A Case of PMM2-CDG Caused by an A108V Mutation Associated With a Heterozygous 70 Kilobases Deletion

Elodie Lebredonchel (  elodie.lebredonchel@chu-lille.fr ) UGSF: Unite de Glycobiologie Structurale et Fonctionnelle https://orcid.org/0000-0001-5119-6005 A. Riquet CHU Lille Pôle Enfant: Centre Hospitalier Universitaire de Lille Pole Enfant D. Neut CH Boulogne sur Mer: Centre Hospitalier de Boulogne sur Mer F. Broly Centre Hospitalier Régional Universitaire de Lille Centre de Biologie Pathologie: Centre Hospitalier Regional Universitaire de Lille Pole de Biologie Pathologie Genetique G. Matthijs KU Leuven Center for Human Genetics: Katholieke Universiteit Leuven Centrum Menselijke Erfelijkheid A. Klein CHRU de Lille CBP: Centre Hospitalier Regional Universitaire de Lille Pole de Biologie Pathologie Genetique F. Foulquier UGSF: Unite de Glycobiologie Structurale et Fonctionnelle


Introduction
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 rst patient cases were reported 40 years ago by Jaeken et al., (2). Mutations in PMM2 (OMIM 601785), a gene on chromosome 16p13 encoding a phosphomannomutase. This enzyme catalyzes the conversion in the cytosol of mannose6-P to mannose-1-P, necessary for the synthesis of donor substrates for glycosylation, GDPmannose, and DolPmannose.
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 rst 4 years of life is about 20%. Beyond childhood, PMM2-CDG patients have a good life expectancy (6). The number of PMM2 mutations classi ed 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 rst-described >70 kb-deletion on the other allele.

Case Report
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 minute and 5 minutes 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. The clinical examination at 9 months of age revealed ataxia, hypotonia, hyperlaxity, strabismus, esotropia, feeding di culties, 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 nally reaches a seated posture at 11 months of age.

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, that is 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 patients are 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.
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). A heterozygous deletion of PMM2 gene was then suspected. 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 70453 bp in position chr16:8,897,826-8,968,278 (Fig. 3). In the HGMD database, the largest deletion reported is 28 kb-long.
The deletion also affects a part of CARHSP1 (Calcium Regulated Hear Stable Protein 1) (OMIM: 616885) 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 identi ed 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 investigation were performed to precise the genetic transmission of the disease.
A108V was rst described in France (7) and its 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 70453 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 con rmed with a parental genetic study. As the disease transmission for the couple is 25%, an antenatal diagnostic can now be proposed for future pregnancies.