Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome

Abstract

A clinically recognizable 9q subtelomeric deletion syndrome has recently been established. Common features seen in these patients are severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, cupid bow or tented upper lip, everted lower lip, prognathism, macroglossia, conotruncal heart defects, and behavioral problems. The minimal critical region responsible for this 9q subtelomeric deletion (9q-) syndrome has been estimated to be <1 Mb and comprises the euchromatin histone methyl transferase 1 gene (EHMT1). Previous studies suggested that haploinsufficiency for EHMT1 is causative for 9q subtelomeric deletion syndrome. We have performed a comprehensive mutation analysis of the EHMT1 gene in 23 patients with clinical presentations reminiscent of 9q subtelomeric deletion syndrome. This analysis revealed three additional microdeletions that comprise the EHMT1 gene, including one interstitial deletion that reduces the critical region for this syndrome. Most importantly, we identified two de novo mutations--a nonsense mutation and a frameshift mutation--in the EHMT1 gene in patients with a typical 9q- phenotype. These results establish that haploinsufficiency of EHMT1 is causative for 9q subtelomeric deletion syndrome.

Figure 1.

Deletions of chromosome 9q34.3 identified in individuals with 9q subtelomeric deletion syndrome. The top bar is the genomic organization of the relevant region in 9q34.3. The sizes and orientations of the known and predicted genes are indicated by gray arrows. The sizes of the deletions reported elsewhere (L1–L3) and the new deletions identified in this study (P1–P3) are represented by the bars. Gray lines are deleted regions, and the thick bars represent the regions with normal copy numbers. The intervening regions are indicated by black lines. Deletions reported elsewhere are redrawn from Stewart et al. (L1), Harada et al. (L2), and Yatsenko et al. (L3). A large deletion of at least 1 Mb, with the proximal border between the tMRPL41 gene and the TRAF2 gene, was identified in patient P1. The deletion in P2 is similar to the critical region indicated by L3. P3 has a smaller interstitial deletion. The size of the deletion was determined by FISH analysis and MLPA (lower panel). Unblackened boxes indicate probes located in exons or introns (i) with reduced (∼50%) signal intensity, whereas the blackened boxes represent probes with normal intensities.

Figure 2.

Mutations in the EHMT1 gene identified in P4 and P5. A, De novo nonsense mutation identified in exon 24 of P4, which predicts premature stop codon R1137X. B, Deletion of 13 bp identified in exon 8 of P5. The deletion is indicated by the box. The endpoints of the deletion carry the same AGAC tetranucleotide sequence (underlined), which suggests that it has arisen as a result of a replication error. Top chromatogram is the sequence from a control individual; P5-S and P5-AS are the sense and antisense (reverse-complement) sequences from patient 5. C, Agarose gel showing that the 13-bp deletion has also occurred de novo. Sizes of the PCR products on the agarose gel: 324-bp normal exon 8 PCR product and 311-bp mutant product. Lane C is a control sample. D, Gene structure of the EHMT1 gene and protein domain structure of the three predicted isoforms that result from alternative splicing. Isoform 1 is the canonical Eu-HMTase1 protein, which has eight ankyrin repeats, a pre-SET domain, and a SET domain. Isoform 2 lacks most of these recognizable C-terminal protein domains because of the use of an alternative exon 15. Isoform 3 is produced by the use of two alternative last exons. As a consequence, this isoform has an incomplete SET domain.

Figure 3.

A, Patient 1 at age 8 years. B, Patient 2 at age 1 year and 6 mo. C, Patient 3 at different ages: neonate (top left), 11 years (top right), and 36 years (bottom panels). D, Patient 4 at different ages: 6 mo (top left), 18 mo (top right), and 4 years (bottom panels). E, Patient 5 at age 16 years. Note the characteristic facial dysmorphisms in all the patients. Written consent to publish these photographs was obtained from the parents of each patient.