Introduction

Meis homeobox 2 (MEIS2; also known as MRG1, NM_170677.4) encodes a homeobox (HOX) protein belonging to the three amino acid loop extension (TALE) superfamily. TALE homeobox proteins are highly conserved transcription factors.1 MEIS functions as a HOX co-factor that cooperatively binds deoxyribonucleic acid (DNA) to pre-B cell leukemia transcription factor (PBX) and/or HOX proteins to increase the affinity and specificity of their DNA binding activity.2, 3 In mice, Meis2 is expressed in embryonic neural crest cells (multipotent and migratory cells), particularly in the heart, central nervous system, upper and lower jaw, and intestinal tract.4 Meis2 null mice are embryonic lethal at E13.5–14.5, with defects of craniofacial cartilage and heart derived from neural crest cells, which is also observed in knockdown zebrafish.4, 5, 6 The detailed phenotype of heterozygous knockout mice is unknown. Human MEIS2 is highly expressed in fetal and adult brain and lymphoid organs including spleen, lymph node, thymus and appendix.7, 8 Further, MEIS2 mutations in humans may lead to cardiac septal defects, cleft palate and intellectual disability (ID), as observed with MEIS2 haploinsufficiency in six sporadic and one familial deletion cases (123 kb–5.6 Mb), and in another family with a 58-kb duplication that results in a frame shift mutation.9, 10, 11, 12 Only one MEIS2 mutation at the nucleotide level has been found in a female patient: c.998_1000del (p.Arg333del).13 This 3-bp deletion at the homeodomain may interfere with DNA binding. Moreover, this female patient also presented with various anomalous features and developmental abnormalities. Here, we report on another MEIS2 mutation case of specific clinical features and discuss the clinical consequence of MEIS2 mutations.

Case Report

The patient is the first child of non-consanguineous healthy French parents. She was born after a full-term uneventful pregnancy. Her birth length was 46 cm (−2.5SD), weight 3270 g (mean) and head circumference 33.5 cm (−1SD). Cleft palate, atrial septal defect (ASD) and ventricular septal defect (VSD) were noted at birth. ASD and VSD were surgically corrected 2 weeks after birth, and her cleft palate was operated on twice (at 7 and 23 months). She presented with mild morphological features including a large forehead, mild trigonocephaly, sparse eyebrow, deeply set eyes, large and low-set ears, full cheeks and thin upper lip vermilion (Figures 1a–c). She had a serious feeding difficulty with severe gastro-esophageal reflux requiring gastrostomy at 3 months. Her motor development was delayed: sitting at 11 months and independent gait at 2 years 9 months. Severe hypermetropia was also noticed. At 2 years 9 months, her height was 95 cm (+1SD), weight 12.5 kg (mean) and head circumference 46.8 cm (−1.5SD). Her speech was delayed with only a few words at 2 years 9 months and severe constipation was noted. At 18 months, brain magnetic resonance imaging and comparative genomic hybridization array (Agilent 60k, Agilent Technologies, Santa Clara, CA, USA) were all normal.

Figure 1
figure 1

Patient’s facial features and MEIS2 mutation. Facial photographs at 13 months (a) and 20 months (b and c). (d) Electoropherograms for the c.611C>G (p.Ser204*) mutation in MEIS2. Arrow indicates the mutation. (e) Schematic representation of MEIS2 protein with its domain and the mutation. Open and filled circles indicate a previously reported mutation and the current mutation, respectively. The protein contains a N-terminal of Homeobox Meis and PKNOX1 (Meis_PKNOX_N) domain and a homeobox (HOX) domain predicted by SMART (http://smart.embl.de/). A full color version of this figure is available at the Journal of Human Genetics journal online.

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Materials and methods

Whole-exome sequencing was performed for the patient only. Blood leukocytes were obtained from the patient and her parents with their informed consent. Genomic DNA was extracted by standard methods, partitioned using the SureSelect Human All Exon v5 Kit (Agilent Technologies), and sequenced by Illumina HiSeq2000 (Illumina, San Diego, CA, USA) with 101-bp paired-end reads. Variants were selected by excluding synonymous variants and variants registered in dbSNP137, our in-house exome database of 575 Japanese individuals, and the NHLBI Exome Sequencing Project (ESP6500, http://evs.gs.washington.edu/EVS/) (MAF≧0.01). The remaining variants were then evaluated depending on whether they fitted an X-linked or autosomal dominant model (de novo occurrence), and were found in the known mutant genes registered in the Human Gene Mutation database Professional (http://www.biobase-international.com/product/hgmd). Candidate variants were confirmed by Sanger sequencing. Parentage was tested using nine microsatellite markers with Gene Mapper software v4.1.1 (Life Technologies Inc., Carlsbad, CA, USA). This study was approved by the institutional review board of Yokohama City University School of Medicine.

Results and Discussion

Mean whole-exome sequencing read depth in protein coding regions of RefSeq was 102 × , with 93.9% of target regions covered by 20 reads or more. Through our variant selection, we successfully narrowed down the variants to a heterozygous nonsense MEIS2 mutation (c.611C>G, p.Ser204*). In addition, using Sanger sequencing we confirmed that the mutation occurred de novo with no discrepant parentage (Figure 1d).

Similar to patients with MEIS2 haploinsufficiency, two patients possessing MEIS2 point mutations commonly show cleft palate, cardiac septal defect and ID (Table 1).9, 10, 11, 12, 13 Therefore, we reconfirm these clinical features as the MEIS2 alteration phenotype. In addition, severe gastro-esophageal reflux requiring gastrostomy was observed commonly in both patients with a MEIS2 mutation (Table 1).13 MEIS proteins play an important role in neural crest cell development.2, 4 Neural crest cell-related diseases are collected under the term “neurocristopathy”, and include the 22q11.2 deletion syndrome, CHARGE syndrome and Hirschsprung disease, which is a congenital intestinal motility disorder.14, 15 Like MEIS2 alterations, digestive symptoms (gastro-esophageal reflux, esophagitis or chronic constipation) are observed in 24% (50/211) of patients with 22q11.2 deletions,16 and gastro-esophageal reflux is observed in 89% (32/36) of patients with CHARGE syndrome.17 Therefore, digestive symptoms may be a part of the phenotype caused by a loss-of-function mutation in MEIS2, and may result from neuronal dysfunction and/or abnormal number of neurons as seen in the neurocristopathy.15, 17 Nevertheless, to fully understand the disease accumulation of patients with MEIS2 aberrations is highly encouraged.

Table 1 Clinical features of patients with MEIS2 alterations
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In summary, we have identified a novel nonsense MEIS2 mutation in a female patient with cleft palate, cardiac septal defect, severe ID, developmental delay and feeding difficulty with gastro-esophageal reflux. Our findings strongly suggest neurocristopathy be included in the clinical features associated with MEIS2 alterations.