PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome

Abstract

Objective: To investigate the genetic and physiologic basis of the neuromuscular symptoms of hypotonia-cystinuria syndrome (HCS) and isolated PREPL deficiency, and their response to therapy.

Methods: We performed molecular genetic, histochemical, immunoblot, and ultrastructural studies, investigated neuromuscular transmission in vitro in a patient with isolated PREPL deficiency, and evaluated the effect of pyridostigmine in this patient and in 3 patients with the HCS.

Results: HCS is caused by recessive deletions involving the SLC3A1 and PREPL genes. The major clinical features of HCS are type A cystinuria, growth hormone deficiency, muscle weakness, ptosis, and feeding problems. The proband with isolated PREPL deficiency had myasthenic symptoms since birth and a positive edrophonium test but no cystinuria. She and 1 of 3 patients with HCS responded transiently to pyridostigmine during infancy. The proband harbors a paternally inherited nonsense mutation in PREPL and a maternally inherited deletion involving both PREPL and SLC3A1; therefore, the PREPL deficiency determines the phenotype. We detected no PREPL expression in the patient's muscle and endplates. Electrophysiology studies revealed decreased quantal content of the endplate potential and reduced amplitude of the miniature endplate potential without endplate acetylcholine receptor deficiency or altered endplate geometry.

Conclusion: Isolated PREPL deficiency is a novel monogenic disorder that causes a congenital myasthenic syndrome with pre- and postsynaptic features and growth hormone deficiency. The myasthenic symptoms in PREPL deficiency with or without cystinuria may respond to pyridostigmine in early life. We attribute the myasthenia to abrogated interaction of PREPL with adaptor protein 1.

Figure 1. Clinical and genetic features

(A, B) Patient at age 6 months. Note marked hypotonia with frog-leg posture and head drop. (C, D) Positive response to pyridostigmine at age 11 weeks. (E) PCR fragments in the patient, the parents, and a control. Upper lane: junction fragment, present only in the patient and her mother; middle lane: PCR with forward primer in the nondeleted and reverse primer in the deleted region of SLC3A1; lower lane: PCR with forward primer in the deleted and reverse primer in the nondeleted region of PREPL. Only the normal allele will produce a band in the 2 lower lanes. (F) Sequence chromatograms show a single base pair (bp) deletion (first bp after the vertical bar) in the patient's genomic DNA (gDNA) and complementary DNA (cDNA) and in the father's gDNA and cDNA, not present in the mother. cDNA primers were designed to amplify only the nondeleted allele so that only the paternal allele is sequenced in the patient's cDNA, clearly demonstrating the single bp deletion. (G) Overview of 2p21 region showing the identified deletions. The patient with isolated PREPL deficiency was compound heterozygous for the nonsense mutation (red arrow) and deletion J. Deletions A, B, and H were homozygous in the patients with hypotonia-cystinuria syndrome.

Figure 2. PREPL expression (A–E) and EP ultrastructure (F–I)

(A, B) PREPL (green signal) and α-bungarotoxin (red signal) localization in control muscle fibers and endplates (EPs). (C) Merged image of A and B. (D) PREPL localization in patient muscle fibers and EPs. Note absence of PREPL expression. (F) Ultrastructure of well-developed EP harboring abundant synaptic vesicles. Asterisks indicate vesicles focused on active zones. (G) Normal density and distribution of the acetylcholine receptor on junctional folds revealed by peroxidase-labeled α-bungarotoxin. (H) Abnormal nerve terminal filled with neurofilaments surrounded by degenerate organelles. (I) An EP harbors autophagic vacuoles in the junctional sarcoplasm (X). A postsynaptic region is denuded of its nerve terminal (x). The asterisk indicates a nearby nerve sprout. Bar in D = 25 μm for panels A–E. Bars in F and G = 0.5 μm; bar in H = 1.3 μm; bar in I = 2 μm.