Abstract Reciprocal balanced translocations associated with clinical features are very rare. This study reports cytogenetic and molecular cytogenetic findings in a 3-year-old patient with mild developmental retardation, slight hypotone with a de novo balanced 46, XX, t(2; 11) (q33; q23) translocation. G-banded chromosomes and FISH-Analysis were used to examine the patient's karyotype as well as her parents'. FISH-probes prepared with specific RP11-BAC clones mapped near 2q33 and 11q23 regions were used to characterize the location of the breakpoints. The FISH results revealed that one of the break points is located within the human NBEAL1-Gene locus on chromosome 2, suggesting a correlation between this gene disruption and the patient’s mild developmental retardation. 

Karim Zad Hagh, Javad

Omrani, Mir Davood

Saleh Gargari, Soraya

English

Journals Portal, Shahid Beheshti University of Medical Sciences

94Revised: 05- Jan -2017Last Revised: 15- March -2017Accepted: 23- May -2017Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point on the Human NBEAL1-GeneHoHow to Cite This Article: Karimzadhagh J, Salehgargari S, Omrani MD. Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point on the Human NBEAL1-GeneHo. Iran J Child Neurol. Winter 2018;12(1):94-100.1. Watson Genetic Laboratory, Tehran, Iran.2. Feto-maternal unit, Shohada Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran.3. Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.Corresponding Author: Omrani MD. MDDepartment of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail: davood_omrani@sbmu.ac.irAbstractReciprocal balanced translocations associated with clinical features are very rare. This study reports cytogenetic and molecular cytogenetic findings in a 3-yr-old female patient with mild developmental retardation, slight hypotone with a de novo balanced 46, XX, t(2; 11) (q33; q23) translocation. Her parent attended private office at Tehran, Iran in 2013. G-banded chromosomes and FISH-Analysis were used to examine the patient’s karyotype as well as her parents. FISH-probes prepared with specific RP11-BAC clones mapped near 2q33 and 11q23 regions were used to characterize the location of the breakpoints. One of the break points is located within the human NBEAL1-Gene locus on chromosome 2, suggesting a correlation between this gene disruption and the patient’s mild developmental retardation. Keywords: Balanced translocation; NBEAL1-Gene, ALS2-gene; ALS; FISH; BACs cloneIntroductionThe Amyotrophic lateral sclerosis (ASL) is a progressive, neurodegenerative disease causing functionless of the motor upper neurons in the brain and lower motor neurons of the spinal cord systems and finally death from respiratory failure, usually within 3 to 5 yr of symptom onset. Over 90% of ALS are sporadic. Totally, 5%-10% is familial amyotrophic lateral sclerosis (FALS) but clinically indistinguishable from the sporadic form of the disease (1). Despite numerous efforts in the last decade, the basic pathophysiology of ALS remains unclear. Familial amyotrophic lateral sclerosis (FALS) is inherited as either autosomal dominant or autosomal recessive pattern. Recessive forms of ALS usually have juvenile onset. About 20% of FALS occur because of mutation found on SOD1 gene, but the causes of about 80% of adult-onset dominantly inherited FALS are not clear yet (2). One of the genes located on the critical region of type two ALS is NBEAL1 gene (ALS2CR17; Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 17 proteins). The NBEAL1-Gene is a novel human neurobeachin-like 1 protein gene located on the chromosome two (2q33.2) and belongs to candidate genes for lateral sclerosis 2 (ALS2) with 25 Exons and two Beige/BEACH domains and 3 WD40 repeats. NBEAL1 gene spans about 73 kb of genomic DNA. The RT-PCR results showed that NBEAL1 is expressed mostly in the human brain, kidney, prostate, and testis. The significant rate of NBEAL1 gene expression in gliomas, compared to normal brain tissue, suggests Javad KARIMZADHAGH PHD1, Soraya SALEHGARGARI MD2, Mirdavood OMRANI PHD3CASE REPORT95normal phenotype (5). The current study was an attempt to map the exact breakpoint on chromosome number two and eleven and to nominate and characterize the disrupted genes and their correlation with clinical findings on this patient.Case ReportThe case is a 3 yr old girl with mild developmental delay. Parents referred to private physicians’ clinics since the mother was over 35 during pregnancy in Tehran, Iran in 2013. The result of amniocentesis showed a balanced translocation in fetus with no other findings in ultrasound scans (46, XX, t(2; 11) (q33; q23) (Figure 1). Parents’ karyotypes were normal. After birth, the baby girl did not show any major or minor clinical anomalies and therefore was discharged from the hospital. After 3 yr, the girl showed some mild developmental delays, therefore, referred for further genetic analysis. Informed consent was taken from the parents.a correlation with gliomas (3). However, there are no reported cases indicating the fact that, disruption of this gene could yield clinical phenotype. Although one of the causes of the gene disruption in neonates is autosomal reciprocal translocations, the frequency of this sort of translocation is very low (0.1%) (4). The majority of balanced translocations (96%) are not associated with clinical features (3). This is due to either translocation disrupting non-coding regions of genome or breakpoint is not on dose sensitive genes. Therefore, characterization of the genetic causes of the cases with de novo balanced translocation and clinical features is still favoured and followed by researchers, which could help to understand correlation mechanism of patient genotype and phenotype.If carrier parent have a balanced rearrangement their offspring is in majority case normal. The risk for phenotypic abnormality is generally very low. Nonetheless, there are several reports for balanced translocation people with abnormal phenotypes, who has parent with the same balanced chromosomes but Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point ...Fig 1. High-resolution GTG banding showed a reciprocal balanced translocation t(2;11)(q33.2;q23.2) in the fetus.  1a: Translocated chromosomes 2 and 11.  2 b: Normal chromosomes 2 and 1196Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point ...Peripheral blood was collected for karyotype and DNA extraction. This procedure was carried out for both parents too. After locating, the primary breakpoint region in chromosome number two and eleven by high-resolution karyotype methods, 30 BACs clones were ordered for further analysis of the regions and then 21 was used for mapping the breakpoint.Preparing Locus Specific probesChromosome preparations and high-resolution chromosomes were made from peripheral blood of the patient and her parents using standard methods. In order to map the breakpoint on the chromosome 2q33 and 11q23 regions, the following BACs (bacterial artificial chromosome) were ordered based on the information obtained from the genome browser part of http://genome.ucsc.edu/ (Table1).Table 1. Used BACs, their position, and characterName Chromosome Start End Length (kb) BandRP11-265N15 2 200002213 200178128 175.916 2q33.1RP11-984A14 2 201531144 201699319 168.176 2q33.1RP11-61B5 2 202446118 202606502 160.385 2q33.1RP11-951H20 2 202897953 203067146 169.194 2q33.1RP11-164J9 2 203452370 203611753 159.384 2q33.1-2q33.2RP11-96E8 2 203558342 203715728 157.387 2q33.2RP11-1133F11 2 203658771 203809833 151.063 2q33.2RP11-771C13 2 203758666 203939481 180.816 2q33.2RP11-963F8 2 203804972 204003029 198.058 2q33.2RP11-916F5 2 208636277 208823743 187.467 2q33.3RP11-14M16 11 112546742 112704340 157.599 11q23.1RP11-1146P22 11 113500406 113657059 156.654 11q23.2RP11-584C22 11 114601776 114812896 211.121 11q23.2RP11-521L22 11 114820119 114986617 166.499 11q23.2RP11-698I1 11 114914151 115091089 176.939 11q23.2RP11-158B3 11 115064956 115220235 155.280 11q23.2RP11-822K7 11 115141419 115327772 186.354 11q23.3RP11-1081G12 11 114385681 114608391 222.711 11q23.2RP11-984C17 11 115326382 115523057 196.676 11q23.2-11q23.3RP11-2020 11 116250688 116416874 166.187 11q23.3RP11-764D19 11 116994343 117176849 182.507 11q23.397Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point ...BACs DNA was labeled by Nick-Translation method using digoxigenin 11-dUTP (Roche) and Biotin (Invitrogen). DNA hybridization was performed as described in standard protocols. FISH slides were analyzed using a Zeiss Axioskop microscope with a cooled charged-coupled-device camera (Photometric) and then Imaging Meta system software was applied.Aminocytic analysis revealed a balanced translocation t (2q;11q) in the fetus. The mapping of breakpoint was carried out using locus-specific FISH probes (LSP) made on with about 21 BACs clones. All the selected FISH probes were hybridized with the long arm of chromosome number two except FISH probes made on BACs RP11-771C13, RP11-963F8, and RP11-916F5 that hybridized with long arm of chromosome 11. In addition, hybridization of BACs RP11-113FI1 was positive, but its signal was divided into two regions in the band 2q33.2 and chromosome 11. In chromosome number 11, all probes were hybridized and signals were obvious except for BACs probes RP11-158B3, Rp11-822K7, Rp11-984C17 and Rp11-2020 that hybridized with long arm of chromosome number 2. Rp11-69811signals was divided between long arm of chromosome number 2 and 11 (Figure 2). DiscussionMore than 90% of the de novo balanced translocation has no clinical impact since it occurs in the non-coding region of the genome or disrupts non-dose dependent genes. In the current study, a baby girl came to attention since she had mild developmental delay. Although her balanced translocation was noticed during her fetal period, her parents did not show any interest for further examination until she becomes 3 yr old and showed 98Fig 2. FISH signals in metaphase and interphase from peripheral blood and a combination of two FISH-probes from split BACs (SB). The split BAC of chromosome 2q (RP11-1133F11) was red labeled and the split BAC of chromosome 11q (RP11-698I1) was green labeled. In normal chromatid, only one signal either green (on Chr. 2) or red (on Chr. 11) were detected. In chromatid with translocation, two signals (Red/Green) or their combination (yellow) was detected. N=normal, T=translocated99Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point ...some symptoms. When locus-specific identifier (LSI) probes were designed to map the breakpoint between bands 2q33.1 and 2q33.3, all probes were hybridized, except the probe made from BAC RP11-771C13, RP11-963F8 and RP11-916F5 that hybridized with long arm of chromosome 11. In addition, hybridization of BAC RP11-113F11 was positive, but its signal was divided into two regions in the band 2q33.2 and chromosome 11. This clone contains about 157kb of chromosome 2q33.2 band. The clone start point is from 203558342bp and end is 203715728bp. When the LSI probe (Rp11-771C13) in the only 42938bp (about 43kb) downstream of this clone was used for hybridization purpose, FISH signal was negative on chromosome 2 but has positive signal on chromosome 11. Rp11-771C13 started from 203758666bp and ended in 203939481bp on the band 2q33.2. In the next step, the researcher chose an LSI probe that overlapped the distal part of Rp11-96E8 BAC and the proximal part of Rp11-771C13 BAC, in addition to covering 43 kb gap between these two probes. The start and end of the BAC Rp11-113F11 were 203658771-203809833bp, respectively. This probe was hybridized but its signals were divided between long arms of chromosome number 2 and 11. The Rp11-113F11 BAC probe signal may indicate that this signal was related to the 43 kb distance between these two BACs Rp11-96E8 and RP11-771C13 (Figure 3).Fig 3. Some of the BACs clones were used for mapping the breakpoint region on chromosome 2 and how RP11-1133F11 probe overlap with NBEAL1 gene. NT=Not translocated, T=Translocated, SB=Spilt BACs100The application of blast for the sequence spanned in this 43kb region indicated that NBEAL1 gene was located in this region. It was for the first time that a correlation with the disruption of NBEAL1 gene and its consequence was shown. Unfortunately, after reporting this finding to the parents of the patient, they did not show interest for further cooperation and, consequently, it was not possible to sequence the NBEAL1 gene in the patient for its further analysis to identify its exact disruption point. In order to evaluate the breakpoint region on the long arm of chromosome 11 (11q23.2 band), among all used FISH probes only probe on BACs RP11-158B3, Rp11-822K7, Rp11-984C17, and Rp11-2020 did not hybridize with the expected region on chromosome 11 and instead hybridized with long arm of chromosome number 2. Rp11-69811 signals were divided between long arm of chromosome number 2 and 11Rp11-698I1. The start point of the BAC 114914151bp and the end is 115091089bp (176939bp). When this region sequence was blasted, no coding sequence was found which means this deletion is in the non-coding region of chromosome number 11 and has no effect on and contribution to the observed patient symptoms. In conclusion, despite this fact that more than 90% of the de novo balanced translocation has no clinical impact, in this special case it was shown that the phenotype of patient is because of her disrupted NBEAL1 gene.  AcknowledgementWe would like to thanks patient family for their cooperation.Author`s contributionCollecting fetus sample and her parents carried out by Dr. saleh gargari, Dr. Karimzad and Dr. Omrani were performed Moecular and FISH analysis.All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Conflict of interestThe authors declare that there is no conflict of interest.References 1. Saberi S, Stauffer JE, Schulte DJ, Ravits J. Neuropathology of Amyotrophic Lateral Sclerosis and Its Variants. Neurol Clin 2015;33(4):855-76.2. Chen S, Sayana P, Zhang X, Le W. Genetics of amyotrophic lateral sclerosis: an update. Mol Neurodegener 2013;13(8):1326-28. 3. Chen J, Lu Y, Xu J, Huang Y, Cheng H, Hu G, et al. Identification and characterization of NBEAL1, a novel human neurobeachin-like 1 protein gene from fetal brain, up regulated in glioma. Brain Res Mol Brain Res 2004;125(1-2):147-55.4. Hook EB, Hamerton JL. Porter IH. Population Cytogenetics – Studies in Humans. In: The frequency of chromosome abnormalities detected in consecutive newborn studies – differences between studies – results by sex and by severity of phenotypic involvement; New York: Academic Press; 1977. pp. 63–79.5.  Warburton D. De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnostics: clinical significance and distribution of breakpoints. Am J Hum Genet 1991;49(5): 995–1013.Characterization of a de Novo Constitutional Balanced Translocation t (2;11)(q33.2;q23.2) with Break Point ...

Characterization of a de novo constitutional balanced translocation

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Characterization of a de novo constitutional balanced translocation

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