A Novel AGRN Mutation Leads to Congenital Myasthenic Syndrome Only Affecting Limb-girdle Muscle.

BACKGROUND: Congenital myasthenic syndromes (CMSs) are a group of clinically and genetically heterogeneous disorders caused by impaired neuromuscular transmission. The defect of AGRN was one of the causes of CMS through influencing the development and maintenance of neuromuscular transmission. However, CMS reports about this gene mutation were rare. Here, we report a novel homozygous missense mutation (c.5302G>C) of AGRN in a Chinese CMS pedigree.
METHODS: We performed a detailed clinical assessment of a Chinese family with three affected members. We screened for pathogenic mutations using a disease-related gene panel containing 519 genes associated with genetic myopathy (including 17 CMS genes).
RESULTS: In the family, the proband showed limb-girdle pattern of weakness with sparing of ocular, facial, bulbar, and respiratory muscles. Repetitive nerve stimulation showed a clear decrement of the compound muscle action potentials at 3 Hz only. Pathological analysis of the left tibialis anterior muscle showed predominance of type I fiber and the presence of scattered small angular fibers. The proband's two elder sisters shared a similar but more severe phenotype. By gene analysis, the same novel homozygous mutation (c.5302G>C, p. A1768P) of AGRN was identified in all three affected members, whereas the same heterozygous mutation was found in both parents, revealing an autosomal recessive transmission pattern. All patients showed beneficial responses to adrenergic agonists.
CONCLUSIONS: This study reports a Chinese pedigree in which all three children carried the same novel AGRN mutation have CMS only affecting limb-girdle muscle. These findings might expand the spectrum of mutation in AGRN and enrich the phenotype of CMS.

INTRODUCTION

Congenital myasthenic syndromes (CMSs [MIM 608931]) represents a group of clinically and genetically heterogeneous disorders caused by impaired neuromuscular junction (NMJ) transmission leading to fatigable weakness.[1] Conventionally, CMS were classified on the basis of the location of a mutated protein as presynaptic, synaptic basal lamina-associated, or postsynaptic. Currently, gene defects that influence the development and maintenance of NMJ are assigned to a separate group of the CMS and rank second in the disease causes following defects of the acetylcholine receptors (AChRs).[2] These genes include RAPSN, DOK7, LRP4, MUSK, and AGRN.[34] Agrin, encoded by AGRN, is a cell-specific heparan sulfate proteoglycan generated by alternative splicing. Motoneuron-derived agrin is secreted from nerve terminals into the synaptic cleft and leads to clustering and synthesis of postsynaptic AChRs through activation of the postsynaptic LRP4-MuSK-Dok-7 complex.[5] There are only a few cases reported about this gene mutation so far.[6789] Here, we report a novel homozygous missense mutation (c.5302G>C) of AGRN in a Chinese CMS pedigree.

METHODS

Ethical approval

The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Chinese People's Liberation Army General Hospital. Informed consent was obtained from all subjects.

Clinical assessment

A detailed history was taken, and a thorough neurological examination was performed. Electrophysiological studies and muscle pathology studies were performed to determine the location and nature of the impairment. Auxiliary examinations included muscular magnetic resonance imaging (MRI), creatine kinase levels and anti-AChR and anti-MuSK antibody tests. The diagnosis of CMS can be suspected when there are clinical symptoms of early onset fatigable muscle weakness, a positive family history, and a decremental response of repetitive nerve stimulation (RNS). Genetic studies are needed to confirm the diagnosis.

Genetic and bioinformatics analyses

Venous blood samples were obtained from the pedigree. Genomic DNA was extracted from peripheral blood using a standard procedure. The amplified DNA of the proband was captured with a disease-related gene panel containing 519 genes associated with genetic myopathy including 17 CMS genes [Supplementary Table S1] using biotinylated oligoprobes (MyGenostics GenCap Enrichment technologies) and sequenced on an Illumina HiSeq 2000. The candidate variant was confirmed by Sanger's sequencing and was evaluated the pathogenicity by three algorithms, namely, SIFT (http://sift.jcvi.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph2/index.shtml) and Mutation Taster (http://mutationtaster.org/) as described previously. Sanger's sequencing was then conducted across the family.

Supplementary Table S1

The list of 519 genes related with genetic myopathy contained in the panel

ABAT	ABCB1	ABCC2	ABCC8	ACOX1	ACY1	ADCK3	ADSL
AGA	AHI1	AKT2	AKT3	ALDH4A1	ALDH5A1	ALDH7A1	ALG1
ALG3	ALG6	ALG8	ALG9	ALG11	ALG12	ALG13	AMT
APIS2	APTX	ARFGEF2	ARG1	ARHGEF15	ARHGEF9	ARL13B	ARSA
ARSB	ARX	ASAH1	ASPA	ATIC	ATN1	ATP13A2	ATP1A2
ATP1A3	ATP2A2	ATP5A1	ATP6AP2	ATP6VOA2	ATP7A	ATPAF2	ATR
ATRX	AVPR1A	B4GALT1	BCKDHA	BCKDHB	BCKDK	BCS1L	BDNF
BLK	BRAF	BRAT1	BRD2	BTD	BUB1B	C12orf57	C12orf65
C12orf12	CACNA1A	CACNA1C	CACNA1H	CACNB4	CASK	CASR	CBL
CC2D2A	CCL2	CDK5RAP2	CDKL5	CDON	CEL	CENPJ	CEP152
CEP290	CHD2	CHD4	CHD7	CHD8	CHRNA2	CHRNA4	CHRNB2
CISD2	CLCN2	CLCN4	CLCNKA	CLCNKB	CLN3	CLN5	CLN6
CLN8	CNTN2	CNTNAP2	COG1	COH4	COG5	COG6	COG7
COG8	COL18A1	COL4A1	COQ2	COQ9	COX15	CP	CPT1A
CPT2	CREBBP	CSTB	CTSA	CTSD	CTSF	CUL4B	CYP1B1
CYP2A6	CYP2B6	CYP2C19	CYP2C9	CYP2D6	CYP2R1	CYP2U1	CYP3A5
DBT	DCAF17	DCX	DDC	DDOST	DEPDC5	DHCR7	DLD
DOLK	DPM1	DPM2	DPM3	DPYD	DYRK1A	EEF1A2	EFHC1
EFHC2	EHMT1	EIF2AK3	EIF2B1	EIF2B2	EIF2B3	EIF2B4	EIF2B5
EMX2	EPM2A	ERCC6	ERCC8	ETFA	ETFB	ETFDH	FA2H
FAAH	FAM126A	FDG1	FGF8	FGFR1	FGFR2	FGFR3	FH
FKRP	FKTN	FLVCR2	FMR1	FOLR1	FOXR1	FOXH1	FOXP1
FOXP2	FOXP3	FTL	FUCA1	GABBR2	GABRA1	GABRA2	GABRA3
GABRD	GABRG2	GALC	GALNS	GAMT	GATA6	GATM	GCDH
GCK	GCSH	GFAP	GLB1	GLDC	GLI2	GLT3	GLIS3
GLRA1	GLRB	GLUD1	GLUL	GNAO1	GNE	GNPTAB	GNPTG
GNS	GOSR2	GPC3	GPHN	GPR56	GRIA3	GRIN1	GRIN2A
GRIN2B	GU2B	HADH	HCN1	HCN4	HDAC8	HEXA	HEXB
HGSNAT	HNF1A	HNF1B	HNF4A	HNRNPU	HOXA1	HPD	HPRT1
HGAS	HSD17B10	HSD17B4	HYAL1	IBA57	IDH2	IDS	IDUA
IER3IP1	INPP5E	INS	INSR	IQSEC2	KAT6B	KCNA1	KCNV2
KCDH7	KDM5C	KIAA1279	KLF11	KRAS	L1CAM	L2HGDH	LARGE
LRB	LGI1	LIAS	LIG4	LRPPRC	MAGI2	MAGT1	MAP2K1
MAP2K2	MAPK10	MBD5	MCOLN1	MCPH 1	ME2	MECP2	MED12
MED17	MEF2C	MET	MFSD8	MGAT2	MID1	MKKS	MLC1
MMACHC	MOCS1	MOCS2	MOGS	MPDU1	MPI	MTHFR	MTR
MTRR	MYBPC1	NAGLU	NDE1	NDUFA1	NDUFA2	NDUFS1	NDUFS3
NDUFS4	NDUFS7	NDUFS8	NDUFV1	NEU1	NEUROD1	NEUROG3	NF1
NGLY1	NHEJ1	NHLRC1	NHS	NIPBL	NKX2-2	NLGN3	NLGN4X
NODAL	NOTCH3	NPC1	NPC2	NPHP1	NRAS	NRXN1	NSD1
OFD1	OPA1	OPHN1	PAFAH1B1	PAK3	PANK2	PAX4	PAX6
PC	PCDH19	PDGFRB	PDHA1	PDHX	PDSS1	PDSS2	PDX1
PEX1	PEX10	PEX12	PEX13	PEX14	PEX16	PEX19	PEX2
PEX26	PEX3	PEX5	PEX6	PEX7	PGK1	PGM1	PHF6
PHFDH	PIGV	PIK3CA	PIK3R2	PLA2G6	PLAGL1	PLCB1	PLP1
PMM2	PNKP	PNPO	POLG	POMGNT1	POMT1	POMT2	PPT1
PQBP1	PRICKLE1	PRICKLE2	PRODH	PRRT2	PSAP	PSAT1	PTCH2
PTEN	PTF1A	PTPN11	QDPR	RAB39B	RAB3GAP1	RAD21	RAF1
RAI1	RARS2	RFT1	RFX6	RNASEH2A	RNASEH2B	RNASEH2C	RPGRIP1L
RPS6KA3	RRP1B	RTTN	SAMHD1	SCARB2	SCN10A	SCN11A	SCN1A
SCN1B	SCN2A	SCN2B	SCN3A	SCN3B	SCN4B	SCN5A	SCN8A
SCN9A	SCO2	SDHA	SERPINI1	SETBP1	SGCE	SGSH	SHANK2
SHANK3	SHH	SHOC2	SIX3	SLC16A2	SLC17A5	SCL19A2	SLC19A3
SLC1A3	SLC2OA2	SLC25A15	SLC25A19	SLC25A22	SLC2A1	SLC35A1	SLC35A2
SLC35C1	SLC46A1	SLC6A4	SLC6A5	SLC6A8	SLC9A6	SMC1A	SMC3
SMN1	SMPD1	SMS	SNAP29	SNIP1	SOS1	SPRED1	SPTAN1
SRD5A3	SRPX2	ST3GAL5	STIL	STRADA	STXBP1	SUCLA2	SUMF1
SUOX	SURF1	SYN1	SYNGAP1	SYP	TACO1	TBC1D24	TBX1
TCF4	TGIF1	TMEM165	TMEM216	TMEM67	TMEM70	TPP1	TREX1
TRPM6	TSC1	TSC2	TSEN2	TSEN34	TSEN54	TUBA1A	TUBA8
YUBB2B	TUSC3	UBE3A	UCP2	VANGL1	VPS13A	VPS13B	VPK1
WDR45	WDR62	WFS1	ZEB2	ZFP57	ZIC2	CHAT*	COLQ*
LAMB2*	CHRNA1*	CHRNB1*	CHRND*	CHRNE*	CHRNG*	AGRN*	DOK7*
MUSK*	RAPSN*	GFPT1*	DPAGT1*	ALG2*	PLEC*	SCN4A*

*The 17 genes are congenital myasthenic syndrome related genes screened in the study.

RESULTS

Clinical features

The proband (II-3, the pedigree shown in Figure 1) was a 27-year-old man who had an apparently normal childhood and adolescence except failing to pass the physical examination of high jump and running. At 21 years old, he began to suffer from fatigable weakness of lower limbs. Gradually, he had difficulty standing up from a squat position, jumping, and running. During the cause of the disease, he had no ptosis, bulbar or facial weakness. Neurological examination at the age of 25 years revealed normal cranial nerves and mild muscle atrophy of lower legs. Muscle strength of lower limbs was Medical Research Council (MRC) Grade 4−/5 in proximal and Grade 4+/5 in distal. Tendon reflexes were preserved except bilateral Achilles reflexes. Ocular, facial, bulbar, and respiratory muscles were not involved. Creatine kinase level was normal and anti-AChR, and anti-MuSK antibody tests were negative. The MRI of lower extremities was normal. The nerve conduction study and needle electromyography were within normal limits. RNS at 3 Hz evoked from common peroneal nerves showed a clear decrement of the compound muscle action potentials, with 16% and 18% decline in left and right tibialis anterior, respectively. No significant changes were recorded of RNS at 10 Hz or 20 Hz. Pathological analysis of the left tibialis anterior muscle under light microscopy showed a predominance of type I fiber and the presence of scattered small angular fibers [Figure 2].

Figure 1

A Chinese congenital myasthenic syndrome pedigree with a novel AGRN mutation only affecting limb-girdle muscle. Arrow indicates the proband. The homozygous AGRN mutation (c.5302G>C) was inherited from parents.

Figure 2

Pathological results of left tibialis anterior muscle from the proband (original magnification, ×100). (a) Presence of scattered small angular fibers (H & E staining). (b) ATPase staining showed predominance of type I fiber (dark).

The other two elder sisters shared a similar but more severe phenotype. The 29-year-old sister (II-2) suffered from lower limb weakness at the age of 7 years. She complained of walking slowly, difficulty in climbing and a tendency to fall. Upper limbs became involved from the age of 9 years. Neurological assessment at 12 years old showed normal cranial nerve function except trapezius muscles weakness (MRC Grade 4/5). Muscle strength of limbs was Grade 4/5 in proximal and Grade 5−/5 in distal. Deep tendon reflexes were decreased. Muscle enzyme levels were normal. Needle electromyography of distal muscles in four extremities showed short duration and low amplitude motor unit potentials with a few abnormal spontaneous potentials. Nerve conduction studies were normal. Pathological analysis of muscle biopsy under light microscopy revealed type II muscle fiber atrophy. Another sibling, a 31-year-old female (II-1), showed a similar manifestation, but she did not undergo evaluation.

Genetic analysis

We identified a novel homozygous missense mutation (c.5302G>C) in exon 31 of AGRN leading to the substitution of alanine to proline in the C-terminal LG2 domain of agrin (p. A1768P; RefSeq: NM_198576). All three siblings were homozygous for the mutation while both parents were heterozygous [Figure 3]. This variation is not found in ExAC population database. SIFT predicted the substitution to affect protein function with a score of 0.03. Polyphen revealed the mutation to be probably damaging with a score of 1.0 and Mutation Taster predicted that this mutation was disease-causing. Therefore, we made the diagnosis of CMS caused by a novel homozygous mutation in AGRN (c.5302G>C) (we have submitted the variant to Leiden Open Variation Database http://databases.lovd.nl/shared/variants/0000128826).

Figure 3

Sanger sequences of AGRN mutation (c.5302G>C) across the family. The red arrow indicated the mutation site.

Treatment and follow-up

First treatment with pyridostigmine only showed a beneficial response during the 1st month, then, the symptoms were aggravated, so we tried ephedrine and acquired an evident symptomatic improvement after only 3 days of treatment. Due to the difficulty in obtaining ephedrine, we changed the treatment to salbutamol and observed a similar therapeutic effect as ephedrine. After treatment, the more severely affected sister (II-1) could walk a much longer distance, improving from <50 m to more than 500 m. All three patients are still receiving treatment and have taken salbutamol (2 mg tid) for more than 1 year, and the movement status is sustained.

DISCUSSION

We report a Chinese pedigree with all three CMS patients harboring the same novel missense pathogenic mutation (c.5302G>C p. A1768P) of AGRN. Genetic analysis revealed both parents were heterozygous carrying one single mutated allele that had been transmitted to their three affected children. The parents denied that they were consanguineous, but both of them were from a small village. To the best of our knowledge, previously, only four reports described CMS caused by defects in AGRN, which displayed heterogeneous clinical features. In 2009, Huzé et al.[6] first reported two siblings from a consanguineous family carrying a homozygous missense mutation (G1709R) and presented with ptosis, mild facial and limb-girdle muscles weakness. The second report described a severe CMS patient who required continuous respiratory support caused by two compound heterozygous mutations (V1727F, Q353X).[7] The third article reported five patients from three unrelated families who shared different phenotypes of distal muscle weakness and atrophy.[8] The latest case reported a 17-month-old boy harboring a homozygous mutation (G1765S) who presented with dropped head in addition to proximal muscle weakness, ptosis, and ophthalmoplegia.[9] Acetylcholinesterase inhibitors were not helpful in most of the cases, while adrenergic agonists provided a positive effect for some of the patients. More detailed, there are three mutations located in the LG2 domain as well as our report. As we know, agrin includes three globular, C-terminal LG domains, an N-terminal (NtA) domain and follistatin-like domains.[10] The NtA domain is responsible for binding to basal laminae. The C-terminal LG3 domain is critical for the aggregation of AChRs and other molecules at the NMJ, whereas LG1 and LG2 domain of agrin are involved in interacting with α-dystroglycan, which is a multimeric transmembrane protein complex and is thought to be associated with structural stability of muscle cell membrane.[11] The interaction seems to promote the binding of agrin to the surface of muscle cells, and hence increase the potency of agrin in inducing AChRs clustering, which is an important event in NMJ development.[12] The way in which the interaction affects neuromuscular transmission remains unclear. Studied about the G1709R substitution in LG2 domain showed that the mutation did not affect agrin's ability to activate MuSK or cluster AChRs, nor does it affect the interaction with a-dystroglycan, it seemed to perturb the endplate maintenance.[6] On the contrary, another analysis showed that V1727Fmutation in LG2 domain significantly reduced AChRs clustering activity by impairing MuSK activation and increased affinity to α-dystroglycan, which mimics non-neural isoform agrin.[7] In our report, the patients showed a typical electrophysiological change in the RNS test. The pathology demonstrated the predominance of type I fiber and a slight myopathic change. The therapeutic effects of adrenergic agonists on all three patients are evident. All these features are in accordance with congenital muscular dystrophy caused by AGRN mutation. However, the clinical manifestations of our patients were somewhat different from those of previously reported cases. They showed a limb-girdle pattern weakness without the involvement of ocular, facial, bulbar, and respiratory muscles. Although bearing the same mutation, the three siblings showed variations in age of onset and in symptom severity. The missense mutation we identified were predicted to affect the function of the protein. However, future investigations are needed to pin down the detailed molecular mechanism how a defect in the C-terminal LG2 domain of agrin influence NMJ.

In conclusion, we report a Chinese CMS pedigree with a novel AGRN mutation only affecting limb-girdle muscle. The study findings might expand the spectrum of mutation in AGRN and enrich the phenotype of CMS.

Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.