Individual Clinically Diagnosed with CHARGE Syndrome but with a Mutation in KMT2D, a Gene Associated with Kabuki Syndrome: A Case Report

We report a Japanese female patient presenting with classic features of CHARGE syndrome, including choanal atresia, growth and development retardation, ear malformations, genital anomalies, multiple endocrine deficiency, and unilateral facial nerve palsy. She was clinically diagnosed with typical CHARGE syndrome, but genetic analysis using the TruSight One Sequence Panel revealed a germline heterozygous mutation in KMT2D with no pathogenic CHD7 alterations associated with CHARGE syndrome. Kabuki syndrome is a rare multisystem disorder characterized by five cardinal manifestations including typical facial features, skeletal anomalies, dermatoglyphic abnormalities, mild to moderate intellectual disability, and postnatal growth deficiency. Germline mutations in KMT2D underlie the molecular pathogenesis of 52–76% of patients with Kabuki syndrome. This is an instructive case that clearly represents a phenotypic overlap between Kabuki syndrome and CHARGE syndrome. It suggests the importance of considering the possibility of a diagnosis of Kabuki syndrome even if patients present with typical symptoms and meet diagnostic criteria of CHARGE syndrome. The case also emphasizes the impact of non-biased exhaustive genetic analysis by next-generation sequencing in the genetic diagnosis of rare congenital disorders with atypical manifestations.

Kabuki syndrome (KS) (OMIM #147920 and #300867) is a rare multiple malformation disorder that was originally reported in Niikawa et al. (1981). Subsequently, the five cardinal manifestations of KS were defined by Niikawa et al. (1988) as typical facial features, skeletal anomalies, dermatoglyphic abnormalities, mild to moderate intellectual disability, and postnatal growth deficiency. However, consensus clinical diagnostic criteria for KS have not been established.
The second causative gene of KS is KDM6A (OMIM * 300128), encoding lysine-specific demethylase 6A, which causes X-linked KS subtype 2 when mutated (OMIM #300867). Germline mutations in KDM6A are relatively rare, and are responsible for fewer than 5% of patients with KS (Lederer et al., 2012;Miyake et al., 2013;Banka et al., 2015). Most KS patients, especially those with typical facial dysmorphism, carry KMT2D mutations (Banka et al., 2012). However, phenotypical variability has been documented in individuals with KMT2D mutations, indicating that such mutations can be detected in KS patients with atypical manifestations.
Kabuki syndrome and CHARGE syndrome are distinct congenital disorders, although phenotypic and molecular links between them have been reported previously (Ming et al., 2003;Genevieve et al., 2004;Schulz et al., 2014;Verhagen et al., 2014;Badalato et al., 2017;Butcher et al., 2017). However, to our knowledge, there are no reports of KS cases that have met both Blake and Verloes diagnostic criteria for CHARGE syndrome (Blake et al., 1998;Verloes, 2005). We herein report a patient clinically diagnosed with typical CHARGE syndrome that fulfilled both Blake and Verloes criteria (Blake et al., 1998;Verloes, 2005), but who was genetically diagnosed with atypical KS based on the presence of a de novo KMT2D mutation and the absence of pathogenic variation in CHD7. This case demonstrates the phenotypic overlap between CHARGE syndrome and KS.

CASE REPORT
The 24-year-old Japanese female patient was born to non-consanguineous parents ( Figure 1A) at 36 weeks and 6 days of gestational age by Cesarean section because of fetal distress. At birth, she had a low body weight (2,100 g) and a short stature (44 cm). She presented with multiple dysmorphic features including choanal atresia, cleft palate, micrognathia, a hypoplastic cupped auricle with atresia of the external auditory meatus, and right facial nerve palsy. Echocardiography revealed no structural abnormalities of the heart, and no coloboma was observed in either eye, together with no abnormalities in the iris, retina, choroid, or optic disk. Chromosome analysis revealed a normal karyotype (46, XX). Mechanical ventilation was required for respiratory distress for 2 weeks, after which an oropharyngeal tube was used to maintain the airways until she was 7 months old. Her choanal atresia was treated surgically at age 1 year. She had tooth hypoplasia with tooth malalignment. She also had entropion of the right upper eyelid, and an epicanthal fold of the left eye that were surgically treated at age 11 years.
She presented at our hospital at age 11 years with severe short stature (111.5 cm, −5.87 standard deviation). At the initial visit, she showed deafness, right facial nerve palsy, cleft palate, malformation of the auricle with atresia of the external auditory meatus, and bilateral hypoplastic nipples. Computed tomography revealed hypoplasia of the pancreatic body and tail (Figure 2A), and severe uterine hypoplasia with the presence of both ovaries ( Figure 2B). It also revealed bilateral atresia of the external auditory meatus, hypoplasia of the right vestibular aqueduct, bilateral hypoplasia of the long limb of incus, and bilateral agenesis of stapes and the posterior semicircular canal ( Figure 2C). Based on these findings, she was clinically diagnosed with typical CHARGE syndrome.
She underwent plastic surgery to correct the malformation of the auricle five times from age 14 years. She also started estrogen replacement therapy from age 15 years. The patient is currently 24 years old and has multiple dysmorphic features ( Figure 1D). She has neither fingertip pads nor hockeystick palmar creases. Genetic testing was performed at age 23 years. Comprehensive DNA sequencing using the TruSight One sequencing panel (Illumina, San Diego, CA, United States) revealed a novel heterozygous mutation, c.10690 C > G (p.L3564V), in KMT2D. The mutation was confirmed by Sanger sequencing (Figure 1B). It was absent from both of her parents, suggesting that it was de novo, and was not found in the public databases (NCBI, Ensembl, dbSNP, or ExAc). It was predicted to be damaging by PolyPhen-2 (probably damaging) and SIFT (damaging) prediction tools, suggesting that it was not an irrelevant polymorphism. A microarray-based comparative genomic hybridization assay revealed no obvious pathogenic DNA copy number aberrations.

MATERIALS AND METHODS
Genomic DNA was extracted from peripheral blood samples using the QIAamp Blood Midi Kit (QIAGEN, Hilden, Germany). We performed trio sequencing using a TruSight One sequencing

DISCUSSION
The diagnostic criteria of CHARGE syndrome are defined by two representative papers (Blake et al., 1998;Verloes, 2005). The current patient met the diagnostic criteria of typical CHARGE syndrome defined by Blake et al. by presenting with three major criteria: choanal atresia, characteristic ear abnormalities and cranial nerve dysfunction, and four minor criteria: developmental delay, growth deficiency, an orofacial cleft and genital hypoplasia (based on the finding of hypogonadotropic hypogonadism). She met the diagnostic criteria of typical CHARGE syndrome defined by Verloes by fulfilling two major signs: choanal atresia and hypoplastic semi-circular canals, and four minor signs: rhombencephalic dysfunction, hypothalamohypophyseal dysfunction, abnormalities of the middle and external ear, and mental retardation. The patient was therefore clinically diagnosed with typical CHARGE syndrome. In contrast, she did not show choanal atresia or heart defects which are frequently identified in patients with CHARGE syndrome (Pagon et al., 1981;Blake et al., 1998;Zentner et al., 2010). Moreover, comprehensive genetic analysis identified a de novo germline mutation, L3564V, in KMT2D, which is a gene associated with KS. No mutations or variants were found in CDH7. Therefore, the patient was genetically diagnosed with KS despite presenting with typical symptoms of CHARGE syndrome.
A phenotypic overlap between CHARGE syndrome and KS has been described in previous studies (Ming et al., 2003;Genevieve et al., 2004;Schulz et al., 2014;Verhagen et al., 2014;Badalato et al., 2017;Butcher et al., 2017). For example, coloboma is a major symptom, found in 65-90% of patients with CHARGE syndrome (Blake et al., 1998;Zentner et al., 2010). However, coloboma is also found in patients with KS who show a phenotypic overlap between KS and CHARGE syndrome (Ming et al., 2003). Three cases of genetically confirmed KS with KMT2D mutations who also met typical CHARGE diagnostic criteria, as defined by either Blake et al. (Schulz et al., 2014;Verhagen et al., 2014) or Verloes (Patel and Alkuraya, 2015), have been previously reported. A detailed comparision of clinical symptoms is shown in Supplementary Tables 4, 5. In contrast, the current case is the first molecularly diagnosed KS patient who simultaneously met two representative diagnostic criteria of typical CHARGE syndrome as defined by Blake et al. and Verloes. It is therefore considered to be an instructive case that clearly indicates the phenotypic overlap between CHARGE syndrome and KS.
The current case presented with several rare KS symptoms. Cranial nerve dysfunction is a typical symptom defined as a major criterion found in 70-92% of patients with CHARGE syndrome (Byerly and Pauli, 1993;Blake et al., 1998Blake et al., , 2008. Specifically, the involvement of cranial nerves I, VII, VIII, IX, and/or X are frequently observed in patients with CHARGE syndrome (Blake et al., 2008). The current case presented with right facial nerve palsy, which occurs in 32-50% of patients with CHARGE syndrome (Blake et al., 2008), but has only been reported in one previous study of a patient with KS (Iida et al., 2006). Choanal atresia is another typical symptom identified in 50-60% of patients with CHARGE syndrome (Blake et al., 1998). However, it is rarely seen in patients with KS, having been reported in four previous studies (Powell et al., 2003;Teissier et al., 2008;Schulz et al., 2014;Badalato et al., 2017). Among those patients, KMT2D mutations were only identified in one familial case (Badalato et al., 2017) of autosomal dominant inheritance associated with the Q3575H mutation in exon 38. Surprisingly, the L3564V mutation of the current patient is located in the same exon and affects an amino acid close to that mutated by Q3575H. Further studies are required to determine whether these two missense mutations affect the development of choanal atresia.
Recent studies revealed the presence of molecular link between CHD7 and KMT2D proteins (Schulz et al., 2014;Butcher et al., 2017). Both CHD7 and KMT2D interact with members of the WAR complex, suggesting that these two molecules function as part of the same chromatin modification machinery (Schulz et al., 2014). Butcher et al. (2017) investigated genome-wide DNA methylation profiles in patients with CHD7 or KMT2D mutations, and found that they showed distinct patterns of epigenetic dysregulation. They also identified common DNA methylation signatures, including a gain of DNA methylation at homeobox A5 (HOXA5), which is shared by the two genetic disorders and may account for some of the clinial overlap between CHARGE syndrome and KS. Therefore, both phenotypic and molecular links are observed in patients with CHARGE syndrome and KS.

CONCLUDING REMARKS
We report an atypical case of KS showing clear phenotypic overlap with CHARGE syndrome. This case highlights the importance of considering a diagnosis of KS even if patients fully meet the diagnostic criteria of typical CHARGE syndrome. Therefore, molecular testing of KMT2D should be considered in patients clinically diagnosed with CHARGE syndrome without CHD7 mutations. It also emphasizes the impact of non-biased exhaustive genetic analysis by next-generation sequencing in the molecular diagnosis of rare congenital disorders with atypical manifestations.

ETHICS STATEMENT
We obtained written informed consent for genomic analysis of the patient and her parents in accordance with the Declaration of Helsinki. The genetic study was approved by the Institutional Review Board of Nagoya City University Graduate School of Medical Sciences (approval no. 130). The mother of the patient provided written informed consent for the publication of the patient's identifiable information.