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Integrated facial analysis and targeted sequencing identifies a novel KDM6A pathogenic variant resulting in Kabuki syndrome

Shi, Weihuia,b; Chen, Yiyaoa,b; Chen, Songchanga,b; Li, Shuyuana,b; Chang, Chunxina,b; Zhang, Lanlana,b; Fei, Hongjuna,b; Huang, Hefenga,b; Zhang, Junyua,b,∗; Xu, Chenminga,b,∗

Author Information
doi: 10.1097/JBR.0000000000000022



Kabuki syndrome (KS), first described in 1981 by Niikawa et al[1] and Kuroki et al,[2] is a rare congenital mental retardation condition with an incidence ranging from 1/32,000 to 1/86,000.[3] Patients with KS typically present with characteristic facial features including long palpebral fissures with ectropion of eversion of the lateral third of the lower eyelids, arched eyebrows, a depressed nasal tip, and prominent ears.

KS is also known as Kabuki make-up syndrome due to the resemblance of the typical facial dysmorphia to actors making up in Japanese traditional theater. Additional manifestations that are frequently associated with KS include a high incidence of intellectual disability, cardiovascular diseases, short stature, and musculoskeletal abnormalities.[4]

Both KMT2D and KDM6A pathogenic variants were associated with KS, resulting in KS type 1 (KS1, OMIM #147920) and KS type 2 (KS2, OMIM #300867), respectively. Approximately 55% to 80% KS patients were attributed to de novo or autosomal dominant KMT2D (12q13.12) variants.[5] Loss-of-function variants of KDM6A (Xq11.3) have been identified in several KMT2D variant-negative patients since Lederer et al[6] reported 3 KS cases in 2012. The KDM6A contains 29 exons and encodes the lysine-specific demethylase 6A (KDM6A), which demethylates the H3K27me3/me2 mark specifically.[7–9] The H3K27me3/me2 mark was notified as a DNA methylation-independent imprinting mark recently.[7–9] Moreover, KDM6A and KMT2D encode methyltransferase activating signal cointegrator-2-containing complex that contains various unique components, contributing to equilibrating the active/repressive histone methylations.[10–13]

Since deletions of KDM6A were initially identified in 3 KMT2D-variant-negative KS patients by array comparative genomic hybridization analysis,[6] 45 X-linked KS2 patients have been reported in various countries during the last 6 years,[3–6,12,14–20] but only 1 case was reported in Chinese population.[21] In terms of reported KDM6A variants, KS2 seems more common in Europe (51.1%, 23/45), where the first 3 KS2 patients were identified.

The present study identified a novel de novo variant of the KDM6A gene in a Chinese patient with characteristic facial features by a combination of facial dysmorphology analysis and NGS-based multigene panel approach. The consequence of the variant was analyzed through RNA assay.

Participants and methods

Participants and facial dysmorphology analysis

Genomic DNA of all the participants, including the patient, her parents and 215 non-relative healthy controls matched for ethnicity, were extracted from their peripheral blood samples according to standard procedures. Face2Gene (, a suite of phenotyping application that contributed to genetic disease evaluation, was used for facial dysmorphology analysis. A cluster of syndromes was suggested after information of the proband (her photo, date of visit, date of birth, gender, height, weight, etc.) were filled out online.

All the participants were recruited from the outpatient department of the International Peace Maternity and Child Health Hospital (IPMCH), Shanghai Jiao Tong University School of Medicine, China. This study was approved by the Ethics Review Committee of IPMCH (No. GKLW2017–149) and performed according to the Declaration of Helsinki. Written informed consent was provided by legal guardians of the KS patient and other participants.

Targeted sequencing and data analysis

Targeted capture of 2181 genes and NGS had been described in detail previously.[22,23] The genomic DNA was isolated from peripheral blood sample and sheared into segments in size from 200 to 250 bp. Sequencing of the captured target regions was performed on Illumina HiSeq2500 Analyzers (Illumina, San Diego, CA, USA). Alignment against the human reference genome hg19/GRCh37 was performed with Burrows–Wheeler Aligner (BWA; version 0.7.12) ([24] Variant calling and annotation were achieved using the Genome Analysis Toolkit (GATK; version 3.5) ( and ANNOVAR,[25] respectively. The resulting variants were prioritized with our in-house developed pipeline (MultiOmics One).[23] The interpretation and classification of variants were based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Guideline.[26]

A total of 3.88 Gb raw data were obtained with an average coverage of 189.04× and 97.3% of bases in the defined target reached the coverage of at least 10×. Subsequent variants were filtered using our in-house MultiOmics One pipeline and intersected with genes associated with the suggested syndromes according to Face2Gene analysis.

Reverse-transcription PCR (RT-PCR), Sanger sequencing and identity testing

RNA extracted from peripheral blood mononuclear cells of the proband and a healthy female control were reversed to cDNA with a PrimeScript™ RT reagent Kit with gDNA Eraser (NO. RR047Q, Takara, Japan). Nonsense-mediated mRNA decay (NMD) was subsequently detected by polymerase chain reaction (PCR) amplification and Sanger sequencing following the standard methods. Primer sequences for verifying KDM6A variant were designed with the Primer 3 Web ([27] Sequence variant was named based on the current Human Genome Variation Society nomenclature (version 15.11) ([28] and checked with the Mutalyzer ([29] The short tandem repeat (STR) typing method was applied for identity testing, as described previously.[23] All primers were listed in supplemental materials (Additional Table 1,


Clinical features of the patient

The patient, a Chinese 24-year-old married woman, came to the outpatient department of IPMCH for pre-pregnancy consultation (Fig. 1A, patient II-1). She had moderate learning difficulties, short stature (140 cm, <1st centile) and skeletal malformations including brachydactyly and hip dysplasia, leading to unsteady walking with unequal legs. She presented with characteristic facial features, including arched and laterally sparse eyebrows, long palpebral fissure, left eye exotropia, eversion of lateral third lower eyelid, long eyelashes, depressed tip of nose with short columella, and dental anomalies (large upper incisors, irregular teeth and malocclusion) (Fig. 1B–E). Her parents and husband, showed in the pedigree chart, were normal in these physical examinations and they denied similar patients in their family.

Figure 1:
Pedigree and clinical features of the Kabuki syndrome patient. (A) The patient (II-1) for pre-pregnancy consultation and her family members. P means the Kabuki syndrome patient. (B) and (C) Facial examination showed arched and laterally sparse eyebrows, long palpebral fissure, left eye exotropia, eversion of lateral third lower eyelid, long eyelashes, depressed tip of nose with short columella and dental anomalies, including large upper incisors, irregular teeth and malocclusion. (D) and (E) The patient is brachydactylic without long halluces.

Candidate syndromes revealed by facial dysmorphology analysis

Considering characteristic facial features of the patient, the phenotyping application Face2Gene was applied to match the molecularly confirmed syndromes by analyzing facial features of the proband. The top four syndromes suggested by Face2Gene were KS, Mucopolysaccharidoses, Prader-Willi syndrome and odontoonychodermal dysplasia (Fig. 2A). Remarkably, the proband has a high gestalt similarity to composite public photos of KS patients (Fig. 2B).

Figure 2:
Facial dysmorphology analysis with Face2Gene. (A) Top four syndromes, matching to phenotypes of the proband, were suggested by Face2Gene. (B) A high gestalt similarity between the proband and composite public photos of Kabuki syndrome patients.

Identification of a novel KDM6A pathogenic variant using NGS-based targeted sequencing

The proband was heterozygous for a novel nonsense variant NM_021140.2(KDM6A):c.3521G>A, predicting to introduce a premature stop codon (p.Trp1174∗) of the KDM6A protein (Fig. 3A).

Figure 3:
Identification of a novel pathogenic KDM6A variant in the KS patient. (A) Intergative Genomics Viewer snapshot of sequence data at KDM6A for the proband, and the corresponding calls made from the data. (B) Sanger sequencing indicates a heterozygous substitution of G with A in KDM6A compared with wild type, which induces a premature termination codon (p.Trp1174∗). (C) Sequencing of KDM6A cDNA products showed only the wild-type KDM6A transcripts of the proband, suggesting that the corresponding variant transcript underwent nonsense-mediated mRNA decay. KDM6A = lysine-specific demethylase 6A, KS=Kabuki syndrome type 2.

Sanger sequencing and variant classification

The result of Sanger sequencing was consistent with NGS, validating the presence of NM_021140.2(KDM6A):c.3521G>A heterozygous variant in the proband (Fig. 3B). The variant was not detected in her parents. Furthermore, identity testing, using short tandem repeat (STR) loci and amelogenin, confirmed the de novo characteristics (Additional Table 2, The variant was also excluded in 1000 Genomes Project (, Exome Aggregation Consortium (ExAC;, database of human single-nucleotide polymorphisms (dbSNP;, as well as 215 unrelated controls.

The nonsense variant in exon 24 was predicted to lead to a premature stop codon (p.Trp1174∗), which might result in the disruption of Jumonji C domain, the most functionally important part of KDM6A in catalyzing the demethylation of H3K27.[30] More importantly, the variant was not located in the last two exons, suggesting the corresponding variant transcript could undergo the NMD.[31]

RT-PCR analysis of KDM6A mRNA

To validate our hypothesis, RT-PCR analysis of RNA extracted from peripheral blood mononuclear cells of the proband and a healthy female control was performed with the primers in exon 22 and exon 27 (Additional Table 1, cDNA sequencing only showed the wild-type KDM6A transcripts of the proband (Fig. 3C), suggesting monoallelic expression of the wild-type allele and NMD of the variant KDM6A transcripts. Together, this variant can be categorized to pathogenic variant according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guideline[26] using pathogenicity very strong and pathogenicity strong evidence.


The integrated phenotype and genotype-based prioritization strategy has been shown to be highly effective for the final diagnosis of KS.[32] However, performance of this strategy also requires detailed phenotypes and clinical experience. Facial dysmorphology technology is becoming more accessible and may help physicians narrow down candidate genetic diagnosis behind disorders associated with characteristic facial features. The Face2Gene has been limited to be applied in an Asian population. Recently, a study indicated ethnic background of the patient could influence the evaluation.[33] In this study, Face2Gene was used to match the genetically confirmed syndromes by analyzing facial features of the proband. Notably, KS was prioritized at the top of suggested syndromes list, consistent with the final molecular diagnosis, which might reflect the face of the KS patient is significantly different from the average, general population face.

KMT2D and KDM6A are the only two genes known to cause KS, among which KDM6A variants account for 3% to 8% of KS cases.[34] A recent study demonstrated that clinical manifestations of two subtypes of KS, KMT2D associated KS1 and KDM6A associated KS2, were so similar that it was hard to distinguish between them.[12] The most evident distinction between KS1 and KS2 is that patients with KDM6A pathogenic variants tend to have sex-specific phenotypic differences, that is, male patients showed much more severe intellectual disability and developmental delay than females.[5,12,16] It is especially evident in the three-generation KS2 family, two inherited sons with severe phenotypes, and their mother and maternal grandmother with attenuated phenotypes.[14] For our patient, the 24-year-old female presented with mild learning difficulties besides a typical face, short stature, and hip dysplasia. She was not aware of the aforementioned features until she considered having a baby, consistent with the previous findings that female patients were mild in intellectual manifestation. Besides, the long halluces, large central incisors and hypertrichosis features in adults and hypoglycemia in children have been more frequently described in KS2 and served as good indicators of KDM6A variants.[5,12] However, only the large central incisors were found in our patient.

To date, only 45 KS2 cases have been reported all over the world, of them 23 (51.1%) patients are from Europe, 11 (24.4%) from Asia, 5 (11.1%) from America, 1 (2.2%) from Australia and 5 (11.1%) cases lack nationality information (Additional Table 3, With regard to updated KS2 cases, a large proportion of KS type 2 patients were from Europe. Nevertheless, disparity in techniques and awareness of KS2 between countries should be taken into account for further prediction of the ethnic or racial predilection of KS2.

The evidence for genotype-phenotype correlations between KDM6A variants and clinical severity is ambiguous with respect to the limited reported variants of KDM6A. Recently, Bögershausen et al[12] indicated that KDM6A splice-site variants are the most common variant type, whereas the updated published variants combined with our identified case showed an equality between the number of nonsense variants and splice-site variants in total (Fig. 4A). It is found that a large proportion of KDM6A variants distributed across exons 16 to 29, which is the same as the observation of Bögershausen et al.[12] Variants in exon 6 are the most frequently observed among these single-exon-involved variants, leaving out variants in exon 17, which contained a three-generation family with four patients (Fig. 4B), suggesting a variant hot spot may locate in exon 6. However, approximately 30% of patients with KS-like phenotypes are negative for KDM6A and KMT2D, implying that there may be other candidate genes associated with KS. Genes similar to KDM6A and/or KMT2D in function or biological processes would be more likely to be new candidate genes for further consideration.

Figure 4:
Exon distribution and variant types of KDM6A. (A) Variant types of all disease-causing variants in KDM6A. Gender information was indicated with different colors. (B) Exon distribution of pathogenic variants in KDM6A. Variants involving more than one exon were excluded. KDM6A = lysine-specific demethylase 6A.

KDM6A is able to escape X-inactivation, and haploinsufficiency of KDM6A is likely to be the pathogenic mechanism of KS2.[17] Thus, a pathogenic variant of KDM6A in females, on either the inactive or the active X chromosome, would be disease-causing. For our patient, the risk for an affected son or daughter is 50%. The successful genetic diagnosis of the proband thus could provide an opportunity for prenatal diagnosis of the family.

In conclusion, we reported a novel de novo KDM6A pathogenic variant associated with KS via integrated facial analysis and targeted sequencing. The novel pathogenic variant is the first KDM6A point variant identified in Chinese patients. Moreover, our results provide evidence that the integrated facial dysmorphology analysis and multigene panel sequencing approach is valuable for the diagnosis of the clinical and genetic variability in KS.



Author contributions

WS, JZ, SC, HF, YC, SL, CC and LZ carried out the experiments; JZ and HH analyzed the data; JZ made the figures; WS and JZ drafted and CX revised the paper. All authors approved the final version of the paper.

Financial support

This work was supported by the National Natural Science Foundation of China (No. 81471506, 81401219, 81501276, and 81771638), the National Key Research and Development Program of China (No. 2016YFC0905103), the Shanghai Municipal Commission of Science and Technology Program, China (No. 15411966700, 15411964000, and 17411972900), the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai, China (No. 2018YQ39), the Shanghai Municipal Commission of Health and Family Planning Program, China (No. 20154Y0039 and 15GWZK0701), the Shanghai Jiao Tong University Program, China (No. YG2017MS39), and the Innovation Foundation of Translational Medicine of Shanghai Jiao Tong University School of Medicine, Shanghai SJTUSM Biobank, China (No. 15ZH4011).

Conflicts of interest

The authors declare that they have no conflicts of interest.

Institutional review board statement

This study was approved by the Ethics Review Committee of IPMCH (No. GKLW2017–149) and performed according to the Declaration of Helsinki.


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facial analysis; Kabuki syndrome type 2; RNA assay; targeted sequencing

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