First case of compound heterozygosity in ALS2 gene in infantile‐onset ascending spastic paralysis with bulbar involvement

To the Editor: Homozygous mutations in the ALS2 gene are causative for autosomal recessive, early-onset forms of upper motor neuron (UMN) diseases described as infantile-onset ascending hereditary spastic paralysis (IAHSP) and juvenile primary lateral sclerosis (JPLS) with overlapping phenotypes (1–7). They are also rarely associated with lower motor neuron (LMN) involvement in juvenile ALS, ALS2 (1, 2, 8). The homogeneous phenotype associated with ALS2 mutation includes onset of stiffness and spasticity in the lower limbs at the age of 1–2 years and progression to upper limbs weakness and dysarthria during childhood. Ultimately, individuals become tetraplegic with preserved intellect (5, 9–11). Here, we describe the first family of infantile ascending spastic paralysis with bulbar involvement with two affected siblings, both showing compound heterozygosity for two novel mutations in ALS2. The parents are non-consanguineous and healthy. The proband (II-1, Fig. 1a) was born at term after a normal pregnancy and delivery. Her development was normal until 10 months of age when stiffness of the lower limbs and tiptoeing were noticed. Severe spasticity of the legs with hyperactive deep tendon reflexes (DTR), sustained clonus, positive Babinski sign and scissoring in the vertical position were observed. She was never able to walk without support. An ascending progression of motor difficulties was then observed, with spasticity becoming evident in the upper extremities after 2 years of age. By 5 years, she developed supranuclear bulbar palsy, with progressive dysarthria and dysphagia with drooling. Brain and spinal cord magnetic resonance imaging (MRI) was normal. Electromyography and nerve conduction studies did not reveal any LMN involvement. Electrophysiological studies including electroencephalography, electroretinography, visual evoked potentials, brainstem auditory evoked potentials and somatosensory evoked potentials were normal. Motor evoked potentials were attempted but could not be recorded. She underwent pharyngeal plastic surgery at 5 years of age and Achilles tenotomy at 6 years of age. She is now 11 years old with preserved intellectual skills; hearing and vision are normal. Her face is expressionless with inability to wrinkle the forehead and close the eyes. Severe dysphagia and dysarthria can be observed with spastic tongue. She is wheelchair bound with severe spastic tetraparesis. Sensation is intact. The affected sister (II-2, Fig. 1a) was born at term (birth weight 3250 g). Her gross motor development was severely delayed. She was never able to walk because of bilateral leg stiffness. Neurological examination at 5 years of age revealed mild dysarthria with preserved tongue movements, chewing and swallowing. The muscle tone and power were normal in the upper limbs; however, severe spastic paresis with brisk DTR and positive Babinski response were observed in the lower extremities. Her sensation was intact. Brain and spinal cord MRI was normal. Electrophysiological studies were not conducted because she presented the same signs and symptoms observed in the elder sister, and the parents refused any further investigation. She is now 6 year old and wheelchair bound. ALS2 mutation analysis of the family here described. (a) The pedigree of the Hungarian family and the ALS2 genotype for each member are shown. The arrows indicate the affected members. Diamond symbols are used for privacy reasons. At the bottom, a representative section of sequence chromatograms of genomic DNA fragments encompassing the mutations in patients II-1 and II-2 (Pt) with respect to control (C). Sequence of the mutant subcloned alleles for both mutations is also shown (Mut allele). Arrows represent the mutation site, while the boxed sequence represents the 5 bp insertion. (b) Schematic representation of alsin protein domain structure with all mutations identified so far. RCC1 (regulator of chromatin condensation), DH/PH (Dbl and pleckstrin homology) and VPS9 (vacuolar protein sorting 9), which have highest sequence similarity to guanine nucleotide exchange factors for the Ran, Rho and Rab5 families of guanosine triphosphatases, respectively; MORN (membrane occupation and recognition nexus motifs) are implicated in the binding of the plasma membrane. Numbers on the top of the domains indicate the amino acid position of each domain along the protein sequence. The novel mutations detected in the family here described are circled, while stars indicate all known ALS2 mutations associated with infantile-onset ascending hereditary spastic paralysis phenotype. Sequencing analysis of the ALS2 gene in the proband revealed the presence of two heterozygous mutations: a 5 bp insertion, c.1825_1826insCAGTG, and a nonsense mutation c.3529G>T (Fig. 1a), both leading to the creation of premature stop codons (p.E609fsX9, p.G1177X) within the regulator of chromatin condensation (RCC1) and the membrane occupation and recognition nexus motifs (MORN) domain of alsin, respectively (Fig. 1b). Both mutations were also detected in the proband sister, while the mother and the father were carriers of the insertion and of the nonsense mutation, respectively. All 12 mutations reported so far in the ALS2 gene are homozygous, and all but two are truncating (4, 12, 13). Most of them determine loss of protein function coupled with protein instability [(12, 13) for review]. The mutations described here generate prematurely truncated alsin protein too, likely leading to loss of protein functions. Alsin contains multiple putative guanine nucleotide exchange factors (GEFs) such as RCC1-like domain (RLD), DH/PH and VPS9 (vacuolar protein sorting 9) (1, 2) and is involved in different processes such as endosome dynamics, cytoskeletal organization, anti-cytotoxicity and neuronal development/maintenance [(12, 13) for review]. The p.E609fs9X mutation in the N-terminal RLD should likely impair most of the interacting activities of alsin-RLD such as association to the membrane compartments and its interaction with the C-terminal portion of alsin [(12, 13) for review]. The p.G1177X mutation in the fifth MORN domain is predicted to generate a protein lacking both its Rab5-GEF activity and its ability to oligomerize (12, 13). Looking at all ALS2 mutations including those here reported (Fig. 1b), it is evident that all those located in the second half of alsin are associated with IAHSP only, while mutations falling within the N-terminus may be associated with ALS2, JPLS or IAHSP. This adds to the previous genotype–phenotype correlation (1, 2) and suggests that specific functions associated with the C-terminal domain such as Rab5 activity, ability to oligomerize and binding to the plasma membranes might be more directly related to UMN integrity and functioning than the rest of the protein. At the genetic and diagnostic level, the compound heterozygosity in ALS2 here reported justifies ALS2 mutation screening in all cases of recessive early-onset upper motor neuron disease regardless of any family consanguinity or homozygous haplotype at the ALS2 locus. The authors wish to thank the family for the co-operation with this study and Dr A. Martinuzzi for critical reading of the manuscript. Drs Sztriha and Túri were supported by a Marie Curie International Reintegration Grant (MIRG-CT-2005-030967) within the 6th European Community Framework Programme. This work was supported by research grants from the Association Francaise contre les Myopathies project number 12769 to M. T. B., from the Italian Ministry of Health, Prog PS-Neuro-ex.Art 56/05/7 to M. T. B. and N. B., and grants numbers RF2007-75 and RF2006-70 to M. T. B.