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The ALDH18A1 gene is located at 10q24.1 and encodes delta-1-pyrroline-5-carboxylate synthetase (P5CS), a mitochondrial bifunctional enzyme that catalyzes the first two steps in de novo biosynthesis of proline, ornithine, citrulline, and arginine. ALDH18A1-related disorders have been classified into four groups, such as autosomal dominant and recessive hereditary spastic paraplegia (SPG9A and SPG9B, respectively), as well as autosomal dominant and recessive cutis laxa (ADCL3 and ARCL3A, respectively). Neurodegeneration is a characteristic feature of all groups.
Here, we report a girl with compound heterozygous disease-causing variants (c.-28-2A>G and c.383G>A, p.Arg128His) in the ALDH18A1 gene, revealed by whole exome sequencing. The c.-28-2A>G variant in intron 1, inherited from the mother, is a novel mutation, while the c.383G>A variant in exon 4, inherited from the father, has already been reported. The patient presented with vigorous infantile tremor preceding progressive spastic paraplegia. Dysmorphic features included elongated face, deep-set ears, upturned nose, long philtrum and pointed chin. Intrauterine and postnatal growth retardation, microcephaly, global developmental delay and profound intellectual disability were also noticed. Blood fasting ammonia level, plasma proline, ornithine and arginine levels were normal, while citrulline level was slightly decreased. Brain MRI revealed moderate hypoplasia of the corpus callosum and reduction of white matter volume.
The patient represents SPG9B, a rare form of autosomal recessive hereditary spastic paraplegias. The early onset tremor, preceding lower limb spasticity appears to be a unique early manifestation of neurodegeneration in this case.
]. Based on genotypic and phenotypic features the ALDH18A1-related disorders have been classified into four groups, such as autosomal dominant and recessive hereditary spastic paraplegia (SPG9A, OMIM 601162 and SPG9B, OMIM 616586, respectively), as well as autosomal dominant and recessive cutis laxa (ADCL3, OMIM 616603 and ARCL3A, OMIM 219150, respectively) [
]. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthetase (P5CS, EC 22.214.171.124 and 126.96.36.199), a mitochondrial bifunctional enzyme that catalyzes the first two steps in the biosynthesis of proline, ornithine, citrulline, and arginine from glutamate. It comprises two domains, with different enzymatic activities: an N-terminal glutamate 5-kinase (G5K) domain, responsible for the glutamate phosphorylation to gamma-glutamyl phosphate, and a C-terminal glutamate 5-phosphate reductase (G5PR) domain, which catalyzes the reduction and conversion to gamma-glutamyl semialdehyde, which is further metabolized to proline and ornithine [
]. Two isoforms of P5CS are generated, differing only by 2 amino acid insert in the G5K domain. The short P5CS isoform has high activity in gut, where it catalyzes an essential step in the arginine biosynthetic pathway. The long isoform of P5CS is expressed in multiple tissues and is necessary for the synthesis of proline from glutamate [
]. We extend the genotypic and phenotypic spectrum of SPG9B by reporting a girl with compound heterozygous ALDH18A1 mutations who had intense tremor in infancy, preceding the development of spastic paraplegia.
2. Case report
The proband, a girl was born from the first uneventful pregnancy to heathy, non-consanguineous Caucasian parents on the 37th gestational week. Her birth weight was 1950 g (−2.2 SD), head circumference 30 cm (−2.0 SD) and length 44 cm (−1.4 SD). Dysmorphic features included elongated face, deep-set ears, upturned nose, long philtrum and pointed chin (Fig. 1A, B). She did not have any cutaneous involvement. At about 2 months of age fast head and hand tremor appeared in the form of rhythmic back-and-forth involuntary movements with low amplitudes both at rest and during action. Fasting did not worsen these movements. The tremor became quite vigorous with waxing amplitudes during infancy and gradually waned later. There was no tremor at rest by the age of 5 years; however, emotional distress, particularly fear still provoked it. She had hypotonia and was unable to sit at age of 10 months, or stand at 12 months of age. She had very short attention span and limited interest in her surroundings. Developmental Quotient (DQ) of 45 was found by Brunet-Lézine test at the age of 2 years.
Fasting ammonia level was normal. Plasma proline, ornithine and arginine levels were also within the normal range, while citrulline level was slightly decreased (9 µmol/L, normal: 10–50 µmol/L, borderline). The results of other blood tests were normal.
Brain MRI at the age of 2 years revealed reduced volume of white matter and moderate hypoplasia of the corpus callosum (Fig. 1C–F).
At the age of 5.5 years her head circumference was 46 cm (−3.5 SD), weight 15 kg, (−1.9 SD), and height 98 cm (−2.8 SD). By this age, marked spasticity through most of the range of motion (Modified Ashworth scale 2) developed in her lower limbs with brisk deep tendon reflexes. Wide based spastic gait was also observed. There was no speech and intellectual disability was evident. She had disruptive behavior hindering us from taking formal Intelligence Quotient (IQ) test.
2.1 Genetic analysis
Routine chromosomal analysis by G-banding showed normal 46,XX karyotype. Genomic DNA was extracted from peripheral blood samples with the Puregene kit (Gentra). Array comparative genomic hybridization (aCGH) showed normal genomic copy number (Quantitative Genomic Medicine Laboratories, S.L., Barcelona, Spain).
Trio analysis by whole exome sequencing (WES) was performed with CentoXome® Gold at Centogene AG (Rostock, Germany) as described earlier [
]. A heterozygous variant in intron 1 (NM_002860.4:c.-28-2A>G) and another heterozygous variant in exon 4 (NM_002860.4:c.383G>A, NP_002851.2:p.Arg128His) of the ALDH18A1 gene were detected (Fig. 2A, B). The c.-28-2A>G variant is likely pathogenic because it changes the acceptor splice site of intron 1 in the 5′-UTR, causing skipping of exon 2 (the start codon is in exon 2). The possible molecular effect of this variant was tested in silico using MutationTaster. It predicted that c.-28-2A>G variant is disease causing (prob: 0.969300883264394); the wild type splice site (tgca|GATA c.-28) has been lost. This variant is absent in the databases [GnomAD (The Genome Aggregation Database), dbSNP, Exome Variant Server, ClinVar]. However, the p.Arg128His variant has been previously reported in compound heterozygous state as disease-causing for autosomal recessive spastic paraplegia [
]. The c.-28-2A>G variant was also detected in the mother (Fig. 2A) in a heterozygous state, whereas the c.383G>A was detected in the father (Fig. 2B) also in a heterozygous state.
The patient in this report is compound heterozygous for two ALDH18A1 disease-causing mutations. Both mutations affect the G5K domain of the P5CS enzyme. The phenotypic characteristics, such as intrauterine growth retardation, dysmorphic features, short statue, microcephaly, global developmental delay, cognitive impairment, progressively developing spastic paraplegia and lack of cutaneous manifestations in association with biallelic ALDH18A1 mutations meet the criteria of SPG9B [
].Fifteen patients in 8 families have been described so far with this disorder, including the patient in this report (Table 1). The compound heterozygous mutations in 5 families and homozygous mutations in 3 families distributed randomly in the ALDH18A1 gene, affecting both the G5K and G5PR domains (Table 1). Corpus callosum hypoplasia and thin white matter, found in our patient, have been described in ALDH18A1-related disorders, however they are rare in SPG9B (Table 1). Autopsy findings or neuropathology have never been reported.
The vigorous infantile tremor, the presenting sign in our patient, was unusual and has never been reported so early in SPG9B. Tremor, reported in other cases of SPG9B, started later, at around the 7th and 15th years of age [
]. A unifying view for these disorders has been hypothesized, claiming that the different presentations conform to a disease continuum of decreasing severity from the cutis laxa forms ARCL3A and ADCL3 to the motor syndromes SPG9B and SPG9A [
]. Global developmental delay and intellectual disability are major manifestations of the central nervous system involvement with equal frequency in both autosomal recessive forms, i.e. in ARCL3A and SPG9B [
Data has been collected and reviewed by Marco-Marin and coworkers in favor of the view that the severity of the various syndromes in ALDH18A-related disorders would correspond to higher or lower degrees of loss of P5CS function [
]. Decreased serum P5CS activity was found in a patient with SPG9B due to homozygous p.Ser242Asn mutation in the G5K domain; however, the P5CS protein level and its mitochondrial localization in HeLa cells transfected with the mutant ALDH18A1 plasmids remained unchanged [
]. Indeed, these findings may suggest that the SPG9B phenotype could be associated with residual P5CS activity, while some patients with biallelic null mutations in ALDH18A1 and cutis laxa phenotype exhibited pronounced reduction or total absence of P5CS protein [
Measurements of blood ammonia and amino acid levels led to inconstant results. Increased blood ammonia and low plasma proline, ornithine, citrulline and arginine were described in both SPG9A and ARCL3A [
]. In contrast, the ammonia and amino acid levels were in the normal or low normal range in SPG9B patients regardless of whether the mutation affected the G5K, as in our case, the G5PR, or both domains [
]. The patient population with ALDH18A1-related disorders is small, hampering the comparison of the sometimes contradictory results gained by various methods in singular cases, or families with different phenotypes.
The pathophysiology of the neurological impairment in ALDH18A-related disorders remains to be clarified. Reduced cerebral proline and/or creatine synthesis might have a role [
]. Further research warranted to elucidate the mechanism of neurodegeneration in these conditions.
We report a girl with a rare form of autosomal recessive hereditary spastic paraplegia (SPG9B) due to compound heterozygous mutations in the ALDH18A1 gene. The c.-28-2A>G variant in intron 1 is a novel mutation; it was inherited from her mother. The other, c.383G>A variant in exon 4, inherited from her father, has already been published. Vigorous infantile tremor preceding progressive spastic paraplegia was a unique clinical manifestation of the disease. Intrauterine and postnatal growth retardation, dysmorphic features, microcephaly, delayed development and intellectual disability were the other characteristic features of the disorder.
The authors are grateful to the patient’s parents for their collaboration.
The authors thank to Professor Eva Morava-Kozicz MD, PhD for the amino acid analysis.
This study was supported by the GINOP-2.3.2-15-2 grant (TK and ZM) provided by the National Research, Development and Innovation Office (Hungary).
Written informed parental consent has been obtained.
Written permission has been gained from the parents to publish the patient’s photos in a scientific journal.
The study was approved by the Human Investigation Review Board at Albert Szent-Györgyi Clinical Centre, University of Szeged, Hungary.
Conflict of Interest Disclosures
The authors declare no competing interests.
Δ1-Pyrroline-5-carboxylate synthetase deficiency: An emergent multifaceted urea cycle-related disorder.