Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

Abstract

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.

Figure 1.

Analysis of Family A (Iranian family): (A) Pedigree. Black-shaded symbols indicate affected individuals (IV:1, IV:4, IV:6 and IV:9). (B) Photos of affected individuals: from left to right: IV:1, IV:4, IV:6 and IV:9. (C) Homozygosity mapping data analysis indicates peaks (LOD = 3.0) on chromosomes 2 and 13. (D) Electropherograms from Sanger confirmation in family members showing NM_006895.2 (HNMT): c.179G>A; p.Gly60Asp WT, heterozygous and homozygous sequence. (E) In silico modeling of p.Leu208Pro within HNMT predicted protein structure for p.Gly60Asp WT and mutant using PDB file 2AOT and Pymol software. The red arrow indicates the location of residue 60 within the protein.

Figure 2.

Analysis of Family B (Kurdish family): (A) Pedigree. Black-shaded symbols indicate affected individuals (III:1, III:3 and III:4). (B) Photos of affected individuals: from left to right: III:1, III:3 and III:4. (C) Homozygosity mapping data analysis indicates peaks (LOD = 2.4) including a large interval (30 Mb) on chromosome 2. (D) Electropherograms from Sanger confirmation in family members showing NM_006895.2 (HNMT):c.632T>C; p.Leu208Pro WT, heterozygous and homozygous sequence. (E) In silico modeling of p.Leu208Pro within HNMT. Modeling was performed with PDB file 2AOT and PyMol software. The red arrow indicates the location of residue 208 within the protein.

Figure 3.

Three-dimensional Protein structure and ClustalW2 analysis: structures of HNMT (pdb 1jqd) at the catalytic domain for (A) Gly60, (B) Asp60, with HA in yellow and S-adenosyl homocysteine (SAH) in pink, and Gly60 and Asp60 in light pink, and (C) ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) alignment/comparison of HNMT across vertebrate species showing conservation at Gly60Asp (highlighted in pink). Structures at the hydrophobic pocket around residue 208 (labeled pink) for (D) Leu208 and (E) Pro208. Leu155, Leu204, Leu211 and Leu213 were labeled in gray. Ile288 and Ile290 were labeled in yellow. Tyr215 and Gly212 were labeled in blue. (F) ClustalW2 alignment of HNMT for the hydrophobic pocket surrounding Leu208, with Leu208 highlighted in pink, Leu155, Leu204, Leu211 and Leu213 labeled in gray, Ile288 and Ile290 labeled in yellow. Tyr215 and Gly212 labeled in blue. Sequences used for the HNMT alignment included human (NP_008826.1), mouse (NP_536710.1), opossum (from N-SCAN and Genscan gene predictions, UCSC browser), chicken (NP_001264802.1), Xenopus laevis (NP_001080614.1), zebrafish (NP_001003636.1), Tetraodon nigroviridis (Q4SBY6.1), lancelet (Branchiostoma floridae: predicted from mRNA XM_002613293.1) and sea urchin (Strongylocentrotus purpuratus: from mRNAs CX698504, CD312314 and CX689147).

Figure 4.

Cell viability using the MTT assay. Lymphoblast cells from 4 patients from Family A and 12 healthy unrelated controls were treated with 125 µM concentration of HA for 2 h. Reconstituted MTT in an amount equal to 10% of the culture medium volume was added to the cells. The plate was read at 570 nm. Values are mean ± SD for three independent experiments, and averaged across all the individuals per group.

Figure 5.

Thermal stability of HNMT (WT) and HNMT (Gly60Asp) using Thermofluor^®. The experiment was performed by mixing 5 μl of 1 mg/mL of HNMT (WT) or HNMT (Gly60Asp) with 5 μl 200-fold diluted SYPRO Orange and a buffer containing 50 mm Tris–HCl and 100 mm NaCl, pH 8.0, in a 98-well RT-PCR plate. Reaction mixtures were heated at 0.5°C/min from 20 to 95°C. A plot between d(fluorescence)/dT versus temperature of HNMT (WT) (red line) and HNMT (Gly60Asp) (blue line) is shown. A melting temperature of 62.3 ± 1.7 and 58.3 ± 1.1°C for WT and the Gly60Asp variant, respectively, was obtained.

Figure 6.

Isothermal calorimetric titration of HNMT (WT) and Gly60Asp variant with SAM and HA. The experiments for HNMT (WT) consisted of 20 consecutive injections of 15 μl of 0.42 mm SAM into 30 μm HMNT (WT) at 25°C. (A) The released heats of injection after baseline correction by subtracting the heat of the reference measurement. (B) Integrated data and data analysis using non-linear least square fitting in Origin 7. The dissociation constant of HNMT (WT) for SAM was 49.2 ± 2.0 μm. (C) Released heats of 20 consecutive injections of 15 μl of 2 mm SAM into 30 μm HNMT (Gly60Asp) at 25°C.

Figure 7.

Catalytic activity of HNMT (WT) and HNMT (Gly60Asp) as determined by isothermal titration calorimetry. (A) Raw calorimetric data for the methylation of HA catalyzed by HNMT (WT) using SAM as a methyl group donor in 50 mm Tris–HCl buffer, pH 8.0, containing 100 mm NaCl at 25°C. The experiment was performed by injection of 2 mm HA (2 μl/injection; 20 injections). The cell contained 1 μm HNMT (WT) and 100 μm SAM. A Michaelis–Menten plot for methylation by HNMT (WT) is shown in the inset of A. (B) Raw calorimetric data for the reaction with the Gly60Asp variant (same conditions as for WT).