Novel biallelic GNE variants identified in a patient with chronic thrombocytopenia without any symptoms of myopathy

doi:10.21203/rs.3.rs-4708622/v1

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Case Report

Novel biallelic GNE variants identified in a patient with chronic thrombocytopenia without any symptoms of myopathy

https://doi.org/10.21203/rs.3.rs-4708622/v1

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[Background] GNE encodes a rate-limiting enzyme that regulates the biosynthesis of a sialic acid precursor. As sialic acids are critical for the platelet membrane and muscle fibers, GNE variants cause GNE-related thrombocytopenia and GNE-related myopathy. Here, we report a neonate with thrombocytopenia that initially met the criteria for neonatal allo-immune thrombocytopenia (NAIT) but was resistant to treatments and then revealed novel biallelic heterozygous GNE variants without any symptoms of myopathy when diagnosed.

[Case] NAIT was initially diagnosed due to alloantibodies against HPA5 and its mismatch between the patient and his mother. However, intravenous immunoglobulin therapy and platelet transfusions showed minimal improvement in the platelet count. Platelet counts remained around 60 × 10⁹/L, suggesting congenital thrombocytopenia. Gene panel sequencing at the age of 13 identified biallelic pathogenic variants of GNE. The patient did not exhibit any symptoms of muscular weakness suggesting GNE-related myopathy.

[Discussion] We demonstrated a GNE-related thrombocytopenia patient with novel biallelic heterozygous GNE variants. Clinical trials have involved the use of sialic acids or their precursors, as well as gene therapy, to treat GNE-related myopathy, which may slow or halt the progression of the disease. Therefore, early diagnosis of this disease may significantly impact its clinical course.

GNE

thrombocytopenia

myopathy

NAIT

Congenital thrombocytopenia in neonates is less likely than the acquired ones [1]. Although it is fairly rare, 10% of treatment-resistant or chronic immune thrombocytopenia (ITP) cases are thought to be congenital [2].

The GNE gene encodes a rate-limiting enzyme that regulates the biosynthesis of a precursor of sialic acid called UDP-N-acetyl glucosamine 2-epimerase/N-acetylmannosamine kinase. The pathogenic variants in this gene reduce the production of sialic acid and cause oxidative stress in the plasma membrane. This oxidative stress causes protein misfolding and the activation of the ubiquitin-proteasome system. The modification of sialic acid of glycoproteins and glycolipids on the cell surface is also significant for its functions, such as cell adhesion and signal transduction [3].

Homozygous or biallelic heterozygous variants in GNE cause thrombocytopenia with or without myopathy (NIM#620757). This thrombocytopenia is an autosomal recessive disorder characterized by congenital thrombocytopenia apparent from infancy or early childhood [3]. Most affected individuals have bleeding episodes, including petechiae, easy bruising, epistaxis, hematomas, and increased bleeding after trauma or surgery.

Although GNE-related thrombocytopenia often occurs during the neonatal or infantile periods, neonates with allo-immune anti-platelet antibodies have not been reported to our knowledge. Here, we report a neonatal case of thrombocytopenia that initially met the criteria for neonatal allo-immune thrombocytopenia (NAIT), but was resistant to treatments. We revealed novel GNE variants without any symptoms of myopathy appearing in the patient at the time of diagnosis.

A 13-year-old boy presented with persistent thrombocytopenia. The patient was born to a healthy mother at full term. The neonate had jaundice 3 days after birth, and blood tests were performed to determine the cause of incidentally noted thrombocytopenia. His parents were not consanguineous and without a history of thrombocytopenia or bleeding symptoms (Fig. 1a). As we proceeded with our diagnosis for the newborn, we suspected NAIT based on alloantibodies against HPA5 and its mismatch between the patient and his mother. Thus, the newborn began treatment with intravenous immunoglobulins (IVIG) and platelet transfusion on day 6, which showed brief improvement, but the platelet count started to decrease later. Therefore, another IVIG and platelet transfusion were given on day 15, which showed a similar result as the first time. Once the platelet count stabilized around 50 × 10⁹/L, the patient was discharged (Fig. 1b). He was carefully observed through outpatient visits as there were no episodes of severe bleeding. At 6 years of age, prednisolone 1.0 mg/kg/day for a week was given for tooth extraction with little improvement in platelet counts. We administered elthrombopag 6.25 mg/day for a week and 12.5 mg/day for four weeks, resulting in an increase in platelet count up to 100 × 10⁹/L.

At 13 years of age, he had purpura, but no hearing loss, cataracts, or hepatosplenomegaly. Neurological examinations showed normal results without muscular weakness, upper/lower motor neuron symptoms, or abnormal reflexes/sensory signs/meningeal signs. We found normal coordinated movement, gait, and autonomic neuron functions. Complete blood count showed white blood cells 6.3 × 10⁹/L, hemoglobin 130 g/L, platelets 150 × 10⁹/L, mean platelet volume unmeasurable, and mean platelet diameter 4.3 µm (control: 2.7 µm) (Table 1, Figs. 1c and 1d). The serum chemistry tests, including liver and renal functions and urine analyses, were all normal. Coagulation studies revealed a Duke bleeding time of 5 min and von Willebrand factor antigen 84%. Bone marrow examination showed normal nucleated cells and megakaryocytes with normal maturity (Figs. 1h and 1i). Flow cytometry showed increased expression of GPIIb and GPIIIa on the platelets (Table 1). Chromosome analysis was also normal. After informed consent was obtained, the genomic DNA of the patient was tested by gene panel sequencing, as previously shown [4]. The gene panel testing revealed novel biallelic variants in GNE, namely, [NM_01128227.3:c.1277del,p.(Asn426IlefsTer13)] and [NM_01128227.3:c.1163 + 5G > T], which were confirmed by Sanger sequencing (Fig. 1j). These variants were classified as pathogenic and likely pathogenic according to the ACMG-AMP guidelines, respectively (Supplementary table).

Table 1

Clinical and laboratory characteristics of the members of the reported family
	Patient	Mother	Father
Age, years	1	55	45
Bleeding tendency	Purpura	No symptoms	No symptoms
White blood cells, 10⁹/L	6.3	4.9	6.1
Hemoglobin, g/L	130	137	131
Platelet count, 10⁹/L	15	187	148
MPV, fL	Unmeasurable	10.6	11.6
MPD, µm	4.3	2.7	2.8
Bleeding time, min	5.0	2.0	2.5
GPIb, %	103.6	103.6	96.4
GPIX, %	129.6	111.1	106.2
GPIIb, %	177.4	124.1	100
GPIIIa, %	218.6	126.5	116.7
GPIIb/IIIa, %	202.7	126.7	120.5
Bleeding time was measured by Duke’s method. Abbreviations: MPV, mean platelet volume; MPD, mean platelet diameter; GP, glycoprotein.

A family study showed that platelet counts were 187 × 10⁹/L in the father and 148 × 10⁹/L in the mother. Platelet sizes and morphologies were consistent with the controls (Figs. 1e and 1f). The father had a heterozygous variant in GNE, [NM_01128227.3:c.1277del,p.(Asn426IlefsTer13)] (Figs. 1j and 1k). The mother had a heterozygous variant in GNE, [NM_01128227.3:c.1163 + 5G > T] (Figs. 1j and 1l).

In this case, a thrombocytopenic neonate met the criteria for the NAIT diagnosis; however, treatment showed little improvement. Hence, congenital thrombocytopenia was subsequently suspected, which led to the diagnosis of GNE-related thrombocytopenia.

This case is unique in a sense that there has not been a report of any case of GNE-related thrombocytopenia meeting the criteria for the diagnosis of NAIT. Immune-mediated thrombocytopenia, such as NAIT, is one of the most common causes of early-onset neonatal thrombocytopenia. The estimated incidence of the thrombocytopenia is 0.3%, half of which is due to NAIT [5, 6]. NAIT was suspected in this case because of the HPA5 mismatch between the patient and his mother. Response to IVIG treatment was poor and platelet transfusion showed only transient improvement in the platelet count. In combination with platelet transfusion, IVIG is thought to be an effective treatment for NAIT. In this case, despite the unstable platelet count, it remained around 60 × 10⁹/L, which led to suspicion of congenital thrombocytopenia.

GNE-related thrombocytopenia is very rare in congenital thrombocytopenia. Sialic acids mediate the prevention of rapid platelet clearance by Ashwell-Morell receptors on the hepatocytes [7]. A variant in the gene causes increased platelet clearance. Platelets are large (macrothrombocytopenia) and circulating immature platelets are increased, suggesting increased production. Patient platelets show hyposialylation due to GNE variants, which causes accelerated removal of platelets from the circulation, shortened platelet lifespan, and resultant thrombocytopenia. Our patient had novel biallelic heterozygous variants in GNE. The mother had mild thrombocytopenia, suggesting that the variant may affect splicing of GNE, which is likely to be pathogenic.

In our patient, GNE-related thrombocytopenia was determined in the low teens. Biallelic variants in GNE also cause GNE-related myopathy. It is an autosomal recessive progressive adult-onset myopathy with a predilection for distal muscle involvement, usually affecting the lower limbs and resulting in gait abnormalities or loss of ambulation. In recent studies, GNE variants have been reported to cause myopathy and thrombocytopenia [8]. There are two large registries of GNE myopathy, one in Japan and one in Israel, 2.5% and 0% of the patients had associated thrombocytopenia, respectively [9]. In contrast to thrombocytopenia, which is present since birth or early childhood, features of myopathy usually do not develop until the mid-twenties. Its average onset age is thought to be 27.7 ± 9.6 years [9]. GNE variants were diagnosed in our patient without clinical symptoms of neurological/muscular impairment. Clinical trials have involved the use of sialic acids or their precursors, as well as gene therapy trials, to treat GNE-related myopathy [10]. These trials are still in the early stages, but some products are thought to have a slight impact on muscle functional measurements without serious adverse effects [11]. Although it still is not clear which metabolic supplementation can affect its clinical course, slowing or halting the progression of the disease is the realistic outcome and this could have an incredible effect if the disease is diagnosed at an early stage [10]. The search for better GNE metabolites or sialic acid compounds [12], drugs to block or modify degenerative processes, and gene or cell-based therapy is ongoing. In the future, these therapies can be combined to provide better outcomes for the disease. To achieve appropriate treatments, we should carefully monitor the patient’s symptoms.

Despite the initial diagnosis of NAIT, biallelic GNE variants were diagnosed for chronic resistant thrombocytopenia in the patient. We conducted the patient’s gene analysis, although the patient had no muscular/neurological symptoms. We believe that gene analysis can be a key component in diagnosing neonatal thrombocytopenia when prolonged thrombocytopenia is found. Early diagnosis of this disease may significantly impact its clinical course by the potential treatments in the clinical trials.

Compliance with ethical standards

This study was approved by the Ethics Committees of the NCCHD in May, 2018 (#1818). Informed consent for this report was obtained from the patient and her parents.

Permission to reproduce material from other sources

In accordance with the regulations of the place where the article was accepted.

Data availability statement

De-identified patient data is available upon request to corresponding author.

Competing interests

The authors declare that they have no competing interests.

Funding sources

This study was supported by The Grants of Japan Agency for Medical Research and Development (18ek0109366h0001, 19ek0109366h0002, and 20ek0109366h003), and The Grant of NCCHD (2021B-3, 2024B-3) to Ishiguro A.

Author contributions

ST, AS, SK, and AI (Ishiguro) contributed to the conception of this study; ST, AS and AI (Ishiguro) created the database and drafted the manuscript; TU, TK, KY, and SK performed gene analyses. HK and others managed patients and collected critical samples. All authors reviewed and approved the final manuscript

Acknowledgments

We express our gratitude to the participating patient and her parents. We are grateful to the senior medical editor at the Center for Postgraduate Education and Training at the National Center for Child Health and Development for English language review.

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No competing interests reported.

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Novel biallelic GNE variants identified in a patient with chronic thrombocytopenia without any symptoms of myopathy

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