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ORIGINAL ARTICLE
Mutations in the histamine N-methyltransferase gene,
HNMT, are associated with nonsyndromic autosomal
recessive intellectual disability
Abolfazl Heidari1,2, Chanakan Tongsook4,†, Reza Najafipour2,†, Luciana
Musante5, Nasim Vasli1, Masoud Garshasbi5,6, Hao Hu5, Kirti Mittal1, Amy J. M.
McNaughton7, Kumudesh Sritharan1, Melissa Hudson7, Henning Stehr9, Saeid
Talebi10, Mohammad Moradi2, Hossein Darvish11, Muhammad Arshad Rafiq1,
Hossein Mozhdehipanah3, Ali Rashidinejad12, Shahram Samiei13, Mohsen
Ghadami14, Christian Windpassinger15, Gabriele Gillessen-Kaesbach16,
Andreas Tzschach5,‡, Iltaf Ahmed1,17,AnnaMikhailov
1, D. James Stavropoulos18,
Melissa T. Carter19, Soraya Keshavarz2,MuhammadAyub
8,
Hossein Najmabadi20,21,XudongLiu
7, Hans Hilger Ropers5,
Peter Macheroux4and John B. Vincent1,22,23,*
1
Molecular Neuropsychiatry and Development (MiND) Lab, The Campbell Family Mental Health Research
Institute, Centre for Addiction and Mental Health (CAMH), Toronto, ON, Canada M5T 1R8,
2
Cellular and Molecular
Research Center,
3
Department of Neurology, Bou Ali Sina Hospital, Qazvin University of Medical Sciences, Qazvin
34197/59811, Iran,
4
Institute of Biochemistry, Graz University of Technology, Graz 8010, Austria,
5
Max Planck
Institute of Molecular Genetics, Berlin D-14195, Germany,
6
Department of Medical Genetics, Faculty of Medical
Sciences, Tarbiat Modares University, Tehran 14117-13116, Iran,
7
Ongwanada Genomics Lab,
8
Division of
Developmental Disabilities, Department of Psychiatry, Queen’s University, Kingston, ON, Canada K7L7X3,
9
Department of Medicine, Stanford University, Stanford, CA 94305-5101, USA,
10
Department of Medical Genetics,
Medical University of Tehran, Tehran 14167-53955, Iran,
11
Department of Medical Genetics, Shahid Beheshti
University of Medical Sciences, Tehran 4739, Iran,
12
Maternal, Fetal and Neonatal Research Center, Tehran
University of Medical Sciences, Tehran 1419733141, Iran,
13
Blood Transfusion Research Center, Tehran
1449613111, Iran,
14
Department of Medical Genetics, Tehran University of Medical Sciences, Tehran 1417613151,
Iran,
15
Institute of Human Genetics, Medical University of Graz, Graz 8010, Austria,
16
Institut für Humangenetik,
Universität zu Lübeck, Lübeck 23562, Germany,
17
Atta-ur-Rehman School of Applied Biosciences, National
University of Sciences and Technology, H-12, Islamabad, Pakistan,
18
Department of Paediatric Laboratory
Medicine, The Hospital for Sick Children, Toronto, ON, Canada,
19
Division of Clinical and Metabolic Genetics,
Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada,
†
These authors contributed equally.
‡
Present address: Institute of Clinical Genetics, Technische Universität Dresden, 01307 Dresden, Germany.
Received: March 9, 2015. Revised and Accepted: July 13, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Human Molecular Genetics, 2015, 1–14
doi: 10.1093/hmg/ddv286
Advance Access Publication Date: 23 July 2015
Original Article
1
HMG Advance Access published July 29, 2015
20
Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran 19857, Iran,
21
Kariminejad-Najmabadi Pathology and Genetics Center, Tehran 14667, Iran,
22
Department of Psychiatry,
University of Toronto, Toronto, ON, Canada M5T 1R8 and
23
Institute of Medical Science, University of Toronto,
Toronto, ON, Canada M5S 1A8
*To whom correspondence should be addressed at: The Campbell Family Mental Health Research Institute, R-32, Centre for Addiction and Mental Health,
250 College Street, Toronto, ON, Canada M5T 1R8. Tel: +1 416 535 8501; Fax: +1 416 979 4666; Email: john.vincent@camh.ca
Abstract
Histamine (HA) acts as a neurotransmitter in the brain, which participates inthe regulation ofmany 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--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 functionalHNMT 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.
Introduction
Intellectual disability (ID) is a neurodevelopmental disorder,
characterized by considerable limitation of intellectual function-
ing, adaptive behavior, or daily living skills, and with an onset
before 18 years of age. It is one of the most important challenges
in healthcare, with significant life-long socio-economic burden.
ID is genetically heterogeneous and may result from chromo-
somal aberrations, or from either autosomal recessive (AR), auto-
somal dominant, X-linked or mitochondrial mutations. With the
prevalence of ∼1% of children worldwide (1), ID can be divided
into two main groups: nonsyndromic (NS) ID, where it might
present as the sole clinical feature, whereas in syndromic ID add-
itional clinical or dysmorphological features may also be present.
Over the past few years, next-generation sequencing technologies
have led to the identification of a number of ID-associated genes,
emphasizing the considerablegenetic heterogeneityof ID (2). Stud-
ies into the molecular basis of autosomal recessive forms of ID
(ARID) are lagging some way behind studies of X-linked ID, in
part because the larger families needed for gene mapping are
rare in North American and European populations. However, a re-
cent review suggests that ARID is not rare, and in outbred popula-
tions as many as 13–24% of ID may be due to AR genes (2).
Histamine (HA), a biogenic amine, plays a key role in the regu-
lation of gastric acid secretion (3), and is a neurotransmitter in the
central nervous system (CNS) (4). HA is produced and stored in
airway mast cells, basophils and in the synaptic vesicles of HAer-
gic neurons. In response to immune allergens, HA releases from
storage granules and rapidly diffuses into surrounding tissues.
Released HA is rapidly inactivated and disappears from the
bloodstream within minutes [reviewed in Schwartz et al.(4)].
Histamine N-methyltransferase (HNMT; MIM 605238) is a
cytoplasmic protein that belongs to the methyltransferase super-
family and is one of two enzymes involved in the metabolism of
HA. N-methylation catalyzed by HNMT and oxidative deamin-
ation catalyzed by diamine oxidase (DAO; encoded by amino oxi-
dase, copper containing 1 (AOC1)) are the two major pathways for
HA biotransformation in mammals (5,6). HNMT catalyzes the
methylation of HA in the presence of S-adenosylmethionine
(SAM), forming N-tele-methylhistamine (7,8). HNMT is widely ex-
pressed in human tissues; with the greatest expression in kidney
and liver, followed by spleen, colon, prostate, ovary, spinal cord
cells, bronchi and trachea. HNMT is the key enzyme for HA deg-
radation in the bronchial epithelium (9). Since DAO is not ex-
pressed in the CNS, N-methylationthroughHNMTisthemain
pathway responsible for termination of the neurotransmitter ac-
tions of HA in the brain (4).
Here, we report the identification of HNMT as a novel gene re-
sponsible for ID and discuss the consequences of the identified
missense mutations on the protein function.
Results
Family A (Iranian family)
Ascertainment and clinical evaluation
We ascertained a consanguineous family with Turkish back-
ground from the Avaj area within Qazvin province in Iran, in
which the first-cousin parents had nine children, four of them
were affected with NS ID; two males and two females (Fig. 1).
The study was approved by the Research and Ethics Board of Qaz-
vin Medical University and appropriate written informed consent
was obtained from the parents. The affected familymembers were
assessed by an experienced neurologist and standard clinical as-
sessment forms were used todocument the findings. The clinical
descriptions of the patients are summarized in Table 1.Theaf-
fected females showed profound to severe ID and their speech
was limitedto just a few words, whereasin affected males the con-
dition was milder. A mild degree of regression after about 5 years
of age was reported for affected members. The patients did not
have any neurological problems, autistic features, congenital mal-
formations or facial dysmorphisms. Body height, weight and head
circumference were normal in all patients. Wechsler Intelligence
Scales for Children (WISC) were used to assess the IQ in patients.
For patient IV:I, we performed a magnetic resonance imaging scan
which revealed no morphological brain abnormalities.
2|Human Molecular Genetics
Homozygosity-by-descent (HBD) mapping and mutation identification
HBD mapping led to the identification of a 14-Mb autozygous locus
on 2q21.3 (single-nucleotide polymorphisms, SNPs: rs1869829–
rs7573156), and a 3-Mb autozygous within region 13q33.1 (SNPs:
rs1336666–rs1475276) with a significant LOD (logarithm (base 10)
of odds) scoreof 3.13 (Fig. 1C). Additionally, existenceof copy num-
ber variations (CNVs) exclusive to theaffected individualswas also
ruled out. Exome target enrichment was performed within the
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.
Human Molecular Genetics |3
linkage intervals by using a custom Agilent SureSelect array, fol-
lowed by sequencing 8.3 Gb of 101 bp paired-end reads using the
Illumina Genome Analyzer II platform with 100% coverage and
average depth of 202 reads. After filtering the variants with
dbSNP130, and 1000 Genome data, we annotated the remaining
mutations with the RefSeq gene model. Analysis of prospective
changes from the critical regions indicated DNA variants in
three genes: METTL21C [chr13:103346806C>G, NM_001010977.1:
c.43G>C; p.Gly60Arg], ZRANB3 [chr2:136107663T>C, NM_032143.
2:c.482A>G; p.Tyr161Cys] and HNMT [chr2:138727776G>A; NM_
006895.2:c.179G>A; p.Gly60Asp] (coordinates used in hg19). The
evolutionary conservation of the relevant nucleotides, as defined
by the PhyloP44 score and the pathogenicity of these variants, as
predicted by PolyPhen2 and SIFT were calculated (Table 2). Ana-
lysis with Condel—an integrated analysis that uses prediction
using five different algorithms, including PolyPhen2, SIFT and
Mutation Assessor (10), was also performed (Table 2). The co-
segregation pattern of the three variants was checked in the fam-
ily, and only the HNMT variant segregated correctly. The parents
of the patients were both heterozygous for the HNMT missense
variant, which was not found in either in a homozygous or
heterozygous form among 100 unrelated healthy Iranian and
200 Pakistani individuals.
We identified a potentially pathogenic missense mutation
(HNMT c.179G>A [ p.Gly60Asp]) in HNMT (RefSeqNC_000002.11),
which encodes a two-domain protein; MTase, which is the larger
domain, is composed of a seven-stranded β-sheet and is mainly
responsible for interaction with cofactor (SAM) and substrate
(HA), and the S domain of HNMT, which may interact with other
proteins for its in vivo function. The homozygous c.179G>A
(p.Gly60Asp) variant occurs in the conserved MTase region I
(Ile56-Gly64), which is part of the SAM-binding pocket (Fig. 3),
highlighting the role of Gly60 in the interaction of HNMT with
its cofactor. The predicted 3D structure of HNMT appears to be
altered by the substitution (Fig. 1E). This variant was not present
in either dbSNP138, nor the Exome Variant Server, NHLBI GO
Exome Sequencing Project (ESP), Seattle, WA, USA (http://evs.
gs.washington.edu/EVS/; accessed January 2014), nor in 1000
Genomes (http://browser.1000genomes.org/index.html), but was
present in 2 of 125604 alleles inthe Exome Aggregation Consortium
database (ExAC; Cambridge, MA, USA; http://exac.broadinstitute.
org; October 2014). Importantly, it was shown to be absent from a
panel of 200 ethnically matched control chromosomes, as well as
an in-house database of 521 exomes/genomes from unrelated indi-
viduals of Middle Eastern origin. The c.179G>A (p.Gly60Asp) muta-
tion showed complete segregation with the disease in all affected
family members; parents were heterozygous carriers and unaffect-
ed children available for genetic screening were either carriers or
did not harbor the mutation.
A known C-to-T SNP (rs11558538) in HNMT changes the amino
acid at position 105 from threonine to isoleucine, with the fre-
quencies of the Thr105 and Ile105 alleles being ∼90 and 10%, re-
spectively. The Ile105 allele is correlated with diminished HNMT
enzymatic activity, which could result in reduced HA inactivation
and increased sensitivity to this amine (11,12). We checked this
polymorphism in Family A and verified that Ile105 allele is not
present in the patients.
Family B (Kurdish family)
Ascertainment and clinical evaluation
We ascertained a three-generation consanguineous Kurdish
family (G016), originally from Iraq, recruited in Germany. The
first-cousin parents had seven children, three of them presented
Table 1. Clinical and biometric features for the Iranian family (Family A) and the Kurdish family (Family B)
Family A: IV:1 Family A: IV:4 Family A: IV:6 Family A: IV:8 Family B: III:1 Family B: III:3 Family B: III:4
HNMT mutation
(NM_006895.2)
c.179G>A
(p.Gly60Asp)
c.179G>A (p.
Gly60Asp)
c.179G>A
(p.Gly60Asp)
c.179G>A
(p.Gly60Asp)
c.632T>C
(p.Leu208Pro)
c.632T>C
(p.Leu208Pro)
c.632T>C
(p.Leu208Pro)
Gender F M F M M F M
Parental consanguinity First cousins First cousins First cousins First cousins First cousins First cousins First cousins
Ethnic origin Turkish Turkish Turkish Turkish Kurdish Kurdish Kurdish
Age at examination (years) 35 33 31 27 18 15 13
Height (cm) (SD) 151 (−2.5 SD) 172 (−0.8 SD) 153 (−2.2 SD) 170 (−1.2 SD) 165 (−1.8 SD) 151 (−2 SD) 138 (−2.1 SD)
Weight (kg) (SD) 65 (+1.5 SD) 71 (+0.7 SD) 63 (+1.2 SD) 69 (+0.6 SD) 56 (+0.2 SD) 44 (+0.2 SD) 40 (+0.8 SD)
Head circumference (cm) (SD) 55 (Mean) 54.5 (−1.46 SD) 55 (Mean) 54 (−1.46 SD) 54 (−1.5 SD) 52 (−1.7 SD) 52 (−1.7 SD)
ID IQ: 28 (severe) IQ: 49 (moderate) IQ: 25 (severe) IQ: 54 (mild) IQ: 20–34 (severe) IQ: 20–34 (severe) IQ: 20-34 (severe)
Height and head circumference are given in cm; weight in kg. Standard deviation (SD) from mean values is given in parentheses.
4|Human Molecular Genetics
with NS ID; the pedigree is shown in Figure 2. The probands were
examined by experienced clinical geneticists who assessed their
physical and mental status. The study was approved by the local
institutional ethics committee, and appropriate informed con-
sent was obtained from the parents. To exclude chromosomal ab-
normalities, karyotype analysis by G-banding was performed in
all affected individuals; karyotypes were found to be normal.
Clinical descriptions of the patients are summarized in Table 1.
On assessment, the 18-year-old boy (III:1) showed severe ID. He
started to walk at 1 year of age. His speech was severely delayed
and he attendedspecial school for intellectually disabled children.
His sister, III:3, a 15-year-old, was also severely intellectually dis-
abled. She had a normal motor development, but active speech
started at the age of about 2 years and she attended, as with the
older brother, a special school for intellectually disabled children.
The younger affected male (III:4), age 13 years, was more delayed
than his older siblings. The mother reported that at birth the
child was thin and hypotonic. He began walking at 2½ years. He
started to speak at 4–5 years of age. He presented with hyperactive
behavior. Atthe age of 12 years, he was diagnosed with myelodys-
plastic syndrome. The patients did not present with congenital
malformations or facial dysmorphisms. Physical measurements
are also reported in Table 1.
HBD mapping and mutation identification
Analysis of genotypes for the available individuals (III:1, III:2, III:3
and II:1) of family G016 revealed a large interval of autozygosity
on 2q21.2–q24.3. This 30 Mb interval was flanked by the heterozy-
gous SNPs, rs10928469 and rs6705268. Four additional autozygous
loci were identified: a 3.4-Mb interval on 10q26 (rs11244548–
rs4751029), a 1.7-Mb HBD on 11p12–p13 (rs672597–rs10836780), a
12.6-Mb region on 11q22.3–q23.3 (rs10895742–rs521609), and finally
an 8.5-Mb locus in 18q12.1–q12.3 (rs1021598–rs12971263; Fig. 2C).
We performed targeted next-generation sequencing by en-
richment of the exonic regions within the linkage intervals.
After filtering the variants, a single homozygous variant in
HNMT [chr2: 138771444T>C; NM_006895.2:c.623T>C; p.Leu208Pro]
(coordinates used in hg19) was ranked as potentially pathogenic.
The mutation has not been reported in dbSNP 138, 1000 Genomes
or the NHLBI Exome Variant Server, and was absent in our in-
house databases of 521 exomes/genomes from unrelated indivi-
duals of Middle Eastern origin, but was present in 1 of 126 358 al-
leles in the ExAC database. Direct Sanger sequencing analysis
was performed for all available family members, and demon-
strated co-segregation with the disease in the family according
to a recessive mode of inheritance. The predicted 3D structure
of HNMT does not appear to be significantly altered by the substi-
tution (Fig. 2E). The mutation affects a highly conserved amino
acid (Fig. 3) and, in line with this, the PhyloP score (13)was
found to be 4425, Grantham score 98 (14). In silico analyses with
SIFT, PolyPhen-2, ConDel and others all predicted the amino
acid substitution to be damaging (Table 2).
ID and ASD cohorts from outbred population
Cohorts of N= 991 ID and N=1000 autism spectrum disorder
(ASD) subjects were screened using a pooled targeted sequencing
approach. No potentially damaging rare homozygous or com-
pound heterozygous variants were identified. A rare heterozy-
gous variant, NM_006895.2:c.430-1G>A; Chr2:138762701G>A,
which would potentially alter splicing of exon 5, was identified
in one ASD and one ID individual. This variant was not present in
theExACdatabase(∼65000exomes),norinadatabaseof521Mid-
dle Eastern exomes. Although potentially damaging heterozygous
Table 2. Analysis of variants identified by targeted exome sequencing in the Iranian and Kurdish families (A and B, respectively)
Family Gene (accession #) Variant SNP? Polyphen2 SIFT PROVEAN PhyloP44
(Mean)
Condel Segregates with
phenotype, Y/N
Family A (Iranian) HNMT (NM_006895.2) Gly60Asp 1 (probably damaging) 0.003 (damaging) −6.303 (damaging) 3.092 1 (deleterious) Y
METTL21C (NM_001010977.1) Gly15Arg rs2390760 0.002 (benign) 0.429 (tolerated) 0.523 (neutral) −0.444 0 (neutral) N
ZRANB3 (NM_032143.2) Tyr161Cys rs181335970 1 ( probably damaging 0.001 (damaging) −6.847 (damaging) 2.268 0.990 (deleterious) N
Family B (Kurdish) HNMT (NM_006895.2) Leu208Pro 1 (probably damaging) 0.001 (damaging) −6.534 (damaging) 2.475 1 (deleterious) Y
Scores and predicted effect are indicated from Polyphen2, SIFT, PROVEAN, PhyloP44 and Condel (which is itself an integration of five separate prediction algorithms).
Human Molecular Genetics |5
single-nucleotide variants (SNVs) and CNVs are reported else-
where for schizophrenia, bipolar disorder, ID andautism spectrum
disorder (Supplementary Material, Fig. S1), loss of function (LoF)
variants are reported for HNMT in more than 40 individuals in
the ExAC control data, and thus, it seems unlikely that heterozy-
gous LoF variants are associated with these disorders.
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) inclu ding 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.
6|Human Molecular Genetics
In silico modeling of HNMT Leu208Pro predicts
functional impairment
To gain insights into the structural consequence of the p.Leu208-
Pro mutation in HMNT, we performed structure-based in silico
three-dimensional modeling of the mutant protein (Fig. 2E).
The non-conservative leucine to proline change at position 208
occurs in one of the six regions with an alpha helix conformation
(α-E) which flanks the seven-stranded β-sheet within the MTase
fold (15). The analysis indicated that although the mutation is
not proximal to the catalytic site, the rigidity introduced by the
proline residue would likely alter the helical conformation, desta-
bilizing the protein and therefore affecting the enzymatic func-
tion. Proline is well known as an alpha helix breaker due to its
side chain and steric constraints. In fact, the hydrogen bonding
network and conformation of the helix would be disrupted by
the side chain forced into the space occupied by the helix
backbone, and by the methyl group at the position normally oc-
cupied by an amide proton (16). We have quantified this effect by
performing in silicoenergy calculations with PoPMuSiC 2.1 and IMu-
tant 2.0 (17), which calculate stability changes upon mutation. In
both cases, the simulation revealed a decrease in stability, 3.73
and 2.09 kcal/mol, respectively. An additional analysis using
FoldX (18) revealed a more stark decrease in stability of 8.98 kcal/
mol. A leucine to proline substitution is causing the most destabil-
izing effect when all permutations are considered. Thus, we hy-
pothesize that the p.Leu208Pro mutant protein is either unstable
or the function is severely impaired.
Gly60Asp and Leu208Pro alterations do not affect
HNMT protein localization
To check if Gly60Asp and Leu208Pro changes disturb HNMT pro-
tein localization, COS-7 cells were transfected with mutant and
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).
Human Molecular Genetics |7
wild-type (WT) HNMT constructs with GFP Tag. Visualization of
the fusion protein was performed using a confocal laser scanning
system (data not shown). HNMT-Gly60Asp HNMT-GFP, like WT, is
localized to the cytoplasm of the cells, suggesting that there is no
difference in cellular localization between mutant and WT form
of the HNMT protein. For Leu208Pro, the transformed cells appear
to show a punctuatedistribution of HNMT-GFP, possibly indicative
of the formation of protein aggregates; however, consistent results
were not achieved (data not shown). We conclude that Gly60Asp
does not impair the proper cellular localization of HNMT.
Patients’lymphoblasts are considerably more
vulnerable to HA than the controls
We examined the vulnerability of patients’lymphoblasts (avail-
able for Family A, but not Family B) to HA when compared with
lymphoblasts from unrelated healthy controls. Cells were first
treated with different concentrations of HA and then using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay, the viability of cells was determined. Cells in both
groups were found to rapidly undergo cell death upon culture in
vitro after treating with high concentrations of HA (at 500 µM),
which is in line with findings suggesting that HA induces neutro-
phil apoptosis at the sites of allergic inflammation (19). While
control cells were able to survive at lower concentration of HA
(125 µM), patients’lymphoblast cells were still not tolerant to
the cytotoxic effects of HA, supporting our hypothesis that
HNMT, as the defense mechanism against HA, is defective in
the patients (P-value, 1 × 10
−5
) (Fig. 4).
No significant difference in HA and N-tele-
methylhistamine levels between patients carrying
Gly60Asp variant and healthy unrelated controls
The primary goal of HA inactivation is its conversion to metabo-
lites that will not activate HA receptors, and this is achieved
either by methylation or by oxidation. HNMT catalyzes the trans-
fer of a methyl group from SAM to the secondary amino group of
the imidazole ring of HA, forming N-tele-methylhistamine.
Therefore, if HNMT is defective, we would expect to detect high
levels of HA and low levels of methylhistamine. We measured
the HA and methylhistamine levels in patients’and controls’
lymphoblast cell lines using an ELISA assay. However, we could
notdetectanysignificant difference between them (data not
shown).
Gly60Asp alteration does not affect HNMT expression
at the RNA and protein level
To examine whether the Gly60Asp alteration disrupts the expres-
sion of HNMT, quantitative RT-PCR and western blotting analysis
were performed using patients’and controls’lymphoblast cell
lines. We did not detect any significant difference in HNMT ex-
pression between patients and controls atboth the RNA and pro-
tein level (data not shown).
Gly60Asp variant affects thermal stability of HNMT
protein
Melting temperatures are the best descriptor of thermal stability.
A difference in the melting temperature corresponds to a dif-
ference in energy between the pair of proteins. As indicated in
Figure 5, glycine to aspartic acid replacement decreases the melt-
ing temperature of the HNMT protein from 62.3 ± 1.7 to 58.3 ± 1.1°C
for WT and the Gly60Asp variant, respectively, suggesting that
amino acid change at position 60 will lead to reduced thermal sta-
bility of HNMT.
Gly60Asp variant disturbs the affinity of HNMT protein
for binding to SAM
The dissociation constant (K
d
) is commonly used to describe the
affinity and binding property between a ligand and a protein, i.e.
how tightly a ligand binds to a particular protein. Figure 6pre-
sents the calorimetric titration of HNMT (WT) and the Gly60Asp
variant with SAM by isothermal titration calorimetry. Figure 6A
indicates the isothermal binding curve for the titration of 30 μ
HNMT (WT) with 0.42 mSAM. The isothermal binding curve
(Fig. 6B) yields a K
d
of 49.2 ± 2.0 μ. Figure 6C demonstrates a sig-
nificant decrease in affinity of the Gly60Asp variant for SAM,
Figure 4. Cell viability using the MTT assay. Lymphoblast cells from 4 patients
from Family A and 12 healthy unrelat ed 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 μlof1mg/mLof
HNMT (WT) or HNMT (Gly60Asp) with 5 μl 200-fold diluted SYPRO Orange and a
buffer containing 50 mTris–HCl and 100 mNaCl, 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.
8|Human Molecular Genetics
providing another line of evidence for the effects of the amino
acid substitution at Gly60.
Gly60Asp variant significantly disrupts catalytic
activity of HNMT protein
The isothermal titration calorimetric (ITC) method was used to
assess the interaction between WT and Gly60Asp variant
HNMT with SAM. As indicated in Table 3,theK
m
(substrate con-
centration at which the reaction rate is half its maximum
value) of WT HNMT lies in the normal range of most enzymes.
However, this value was not measurable for Gly60Asp variant
(Fig. 7). This can be explained by the failure of mutant HNMT
to bind with SAM due to the dramatic change of its substrate-
binding pocket.
Leu208Pro destabilizes HNMT
Attempts were made to generate the Pro208 variant of HNMT;
however, soluble protein was not detectable from the constructs,
suggesting that misfolding occurs, leading to rapid degradation
of the protein. Addition of N-terminal GST tag, or expression in
the presence of chaperones, was not successful, and the variant
protein was only detectable in the inclusion body fraction (data
not shown). Interestingly, constructs for additional variants at
Leu208 (Leu208Val, Leu208Phe, Leu208Arg, Leu208Thr, Leu208Asp
and Leu208Asn) have also been generated in Escherichia coli, and
only substitutions with the first two (apolar) amino acids, valine
and phenylalanine, and to a much lower extent the polar, posi-
tively charged arginine (i.e. 5 mg/20 g cells instead of 20 mg/20 g
cells), lead to soluble and active HNMT protein (P. Macheroux and
C. Tongsook, personal communication). For this reason, we were
unable to perform parallel experimentation to the Gly60Asp mu-
tation for thermal stability, SAM-binding affinity and ITC mea-
surements, and would also be a plausible explanation for the
punctate cellular distribution of Leu208Pro-HNMT-GFP.
Discussion
HA in adult brain acts as a neurotransmitter in several biological
processes. Moreover, it has been reported that, during the devel-
opment of the rat brain in the fetus, HA concentration reaches its
maximum level throughout the period where neuronal differen-
tiation takes place in several brain regions, suggesting that HA,
acting as a neurogenic factor, is an important modulator in the
developing brain (20,21). Even at very low concentrations, HA
has strong pharmacological activity (22). HA-induced apoptosis
is mediated by caspase activation and PKC-γsignaling (23).
Therefore, synthesis, release and degradation of HA have to be
carefully regulated to avoid undesirable reactions. HAergic
neurons synthesize substantial quantities of HA and store it in
special storage granula inside the cell (24). Basal plasma HA con-
centrations of 0.3–1.0 ng/ml are considered normal and exceed-
ing these HA levels give rise to concentration-dependent HA-
mediated symptoms (25). The level of HA in brain is slightly
lower than that of other biogenic amines; however, its turnover
is considerably faster and once released, it must be inactivated
within a few minutes (19).HNMTisthesoleenzymeinthe
CNS, inactivating the neurotransmitter actions of HA (26).
We identified two homozygous missense mutations in the
HNMT gene segregating with NS ID in an Iranian family of Turkish
origin (Family A) and in a Kurdish family (Family B). For Family
A, the condition had a relatively similar pattern during the child-
hood of the patients, starting at around 5 years of age and grad-
ually worsening. Males were relatively mildly affected, but the
condition in females was profound. This could be at least in
part due to gender differences in neurotransmitter activity of
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 μlof0.42mSAM into 30 μHMNT (WT) at 25°C. (A) The
released heats of injection aft er 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 μ.(C) Released heats of 20 consecutive injections of
15 μlof2mSAM into 30 μHNMT (Gly60Asp) at 25°C.
Table 3. Apparent K
m
for HA from HNMT reaction
Enzyme K
m
(μ)V
max
(μs
−1
)k
cat
(s
−1
)
HNMT (WT) 5.47 ± 1.41 8.21 ± 0.16 × 10
−3
8.21 ± 0.16 × 10
−3
WT and Gly60Asp HNMT constructs were used; however, only results for WT are
given, as the catalytic activity of HNMT-Gly60Asp was too low to record using the
VP-ITC system (MicroCal, GE Healthcare).
Human Molecular Genetics |9
HA. It has been reported that in certain areas of the brain, neuro-
transmitter synthesis, content and metabolism are sexually
differentiated and under the influence of sex steroids in develop-
ment and adulthood (27,28). As mentioned above, once HA is
released in the brain, it would typically be cleared from the intra-
cellular space within a few minutes by HNMT. Therefore, mal-
functioning HNMT would be expected to result in a high
residual level of HA in the region. Using ELISA, we tested to see
if there is any difference in the level of HA between patients’
and controls’lymphoblast cell lines. However, no significant dif-
ference was detected. Since the alternative pathway of HA inactiva-
tion, i.e. DAO is active in the lymphoid lineage (but not in the CNS),
therefore we measured HA level after blocking the DAO pathway
and again no difference was detected (data not shown). As dis-
cussed above, during rat embryonic development, HA concentra-
tion reaches its maximum level at embryo days 14–16 when
neuronal differentiation takes place and then steadily decreases
until birth, yielding the concentration present in the adult organ-
ism. This may explain why differences in the level of HA among
the patients’and controls’lymphoblast cell lines were not detected.
HNMT has a two-domain structure—the MTase domain and
the S domain. The MTase domain primarily carries out cofactor
binding and probably catalysis, while both domains contribute
to HA binding (11,26). The Gly60Asp variant occurs within the
conserved MTase region I (Ile56-Gly64), which is part of the
SAM-binding pocket, highlighting the role of Gly60 in the inter-
action of HNMT with its cofactor. From our protein modeling an a-
lysis, we predict that the Gly60Asp variant does not bind SAM.
Inspection of the active site also shows that the aspartate side
chain would interfere with the ribose ring of SAM, rationalizing
the lack of binding. Therefore, Gly60Asp does not affect overall
protein stability, but compromises SAM in the active site of the
enzyme. This interpretation is fully supported by the results ob-
tained with the recombinant HNMT-Gly60Asp variant.
Leu208, as part of a very compact hydrophobic pocket, has
several contacts with the residues in the same α-helix, the adja-
cent α-helix and the two β-strands. As a result, the leucine-to-
proline substitution has a disruptive effect on the hydrophobic
pocket, and it is likely that the substitution breaks the α-helix,
and this in turn disrupts the hydrophobic packing in this area
leading to exposure of hydrophobic residues, and thus favoring
misfolding and aggregation of the protein.
Expression studies of the Gly60Asp variant revealed that the
mutation does not disrupt the expression of HNMT at either the
mRNA or protein level, suggesting that HNMT malfunction is not
a result of reduced enzyme concentration. Moreover, observations
from protein localization studies of HNMT proved that Gly60Asp
and Leu208Pro variants do not disrupt the normal distribution of
protein throughout the cytoplasmof cell. Results fromthermal sta-
bility,binding affinityand catalytic activity investigations of HNMT
WT and the Gly60Asp variant, and failed attempts to stabilize and
solubilize Leu208Pro, support the deleterious effects of these sub-
stitutions on HNMT function in these two ID families.
A short isoform of HNMT has also been reported that includes
the N-terminal 126 amino acids (Genbank ID: NM_001024075.1;
NP_001019246.1 (Q8IU56)). The function of this isoform is un-
known. The mutation identified in Family A (Gly60Asp) would
potentially affect this as well as the long isoform, whereas the
mutation identified in Family B (Leu208Pro) would only impact
the longer isoform. Also, it is predicted that the Leu208Pro substi-
tution is extremely destabilizing for the protein. The effects of
different mutations on protein stability and function, as well as
on different isoforms, could account for different clinical aspects
and severities for the two families.
Decreased HA levels have been found in the brain of patients
with Alzheimer disease (AD) (29), including reduction in the
neuronal pool of hippocampus, hypothalamus and temporal cor-
tex of AD patients (30), but, in contradicting reports, increased HA
levels in cerebrospinal fluid and brain tissue were demonstrated
(31,32). HA measurements per se may be, however, unreliable due
to many confounding factors as, e.g. HA levels increase with post-
mortem interval (30), but may be valuable when accompanied by
determination of activity or levels of HA-related enzymes.
While we can postulate about the effect of these mutations on
levels of HA in the CNS, and possible detrimental effects on neu-
rodevelopment, it cannot be ignored that a decrease in levels of
the catabolite and product of HNMT activity, N-tele-methylhista-
mine may also play an important role in the disease etiology in
these families. It has been suggested that there are HA/glutamate
functional interactions in the brain (33), and it has already been
shown that HA can potentiate NR2B-type N-methyl--aspartate
(NMDA) receptors in hippocampal neurons, and that N-tele-
methylhistamine also produces an equipotent enhancement of
NMDA currents (34). In addition, N-tele-methylhistamine has
been reported to be an agonist for β
3
γ-butyric acid (GABA) recep-
tors (35). HA has recently been reported to increase neural differ-
entiation to FOXP2 neurons in cultured cells (36). Mutation of the
FOXP2 gene has been reported as the cause of a rare speech/
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 mTris–HCl buffer, pH 8.0, containing 100 mNaCl at 25°C. The experiment was performed by
injection of 2 mHA (2 μl/injection; 20 injections). The cell contained 1 μHNMT (WT) and 100 μSAM. 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).
10 |Human Molecular Genetics
language disorder (MIM 602081) (37). Given the potential for
N-tele-methylhistamine to act as an analog of HA, it would be in-
teresting to see whether it would have a similar effect, in which
case one could postulate that some of the deficits identified in the
patients reported here could be the result of such a mechanism.
Conclusion
In the current study, we have shown that mutations resulting in
HNMT LoF are associated with a NS form of ARID. We also con-
firmed that the p.Gly60Asp substitution results in complete loss
of HNMT enzymatic activity, resulting in reduced HA inactivation
and increased sensitivity to this amine. For p.Leu208Pro, al-
though the mutation does not lie in the catalytic region of the
protein, the rigidity introduced by the proline residue would
most likely alter the helix conformation, destabilizing the protein
and therefore affecting protein stability and the substrate-binding
site.
Collectively, these findings indicate that HNMT plays an im-
portant role in human neurodevelopment. Our results indicate
that HNMT should be included in genetic testing of individuals
presenting with ID in consanguineous populations and, given esti-
matesof a role for AR genes in 13–24% of ID in non-consanguineous
populations (2), in outbred populations.
Materials and Methods
This study was approved by the Research Ethics Committees of
the Qazvin University of Medical Sciences, Qazvin, Iran, Univer-
sity of Social Welfare and Rehabilitation Sciences, Tehran, Iran,
Max Planck Institute of Molecular Genetics, Berlin, Germany,
the Centre for Addiction and Mental Health in Toronto, Canada,
and Queen’s University, Kingston, Canada. Informed written
consent was obtained for all participating subjects.
Gene mapping
Family A (Iranian family)
Genomic DNA was extracted from peripheral blood leukocytes by
standard methods. We used the Affymetrix GeneChip Mapping
SNP 6.0 array (950K SNPs and 950 K CNV markers) to analyze
DNA samples of all affected individuals, parents and one healthy
sibling. Approximately 200 000 markers with good quality geno-
types (based on their array hybridization confidence score) were
selected for linkage analysis. Appropriate input files for the link-
age analysis programs Merlin (38) and Allegro (39) were generated
by ALOHOMORA software (40) with subsets of 300–500 markers in
a sliding window mode based on mapping information from
DeCode and Caucasian allele frequencies. Quality control checks
such as gender check and verifying the relationships between in-
dividuals within the family were performed (38). Mendelian in-
consistencies and unlikely genotypes were detected by the
PedCheck (41) and Merlin (38) programs, respectively, and they
were excluded from genotyping data prior to linkage analysis.
Parametric linkage analysis was carried out based on an AR
mode of inheritance, and assuming complete penetrance.
Family B (Kurdish family)
Genomic DNA was extracted from blood samples using standard
protocols. Genotyping (SNP analysis) was performed using the Af-
fymetrix 250k Genome-Wide Human SNP Array (Affymetrix, Santa
Clara, CA, USA) for available individuals. We used ALOHOMORA
software (40) for SNP arrayquality controls,as described previously
(42). The program Merlin was applied for parametric multipoint
linkage analysis, consistent with an AR modeof inheritance, a dis-
ease allele frequency of 10
−3
and complete penetrance.
Exon enrichment and high-throughput sequencing
Custom-made Agilent SureSelect DNA Capture Arrays (Agilent
Technologies, Inc., Santa Clara, CA, USA) were used for the en-
richment of exons from homozygous intervals, including, on
average, 60 bp of flanking sequences on either side of the exon.
Enriched exons were sequenced on an Illumina Genome Analyz-
er II, generating 76-bp single reads with 98.9% coverage.
Sequence alignment, variant calling, annotation and
verification
Raw sequence reads were prescreened to remove low-quality
reads, and then aligned to the human reference genome (hg19,
GRCh37) with SOAP (version 2.20). Aligned and unaligned reads
were used to call the SNVs and Indels, respectively. Variant lists
were filtered against dbSNP137, whole genomes from 185 healthy
individuals (1000 Genomes Project), and 200 exomes from Danish
individuals, and 6500 exomes present in the Exome Variant Ser-
ver (NHLBI GO Exome Sequencing Project, Seattle, WA, USA; http
://evs.gs.washington.edu/EVS/; 30 August 2013; v.0.0.21). In add-
ition, variants were compared with an in-house database con-
taining more than 521 exomes from individuals of Middle
Eastern origin. Variants were ranked as potential candidates as
previously described (43), using an improved version of Medical
Re-sequencing Analysis Pipeline (MERAP) (44). The OMIM catalog
(http://www.ncbi.nlm.nih.gov/omim) and the Human Gene
Mutation Database (HGMD, http://www.hgmd.org/) were used
as a filter to identify all previously described pathogenic changes.
Sanger sequencing was used to confirm the co-segregation of the
final candidate variants in the family.
Targeted exome sequencing for HNMT
Ninety-nine genes were included in the custom design: 64 for
known or suspected NS ARID genes, including HNMT;4for
known or suspected NS AR autism genes; 7 for known or sus-
pected NS X-linked ID or ASD genes; 7 genes for known syn-
dromic ARID, also reported in ASD cohorts and 17 known or
suspected NS autosomal dominant ID or ASD genes. N= 1000
ASD and N= 991 ID-unrelated individuals from an outbred
population (Canada) were included. Samples were measured
using the RNaseP assay by quantitative PCR, using ViiA™7
Real-Time PCR (Life Technologies, Carlsbad, CA, USA), and
pooled at equimolar concentrations in pools of 20. Seven
pools were barcoded and run using the custom Ampliseq (Life
Technologies) primer pools on a single Proton P1v2 chip. Sam-
ples were analyzed using the Ion Torrent software (Life
Technologies), and Bam files generated and runs visualized
using the Integrated Genome Viewer (Broad Institute: http://
www.broadinstitute.org/igv/), and for the purpose of this study,
focusing just on HNMT coding regions.
Expression studies of HNMT
Quantitative reverse transcriptase-PCR
Primers were designed to amplify the coding sequence of the
HNMT gene. Coding sequence primers for β-Actin (ACTB)and
HPRT were used as an internal reference for all the runs. Reverse
transcriptase (RT)-PCR was performed in quadruplicates using a
384-well optical plate, with a final reaction volume of 16 µl. Uni-
versal SYBR Green PCR conditions were used, consisting of 95°C
Human Molecular Genetics |11
for 2 min and 30 s and 40 cycles at 95°C for 4 s and 60°C for 20 s.
Each reaction contained 2 µl of cDNA in a 16-µl volume run in
384-well optical plates on a ViiA™7 (Life Technologies). For
each gene analyzed, all samples were run on one plate, to avoid
interplate variability, and in quadruplicate. Furthermore, each
plate contained H
2
O, RT-minus and RNA-minus as negative con-
trols. The C
t
for all reactions was calculated automatically by the
ViiA™7 (Life Technologies) software. Gene expression analysis
was calculated using the comparative C
t
method.
Western blot analysis
Protein samples from patients and controls were separated by
SDS page (4–20% Mini-PROTEAN TGX polyacrylamide gel, Bio-
Rad Laboratories, Hercules, CA, USA) and electrically transferred
to a nitrocellulose membrane (BioTrace NT nitrocellulose mem-
brane, PALL Life Sciences, Ann Arbor, MI, USA). The membrane
was blocked for 1 h using 5% skimmed milk in TBS-Tween, incu-
bated overnight with the primary rabbit anti-HNMT (1 : 1000,
Bethyl Laboratories, Inc., Montgomery, TX, USA) and then incu-
bated with the secondary antibody horseradish peroxidase-
conjugated donkey anti-rabbit IgG (1 : 1000, G E Healthcare UK
Limited, Little Chalfont, UK). The immunoblots were developed
by an enhanced chemiluminescence western blot detection
system (GE Healthcare).
Heterologous expression of WT HNMT, p.Gly60Asp
and p.Leu208Pro variants in E. coli
The open reading frames encoding the target proteins (WT
HNMT; p.Gly60Asp and p.Leu208Pro variants) were amplified by
PCR from the pcDNATM3.3-TOPO TA vector using primers
5′-TCTCATATGATGGCATCTTCCATGAGGAGC-3′(sense strand)
and 5′-TCAGCGGCCGCTTAATGATGATGATGATGATGTGCCTCA
ATCACTATGAAACTCAGA G-3′(anti-sense strand) using Phusion
High-Fidelity DNA polymerase (Thermo Scientific, Waltham, MA,
USA). The amplified genes were then cloned into the pET-21a
vector using the NdeI and NotI restriction sites for expression as
C-terminally hexa-histidine tagged proteins. Similarly, glutathi-
one S-transferase (GST) fusions were generated by cloning the
open reading frames into pGEX-6p2 using the BamHI and NotI
restriction sites. Induction of expression of HNMT WT, and
Gly60Asp, and Leu208Pro variants with C-terminal His6 or N-
terminal GST tag was carried out at an OD
600
∼1, by addition of
0.1 mIPTG ( final concentration). The culture was maintained
at 18°C for 14 h prior to harvest, resulting in a yield of 5 g of cell
paste per liter of cell culture.
Purification of HNMT WT and the Gly60Asp variant
Frozen cell paste (∼20 g) was thawed and resuspended in lysis
buffer (50 mTris–HCl, pH 8.0 containing 100 mNaCl,
10 mimidazole, 1 mDTT and 100 μPMSF). Cells were dis-
rupted by ultrasonication with 50% amplitude for 15 min.
After ultrasonication, the suspension was centrifuged at 18 000
rpm at 4°C for 1 h and the pellet was discarded. The clear crude
extract was filtered (0.22 µm) prior to loading onto a 5-ml Ni-
Sepharose™High-Performance HisTrap™HP column equili-
brated with lysis buffer. The column was washed with 100 ml
of 50 mTris–HCl buffer, pH 8.0, containing 100 mNaCl and
20 mimidazole. Elution of HNMT was initiated using 50 m
Tris–HCl buffer, pH 8.0, containing 100 mNaCl and 300 m
imidazole. Fractions containing HNMT were pooled, concen-
trated using a Centricon YM10 and loaded onto a Superdex
200 prep grade column previously equilibrated with 50 m
Tris–HCl buffer, pH 8.0, containing 100 mNaCl. Fractions con-
taining target protein were pooled and concentrated as before.
The purified protein was stored at −80°C.
Determination of melting temperature of HNMT (WT)
and the Gly60Asp variant
To investigate the thermal stability of HNMT (WT) and the
Gly60Asp variant, the melting temperature was determined
using a fluorescence-based thermal shift assay. Experiments
were performed in a real-time PCR detection system (Bio-Rad)
with a 96-well plate in a FRET scan mode. In the experiments,
5 µl of 1 mg/ml HNMT (WT) or the Gly60Asp variant was mixed
with 5 µl of 200-fold diluted SYPRO Orange solution and 15 µl of
a buffer containing 50 mTris–HCl, pH 8.0 and 100 mNaCl to
afinal volume of 25 µl in a 96-well plate. Thermal unfolding of
the protein was monitored using a temperature gradient from
20 to 95°C, measuring fluorescence emission at 0.5°C increments
with a 60-s hold for signal stabilization. The melting temperature
of the protein was derived from the peak of the derivatives of the
experimental data.
Determination of dissociation constant (K
d
) of HNMT
(WT) and Gly60Asp variant
Dissociation consta nts for binding of SAM and H A to HNMT (WT)
and Gly60Asp variant were determined using the VP-ITC system
(MicroCal, GE Healthcare). Experiments were performed at 25°C
in 50 mTris–HCl buffer, pH 8.0, containing 100 mNaCl. All
solutions were degassed before measurements. Titration ex-
periments for SAM and HA were performed by using 20 injec-
tionsof15µlof0.42mSAM or 4 mHA (duration time 29.9 s
and spacing time 250 s) into the cell containing 30 µM HNMT
(WT). In the case of the Gly60Asp variant, 30 µM was titrated
with SAM (2 m, 20 injection, 15 µl/injection, duration time
29.9 s and spacing time 250 s) or HA (200 µM, 20 injection,
15 μl/injection, duration time 29.9 s and spacing time 250 s). To
determine K
d
values of HNMT (WT) and Gly60Asp variant, one
set of sites fitting with Origin version 7.0 (MicroCal) for ITC
data analysis was used.
Determination of catalytic activity of HNMT
WT and Gly60Asp variant
Kinetic parameters for HNMT (WT) and the Gly60Asp variant
were determined using the VP-ITC system (MicroCal) at 25°C in
50 mTris–HCl buffer, pH 8.0, containing 100 mNaCl in
multi-injection mode. All solutions were degassed before the
measurements. The HNMT analysis consisted of 20 injections
of 2 µl (duration time 4 s and spacing time 250 s) of 2 mHA
into the cell containing 1 µM of HNMT (WT) or Gly60Asp variant
and 100 µM SAM. The experimental data were fitted with Origin
version 7.0 (MicroCal) for ITC data analysis to obtain kinetic
parameters.
In vitro toxicology assay
The cytotoxic effects of HA on patients’lymphoblast cells carry-
ing the p.Gly60Asp variation and lymphoblasts from unrelated
healthy controls were determined using the MTT assay. This
method measures the metabolic reduction of MTT to a colored
water-insoluble formazan salt by mitochondrial dehydro-
genases. Lymphoblast cells were available for all four affected
individuals from Family A. For comparison, lymphoblast cell
lines from 12 unrelated unaffected individuals were selected,
12 |Human Molecular Genetics
and matched for age, gender and number of passages for the
cells. Cells for each line were seeded at 10 000 cells per well in
96-well plates (in triplicates for each line) and cultured in
serum-free DMEM for 2 h. Cells were subsequently incubated
for 2 h with 125 µM HA after which reconstituted MTT in an
amount equal to 10% of the culture medium volume was
added. After incubation of the plates at 37°C for 1 h, the cells
were then washed with phosphate-buffered saline and the for-
mazan salts dissolved in 200 ml of dimethyl sulfoxide with gentle
shaking for 10 min at room temperature. The plates were read at
570 nm using a Tecan Spectra Fluor plate reader, and data were
averaged across replicates and across both the affected and un-
affected group.
HNMT protein localization
We generated two different sets of genetic constructs for HNMT
protein localization. RNA was extracted from one of the patients’
(Family A) fibroblast cells, and then HNMT full-length cDNA
(CCDS2181.1) was PCR amplified and cloned into the pcDNA3.1/
CT-GFP-TOPO expression vector (Invitrogen, Carlsbad, CA, USA).
Using the Q5
®
Site-Directed Mutagenesis Kit (New England Bio-
Labs, Ipswich, MA, USA), an A>G change was introduced to the
HNMT mutant plasmid (c.179G>A) in order to generate the
HNMT WT construct. Subsequently, a T>C change was introduced
to the WT construct to generate a mutant HNMT construct for the
c.623T>C, ( p.Leu208Pro) substitution. Thus, we generated three
constructs that are identical except for the mutation changes,
in order that we would be able to exclude any potential effects
of intervening SNPs, as it has been well documented that genetic
variation among individuals can result in as much as 5-fold dif-
ferences in HNMT activity (20). Orientation of the inserts and cor-
rect sequences were finally confirmed by Sanger sequencing.
To examine the consequences of mutations on cellular localiza-
tion of the HNMT protein, we transiently transfected COS-7 cells
with 2 µg of purified constructs (p.Gly60Asp, p.Leu208Pro and WT)
with Polyfect (Qiagen, Germantown, MD, USA). Twenty-four h after
transfection, we visualized the HNMT-GFP fusion protein in trans-
fected cells using a Zeiss Axioplan 2 imaging microscope (Carl Zeiss
AG, Oberkochen, Germany), equipped with the LSM510 array con-
focal laser scanning system, and the Zeiss LSM510 version 3.2 SP2
software package.
Measurement of HA/methylhistamine levels in patients’
and controls’lymphoblast cells’by ELISA
Cell-free supernatants were used to measure the HA and N-tele-
methylhistamine levels in patients’lymphoblast cell lines and
in a group of unrelated healthy controls using an ELISA assay,
performed according to the manufacturer’s instructions (GenWay
Biotech, Inc., San Diego, CA, USA) through a Fluoroskan Ascent
microplate reader (Thermo Scientific) at 450 nm. Standard re-
agents were employed to draw a calibration curve. The samples
OD values and the calibration curvewereusedtocalculatetheHA
and N-tele-methylhistamine concentrations in cases and controls.
Bioinformatic analysis of the HNMT protein
Protein structure modeling was conducted with the Phyre2 server
with the 3D structure image realized by JMOL. In addition, mu-
tant protein structures were modeled with the FoldX package
Version 3.0 Beta3 (18). Models were built based on the WT crystal
structure [PDB 2AOT (26); available in the Protein Data Bank PDB]
as the template using the BuildModel function after energy
minimization of the WT structure using the RepairPDB function.
Figures were generated with PyMOL (The PyMOL Molecular
Graphics System, Version 1.5.0.1, Schrodinger, LLC).
Supplementary Material
Supplementary Material is available at HMG online.
Acknowledgments
We thank all family members and affected individuals for partici-
pating in this study. We also thank the Qazvin University of
Medical Sciences and Qazvin Rehabilitation Organization for
their support in this study. We also thank the Austrian Academic
Exchange Service (ÖAD) for a fellowship to C.T. and the Austrian
Science Fund (FWF) for the support through the doctoral school
“Molecular Enzymology”(W901).
Conflict of Interest statement. None declared.
Funding
This research was supported by a grant from the Canadian Insti-
tutes of Health Research (#MOP-102758) and Max Planck Society
and EU FP 7 project GENCODYS, grant # 241995.
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