MLL2 mosaic mutations and intragenic deletion duplications in patients with Kabuki syndrome

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1 Clin Genet 2013: 83: Printed in Singapore. All rights reserved Short Report 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd CLINICAL GENETICS doi: /j x MLL2 mosaic mutations and intragenic deletion duplications in patients with Kabuki syndrome Banka S, Howard E, Bunstone S, Chandler KE, Kerr B, Lachlan K, McKee S, Mehta SG, Tavares ALT, Tolmie J, Donnai D. MLL2 mosaic mutations and intragenic deletion duplications in patients with Kabuki syndrome. Clin Genet 2013: 83: John Wiley & Sons A/S. Published by Blackwell Publishing Ltd, 2012 Kabuki syndrome (KS) is a rare multi-system disorder that can result in a variety of congenital malformations, typical dysmorphism and variable learning disability. It is caused by MLL2 point mutations in the majority of the cases and, rarely by deletions involving KDM6A. Nearly one third of cases remain unsolved. Here, we expand the known genetic basis of KS by presenting five typical patients with the condition, all of whom have novel MLL2 mutation types two patients with mosaic small deletions, one with a mosaic whole-gene deletion, one with a multi-exon deletion and one with an intragenic multi-exon duplication. We recommend MLL2 dosage studies for all patients with typical KS, where traditional Sanger sequencing fails to identify mutations. The prevalence of such MLL2 mutations in KS may be comparable with deletions involving KDM6A. These findings may be helpful in understanding the mutational mechanism of MLL2 and the disease mechanism of KS. Conflictofinterest The authors declare no conflict of interest. S Banka a, E Howard a, S Bunstone a, KE Chandler a, BKerr a, K Lachlan b, S McKee c, SG Mehta d, ALT Tavares d, J Tolmie e and D Donnai a a Department of Genetic Medicine, St Mary s Hospital, Manchester Academic Health Sciences Centre (MAHSC), University of Manchester, Manchester, UK, b Wessex Clinical Genetics Service, University Hospital Southampton NHS Trust and Human Genetics and Genomic Medicine, Faculty of Medicine, University of Southampton, Southampton, UK, c Northern Ireland Regional Genetics Service, Belfast City Hospital, Belfast, UK, d Department of Clinical Genetics, East Anglian Medical Genetics Service, Addenbrooke s Hospital, Cambridge, UK, and e Clinical Genetics Department, Yorkhill Hospitals, Glasgow, UK Key words: intragenic deletion duplication Kabuki syndrome KDM6A MLL2 mosaic Corresponding author: Dr Siddharth Banka, Department of Genetic Medicine, St Mary s Hospital, Oxford Road, Manchester M13 9WL, UK. Tel.: ; fax: ; Siddharth.Banka@manchester.ac.uk Received 28 June 2012, revised and accepted for publication 13 August 2012 Kabuki syndrome (KS or Niikawa Kuroki syndrome, OMIM ) is an autosomal dominant condition that is characterized by a recognizable facial phenotype, a high incidence of internal malformations, feeding difficulties in infancy, postnatal failure to thrive, hypotonia and mild to moderate developmental delay. It is caused by point mutations in MLL2, that mostly arise de novo in 55 80% of cases (1 6). We recently undertook a systematic molecular and phenotype study of KS and showed that the majority of, but not all, patients who have typical features of KS, have MLL2 mutations (2). We proposed that failure to identify MLL2 mutations by traditional Sanger sequencing, in approximately one third of cases, could be attributed 467

2 Banka et al. to clinical misdiagnosis, genetic heterogeneity, deepintronic mutations, large intragenic deletions or duplications or changes affecting the regulatory elements (2). Genetic heterogeneity has been confirmed recently by the discovery of deletions involving KDM6A in three patients with KS (7). In this study, we expand the known genetic basis of the disorder by showing mosaic mutations and large intragenic deletions and duplications of MLL2 in patients with KS. Methods and results The study was approved by the Central Manchester Research Ethics Committee (02/CM/238) and the University of Manchester Ethics Committee and was performed in accordance with the declaration of Helsinki protocol. Fully informed signed consents were taken from legal guardians of all the patients described here. A service, offering sequencing of all exons of MLL2, including the intron exon boundaries, has been recently set up at the Regional Molecular Genetics Laboratory in Manchester, UK. Over a period of 10 months, as part of this new service, we analysed samples of 64 patients, clinically suspected to have KS, via bi-directional Sanger sequencing of MLL2, as described previously (2). Of note, these patients were not part of our previously reported study (2). We identified pathogenic MLL2 mutations in 31 patients (48%). The mutation detection rate is lower than what has been described previously (2). This, probably, represents the true mutation detection rate in KS in a clinical setting. In two patients, out of these 31, the mutated alleles appeared to have weaker sequence chromatograms, leading us to suspect that they may harbour mosaic mutations. Patient 1 has a mosaic single base deletion, c.9494dela (Fig. 1a), which was confirmed by pyrosequencing (Appendix S1 and Figure S1, Supporting Information) to rule out the possibility of a sequencing artefact. Pyrosequencing showed that less than 15% of the alleles were mutated in the tested sample (Appendix S1, Supporting Information). Patient 2 has a mosaic 13 base deletion, c.8463_8475del13 (Fig. 1b) that was confirmed via bi-directional Sanger sequencing using two additional sets of primers (Appendix S2 and Figure S2, Supporting Information). To explore the possibility of intragenic deletions and duplications, we studied dosage of exons 1, 4, 7, 10, 11, 14, 15, 19, 22, 26, 30, 31, 33, 34, 35, 39, 41, 43, 45, 47, 49, 51 and 54 of MLL2 by multiplex ligand probe amplification (MLPA) using the MLL2 V2 P389- A1 kit (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer s instructions. MLL2 MLPA was performed in 58 KS patients found to be MLL2 mutation negative following Sanger sequencing. Out of these, 24 samples were from our MLL2 sequencing service and 34 samples were from our recently reported study (2). MLL2 MLPA led to identification of three patients with copy number changes that were not detected via array comparative genomic hybridization (a-cgh). Patient 3 was found to have a mosaic deletion that included the whole MLL2 gene (Fig. 1c). From the MLPA results, the level of mutated alleles could be estimated to be between 10% and 20%. Patient 4 was detected to have a terminal multi-exon deletion from exon 43 to 54 (Fig. 1d). Of note, a MLPA probe for exon 42 is not available and exon 54 is the last exon of MLL2. Therefore, it is possible that this deletion involves exon 42 and extends beyond the MLL2 gene. Irrespective of the extent of the deletion, the mutation is predicted to result in a truncated or absent protein. Patient 5 (Fig. 1e) has a de novo intragenic duplication of exons This mutation is predicted to result in an absent or abnormal protein. All three findings were confirmed by repeating the MLPA assay with two different DNA concentrations. Discussion We have identified one patient with a mosaic MLL2 point mutation, one with a mosaic 13 base-pair deletion, one with a mosaic whole gene deletion, one with a partial multi-exon deletion and one with a partial intragenic duplication. Mosaic MLL2 mutations, whole gene deletions encompassing MLL2, or intragenic deletion and duplications of the MLL2 gene have not been described till date. We examined the clinical features of all five patients to investigate the phenotype and the disease course in detail. We had shown earlier that facial dysmorphism is a strong predictor of MLL2 mutation status (2). In this study, facial phenotypes of all five patients, although not entirely typical, are consistent with KS (Fig. 1). Long palpebral fissures and sparse lateral eyebrows were present in all five patients. Table 1 provides an overview of other clinical features in all the patients. Feeding difficulties were found in 4 of 5 patients in infancy or childhood, 3 of 5 patients were hypotonic in infancy and all patients had motor delay and mild or moderate learning disability. Minor cardiac malformations were found in 2 of 5 patients, 1 of 5 patients had cleft lip and palate and 2 of 5 patients had renal malformations. Susceptibility to infections was noted in 3 of 5 patients. The only male patient in this series had delayed puberty, while one female patient had precocious puberty. All patients had foetal pads. Although a statistical analysis is not possible because of the small numbers, the patients with mosaic mutations (patients 1, 2 and 3) do not have milder facial phenotypes or less severe clinical features. However, heights of all the three patients with mosaic mutations were between 50th and 75th centile. It is, therefore, possible that mosaic mutations have a milder effect on stature of the patients. Notably, the levels of mutated alleles in patients 1 and 3 were comparatively small. We were unable to quantify the proportion of mutated alleles in patient 2. DNA from all three patients with mosaic mutations was extracted from blood samples and thus the possibility of higher levels of mutated alleles in other tissues cannot be ruled out. KS is a systemic disorder that is not limited to a single organ or tissues of a common 468

3 MLL2 mosaic mutations and intragenic deletion duplications in patients with KS (a) (b) (c) (d) (e) Fig. 1. Facial phenotypes and genotypes, (a) Patient 1 (outlined with red): Photographs taken at age 3 and 11 years, showing mildly arched eyebrows, long palpebral fissures and eversion of lateral lower eyelids and squint. Features are harder to recognize in the older photograph. Upper sequence trace is from a control sample and the lower trace is from patient s sample showing mosaic single base deletion. (b) Patient 2 (outlined with blue): Photograph showing typical facial phenotype with arched eyebrows that are sparse laterally, ptosis, long palpebral fissures, broad nasal tip and thin upper lip. Upper sequence trace shows a control trace and the lower trace is from patient s sample showing the 13 base mosaic deletion. (c) Patient 3 (outlined with green): Photograph showing slightly atypical facial phenotype with a broad forehead, rather straight eyebrows with lateral sparseness, narrow-long palpebral fissures, prominent nasal bridge, repaired cleft lip and palate and a bow-shaped thin upper lip. The MLPA results are represented in the accompanying bar graph showing the mosaic deletion of whole gene. The bright green columns represent dose of reference probes and the blue columns represent the dose of MLL2 probes. Together they illustrate the relative dose of MLL2 exons compared with the control probes. The represented exons are marked within each column. (d) Patient 4 (outlined with maroon): Photographs taken at 16 and 25 years, showing facial phenotype with arched eyebrows and long palpebral fissures. The photograph at the older age shows worsening of prognathism. The MLPA result shows the partial intragenic deletion from exon 43 to 54. (e) Patient 5 (outlined with purple): The MLPA result showed duplication from exon 15 to 34. Consent to publish photograph of this patient was not available but she has arched eyebrows, depressed nasal bridge, prominent ears, pillowed lower lip and prominent areola. embryonic origin. Thus, mosaic MLL2 mutations in patients with KS most probably represent early events during embryogenesis. Another less probable possibility is that of somatic mosaicism because of the reversion (8). Further, the presence of somatic mosaicism raises the possibility of gonadal mosaicism, although there are as yet no reported cases of more than one affected child with KS born to unaffected parents. However, possibility of gonadal mosaicism may have important implications in genetic counselling. Our findings indicate that MLL2 haploinsufficiency in some, and not all, cells may be enough to produce deleterious phenotypic effects. The majority of MLL2 mutations in KS are truncating (2), therefore, the finding of larger deletions or duplications is not surprising. Notably the whole gene deletion in patient 3 was found to be mosaic. It is possible that non-mosaic whole gene MLL2 deletions may be embryonic lethal. Importantly, our results show that MLL2 dosage studies, such as those possible by MLPA, must be considered in KS patients in whom mutation is not identified by traditional sequencing and where the clinical phenotype is strongly suggestive of the diagnosis. Furthermore, small or mosaic deletions or duplications may not be detected with a-cgh. Notably, till date, only three patients with KDM6A deletions have been 469

4 Banka et al. Table 1. Genotype and phenotype of patients with MLL2 mosaic mutations and intragenic deletion or duplications Patient number Gender Female Female Female Male Female Genotype c.9494dela c.8463_8475 del13 Whole gene deletion Deletion of exons Duplication of exons Mosaicism Yes Yes Yes No No Current age (years) Feeding Normal Feeding difficulties in infancy Feeding difficulties in infancy Feeding difficulties in infancy Initially on nasogastric tube feeds, now gastrostomy fed. Severe reflux and unsafe swallow Hypotonia in infancy No Yes Yes Yes No Motor Delay Mild Yes Mild Mild Mild Development and learning Mild delay Moderate delay Severe retardation. Does not have speech Vision Hypermetropia and right convergent squint. Right convergent squint and hypermetropia Hearing No problems Decreased hearing at low frequencies. Sensitive to loud noises Delayed speech and needed speech therapy. School for special educational needs Pale optic discs No problems No problems No problems No problems No problems Motor skills at 6 9 month level when assessed at 14 months chronological age Cardiac defects Normal echocardiogram Normal echocardiogram None Bicuspid aortic valve Patent foramen ovale (closed spontaneously) Palate Normal V-shaped Cleft lip and palate High arched with nasal speech Normal Urogenital defects None Right ectopic kidney None known None Horse-shoe kidney Endocrine abnormalities Precocious puberty at the age of 8 years Joint dislocations Congenital left hip dislocation needing open reduction. Postnatal hypoglycaemia requiring dextrose infusion- resolved None Delayed puberty (stages 2 3 at the age of 15 years). None None None None Immunity related problems Recurrent otitis media Recurrent otitis media None None Recurrent respiratory infections Foetal pads Yes Yes Yes Yes Yes Head circumference (age; centile) 55.2 cm (11 years 2 months; 90th) Height (age; centile) cm (11 years 2 months; Weight (age; centile) 64.6 (11 years 2 months; 99th) Other problems Joint hyperextensibility with leg pains and small lateral incisors 48.7 cm (5 years 2 months; 0.4th) 52.5 cm (7 years 9 months; 25th 50th) cm (5 years 2 months; 23.3 kg (5 years 2 months; 91st 98th) Constipation requiring anal dilation, broad thumbs and toes, anteriorly placed anus, dimple proximal to anus and irregularly spaced teeth None 54 cm (16 years; 50th 25th) 43.1 cm (12 months; 0.4th) 128 cm (7 years 9 months; cm (16 years; <0.4th). (Final 71.3 cm (12 months; 9th 25th) height cm) Not known 45.7 kg (16 years; 2nd) 7.35 kg (12 months; 2nd 9th) Haemagiomatous lesion on right thigh and epilepsy Joint hyperextensibility, hypoplastic clavicles, 5th finger clinodactyly, multiple moles on trunk and rough patch of alopecia at vertex arising from a congenital skin defect Had a two vessel cord 470

5 MLL2 mosaic mutations and intragenic deletion duplications in patients with KS reported (7). Therefore, in patients with KS the prevalence of intragenic MLL2 deletion duplications may be comparable with deletions involving KDM6A. We, therefore, suggest that the molecular diagnostic strategy for KS should be MLL2 sequencing followed by dosage analysis of MLL2 and KDM6A. In summary, our findings expand the known genetic basis of KS. Additionally, we have shown that there are no marked differences in the pattern of problems in these patients when compared with what is known in KS and that MLL2 dosage studies must be included in the diagnostic screen of KS. Future studies may help to uncover the frequency of mutation types described here. Further studies to understand the mechanism of mosaic MLL2 mutations in KS are warranted, especially because somatic MLL2 mutations have been identified in a number of cancers (9). Our findings may be helpful in understanding the mutational mechanism of MLL2 and disease mechanism of KS. Supporting Information The following Supporting information is available for this article: Fig S1. Pyrosequencing results: pyrogram showing a quantitative peak profile of patient 1 compared to a control sample. Fig S2. Chromatograph from bi-directional Sanger sequencing of exon 34 of MLL2 of patient 2, using two additional primer sets. Table S1. Sequences of the primer used for pyrrosequncing and sequencing. Appendix S1. Pyrrosequencing. Appendix S2. Sequencing. Additional Supporting information may be found in the online version of this article. Acknowledgements We are grateful to Mr and Mrs P Scales, their family and friends for funding and support received for this project through the Central Manchester University Hospitals NHS Foundation Trust, Kabuki Research Fund no We also acknowledge the support of Manchester Biomedical Research Centre. We thank all our patients, families and their clinicians for help in the research. References 1. Ng SB, Buckingham KJ, Hannibal MC et al. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 2010: 42 (9): Banka S, Veeramachaneni R, Reardon W et al. How genetically heterogeneous is Kabuki syndrome?: MLL2 testing in 116 patients, review and analyses of mutation and phenotypic spectrum. Eur J Hum Genet 2011: 20 (4): Hannibal MC, Buckingham KJ, Ng SB et al. Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome. Am J Med Genet A 2011: 155 (7): Paulussen ADC, Stegmann APA, Blok MJ et al. MLL2 mutation spectrum in 45 patients with Kabuki syndrome. Hum Mutat 2011: 32 (2): E2018 E Micale L, Augello B, Fusco C et al. Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients. Orphanet J Rare Dis 2011: 6 (1): Li Y, Bögershausen N, Alanay Y et al. A mutation screen in patients with Kabuki syndrome. Hum Genet 2011: 130 (6): Lederer D, Grisart B, Digilio MC et al. Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with Kabuki syndrome. Am J Hum Genet 2012: 90 (1): Hirschhorn R. In vivo reversion to normal of inherited mutations in humans. J Med Genet 2003: 40 (10): Cross NC. Histone modification defects in developmental disorders and cancer. Oncotarget 2012: 3 (1):