Background

Congenital Disorders of Glycosylation (CDG) are a rapidly expanding family of genetic diseases. Today, 140 different CDG subtypes have been reported [1] categorized in 2 groups: CDG-I, affecting steps before the oligosaccharide precursor transfer in the endoplasmic reticulum, and CDG-II, affecting the steps following the transfer, mostly in the Golgi apparatus. The first patient cases were reported 40 years ago by Jaeken et al., [2]. Mutations in PMM2 (OMIM 601,785), a gene on chromosome 16p13 encoding a phosphomannomutase were shown to be responsible for the disease. This enzyme catalyzes the conversion in the cytosol of mannose-6-P to mannose-1-P, necessary for the synthesis of donor substrates for glycosylation, GDP-mannose, and Dol-P-mannose.

First named CDG-Ia then changed into PMM2-CDG in 2009 [3], the disease is thought to represent 70% of the total CDG cases, with an estimated incidence around 1:20 000 [4]. The spectrum of clinical phenotypes and severity is broad and is characterized with mainly a psychomotor development impairment associated with cerebellar hypoplasia, hypotonia, dysmorphia, and coagulopathy [5]. The lethality rate in the first 4 years of life is about 20%. Beyond childhood, PMM2-CDG patients have a good life expectancy [6]. The number of PMM2 mutations classified in HGMD (https://my.qiagendigitalinsights.com) is 142, most of these, 113, are missense mutations and the most frequent is p.Arg141His (R141H) [7]. We report the particular genotype of a PMM2-CDG patient with a heterozygous p.Ala108Val (A108V) mutation on one allele and a first-described > 70 kb-deletion on the other allele.

Case presentation

We report the case of a PMM2-CDG French child of Caucasian descent. She was born at term with a length of 48 cm for 3.110 kg, after uncomplicated pregnancy with vaginal delivery. The AGPAR score was 10 both at 1 min and 5 min and the newborn screening results were normal. The parents are unrelated and the 6-years older brother is healthy. Parents reported abnormal abrupt movements of the child after birth that stopped spontaneously.

Biologically, at 9 months of age, elevated transaminases TGO = 50 UI/ L, TGP = 51 [normal values 10–35 UI/ L] and normal coagulation parameters with ATIIIA = 63% [normal values 80–120%] and FXI 53% at the lower limit [normal values 50–150%] were assayed. The cytology and the thyroid function were normal.

The clinical examination at 9 months of age revealed ataxia, hypotonia, hyperlaxity, strabismus, esotropia, feeding difficulties, and inverted nipples. The child was calm and exclusively breastfed with an absence of facial dysmorphia and no sleeping disorders. At that time, the girl presented an inability to reach a seated posture. The diagnosis of CDG was oriented by an abnormal pattern in serum transferrin isoelectrofocusing [8] with an elevation of asialo- and disialo-transferrin, typical from a type I CDG (Fig. 1). Brain MRI revealed cerebellar abnormalities with vermis hypoplasia. The child finally reaches a seated posture at 11 months of age.

Fig. 1
figure 1

Distribution of transferrin glycoforms via transferrin isoelectrofocusing of the patient compared to a control. Numbers 0, 2, 3, 4, 5, and 6 indicate the migration position of the asialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin forms respectively

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Genetic testing

Direct Sanger sequencing of the 8 exons of PMM2 reported a seemingly homozygous variant rs200203569 NM_000303.3(PMM2): c.323C > T in exon 4. The variant leads to a missense substitution of alanine 108 to proline (p.Ala108Pro), commonly named A108V, known to be pathogenic (ClinVar, SIFT, Mutation Taster). The A108V mutation is quite rare and is often associated with R141H, the most common deleterious PMM2 mutation, in compound heterozygous patients [9]. A homozygous presentation of R141H variant is thought to be incompatible with life as no case was reported so far [10]. For the A108V variant, the gnomAD (2.1) website reports a frequency of 0.0012% in the overall population. To our knowledge, no homozygous A108V patient is reported in the literature and, as the parents were unrelated, further genetic explorations were conducted. Direct Sanger sequencing of PMM2 of the paternal DNA reported a heterozygous A108V mutation while no mutation was found in the mother. At the time of the genetic exploration, the seemingly second variant could have been either due to the de novo variant or to the absence of sequence at the same location on the other allele. A quantitative PCR (qPCR) of the 13 exons was performed, showing a reduction of the DNA of the gene by 50% in the mother and the proband from exon 3 to exon 8, the last exon of PMM2 (Fig. 2). The hypothesis of the de novo variant was rejected as the heterozygous deletion in PMM2 gene was found. To evaluate the extent of the deletion that goes beyond PMM2 gene, Whole Genome Sequencing (WGS) was performed. WGS was preferred to CGH array to accurately determine the exact position of the breakpoints. WGS allowed to delineate the deletion of 70,453 bp in position chr16:8,897,826–8,968,278 (Fig. 3). In the HGMD database, the largest deletion reported is 28 kb-long.

Fig. 2
figure 2

PCR quantification of PMM2 exons. qPCR of exons 2 to 8 of PMM2 in the proband and the parents compared to standard and control samples

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Fig. 3
figure 3

Deletion breakpoint localization. Reads quantification in position chr16:8,897,826–8,968,278 of the family members. A deletion of 70453pb is seen for the proband and the mother. The genes affected by the deletion are named on the bottom of the figure

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The deletion also affects a part of CARHSP1 (Calcium Regulated Hear Stable Protein 1) (OMIM: 616,885) gene that plays a role in TNF mRNA stabilization, seemingly not affecting the phenotype.

Discussion and conclusions

In the present study, we described the case of a PMM2-CDG patient with congenital ataxia. The genotype identified in the child is novel. The clinical course was relatively mild for a child with PMM2-CDG as the child does not present facial dysmorphia. Given that the maternal mutation could not be detected upon Sanger sequencing, further investigations were performed to precise the genetic transmission of the disease.

A108V mutation was first described in France [7] and the effect on the enzyme activity is unknown. When associated to a mutation in R141H the remaining phosphomannomutase activity in leucocytes is 0.09% [11].

Quantitative PCR and WGS allowed to identify a large deletion on the maternal allele. A new deletion of 70,453 bp in position chr16:8,897,826–8,968,278 could be accurately detected with WGS including 6 exons of PMM2 and a part of CARHSP1 gene. Knockout of CARHSP1 has demonstrated the role of CARHSP1 as a TNF-α mRNA stability enhancer [12]. GnomAD database reports various loss of function heterozygote mutation for CARHSP1, indicating that the observed pathology is mainly due to the phosphomannomutase defect.

To our knowledge, this is the largest PMM2 deletion reported so far. Our example illustrates the usefulness of WGS in the case of an apparent homozygous variant in an unrelated family. Wherever possible, compound heterozygosity has to be confirmed with a parental genetic study. As the disease transmission for the couple is 25%, an antenatal diagnostic can now be proposed for future pregnancies.

This case underlines the importance of the correlation between the phenotype description and the genetic study, especially in disorders with a wide phenotypic spectrum like PMM2-CDG [13], where it is essential for a correct diagnosis to search thoroughly over the appearances.

In case of strong suspicion of PMM2-CDG based on the clinical phenotypes and despite the absence of transferrin abnormalities, the molecular analysis should be performed to avoid any diagnostic deadlocks.