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Array CGH detection of a novel cryptic deletion at 3q13 in a complex chromosome rearrangement
Genomics 103 (2014) 288–291
Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
Short Communication
Array CGH detection of a novel cryptic deletion at 3q13 in a complex chromosome rearrangement Isabel López-Expósito a,d,⁎, María Juliana Ballesta-Martinez b,d, Juan Antonio Bafalliu a,d, Ascensión Vera-Carbonell a,d, Rosario Domingo-Jiménez c,d, Vanesa López-González b,d, Asunción Fernández a, Encarna Guillén-Navarro b,d,e a
Sección de Citogenética, Centro de Bioquímica y Genética Clínica, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Unidad de Genética Médica, Servicio de Pediatría, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain c Sección de Neuropediatría, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain d Centro de Investigación Biomédica de Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain e Cátedra de Genética Médica, Universidad Católica de San Antonio (UCAM), Murcia, Spain b
a r t i c l e
i n f o
Article history: Received 3 October 2013 Accepted 24 February 2014 Available online 4 March 2014 Keywords: Complex chromosome rearrangement Oligo array-CGH 3q13 deletion
a b s t r a c t Complex chromosome rearrangements (CCRs) are extremely rare in humans. About 20% of the apparently balanced CCRs have an abnormal phenotype and the degree of severity correlates with a higher number of breakpoints. Several studies using FISH and microarray technologies have shown that deletions in the breakpoints are common although duplications, insertions and inversions have also been detected. We report a patient with two simultaneous reciprocal translocations, t(3;4) and t(2;14;18), involving five chromosomes and six breakpoints. He showed dysmorphic features, preaxial polydactyly in the left hand, brachydactyly, postnatal growth retardation and developmental delay. The rearrangement was characterized by FISH analysis which detected an interstitial segment from chromosome 14 inserted in the derivative chromosome 2, and by whole genome array which revealed an interstitial deletion of approximately 4.5 Mb at the breakpoint site on chromosome 3. To our knowledge this microdeletion has not been previously reported and includes ~ 12 genes. The haploinsufficiency of one or several of these genes is likely to have contributed to the clinical phenotype of the patient. © 2014 Elsevier Inc. All rights reserved.
1. Introduction There are different definitions of complex chromosome rearrangement (CCR) and all of them are based on the first report by Pai et al. [1]. The origin of constitutional CCRs is still unknown but several mechanisms have been proposed, most of them based on DNA replication [2]. Recently, it has been proposed that they may be formed as a ‘one-off’ event similar to the phenomenon termed chromothripsis seen in malignancy [3]. Although CCRs are extremely rare in humans with approximately 250 cases reported [4], their identification is important because CCR carriers can have variable phenotypes, which include clinically normal individuals, male infertility and patients with intellectual disability (ID) and/or multiple congenital abnormalities (MCA). Around 70% of CCRs have been detected in normal individuals referred for reproductive
⁎ Corresponding author at: Centro de Investigación Biomédica de Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Fax: +34 968 381222. E-mail address: [email protected] (I. López-Expósito).
http://dx.doi.org/10.1016/j.ygeno.2014.02.008 0888-7543/© 2014 Elsevier Inc. All rights reserved.
problems and/or having had a malformed child, 20–25% in patients with ID and/or MCA and 5–10% in prenatal diagnosis [5–7]. Approximately 70% of CCRs originate de novo and the remainder have a hereditary origin whose transmission is usually through the mother [4]. These cases tend to be less complex, with fewer breakpoints and chromosomes involved than in de novo cases. In both, the CCR can be either balanced or unbalanced. It has been estimated that about 20% of patients with apparently balanced CCRs have an abnormal phenotype and the probability that a de novo CCR is associated with an abnormal phenotype increases with the number of breakpoints [4]. Most CCRs detected by conventional cytogenetics may be more complex than initially suspected and, although FISH and derivative techniques such as multicolor banding facilitate the characterization of CCR, the application of microarray techniques based on comparative genomic hybridization (aCGH) is essential for identifying its molecular nature. Several studies with FISH and aCGH have shown that, at the breakpoints, deletions are common although duplications, insertions or inversions have also been detected [6,8]. De Gregori et al. [6], applying aCGH to patients with abnormal phenotype and apparently balanced chromosomal abnormalities, observed that 40% of the reciprocal
I. López-Expósito et al. / Genomics 103 (2014) 288–291
translocations and 90% of CCRs presented cryptic deletions. Furthermore, 18% of simple reciprocal translocations were more complex rearrangements. Although different classifications have been proposed for CCRs [9], recently Madan [10] proposed a new classification based on four types: I) CCRs with a number of breakages equal to the number of chromosomes involved, II) the number of breaks is one more than the number of chromosomes and includes an inversion, III) the number of breaks is higher than the number of chromosomes and includes one or more insertions and IV) the number of breakages is higher than the number of chromosomes and there is a “middle segment”, at least one derivative chromosome being composed of segments from at least three chromosomes. Balanced CCR carriers have an overall empirical risk of 50% for recurrent spontaneous abortions and 20% for having offspring with a chromosomal imbalance [7], although this risk depends on the type of CCR [10]. Thus, in Type IV, the probability of recombination in the intermediate segment is about 35% and this can result in balanced and unbalanced gametes. In most cases, the complex rearrangement changes to one single translocation [11,12] and in those CCRs with double or triple simultaneous translocations, the empirical risk is calculated by adding the estimated risk for each separate translocation [4]. As previous studies have indicated, when an apparently balanced de novo CCR is detected, all molecular methods available should be used to look for a chromosomal imbalance, especially prenatally [8]. If no imbalance is detected after performing these analyses, the only available guidelines to estimate the risk of the child developing phenotypic abnormalities are those from Warburton [13] and this risk is about 3.5% per breakpoint. We report a patient with dysmorphic features, polydactyly and psychomotor retardation in whom a CCR involving five chromosomes and six breakpoints was detected. The rearrangement was characterized by FISH and aCGH, identifying a cryptic 3q13 deletion not previously reported. 2. Clinical report The patient, a 3-year-old boy, was referred for genetic evaluation because of intellectual disability and multiple congenital anomalies. He was the first child of non-consanguineous parents. His 40-year-old mother had a medical history of diabetes, hypertension, hypercholesterolemia and asthma. His father, who died at age 45 of pancreatic cancer, had depressive disorder and blindness secondary to meningitis. The patient had a paternal half-brother aged 21 with a behavioural disorder but apparently without intellectual disability or dysmorphism. The paternal grandmother and grandfather both died of Alzheimer's Disease. The patient's prenatal history was remarkable since the pregnancy was achieved by In Vitro Fertilisation (IVF) using the parents' gametes and the mother had both gestational hypertension and diabetes, treated with insulin. Prenatal ultrasonograms were normal. He was born at 34 week gestation by Caesarian section because of preclampsia. Birth somatometry was as follows: Weight: 2600 g (p90). Length: 44 cm (p30). OFC: 35 cm (p N 99). Apgar score was 7/8/9. At birth, he was admitted to the neonatology ward due to prematurity and preaxial polydactyly of the left hand was detected. During his hospital admission, the only abnormal findings were transient hypocalcemia and swallowing difficulties. Brain and cardiac ultrasonography, as well as metabolic and neonatal hearing screening, were all normal. He was referred again at 2 1/2 years to a Neuropediatrician due to psychomotor retardation. He held up his head at 12 months of age, sat down at 17 months and started walking alone at 22 months. Poor language development and cognitive impairment were also detected. Physical examination revealed short stature (89 cm at 3 years of age; bP1) with relative macrocephaly (PC: 51.5 cm; P52), short neck and dysmorphic facial features (prominent forehead, hypertelorism, epicanthus and lower palpebral deviation, broad and flat nasal root with short nose,
289
Fig. 1. Patient at the age of 3 years old. Note prominent forehead, hypertelorism, lower palpebral deviation, broad and flat nasal root, short nose and short neck.
bulbous tip and anteverted nares, low-set ears) (Fig. 1). Chest and abdominal examination were normal, as were his genitalia. There were no focal neurological findings apart from psychomotor retardation. He also had bilateral single palmar tranverse crease, scar in the radial side of the left hand (due to extra digit removal), 5th finger clinodactyly in his hands and feet as well as brachydactyly of his toes. Further investigations including audiometric and ophthalmological examinations and magnetic resonance imaging (MRI) scan of the brain were all normal. High resolution GTG banding analysis revealed an CCR apparently balanced with five breakpoints that involved two independent translocations, a two-way (3;4) and a three- way translocation (2;14;18) (Fig. 2a). FISH studies allowed us to define the complex rearrangement detecting an insertion of 14q22q24.3 segment on the long arm of derivated chromosome 2, so the CCR had 6 breakpoints (Figs. 2c,d). His mother's karyotype was normal but his father's could not be performed. Based on these data, we concluded that the patient's karyotype was: 46,XY,t(3;4)(3pter → 3q13.3::4q23 → 4qter;4pter → 4q23::3q13.3 → 3qter),t(2;14;18)(2pter → 2q33:: 14q22q24.3::18q23 → qter;14pter → 14q22::2q33 → 2qter;18pter → 18q23::14q24.3 → 14qter). Subsequently, an Agilent 105 K oligo aCGH (Feature Extraction software v10.1 and Genomic Workbench software v6.5) identified a deletion in the breakpoint on chromosome 3 involved in the translocation (Fig. 2b). The size of the deletion was 4.5–4.6 Mb, in the region 3q13.31q13.33 (chromosome position of the minimum boundaries: chr 3:115,492,890–120,031,896 and maximum boundaries: chr 3:115,440,032–120,075,357) and affected the dose of 13 OMIM genes: GAP43 (MIM 162060), LSAMP (MIM 603241), UPK1B (MIM 602380), B4GALT4 (MIM 604015), ARHGAP31 (MIM 610911), CD80 (MIM 112203), PLA1A (MIM 607460), POPDC2 (MIM 605823), COX17 (MIM 604813), MAATS1 (MIM 609910), NR1I2 (MIM 603065), GSK3B (MIM 605004) and GPR156 (MIM 610464). Other CNVs identified in the aCGH were also searched in databases and considered unlikely to have contributed to the phenotype of the patient. 3. Discussion The combination of FISH and aCGH has greatly facilitated the characterization of CCRs. Prior to array CGH technology, detection of genomic imbalances associated with chromosomal rearrangements was performed by a sequencing technique through the breakpoints and only a few cases had been published due to the complexity of the
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Fig. 2. Karyotype, FISH and microarray analysis of the patient. a: Chromosomes with GTG bands. Arrows are used to label the derivative chromosomes resulting from the two simultaneous reciprocal translocations, t(3;4) (red arrows) and t(2;14;18) (green arrows). b: Oligo array-CGH log2 ratio plot of whole chromosome 3 with ideogram showing deletion. c: Diagram illustrating how the segments of chromosome 14 are rearranged on chromosomes 2 and 18 (left: normal, right: derivatives). d: FISH analysis with whole chromosome painting 14 probe confirming the insertion of 14q22q24.3 segment on the derivated chromosome 2.
technique [14,15]. Feenstra et al. [8], in a meta-analysis of 171 patients with developmental delay (DD) and/or MCA and de novo balanced rearrangements by conventional cytogenetic, applying aCGH, detected genomic imbalance in 37% of patients with two breakpoints per rearrangement (translocations and inversions) and in 90% of patients with CCRs. In this last group, approximately 79% were due to deletions at the breakpoints and, in the rest, the imbalance was found apart from the breakpoints (similar to the general population with DD/MCA and normal karyotype). The CCR described in the present study was interpreted with GTG bands as balanced and implied two independent reciprocal translocations, one involving two chromosomes (3 and 4) and another involving three (2, 14 and 18). Our literature search found only three published cases with the coincidental presence of a two-way and a three-way translocation and all of the carrier individuals had been detected due to affected offspring with a chromosomal imbalance (for references see Madan [10]). However, in our case, the FISH technique showed a more complex rearrangement than was initially thought because it included an insertional translocation of chromosome 14 to chromosome 2. Therefore, based on this latter finding and on the classification proposed by Madan [10], our CCR was considered to be Type IV. In addition, aCGH analysis detected a cryptic deletion of 4.5–4.6 Mb at the breakpoint on chromosome 3 involved in the rearrangement, in the region 3q13.31q13.33, which affects the dose of about 12 genes. In our literature review, we have not found cases with the same deletion as that of our patient. However, recently a new microdeletion syndrome
at 3q13.13 has been described [16]. These authors reviewed a total of 27 patients with deletions of various sizes within the 3q12.3–3q21.3 region and concluded that patients with deletions that included the critical region 3q13.31 had common characteristics such as DD, abnormal male genitalia, postnatal overgrowth and dysmorphic features. The smallest overlapping deletion was 580 kb located in 3q13.31 and included two candidate genes for developmental delay, Dopamine receptor D3 (DRD3; MIM 126451) and Zinc finger-and BTB domaincontainig protein 20 (ZBTB20; MIM 606025). Our deletion does not include this critical region and the phenotypic characteristics do not overlap those described by Molin et al. [16]. More than 12 genes were deleted in our patient and, although the phenotypic effect due to haploinsufficiency for these genes is unknown, their functions are so broad that they may have a possible role in the abnormal phenotype. We propose that haploinsufficiency of one or more genes, such as Rho GTPase activating protein 31 (ARHGAP31; MIM 610911), Limbic system-associated membrane protein (LSAMP; MIM 603241), Growth-associated protein 43 (GAP43; MIM 162060), Popeye domain-containing protein 2 (POPDC2; MIM 605823) and Glycogen synthase kinase 3-beta (GSK3B; MIM 605004), included in the deletion region could influence the phenotype. Importantly, ARHGAP31 is a regulator of Cdc42 and Rac1 signalling to the cytoskeleton and thereby plays a key role in controlling the temporal and spatial cytoskeletal remodelling necessary for the precise control of cell morphology and migration [17]. Arhgap31 expression in mice is substantially restricted to the terminal limb buds and craniofacial
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processes during early development. Recently, Southgate et al. [18] have demonstrated that heterozygous gain-of-function mutations in ARHGAP31 cause an autosomal-dominant form of Adams–Oliver Syndrome-1(AOS1; MIM 100300), characterized by congenital aplasia cutis and terminal transverse limb defects. LSAMP guides the development of specific patterns of neuronal connections and may be relevant in the context of human neuropsychiatric and neurological disorders [19,20]. GAP43 is involved in neurone outgrowth, neurotransmission and synaptic plasticity, among other functions, and was also recently identified as a candidate gene for autism and autistic-like features in humans and mice [21,22]. POPDC2 is a membrane-associated protein predominantly expressed in skeletal and cardiac muscle and may have an important function in these tissues [23]. Finally, GSK3B is a serinethreonine kinase gene involved in energy metabolism and neuronal cell development. Polymorphisms in this gene have been implicated in modifying the risk of Parkinson's disease and studies in mice show that overexpression of this gene may be relevant in the pathogenesis of Alzheimer's disease [provided by RefSeq, Sep 2009]. However, besides the haploinsufficiency of these genes included in the 3q13.3 deletion, it is likely that, due to the complexity of the rearrangement in our patient, other possible mechanisms including gene disruption, fusion gene creation, position effects or dysregulation of genes located at one or more breakpoints, or even a combination of different causes, may be responsible for his phenotype. In summary, this study shows that the use of aCGH is essential for detecting possible chromosomal imbalances in apparently balanced rearrangements, especially in CCRs, leading to improved management and genetic counselling of the patient and family members. Moreover, the contribution of new cases with the same genomic imbalance allows us to establish a better genotype/phenotype correlation and a greater understanding of the function of genes involved in the rearrangement.
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
[16]
Acknowledgments We are grateful to Dr M V Tobin for critically reviewing the manuscript.
[17]
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Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
Short Communication
Array CGH detection of a novel cryptic deletion at 3q13 in a complex chromosome rearrangement Isabel López-Expósito a,d,⁎, María Juliana Ballesta-Martinez b,d, Juan Antonio Bafalliu a,d, Ascensión Vera-Carbonell a,d, Rosario Domingo-Jiménez c,d, Vanesa López-González b,d, Asunción Fernández a, Encarna Guillén-Navarro b,d,e a
Sección de Citogenética, Centro de Bioquímica y Genética Clínica, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Unidad de Genética Médica, Servicio de Pediatría, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain c Sección de Neuropediatría, Hospital Clínico Universitario “Virgen de la Arrixaca”, El Palmar, Murcia, Spain d Centro de Investigación Biomédica de Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain e Cátedra de Genética Médica, Universidad Católica de San Antonio (UCAM), Murcia, Spain b
a r t i c l e
i n f o
Article history: Received 3 October 2013 Accepted 24 February 2014 Available online 4 March 2014 Keywords: Complex chromosome rearrangement Oligo array-CGH 3q13 deletion
a b s t r a c t Complex chromosome rearrangements (CCRs) are extremely rare in humans. About 20% of the apparently balanced CCRs have an abnormal phenotype and the degree of severity correlates with a higher number of breakpoints. Several studies using FISH and microarray technologies have shown that deletions in the breakpoints are common although duplications, insertions and inversions have also been detected. We report a patient with two simultaneous reciprocal translocations, t(3;4) and t(2;14;18), involving five chromosomes and six breakpoints. He showed dysmorphic features, preaxial polydactyly in the left hand, brachydactyly, postnatal growth retardation and developmental delay. The rearrangement was characterized by FISH analysis which detected an interstitial segment from chromosome 14 inserted in the derivative chromosome 2, and by whole genome array which revealed an interstitial deletion of approximately 4.5 Mb at the breakpoint site on chromosome 3. To our knowledge this microdeletion has not been previously reported and includes ~ 12 genes. The haploinsufficiency of one or several of these genes is likely to have contributed to the clinical phenotype of the patient. © 2014 Elsevier Inc. All rights reserved.
1. Introduction There are different definitions of complex chromosome rearrangement (CCR) and all of them are based on the first report by Pai et al. [1]. The origin of constitutional CCRs is still unknown but several mechanisms have been proposed, most of them based on DNA replication [2]. Recently, it has been proposed that they may be formed as a ‘one-off’ event similar to the phenomenon termed chromothripsis seen in malignancy [3]. Although CCRs are extremely rare in humans with approximately 250 cases reported [4], their identification is important because CCR carriers can have variable phenotypes, which include clinically normal individuals, male infertility and patients with intellectual disability (ID) and/or multiple congenital abnormalities (MCA). Around 70% of CCRs have been detected in normal individuals referred for reproductive
⁎ Corresponding author at: Centro de Investigación Biomédica de Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Fax: +34 968 381222. E-mail address: [email protected] (I. López-Expósito).
http://dx.doi.org/10.1016/j.ygeno.2014.02.008 0888-7543/© 2014 Elsevier Inc. All rights reserved.
problems and/or having had a malformed child, 20–25% in patients with ID and/or MCA and 5–10% in prenatal diagnosis [5–7]. Approximately 70% of CCRs originate de novo and the remainder have a hereditary origin whose transmission is usually through the mother [4]. These cases tend to be less complex, with fewer breakpoints and chromosomes involved than in de novo cases. In both, the CCR can be either balanced or unbalanced. It has been estimated that about 20% of patients with apparently balanced CCRs have an abnormal phenotype and the probability that a de novo CCR is associated with an abnormal phenotype increases with the number of breakpoints [4]. Most CCRs detected by conventional cytogenetics may be more complex than initially suspected and, although FISH and derivative techniques such as multicolor banding facilitate the characterization of CCR, the application of microarray techniques based on comparative genomic hybridization (aCGH) is essential for identifying its molecular nature. Several studies with FISH and aCGH have shown that, at the breakpoints, deletions are common although duplications, insertions or inversions have also been detected [6,8]. De Gregori et al. [6], applying aCGH to patients with abnormal phenotype and apparently balanced chromosomal abnormalities, observed that 40% of the reciprocal
I. López-Expósito et al. / Genomics 103 (2014) 288–291
translocations and 90% of CCRs presented cryptic deletions. Furthermore, 18% of simple reciprocal translocations were more complex rearrangements. Although different classifications have been proposed for CCRs [9], recently Madan [10] proposed a new classification based on four types: I) CCRs with a number of breakages equal to the number of chromosomes involved, II) the number of breaks is one more than the number of chromosomes and includes an inversion, III) the number of breaks is higher than the number of chromosomes and includes one or more insertions and IV) the number of breakages is higher than the number of chromosomes and there is a “middle segment”, at least one derivative chromosome being composed of segments from at least three chromosomes. Balanced CCR carriers have an overall empirical risk of 50% for recurrent spontaneous abortions and 20% for having offspring with a chromosomal imbalance [7], although this risk depends on the type of CCR [10]. Thus, in Type IV, the probability of recombination in the intermediate segment is about 35% and this can result in balanced and unbalanced gametes. In most cases, the complex rearrangement changes to one single translocation [11,12] and in those CCRs with double or triple simultaneous translocations, the empirical risk is calculated by adding the estimated risk for each separate translocation [4]. As previous studies have indicated, when an apparently balanced de novo CCR is detected, all molecular methods available should be used to look for a chromosomal imbalance, especially prenatally [8]. If no imbalance is detected after performing these analyses, the only available guidelines to estimate the risk of the child developing phenotypic abnormalities are those from Warburton [13] and this risk is about 3.5% per breakpoint. We report a patient with dysmorphic features, polydactyly and psychomotor retardation in whom a CCR involving five chromosomes and six breakpoints was detected. The rearrangement was characterized by FISH and aCGH, identifying a cryptic 3q13 deletion not previously reported. 2. Clinical report The patient, a 3-year-old boy, was referred for genetic evaluation because of intellectual disability and multiple congenital anomalies. He was the first child of non-consanguineous parents. His 40-year-old mother had a medical history of diabetes, hypertension, hypercholesterolemia and asthma. His father, who died at age 45 of pancreatic cancer, had depressive disorder and blindness secondary to meningitis. The patient had a paternal half-brother aged 21 with a behavioural disorder but apparently without intellectual disability or dysmorphism. The paternal grandmother and grandfather both died of Alzheimer's Disease. The patient's prenatal history was remarkable since the pregnancy was achieved by In Vitro Fertilisation (IVF) using the parents' gametes and the mother had both gestational hypertension and diabetes, treated with insulin. Prenatal ultrasonograms were normal. He was born at 34 week gestation by Caesarian section because of preclampsia. Birth somatometry was as follows: Weight: 2600 g (p90). Length: 44 cm (p30). OFC: 35 cm (p N 99). Apgar score was 7/8/9. At birth, he was admitted to the neonatology ward due to prematurity and preaxial polydactyly of the left hand was detected. During his hospital admission, the only abnormal findings were transient hypocalcemia and swallowing difficulties. Brain and cardiac ultrasonography, as well as metabolic and neonatal hearing screening, were all normal. He was referred again at 2 1/2 years to a Neuropediatrician due to psychomotor retardation. He held up his head at 12 months of age, sat down at 17 months and started walking alone at 22 months. Poor language development and cognitive impairment were also detected. Physical examination revealed short stature (89 cm at 3 years of age; bP1) with relative macrocephaly (PC: 51.5 cm; P52), short neck and dysmorphic facial features (prominent forehead, hypertelorism, epicanthus and lower palpebral deviation, broad and flat nasal root with short nose,
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Fig. 1. Patient at the age of 3 years old. Note prominent forehead, hypertelorism, lower palpebral deviation, broad and flat nasal root, short nose and short neck.
bulbous tip and anteverted nares, low-set ears) (Fig. 1). Chest and abdominal examination were normal, as were his genitalia. There were no focal neurological findings apart from psychomotor retardation. He also had bilateral single palmar tranverse crease, scar in the radial side of the left hand (due to extra digit removal), 5th finger clinodactyly in his hands and feet as well as brachydactyly of his toes. Further investigations including audiometric and ophthalmological examinations and magnetic resonance imaging (MRI) scan of the brain were all normal. High resolution GTG banding analysis revealed an CCR apparently balanced with five breakpoints that involved two independent translocations, a two-way (3;4) and a three- way translocation (2;14;18) (Fig. 2a). FISH studies allowed us to define the complex rearrangement detecting an insertion of 14q22q24.3 segment on the long arm of derivated chromosome 2, so the CCR had 6 breakpoints (Figs. 2c,d). His mother's karyotype was normal but his father's could not be performed. Based on these data, we concluded that the patient's karyotype was: 46,XY,t(3;4)(3pter → 3q13.3::4q23 → 4qter;4pter → 4q23::3q13.3 → 3qter),t(2;14;18)(2pter → 2q33:: 14q22q24.3::18q23 → qter;14pter → 14q22::2q33 → 2qter;18pter → 18q23::14q24.3 → 14qter). Subsequently, an Agilent 105 K oligo aCGH (Feature Extraction software v10.1 and Genomic Workbench software v6.5) identified a deletion in the breakpoint on chromosome 3 involved in the translocation (Fig. 2b). The size of the deletion was 4.5–4.6 Mb, in the region 3q13.31q13.33 (chromosome position of the minimum boundaries: chr 3:115,492,890–120,031,896 and maximum boundaries: chr 3:115,440,032–120,075,357) and affected the dose of 13 OMIM genes: GAP43 (MIM 162060), LSAMP (MIM 603241), UPK1B (MIM 602380), B4GALT4 (MIM 604015), ARHGAP31 (MIM 610911), CD80 (MIM 112203), PLA1A (MIM 607460), POPDC2 (MIM 605823), COX17 (MIM 604813), MAATS1 (MIM 609910), NR1I2 (MIM 603065), GSK3B (MIM 605004) and GPR156 (MIM 610464). Other CNVs identified in the aCGH were also searched in databases and considered unlikely to have contributed to the phenotype of the patient. 3. Discussion The combination of FISH and aCGH has greatly facilitated the characterization of CCRs. Prior to array CGH technology, detection of genomic imbalances associated with chromosomal rearrangements was performed by a sequencing technique through the breakpoints and only a few cases had been published due to the complexity of the
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Fig. 2. Karyotype, FISH and microarray analysis of the patient. a: Chromosomes with GTG bands. Arrows are used to label the derivative chromosomes resulting from the two simultaneous reciprocal translocations, t(3;4) (red arrows) and t(2;14;18) (green arrows). b: Oligo array-CGH log2 ratio plot of whole chromosome 3 with ideogram showing deletion. c: Diagram illustrating how the segments of chromosome 14 are rearranged on chromosomes 2 and 18 (left: normal, right: derivatives). d: FISH analysis with whole chromosome painting 14 probe confirming the insertion of 14q22q24.3 segment on the derivated chromosome 2.
technique [14,15]. Feenstra et al. [8], in a meta-analysis of 171 patients with developmental delay (DD) and/or MCA and de novo balanced rearrangements by conventional cytogenetic, applying aCGH, detected genomic imbalance in 37% of patients with two breakpoints per rearrangement (translocations and inversions) and in 90% of patients with CCRs. In this last group, approximately 79% were due to deletions at the breakpoints and, in the rest, the imbalance was found apart from the breakpoints (similar to the general population with DD/MCA and normal karyotype). The CCR described in the present study was interpreted with GTG bands as balanced and implied two independent reciprocal translocations, one involving two chromosomes (3 and 4) and another involving three (2, 14 and 18). Our literature search found only three published cases with the coincidental presence of a two-way and a three-way translocation and all of the carrier individuals had been detected due to affected offspring with a chromosomal imbalance (for references see Madan [10]). However, in our case, the FISH technique showed a more complex rearrangement than was initially thought because it included an insertional translocation of chromosome 14 to chromosome 2. Therefore, based on this latter finding and on the classification proposed by Madan [10], our CCR was considered to be Type IV. In addition, aCGH analysis detected a cryptic deletion of 4.5–4.6 Mb at the breakpoint on chromosome 3 involved in the rearrangement, in the region 3q13.31q13.33, which affects the dose of about 12 genes. In our literature review, we have not found cases with the same deletion as that of our patient. However, recently a new microdeletion syndrome
at 3q13.13 has been described [16]. These authors reviewed a total of 27 patients with deletions of various sizes within the 3q12.3–3q21.3 region and concluded that patients with deletions that included the critical region 3q13.31 had common characteristics such as DD, abnormal male genitalia, postnatal overgrowth and dysmorphic features. The smallest overlapping deletion was 580 kb located in 3q13.31 and included two candidate genes for developmental delay, Dopamine receptor D3 (DRD3; MIM 126451) and Zinc finger-and BTB domaincontainig protein 20 (ZBTB20; MIM 606025). Our deletion does not include this critical region and the phenotypic characteristics do not overlap those described by Molin et al. [16]. More than 12 genes were deleted in our patient and, although the phenotypic effect due to haploinsufficiency for these genes is unknown, their functions are so broad that they may have a possible role in the abnormal phenotype. We propose that haploinsufficiency of one or more genes, such as Rho GTPase activating protein 31 (ARHGAP31; MIM 610911), Limbic system-associated membrane protein (LSAMP; MIM 603241), Growth-associated protein 43 (GAP43; MIM 162060), Popeye domain-containing protein 2 (POPDC2; MIM 605823) and Glycogen synthase kinase 3-beta (GSK3B; MIM 605004), included in the deletion region could influence the phenotype. Importantly, ARHGAP31 is a regulator of Cdc42 and Rac1 signalling to the cytoskeleton and thereby plays a key role in controlling the temporal and spatial cytoskeletal remodelling necessary for the precise control of cell morphology and migration [17]. Arhgap31 expression in mice is substantially restricted to the terminal limb buds and craniofacial
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processes during early development. Recently, Southgate et al. [18] have demonstrated that heterozygous gain-of-function mutations in ARHGAP31 cause an autosomal-dominant form of Adams–Oliver Syndrome-1(AOS1; MIM 100300), characterized by congenital aplasia cutis and terminal transverse limb defects. LSAMP guides the development of specific patterns of neuronal connections and may be relevant in the context of human neuropsychiatric and neurological disorders [19,20]. GAP43 is involved in neurone outgrowth, neurotransmission and synaptic plasticity, among other functions, and was also recently identified as a candidate gene for autism and autistic-like features in humans and mice [21,22]. POPDC2 is a membrane-associated protein predominantly expressed in skeletal and cardiac muscle and may have an important function in these tissues [23]. Finally, GSK3B is a serinethreonine kinase gene involved in energy metabolism and neuronal cell development. Polymorphisms in this gene have been implicated in modifying the risk of Parkinson's disease and studies in mice show that overexpression of this gene may be relevant in the pathogenesis of Alzheimer's disease [provided by RefSeq, Sep 2009]. However, besides the haploinsufficiency of these genes included in the 3q13.3 deletion, it is likely that, due to the complexity of the rearrangement in our patient, other possible mechanisms including gene disruption, fusion gene creation, position effects or dysregulation of genes located at one or more breakpoints, or even a combination of different causes, may be responsible for his phenotype. In summary, this study shows that the use of aCGH is essential for detecting possible chromosomal imbalances in apparently balanced rearrangements, especially in CCRs, leading to improved management and genetic counselling of the patient and family members. Moreover, the contribution of new cases with the same genomic imbalance allows us to establish a better genotype/phenotype correlation and a greater understanding of the function of genes involved in the rearrangement.
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
[16]
Acknowledgments We are grateful to Dr M V Tobin for critically reviewing the manuscript.
[17]
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