ALG3-CDG: a patient with novel variants and review of the genetic and ophthalmic findings

Background ALG3-CDG is a rare autosomal recessive disease. It is characterized by deficiency of alpha-1,3-mannosyltransferase caused by pathogenic variants in the ALG3 gene. Patients manifest with severe neurologic, cardiac, musculoskeletal and ophthalmic phenotype in combination with dysmorphic features, and almost half of them die before or during the neonatal period. Case presentation A 23 months-old girl presented with severe developmental delay, epilepsy, cortical atrophy, cerebellar vermis hypoplasia and ocular impairment. Facial dysmorphism, clubfeet and multiple joint contractures were observed already at birth. Transferrin isoelectric focusing revealed a type 1 pattern. Funduscopy showed hypopigmentation and optic disc pallor. Profound retinal ganglion cell loss and inner retinal layer thinning was documented on spectral-domain optical coherence tomography imaging. The presence of optic nerve hypoplasia was also supported by magnetic resonance imaging. A gene panel based next-generation sequencing and subsequent Sanger sequencing identified compound heterozygosity for two novel variants c.116del p.(Pro39Argfs*40) and c.1060 C > T p.(Arg354Cys) in ALG3. Conclusions Our study expands the spectrum of pathogenic variants identified in ALG3. Thirty-three variants in 43 subjects with ALG3-CDG have been reported. Literature review shows that visual impairment in ALG3-CDG is most commonly linked to optic nerve hypoplasia.


Background
Congenital disorders of glycosylation (CDG) are a rapidly expanding group of genetic multisystem diseases characterized by hypoglycosylation of glycoproteins and glycolipids. Based on the type of glycosidic bond, N-and O-glycosylation disorders are distinguished as two main subclasses of CDG [1].
In this study, we characterize in detail the first Czech patient with ALG3-CDG and provide a review of the literature on the identified disease-causing variants and reported ocular findings.

Case presentation
The study was approved by the Ethics committee of the General University Hospital in Prague and adhered to the tenets of the Helsinki Declaration.
The patient was born at 34 6/7 weeks of gestation, with a birth weight of 2.190 kg (34th percentile, -0.41 SD), length of 45 cm (46th percentile, -0.1 SD) and head circumference of 30 cm (16th percentile, -1 SD), following a pregnancy with polyhydramnion and discrete pericardial effusion. She is the first child of non-consanguineous Czech parents. Her early postnatal adaptation was uneventful (Apgar score 8-9-9), but already at birth multiple joint contractures and craniofacial dysmorphic features were noticed. The otoacoustic emissions were absent. Abdominal ultrasound documented multiple renal cysts.
Neurological examination showed spastic tetraplegia, hyporeflexia and developmental delay. Although clinical seizures were not noticed, electroencephalography (EEG) was severely abnormal showing high amplitude slow spike-wave complexes (SSWC) and slow background activity. Anticonvulsant therapy was therefore initiated. The brain magnetic resonance imagining showed cerebral and cerebellar atrophy, cavum septum pellucidum, enlarged 4th ventricle and cisterna magna, delayed myelinization, and optic nerve hypoplasia.
Ophthalmic examination showed hypopigmentation of the fundus and pale optic discs. Visual and brain stem auditory evoked potentials (VEP, BAEP) were both pathological with prolonged latencies of wave N70 and waves III, V, respectively. Cardiological examination was normal apart from insignificant patent arterial duct and patent foramen ovale.
At 12 months of age, epilepsy was not compensated despite combined therapy with levetiracetam and phenobarbital. Add-on therapy with topamirate was started. At that time, although the development was severely delayed, the patient began to fix objects and to react to sounds and voices of family members, gurgle and lift up her head.
We performed esophagogastroduodenoscopy that revealed esophagitis and duodenum ulcer. Repeated abdominal ultrasonography documented multiple cysts in both kidneys which were of stable size since birth, other abdominal organs were without abnormalities.
Ocular examination including spectral-domain optical coherence tomography (SD-OCT) (Spectralis, Heidelberg Engineering GmbH, Heidelberg, Germany) revealed profound loss of retinal ganglion cells and inner retinal layer thinning with preservation of the external limiting membrane, ellipsoid zone and interdigitation zone ( Fig. 1 F-1I). Fundus appeared hypopigmented and optic discs were pale as documented using ultra-wide field camera (Clarus 700, Carl Zeiss Meditec AG, Jena, Germany). Visual activity could not be measured.
Transferrin isoelectric focusing was performed as described [19] and showed a type 1 pattern. DNA from the proband and her parents was extracted from peripheral leucocytes. Targeted sequencing of 260 genes related to neuromuscular diseases and arthrogryposis was performed by hybrid capture enrichment (Roche NimbleGen, Pleasonton, CA, USA) and massive parallel sequencing (NextSeq, Illumina, San Diego, CA, USA) as described [20]. Sequencing data were analysed with Sequence Pilot software (JSI medical systems, Ettenheim, Germany). Conventional Sanger sequencing Analysis of the ganglion cell layer using automatic segmentation documents profound loss (H) when compared to a scan obtained from a similarly aged healthy female child (I). The quality of retinal imaging was limited by compliance, images of the left eye could not be obtained was used to verify the presence of presumed causal variants and for targeted screening in first-degree relatives. The effect of one missense mutation was predicted using five computational tools: Sorting Intolerant from Tolerant (SIFT) [21], Polyphen-2 [22], Mutation Taster2 [23], M-CAP [24] and CADD [25]. The pathogenicity of the detected variants was evaluated according to the American College of Medical Genetics and Genomics (ACMG) recommendations [26].
Two ALG3 variants (NM_005787.6) c.116del p.(Pro39Argfs*40) and c.1060 C > T p.(Arg354Cys) were identified, neither of them has been reported. Segregation analysis confirmed their trans position, the c.116del was inherited from the mother while the c.1060 C > T from the father. All five prediction tools used evaluated the missense variant as deleterious or likely deleterious. Based on the available evidence both variants were classified as pathogenic according to the ACMG criteria.

Discussion
In this study we describe clinical, biochemical, and molecular genetic findings of an unreported ALG3-CDG patient.    We also review ocular and molecular genetic findings in all 43 individuals with ALG3-CDG identified to date.
Our case adds to the previously published phenotype data several novel aspects. In earlier reports pale or small optic disc was observed in 12 patients suggesting optic nerve atrophy or hypoplasia [2,5,6,8,9,11,14,18]. Our case report is however the first clearly documenting optic nerve and retinal layers pathology by ocular imaging methods.

Clinical course
Out of the 43 reported individuals 17 had a lethal phenotype with prenatal manifestation, medical termination of pregnancy and/or early neonatal death. Three further patients died of respiratory failure during pneumonia in childhood (aged 1.8; 3.5 and 6 years) [11,12,14] and one of multiorgan failure (aged 1 year) [18]. Neurological symptoms have recently been summarized in 26 patients, and a ketogenic diet was suggested as a therapeutic option for intracrable epilepsy [16]. Dysmorphic and musculoskeletal features have been also recently reviewed in 19 patients [15].

Ocular phenotype
Presumably because of poor compliance due to young age at examination and developmental delay available ophthalmic data in ALG3-CDG are limited (Table 1). Most often poor eye fixation, strabismus and/or nystagmus have been noted. However, the likely underlying cause was only reported in few patients and comprised optic nerve atrophy/hypoplasia observed in 11 patients including the current study confirming deficiency of retinal ganglion cells by SD-OCT, and signs of chorioretinal dystrophy in three patients, of these one had also optic atrophy [11]. Electroretinography was reported in three patients; decreased amplitudes of both rod and cone responses were observed [4,7].
Less common ocular features of ALG3-CDG were congenital cataract found in one individual [10] and iris coloboma present in another patient [2]. One fetus examined after pregnancy termination at week 25 was noted to have proptosis and corneal opacities [10]. Another fetus terminated in week 28 had eyelid ptosis [15].

Conclusions
CDG pose a diagnostic challenge due to their high phenotypic variability. Combination of severe neurologic and visual impairment with dysmorphia, arthrogryposis and other congenital malformations should raise suspicion of a CDG syndrome. Authors' contributions MF and AC conducted the review and wrote a substantial part of the manuscript including the figure and tables. TH, MF, BK, and PL examined the patient. TH and PL contributed to the manuscript with advice and critical comments, supervised the manuscript preparation. HH and NO performed the biochemical analyses. JZ did the molecular genetic study. The manuscript was reviewed and revised by all the authors. The author(s) read and approved the final manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics committee of the General University Hospital in Prague (reference no. 34/19; date 23.5.2019). Written informed consent was obtained from the patient's parents before inclusion.

Consent for publication
We obtained from the patient's parents written informed consent for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Competing interests
The authors declare that they have no competing interests.