A Novel Missense Variant in the AGRN Gene; Congenital Myasthenic Syndrome Presenting With Head Drop

Abstract

Congenital myasthenic syndromes (CMS) are a heterogeneous group of diseases of the neuromuscular junction caused by compromised synaptic transmission. Clinical features include early-onset weakness of limbs and oculobulbar muscles resulting in hypotonia, bulbar paresis, ptosis, and hypoventilation. The first dropped head syndrome in children were detected in 2 patients with LMNA and SEPN1 mutations. We report a 17-month-old boy with dropped head and limb–girdle weakness, who had no ptosis or ophthalmoplegia at presentation. We performed whole exome sequencing, which revealed a homozygous missense variant in the AGRN gene c.5023G>A, p.Gly1675Ser in the LG2 domain, which is predicted to be likely disease causing by in silico tools. Agrin is known to play a critical role in the development and maintenance of the neuromuscular junction. Agrinrelated CMS is one of the rarest subtypes. Of note, our patient is the first described patient with agrinrelated CMS with dropped head phenotype.

INTRODUCTION

Congenital myasthenic syndromes (CMS) are a group of inherited diseases of the neuromuscular junction (NMJ), where the transmission is compromised at the motor endplate.¹ Typical presentations of CMS include early-onset weakness and/or fatigability of limbs and oculobulbar muscles resulting in hypotonia, bulbar paresis, ptosis, hypoventilation, and apneic episodes.² The first dropped head syndrome defined in children were in 2 patients with LMNA and SEPN1 mutations.³ In the literature, no CMS patient with dropped head phenotype has been reported.

CASE REPORT

Informed consent was obtained from the parents for genetic investigations, recording, and publishing the disease-related information. The proband of Turkish origin, from a consanguineous marriage between first cousins, was born with normal weight and APGAR scores. There was no described hypoventilation or apneic episodes during the early postnatal period. He was able to hold his head, sit unsupported, stand and walk unsupported a few steps by 1 year of age, but beginning at age 1, his parents noticed frequent head droppings and neck weakness throughout the day. First visit to pediatric neurology clinic at 17 months of age revealed prominent neck extensor muscle weakness along with mild limb–girdle weakness. There was no ptosis, ophthalmoplegia, or distal muscle weakness at presentation. Creatine kinase level was 84 U/L. His language and social skills were consistent with his age. Over the following year, he was followed with a provisional clinical diagnosis of nonprogressive congenital myopathy with neck weakness (Fig. 1).

FIGURE 1.

Phenotype and genetic analysis. A–C, Patient at 4 years of age. Prominent neck weakness, ophthalmoplegia, and bilateral ptosis were noted. Increased lumbar lordosis is notably seen. Head dropping despite adequate ambulation is noted. D, Sanger confirmation of the variant in the family. The patient was homozygote, and parents were heterozygotes for the Gly1675Ser variant. The variant was present only in heterozygous state and at extremely low frequencies in control populations. E, The conservation of glycine at position 1675 within various species shown by homologene-generated amino acid alignment (http://www.ncbi.nlm.nih.gov/homologene).

At 30 months of age, he presented with bilateral ptosis and restrictive eye movements deteriorating during the day. His weakness of extensor neck muscles and lordotic gait were still present. At this point, the clinical picture was more consistent with myasthenia, thus screens for acetylcholine receptor antibodies and muscle-specific receptor tyrosine kinase (MuSK) were done, with negative results. Electromyographic study showed 10%–30% electrodecremental response during repetitive stimulation at 3.5 Hz stimulation of the right ulnar nerve, whereas no decline was observed at 20 Hz stimulation. His initial presentation with prominent neck weakness sparing the eyes was not considered as a typical presentation of CMS. Thus, we considered LMNA -or SEPN1-related myopathies in the differential diagnosis. In the interim, pyridostigmine and salbutamol treatment was started based on the progression of symptoms consistent with CMS. During his most recent visit at 5 years of age, he still had difficulty in holding head in an upright position. However, it was observed that the patient’s symptoms typically improved after salbutamol and the first few hours of pyridostigmine administration.

As the yield achieved from all diagnostic tests was unsatisfactory, whole exome sequencing (WES) was performed. WES of the proband’s DNA isolated from peripheral blood revealed a homozygous missense variant in the AGRN gene in exon 29, g.chr1:985853G>A, c.5023G>A, p.Gly1675Ser (NM_198576), which encodes agrin. This variant was not present in the Exome Variant Server databases but was detected in the heterozygous state in 3/8548 East Asian and 1/64,402 European chromosomes by the Exome Aggregation Consortium. Glycine at 1675 is conserved in vertebrates, and analysis of the serine variant at this position is predicted to be disease causing by Mutation Taster. Review of the WES data did not reveal any potential pathogenic mutation in SEPN1, LMNA, or any other known muscle disease-related genes. The AGRN Gly1675Ser variant was confirmed by Sanger sequencing in the proband and his parents. As predicted from the WES results, the proband appeared homozygous for the chr1:985853A variant. The sequence chromatograms for DNA from both parents clearly contained both the normal (control) peak representing a G at this position and an overlapping second A peak, demonstrating that both are heterozygotes for the same mutation (Fig. 1).

DISCUSSION

The patient described in this report harbor a homozygous Gly1675Ser mutation in the laminin globular domain (LG2) of the agrin protein. Agrin is a heparan sulfate proteoglycan known to play a critical role in the development and maintenance of the NMJ.⁴ Together with the low-density lipoprotein receptor–related protein 4 (LRP4) and MuSK, agrin forms agrin–MuSK–LRP4 signaling system, which is crucial for the development and maintenance of NMJ.⁵ Secreted by the nerve terminal into the synaptic space, agrin plays the trigger role in activation of MuSK and clustering of MuSK and LRP4.⁵

Agrin-related CMS is very uncommon. A recent study from the United Kingdom assessing the prevalence of genetic defects in childhood myasthenia revealed CHRNE, RAPSN, and DOK7 as the most common genes identified in the patients, and no AGRN mutation was detected in the study.⁶ However, we acknowledge that the prevalence of subtypes in the United Kingdom may not represent the prevalence of the subtypes in Turkey.

Nicole et al described 5 patients from 3 different kinship in different range of age groups. Homozygous and heteroallelic mutations dispersed through the entire gene resulted in a distinct phenotype associated with slowly progressive distal muscle weakness and muscle atrophy with fat replacement sparing axial and ocular muscles (Table 1).⁸ Maselli et al⁴ reported a different and more severe phenotype in a 39-year-old man, including respiratory failure, ophthalmoplegia, and facial weakness, which were caused by 2 heteroallelic mutations (p.Gln353* and p.Val1727Phe). The mutations were shown to be located in follistatin-like domain and laminin globular domain (LG2) of the protein (Table 1). Huze et al⁷ reported a milder phenotype in 42- and 36-year-old siblings resulting from a missense homozygous mutation (Gly1709Arg), in which bilateral ptosis, mild external ophthalmoplegia, and thin thorax were prominent features. The mutation was located in the LG2 domain, and in vitro expression studies revealed that the mutation perturbs endplate maintenance without interfering postsynaptic differentiation.⁷ The mutation identified in our patient is predicted to be located in LG2 domain based on the known structure of the mouse agrin.⁴ These data suggest features like ophthalmoplegia and limb–girdle weakness that can be common findings resulting from the mutations in LG2 domain, which interacts with alpha-dystroglycan and integrins.^4,7 Our case with typical head drop shows a distinctive presentation among other reported AGRN-related CMS cases.

TABLE 1.

Clinical and Genetic Overview of the Patients Reported in the Literature

Article	Clinical Phenotype	Age at Presentation, yrs	Mutation	Exon	cDNA Change	Protein Alteration	Altered Domain
Huze et al7	Ptosis, facial weakness, ophthalmoplegia, thin thorax, and pelvis	Patient 1: 42	Missense homozygous	29	c.5125G>C	p.Gly1709Arg	LG2
		Patient 2: 36
Maselli et al4	Ptosis, ophthalmoplegia, respiratory insufficiency	39	Compound heterozygous	6	N/A	Gln353*	FS
				30		Val1727Phe	LG2
Nicole et al8	Distal muscle weakness, distal muscle atrophy	Patient 1: 45	Compound heterozygous	2	c.226G>A	Gly76Ser	Nta
		Patient 2: 43			Chr1 deletion	Chr1 deletion
		Patient 3: 32	Compound heterozygous	2	c.314A>T	Asn105Ile	Nta
		Patient 4: 23		7	c.1362dupC	Ser455Glnfs*8	Kazal-like 4
		Patient 5: 25	Missense homozygous	32	c.5611G>A	Gly1871Arg	LG3
This study	Dropped head phenotype, proximal muscle weakness	1.5	Missense homozygous	29	c.5023G>A	Gly1675Ser	LG2

All reported disease-causing AGRN mutations are shown to be recessive inheritance.^4,7,8

cDNA, complementary DNA; FS, follistatin-like domain; LG2, laminin G-like domain 2; LG3, laminin G-like domain 3; Nta, N-terminal domain.

Salbutamol treatment is recommended to patients with mutations in the agrin–LRP4–MUSK–DOK7 pathway,⁹ thus we included salbutamol in the treatment protocol. Of note, we observed no deterioration in the symptoms after pyridostigmine was added to the protocol.

Although there is evidence suggesting pathogenicity of the homozygous Gly1675Ser variant in our patient, additional studies are needed to clarify its clinical significance. Additional data demonstrating a damaging effect on agrin function would be needed to clarify the significance of this variant.

CONCLUSIONS

Our case demonstrated a clinical picture of CMS with dropped head phenotype. WES identified a homozygous missense variant of the AGRN gene, strongly suggesting the molecular defect behind this phenotype. Sequence analysis of additional similar cases, and functional studies on protein levels and function, will be important to confirm this variant as a disease causing mutation and to further define the clinical spectrum of findings associated with AGRN mutations.