Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Research Recurrence, submicroscopic complexity, and potential clinical relevance of copy gains detected by array CGH that are shown to be unbalanced insertions by FISH Nicholas J. Neill,1 Blake C. Ballif,1 Allen N. Lamb,1 Sumit Parikh,2 J. Britt Ravnan,1 Roger A. Schultz,1 Beth S. Torchia,1 Jill A. Rosenfeld,1 and Lisa G. Shaffer1,3 1Signature Genomic Laboratories, Spokane, Washington 99207, USA; 2Center for Pediatric Neurology, Cleveland Clinic, Cleveland, Ohio 44195, USA Insertions occur when a segment of one chromosome is translocated and inserted into a new region of the same chro- mosome or a non-homologous chromosome. We report 71 cases with unbalanced insertions identified using array CGH and FISH in 4909 cases referred to our laboratory for array CGH and found to have copy-number abnormalities. Al- though the majority of insertions were non-recurrent, several recurrent unbalanced insertions were detected, including three der(Y)ins(Y;18)(q?11.2;p11.32p11.32)pat inherited from parents carrying an unbalanced insertion. The clinical sig- nificance of these recurrent rearrangements is unclear, although the small size, limited gene content, and inheritance pattern of each suggests that the phenotypic consequences may be benign. Cryptic, submicroscopic duplications were observed at or near the insertion sites in two patients, further confounding the clinical interpretation of these insertions. Using FISH, linear amplification, and array CGH, we identified a 126-kb duplicated region from 19p13.3 inserted into MECP2 at Xq28 in a patient with symptoms of Rett syndrome. Our results demonstrate that although the interpretation of most non-recurrent insertions is unclear without high-resolution insertion site characterization, the potential for an otherwise benign duplication to result in a clinically relevant outcome through the disruption of a gene necessitates the use of FISH to determine whether copy-number gains detected by array CGH represent tandem duplications or unbalanced insertions. Further follow-up testing using techniques such as linear amplification or sequencing should be used to de- termine gene involvement at the insertion site after FISH has identified the presence of an insertion. [Supplemental material is available for this article. The microarray data from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). A full list of accession numbers can be found in Sup- plemental Table 2.] Insertions occur when a segment of one chromosome is trans- form of the rearrangement, with either partial trisomy or partial located and inserted into an interstitial region of another non- monosomy, in progeny (Fogu et al. 2007). homologous chromosome (interchromosomal) (Abuelo et al. 1988; Here, we report the identification and characterization of Van Hemel and Eussen 2000) or into a different region of the same insertions in 71 probands in a diagnostic setting by array CGH and chromosome (intrachromosomal) (Madan and Menko 1992). In- FISH. Our results demonstrate that although the interpretation of sertions may occur in a direct fashion, in which the inserted seg- most non-recurrent insertions is unclear without high-resolution ment maintains its orientation with respect to the centromere, or insertion site characterization, follow-up techniques such as FISH may be inverted. Estimates of the incidence of insertions range and linear amplification coupled with array CGH may determine from 1:10,000 to 1:80,000 live births by cytogenetic techniques whether copy-number gains detected by array CGH represent (Van Hemel and Eussen 2000). tandem duplications or unbalanced insertions. High-resolution Many factors affect the phenotypic consequences of in- insertion-site characterization may also determine gene involve- sertions, including the size and gene content of the inserted seg- ment at the insertion site. ment, which may cause functional aneusomy of a dosage-sensitive gene(s); the orientation of the insertion; position effects exerted on Results genes in the inserted segment and/or at the site of insertion; dis- ruption of a gene by the insertion; and the presence or absence of During the study period from March 2004 to February 2010, we additional alterations around the breakpoints of the insertion tested more than 40,000 patients in our laboratory by array CGH (Baptista et al. 2005, 2008). Although balanced insertions are less and reported 8861 copy-number alterations in 7441 patients. Of likely to have clinical consequences, malsegregation of a balanced these alterations, 4648 involved copy-number gain, whereas 4213 insertion present in a carrier parent can result in an unbalanced involved loss of material. Metaphase FISH analysis was performed on a total of 3884 copy-number gains and 1644 losses, and pa- rental FISH was performed for 1982 copy-number gains and 1759 losses. In total, 5643 abnormalities (3884 gains and 1759 losses) 3 Corresponding author. in 4909 patients were further investigated by performing FISH E-mail [email protected]; fax (509) 474-6839. Article published online before print. Article, supplemental material, and pub- analysis and/or obtaining parental samples after array CGH iden- lication date are at http://www.genome.org/cgi/doi/10.1101/gr.114579.110. tified a copy-number alteration potentially resulting from an 21:000–000 Ó 2011 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/11; www.genome.org Genome Research 1 www.genome.org 2 Genome Research Table 1. Thirty-six probands with unbalanced insertions Neill et al. Array CGH Estimated Number of Indication for Balanced www.genome.org # Sex Classification result (hg18) size genes study Inheritance parent? 1 F der(1)ins(1;X)(p36.32;q22.2q22.2) arr Xq22.2(102675779–102986217)x3 dn,1p36. 310,438 8 DD, large head, nystagmus De novo N/a Downloaded from dup(1)(p36.32p36.32) 32(3323541–4052757)x3 dn 2 M der(21)ins(21;X)(q21.1;q28q28) arr Xq28(152731938–153061110)x3 dn, 21q21. 329,172 13 DD, hypertonia De novo N/a dup(21)(q21.1q21.1) 1(22347877–22623043)x3 3 M der(4)ins(4;10)(q?;p11.23p11.22) arr 10p11.23p11.22(29468548–33536513)x3 dn 4,067,965 20 46,XY,t(4;8)(q25;p21) De novo N/a 4 M der(12)ins(12;21)(p?12;q21.2q21.3) arr 21q21.2q21.3(24932718–28487546)x3 dn 3,554,828 14 DD, DF, small mouth De novo N/a 5 M der(7)ins(7;2)(q36.1;p25.3p25.3) arr 2p25.3(402953–774426)x3 dn 371,473 2 DD De Novo N/a 6 F der(X)ins(X;19)(q28;p13.3p13.3) arr 19p13.3(4845920–4971768)x3 dn 125,848 3 Partial epilepsy with De novo N/a intractable epilepsy genome.cshlp.org 7 F der(C)ins(C;7)(?;q11.2q11.2) arr 7q11.2(71245134–71417110)x3 mat 171,976 1 DD, DF Maternal Unbalanced 8 F der(9)ins(9;X)(p11?;p21.1p21.1) arr Xp21.1(32772868–32818426)x3 mat 45,558 1 Idiopathic peripheral Maternal Unbalanced neuropathy 9 M der(2)ins(2;11)(p?14;q23.1q23.1) arr 11q23.1(111095142–111527763)x3 mat 432,621 15 Abnormal maternal serum Maternal Balanced screen 10 F der(15)ins(15;13)(q11.2;q12.11q14.3) arr 13q12.11q14.3(18448674–49406099)x3 mat 30,957,425 191 Hypoglycemia Maternal Balanced 11 F der(9)ins(9;2)(p13.?2;q14.2q14.3) arr 2q14.2q14.3(121124717–123291212)x3 mat 2,166,495 6 DD, DF Maternal Balanced onSeptember29,2021-Publishedby 12 M der(21)ins(21;X)(p1?1;q25q25) arr Xq25(127016331–127285027)x3 mat 268,696 0 Encephalopathy Maternal Unbalanced 13 M der(12)ins(12;8)(q21.?1;p12p11.22) arr 8p12p11.22(38289074–39797061)x3 mat 1,507,987 15 congenital heart defect Maternal Balanced 14 F der(9)ins(9;5)(p?;p15.33p15.33) arr 5p15.33(1169180–1562887)x3 mat 393,707 6 Estropia, cleft palate Maternal Unbalanced 15 M der(16)ins(16;9)(q12?;p23p23) arr 9p23(9839207–10096124)x3 mat 256,917 1 DD Maternal Unbalanced 16 M der(X)ins(X)(q28p22.33p22.33) arr Xp22.33(249940–488150)x3 mat,Xq28 238,210 1 DD Maternal Unbalanced dup(X)(q28q28) (154396893–154429972)x3 mat 17 M der(X)ins(X)(q28p22.33p22.33) arr Xp22.33(300092–669611)x3 mat,Xq28 369,519 1 MCA Maternal Unbalanced dup(X)(q28q28) (154396893–154429972)x3 18 M der(X)ins(X;Y)(p22.?3;p11.2p11.2) arr Yp11.2(3780840–7082099)x3 mat 3,301,259 16 DD, delayed milestones Maternal Unbalanced 19 F der(15)ins(15;8)(?q11.2;p11. arr 8p11.21(41099138–41374290)x3 mat 275,152 1 2 vessel cord/single umbilical Maternal Unbalanced 21p11.21) artery 20a F der(14)ins(14;11)(p11.2;p11.2p11.2) DD Maternal Unbalanced 21 F der(13)ins(10;13)(q2?4;q13.3q21.1) arr 13q13.3q21.1(36064940–57255210)x1 mat 21,190,270 147 DD, DF, retinoblastoma Maternal Balanced 22 M der(5)ins(5;3)(q13?;p26.3p26.3) arr 3p26.3(261299–348311)x3 mat 87,012 1 DF Maternal Unbalanced 23 F der(1)ins(1;X)(q12?;q21.33q21.33) arr Xq21.33(96506690–97403811)x3 mat 897,121 1 interruption of aortic arch Maternal Unbalanced 24 F der(7)ins(7;9)(q?;q34.3q34.3) arr 9q34.3(138298382–140031595)x3 mat 1,733,213 81 DD, pituitary dwarfism Maternal Unbalanced 25 F rec(9)del(9)ins(q?13q34.11q34.11) arr 9q34.11(130064648–132004156)x1 mat 1,939,508 43 DD, MCA, microcephalus Maternal Balanced Cold SpringHarborLaboratoryPress 26 M rec(9)dup(9)(q21.13q21.32)del(9) arr 9q21.13q21.32(75805226–84795201)x3 8,989,975 26 MR, MCA, reduction Paternal Balanced (q31.1q31.3)rcp ins(9)(q21.13q21.