CLINICAL REPORT
Homozygous 16p13.11 Duplication Associated with Mild Intellectual Disability and Urinary Tract Malformations in Two Siblings Born from Consanguineous Parents N. Houcinat,1,2* B. Llanas,3 S. Moutton,1,2 J. Toutain,1 D. Cailley,1 B. Arveiler,1,2 C. Combe,4,5 D. Lacombe,1,2 and C. Rooryck1,2 1
Genetique medicale, CHU, Bordeaux, France
2
Univ. Bordeaux, Maladies Rares : Genetique et Metabolisme (MRGM), EA 4576,, F-33000 Bordeaux, France
3
Nephrologie pediatrique, CHU, Bordeaux, France Nephrologie Transplantation Dialyse, CHU, Bordeaux, France
4 5
Univ. Bordeaux, Unite INSERM 1026, F-33000 Bordeaux, France
Manuscript Received: 4 February 2014; Manuscript Accepted: 4 June 2015
The use of array-comparative genomic hybridization (arrayCGH) in routine clinical work has allowed the identification of many new copy number variations (CNV). The 16p13.11 duplication has been implicated in various congenital anomalies and neurodevelopmental disorders, but it has also been identified in healthy individuals. We report a clinical observation of two brothers from related parents each carrying a homozygous 16p13.11 duplication. The propositus had mild intellectual disability and posterior urethral valves with chronic renal disease. His brother was considered a healthy child with only learning disabilities and poor academic performances. However, a routine medical examination at 25-years-old revealed a mild chronic renal disease and ureteropelvic junction obstruction. Furthermore, the father presented with a unilateral renal agenesis, thus it seemed that a “congenital anomalies of kidney and urinary tract” (CAKUT) phenotype segregated in this family. This may be related to the duplication, but we cannot exclude the involvement of additional genetic or non-genetic factors in the urological phenotype. Several cohort studies showed association between this chromosomal imbalance and different clinical manifestations, but rarely with CAKUT. The duplication reported here was similar to the larger one of 3.4 Mb previously described versus the more common of 1.6 Mb. It encompassed at least 11 known genes, including the five ohnologs previously identified. Our observation, in addition to expanding the clinical spectrum of the duplication provides further support to understanding the underlying pathogenic mechanism. © 2015 Wiley Periodicals, Inc.
How to Cite this Article: Houcinat N, Llanas B, Moutton S, Toutain J, Cailley D, Arveiler B, Combe C, Lacombe D, Rooryck C. 2015. Homozygous 16p13.11 duplication associated with mild intellectual disability and urinary tract malformations in two siblings born from consanguineous parents. Am J Med Genet Part A 167A:2714–2719.
Key words: 16p13.11; duplication; ohnologs; CAKUT; arrayCGH; intellectual disability
INTRODUCTION The use of array-comparative genomic hybridization (array-CGH) in routine clinical work has led to the identification of many new copy number variations (CNVs) [Cooper et al., 2011; Girirajan et al., 2012]. Indeed, the 16p13.11 locus is considered as a CNV hotspot mediated by non-homologous recombination between chromosome 16 low copy repeats (LCRs) [Ingason et al., 2011]. Deletions and reciprocal duplications in this region have been
Correspondence to: Nada Houcinat, CHU Bordeaux, place amelie Raba leo, 33000 Bordeaux, Bordeaux 33000 France. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 26 June 2015 DOI 10.1002/ajmg.a.37212
© 2015 Wiley Periodicals, Inc.
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implicated in multiple congenital anomalies and neurodevelopmental disorders such as epilepsy, autism, and intellectual disability [Ullmann et al., 2007; Hannes et al., 2009; Nagamani et al., 2011; Ramalingam et al., 2011; Wisniowiecka-Kowalnik et al., 2013]. The 16p13.11 duplication has also been associated in many studies with varied phenotypes such as radial ray deficiency [Vergult et al., 2013], aortic dissections, schizophrenia, and attention deficit hyperactivity disorder [Sahoo et al., 2011]. The 16p13.11 CNV is thought to be a susceptibility locus for neurocognitive disease, as several authors found the duplication statistically enriched in the patient versus the control populations [Mefford et al., 2009; Girirajan et al., 2012; Tropeano et al., 2013; Coe et al., 2014]. Here, we report a clinical observation of two brothers carrying a homozygous 16p13.11 duplication and presenting with mild intellectual disability and urological malformations. We discuss the implication of this chromosomal imbalance in their phenotype especially in the anomalies of the renal system.
MATERIALS AND METHODS Oligonucleotides array-CGH was performed using a 44 K whole genome microarray (Agilent Technologies, Santa Clara, California) according to manufacturer’s instructions, after DNA extraction from the patient’s peripheral blood leukeocytes using standard procedures. DNA from the patient was analyzed in comparative genomic hybridization experiments with fluorochrome swapping in a trio along with DNA from two patients with different phenotype (serving as reference DNAs) [Rooryck et al., 2010]. Fluorescence ratio was calculated to detect chromosomal imbalances. Deletions and duplications were taken into consideration when a minimum of three consecutive variant points were detected by the statistical algorithm ADM2 with a threshold of 5 (Agilent Genomic Workbench Lite Edition 6.5, Agilent Technologies, Santa Clara, California). To confirm the rearrangement identified by array-CGH, FISH technique was carried out on metaphase spreads using specific probe. Mutational screening of candidate gene was performed on genomic DNA extracted from peripheral blood lymphocytes. The coding regions and exon-intron boundaries of the gene were amplified by PCR. The amplicons were then sequenced using the Big Dye Terminator Cycle Sequencing Kit v3, and run on an automated sequencer ABI 3500 xL Dx (Applied Biosystems). The electrophoretic profiles were analyzed with Seqscape v2.7 (Applied Biosystems).
CLINICAL REPORTS The propositus was a 32-year-old man, he was the first child born from consanguineous parents (Fig. 1). He came to medical attention on day 1 of life for urinary tract distension due to posterior urethral valves, corrected surgically at day 15 of life. Many complications occurred after surgery: multiple infections of the urinary tract and interstitial nephropathy. The patient developed a chronic renal disease and an uretero-hydronephrosis. He had developmental delay and intellectual disability, could not read or write, or live independently. He had normal motor development, and otherwise normal neurological examination. His brother was healthy as a
FIG. 1. Pedigree of the family. The father had a unilateral renal agenesis; the two sons had both renal anomalies and cognitive impairment.
child and as a young adult until age 25 when a routine blood test revealed a mild chronic renal disease. Ultrasound examination showed an ureteropelvic junction obstruction. He had no intellectual deficiency, he worked as a car mechanic, and his history revealed only learning disability. Indeed, he had poor overall academic performance and he needed a specialized teaching class and tutoring. The investigations of the parents revealed a unilateral renal agenesis in the father who otherwise had normal intellectual abilities. The mother was a healthy woman. Clinical morphological examination also revealed that the two brothers shared common dysmorphic features; prominent supra-orbital ridges, deep-set eyes, and prognathism (Fig. 2). Parents were normal without dysmorphic features. Chromosome analysis showed a 46,XY normal karyotype in the propositus.
RESULTS Array-CGH performed in the propositus identified a 16p13.11– p12.3 duplication with a minimal size of 3.2 Mb (chr16:15, 256, 686–18, 546, 759) and a maximal size of 3.6 Mb (chr16:15, 154, 746–18, 794, 446) (Hg19). The log2 ratio average of fluorescence’ signals was 0.8 which was consistent with a homozygous state of the duplication (four copies). This duplicated region contained at least 11 known genes (Fig. 3). Familial segregation study of this rearrangement was performed by FISH analysis, using specific probe (RP11-120O23) targeting this region. It confirmed the homozygous duplication in the propositus and also in his brother. Parents were each heterozygous for the duplication.
DISCUSSION Since the advent of array-CGH, several cohort studies of patients carrying heterozygous genomic 16p13.11 imbalances have been published. Ullman et al. [2007] first reported a recurrent 1.65 Mb
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
FIG. 2. Patients’ pictures (sons), A: propositus, B: propositus’ brother.
duplication at the 16p13.11 locus (chr16:14.9–16.4 Mb) (Hg19) in three individuals, among a cohort of 182 patients, presenting with autism or intellectual disability. Hannes et al. [2008], detected a 16p13.11 duplication in 7 out of 1,027 patients, five with the recurrent 1.65 Mb duplication and two with a larger one
FIG. 3. Array CGH results.
3.4Mb (chr16: 15.1–18.5 Mb) (Hg19). These patients presented with intellectual disability and variable congenital anomalies. Only one of the patients carrying the larger duplication had an anomaly of the kidney and urinary tract (unilateral renal agenesis). However, his phenotype, resembling a CHARGE syndrome, was more
HOUCINAT ET AL. complex than the one usually described in patients carrying the 16p13.11 rearrangement. The duplication was inherited from a healthy parent in two cases and appeared de novo in one case. Furthermore, the duplication was found in five asymptomatic individuals out of 2,014 controls in the same study. Mefford et al. [2009] detected the duplication in 11 of 1,010 affected individuals contrasting with only two of 2,493 controls. Nagamani et al. [2011] described 10 patients carrying this duplication, they had varied clinical features including behavior abnormalities, developmental delay, skeletal manifestations, and congenital heart defects but no anomaly of the renal system. Ramalingam et al. [2011] described the clinical phenotype of eight patients with the duplication, only one of them had unspecified kidneys anomalies. Overall, studies showed that the duplication was significantly enriched in patients with intellectual disabilities and/or autism versus controls populations, and the kidney anomalies have been seen in only 2 of 39 cases with phenotypic information, one with unspecified kidney anomalies and one unilateral renal agenesis [Cooper et al., 2011; Girirajan et al., 2012; Tropeano et al., 2013; Coe et al., 2014]. The urological malformations segregating in our family were varied. They belong to the spectrum of CAKUT phenotype (Congenital anomalies of kidney and urinary tract). Human CAKUT are a group of heterogeneous diseases characterized by a wide range of structural and functional malformations of kidneys and lower urinary tract. Genetic forms may be syndromic, associated with multi-organ malformations, or isolated. Animal model studies in knock-out mice provided a better understanding of the molecular basis of the renal system morphogenesis, allowing the identification of many genes involved in renal development [Costantini and Kopan 2010; Men-
2717 delsohn 2009]. Only few of them were found to be mutated in recessive and dominant forms of human CAKUT. Therefore, the pathological phenotype seemed to arise from interactions between modifier genes and epigenetic factors [El-Dahr et al., 2000; Song and Yosypiv 2011]. The association between the duplication and CAKUT phenotype appeared to be not so rare. Indeed, it has been reported in 2 out of 39 heterozygous carriers with phenotypic information (3 of 40 cases if we consider the father in our family). However, the patient with renal agenesis reported by Hannes et al. [2008]) had a complex phenotype and presented with aplasia of semicircular canals, asymmetric face with left facial nerve paresis, coloboma, atresia of right choana, and unique right kidney with double ureters. All these features are strongly suggestive of CHARGE syndrome. Thus, one must be careful with conclusions about the implication of the duplication in this case. Concerning this observation, we cannot exclude the implication of additional genetic or non-genetic factors in the urological malformations. There is no evident candidate gene for urological malformations in this duplicated region, and it is well-known that environmental and epigenetic factors play an important role in the renal development [ElDahr et al., 2000; Song and Yosypiv 2011]. Therefore, it may be interesting to search for the 16p13.11 duplication in patients with a CAKUT phenotype to see if this CNV is enriched in patients with renal anomalies. In view of the family pedigree (the father was also the mother’s uncle) and the preferential affected males, an X-linked inheritance has been considered even if the pedigree was small. We also evaluated the only CAKUT gene located on the X chromosome: AGTR2, coding for Angiotensin type-2 receptor R. This gene was implicated in the morphogenesis of the renal system in mouse models and AGTR2 knock-out (KO) mice developed a CAKUT
FIG. 4. DNA copy number variants and LCRs in the16p13 region. A screenshot from the University of California, SantaCruz (UCSC) Genome Browser: the duplication identified in our family is in blue and the two duplications described in the literature (red bar), the classical and the larger one.
2718 phenotype [Nishimura et al., 1999; Song et al., 2010]. In addition, association between CAKUT and a single polymorphism, c.1332A>G transition, in the human AGTR2 gene, have been reported in several, but not all, studies [Hahn et al., 2005; Rigoli et al., 2004]. Moreover, mutations in AGTR2 gene have been found in several cases of X-linked intellectual disability [Vervoort et al., 2002; Ylisaukko-oja et al., 2004]. Unfortunately, the mutational screening of AGTR2 coding regions proved negative. The duplication reported here was similar to the large one previously described [Hannes et al., 2009] overlapping the typical and most frequently described smaller duplication (Fig. 4). It encompassed at least 11 known genes, including the five ohnologs (NDE1, MYH11 ABCC1, ABCC6 and XYLT1) previously identified [Tropeano et al., 2013]. Four of these genes (NDE1, MYH11 ABCC1, ABCC6) are common between the large duplication (3.4Mb) and the more commonly described smaller duplication (1.65 Mb) (Fig. 4). One increasingly popular hypothesis is that ohnologs, genes retained after ancestral whole genome duplication events, are particularly sensitive to dosage change. They are thought to be responsible for the major pathological phenotype associated with duplications [Makino and McLysaght 2010; Kasahara 2007]. The duplication in a homozygous state did not appear to increase the phenotypic severity, including the cognitive aspect as these two brothers, especially the second son, presented with a milder phenotype than described for some heterozygous carriers in the literature. Thus, the clinical severity was not proportional to the number of copies of the region and perhaps for this region any dosage imbalance could have a pathogenic effect, regardless of the copy number variation. This underlines the importance of the role played by the dosage imbalance itself, in the pathogenic effect, whatever the degree of the imbalance. Varied genetic, epigenetic and environmental factors appear to influence the pathological presentation more than the number of copies. All these considerations make interpretation of CNVs found by array-CGH difficult. Indeed, regarding our patients, it is difficult to conclude about the mental prognosis of their future offspring that have 100% risk of being a heterozygous carrier of the 16p13.11 duplication.
ACKNOWLEDGMENT We thank Drs Alfio de Marin and Patrick Pauly, Cabinet de Nephrologie, Polyclinique de Navarre, 64075 Pau, France, for referring the patients.
REFERENCES Coe BP, Witherspoon K, Rosenfeld JA, van Bon BW, Vulto-van Silfhout AT, Bosco P, Friend KL, Baker C, Buono S, Vissers LE, Schuurs-Hoeijmakers JH, Hoischen A, Pfundt R, Krumm N, Carvill GL, Li D, Amaral D, Brown N, Lockhart PJ, Scheffer IE, Alberti A, Shaw M, Pettinato R, Tervo R, de Leeuw N, Reijnders MR, Torchia BS, Peeters H, O’Roak BJ, Fichera M, Hehir-Kwa JY, Shendure J, Mefford HC, Haan E, Gecz J, de Vries BB, Romano C, Eichler EE. 2014. Refining analyses of copy number variation identifies specific genes associated with developmental delay. Nat Genet 46:1063–1071. Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, Williams C, Stalker H, Hamid R, Hannig V, Abdel-Hamid H, Bader P, McCracken E, Niyazov D, Leppig K, Thiese H, Hummel M, Alexander N, Gorski J,
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Kussmann J, Shashi V, Johnson K, Rehder C, Ballif BC, Shaffer LG, Eichler EE. 2011. A copy number variation morbidity mao of developmental delay. Nat Genet 2011/08/16 43:838–846. Costantini F, Kopan R. 2010. Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Cell 18:698–712. El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, Meleg-Smith S. 2000. Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development. Physiol Genomics 3:121–131. Girirajan S, Rosenfeld JA, Coe BP, Parikh S, Friedman N, Goldstein A, Filipink RA, McConnell JS, Angle B, Meschino WS, Nezarati MM, Asamoah A, Jackson KE, Gowans GC, Martin JA, Carmany EP, Stockton DW, Schnur RE, Penney LS, Martin DM, Raskin S, Leppig K, Thiese H, Smith R, Aberg E, Niyazov DM, Escobar LF, El-Khechen D, Johnson KD, Lebel RR, Siefkas K, Ball S, Shur N, McGuire M, Brasington CK, Spence JE, Martin LS, Clericuzio C, Ballif BC, Shaffer LG, Eichler EE. 2012. Phenotypic heterogeneity of genomic disorders and rare copy-number variants. N Engl J Med 367:1321–1331. Hahn H, Ku SE, Kim KS, Park YS, Yoon CH, Cheong HI. 2005. Implication of genetic variations in congenital obstructive nephropathy. Pediatr Nephrol 20:1541–1544. Hannes FD, Sharp AJ, Mefford HC, de Ravel T, Ruivenkamp CA, Breuning MH, Fryns JP, Devriendt K, Van Buggenhout G, Vogels A, Stewart H, Hennekam RC, Cooper GM, Regan R, Knight SJ, Eichler EE, Vermeesch JR. 2009. Recurrent reciprocal deletions and duplications of 16p13.11: The deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant. J Med Genet 46:223–232. Ingason A, Rujescu D, Cichon S, Sigurdsson E, Sigmundsson T, Pietilainen OP, Buizer-Voskamp JE, Strengman E, Francks C, Muglia P, Gylfason A, Gustafsson O, Olason PI, Steinberg S, Hansen T, Jakobsen KD, Rasmussen HB, Giegling I, Moller HJ, Hartmann A, Crombie C, Fraser G, Walker N, Lonnqvist J, Suvisaari J, Tuulio-Henriksson A, Bramon E, Kiemeney LA, Franke B, Murray R, Vassos E, Toulopoulou T, Muhleisen TW, Tosato S, Ruggeri M, Djurovic S, Andreassen OA, Zhang Z, Werge T, Ophoff RA, Rietschel M, Nothen MM, Petursson H, Stefansson H, Peltonen L, Collier D, Stefansson K, St Clair DM. 2011. Copy number variations of chromosome 16p13.1 region associated with schizophrenia. Mol Psychiatry 16:17–25. Kasahara M 2007. The 2R hypothesis: An update. Curr Opin Immunol 19:547–552. Makino T, McLysaght A. 2010. Ohnologs in the human genome are dosage balanced and frequently associated with disease. Proc Natl Acad Sci USA 107:9270–9274. Mefford HC, Cooper GM, Zerr T, Smith JD, Baker C, Shafer N, Thorland EC, Skinner C, Schwartz CE, Nickerson DA, Eichler EE. 2009. A method for rapid, targeted CNV genotyping identifies rare variants associated with neurocognitive disease. Genome Res 19:1579–1585. Mendelsohn C 2009. Using mouse models to understand normal and abnormal urogenital tract development. Organogenesis 5:306–314. Nagamani SC, Erez A, Bader P, Lalani SR, Scott DA, Scaglia F, Plon SE, Tsai CH, Reimschisel T, Roeder E, Malphrus AD, Eng PA, Hixson PM, Kang SH, Stankiewicz P, Patel A, Cheung SW. 2011. Phenotypic manifestations of copy number variation in chromosome 16p13.11. Eur J Hum Genet 19:280–286. Nishimura H, Yerkes E, Hohenfellner K, Miyazaki Y, Ma J, Hunley TE, Yoshida H, Ichiki T, Threadgill D, Phillips JA3rd, Hogan BM, Fogo A, Brock JW3rd, Inagami T, Ichikawa I. 1999. Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men. Mol Cell 3:1–10. Ramalingam A, Zhou XG, Fiedler SD, Brawner SJ, Joyce JM, Liu HY, Yu S. 2011. 16p13.11 duplication is a risk factor for a wide spectrum of neuropsychiatric disorders. J Hum Genet 56:541–544.
HOUCINAT ET AL. Rigoli L, Chimenz R, di Bella C, Cavallaro E, Caruso R, Briuglia S, Fede C, Salpietro CD. 2004. Angiotensin-converting enzyme and angiotensin type 2 receptor gene genotype distributions in Italian children with congenital uropathies. Pediatr Res 56:988–993. Rooryck C, Souakri N, Cailley D, Bouron J, Goizet C, Delrue MA, Marlin S, Lacombe FD, Arveiler B. 2010. Array-CGH analysis of a cohort of 86 patients with oculoauriculovertebral spectrum. Am J Med Genet A 152A:1984–1989. Sahoo T, Theisen A, Rosenfeld JA, Lamb AN, Ravnan JB, Schultz RA, Torchia BS, Neill N, Casci I, Bejjani BA, Shaffer LG. 2011. Copy number variants of schizophrenia susceptibility loci are associated with a spectrum of speech and developmental delays and behavior problems. Genet Med 13:868–880. Song R, Spera M, Garrett C, El-Dahr SS, Yosypiv IV. 2010. Angiotensin II AT2 receptor regulates ureteric bud morphogenesis. Am J Physiol Renal Physiol 298:F807–F817. Song R, Yosypiv IV. 2011. Genetics of congenital anomalies of the kidney and urinary tract. Pediatr Nephrol 26:353–364. Tropeano M, Ahn JW, Dobson RJ, Breen G, Rucker J, Dixit A, Pal DK, McGuffin P, Farmer A, White PS, Andrieux J, Vassos E, Ogilvie CM, Curran S, Collier DA. 2013. Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders. PLoS ONE 8: e61365.
2719 Ullmann R, Turner G, Kirchhoff M, Chen W, Tonge B, Rosenberg C, Field M, Vianna-Morgante AM, Christie L, Krepischi-Santos AC, Banna L, Brereton AV, Hill A, Bisgaard AM, Muller I, Hultschig C, Erdogan F, Wieczorek G, Ropers HH. 2007. Array CGH identifies reciprocal 16p13.1 duplications deletions that predispose to autism and/or mental retardation.. Hum Mutat 28:674–682. Vergult S, Hoogeboom AJ, Bijlsma EK, Sante T, Klopocki E, De Wilde B, Jongmans M, Thiel C, Verheij JB, Perez-Aytes A, Van Esch H, Kuechler A, Barge-Schaapveld DQ, Sznajer Y, Mortier G, Menten B. 2013. Complex genetics of radial ray deficiencies: screening of a cohort of 54 patients. Genet Med 15:195–202. Vervoort VS, Beachem MA, Edwards PS, Ladd S, Miller KE, de Mollerat X, Clarkson K, DuPont B, Schwartz CE, Stevenson RE, Boyd E, Srivastava AK. 2002. AGTR2 mutations in X-linked mental retardation. Science 296:2401–2403. Wisniowiecka-Kowalnik B, Kastory-Bronowska M, Bartnik M, Derwinska K, Dymczak-Domini W, Szumbarska D, Ziemka E, Szczaluba K, Sykulski M, Gambin T, Gambin A, Shaw CA, Mazurczak T, Obersztyn E, Bocian E, Stankiewicz P. 2013. Application of custom-designed oligonucleotide array CGH in 145 patients with autistic spectrum disorders. Eur J Hum Genet 21:620–625. Ylisaukko-oja T, Rehnstrom K, Vanhala R, Tengstrom C, Lahdetie J, Jarvela I. 2004. Identification of two AGTR2 mutations in male patients with non-syndromic mental retardation. Hum Genet 114:211–213.
Homozygous 16p13.11 Duplication Associated with Mild Intellectual Disability and Urinary Tract Malformations in Two Siblings Born from Consanguineous Parents N. Houcinat,1,2* B. Llanas,3 S. Moutton,1,2 J. Toutain,1 D. Cailley,1 B. Arveiler,1,2 C. Combe,4,5 D. Lacombe,1,2 and C. Rooryck1,2 1
Genetique medicale, CHU, Bordeaux, France
2
Univ. Bordeaux, Maladies Rares : Genetique et Metabolisme (MRGM), EA 4576,, F-33000 Bordeaux, France
3
Nephrologie pediatrique, CHU, Bordeaux, France Nephrologie Transplantation Dialyse, CHU, Bordeaux, France
4 5
Univ. Bordeaux, Unite INSERM 1026, F-33000 Bordeaux, France
Manuscript Received: 4 February 2014; Manuscript Accepted: 4 June 2015
The use of array-comparative genomic hybridization (arrayCGH) in routine clinical work has allowed the identification of many new copy number variations (CNV). The 16p13.11 duplication has been implicated in various congenital anomalies and neurodevelopmental disorders, but it has also been identified in healthy individuals. We report a clinical observation of two brothers from related parents each carrying a homozygous 16p13.11 duplication. The propositus had mild intellectual disability and posterior urethral valves with chronic renal disease. His brother was considered a healthy child with only learning disabilities and poor academic performances. However, a routine medical examination at 25-years-old revealed a mild chronic renal disease and ureteropelvic junction obstruction. Furthermore, the father presented with a unilateral renal agenesis, thus it seemed that a “congenital anomalies of kidney and urinary tract” (CAKUT) phenotype segregated in this family. This may be related to the duplication, but we cannot exclude the involvement of additional genetic or non-genetic factors in the urological phenotype. Several cohort studies showed association between this chromosomal imbalance and different clinical manifestations, but rarely with CAKUT. The duplication reported here was similar to the larger one of 3.4 Mb previously described versus the more common of 1.6 Mb. It encompassed at least 11 known genes, including the five ohnologs previously identified. Our observation, in addition to expanding the clinical spectrum of the duplication provides further support to understanding the underlying pathogenic mechanism. © 2015 Wiley Periodicals, Inc.
How to Cite this Article: Houcinat N, Llanas B, Moutton S, Toutain J, Cailley D, Arveiler B, Combe C, Lacombe D, Rooryck C. 2015. Homozygous 16p13.11 duplication associated with mild intellectual disability and urinary tract malformations in two siblings born from consanguineous parents. Am J Med Genet Part A 167A:2714–2719.
Key words: 16p13.11; duplication; ohnologs; CAKUT; arrayCGH; intellectual disability
INTRODUCTION The use of array-comparative genomic hybridization (array-CGH) in routine clinical work has led to the identification of many new copy number variations (CNVs) [Cooper et al., 2011; Girirajan et al., 2012]. Indeed, the 16p13.11 locus is considered as a CNV hotspot mediated by non-homologous recombination between chromosome 16 low copy repeats (LCRs) [Ingason et al., 2011]. Deletions and reciprocal duplications in this region have been
Correspondence to: Nada Houcinat, CHU Bordeaux, place amelie Raba leo, 33000 Bordeaux, Bordeaux 33000 France. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 26 June 2015 DOI 10.1002/ajmg.a.37212
© 2015 Wiley Periodicals, Inc.
2714
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implicated in multiple congenital anomalies and neurodevelopmental disorders such as epilepsy, autism, and intellectual disability [Ullmann et al., 2007; Hannes et al., 2009; Nagamani et al., 2011; Ramalingam et al., 2011; Wisniowiecka-Kowalnik et al., 2013]. The 16p13.11 duplication has also been associated in many studies with varied phenotypes such as radial ray deficiency [Vergult et al., 2013], aortic dissections, schizophrenia, and attention deficit hyperactivity disorder [Sahoo et al., 2011]. The 16p13.11 CNV is thought to be a susceptibility locus for neurocognitive disease, as several authors found the duplication statistically enriched in the patient versus the control populations [Mefford et al., 2009; Girirajan et al., 2012; Tropeano et al., 2013; Coe et al., 2014]. Here, we report a clinical observation of two brothers carrying a homozygous 16p13.11 duplication and presenting with mild intellectual disability and urological malformations. We discuss the implication of this chromosomal imbalance in their phenotype especially in the anomalies of the renal system.
MATERIALS AND METHODS Oligonucleotides array-CGH was performed using a 44 K whole genome microarray (Agilent Technologies, Santa Clara, California) according to manufacturer’s instructions, after DNA extraction from the patient’s peripheral blood leukeocytes using standard procedures. DNA from the patient was analyzed in comparative genomic hybridization experiments with fluorochrome swapping in a trio along with DNA from two patients with different phenotype (serving as reference DNAs) [Rooryck et al., 2010]. Fluorescence ratio was calculated to detect chromosomal imbalances. Deletions and duplications were taken into consideration when a minimum of three consecutive variant points were detected by the statistical algorithm ADM2 with a threshold of 5 (Agilent Genomic Workbench Lite Edition 6.5, Agilent Technologies, Santa Clara, California). To confirm the rearrangement identified by array-CGH, FISH technique was carried out on metaphase spreads using specific probe. Mutational screening of candidate gene was performed on genomic DNA extracted from peripheral blood lymphocytes. The coding regions and exon-intron boundaries of the gene were amplified by PCR. The amplicons were then sequenced using the Big Dye Terminator Cycle Sequencing Kit v3, and run on an automated sequencer ABI 3500 xL Dx (Applied Biosystems). The electrophoretic profiles were analyzed with Seqscape v2.7 (Applied Biosystems).
CLINICAL REPORTS The propositus was a 32-year-old man, he was the first child born from consanguineous parents (Fig. 1). He came to medical attention on day 1 of life for urinary tract distension due to posterior urethral valves, corrected surgically at day 15 of life. Many complications occurred after surgery: multiple infections of the urinary tract and interstitial nephropathy. The patient developed a chronic renal disease and an uretero-hydronephrosis. He had developmental delay and intellectual disability, could not read or write, or live independently. He had normal motor development, and otherwise normal neurological examination. His brother was healthy as a
FIG. 1. Pedigree of the family. The father had a unilateral renal agenesis; the two sons had both renal anomalies and cognitive impairment.
child and as a young adult until age 25 when a routine blood test revealed a mild chronic renal disease. Ultrasound examination showed an ureteropelvic junction obstruction. He had no intellectual deficiency, he worked as a car mechanic, and his history revealed only learning disability. Indeed, he had poor overall academic performance and he needed a specialized teaching class and tutoring. The investigations of the parents revealed a unilateral renal agenesis in the father who otherwise had normal intellectual abilities. The mother was a healthy woman. Clinical morphological examination also revealed that the two brothers shared common dysmorphic features; prominent supra-orbital ridges, deep-set eyes, and prognathism (Fig. 2). Parents were normal without dysmorphic features. Chromosome analysis showed a 46,XY normal karyotype in the propositus.
RESULTS Array-CGH performed in the propositus identified a 16p13.11– p12.3 duplication with a minimal size of 3.2 Mb (chr16:15, 256, 686–18, 546, 759) and a maximal size of 3.6 Mb (chr16:15, 154, 746–18, 794, 446) (Hg19). The log2 ratio average of fluorescence’ signals was 0.8 which was consistent with a homozygous state of the duplication (four copies). This duplicated region contained at least 11 known genes (Fig. 3). Familial segregation study of this rearrangement was performed by FISH analysis, using specific probe (RP11-120O23) targeting this region. It confirmed the homozygous duplication in the propositus and also in his brother. Parents were each heterozygous for the duplication.
DISCUSSION Since the advent of array-CGH, several cohort studies of patients carrying heterozygous genomic 16p13.11 imbalances have been published. Ullman et al. [2007] first reported a recurrent 1.65 Mb
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
FIG. 2. Patients’ pictures (sons), A: propositus, B: propositus’ brother.
duplication at the 16p13.11 locus (chr16:14.9–16.4 Mb) (Hg19) in three individuals, among a cohort of 182 patients, presenting with autism or intellectual disability. Hannes et al. [2008], detected a 16p13.11 duplication in 7 out of 1,027 patients, five with the recurrent 1.65 Mb duplication and two with a larger one
FIG. 3. Array CGH results.
3.4Mb (chr16: 15.1–18.5 Mb) (Hg19). These patients presented with intellectual disability and variable congenital anomalies. Only one of the patients carrying the larger duplication had an anomaly of the kidney and urinary tract (unilateral renal agenesis). However, his phenotype, resembling a CHARGE syndrome, was more
HOUCINAT ET AL. complex than the one usually described in patients carrying the 16p13.11 rearrangement. The duplication was inherited from a healthy parent in two cases and appeared de novo in one case. Furthermore, the duplication was found in five asymptomatic individuals out of 2,014 controls in the same study. Mefford et al. [2009] detected the duplication in 11 of 1,010 affected individuals contrasting with only two of 2,493 controls. Nagamani et al. [2011] described 10 patients carrying this duplication, they had varied clinical features including behavior abnormalities, developmental delay, skeletal manifestations, and congenital heart defects but no anomaly of the renal system. Ramalingam et al. [2011] described the clinical phenotype of eight patients with the duplication, only one of them had unspecified kidneys anomalies. Overall, studies showed that the duplication was significantly enriched in patients with intellectual disabilities and/or autism versus controls populations, and the kidney anomalies have been seen in only 2 of 39 cases with phenotypic information, one with unspecified kidney anomalies and one unilateral renal agenesis [Cooper et al., 2011; Girirajan et al., 2012; Tropeano et al., 2013; Coe et al., 2014]. The urological malformations segregating in our family were varied. They belong to the spectrum of CAKUT phenotype (Congenital anomalies of kidney and urinary tract). Human CAKUT are a group of heterogeneous diseases characterized by a wide range of structural and functional malformations of kidneys and lower urinary tract. Genetic forms may be syndromic, associated with multi-organ malformations, or isolated. Animal model studies in knock-out mice provided a better understanding of the molecular basis of the renal system morphogenesis, allowing the identification of many genes involved in renal development [Costantini and Kopan 2010; Men-
2717 delsohn 2009]. Only few of them were found to be mutated in recessive and dominant forms of human CAKUT. Therefore, the pathological phenotype seemed to arise from interactions between modifier genes and epigenetic factors [El-Dahr et al., 2000; Song and Yosypiv 2011]. The association between the duplication and CAKUT phenotype appeared to be not so rare. Indeed, it has been reported in 2 out of 39 heterozygous carriers with phenotypic information (3 of 40 cases if we consider the father in our family). However, the patient with renal agenesis reported by Hannes et al. [2008]) had a complex phenotype and presented with aplasia of semicircular canals, asymmetric face with left facial nerve paresis, coloboma, atresia of right choana, and unique right kidney with double ureters. All these features are strongly suggestive of CHARGE syndrome. Thus, one must be careful with conclusions about the implication of the duplication in this case. Concerning this observation, we cannot exclude the implication of additional genetic or non-genetic factors in the urological malformations. There is no evident candidate gene for urological malformations in this duplicated region, and it is well-known that environmental and epigenetic factors play an important role in the renal development [ElDahr et al., 2000; Song and Yosypiv 2011]. Therefore, it may be interesting to search for the 16p13.11 duplication in patients with a CAKUT phenotype to see if this CNV is enriched in patients with renal anomalies. In view of the family pedigree (the father was also the mother’s uncle) and the preferential affected males, an X-linked inheritance has been considered even if the pedigree was small. We also evaluated the only CAKUT gene located on the X chromosome: AGTR2, coding for Angiotensin type-2 receptor R. This gene was implicated in the morphogenesis of the renal system in mouse models and AGTR2 knock-out (KO) mice developed a CAKUT
FIG. 4. DNA copy number variants and LCRs in the16p13 region. A screenshot from the University of California, SantaCruz (UCSC) Genome Browser: the duplication identified in our family is in blue and the two duplications described in the literature (red bar), the classical and the larger one.
2718 phenotype [Nishimura et al., 1999; Song et al., 2010]. In addition, association between CAKUT and a single polymorphism, c.1332A>G transition, in the human AGTR2 gene, have been reported in several, but not all, studies [Hahn et al., 2005; Rigoli et al., 2004]. Moreover, mutations in AGTR2 gene have been found in several cases of X-linked intellectual disability [Vervoort et al., 2002; Ylisaukko-oja et al., 2004]. Unfortunately, the mutational screening of AGTR2 coding regions proved negative. The duplication reported here was similar to the large one previously described [Hannes et al., 2009] overlapping the typical and most frequently described smaller duplication (Fig. 4). It encompassed at least 11 known genes, including the five ohnologs (NDE1, MYH11 ABCC1, ABCC6 and XYLT1) previously identified [Tropeano et al., 2013]. Four of these genes (NDE1, MYH11 ABCC1, ABCC6) are common between the large duplication (3.4Mb) and the more commonly described smaller duplication (1.65 Mb) (Fig. 4). One increasingly popular hypothesis is that ohnologs, genes retained after ancestral whole genome duplication events, are particularly sensitive to dosage change. They are thought to be responsible for the major pathological phenotype associated with duplications [Makino and McLysaght 2010; Kasahara 2007]. The duplication in a homozygous state did not appear to increase the phenotypic severity, including the cognitive aspect as these two brothers, especially the second son, presented with a milder phenotype than described for some heterozygous carriers in the literature. Thus, the clinical severity was not proportional to the number of copies of the region and perhaps for this region any dosage imbalance could have a pathogenic effect, regardless of the copy number variation. This underlines the importance of the role played by the dosage imbalance itself, in the pathogenic effect, whatever the degree of the imbalance. Varied genetic, epigenetic and environmental factors appear to influence the pathological presentation more than the number of copies. All these considerations make interpretation of CNVs found by array-CGH difficult. Indeed, regarding our patients, it is difficult to conclude about the mental prognosis of their future offspring that have 100% risk of being a heterozygous carrier of the 16p13.11 duplication.
ACKNOWLEDGMENT We thank Drs Alfio de Marin and Patrick Pauly, Cabinet de Nephrologie, Polyclinique de Navarre, 64075 Pau, France, for referring the patients.
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