Clinical, molecular and glycophenotype insights in SLC39A8-CDG

SLC39A8, a gene located on chromosome 4q24, encodes for the manganese (Mn) transporter ZIP8, which plays a major role in regulating Mn homeostasis in blood and tissues [1]. Mn is an essential trace element and has a major role as cofactor for various enzymes (e.g. pyruvate carboxylase, arginase, superoxide dismutase-2, glutamine synthase and glycosyltransferases) [2, 3]_.

Since Mn is a cofactor for beta-1,4-galactosyltransferase [2], SLC39A8 variants are associated with a congenital disorder of glycosylation (CDG), characterized by deficient galactosylation of glycan chains of glycoproteins. Moreover, the common SLC39A8 variant rs13107325 (C → T) in exon 8 (A391T), is associated with increased risk for other neurological and systemic disorders, and with decreased serum Mn.

CDG are inborn errors of metabolism characterized by improper glycosylation of glycoconjugates. Protein glycosylation defects are caused by genetic variants in the N- and/or O-glycosylation pathways. Serum transferrin isoelectric focusing (IEF) and capillary electrophoresis (CDT) are the conventional laboratory tests for the N-glycosylation defects associated with sialic acid deficiency. These blood measurements enable to distinguish between CDG-I (cytosol and endoplasmic reticulum defects of N-glycan assembly) and CDG-II (Golgi processing defects). So far fourteen subjects with SLC39A8-CDG have been reported [2‐5]. Most patients exhibited a serum Tf IEF type 2 pattern and/or low to undetectable blood Mn levels and elevated Mn urine levels, consistent with renal wasting [3, 6]. The variability in Mn blood levels among the affected patients is likely due to partial compensation by other transporters, to different effects of SLC39A8 variants on Mn homeostasis and to variations of Mn levels as result of the dietary intake [3].

SLC39A8-CDG presents with developmental delay, severe intellectual disability, seizures, and cerebral and/or cerebellar atrophy [6]. Dystonia and basal ganglia T2-hyperintensities on magnetic resonance imaging (MRI) are also part of the neurological spectrum [4, 5]. SLC39A8-CDG features have been related to both Mn deficiency and hypoglycosylation [2]. In particular, SLC39A8-CDG may present with the clinical, biochemical and neuroradiological features of Leigh syndrome-like mitochondrial disease, probably due to reduced activity of Mn-dependent superoxide dismutase (SOD), a reactive species scavenger in mitochondria [4]. Since hypoglycosylation in SLC39A8 deficiency is secondary to a defect of galactosylation, galactose supplementation (in combination with uridine) was reported to improve transferrin glycosylation [2, 4]. More recently, Mn-II-sulfate supplementation was found to ameliorate both biochemical (e.g., improvement of glycosylation of serum transferrin and normalization of blood and urinary Mn levels) and clinical features (e.g. improved motor abilities, ataxia, muscle strength, postural control, frequency and severity of epilepsy, vision, hearing and swallowing) [7]. Appropriate Mn supplementation was not associated with clinical or neuroradiological signs of manganism (e.g. psychiatric symptoms or Mn deposition in basal ganglia with associated dystonia) [7]; however, close monitoring of the patients is recommended. Transferrin glycosylation analyses may represent a useful marker to establish the therapeutic dose of Mn [7].

Due to the paucity of studies on clinical, glycosylation and molecular correlates of SLC39A8-CDG, further insights are warranted to better characterize this disorder, also in light of possible therapeutic interventions.

We describe clinical and neuroradiological features, and the glycophenotypes associated with novel SLC39A8 variants (Family 1, Patient-1 and Patient-2) and with the already known SLC39A8 homozygous missense variant p.Ala391Thr (Family 2, Patient-3).

The study was based solely on information and investigations that were carried out as part of the routine clinical care of CDG patients. Informed consent was signed by the parents.

Here, we report the identification of novel SCL39A8 variants and homozygosity for a common missense variant associated with clinical, neuroradiological and glycosylation findings consistent with SLC39A8-CDG. So far, a paucity of SLC39A8-CDG studies have been reported, including 14 patients with different clinical and neuroradiological characteristics and variable transferrin glycosylation changes [2‐5] (Table 1). Common clinical features of SLC39A8-CDG patients are hypotonia, poor postural control, increased peripheral tone, global developmental delay, intellectual disability, and failure to thrive. Strabismus is a recurring feature [2‐5], also in this study (Patient-2). Hearing impairment was described in only one patient [2]. Dysmorphic features (broad forehead, hirsutism, anteverted nostrils, thin lips, smooth philtrum) were reported in a few subjects [2, 4, 5, 10], including one infant with disproportionate dwarfism and craniosynostosis. Microcephaly was reported in one instance and cranial asymmetry was observed in Patient-2. Some patients affected by SLC39A8-CDG presented with scoliosis, as observed in the Patient-1, or with additional skeletal abnormalities [2‐4]. In the present study, all patients were affected by dystonia and they also presented episodes of eye rolling and dyskinetic movements of the oral region. Due to feeding difficulties and poor weight gain, gastrostomy and fundoplication were performed in two study patients. Consistently, previous studies described patients with SLC39A8-CDG and prominent dystonic postural pattern who underwent gastrostomy [3‐6]. According to what has been observed in the patients of this study and in others reported previously [5, 6], SLC39A8-CDG should be considered among CDG presenting with major hyperkinetic movement disorders such as global and segmental dystonia and dyskinesia, particularly at the orofacial region, accompanied by dysphagia [11]. Epilepsy is another common feature in SLC39-A8-CDG [2‐5, 10] and it is often refractory to most anticonvulsant drugs [2, 4, 5]. Differently from Patient-1, who displayed atypical absences and clonic seizures, spasms with hypsarrhythmia [2, 5], tonic [5] and myoclonic seizures [3] have been reported.

Brain MRI patterns are highly variable in documented cases of SLC39A8-CDG (Table 1). Our patients presented two different distributions of lesions. Brain MRI in Patient-1 was characterized by a Leigh syndrome-like pattern with bilateral symmetrical basal ganglia hyperintensities and multiple focal signal alterations in subcortical white matter; a similar pattern was identified in the older sister as well (Patient-2) and in four other patients with SLC39A8-CDG irrespective of the underlying genetic variants [4, 5]. However, differently from previously described patients with deep grey matter lesions, Patient-1 and -2 also presented progressive cerebellar atrophy. Cerebellar atrophy is typical of several CDG and it was also found in seven of the eight cases reported by Boycott and colleagues [3] and in a patient described by Park and colleagues [2]. Interestingly, in Patient-1, MRS showed a lactate peak similar to that described in a patient with progressive atrophy of the cerebellar vermis and hemispheres [3]. Cerebellar involvement was not found in Patient-3, whose brain MRI documented thalamic volume loss, subcortical nonspecific alterations of signal, thinning of the corpus callosum and thickening of the anterior commissure but adequate progression of myelinisation and absence of cortical atrophy. Park and colleagues also described a CDG patient without cerebellar atrophy [2]; however, differently from Patient-3, in that case the neuroimaging was also characterized by brain asymmetry with cerebral atrophy of the left hemisphere and enlarged ventricles, especially on the left side of the brain [2]. So, judging from our cases and those already documented in literature, cerebral atrophy, cerebellar atrophy and a Leigh syndrome-like pattern are common features in SLC39A8-CDG and they can be present alone or in different combinations. However, no MRI finding can be considered pathognomonic of this CDG.

Muscular biopsies were performed in both Patient-1 and -3 for a suspected mitochondrial disorder, suggested by their clinical and radiological presentation. In Patient-1 a reduction of respiratory chain complex I activity was found, while respiratory chain enzymes in Patient-3’s skeletal muscle were suggestive of a low activity of complex I + III and an elevated activity of citrate synthetases. However, in both patients, the analysis of mitochondrial and nuclear genome did not show any variant related to mitochondrial disorders. Mitochondrial involvement in SLC39A8-CDG has not been systematically investigated by respiratory chain enzymology so far. However, low muscle complex IV and pyruvate dehydrogenase activity and low liver complexes IV and II + III were identified in a reported patient with Leigh syndrome-like presentation [4]. Thus SCL39A8-CDG should be sought in patients with features of Leigh syndrome-like mitochondrial disease that remain without a genetic diagnosis.

Extensive research on inducible global Slc39a8-knockout mice and Slc39a8 liver-specific-knockout mice unequivocally showed that the hepatocyte ZIP8 transporter reclaims Mn from the bile, decreasing Mn biliary excretion and preserving Mn homeostatic levels in blood and tissues [1]. Disease-associated SLC39A8 mutations caused retention of the ZIP8 transporter in the endoplasmic reticulum thus explaining the inability to localize at the plasma membrane and to transport Mn into cells [12]. Previous studies have investigated the reasons behind the reductions in respiratory chain complex activity in manganese-deficient conditions and have demonstrated that manganese deficiency leads to reduced activity of the reactive oxygen species (ROS) scavenger in mitochondria, MnSOD, manganese being its cofactor [13] and hence to increased levels of superoxide [14]. A direct study, however, demonstrated that SLC39A8 disease-mutations reduced Mn levels in the mitochondria and, in turn, reduced mitochondrial MnSOD activity as well as mitochondrial function. Moreover, SLC39A8 directly promotes the expression of several respiratory chain proteins and ATP production whereas SLC39A8 mutations abolish these functions and enhance ROS generation [12]. ROS could damage mtDNA which encodes some subunits of complex I, III, IV and V of the respiratory chain, and could directly impair the activity of enzymes containing Fe-S clusters, as complex I, II and III [13].

Out of 14 patients with SLC39A8-CDG currently known, transferrin glycoforms analyses by IEF, CE or HPLC showed a CDG type 2 pattern in six [2‐5] and normal patterns in two thus indicating that SLC39A8-CDG diagnosis may be missed by standard transferrin glycosylation analyses. In the patients of this study the sialotransferrin glycosylation patterns were characterized by variable and inconstant increases of trisialotransferrins and mild reductions of tetrasialotransferrins, consistent with the CDG type 2 pattern. We show for the first time in SLC39A8-CDG, age-related transferrin glycosylation changes documented by MALDI-MS in Patient-1. Having monitored transferrin glycosylation along the disease course we found an amelioration of hypoglycosylation in a 4-year period with spontaneous decrease of truncated glycans such as the hypogalactosylated form A2G1S1 at m/z 2227. Spontaneous amelioration of glycosylated biomarkers including transferrin has been reported in some other CDG such as PMM2-CDG [15] and SLC35A2-CDG [16]. Nevertheless, the findings of the present study should be taken into account when using transferrin hypoglycosylation changes to monitor the effectiveness of galactose and/or Mn supplementation therapy in patients with SLC39A8-CDG. Although transferrin glycosylation may only slightly abnormal (Patient-2 and Patient-3) or even normal in patients with SLC39A8-CDG [5], serum N-glycome analyses by MALDI-MS showed a distinct pattern in some previously studied patients [17, 18] (n:4) and in the patients here reported. In particular, total plasma N-glycome profiles are characterized by an increase of undergalactosylated and undersialylated precursors of fully sialylated biantennary glycans, especially the monosialo-monogalacto-biantennary glycan (A2G1S1, m/z 2227). In sum, comprehensive clinical, neuroradiological and glycosylation features are consistent with SLC39A8-CDG, supporting pathogenicity of the SLC39A8 variants in our patients. In Family-1 we identified compound heterozygous Chr4(GRCh37): c.1048G > A (p.Gly350Arg) and c.131C > G (p.Ser44Trp) variants in SLC39A8 that are not yet annotated in ClinVar. The p.Gly350Arg variant most likely modifies the splicing of on the 5’ splice site in exon 7. The p.Ser44Trp variant in exon 2 is located in the same position as the detrimental variant p.Ser44Leu according to GnomAD. Sanger sequencing confirmed the SLC39A8 variants in both affected children and heterozygous in both parents. We here show, for the first time, that the clinical and glycophenotype of a homozygous A391T carrier (Patient-3) are consistent with SLC39A8-CDG. Taking into account the high allele frequency of the A391T (p.Ala391Thr) variant we can thus postulate a spectrum with a wide clinical variability among the variant carriers, ranging from undetectable asymptomatic/paucisymptomatic cases to more severe phenotypes likely due to a compound effect of modulator genes on a homozygous state.

The analysis of plasma protein N-glycosylation showed some degree of dysglycosylation in A391T (p.Ala391Thr) carriers, consistent with a significant increase of biantennary, and decrease of larger triantennary N-glycans and a trend towards less sialylated species than controls. An increase of A2G1S1 monosialo-monogalacto biantennary N-glycan (m/z 2227), consistently found in SLC39A8-CDG, was also observed in A391T homozygous carriers, although a complete clinical information on these subjects was not reported [1, 18]. The rs13107325 (C → T), was found to affect SLC39A8 transcript levels in various tissues and may affect RNA levels in the brain [6, 18]. The A391T mutation is predicted to be disruptive to transporter function, though its precise location is unknown [19, 20].

To date there is little information about mutations related to SLC39A8-CDG (Table 1). Out of fourteen patients, SLC39A8 variant (c.112G > C, p.Gly38Arg) homozygosity was found in nine individuals from three unrelated Hutterite families which establishes p.G38R as a pathogenic founder variant in the Hutterite population. Evolutionary alignment of SCL39A8 amino-acid sequence showed a strict conservation of Gly38 amino-acid in the protein. Several SLC39A8 mutations such as G38R were found to affect the sequence motifs that control subcellular localization and were associated with retention of the ZIP8 transporter in the endoplasmic reticulum explaining the inability to localize at the plasma membrane and transport Mn into cells [12].