CM using molecular cytogenetics techniques. The clinician’s suspicion of a chromosomal etiology for the patient’s condition, despite a normal conventional karyotype, is fundamental to support the need and demand the funding to perform further molecular cytogenetic testing that could positively impact the patient’s diagnosis, and to provide information regarding the biology of the disease.
The aCGH was carried out at Genetadi Laboratory (Bilbao, Spain). Many thanks to Dr. Ariadna Gonzalez del Angel and Dr. Esther Lieberman for the referral of patients.
This study was partially supported by CONACYT FOSSIS-S0008–2008-C01–87792 and Fondos Federales 2013, project 2009/06, Instituto Nacional de Pediatría (México). The funding body did not participate in the design of the study, collection, analysis, or interpretation of data, or in writing the manuscript. EY received a fellowship, CONACYT-176523 (Doctorado en Ciencias Médicas, Odontológicas y de la Salud, UNAM).
Availability of data and materials
The authors declare that all relevant data are included in the article.
EY and SF contributed to the design and development of the protocol, analyzed the results, wrote the manuscript, and designed the figures. VC contributed to the design of the protocol, clinical description and analyzed results. BM, SS and SR performed the G-banded karyotype and FISH technique. LT contributed with the analysis of the aCGH results. MJN wrote the first draft of the manuscript and interpreted results. SA and JLC performed the aCGH. BGT and BA contributed in the clinical description and revised the manuscript for the English language. All authors read and approved the final version and are responsible for all aspects of the manuscript.
Ethics approval and consent to participate
This study has been performed in accordance with the Declaration of Helsinki and was approved by the ethics and research committees of the National Institute of Pediatrics (Mexico) (Project No. 06/2009). Written informed consent was obtained from the patients’ parents for participating in this study.
Consent for publication
Written informed consent was obtained from the patients’ parents for publication of photos and any accompanying images.
The authors declare that they have no competing interests.
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