# GM2 gangliosidosis AB variant: first case of late onset and review of the literature

Benjamin Ganne, Benjamin Dauriat, Laurence Richard, Foudil Lamari, Karima Ghorab, Laurent Magy, Mehdi Benkirane, Alexandre Perani, Valentine Marquet, Patrick Calvas, et al.

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## To cite this version:

Benjamin Ganne, Benjamin Dauriat, Laurence Richard, Foudil Lamari, Karima Ghorab, et al., GM2 gangliosidosis AB variant: first case of late onset and review of the literature. Neurological Sciences, 2022, 43 (11), pp.6517-6527. 10.1007/s10072-022-06270-x. hal-03750585

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
GM2 gangliosidosis AB variant: first case of late onset and review
of the literature

Benjamin Ganne1 · Benjamin Dauriat1 · Laurence Richard2 · Foudil Lamari3 · Karima Ghorab2 · Laurent Magy2 · Mehdi Benkirane4 · Alexandre Perani1 · Valentine Marquet1 · Patrick Calvas5 · Catherine Yardin1 · Sylvie Bourthoumieu1

1 Cytogenetic, medical genetic and reproductive biology department, Hopital de la Mere et de l'Enfant, CHU Dupuytren, 87042 Limoges, France, Limoges University, CNRS, XLIM, UMR 7252, F-87000 Limoges, France

2 Department of Neurology, National Reference Center for “Rare Peripheral Neuropathies”, Dupuytren University Hospital (CHU Limoges), University of Limoges,

2 Avenue Martin Luther King 87042 Limoges Cedex, France

3 APHP Sorbonne University; Pitié-Salpétrière University Hospital, DMU Biogem; Metabolic Biochemistry Department, 75013 Paris, France

4 Rare Diseases Laboratory of Genetics EA7402, University Institute of Clinical Research, Montpellier University, CHU Montpellier, Montpellier, France, PhyMedExp UMR9214 CNRS-UM U1046 INSERM, Montpellier, France

5 Reference Centre for Rare Genetics Diseases in Ophthalmology, Purpan Hospital, Toulouse University, 31052 Toulouse, France

**Keywords**

GM2 gangliosidosis · AB Variant · Late-onset form · GM2A · GM2-AP

**E-mail :**

Benjamin Ganne

benjamin.ganne@chu-limoges.fr; benjamin.ganne.92@gmail.com
Abstract

AB variant is the rarest form of GM2 gangliosidosis, neurodegenerative diseases caused by lysosomal accumulation of GM2 gangliosides. Less than thirty cases are referenced in the literature, and to date, no late-onset form has been described. Our proband is a 22-year-old male with spinocerebellar ataxia and lower limbs motor deficiency. His symptoms started at the age of 10. A genetic analysis revealed two mutations in the *GM2A* gene encoding the GM2 activator protein (GM2-AP), an essential co-factor of hexosaminidase A. Both mutations, *GM2A:c.79A > T:p.Lys27* and *GM2A:c.415C > T:p.Pro139Ser*, were inherited respectively from his father and his mother. The nonsense mutation was predicted to be likely pathogenic, but the missense mutation was of unknown significance. To establish the pathogenicity of this variant, we studied GM2 accumulation and *GM2A* gene expression. Electron microscopy and immunofluorescence performed on patient's fibroblasts did not reveal any lysosomal accumulation of GM2. There was also no difference in *GM2A* gene expression using RT-qPCR, and both mutations were found on cDNA Sanger sequencing. Measurement of plasma gangliosides by liquid-phase chromatography-tandem mass spectrometry showed an accumulation of GM2 in our patient's plasma at 83.5 nmol/L, and a GM2/GM3 ratio at 0.066 (median of negative control at 30.2 nmol/L [19.7-46.8] and 0.019 respectively). Therefore, the association of both p.Lys27* and p.Pro169Ser mutations leads to a GM2-AP functional deficiency. Whereas the first mutation is more likely to be linked with infantile form of GM2 gangliosidosis, the hypomorphic p.Pro169Ser variant may be the first associated with a late-onset form of AB variant.

Introduction

Gangliosides are amphiphilic molecules, discovered by Ernst Klenk in the 1930s [1], and
more than 180 different types have been described in vertebrates [2].

These glycosphingolipids are metabolized through the trans-Golgi network from a
disaccharide lactosylceramide core (LacCer), on which N-acetylgalactosamine (GalNac) and
galactose (Gal) can be added. They differ by the length of the fatty acid and saccharide chain
as well as the number and position of the N-acetylneuraminic substrate (NANA or Neu5Ac).

Their degradation into ceramide requires lysosomal hydrolases and lipid-binding protein, the
"sphingolipid activator proteins" (SAPs).

In humans, gangliosides are mainly found in the nervous system, and concentrated in the plasma membrane of neurons, in domains called lipid rafts [3]. They are involved in different cellular functions, such as intercellular recognition, signal transduction, myelin stabilization, or even cell adhesion [4, 5].
GM2 represents only 4-5% of brain gangliosides, and their degradation into GM3 requires hexosaminidase A (HexA), a heterodimer composed of an α subunit, encoded by HEXA (15q23, OMIM*606,869#272,800), and a β subunit, encoded by HEXB (5q13.3, OMIM*606,873#268,800).

Dimerization of α subunits (αα) or β subunits (ββ) forms respectively hexosaminidase S (HexS) and hexosaminidase B (HexB). These homodimeric hexosaminidases cannot substitute for hexosaminidase A in GM2 ganglioside degradation.

While HexA can cleave both neutral and anionic substrates, HexB can only degrade neutral substrates and catabolizes globosides into globotriaosylceramides [6]. HexS, however, does not participate in GM2 degradation in vivo [7, 8].

The hydrolysis of GM2 also requires a non-enzymatic protein, the GM2 activator protein (GM2-AP), encoded by *GM2A* (5q33.1, OMIM*613,109#272,750). GM2-AP forms a complex with GM2 ganglioside allowing interaction between hexosaminidase A and GM2 ganglioside, its membrane-embedded substrate.

Pathogenic mutations of HEXA, HEXB, and GM2A are responsible for autosomal recessive diseases known as GM2 gangliosidosis, caused by excessive intralysosomal accumulation of GM2 gangliosides, especially in neuronal cells.

Mutations in HEXA, encoding the α chain of HexA, lead to Tay-Sachs disease or B variant, while mutations in HEXB, encoding the β chain, lead to deficiency of both HexA and HexB causing Sandhoff disease or O variant. Mutations of GM2A, encoding GM2-AP, lead to AB variant, which is rarely encountered and indistinguishable from Tay-Sachs disease.

Different clinical GM2 gangliosidosis subtypes have been described according to the age of onset. The infantile form is mainly characterized by hypotonia and seizures that appear in the first year, with premature death around 4-5 years [9]. Juvenile forms have an onset between 2 and 10 years old, with ataxia, psychomotor deterioration, spasticity, and seizures, leading to death before adulthood [10]. Late-onset forms (or adult forms) present with a heterogeneous phenotype and a better prognosis than the previously described forms, with inferior motoneuron deficiency, spinocerebellar ataxia, muscular atrophy, and psychiatric disorders [11].

A particular feature of the infantile form is the presence of a macular cherry-red spot, which was the first characteristic sign described in Tay-Sachs disease, previously called “familial amaurotic idiocy” [12].

AB variant is the rarest form, with less than 30 cases described in the literature, with no reported adult forms [1].

We describe a 22-year-old patient presenting a phenotype close to the late-onset form of GM2 gangliosidosis, in whom were detected two mutations in the *GM2A* gene, one predicted to be likely pathogenic, whereas the other was of unknown significance. We therefore studied GM2 accumulation in his plasma and fibroblasts as well as *GM2A* gene expression and cDNA Sanger sequencing, in order to conclude on the pathogenicity of these variants.

## Patient
Proband is a young man born in 1998 from non-consanguineous parents in good health, as well as his younger brother.

At the age of 10 years, he consulted for gait disorders, length inequality of the lower limbs, calf hypotrophy, and claw toes. After a left foot arthrodesis at 16 years old, his gait worsened, leading to a clinical presentation of lower motor neuronopathy with motor deficit of the lower limb, and a spinocerebellar ataxia progressively settled.

No psychomotor development delay was noted during his childhood. He walked at 13 months, but had poor motor skills. He had no dysphagia, extra-pyramidal symptom, or sensory disorder.

At clinical examination, Achilles and patellar osteotendinous reflexes were absent, walking required assistance and was impossible on tiptoes or heels. In addition to the calf amyotrophy, there was interosseous amyotrophy, associated with slowness of writing without dyspraxia and without other motor deficit of the upper limbs. No fasciculation was noted.

A neuropsychological workup was carried out at the age of 22 and showed a minimal global alteration of cognitive functions, and slight autistic traits. The deficits were predominant in memory and executive functioning. An electromyogram was performed at the same time, showing pure motor involvement and chronic neurogenic pattern, mainly of the lower limbs, with both motor amplitude and conduction speed reduced.

A brain MRI performed at the age of 19 showed discrete atrophy of the cerebellar vermis, slight hyperintensity of posterior horn of the lateral ventricles, without cortical atrophy.

Hexosaminidase A and B enzymatic activities in blood were normal.

A NGS panel for hereditary ataxia showed two mutations in *GM2A* gene (NM_000405):

- c.79A > T:p.Lys27*; nonsense variant inherited from his father, not reported in healthy population databases and predicted as likely pathogenic;

- c.415C > T:p.Pro139 Ser; missense variant, inherited from his mother, reported in the databases (rs767374072) and considered as a variant of undetermined significance (VOUS).

To establish the pathogenicity of this last variant with conflicting interpretations, we studied (1) accumulation of GM2 in our patient's fibroblasts using electron microscopy (EM) and immunofluorescence (IF), and in our patient's plasma using liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS) and (2) *GM2A* gene expression by RT-qPCR, completed with cDNA Sanger sequencing.
# Material and methods

## NGS analysis

Clinical exome sequencing was performed prior to our study using the TruSight One Expanded kit (TSOex, www.illumina.com/trusight) that targets approximately 6700 genes, most of them involved in human inherited diseases. Sequencing was performed using Illumina MiSeq or Next-Seq technology with 150-bp paired-end reads (Montpellier CHRU NGS platform).

Sequence alignment and variant calling were performed following the Broad Institute's Genome Analysis Toolkit (GATK) best practices, using the in-house pipeline Nenufaar (https://github.com/beboche/nenufaar). Sequence reads were aligned against the reference hg19 human genome.

Variants were annotated with Annovar (http://annovar.openbio.io/ormatICS.org/) and Variant Studio (www.illumina.com/variantStudio).

Copy number variation (CNV) analysis was performed with in-house algorithms that compare exon coverage ratios between patients of the same run (https://github.com/Mobidic/MobiCNV).

In silico filtering was performed on a panel of 559 genes, corresponding to the majority of known ataxia and related disorder genes, using American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for clinical sequence interpretation [13].

## Fibroblast cultures

Fibroblast cultures were performed from our patient's cryopreserved fibroblasts and from three healthy negative controls, using Chang medium (CliniSciences, Nanterre, France, Ref T105-100ML) with antibiotics (penicillin-streptomycin mixture; Sigma-Aldrich, Darmstadt, Germany, Ref P0781-20ML) in 25 cm² flasks, under 5% CO₂ at 37 °C in an incubator.

Positive controls for GM2 gangliosidosis were obtained by exposing fibroblast cultures of healthy negative controls to imipramine, a tricyclic antidepressant, causing HexA inhibition and GM2 accumulation [14, 15].

Imipramine (Sigma-Aldrich, Darmstadt, Germany, Ref I7379) was reconstituted in PBS at 37°C to obtain a final concentration of 100 µM in the culture medium.

## Gene expression by RT-qPCR

RNA extraction of fibroblast cultures was performed with the miRNeasy Mini kit (Qiagen, Hilden, Germany, Ref 217,004), and reverse transcription with the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany, Ref 205,311), according to the supplier's recommendations. cDNA of negative controls ($n = 6$ fibroblast cultures, 2 for each negative control) and proband ($n = 4$ fibroblast cultures) were amplified by real-time PCR using the
Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden, Germany, Ref 204,074) and the Corbett Research RG-6000 thermal cycler (Corbett Research, Australia).

Primers were designed using the Primer Blast software (National Center for Biotechnology Information, Rockville Pike, Bethesda, USA): 3 primer pairs for *GM2A* (one for exons 1–2, and two for exons 2–3), and one primer pair for each reference gene, all supplied by Sigma-Aldrich (sequences available on request).

All qPCRs were performed in duplicate for *GM2A* and in triplicate for reference genes (*HPRT* and *GUSB*). *GM2A* gene expression (Qn) was determined using the ΔCt method and the patient/control ratio by ΔΔCt method [16, 17].

## Sanger sequencing

Previous cDNA were amplified using primers targeting exons 1 and 3 of *GM2A* and Taq’Ozyme Purple Mix 2 (Ozyme, Paris, France, Ref OZYA007-200), and then purified with ExoSAP-IT (Ozyme, Paris, France, Ref 78,200–200-UL). The purified PCR products were sequenced bi-directionally using forward and reverse primers and BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, USA, Ref 4,337,452). Sequenced products were purified using DyeEx 2.0 Spin Kit (Qiagen, Hilden, Germany, Ref 63,206) and then subjected to capillary electrophoresis using a 3130xl Genetic Analyzer. Sequences were analyzed with Unipro UGENE V.40.1 software (Unipro, Novosibirsk, Russia).

## Ultrastructure of fibroblasts by electron microscopy (EM)

Fibroblast cultures of our patient, negative controls, and positive controls were fixed in 2.5% glutaraldehyde, stained with 2% osmium tetroxide (OsO4), and embedded in epoxy resin. The microscope was a JEOL JEM-1400 Flash coupled to a Morada G3 camera (Electron Optic Instrument, West Orange, NJ).

## Immunofluorescence (IF)

Fibroblast cultures of our patient, negative controls, and positive controls were washed twice with PBS for 5 min, then fixed with 1 mL of 4% paraformaldehyde for 15 min, and rinsed twice with PBS for 5 min, and once for 10 min. Fibroblasts were permeabilized with 0.1% Triton X-100 for 2 min and then saturated with 1% BSA (bovine serum albumin) for 45 sec. They were then stained overnight using a GMB28 mouse monoclonal anti-GM2 (0.5 mg/mL, EMD Milipore Corporation, Temecula, USA, Ref MABN2550-100UL) as primary antibody, at 1:100 and 1:200. The secondary antibody was an Alexa Fluor 488 Goat anti-mouse IgM μ chain (2 mg/mL, Thermo Fisher Scientific, OR, USA, Ref A21042) used at 1:1000.

Counterstaining of nuclei was performed with DAPI, and we observed our slides with a Metafer epifluorescence microscope (MetaSystems), coupled with Isis software (MetaSystems), to calculate a percentage of fluorescent cells on each.
Liquid-phase chromatography-tandem mass spectrometry assay (LC-MS/MS)

GM2 concentration in our patient's plasma was determined by LC-MS/MS, carried out by Dr Lamari's team at the Pitie-Salpetriere Hospital in Paris, France. Plasma was mixed with a mixture of deuterated gangliosides (Cd3-GM2 and Cd3-GM3) as internal standards and proteins precipitated by ethanol. After mixing and centrifugation, the supernatant was injected to an Acquity UPLC-TQD system (Waters). Gangliosides were detected and quantified in a positive mode with multiple reaction monitoring. This method presents a sensitivity and specificity of 100% for GM2 gangliosidosis diagnosis, using as cutoff 58.9 nM for GM2 34:1, and 0.032 for the GM2 34:1/GM3 34:1 ratio.

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Sanger sequencing of our patient's cDNA showing the presence of both p.Lys27* and p.Pro139Ser mutations</figcaption></figure>

Results

Analysis by RT-qPCR did not reveal a decrease in *GM2A* gene expression in our patient in comparison to our negative controls, with a mean Qn at 0.459 (0.452–0.467) for our patient against 0.474 (0.461–0.483) for the negative controls. The patient/control ratio at 0.97 corresponds to a normal *GM2A* gene expression. Moreover, the Sanger sequencing showed that both mutated alleles were expressed, even if the nonsense mutation seemed to be at a slightly lower level on the chromatograms (Fig. 1).

EM performed on positive controls revealed numerous concentric lamellar inclusions within lysosomes, characteristic of lysosomal storage diseases [18]. In contrast, ultrastructure of patient's fibroblasts did not show a significant increase in these structures compared to negative controls, not allowing them to be reliably distinguished.

The average number of cells labelled by the anti-GM2 antibody (1:100 and 1:200) is 26.1% (n = 4 fibroblast cultures) for our patient, 26.8% (n = 2 fibroblast cultures) for the negative control, and 42.6% (n = 2 fibroblast cultures) for the positive control. There was no difference between our patient and negative controls (Fig. 2).
However, the LC-MS/MS analysis showed a slight increase in GM2 34:1, measured at 83.5 nmol/L (median control 30.2 nmol/L (19.7-46.8)), as well as in the GM2 34:1/GM3 34:1 ratio (0.066 for a median control at 0.019). Compared to the pool of patients with GM2 gangliosidosis available to Dr Lamari's team, plasma GM2 level of our patient is found close to late-onset forms, corresponding to the first quartile of patients with Tay-Sachs or Sandhoff disease (Fig. 3).

<figure><img src="image_4.png" /><figcaption>**Fig. 3** Plasma concentrations of GM2 34:1 and GM2 34:1/GM3 34:1 ratio, determined by LC-MS/MS. Results are classified according to clinical forms (both Tay-Sachs and Sandhoff disease), with our proband (AB variant) at the left of the figure</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Plasma concentrations of GM2 34:1 and GM2 34:1/GM3 34:1 ratio, determined by LC-MS/MS. Results are classified according to clinical forms (both Tay-Sachs and Sandhoff disease), with our proband (AB variant) at the left of the figure</figcaption></figure>
<table><caption>Table 1 Review of the literature of patients with GM2 gangliosidosis AB variant and available genomic data (HGVS Nonallelicure 2016). n.a. not available, mo months' old, yo years' old</caption><thead><tr><th>Genotype</th><th>Gene</th><th>Protein</th><th>Mutation type</th><th>GM2-AP</th><th>mRNA</th><th>Cerebral MRI</th><th>Age of onset</th><th>Cherry-red spot</th><th>Reference</th></tr></thead><tbody><tr><td>Homozygous</td><td>c.412 T > C</td><td>p.Cys138Arg</td><td>Missense</td><td>Absent</td><td>Normal</td><td>n.a</td><td>9mo</td><td>Yes</td><td>Xie B, 1992 [23]</td></tr><tr><td>Homozygous</td><td>c.262_264delAGA</td><td>p.Lys88Del</td><td>InframeDeletion</td><td>Absent</td><td>Normal</td><td>n.a</td><td>8mo</td><td>Yes</td><td>Scheers U, 1996 (Patient A) [21]</td></tr><tr><td>Homozygous</td><td>c.410delA</td><td>p.His137Profs*34</td><td>Frameshift</td><td>Weak</td><td>Normal</td><td>n.a</td><td>7mo</td><td>Yes</td><td>Scheers U, 1996 (Patient B) [21]</td></tr><tr><td>Homozygous</td><td>c.506G > C</td><td>p.Arg169Pro</td><td>Missense</td><td>Weak</td><td>Normal</td><td>n.a</td><td>5mo</td><td>Yes</td><td>Schroder M, 1993 [22]</td></tr><tr><td>Homozygous</td><td>c.262_264delAGA</td><td>p.Lys88Del</td><td>InframeDeletion</td><td>No quantification</td><td>No quantification</td><td>Periventricular and basal ganglia hyperintensity</td><td>9mo</td><td>Yes</td><td>Backmann F, 2017 (Patient 1)</td></tr><tr><td>Homozygous</td><td>c.369_371delinsATTTAA</td><td>p.Pro124Leufs*48</td><td>Famneshift</td><td>No quantification</td><td>No quantification</td><td>Basal ganglia and posterior part of the thalamus hyperintensity, delayed myelination</td><td>10mo</td><td>Yes</td><td>Backmann F, 2017 (Patient 2) [27]</td></tr><tr><td>Homozygous</td><td>c.100G > T</td><td>p.Glu54*</td><td>Nonsense</td><td>Absent</td><td>Absent</td><td>Periventricular hyperintensity, and akered signal density in the basal ganglia</td><td>5mo</td><td>Yes</td><td>Chen B, 1999 [24]</td></tr><tr><td>Homozygous</td><td>c.472G > T</td><td>p.Glu158*</td><td>Nonsense</td><td>No quantification</td><td>No quantification</td><td>Puraminal hyperintensity, thalamic hypointensity, some unmyelinated white matter</td><td>12mo</td><td>Yes</td><td>Sheth J, 2016 [28]</td></tr><tr><td>Homozygous</td><td>c.160G > T</td><td>p.Glu54*</td><td>Nonsense</td><td>No quantification</td><td>No quantification</td><td>Hyperintensity of the posterior part of the thalamus, hypointensity in the anterior part, delayed myelination</td><td>4mo</td><td>Yes</td><td>Reardan D, 2016 [26]</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Genotype</th><th>Gene</th><th>Protein</th><th>Mutation type</th><th>GM2-AP</th><th>mRNA</th><th>Cerebral MRI</th><th>Age of onset</th><th>Cherry-red spot</th><th>Reference</th></tr></thead><tbody><tr><td>Compound heterozygote</td><td>c.298G&gt;T + c.164C&gt;T</td><td>p.Glu87* + p.Pro55Leu</td><td>Missense + Missense</td><td>Absent</td><td>No quantification</td><td>Diffuse cortical and subcortical atrophy, ventricular enlargement, no signal abnormalities</td><td>12mo</td><td>No</td><td>Martins C, 2017 [19]</td></tr><tr><td>Homozygous</td><td>c.164C&gt;T</td><td>p.Pro55Leu</td><td>Missense</td><td>No quantification</td><td>No quantification</td><td>Cortical atrophy</td><td>Tyo</td><td>No</td><td>Salih MA, 2015 (Patient 1) [20]</td></tr><tr><td>Homozygous</td><td>c.164C&gt;T</td><td>p.Pro55Leu</td><td>Missense</td><td>No quantification</td><td>No quantification</td><td>Cortical atrophy</td><td>8yo</td><td>No</td><td>Salih MA, 2015 (Patient 2) [20]</td></tr><tr><td>Homozygous</td><td>c.164C&gt;T</td><td>p.Pro55Leu</td><td>Missense</td><td>No quantification</td><td>No quantification</td><td>Cortical atrophy</td><td>8yo</td><td>No</td><td>Salih MA, 2015 (Patient 3) [20]</td></tr><tr><td>Homozygous</td><td>c.522T&gt;G</td><td>p.Leu174Arg</td><td>Missense</td><td>No quantification</td><td>No quantification</td><td>n.a</td><td>11mo</td><td>Yes</td><td>Kolodny E, 2008</td></tr><tr><td>Homozygous</td><td>c.312delC</td><td>p.Ser105Alafs*14</td><td>Frameshift</td><td>n.a</td><td>n.a</td><td>n.a</td><td>n.a</td><td>n.a</td><td>Encarnçio M, 30/30</td></tr><tr><td>Homozygous</td><td>c.333delC</td><td>p.Cys112Valfs*7</td><td>Frameshift</td><td>n.a</td><td>n.a</td><td>n.a</td><td>12mo</td><td>n.a</td><td>Foranquez-Marmise A, 2014</td></tr><tr><td>Homozygous</td><td>c.412T&gt;C</td><td>p.Cys138Arg</td><td>Missense</td><td>n.a</td><td>n.a</td><td>n.a</td><td>n.a</td><td>n.a</td><td>Schroder M, 1991</td></tr><tr><td>Homozygous</td><td>c.369_371delGCCInsTAA</td><td>p.Glu123_ Pro124delinsA+ p.Asn</td><td>Missense</td><td>No quantification</td><td>No quantification</td><td>No abnormality</td><td>8mo</td><td>Yes</td><td>Inci A, 2021</td></tr></tbody></table>
Discussion

We have described the case of a 22-year-old young man suspected to have a compound heterozygous form of GM2 gangliosidosis AB variant, with lower motor neuronopathy and spinocerebellar ataxia.

Electron microscopy and immunofluorescence did not reveal a lysosomal accumulation of GM2, compared to negative and positive controls. There was also no difference in *GM2A* gene expression using RT-qPCR, and both alleles were expressed. Only a liquid-phase chromatography-tandem mass spectrometry assay showed an accumulation of GM2 in our patient's plasma, corresponding to an adult form of Tay-Sachs or Sandhoff disease.

Only a few cases of AB variant gangliosidosis have been described in the literature, and almost all of them are infantile forms where the symptoms appear before the first year of life with a macular cherry-red spot, psychomotor retardation, hyperacusis, seizures, and axial hypotonia.

A review of the literature of patients with AB variant GM2 gangliosidosis with available genomic data is presented in Table 1.

The only patient who presented symptoms at 12 months of life without macular cherry-red spot, and with axial hypertonia and ataxia in addition to the above "classic" clinical signs, was compound heterozygous for a nonsense variant (p.Glu87*) and a missense variant (p.Pro55Leu) [19]. The latter was previously described as pathogenic in the literature, in an Indian family, for which three homozygous relatives developed chorea in childhood (7-8 years), but without cherry-red spot [20].

GM2A gene expression was reported in five of the presented patients. Four had normal GM2A expression [21–23], and only the patient from Chen had no mRNA on Northern blot [24]. This last patient was homozygous for the nonsense mutation p.Glu54*, thus considerably shortening the final protein, compared to other homozygous patients for a missense mutation (p.Cys138Arg or p.Arg169Pro), an inframe deletion mutation (p.Lys88Del), or a frameshift mutation (p.His137Profs*34). The absence of mRNA may be explained by the presence of this premature stop codon, leading to mRNA degradation through the "Nonsense-Mediated mRNA Decay" (NMD) system [25]. However, p.Lys27* mutation in our patient's cDNA might escape the NMD system, and only homozygous nonsense mutations would lead to a complete absence of mRNA. Gene expression studies in patients with a homozygous nonsense mutation would have allowed us to verify this hypothesis, but was not performed [26].

The visible lesions on brain MRI were extremely similar between patients. Among those for whom clinical signs started at or before 12 months, two showed periventricular and basal ganglia T2 hyper-signals [24, 27], three had basal ganglia hypersignal with delayed myelination [26–28], and only one did not show any abnormality [29]. Only patients carrying the p.Pro55Leu mutation, in the heterozygous [19] or homozygous state, had brain atrophy without signal alteration [20].
Our patient's MRI showed no brain atrophy, but presented a localized atrophy of the cerebellar vermis comparable to that seen in adult forms of Tay-Sachs disease [11, 30], and a slight aspecific hyperintensity of posterior horn of the lateral ventricles.

<figure><img src="image_6.png" /><figcaption>Fig. 4 GM2A conservation across different species, and representation of pathogenic truncating (top line) and missense (lower line) mutations reported. Blue boxes represent membrane interacting hydrophobic loops domains (V90-W94/V153-L163)</figcaption></figure>

A review of the literature on late-onset forms of GM2 gangliosidosis had clarified their clinical profile and evolution [11]. Almost half of the patients had small signs during childhood, such as clumsiness or poor motor skills, which remained stable over time. The most frequent and earliest neurological manifestation was lower limb weakness in 81% of patients, with preferential involvement of the psoas and quadriceps due to lower motor neuronopathy.

Half of the patients had to use a wheelchair after 20 years of disease progression, with a very heterogeneous pattern of gait degradation. Upper limb involvement has never been reported as the first sign of the disease and affects 53% of the patients, especially the triceps and interosseous muscles.

More than half of the patients had a cerebellar syndrome, which was not proportional to the cerebellar atrophy, present in 73% of the patients, and only 15% had cerebral atrophy.

Psychiatric disorders (depression, maniac disorder, psychosis) affected about 30% of patients in Masingue's study, 94% of whom had Tay-Sachs disease, and could either correspond to the initial disorder of the disease or be isolated.

Neudorfer's study found the same proportion of clinical manifestations in their population of adult Tay-Sachs patients, except for psychiatric disorders, which were much more frequent (85.7% versus 30%) [30]. None of these studies reported ophtalmological disorders in late onset forms.

Masingue's study identified four different initial presentations of GM2 gangliosidosis in adults [11]:
- Lower motoneuron disorder responsible for proximal lower limb weakness, with progression to the upper limbs;

- Cerebellar ataxia (more frequently isolated in Sandhoff patients);

- Psychiatric disorder (Tay-Sachs disease only);

- Complex clinical presentation mixing the three above phenotypes.

The clinical course of our patient resembles that of an adult form of Tay-Sachs: initial involvement of the lower limbs with discrete signs in childhood of poor motor skills, progression to the upper limbs with involvement of the ulnar nerve and interosseous amyotrophy, cerebellar atrophy and slight cognitive impairment. The long-term evolution of the disease is mainly marked by the need to use a wheelchair to move around, the risk of developing psychiatric symptoms, and the occurrence of progressive cognitive decline.

Although all three clinical forms (infantile, juvenile, adult) have been described for Tay-Sachs and Sandhoff diseases, this was not the case for the AB variant, of which we report here the first late-onset case.

Neither EM nor IF allowed us to demonstrate a lysosomal accumulation of GM2 by functional deficiency of GM2-AP. RT-qPCR did not show a reduction in gene expression compared to controls, and cDNA Sanger sequencing revealed the presence of both alleles, with the p.Lys27* mutation potentially being slightly less expressed on chromatogram. On the other hand, the newly developed LC-MS/MS technique allowed us to show a slight increase of GM2 in our patient's plasma, with a concentration close to adult forms of Tay-Sachs and Sandhoff disease, allowing us to classify our *GM2A:c.79A > T:p.Lys27* variant, not previously reported in healthy individual databases, as pathogenic. On the other hand, the *GM2A:c.415C > T:p.Pro139Ser* variant was only reported once in the Latin American population, with no known homozygous subject in the gnomAD V2.1.1, ExAC, or TOPMED databases. Alignment of *GM2A* gene across different species confirmed the conservation of Proline 139 through Metazoa, and interestingly, most of previously reported pathogenic missenses occurred on a well-conserved amino acid (Fig. 4). This study allows us to validate the PS3 ACMG criteria for this variant too and reclassify it from VOUS to be likely pathogenic (PM1/PM2/PP3/PS3). This situation highlights the necessity of functional analysis in unusual clinical presentations, allowing for confirmation of both mutations pathogenicity and diagnosis.

Some pathogenic missenses appear to be hypomorphic and are much more represented in adult forms of Tay-Sachs disease, as *HEXA:c.805G > A:p.Gly269Ser* (rs121907954) [31, 32]. Moreover, it has been described that the phenotype is often determined by the second allele in GM2 gangliosidosis, when a premature stop codon is present [8].

Similarly, the *GM2A:c.415C > T:p.Pro139Ser* variant seems to be hypomorphic and may result in an adult or delayed form of AB variant when coupled to other nonsense mutation, or if present in the homozygous state. However, further mildly affected patients are required to confirm this hypothesis.
Conclusion

Although late-onset AB variant was not described to date in the literature, our case demonstrates that this pathology indeed follows the same rules as other GM2 gangliosidosis, with the existence of a clinical adult form, more likely to be linked to our *GM2A:c.415C > T:p.Pro139Ser* variant, which we were able to reclassify as likely pathogenic. Finally, the clinical course of our patient would be similar to that of the adult forms of Tay-Sachs, with foremost a progression of the lower limb deficiency and the necessity to use a wheelchair to move around.

Declarations

Conflict of interest The authors declare no competing interests.

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