CLINICAL REPORT
An Unusual Presentation of Kabuki Syndrome with Orbital Cysts, Microphthalmia, and Cholestasis with Bile Duct Paucity Nina B€ogershausen,1 Umut Altunoglu,2 Filippo Beleggia,3 G€okhan Yigit,1 H€ulya Kayserili,4 Peter N€urnberg,5 Yun Li,1 Janine Altm€uller,5,6 and Bernd Wollnik1* 1
Institute of Human Genetics, University Medical Center Goettingen, Goettingen, Germany Istanbul Medical Faculty, Department of Medical Genetics, Istanbul University, Istanbul, Turkey
2 3
Institute of Human Genetics, University of Duesseldorf, Duesseldorf, Germany
4
Department of Medical Genetics, KoSc University School of Medicine, Istanbul, Turkey Cologne Center for Genomics, University of Cologne, Cologne, Germany
5 6
Institute of Human Genetics, University of Cologne, Cologne, Germany
Manuscript Received: 13 April 2016; Manuscript Accepted: 3 August 2016
Kabuki syndrome (KS) is a rare developmental disorder characterized by multiple congenital malformations, postnatal growth retardation, intellectual disability, and recognizable facial features. It is mainly caused by mutations in either KMT2D or KDM6A. We describe a 14-year-old boy with KS presenting with an unusual combination of bilateral microphthalmia with orbital cystic venous lymphatic malformation and neonatal cholestasis with bile duct paucity, in addition to the typical clinical features of KS. We identified the novel KMT2D mutation c.10588delC, p.(Glu3530Serfs 128) by Mendeliome (Illumina TruSight OneW) sequencing, a next generation sequencing panel targeting 4,813 genes linked to human genetic disease. We analyzed the Mendeliome data for additional mutations which might explain the exceptional clinical presentation of our patient but did not find any, leading us to suspect that the above named symptoms might be part of the KMT2D-associated spectrum of anomalies. We thus extend the range of KS-associated malformations and propose a hypothetical connection between KMT2D and Notch signaling. Ó 2016 Wiley Periodicals, Inc.
Key words: Kabuki syndrome; KMT2D; cyst; microphthalmia; venous lymphatic malformation; vascular malformation; cholestasis; bile duct paucity; notch signaling
INTRODUCTION Kabuki syndrome (KS; OMIM 147920) is a rare genetic disorder characterized by postnatal growth retardation, intellectual disability, and organ and skeletal malformations in addition to a distinctive facial gestalt. KS is usually caused by mutations in the genes KMT2D (MLL2; OMIM 602113) or KDM6A (UTX; OMIM 300128) [Ng et al., 2010; Miyake et al., 2013], two major components of the ASCOM methyltransferase complex
Ó 2016 Wiley Periodicals, Inc.
How to Cite this Article: B€ ogershausen N, Altunoglu U, Beleggia F, Yigit G, Kayserili H, N€ urnberg P, Li Y, Altm€ uller J, Wollnik B. 2016. An unusual presentation of Kabuki syndrome with orbital cysts, microphthalmia, and cholestasis with bile duct paucity. Am J Med Genet Part A 170A:3282–3288.
[Goo et al., 2003]. However, mutations in the genes RAP1A and RAP1B have recently been described as rare causes of KS [B€ ogershausen et al., 2015]. Multiple malformations including cleft lip/palate, congenital heart defects, gastrointestinal abnormalities, cryptorchidism, and congenital hip dysplasia have been Nina B€ ogershausen and Umut Altunoglu contributed equally to this work. Conflicts of interest: None. Patient consent: Written informed consent for the genetic investigation and the publication of results and photographs were obtained from the patient’s parents. Ethics approval: The ethical committee of the University Hospital Goettingen, Goettingen, Germany. Grant sponsor: German Federal Ministry of Education and Research (BMBF); Grant number: 01GM0801. Correspondence to: Bernd Wollnik, M.D., Institute of Human Genetics; University Medical Center Goettingen; Heinrich-D€ uker-Weg 12, Goettingen 37073, Germany. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 17 August 2016 DOI 10.1002/ajmg.a.37931
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€ BOGERSHAUSEN ET AL. associated with KS [B€ ogershausen and Wollnik, 2013]. We report a patient with typical clinical findings of KS, along with the unusual findings of microphthalmia in combination with congenital bilateral orbital cysts and neonatal cholestasis with bile duct paucity. The identification of the de novo one basepair (bp) deletion c.10588delC, p.(Glu3530Serfs 128) in exon 38 of KMT2D by clinical exome sequencing confirmed the diagnosis of KS. We discuss this unique presentation in the context of the current literature and present an extensive analysis of the Mendeliome data in search of an explanation for the unusual clinical presentation.
CLINICAL REPORT We describe a 14-year-old boy (Fig. 1A), the first child of healthy consanguineous parents (Fig. 1B) of Turkish origin, born after an uneventful pregnancy via normal spontaneous delivery at 36 weeks of gestation, with a birth weight of 3,190 g (84th percentile) and birth length of 50 cm (90th percentile). Head circumference was not recorded. He did not experience perinatal adaptation problems. Large cystic lesions of the eyes were noted at birth. Orbital T2-weighted MRI evaluation at 2 days of age showed bilateral microphthalmia with globe dimensions of 8 11 6 mm on the
3283 right and 12 11 8 mm on the left, and extraconal hyperintense multicystic lesions filling both orbital cavities with dimensions of 41 26 25 mm on the left and 28 21 20 mm on the right. Both lesions contained fluid–fluid levels within cysts, and postcontrast scans did not detect marked enhancement within the lesions. This constellation was consistent with a radiologic diagnosis of venous lymphatic malformation. The child experienced feeding difficulties and had progressive jaundice after 10 days of age. Clinical examination was normal except for mild petechial rash and bilateral cryptorchidism. Abdominal ultrasound revealed minimally dilated intrahepatic bile ducts, and the gallbladder could not be visualized. A laboratory work-up showed mixed hyperbilirubinemia and mild hepatitis with coagulopathy (Supplementary Table SI). Serology for hepatitis A, B, and C, and IgM antibodies for toxoplasma, rubella, cytomegalovirus, and herpes was negative. Urine was negative for reducing substances and organic acid levels were within the normal range. Cranial MRI revealed multifocal hemorrhagic lesions of diminutive size in a 1 1.5-cm area superior to the frontal horn of the left lateral ventricle. Echocardiographic examination was normal. A liver biopsy at 40 days of age showed absence of the bile ducts and moderate bile accumulation. At 2 months of age, a laparotomy with intraoperative cholangiogram was
FIG. 1. (A) Face of the index patient at ages 8 months, 15 months, and 1310/12 years (from left to right): note the facial features reminiscent of Kabuki syndrome in addition to large intra- and preorbital cystic lesions, deforming and obstructing the palpebral fissures. (B) Pedigree of the family: the patient is a simplex case, his parents are second degree cousins. (C) Electropherograms of the identified KMT2D mutation in exon 38: The 1-bp deletion c.10588delC; p.(Glu3530Serfs 128) was confirmed in the patient by Sanger sequencing and was not present in either of his parents. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/ajmga].
3284 performed to rule out biliary atresia. Liver biopsy revealed paucity of the bile ducts with bile duct proliferation, prompting a diagnosis of idiopathic cholestasis with bile duct paucity. Jaundice and liver function gradually ameliorated, and cholestasis resolved by the age of 6 months. The boy was hospitalized at 7 months of age due to bronchopneumonia and referred to the genetics outpatient clinics because of his dysmorphic features. At 8 months of age, his weight was 5,200 g ( 3.80 SD), his length 68 cm ( 1.13 SD), and the occipitofrontal circumference (OFC) 38.5 cm ( 4.8 SD). Large bilateral hemangiomatous, cystic lesions extended preorbitally to the area of the lower lids and were more pronounced on the left side. Globes could not be located on examination, and the child had no vision. Facial features of Kabuki syndrome, such as sparse eyebrows, long palpebral fissures with ectropion of lower eyelids, long eyelashes, short columella, thin and mildly tented vermilion of the upper lip, lower lip pits, highly arched palate, and prominent ears were noted. He had mild joint hyperlaxity, prominent fetal fingerpads, and a small sacral dimple. Testes were not palpable, and the scrotum was hypoplastic with a normal penile length of 3.5 cm. Head control was not achieved, and truncal hypotonia was present. He responded to sounds, but had no vocalizations. At the age of 10 months, he presented with small calcium stones, secondarily to hypercalciuria, in the lower pole of the left kidney, which were successfully treated with good fluid intake and temporary cessation of oral vitamin D3. He underwent bilateral orchiopexy at the age of 13 months. At 15 months of age, his weight was 7,450 g ( 3.03 SD), length 78 cm (0.65 SD), and OFC 44 cm ( 2.56 SD). Developmental milestones were severely delayed, in part due to blindness. He sat unsupported at 12 months of age and could walk independently with short steps and an out-toeing gait due to pes valgus after 4 years of age. He started uttering a few meaningful words at the age of 7 years, but currently does not speak in sentences. He needed realignment osteotomy for bilateral congenital hip dysplasia at ages 9 and 13 years. The child was recently re-evaluated at the age of 1310/12 years. He had been attending a rehabilitation program for blind children with special education needs for 10 years. He understood and followed basic commands, such as sitting or putting an object down, and had a vocabulary of approximately 5–10 words. He remained agitated and hypervigilant during the examination. He had a height of 132 cm ( 4.07 SD), weight of 38 kg ( 1.89 SD), and OFC of 51 cm ( 3.17 SD). The cystic lesions of the eyes had regressed to a great degree, but he was blind. Dysmorphic features became more pronounced, with a long face, highly arched, nicked eyebrows with lateral sparseness, a depressed nasal root, widely spaced eyes, midface retrusion, a long, bulbous nose with a depressed nasal tip, and protruding ears (Fig. 1A). Additionally, widely spaced teeth, and gynecomastia compatible with Tanner stage III were present. Routine blood biochemistry screening and thyroid hormones were within normal range. Testosterone levels at 13 10/12 years were 0.07 ng/ml, which is in the low normal range [0.07–12 ng/ml]. LH of 1.2 U/L [0.5–10.5 U/L] and FSH of 2.4 U/L [0.5–7.9 U/L] at the same time ruled out hypergonadotropic hypogonadism and indicated normal prepubertal hormone levels.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
EXPERIMENTAL PROCEDURES Mendeliome Sequencing (MS) After informed consent, peripheral blood samples were taken from patients and parents and DNA was extracted from EDTA blood by standard methods. About 50 ng of patient DNA were enriched using the TruSight One1 chemistry (Illumina, version 1; 4,813 disease-associated genes) followed by next-generation sequencing on a MiSeq sequencer (Illumina). We obtained 20 minimum depth of coverage at more than 95% of targets. MS data analysis and filtering of variants were carried out using the exome analysis pipeline “varbank” of the Cologne Center for Genomics (CCG). We applied the following criteria for filtering of MS variants: coverage of more than six reads, a minimum quality score of 10, an allele frequency between 30% and 70%, and a minor allele frequency (MAF) T, p.(Pro2550Leu) in the FLNA gene (OMIM 300017) was identified, which is annotated with a frequency of 0.00005809 in the ExAC browser. Three healthy individuals are annotated as hemizygous mutation carriers in the ExAC browser. Additionally, no pathogenic variants were found in the genes FLT4 (OMIM 136352), VEGFR2 (KDR; OMIM 191306), AKT1 (OMIM 164730), and TEM8 (ANTXR1; OMIM 606410), associated with a susceptibility for capillary hemangioma or lymphangioma, or CHD7 (OMIM 608892), the gene known to cause CHARGE syndrome. We also analyzed the genes PAX2 (OMIM 167409), PAX6 (OMIM 607108), and SIX6 (OMIM 606326), associated with optic nerve or chorioretinal coloboma, and found no pathogenic variant. The TEM8 gene (ANTXR1; OMIM 606410) contained the rare intronic heterozygous sequence variation g.159196_159196delA with a minor allele frequency (MAF) of 0.0002767 (ExAC browser). The Mendeliome data also contained no mutations or copy number changes in a specific set of 22 genes associated with syndromic and isolated microphthalmia, with or without coloboma, determined by a search of the OMIM database (Supplementary Table SII). We also analyzed the genes JAG1 (OMIM 601920) and NOTCH2 (OMIM 600275), known to be involved in the pathogenesis of Alagille syndrome, but did not find any mutations.
Sanger Sequencing We amplified exon 38 of KMT2D from DNA of the index patient and both parents by PCR and sequenced the PCR products by BigDye Terminator method on an ABI 3100 sequencer. De novo
€ BOGERSHAUSEN ET AL. occurrence of the KMT2D mutation identified by MS was confirmed (Fig. 1C). PCR and sequencing primers for KMT2D were obtained from the UCSC genome browser and purchased from IDT DNA (Leuven, Belgium). Primer sequences are available on request.
DISCUSSION We describe a boy with Kabuki syndrome who presented with an unusual combination of bilateral microphthalmia with orbital cysts, neonatal cholestasis with bile duct paucity, cryptorchidism, and congenital hip dysplasia. We performed Mendeliome sequencing using the Illumina TruSight One chemistry, which covers 4,813 genes associated with Mendelian disease, and discovered the 1-bp deletion c.10588delC, p.(Glu3530Serfs 128) in exon 38 of KMT2D. This mutation has not been published before and is not annotated in the HGMD database as a known disease-causing mutation, nor is it annotated in any of the current databases of human genetic variation as a polymorphic variant (ExAC, EVS, dbSNP). Truncating mutations in exon 38 of KMT2D have previously been described in other patients with Kabuki syndrome [B€ ogershausen and Wollnik, 2013; B€ ogershausen et al., 2016]. Cryptorchidism and congenital hip dysplasia are known to be common symptoms of Kabuki syndrome with frequencies of up to 25% and 37%, respectively [Adam and Hudgins, 2005; B€ ogershausen and Wollnik, 2013]. In contrast, the lymphatic vascular or bile duct malformations observed in our patient have never been reported. The Mendeliome data were analyzed for homozygous variants that could possibly explain the severe presentation and unusual phenotype in our patient, but none were found. The hemizygous rare variant c.7649C>T, p.(Pro2550Leu) in FLNA is annotated once in the Exome Variant Server and five times in the ExAC browser (three times as a hemizygous variant), leading to the interpretation that it is unlikely to have a significant effect on the phenotype. Mutations in FLNA cause a phenotypic spectrum that includes X-linked cardiac valvular dysplasia (OMIM 314400), congenital short bowel syndrome (OMIM 300048), FG syndrome 2 (OMIM 300321), frontometaphyseal dysplasia (OMIM 305620), periventricular heterotopia (OMIM 300049), Melnick–Needles syndrome (OMIM 309350), otopalatodigital syndrome, type I (OMIM 311300), otopalatodigital syndrome, type II (OMIM 304120), and terminal osseous dysplasia (OMIM 300244). None of the above-mentioned diseases are compatible with the phenotype described herein. Other than aortic coarctation, which is known to be a frequent arteriovascular malformation in KS, venous or arterial vascular malformations of the cardiopulmonary system, such as anomalous pulmonary venous drainage or double aortic arch, are rare, reported only in six patients [Dyamenahalli et al., 2007; Shahdadpuri et al., 2008; Moral et al., 2009; Bhat et al., 2012; Oswal et al., 2014; Giudici et al., 2015]. Vascular malformations of the central nervous system or the eyes are even rarer. Sanchez-Carpintero et al. [2012] reported a dilated vein of Galen in a child with KS. Tortuous vessels and telangiectasia of the retina were reported by Chuah et al. [2009] and Anandan et al. [2005], respectively. In our patient, MRI studies of the orbital lesions showed bilateral microphthalmia and extraconal hyperintense multicystic lesions filling both orbital
3285 cavities. The extraconal location and fluid–fluid levels within the multicystic lesion supported the diagnosis of venous-lymphatic malformation. No mutations or copy number changes were present in the known hereditary lymphedema gene FLT4 (OMIM 136352), the known capillary hemangioma gene VEGFR2 (KDR; OMIM 191306) or in the known lymphatic overgrowth gene AKT1 (OMIM 164730). The TEM8 gene (ANTXR1; OMIM 606410), associated with a susceptibility for capillary hemangioma, contained the rare intronic heterozygous sequence variation g.159196_159196delA. Considering its deep intronic location and the frequency noted in the ExAC browser, we assume it to be a rare variant without functional consequence, but cannot fully rule out a minor influence on the phenotype. As we did not find any additional genetic cause of the lymphatic malformation in our patient, we suspect that the lymphatic vascular malformation may have been caused by the KMT2D mutation. However, we cannot fully exclude a possible influence of genes that are not covered by the Mendeliome, that is, genes that have not yet been linked to a human vascular phenotype or somatic mutations that would escape this analysis. For example, somatic mutations in the genes GNAQ, GNA11, PIK3CA, and RASA1 have recently been shown to underlie different capillary or venous malformations [Limaye et al., 2015; Macmurdo et al., 2016; Ayturk et al., 2016]. Due to the parental consanguinity, the possibility of a homozygous variant in a gene not covered by the Mendeliome remains. Initially, a differential diagnosis of colobomatous cysts was also considered. Colobomatous cysts are congenital orbital lesions which are often associated with microphthalmia [Shields and Shields, 2004] and can either occur sporadically or be inherited in an autosomal recessive manner [Porges et al., 1992]. The clinical presentation of our patient with bilateral congenital orbital cysts extending to the lower eyelid, associated with microphthalmia might point toward this diagnosis, but the extraconal location of the lesion makes it less likely. Colobomatous cysts have been described in patients with syndromic conditions such as trisomy 13 [Magni et al., 1991], Aicardi syndrome [Lorenz et al., 1991], and CHARGE syndrome [De Krijger et al., 1999; Pushker et al., 2013]. We did not find any mutation or copy number change in the CHD7 gene (OMIM 608892) in the Mendeliome data of the patient and could thus rule out the most frequent cause of CHARGE syndrome (OMIM 214800). We also ruled out pathogenic variants in the genes PAX2 (OMIM 167409), PAX6 (OMIM 607108), and SIX6 (OMIM 606326), which are associated with coloboma of the optic nerve and/or the chorioretina. Moreover, the Mendeliome data were analyzed for mutations and copy number changes in a set of 22 genes associated with syndromic and isolated microphthalmia, with or without coloboma (Supplementary Table SII), yielding a negative result. Chen et al. [2014] described colobomatous microphthalmia and a retrobulbar cystic mass in a patient with a KMT2D mutation, whose facial features also included ptosis and esotropia, resembling the ocular features in our patient. Microphthalmia and anophthalmia have also been reported in five patients with KS [McVeigh et al., 2015], emphasizing the role of ophthalmic malformation in this disorder. Additionally, various patients with coloboma have been published: Ming et al. [2003] described three patients with KS and retinal coloboma and identified 11 further patients in the
3286 literature with colobomas affecting the iris, choroid, retina, and/or the optic nerve. They pointed out the clinical overlap with CHARGE syndrome, an observation that has also been made by Verhagen et al. [2014] and is supported by the recent finding of a direct interaction between the proteins KMT2D and CHD7 [Schulz et al., 2014]. We, therefore, would like to recommend that an ophthalmological consultation be part of the routine medical care of patients with Kabuki syndrome [Adam et al., 2011; McVeigh et al., 2015]. Hepatic malformations have been rarely described in Kabuki syndrome, with extrahepatic biliary atresia in three patients [McGaughran et al., 2000; Van Haelst et al., 2000; Selicorni et al., 2001] and hepatic fibrosis in one [Nobili et al., 2004]. Cholestasis with bile duct paucity is an extremely rare bile duct malformation, and has never been reported in patients with Kabuki syndrome. There are rare reports of patients with Williams syndrome who have biliary hypoplasia [O’Reilly et al., 2006; Shah et al., 2008; Honeywell et al., 2009; Sakhuja et al., 2015], but syndromic bile duct paucity is generally considered to be pathognomonic for Alagille syndrome (OMIM 118450 and 610205 for types 1 and 2, respectively). The major clinical features of Alagille syndrome have been defined as cholestasis, cardiac defect (pulmonary stenosis), skeletal abnormalities (butterfly vertebrae), ophthalmologic abnormalities (posterior embryotoxon), and characteristic facial features [Spinner et al., 2000]. Apart from cholestasis, none of these features were present in our patient. We searched the Mendeliome data for mutations and copy number changes of the genes known to cause Alagille syndrome but did not find any likely pathogenic variants or copy number changes in either JAG1 (OMIM 601920) or NOTCH2 (OMIM 600275). A combination of hepatic malformation, pigmentary retinopathy, congenital heart disease, intestinal malformations, and renal abnormalities has been described in Hardikar syndrome (HS; OMIM 612726), a rare entity for which the molecular cause is unknown. Nydegger et al. [2008] compared the features of KS and HS and concluded that the two entities were distinct. Ejarque et al. [2011] suggested an overlap of Hardikar and Kabuki syndromes in a patient with hepatoblastoma and pigmentary retinopathy with patchy pattern (cat’s paw) in whom a mutation in KMT2D was identified. However, this patient did not have malformation of the bile ducts, a hallmark feature of HS. Patients with HS have dilatation of the intrahepatic bile ducts in combination with stenosis of the large bile ducts rather than intrahepatic bile duct paucity, thus distinguishing HS from Allagille syndrome [Nydegger et al., 2008] and from the biliary malformation seen in the patient presented here. To date, there is no known connection between KMT2D and the Notch signaling pathway, but the findings in our patient may point in this direction. Notch signaling plays a crucial role in vascular development and is required for lymphatic endothelial cell fate determination [Geudens et al., 2010]. Each of Notch 1–4 has specific roles in the differentiation of biliary epithelial cells, and determination of vascular smooth muscle cell behavior [Baeten and Lilly, 2015], and they are differentially regulated in central nervous vascular malformations [Hill-Felberg et al., 2015]. It is conceivable that the orbital vascular and bile duct
AMERICAN JOURNAL OF MEDICAL GENETICS PART A malformations in our patient may be the result of the same embryological signaling defect. Our findings extend the recognized KS phenotype and suggest a possible overlap of KS with Alagille syndrome in the form of bile duct hypoplasia. This clinical observation leads us to hypothesize that the KMT2D mutation in our patient might impact on transcriptional regulation of one or several key Notch signaling components. Further clinical reports, genomic investigations, and functional studies, however, will be needed to explore a possible connection between KMT2D and the Notch signaling pathway.
ACKNOWLEDGMENTS We are grateful to the family for participating in this study, to Esther Milz for excellent technical assistance, and to Karin Boss for critically reading the manuscript. This work was supported by the German Federal Ministry of Education and Research (BMBF) by grant number 01GM0801 (E-RARE network CRANIRARE-2).
WEB RESOURCES 1000Genomes: http://browser.1000genomes.org/index.html Online Mendelian Inheritance in Man (OMIM): http://www.ncbi. nlm.nih.gov/omim Database of Single Nucleotide Polymorphisms (dbSNP): http:// www.ncbi.nlm.nih.gov/SNP/ DECIPHER: https://decipher.sanger.ac.uk/index Exome variant server (EVS): http://evs.gs.washington.edu/EVS/ Exome Aggregation Consortium (ExAC): http://exac. broadinstitute.org/ Human Gene Mutation Database (HGMD): http://www.hgmd.org/
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Miyake N, Mizuno S, Okamoto N, Ohashi H, Shiina M, Ogata K, Tsurusaki Y, Nakashima M, Saitsu H, Niikawa N, Matsumoto N. 2013. KDM6A point mutations cause Kabuki syndrome. Hum Mutat 34:108–110. Moral S, Zuccarino F, Loma-Osorio P. 2009. Double aortic arch: An unreported anomaly with Kabuki syndrome. Pediatr Cardiol 30:82–84. Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, Beck AE, Tabor HK, Cooper GM, Mefford HC, Lee C, Turner EH, Smith JD, Rieder MJ, Yoshiura K, Matsumoto N, Ohta T, Niikawa N, Nickerson DA, Bamshad MJ, Shendure J. 2010. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 42:790–793. Nobili V, Marcellini M, Devito R, Capolino R, Viola L, Digilio MC. 2004. Hepatic fibrosis in Kabuki syndrome. Am J Med Genet Part A 124A:209–212. Nydegger A, Van Dyck M, Fisher RA, Jaeken J, Hardikar W. 2008. Hardikar syndrome: Long term outcome of a rare genetic disorder. Am J Med Genet Part A 146A:2468–2472. O’Reilly K, Ahmed SF, Murday V, McGrogan P. 2006. Biliary hypoplasia in Williams syndrome. Arch Dis Child 91:420–421. Oswal N, Christov G, Sridharan S, Khambadkone S, Bull C, Sullivan I. 2014. Aberrant subclavian artery origin in tetralogy of Fallot with pulmonary stenosis is associated with chromosomal or genetic abnormality. Cardiol Young 24:478–484. Porges Y, Gershoni-Baruch R, Leibu R, Goldscher D, Zonis S, Shapira I, Miller B. 1992. Hereditary microphthalmia with colobomatous cyst. Am J Ophthalmol 114:30–34. Pushker N, Tinwala S, Khurana S, Sen S. 2013. Bilateral microphthalmos with unilateral superior cyst in a child with autism and CHARGE syndrome. Int Ophthalmol 33:195–198.
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SUPPORTING INFORMATION
Spinner NB, Leonard LD, Krantz ID. 2000 [Updated 2013]. Alagille syndrome. In: Pagon RA, Adam MP, Ardinger HH, et al., editors.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
An Unusual Presentation of Kabuki Syndrome with Orbital Cysts, Microphthalmia, and Cholestasis with Bile Duct Paucity Nina B€ogershausen,1 Umut Altunoglu,2 Filippo Beleggia,3 G€okhan Yigit,1 H€ulya Kayserili,4 Peter N€urnberg,5 Yun Li,1 Janine Altm€uller,5,6 and Bernd Wollnik1* 1
Institute of Human Genetics, University Medical Center Goettingen, Goettingen, Germany Istanbul Medical Faculty, Department of Medical Genetics, Istanbul University, Istanbul, Turkey
2 3
Institute of Human Genetics, University of Duesseldorf, Duesseldorf, Germany
4
Department of Medical Genetics, KoSc University School of Medicine, Istanbul, Turkey Cologne Center for Genomics, University of Cologne, Cologne, Germany
5 6
Institute of Human Genetics, University of Cologne, Cologne, Germany
Manuscript Received: 13 April 2016; Manuscript Accepted: 3 August 2016
Kabuki syndrome (KS) is a rare developmental disorder characterized by multiple congenital malformations, postnatal growth retardation, intellectual disability, and recognizable facial features. It is mainly caused by mutations in either KMT2D or KDM6A. We describe a 14-year-old boy with KS presenting with an unusual combination of bilateral microphthalmia with orbital cystic venous lymphatic malformation and neonatal cholestasis with bile duct paucity, in addition to the typical clinical features of KS. We identified the novel KMT2D mutation c.10588delC, p.(Glu3530Serfs 128) by Mendeliome (Illumina TruSight OneW) sequencing, a next generation sequencing panel targeting 4,813 genes linked to human genetic disease. We analyzed the Mendeliome data for additional mutations which might explain the exceptional clinical presentation of our patient but did not find any, leading us to suspect that the above named symptoms might be part of the KMT2D-associated spectrum of anomalies. We thus extend the range of KS-associated malformations and propose a hypothetical connection between KMT2D and Notch signaling. Ó 2016 Wiley Periodicals, Inc.
Key words: Kabuki syndrome; KMT2D; cyst; microphthalmia; venous lymphatic malformation; vascular malformation; cholestasis; bile duct paucity; notch signaling
INTRODUCTION Kabuki syndrome (KS; OMIM 147920) is a rare genetic disorder characterized by postnatal growth retardation, intellectual disability, and organ and skeletal malformations in addition to a distinctive facial gestalt. KS is usually caused by mutations in the genes KMT2D (MLL2; OMIM 602113) or KDM6A (UTX; OMIM 300128) [Ng et al., 2010; Miyake et al., 2013], two major components of the ASCOM methyltransferase complex
Ó 2016 Wiley Periodicals, Inc.
How to Cite this Article: B€ ogershausen N, Altunoglu U, Beleggia F, Yigit G, Kayserili H, N€ urnberg P, Li Y, Altm€ uller J, Wollnik B. 2016. An unusual presentation of Kabuki syndrome with orbital cysts, microphthalmia, and cholestasis with bile duct paucity. Am J Med Genet Part A 170A:3282–3288.
[Goo et al., 2003]. However, mutations in the genes RAP1A and RAP1B have recently been described as rare causes of KS [B€ ogershausen et al., 2015]. Multiple malformations including cleft lip/palate, congenital heart defects, gastrointestinal abnormalities, cryptorchidism, and congenital hip dysplasia have been Nina B€ ogershausen and Umut Altunoglu contributed equally to this work. Conflicts of interest: None. Patient consent: Written informed consent for the genetic investigation and the publication of results and photographs were obtained from the patient’s parents. Ethics approval: The ethical committee of the University Hospital Goettingen, Goettingen, Germany. Grant sponsor: German Federal Ministry of Education and Research (BMBF); Grant number: 01GM0801. Correspondence to: Bernd Wollnik, M.D., Institute of Human Genetics; University Medical Center Goettingen; Heinrich-D€ uker-Weg 12, Goettingen 37073, Germany. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 17 August 2016 DOI 10.1002/ajmg.a.37931
3282
€ BOGERSHAUSEN ET AL. associated with KS [B€ ogershausen and Wollnik, 2013]. We report a patient with typical clinical findings of KS, along with the unusual findings of microphthalmia in combination with congenital bilateral orbital cysts and neonatal cholestasis with bile duct paucity. The identification of the de novo one basepair (bp) deletion c.10588delC, p.(Glu3530Serfs 128) in exon 38 of KMT2D by clinical exome sequencing confirmed the diagnosis of KS. We discuss this unique presentation in the context of the current literature and present an extensive analysis of the Mendeliome data in search of an explanation for the unusual clinical presentation.
CLINICAL REPORT We describe a 14-year-old boy (Fig. 1A), the first child of healthy consanguineous parents (Fig. 1B) of Turkish origin, born after an uneventful pregnancy via normal spontaneous delivery at 36 weeks of gestation, with a birth weight of 3,190 g (84th percentile) and birth length of 50 cm (90th percentile). Head circumference was not recorded. He did not experience perinatal adaptation problems. Large cystic lesions of the eyes were noted at birth. Orbital T2-weighted MRI evaluation at 2 days of age showed bilateral microphthalmia with globe dimensions of 8 11 6 mm on the
3283 right and 12 11 8 mm on the left, and extraconal hyperintense multicystic lesions filling both orbital cavities with dimensions of 41 26 25 mm on the left and 28 21 20 mm on the right. Both lesions contained fluid–fluid levels within cysts, and postcontrast scans did not detect marked enhancement within the lesions. This constellation was consistent with a radiologic diagnosis of venous lymphatic malformation. The child experienced feeding difficulties and had progressive jaundice after 10 days of age. Clinical examination was normal except for mild petechial rash and bilateral cryptorchidism. Abdominal ultrasound revealed minimally dilated intrahepatic bile ducts, and the gallbladder could not be visualized. A laboratory work-up showed mixed hyperbilirubinemia and mild hepatitis with coagulopathy (Supplementary Table SI). Serology for hepatitis A, B, and C, and IgM antibodies for toxoplasma, rubella, cytomegalovirus, and herpes was negative. Urine was negative for reducing substances and organic acid levels were within the normal range. Cranial MRI revealed multifocal hemorrhagic lesions of diminutive size in a 1 1.5-cm area superior to the frontal horn of the left lateral ventricle. Echocardiographic examination was normal. A liver biopsy at 40 days of age showed absence of the bile ducts and moderate bile accumulation. At 2 months of age, a laparotomy with intraoperative cholangiogram was
FIG. 1. (A) Face of the index patient at ages 8 months, 15 months, and 1310/12 years (from left to right): note the facial features reminiscent of Kabuki syndrome in addition to large intra- and preorbital cystic lesions, deforming and obstructing the palpebral fissures. (B) Pedigree of the family: the patient is a simplex case, his parents are second degree cousins. (C) Electropherograms of the identified KMT2D mutation in exon 38: The 1-bp deletion c.10588delC; p.(Glu3530Serfs 128) was confirmed in the patient by Sanger sequencing and was not present in either of his parents. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/ajmga].
3284 performed to rule out biliary atresia. Liver biopsy revealed paucity of the bile ducts with bile duct proliferation, prompting a diagnosis of idiopathic cholestasis with bile duct paucity. Jaundice and liver function gradually ameliorated, and cholestasis resolved by the age of 6 months. The boy was hospitalized at 7 months of age due to bronchopneumonia and referred to the genetics outpatient clinics because of his dysmorphic features. At 8 months of age, his weight was 5,200 g ( 3.80 SD), his length 68 cm ( 1.13 SD), and the occipitofrontal circumference (OFC) 38.5 cm ( 4.8 SD). Large bilateral hemangiomatous, cystic lesions extended preorbitally to the area of the lower lids and were more pronounced on the left side. Globes could not be located on examination, and the child had no vision. Facial features of Kabuki syndrome, such as sparse eyebrows, long palpebral fissures with ectropion of lower eyelids, long eyelashes, short columella, thin and mildly tented vermilion of the upper lip, lower lip pits, highly arched palate, and prominent ears were noted. He had mild joint hyperlaxity, prominent fetal fingerpads, and a small sacral dimple. Testes were not palpable, and the scrotum was hypoplastic with a normal penile length of 3.5 cm. Head control was not achieved, and truncal hypotonia was present. He responded to sounds, but had no vocalizations. At the age of 10 months, he presented with small calcium stones, secondarily to hypercalciuria, in the lower pole of the left kidney, which were successfully treated with good fluid intake and temporary cessation of oral vitamin D3. He underwent bilateral orchiopexy at the age of 13 months. At 15 months of age, his weight was 7,450 g ( 3.03 SD), length 78 cm (0.65 SD), and OFC 44 cm ( 2.56 SD). Developmental milestones were severely delayed, in part due to blindness. He sat unsupported at 12 months of age and could walk independently with short steps and an out-toeing gait due to pes valgus after 4 years of age. He started uttering a few meaningful words at the age of 7 years, but currently does not speak in sentences. He needed realignment osteotomy for bilateral congenital hip dysplasia at ages 9 and 13 years. The child was recently re-evaluated at the age of 1310/12 years. He had been attending a rehabilitation program for blind children with special education needs for 10 years. He understood and followed basic commands, such as sitting or putting an object down, and had a vocabulary of approximately 5–10 words. He remained agitated and hypervigilant during the examination. He had a height of 132 cm ( 4.07 SD), weight of 38 kg ( 1.89 SD), and OFC of 51 cm ( 3.17 SD). The cystic lesions of the eyes had regressed to a great degree, but he was blind. Dysmorphic features became more pronounced, with a long face, highly arched, nicked eyebrows with lateral sparseness, a depressed nasal root, widely spaced eyes, midface retrusion, a long, bulbous nose with a depressed nasal tip, and protruding ears (Fig. 1A). Additionally, widely spaced teeth, and gynecomastia compatible with Tanner stage III were present. Routine blood biochemistry screening and thyroid hormones were within normal range. Testosterone levels at 13 10/12 years were 0.07 ng/ml, which is in the low normal range [0.07–12 ng/ml]. LH of 1.2 U/L [0.5–10.5 U/L] and FSH of 2.4 U/L [0.5–7.9 U/L] at the same time ruled out hypergonadotropic hypogonadism and indicated normal prepubertal hormone levels.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
EXPERIMENTAL PROCEDURES Mendeliome Sequencing (MS) After informed consent, peripheral blood samples were taken from patients and parents and DNA was extracted from EDTA blood by standard methods. About 50 ng of patient DNA were enriched using the TruSight One1 chemistry (Illumina, version 1; 4,813 disease-associated genes) followed by next-generation sequencing on a MiSeq sequencer (Illumina). We obtained 20 minimum depth of coverage at more than 95% of targets. MS data analysis and filtering of variants were carried out using the exome analysis pipeline “varbank” of the Cologne Center for Genomics (CCG). We applied the following criteria for filtering of MS variants: coverage of more than six reads, a minimum quality score of 10, an allele frequency between 30% and 70%, and a minor allele frequency (MAF) T, p.(Pro2550Leu) in the FLNA gene (OMIM 300017) was identified, which is annotated with a frequency of 0.00005809 in the ExAC browser. Three healthy individuals are annotated as hemizygous mutation carriers in the ExAC browser. Additionally, no pathogenic variants were found in the genes FLT4 (OMIM 136352), VEGFR2 (KDR; OMIM 191306), AKT1 (OMIM 164730), and TEM8 (ANTXR1; OMIM 606410), associated with a susceptibility for capillary hemangioma or lymphangioma, or CHD7 (OMIM 608892), the gene known to cause CHARGE syndrome. We also analyzed the genes PAX2 (OMIM 167409), PAX6 (OMIM 607108), and SIX6 (OMIM 606326), associated with optic nerve or chorioretinal coloboma, and found no pathogenic variant. The TEM8 gene (ANTXR1; OMIM 606410) contained the rare intronic heterozygous sequence variation g.159196_159196delA with a minor allele frequency (MAF) of 0.0002767 (ExAC browser). The Mendeliome data also contained no mutations or copy number changes in a specific set of 22 genes associated with syndromic and isolated microphthalmia, with or without coloboma, determined by a search of the OMIM database (Supplementary Table SII). We also analyzed the genes JAG1 (OMIM 601920) and NOTCH2 (OMIM 600275), known to be involved in the pathogenesis of Alagille syndrome, but did not find any mutations.
Sanger Sequencing We amplified exon 38 of KMT2D from DNA of the index patient and both parents by PCR and sequenced the PCR products by BigDye Terminator method on an ABI 3100 sequencer. De novo
€ BOGERSHAUSEN ET AL. occurrence of the KMT2D mutation identified by MS was confirmed (Fig. 1C). PCR and sequencing primers for KMT2D were obtained from the UCSC genome browser and purchased from IDT DNA (Leuven, Belgium). Primer sequences are available on request.
DISCUSSION We describe a boy with Kabuki syndrome who presented with an unusual combination of bilateral microphthalmia with orbital cysts, neonatal cholestasis with bile duct paucity, cryptorchidism, and congenital hip dysplasia. We performed Mendeliome sequencing using the Illumina TruSight One chemistry, which covers 4,813 genes associated with Mendelian disease, and discovered the 1-bp deletion c.10588delC, p.(Glu3530Serfs 128) in exon 38 of KMT2D. This mutation has not been published before and is not annotated in the HGMD database as a known disease-causing mutation, nor is it annotated in any of the current databases of human genetic variation as a polymorphic variant (ExAC, EVS, dbSNP). Truncating mutations in exon 38 of KMT2D have previously been described in other patients with Kabuki syndrome [B€ ogershausen and Wollnik, 2013; B€ ogershausen et al., 2016]. Cryptorchidism and congenital hip dysplasia are known to be common symptoms of Kabuki syndrome with frequencies of up to 25% and 37%, respectively [Adam and Hudgins, 2005; B€ ogershausen and Wollnik, 2013]. In contrast, the lymphatic vascular or bile duct malformations observed in our patient have never been reported. The Mendeliome data were analyzed for homozygous variants that could possibly explain the severe presentation and unusual phenotype in our patient, but none were found. The hemizygous rare variant c.7649C>T, p.(Pro2550Leu) in FLNA is annotated once in the Exome Variant Server and five times in the ExAC browser (three times as a hemizygous variant), leading to the interpretation that it is unlikely to have a significant effect on the phenotype. Mutations in FLNA cause a phenotypic spectrum that includes X-linked cardiac valvular dysplasia (OMIM 314400), congenital short bowel syndrome (OMIM 300048), FG syndrome 2 (OMIM 300321), frontometaphyseal dysplasia (OMIM 305620), periventricular heterotopia (OMIM 300049), Melnick–Needles syndrome (OMIM 309350), otopalatodigital syndrome, type I (OMIM 311300), otopalatodigital syndrome, type II (OMIM 304120), and terminal osseous dysplasia (OMIM 300244). None of the above-mentioned diseases are compatible with the phenotype described herein. Other than aortic coarctation, which is known to be a frequent arteriovascular malformation in KS, venous or arterial vascular malformations of the cardiopulmonary system, such as anomalous pulmonary venous drainage or double aortic arch, are rare, reported only in six patients [Dyamenahalli et al., 2007; Shahdadpuri et al., 2008; Moral et al., 2009; Bhat et al., 2012; Oswal et al., 2014; Giudici et al., 2015]. Vascular malformations of the central nervous system or the eyes are even rarer. Sanchez-Carpintero et al. [2012] reported a dilated vein of Galen in a child with KS. Tortuous vessels and telangiectasia of the retina were reported by Chuah et al. [2009] and Anandan et al. [2005], respectively. In our patient, MRI studies of the orbital lesions showed bilateral microphthalmia and extraconal hyperintense multicystic lesions filling both orbital
3285 cavities. The extraconal location and fluid–fluid levels within the multicystic lesion supported the diagnosis of venous-lymphatic malformation. No mutations or copy number changes were present in the known hereditary lymphedema gene FLT4 (OMIM 136352), the known capillary hemangioma gene VEGFR2 (KDR; OMIM 191306) or in the known lymphatic overgrowth gene AKT1 (OMIM 164730). The TEM8 gene (ANTXR1; OMIM 606410), associated with a susceptibility for capillary hemangioma, contained the rare intronic heterozygous sequence variation g.159196_159196delA. Considering its deep intronic location and the frequency noted in the ExAC browser, we assume it to be a rare variant without functional consequence, but cannot fully rule out a minor influence on the phenotype. As we did not find any additional genetic cause of the lymphatic malformation in our patient, we suspect that the lymphatic vascular malformation may have been caused by the KMT2D mutation. However, we cannot fully exclude a possible influence of genes that are not covered by the Mendeliome, that is, genes that have not yet been linked to a human vascular phenotype or somatic mutations that would escape this analysis. For example, somatic mutations in the genes GNAQ, GNA11, PIK3CA, and RASA1 have recently been shown to underlie different capillary or venous malformations [Limaye et al., 2015; Macmurdo et al., 2016; Ayturk et al., 2016]. Due to the parental consanguinity, the possibility of a homozygous variant in a gene not covered by the Mendeliome remains. Initially, a differential diagnosis of colobomatous cysts was also considered. Colobomatous cysts are congenital orbital lesions which are often associated with microphthalmia [Shields and Shields, 2004] and can either occur sporadically or be inherited in an autosomal recessive manner [Porges et al., 1992]. The clinical presentation of our patient with bilateral congenital orbital cysts extending to the lower eyelid, associated with microphthalmia might point toward this diagnosis, but the extraconal location of the lesion makes it less likely. Colobomatous cysts have been described in patients with syndromic conditions such as trisomy 13 [Magni et al., 1991], Aicardi syndrome [Lorenz et al., 1991], and CHARGE syndrome [De Krijger et al., 1999; Pushker et al., 2013]. We did not find any mutation or copy number change in the CHD7 gene (OMIM 608892) in the Mendeliome data of the patient and could thus rule out the most frequent cause of CHARGE syndrome (OMIM 214800). We also ruled out pathogenic variants in the genes PAX2 (OMIM 167409), PAX6 (OMIM 607108), and SIX6 (OMIM 606326), which are associated with coloboma of the optic nerve and/or the chorioretina. Moreover, the Mendeliome data were analyzed for mutations and copy number changes in a set of 22 genes associated with syndromic and isolated microphthalmia, with or without coloboma (Supplementary Table SII), yielding a negative result. Chen et al. [2014] described colobomatous microphthalmia and a retrobulbar cystic mass in a patient with a KMT2D mutation, whose facial features also included ptosis and esotropia, resembling the ocular features in our patient. Microphthalmia and anophthalmia have also been reported in five patients with KS [McVeigh et al., 2015], emphasizing the role of ophthalmic malformation in this disorder. Additionally, various patients with coloboma have been published: Ming et al. [2003] described three patients with KS and retinal coloboma and identified 11 further patients in the
3286 literature with colobomas affecting the iris, choroid, retina, and/or the optic nerve. They pointed out the clinical overlap with CHARGE syndrome, an observation that has also been made by Verhagen et al. [2014] and is supported by the recent finding of a direct interaction between the proteins KMT2D and CHD7 [Schulz et al., 2014]. We, therefore, would like to recommend that an ophthalmological consultation be part of the routine medical care of patients with Kabuki syndrome [Adam et al., 2011; McVeigh et al., 2015]. Hepatic malformations have been rarely described in Kabuki syndrome, with extrahepatic biliary atresia in three patients [McGaughran et al., 2000; Van Haelst et al., 2000; Selicorni et al., 2001] and hepatic fibrosis in one [Nobili et al., 2004]. Cholestasis with bile duct paucity is an extremely rare bile duct malformation, and has never been reported in patients with Kabuki syndrome. There are rare reports of patients with Williams syndrome who have biliary hypoplasia [O’Reilly et al., 2006; Shah et al., 2008; Honeywell et al., 2009; Sakhuja et al., 2015], but syndromic bile duct paucity is generally considered to be pathognomonic for Alagille syndrome (OMIM 118450 and 610205 for types 1 and 2, respectively). The major clinical features of Alagille syndrome have been defined as cholestasis, cardiac defect (pulmonary stenosis), skeletal abnormalities (butterfly vertebrae), ophthalmologic abnormalities (posterior embryotoxon), and characteristic facial features [Spinner et al., 2000]. Apart from cholestasis, none of these features were present in our patient. We searched the Mendeliome data for mutations and copy number changes of the genes known to cause Alagille syndrome but did not find any likely pathogenic variants or copy number changes in either JAG1 (OMIM 601920) or NOTCH2 (OMIM 600275). A combination of hepatic malformation, pigmentary retinopathy, congenital heart disease, intestinal malformations, and renal abnormalities has been described in Hardikar syndrome (HS; OMIM 612726), a rare entity for which the molecular cause is unknown. Nydegger et al. [2008] compared the features of KS and HS and concluded that the two entities were distinct. Ejarque et al. [2011] suggested an overlap of Hardikar and Kabuki syndromes in a patient with hepatoblastoma and pigmentary retinopathy with patchy pattern (cat’s paw) in whom a mutation in KMT2D was identified. However, this patient did not have malformation of the bile ducts, a hallmark feature of HS. Patients with HS have dilatation of the intrahepatic bile ducts in combination with stenosis of the large bile ducts rather than intrahepatic bile duct paucity, thus distinguishing HS from Allagille syndrome [Nydegger et al., 2008] and from the biliary malformation seen in the patient presented here. To date, there is no known connection between KMT2D and the Notch signaling pathway, but the findings in our patient may point in this direction. Notch signaling plays a crucial role in vascular development and is required for lymphatic endothelial cell fate determination [Geudens et al., 2010]. Each of Notch 1–4 has specific roles in the differentiation of biliary epithelial cells, and determination of vascular smooth muscle cell behavior [Baeten and Lilly, 2015], and they are differentially regulated in central nervous vascular malformations [Hill-Felberg et al., 2015]. It is conceivable that the orbital vascular and bile duct
AMERICAN JOURNAL OF MEDICAL GENETICS PART A malformations in our patient may be the result of the same embryological signaling defect. Our findings extend the recognized KS phenotype and suggest a possible overlap of KS with Alagille syndrome in the form of bile duct hypoplasia. This clinical observation leads us to hypothesize that the KMT2D mutation in our patient might impact on transcriptional regulation of one or several key Notch signaling components. Further clinical reports, genomic investigations, and functional studies, however, will be needed to explore a possible connection between KMT2D and the Notch signaling pathway.
ACKNOWLEDGMENTS We are grateful to the family for participating in this study, to Esther Milz for excellent technical assistance, and to Karin Boss for critically reading the manuscript. This work was supported by the German Federal Ministry of Education and Research (BMBF) by grant number 01GM0801 (E-RARE network CRANIRARE-2).
WEB RESOURCES 1000Genomes: http://browser.1000genomes.org/index.html Online Mendelian Inheritance in Man (OMIM): http://www.ncbi. nlm.nih.gov/omim Database of Single Nucleotide Polymorphisms (dbSNP): http:// www.ncbi.nlm.nih.gov/SNP/ DECIPHER: https://decipher.sanger.ac.uk/index Exome variant server (EVS): http://evs.gs.washington.edu/EVS/ Exome Aggregation Consortium (ExAC): http://exac. broadinstitute.org/ Human Gene Mutation Database (HGMD): http://www.hgmd.org/
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SUPPORTING INFORMATION
Spinner NB, Leonard LD, Krantz ID. 2000 [Updated 2013]. Alagille syndrome. In: Pagon RA, Adam MP, Ardinger HH, et al., editors.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
