1 INTRODUCTION

When a de novo balanced reciprocal translocation is identified, the disruption of haploinsufficiency-sensitive gene(s) in the proximity of the breakpoints is likely to be responsible. Whether the phenotypic features of the patient are similar to those of patients with truncating variants of such gene(s) disrupted by the translocation should be evaluated. However, precise mapping and the accurate definition of breakpoints have been difficult and time-consuming tasks until the recent availability of long-read sequencers.¹

Here, we report a 3-year-old patient with developmental delay, autism spectrum disorder, and distinctive facial features who had an apparently balanced translocation between chromosome 3q26 and chromosome 7q36. Nanopore long-read sequencing allowed us to document that the balanced translocation disrupted the KMT2C gene, the haploinsufficiency of which leads to Kleefstra syndrome 2 (OMIM #617768) characterized by delayed psychomotor development, variable intellectual disability, and mild dysmorphic features.

The KMT2C gene is highly expressed in the brain and encodes histone-lysine N-methyltransferase 2C, which monomethylates histone H3 lysine 4 (H3K4me1), altering the chromatin structure and causing transcriptional activation that controls gene transcription.² The haploinsufficiency of genes encoding regulators of H3K4 methylation, including KMT2A and KMT2D, leads to genetic syndromes including Wiedemann–Steiner syndrome (MIM #159555) and Kabuki syndrome (MIM #602113), both of which are characterized by intellectual disability.

2 MATERIALS AND METHODS

The patient was a 3-year-old male with developmental delay and dysmorphic facial features including a flattened midface, prominent eyebrows, a high palate, a short nose, and down-slanting palpebral fissures. He was born at 38 weeks of gestation to non-consanguineous couples with no relevant family history. His birthweight was 2840 g (−0.1 SD), his body length was 48.0 cm (−0.2 SD), and his head circumference was 32.0 cm (−0.8 SD). He was able to roll over at 14 months, sat without support at 16 months, and walked independently at 2 years and 4 months. His speech development was also delayed. He was able to express meaningful words at 2 years and 11 months. Sensory sensitivity, picky eating, hyperactivity, and restlessness were also observed. His full-scale intelligence quotient on the Tanaka–Binet intelligence test V score was 54, and he was diagnosed as having intellectual disability. His M-CHAT (Modified Checklist for Autism in Toddlers) score was 5, and he was diagnosed as having autism.

3 RESULTS

After obtaining ethical approval from an institutional review board and informed consent from the patient's guardians, we conducted the molecular study. A blood chromosome analysis by G-banding showed a balanced translocation, t(3;7)(q26.2;q36.1) (Figure 1A). Blood chromosome analyses of the parents were normal. DNA was extracted from the peripheral blood samples by phenol extraction method. Array-CGH testing was performed as described previously.¹ No deletion was detected at 3q26.2 or 7q36.1 but a 5.9-Mb deletion of 11q22.3 was present: Arr[GRCh37]11q22.3(104372190_110262427x1) (Figure 1B). Three genes, namely, GUCY1A2, CUL5, ZC3H12C, with pLI scores of greater than 0.9 were found to reside within the deleted interval of chromosome 11. None of the three genes is known to cause any disease in humans. Human disease-causative genes within the deletion range on chromosome 11 included GRIA4 (OMIM #617864), which causes neurodevelopmental disorder with or without seizures, and gait abnormalities. However, the pLI score of GRIA4 is as low as 0.02, indicating that GRIA4 is unlikely to be haplo-sensitive and that deletion of GRIA4 would have contribute to the patient's symptoms.

The breakpoints were precisely defined by the whole genome long-read sequencing according to a standard protocol as described previously (PromethION system from Oxford Nanopore technologies).¹ We obtained 12 352 829 reads with a total read length of 59 695 286 358 (average read length 4833) using one PromethION flow cell. Base calls were achieved by Guppy 4.0.11. Mapping and alignment to the human reference genome (GRCh37) were achieved by NGMLR. Structural variants were detected by Sniffles2. Analysis using Sniffles2 revealed thar a total of 32 208 structural variants in the proband. Of the 32 208 variants, 20 006 had allele frequencies of less than 0.05. We further filtered out variants in the intergenic regions, regions upstream and downstream of the transcriptional start/end, and kept de novo variants mapped to chromosome 3 or 7 and supported by 10 or more reads. Only one variant, a translocation spanning chromosome 3 and 7, remained. The sequence reads mapped to the proximity of the breakpoints were aligned and merged into a consensus sequence, as described previously,¹ using the dnarrange software package.³ The resultant order and orientation of the rearrangements were represented as a dot plot (Figure S1). The exact breakpoint was confirmed by Sanger sequencing using primers designed to include the estimated breakpoint. The breakpoint of the t(3;7)(q26.2;q36.1) reciprocal translocation was found to be at chr3(GRCh37):g.170183554 on the SLC7A14 gene, and at chr7 (GRCh37):g.151857653 on the KMT2C gene (Figure 1C).

4 DISCUSSION

Here, we report a patient with an apparently balanced translocation t(3;7)(q26.2;q36.1) involving the KMT2C gene and developmental delay, autism spectrum disorder, short stature, microcephaly, and some dysmorphic features. Most, if not all of the clinical features could be ascribed to haploinsufficiency of the KMT2C gene. On the other hand, the SLC7A14 gene, located at the break of chromosome 3, is the causative gene for retinitis pigmentosa 68, an autosomal recessive inheritance form of the disease.

Phenotypic comparisons between this patient and previously reported patient with KMT2C pathogenic variants showed variability in the physical characteristics, but intellectual disability was inevitable to varying degrees and autistic symptoms were frequent (Table S1).^{2, 4-7} In view of these rather nonspecific dysmorphic features of Kleefstra syndrome 2, as reviewed in this study, the contribution of KMT2C disruption would never have been confirmed on clinical grounds alone without the genomic analysis.

A de novo balanced translocation in a symptomatic patient is presumed to have a disease-causing gene at the translocation breakpoint. In the analysis of the structural abnormalities at breakpoints, it is known that conventional short-read sequencer is inadequate for analysis, while long-read sequencing enables more accurate detection of structural changes in a wide range of chromosome regions.

The presently reported patient illustrates the value of long-read sequencing in elucidating the precise genetic etiology of patients with relatively nonspecific intellectual disabilities and chromosomal translocation that are beyond the resolution of conventional G-band chromosome analysis and array-CGH testing.

AUTHOR CONTRIBUTIONS

Mamiko Yamada and Kenjiro Kosaki performed the clinical picture evaluation. Kiyotaka Kosugiyama, Takeshi Ujiie, and Hidefumi Tonoki performed the clinical evaluation of the patient. Mamiko Yamada, Hisato Suzuki, Fuyuki Miya, and Kenjiro Kosaki performed the genetic analysis and interpretated the results. Mamiko Yamada wrote the original draft; all the authors revised the final manuscript. Kenjiro Kosaki supervised the work. Mamiko Yamada and Kenjiro Kosaki financed the work.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.