Clinical and molecular spectrum of a large Egyptian cohort with ALS2‐related disorders of infantile‐onset of clinical continuum IAHSP/JPLS

This study presents 46 patients from 23 unrelated Egyptian families with ALS2‐related disorders without evidence of lower motor neuron involvement. Age at onset ranged from 10 months to 2.5 years, featuring progressive upper motor neuron signs. Detailed clinical phenotypes demonstrated inter‐ and intrafamilial variability. We identified 16 homozygous disease‐causing ALS2 variants; sorted as splice‐site, missense, frameshift, nonsense and in‐frame in eight, seven, four, three, and one families, respectively. Seven of these variants were novel, expanding the mutational spectrum of the ALS2 gene. As expected, clinical severity was positively correlated with disease onset (p = 0.004). This work provides clinical and molecular profiles of a large single ethnic cohort of patients with ALS2 mutations, and suggests that infantile ascending hereditary spastic paralysis (IAHSP) and juvenile primary lateral sclerosis (JPLS) are belonged to one entity with no phenotype–genotype correlation.

Nevertheless, JALS differs clinically with later age of onset, more severe presentation and shorter lifespan, due to respiratory failure. 2 ALS2 gene comprises 34 exons (exon 1 is non-coding) spanning 83 kilobases at chromosome 2q33.1. The encoding protein, alsin, is ubiquitously expressed, with maximal expression in brain and spinal cord, particularly in motor neurons. It acts as a guanine exchange factor (GEF) activating the small GTPase Rab5, thus modulating endosome fusion and trafficking. Alsin mediates numerous cell processes including endocytosis, cytoskeletal organization and membrane dynamics. 3 In this study, we reported the detailed neurological phenotype and molecular findings of 46 patients with ALS2 mutations. Degeneration of motor neurons was confined to UMNs, excluding the diagnosis of JALS.

| Clinical assessment
Patients were subjected for thorough clinical and neurological assessment and comprehensive neuroimaging analysis. We adopted a severity score ranging from 0 to 8 that was calculated for each patient at their last examination (Table S1). The correlation between patients' severity score and their age at last examination and disease-onset were assessed using the Pearson correlation coefficient.

| Molecular analysis
Whole exome sequencing for extracted DNA was carried out using Illumina platform according to the manufacturer's protocol. Identified variants met the internal QC criteria based on extensive validation processes. They were checked in different databases and their possible effects were predicted using different in silico algorithms. Familial segregation was performed using Sanger sequencing. The chi-square test was performed in an attempt to establish possible phenotype-genotype correlation.

| Clinical findings
The study included 46 Egyptian patients (25 males and 21 females) from 23 unrelated families with consanguinity rate of 95.7%. Patients' anthropometric measurements displayed underweight (> À2SD) in eight patients (17.4%), short stature (> À2SD) in 14 (30.4%) and normocephaly (< À3SD) in all of them. The first disease symptom was recorded between the age of 10 and 30 months as tip toe walking (n = 27) (58.7%) or delayed and difficulty in walking (n = 19) (41.3%).   Statistical analysis showed that disease severity was significantly associated with increased age ( p = <0.001) and a positive correlation was documented between the severity score and age of disease onset ( p = 0.004) ( Figure S1). Due to disparity of age among our patients, we could not estimate an accurate correlation within similar ages.

| Molecular results
Sixteen homozygous pathogenic variants were detected in the enrolled subjects; six missense, four frameshift, three splice-site, two nonsense and one in-frame (Figure 2A).  JALS. [5][6][7][8][9] In this context, patients without involvement of LMNs were either described as JPLS (few cases) or as IAHSP (the majority of cases) 2 with no clear difference in the disease onset between the two entities 6,7,9,10 where the same entity may be referred to either JPLS or IAHSP. 1  To date, about 120 pathogenic ALS2 variants have been reported in patients with MNDs. The majority were small deletions and nonsense variants, followed by missense mutations. 12 In the current study, 16 homozygous pathogenic ALS2 variants were identified, seven of them were novel. The variants were mainly missense and splice-site in seven and eight families, respectively. However, splicesite mutations seem to be relatively rare in the literature. 12 Significant proportions of our reported missense variations (83.3%) involved glycine residues. It should be noted that glycine residues are the most flexible protein residues, where loss of this flexibility most probably affect the proper protein's structure and/or function. 13 Four frameshift, two nonsense and an in-frame variants were also detected in four, three, and one families, respectively. It was demonstrated that mutant alsin molecules with in-frame deletions existed as abnormally higher molecular weight complexes. 14 All our variants were detected in a homozygous state similar to the majority of the reports 2 that highlighted the significant effect of consanguinity on disease origination. Reviewing the manuscript.

ACKNOWLEDGMENTS
The work was partially supported by the NRC grants 12060178 and 11010167. We are thankful to Dr. Reza Maroofian, UCL Queen Square Institute of Neurology, UK, for his unlimited help to complete this work.