ErbB4 Mutation that Decreased NRG1-ErbB4 Signaling Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis/Frontotemporal Dementia

Abstract

Background:

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) includes a large spectrum of neurodegenerative disorders.

Objective:

To identify the relationship of ErbB4 mutation and ALS/FTD.

Methods:

Here, we report an atypical case of frontal variant behavioral abnormalities at the initial stage, a stable plateau stage of 5 years, and paralysis involving both upper and lower motor neurons followed by progressive cognitive dysfunction at the advanced stage. The clinical findings suggested a diagnosis of ALS/FTD, and genetic testing revealed erb-b2 receptor tyrosine kinase 4 (ErbB4) heterozygous mutation (c.2136 T>G, p.I712M), identified in an ALS pedigree previously. We modeled mutant ErbB4 protein through the SWISS-MODEL Server, and speculated on the structural change caused by the mutation. We also identified that ErbB4 (I712M) mutation led to reduced auto-phosphorylation of ErbB4 upon neuregulin-1 (NRG1) stimulation.

Results:

A functional analysis of ErbB4 mutation demonstrated an obviously decreased auto-phosphorylation of ErbB4 involving in the pathogenesis of ALS/FTD.

Conclusion:

We firstly found ErbB4 mutation to be identified in ALS/FTD.

Keywords

Amyotrophic lateral sclerosis frontotemporal dementia I712M neuregulin-1

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is characterized by selective destruction of both upper and lower motor neurons, causing progressive paralysis and muscular atrophy, with eventually respiratory failure [1]. Frontotemporal dementia (FTD) is characterized by widespread degeneration of neurons in frontal temporal cortex, which is clinically classified into three subtypes including behavioral variant FTD, semantic dementia, and progressive non-fluent aphasia [2]. Detectable cognitive impairment presents in about 50% of ALS cases, 15–20% of ALS patients receive concomitant diagnosis of FTD [3], and this combination has a worse prognosis and reduction in survival time. Mutations in multiple genes can cause ALS/FTD, indicating that the two classifications are varying manifestations of one pathological mechanism [4]. The genes commonly affected include Chromosome 9 open reading frame 72 (C9ORF72), Charged multivesicular body protein in 2B (CHMP2B), TAR DNA binding protein 43 (TARDBP), Valosin containing protein (VCP), Ubiquitin 2 (UBQLN2), Tubulin alpha 4A protein (TUBA4A), Sequestosome 1 (SQSTM1), Tank-binding kinase 1 (TBK1), and Fused in sarcoma (FUS) [4 –7].

In 2013, Takahashi et al. firstly identified an erb-b2 receptor tyrosine kinase 4 (ErbB4) gene mutation in a Japanese family affected by autosomal-dominant ALS with incomplete penetrance, and an extensive mutational analysis revealed the ErbB4 mutation in a Canadian individual with familial ALS [8]. ErbB4 is a very rare causative gene in ALS and is a member of the epidermal growth factor subfamily of receptor tyrosine kinases. ErbB4 forms a homodimer or heterodimer with ErbB2 or ErbB3 and is activated by binding to neuregulin-1 (NRG1), an epidermal growth factor-like trophic factor [8]. Here we report a case covering a 6-year follow-up of a female patient presenting with behavioral abnormalities at the age of 55, paralysis involving both upper and lower motor neurons after 6 years of onset, followed by progressive cognitive impairment. Whole exome sequencing revealed the mutation c.2136 T>G (p.I712M) in the ErbB4 gene in the patient and her unaffected mother, suggesting incomplete penetrance.

MATERIALS AND METHODS

Genetic procedures

Total genomic DNA was extracted with the phenol/chloroform method. The quality of DNA was assessed by Qubit 3.0 (Thermofisher, USA) and agarose gel electrophoresis. Then the sequencing library was carried out for 150 probands according to the SureSelect^XT Target Enrichment System Manual (Agilent, USA), and whole exome sequencing was performed by HiSeq X Ten (Illumina, USA). The sequencing raw data was aligned to the human reference genome hg19. Single nucleotide polymorphism (SNP) and insert and deletion (INDEL) screenings were based on genome analysis toolkit (GATK) best practice. The quality control (QC) of the whole exome sequencing demonstrated that the total reads number was 79.29 M, capture efficiency rate on target regions was 83.59% and mean coverage sequencing depth on official target was 134.18. All common variants, in which the minor allele frequency (MAF) was higher than 1% based on public databases of human variation (1000 Genomes Project, ExAC, ESP6500, dbSNP and genomAD), were eliminated. We excluded single nucleotide variations (SNVs) including intergenic, intronic, upstream/downstream, and non-coding RNA (ncRNA) according to Func.refGene, and variants that were synonymous according to ExonicFunc.refGene. We focused on variants of known pathogenic genes of dementia and ALS (detailed genes are listed in Table 1). After bioinformatic analysis of the sequencing data, we found that there was a missense mutation (c.2136 T>G, p. I712M) in ErbB4 gene. The missense mutation of ErbB4 gene was analyzed by Sanger sequencing using specific ErbB4 primers (forward primer: 5^′-AGAGAGGACTGACT ATCGGACTGAA-3^′ and reverse primer: 5^′-TGTATCCGTCCCAGC TCATTC-3^′, the values of Tm were 60.7°C and 59.8°C, respectively, and the length of amplified product is 368 bp). The entire coding region successfully sequenced. The software of Mutation Taster was applied to predict the pathogenicity of the detected mutations (http://www.mutationtaster.org/).

Table 1

Known pathogenic genes of dementia and ALS

A2M	BSCL2	DNAJC5	GRIA2	MAP3K14	PLEKHG5	SLC5A7	TRPV4
ABCA7	C19orf12	DNMT1	GRID2	MAPT	PNPLA6	SMN1	TUBA4A
ABCD1	C9orf72	DPP6	GRIK1	MAPT	PRICKLE1	SMN2	TYROBP
ACE	CAMK1G	DYNC1H1	GRN	MATR3	PRKAR1B	SNCA	UBA1
ADAM10	CASP3	EFHD2	GSK3B	MEF2C	PRKN	SNCB	UBQLN2
ALS2	CD2AP	ELP3	HFE	MOB3B	PRNP	SOD1	UNC13A
ANG	CD33	EPHA1	HLA-DQB1	MPO	PRPH	SORL1	UNC5C
APBB2	CELF1	EPM2A	HNRNPA1	MS4A4E	PRPH2	SPAST	VAPB
APEX1	CHCHD10	ERBB4	HNRNPA2B1	MS4A6A	PSEN1	SPG11	VCP
APOE	CHMP2B	ETS1	HSPB1	MYH14	PSEN2	SQSTM1	VEGFA
APP	CLU	EWSR1	HSPB3	NEFH	PTK2B	SUSD2	VPS54
AR	COQ2	FBXO38	HSPB8	NHLRC1	RAB38	TAF15	WDR45
ASAH1	CR1	FERMT2	HTR7	NME8	REEP1	TARDBP	ZNF512B
ASCC1	CSF1R	FGGY	IGHMBP2	NOS3	REST	TBK1	TRPM7
ATP13A2	CTSC	FIG4	INPP5D	NOTCH3	SETX	TFG	SLC52A3
ATP7A	CYP27A1	FMNL1	ITM2B	NPC1	SIGMAR1	TMEM106B	PLAU
ATXN2	CYP2C19	FUS	ITPR2	OPTN	SLC1A1	TREM2	LRRK2
BICD2	DAO	GARS	KIF20B	PFN1	SLC1A2	TRIB3	GRB2
BIN1	DCTN1	GBA	LRP1	PICALM	SLC52A2	TRIP4	DNAJB2
BLMH

The characterization of ErbB4 mutation I712M transcript by cDNA pyrosequencing and Sanger sequencing

The mutation I712M (c.2136 T>G) amplicons of ErbB4 cDNA were obtained from the patient’s mRNA sample by RT-PCR reaction. The cycling parameters of RT-PCR were 3 min at 95°C, followed by 30 s at 94°C, followed by 35 cycles of 60°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 5 min. The target sequence was cttcgtatKttgaaa (the base with BOX was variant site). The pair of PCR primer sequence included the forward primer 5’-CTTGGAAACAGAGTTG GTGGA-3’ and reverse primer 5’-CGTTCCAAAAGCACCT GAG-3’. The pyrosequencing and Sanger sequencing were performed following the manufacturer’s protocols (Sangon, China). The analysis was used through PyroMark Software 1.0.11 software environment (Sangon, China).

ErbB4 structure model prediction

The structure of ErbB4 was acquired from Protein Data Bank server (Research Collaboratory for Structural Bioinformatics, USA) with ID code 3BCE. The p.I712M mutant structure of ErbB4 was calculated by SWISS-MODEL Server (Swiss Institute of Bioinformatics, Lausanne, Switzerland) [9] using its wild-type structure as the starting template. Molecular graphics were performed using Swiss-Pdb viewer 4.1.0 software [10].

Transfection and western blotting assay

Plasmid DNA for wild type ErbB4 was prepared by inserting coding sequence of the human ErbB4 gene (NM_005235) into the pBABE-puro vector (Addgene, USA), and the ErbB4 I712M mutation was obtained by PCR-based site-directed mutagenesis (Transgen, FM111) with c.2136 T>G. After mutagenesis, all the constructs were verified by sequencing. Human embryonic kidney cells (HEK 293T) were grown to 80% confluence and transfected with plasmids by TurboFect transfection reagent (Thermo, USA) according to the manufacturer’s instructions. Medium were then replaced with serum-free DMEM overnight for 16 h, and then stimulated with or without 100 ng/ml NRG1 (RnD, USA) for 10 min at 37°C. After stimulation, the cells were harvested for western blotting. Equal amounts of total proteins (20μg) were subjected to SDS-PAGE and transferred to PVDF membrane (Millipore, USA). Membranes were incubated with antibodies specific for HER4/ErbB4 (111B2) (CST, 4795, USA), phospho-HER4/ErbB4 (Tyr984) (CST, 3790, USA), PI3 Kinase p85 (CST, 4292, USA), phospho-PI3 Kinase p85 (Tyr458) / p55 (Tyr199) (CST, 4228, USA), phospho-Akt (Ser473) (D9E) (CST, 4060, USA), or GAPDH [EPR16891] (abcam, ab181602, USA). Immuno-reactive bands were analyzed from three independent experiments using ImageJ Software.

Ethics and patient consent

We received approval from the regional ethical standards committee from Shanghai Mental Health Center on human experimentation for our experiments using human materials. We also received written informed consent for research from the participants and guardians.

Data availability statement

Any data not published within the article will be shared by request from any qualified investigator.

RESULTS

Case report

In August 2010, a right-handed 55-year-old female patient with 12 years of education put food on to cook and went out, but forgot her key, so the pressure cooker and the food in it were scorched. The patient suddenly appeared inert, showed less facility with language and less movement, and gradually showed impairment in activities of daily living function including the ability to cook and clean. At 2 months post-onset, the patient was talkative and repeated phrases such as “have a meal” for hundreds of times per day. Her memory was in decline, and she got lost twice. In early 2011, the patient showed apparent personality changes including gluttony, slovenliness, and poor manners. Her pants were still pulled down when she was leaving the washroom, and she picked up things to eat from the ground. In May 2011, she was first referred to our department.

The patient underwent a clinical evaluation at our institution and was then enrolled in the Foundation of China Alzheimer’s disease and related disorders study (2008ZX09312). Additional data from her relatives were collected and analyzed.

Her initial standard blood investigations were normal, and the results of the remainder of her neurological examination were unremarkable. Her MMSE (Mini-Mental State Examination) and MoCA (Montreal Cognitive Assessment) scores in October 2011 were 24/30 and 18/30 respectively (Fig. 1A, B). Cerebral MRI in February 2012 revealed mild enlargement of the ventricular system (Fig. 2A, E). An ¹⁸F-FDG PET was performed for clarification of the diagnosis, which revealed nonspecific decreased metabolism within the whole brain (Fig. 1E). The clinical manifestations, except for the imaging features, led to a suspicion of FTD; subsequently, therapy with Memantine and Risperidone was initiated. In the follow-up period, cerebral MRI in June 2013 revealed mild enlargement of frontotemporal sulci and lateral ventricles (Fig. 2B, F). By June 2015, the patient showed a continuation of repetitive speech, gluttony, and impairment in daily function; however, no decline in cognitive function was apparent within the previous five years. Cerebral MRI showed no signs of obvious cerebral atrophy compared with the previous MRI (Fig. 2C). However, the patient began to suffer from weakness and stiffness of her left lower extremity, and did not benefit from acupuncture therapy. In February 2016, the patient developed weakness and stiffness of both lower extremities, dysphagia, and difficulty in speech. Standard blood investigations were normal. The electromyogram showed nerve root damage in the lumbar 4-5 and sacral 1 segment. The orthopedist excluded disabling paralysis caused by orthopedic disease through MRI of cervical, thoracic, and lumbar vertebrae, which showed intervertebral disc protrusion within cervical and lumbar disc spaces (Fig. 1F–H). Therapy with Baclofen, Madopar, and Eperisone were initiated, but there was no obvious improvement.

Fig.1

MMSE and MoCA results, images of the patient’s tongue and hands, ¹⁸F-FDG PET image, cervical MRI, thoracic MRI, and lumbar MRI. A, B) Follow-up summary of MMSE and MoCA scores from Oct 2011 to Mar 2017 displayed a mild trend of decline in cognitive function. C, D) Atrophy of tongue (September 2016) and left muscle of thenar (March 2017). The atrophic areas are indicated by red markers. E) ¹⁸F-FDG PET revealed nonspecific decreased metabolism. F–H) The cervical, thoracic, and lumbar MRI results revealed chronic cervical and lumbar disc protrusion.

Fig.2

Follow-up brain MRI. Brain MRI in February 2012 (1.5 years post-onset) (A), June 2013 (3 years post-onset) (B), June 2015 (5 years post-onset) (C), and November 2016 (6 years post-onset) (D) showed gradual enlargement of frontotemporal sulci and lateral ventricles on transverse and sagittal T1-weighted sequences. Brain MRI in February 2012 (1.5 years post-onset) (E), June 2013 (3 years post-onset) (F), November 2016 (6 years post-onset) (G), and March 2017 (6.5 years post-onset) (H) showed increased atrophy of frontal/temporal lobes and both hippocampi on coronal FLAIR-weighted sequences.

In September 2016, she was referred to our department again. The neurological evaluation showed mild dysarthria, slow speech, dysphagia, pharyngeal reflex retardation, mild glossal amyotrophy (Fig. 1C), mild weakness of her left upper extremity (level IV), a positive bilateral Rossolimo sign, spastic paraplegia and myoclonus of both lower extremities (level I), and a positive bilateral Babinski sign. Her MMSE and MoCA scores were 26/30 and 17/30, respectively (Fig. 1A, B). These findings led to a suspicion of ALS/FTD, so therapy with Memantine, Baclofen, and Madopar was initiated. In November 2016, the patient developed further dysarthria and inability to speak a complete sentence. Due to dysarthria, she completed some tests in the cognitive examination by handwriting. Her MMSE score was 23/30 and her MoCA score was 15/30 (Fig. 1A, B). Cerebral MRI revealed obvious atrophy of frontal/temporal lobes and both hippocampi (Fig. 2D, G). In March 2017, the patient increasingly showed bulbar paralysis and weakness of extremities, exhibiting near-inability to speak, obvious dysdipsia, left muscle of thenar atrophy (Fig. 1D), and declined strength of extremities (left upper extremity level III, right upper extremity level IV, bilateral lower extremities level 0). Her MMSE score was 22/30 and MoCA score 16/30 (Fig. 1A, B). Cerebral MRI revealed increased atrophy of frontal and temporal lobes compared to previous images (Fig. 2H). The myoelectrography revealed lower motor neuron injury in cervical spinal cord including cervical enlargement and lumbar spinal cord. Spontaneous potential can be seen in a resting state, and motor unit action potential (MUAP) is too large with light contraction.

The family history showed no similar presentations in relatives of the patient (Fig. 3D). We identified the presence of a gene mutation in the ErbB4 sequence of the patient (II6) at the c.2136T>G SNP site, causing isoleucine to methionine substitution at codon 712 based on whole exome sequencing. The ErbB4 gene was assessed by evaluating two other members in the family, I2 and II4. The same mutation was identified in the patient’s unaffected mother (I2) (Fig. 3A).

Fig.3

DNA sequence, gene conservation, and pedigree of the family. DNA sequence at codon 712 of ErbB4 gene from the patient (A) and a control (B). The arrows indicate the mutated hemizygous site and a normal homozygous site. C) The p.I712M heterozygous missense mutation occurs at a highly conserved position in ErbB4, as shown by a comparison of the corresponding sequences of ten vertebrates. The bases identical to those in Homo sapiens are indicated in blue. D) The proband is indicated by an arrow (II-6). The patient’s mother (I-2) also received the genetic test and carried the mutation.

Genetic analysis

The mutation c.2136T>G, p.I712M of ErbB4 was detected and has not been reported as pathogenic elsewhere (Fig. 3A). The mutation led to isoleucine to methionine substitution at codon 712 in the ErbB4 sequence. The pathogenicity prediction of the missense mutation by Mutation Taster software was disease causing, with a probability equal to 0.99995. The mutation was not found in the dbSNP, 1000 G, or HGMD database, but was found 1 mutation carrier in the ExAC database and 3 mutants in the gnomAD database. The ErbB4 gene was also assessed by evaluating samples of buccal mucosal cells from the patient’s mother and sister, and the same mutation was detected in her unaffected mother, but not her sister (Fig. 3D). APOE genotyping of the patient was performed at the rs429358 and rs7412 loci, and direct sequencing identified the APOE ɛ3/ɛ3 genotype. The p.I712M heterozygous missense mutation is at a highly conserved position, as shown by a comparison of the corresponding sequences of ten vertebrates (Fig. 3C).

Analysis of ErbB4 mutation I712M cDNA by pyrosequencing and Sanger sequencing

ErbB4 mutation I712M (c.2136T>G) cDNA was amplified by RT-PCR from mRNA extracted from peripheral blood leucocytes of the patient. We found the same mutation in ErbB4 cDNA by Sanger sequencing (Fig. 4A) and pyrosequencing (Fig. 4B), which was consistent with the DNA sequencing results. Due to the unavailability of peripheral blood leucocytes from the patient’s mother, the cDNA genotype of the unaffected carrier was not analyzed.

Fig.4

Transcription of the ErbB4 mutation and predicted 3D structure of ErbB4 protein. cDNA of the ErbB4 variant (c.2136T>G) was analyzed by Sanger sequencing (A) and pyrosequencing (B). The mutation was detected in ErbB4 cDNA from peripheral blood leucocytes in a sample from the patient. The arrow indicates the mutated site. C) Predicted 3D structure of ErbB4 protein by SWISS-MODEL Server. In wild-type ErbB4 protein, the 712th amino acid is within a short β-pleated sheet structure, which was replaced by a random coil in the mutant ErbB4 protein.

Protein modeling

The known crystal structure of ErbB4 kinase (ID: 3BCE), containing 328 amino acids, is from the central part of the full-length ErbB4 protein. The mutant ErbB4 kinase structure was calculated according to the crystal structure of ErbB4 kinase. In the predicted mutant structure, substitution of an isoleucine residue to a methionine at the 11th amino acid resulted in the formation of a β-pleated sheet, an obvious structural change at the mutated site compared to the wild-type structure (Fig. 4D).

Functional analysis of ErbB4 mutant protein

HEK 293T cells were transfected with an empty-vector control or plasmids encoding either wild type or I712M mutant ErbB4 for 24 h, then starved of serum overnight, and stimulated with 0 or 100 ng/ml NRG1 for 10 min. For detection of phospho-ErbB4 and total ErbB4 protein levels, western blotting was performed antibodies against phospho-ErbB4 and ErbB4, respectively. Our research demonstrated that the levels of phospho-ErbB4 protein in the I712M mutation group stimulated with NRG1 was significantly decreased compared to wild type, whereas there were no obvious difference in the levels of total ErbB4, and NRG1-ErbB4 down-pathway signaling including phospho phosphoinositide 3 kinase (pPI3K) and phospho protein kinase B (pAkt) proteins between wild type and mutant ErbB4 groups (Fig. 5).

Fig.5

Functional analysis of wild type and mutant ErbB4 upon NRG1 stimulation. HEK 293T cells transfected with an empty-vector (Vehicle), plasmids encoding either wild type (WT), or mutant ErbB4 (I712M), were stimulated with or without 100 ng/ml NRG1. There was significantly decreased expression of phospho-ErbB4 when stimulated by NRG1 in the I712M mutation group compared to wild type, however, the expression of ErbB4, phospho-PI3K and phospho-Akt protein did not change in I712M mutation groups response to NRG1. All quantified data represent a mean ± SEM. Statistical significance was determined by two-way analysis of variance (ANOVA) test (GraphPad Prism 7.0). *p < 0.05 was considered significant. NS, not significant.

DISCUSSION

The patient exhibited frontal variant dystrophy after a stressful event as the initial symptom, a lack of obvious cognitive dysfunction, and non-obvious abnormities in MRI, which was similar to hysteria or stress-related disorders. After a relatively stationary stage of 5 years, the patient began showing spastic paraplegia and true bulbar paralysis with obvious decline of cognitive function and atrophy of frontal-temporal lobes in MRI. These findings suggested a clinical diagnosis of ALS/FTD. According to whole exon sequencing, an unreported pathogenic mutation of c.2136T>G (p.I712M) in ErbB4 gene was found in the patient. According to the American College of Medical Genetics and Genomics guidelines [11], the mutation c.2136 T>G (p.I712M) of ErbB4 gene is possibly pathogenic. However, due to lack of co-segregation, we could not identify that ErbB4 I712M mutation was completely responsible for ALS/FTD.

Due to the difficulty in obtaining central nervous tissue, we chose peripheral blood leucocyte of the patient to analyze the variant site of ErbB4 cDNA by Sanger sequencing and pyrosequencing. We detected the mutation in the patient’s cDNA sample through both methods. Unfortunately, peripheral blood from the patient’s mother and other family members was not available to transcription of the mutation. In a previous study, we did not detect a genomic mutation in cDNA from peripheral blood leucocyte from an early-onset FTD pedigree [12]. This may be the reason that ALS/FTD pathology involves central and peripheral tissues. To determine how the mutation identified in the ALS/FTD individual affected ErbB4 protein function, we investigated the auto-phosphorylation of ErbB4 in cells expressing either wild type or mutant (c.2136T>G) ErbB4 in the presence of NRG1. NRG1 is a neurotrophic factor localized in the cholinergic synapses. ErbB4 is activated by increased tyrosine kinase activity upon NRG1 binding, resulting in auto-phosphorylation of the C-boutons [8, 13]. NRG1-ErbB4 signaling activated numerous downstream pathways such as PI3K, Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and so on, crucial to neuronal development, neuronal migration, axonal navigation, and synaptic function. NRG1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS [14]. Takahashi et al. firstly demonstrated that ErbB4 (A1275W) mutation led to reduced auto-phosphorylation of ErbB4 upon NRG1 stimulation involved in the pathogenesis of ALS [8]. NRG1 in the post synaptic face of C-boutons and ErbB4 in the motor neurons were both significantly reduced in Superoxide dismutase 1 (SOD1)-ALS mice [13]. Similar to the above studies, we found a significantly decreased level of phospho-ErbB4 protein in ErbB4 (I712M) mutation compared to wild type, which suggested an abnormally reduced NRG1-ErbB4 signaling identified in ALS/FTD. At present, there are few researches about the effect and mechanism of NRG1 and ErbB4 protein on ALS or FTD pathology. ErbB4 null mice displayed the derangement of axon guidance and path-finding of motor neuron during embryogenesis [15], and also showed reduced GABA release and impaired behavior in various paradigms [16]. Disruption of NRG1-ErbB4 signaling in the parvalbumin interneurons might lead to cognitive impairment in a mouse model of sepsis-associated encephalopathy [16], and the abnormality due to ErbB4 deficiency during development were alleviated by restoring ErbB4 expression at the adult stage in mice [16]. NRG1 intramuscular injection enhanced muscle reinnervation by collateral sprouting, and administration of lapatinib (ErbB receptor inhibitor) completely blocked it, which demonstrated that NRG1-ErbB4 pathway played a crucial role in the collateral reinnervation process [16]. Based on previous studies, the deficiency in NRG1-ErbB4 signaling had important effects on neurological disorders. In the present research, we did not find significant changes of phospho-PI3K and phospho-Akt proteins levels due to ErbB4 I712M mutation, which might be related to other NRG1-ErbB4 downstream pathways involved in ALS/FTD pathology, and it needs future study.

ErbB4 was reported as a pathogenic gene of ALS type 19 (MIM: 615515), but has never previously been identified as pathogenic gene for FTD. According to the McKhann clinical criteria for FTD [17], our case fit the criteria of an early change in behavioral deficits, and these deficits were not due to other pathological conditions. This case is the first to suggest an ErbB4 mutation associated with clinical diagnosis of FTD and concomitant diagnosis of ALS. The patient’s unaffected mother carried the same mutation, and 1 healthy mutation carrier in the ExAC database and 3 mutants in the gnomAD database, were also identified, suggesting incomplete penetrance, similar to the first identified Japanese family of ALS with an ErbB4 mutation [8]. It is known that not all loss of function genetic mutants developed phenotypic changes, and many deleterious mutations only produced a phenotype in a subset of mutant individuals. Nichols et al. also demonstrated that penetrance was inherited as a liability-threshold trait [18].

In summary, we firstly identified ErbB4 mutation (I712M) in an ALS/FTD patient, and the mutation caused significantly decreased auto-phosphorylation of ERBB4 protein in the presence of NRG1, which was identified as a deleterious mutation. In addition, there was a factor that limited the findings of the present study. Without a large family showing dementia and paralysis, and enough gene samples from family members, it was difficult to demonstrate that ErbB4 I712M was fully responsible for ALS/FTD. However, we have provided a clue for firstly discussing the pathogenicity significance of ErbB4 I712M mutation in FTD and concomitant diagnosis of ALS, which should foster an understanding of the effect of ErbB4 mutation on ALS/FTD when the mutation can be verified in a large family.

Footnotes

ACKNOWLEDGMENTS

This study was supported by grants of National Key R&D Program of China (2017YFC1310501500), Clinical research center project of Shanghai Mental Health Center (CRC2017ZD02), Western medical guidance project of Shanghai science and Technology Commission (17411970100), National Natural Science Foundation of China (81301139, 91949129 and 81771164), and Precision medical research project of Shanghai Jiao Tong University School of Medicine (15ZH4010). This study was also supported by the Open Research Funds of State Key Laboratory of Cellular Stress Biology, Xiamen University (SKLCSB2019KF014) and Natural Science Foundation of Fujian Province (2018D0022). We thank the patients and family members for their participation in this study.

Authors’ disclosures available online ().

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