Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report

The number of identified genes in which mutations have been shown to induce immunodeficiency has increased considerably as a result of the use of next generation sequencing techniques and the possibility of carrying out whole exome and whole genome analysis [1‐3]. A newly identified gene causing primary immunodeficiency is PGM3 that encodes phosphoglucomutase 3 (PGM3) [4, 5]. Although there are still no unique phenotype characteristics associated with mutations in the PGM3 gene, the majority, but not all patients with PGM3 mutations, present with a pronounced high serum IgE, in combination with recurrent staphylococcal skin abscesses, sinopulmonary infections, and severe eczema [4‐8]. Recently two patients with severe immunodeficiency but without eczema were also described [9]. Other genes causing symptoms of Hyper-IgE syndrome when mutated are STAT3, which is the predominating cause, DOCK8 and TYK2, reviewed in [10, 11].

PGM3 is a key enzyme in the uridine diphosphate N-acetylglucosamine (UDP-GlcNac) synthesis pathway, in which it converts GlcNAc-6-phosphate to GlcNAc-1-phosphate. UDP-GlcNac, is an important building block for both N- and O-linked glycosylation. Congenital disorders in glycosylation can influence many cellular functions, induce developmental changes such as skeletal and mental disturbance and cause severe immunological defects [10, 12]. In mice, it was found that a null mutation in the Pgm3 gene is lethal [13]. Furthermore, until recently all reported studied patients presenting with PGM3 mutations carry homozygous or compound heterozygous missense mutations [4‐9], or a missense mutation in combination with a null mutation [6]. Recently, also patients with a homozygous deletion, causing the loss of a single amino acid, were reported [14]. These mutations all lead to reduced protein stability and reduced enzyme capacity. None of the reported patients were totally devoid of enzyme activity. Altogether, this indicates that a total loss of PGM3 is also likely to be lethal in humans.

The PGM3 mRNA exists in four major transcript isoforms, variants 1 and 4 with 14, and variants 2 and 3 with 13 exons, respectively. In this report, we describe a patient with a homozygous, hypomorphic splice-modifying mutation in an intron of the PGM3 gene. This is the first patient described with altered splicing in PGM3, enabling estimation of the minimum levels of PGM3 necessary for survival.

There are still no established phenotypic markers for PGM3 deficiency, and the number of reported patients is low. In most of the described cases the patients present a phenotype found in glycosylation disorders, including neurologic defects, dysmorphic features, and malformations. In the majority of PGM3 deficient patients very high serum IgE is also found [4, 5, 11]. However, while many patients develop severe immunodeficiency in combination with high serum IgE levels there are cases where patients lack this serological marker [7]. Most PGM3 deficient patients also display severe eczema, but recently two patients with severe immunodeficiency without eczema were described [9]. The patient described in this report, to our knowledge the first case with a hypomorphic splice-altering mutation in the PGM3 gene, also displayed highly elevated serum IgE in combination with immune abnormalities. Still, unlike many of the other described patients this girl did not show any skeletal dysplasia or delayed neurocognitive development. In a few of the recently described cases, cardiovascular changes were reported [8, 9], and cardiovascular abnormalities in combination with hyper-IgE have been described earlier in STAT3 deficient patients [20]. In our patient, vascular abnormalities were found but no heart malformations. In addition, she suffered from a severe, mainly T-cell related, immunodeficiency. While earlier described cases suffered from severe combined B and T cell deficiency [6, 8, 9], the number and distribution of B cells in this patient were within the normal range, although total CD3⁺ T cells were reduced in number. T cell proliferation and responses to recall antigens were also diminished despite increased levels of activated memory T cells. Notably, both the total level and the ratio of recent thymic emigrants, naïve Treg, naïve CD4⁺ and CD8⁺ T cells and Th17 cells were reduced.

The mutation identified here was not found in any of the public databases 1000G and ExAC, and was not present either in a local database of 638 sequenced patients. The region seems well covered both in ExAc and gnomAD. Thus, poor coverage in this region cannot explain that this mutation was not identified earlier. While unlikely, since we do not have access to a collection of DNA samples from a Pakistani control population, we cannot on statistical grounds rule out the possibility that the identified splice site mutation exists as a normal variant in this population. Analyzing the mRNA by RT-PCR confirmed miss-splicing associated with the presence of a single base substitution (A > G) in position + 3 of intron 7. Finding a G in + 3 position of an intron 5′ splice site, as in the patient sequence is not unusual. However, the wild type sequence of the 5′ splice site (ACAGG/GUAUGUCG) is still relatively uncommon due to the U in intron position + 4 [21]. Beside the actual splice-site motif, splicing is influenced by additional sequences within exons as well as in introns, so called splice enhancer (SSE) or splice silencing sequences. As can be seen in Additional file 1: Figure S4, the mutation increases the number of exonic splice silencing sites in this exon/intron border. The mutated splice-site is now so weak that an alternative cryptic splice site further down in intron 7 is also used. Similar erroneous splicing events have been reported in X-linked agammaglobulinemia for instance, where an intact splice-site has been skipped and replaced by an intronic cryptic, suboptimal splice-site due to a change in a SSE sequence [22].

It is always hard to predict the in vivo capacity of mutated enzymes due to a combination of reduced activity and stability. Recombinant PGM3 enzymes with identified mutations have been tested in in vitro enzyme assays [6, 7]. For one of these mutations the enzyme was shown to have around 50% specific enzyme activity while still causing immunodeficiency in the homozygous patient, possibly due to protein instability. Earlier reports of patients with 50% residual enzyme activity might also be overestimated due to in vitro analysis methods, since heterozygous carriers with a null mutation in the PGM3 gene are healthy [6]. It should also be kept in mind that while the existing in vitro assays provide information, it is not known whether they reflect the natural activity of PGM3 in humans. In the patient reported here, the only existing PGM3 protein is derived from the fraction of correctly spliced mRNA, yielding a stable and fully functional wild type protein. Thus, it was for the first time possible to calculate the residual enzyme activity in vivo. By quantitative RT-PCR the wild type mRNA level was found to be 5% of the level in controls. However, according to the Western blot protein quantification the total amount of protein was around 11% of the level found in healthy controls (n = 4). Although mRNA quantification is more precise, there is not always a conserved ratio between mRNA and protein levels. It is known that protein levels can be “buffered” to maintain a stable intracellular level [23], compatible with the higher ratio found in the patient cells.