Severe congenital neutropenia due to G6PC3 deficiency: Case series of five patients and literature review

Glucose‐6‐phosphate catalytic subunit 3 (G6PC3) deficiency is characterized by severe congenital neutropenia with recurrent pyogenic infections, a prominent superficial venous pattern and cardiovascular and urogenital malformations caused by an alteration of glucose homeostasis, with increased endoplasmic reticulum stress and cell apoptosis.


| INTRODUCTION
While rare diseases are individually infrequent, collectively they afflict around 10% of the world population. Patients with rare diseases typically must endure a 'diagnostic odyssey' that may take several years before receiving a correct diagnosis and treatment. Inborn errors of immunity (IEI) are a group of rare congenital diseases caused by monogenic germline variants that result in the modification of protein expression or function, affecting the development, function and homeostasis of the immune system. 1 IEI comprise a wide spectrum of disorders that may manifest not only with increased susceptibility to infections but also with inflammation, autoimmunity, allergy or malignancy.
In 1950, Rolf Kostmann first described 'hereditary agranulocytosis', or severe congenital neutropenia (SCN), characterized by an early onset of recurrent bacterial and fungal infections of the mouth, umbilical stump, skin, gastrointestinal tract, bones, lungs and lymph nodes. 2 Although pathogenic variants in ELANE are the most common genetic aetiology of autosomal dominant (AD) SCN, several others have been described in the last 2 decades: X-linked WAS gain of function (GOF); autosomal recessive (AR) HAX1, GFI1, CSF3R and G6PC3 loss of function (LOF) and AD TCIRG1 haploinsufficiency. The molecular cause is unknown in about 30% of patients with SCN.
In 2009, Boztug and Klein reported 3 a series of 12 patients with congenital neutropenia and various cardiovascular and urogenital developmental anomalies, who had homozygous and compound heterozygous variants in the gene-encoding glucose-6-phosphatase catalytic subunit 3 (G6PC3 deficiency, also known as type 4 SCN, OMIM #612541), as well as prominent superficial veins (mainly in trunk and limbs). G6PC3 is located on chromosome 17q21 and spans 6 exons.
Independently, a pair of Turkish siblings were reported with pulmonary arterial hypertension, and other abnormalities including cardiac, haematological and skeletal defects. 4 Over the last decade, the spectrum of the disease has continuously widened to include non-syndromic forms and new features. 5 Here, we describe 5 patients from Mexico, and review the available literature for clinical features, genetic variants, treatment and outcome of 89 more patients with G6PC3 deficiency.

| Patient 1
A 3-month-old male, born to non-consanguineous parents from rural central Mexico, was born prematurely at 35 weeks and admitted to the neonatal intensive care unit for extensive oedema and ecchymoses, with respiratory distress that required mechanical ventilation. He was started on intravenous antibiotics for pneumonia and sepsis.
Physical examination found low weight, a low-grade systolic murmur, hepatomegaly and cryptorchidism; prominent superficial veins in thorax, abdomen and all 4 limbs were later noted. Laboratory workup reported transient-variable anaemia, lymphopaenia and thrombocytopenia; as well as severe persistent neutropenia and pan-hypogammaglobulinaemia. An echocardiogram showed a persistent foramen ovale (4 mm) with bidirectional shunt and pulmonary arterial hypertension at 58 mm Hg. Abdominal ultrasound revealed pyloric stenosis and severe bilateral hydronephrosis.
Filgrastim (granulocyte colony-stimulating factor, G-CSF) was started at 3-5 µg/kg/d, with a spectacular but transient increase in neutrophil count, as well as prophylactic antibiotics (trimethoprim/sulfamethoxazole and itraconazole) and monthly intravenous immunoglobulin | 3 of 27 VELEZ-TIRADOetal. (IVIG) at 1g/kg. A homozygous single-nucleotide deletion in exon 1 of G6PC3 (c.210delC, p. Phe71SerfsTer46) was identified through Sanger sequencing, a variant previously reported. 6 In time, the dosage of filgrastim had to be increased up to 22 mg/kg/d to achieve an acceptable neutrophil count. Due to this severe and early presentation, haematopoietic stem cell transplantation (HSCT) was performed in this patient at the age of 15 months. Unfortunately, he developed neutropenic colitis and died of sepsis on day +29.

| Patient 2
A 15-year-old female, born to non-consanguineous parents from a small rural community (300 inhabitants), was admitted for a 6-year history of episodic diarrhoea and intense generalized abdominal pain, each episode lasting between 7 and 14 days, for which she had received multiple antibiotic treatments with slight improvement. Her past medical history also included pneumonia at 4 months old, as well as recurrent otitis media and other upper airway infections.
On admission, we found a severely undernourished patient, with prominent superficial veins, finger clubbing, brachymetatarsia of the third toes and bilateral sensorineural hearing loss. Laboratory workup revealed anaemia, intermittent neutropenia and lymphopaenia. Colonoscopy and histopathology were compatible with Crohn's disease; echocardiography revealed mild tricuspid insufficiency.
At age 16, she developed bronchopulmonary aspergillosis and was treated with voriconazole. Despite good adherence, complete remission of IBD was never achieved. At 17 years, she presented with acute abdominal pain, fever and vomit; she died with abdominal sepsis after intestinal perforation. Remarkably, prior to this catastrophic event, she had been asymptomatic. The autopsy revealed severe extensive bowel inflammation, which was at odds with her clinical symptoms and signs.

| Patient 3
A 3-month-old female patient, born to non-consanguineous Mexican parents, with a family history of 3 paternal uncles who died before the age of 5 years. She first presented with neonatal sepsis requiring intravenous antibiotics, after which she had 5 episodes of pneumonia and a surgically corrected rectovaginal fistula.
On physical examination, we found severe chronic malnutrition, prominent superficial veins in her abdominal wall, bilateral sensorineural hearing loss and supplemental oxygen dependence. Her clinical assessment revealed severe neutropenia, atrial septal defect and severe pulmonary damage due to multiple atelectasis and bronchiectasis, associated with pulmonary arterial hypertension. Whole-exome sequencing identified the same homozygous single-nucleotide deletion in G6PC3 (c.210delC; p. Phe71SerfsTer46) as in patients 1 through 4. Patient 3 is currently 3 years old, alive and well, under treatment with granulocyte colony-stimulating factor (G-CSF, filgrastim-30 mcg/kg/d).

| Patient 4
A 9-year-old boy from northern Mexico, with no known consanguinity or family history of infections or early deaths. He started at age 3 months with suppurative otitis media and pneumonia that required admission to the intensive care unit (ICU) and was hospitalized for a month. Neutropenia was recorded since then, with absolute neutrophil counts (ANC) within the range 450-790/mm 3 , which responded well to filgrastim (G-CSF).
However, despite the increase in neutrophil numbers, the patient persisted with multiple respiratory tract infections requiring frequent hospitalizations and parenteral antibiotics up to 5 years of age. He also presented with oral, finger and perianal abscesses, without any isolates. From 6 years on, the frequency of infections decreased to 1-2 per year. On physical examination, weight and height were normal; he had a prominent superficial venous pattern in trunk and limbs, as well as redundant skin folds in the neck and a left inguinal hernia.
Laboratory workup reported intermittent thrombocytopenia and persistent lymphopaenia; bone marrow aspirate showed myeloid cell hypoplasia and non-specific findings. An echocardiogram revealed persistent foramen ovale (corrected percutaneously with an Amplatzer device), and pulmonary hypertension of 45mm/Hg, whereas a Doppler ultrasound of the liver documented compensated portal hypertension. Percutaneous liver biopsy reported sinusoid fibrosis and steatosis.
Sanger sequencing identified a compound heterozygous genotype, with variants in exons 1 and 4 of G6PC3: the same single-nucleotide deletion (c.210delC, p. Phe71SerfsTer46) and a nonsense transition (c.481C>T, p. Arg161*) respectively. The patient is currently alive and well, under treatment with G-CSF 5 mcg/kg/day.

| Patient 5
A 2-year-old female, born to non-consanguineous Mexican parents, was referred to our care for a history of cow's milk allergy, allergic asthma and rhinitis, recurrent otitis media, sepsis, urinary tract infection, autoimmune colitis and disseminated herpes zoster.
On physical examination, she did not have any syndromic features. Complete blood count reported severe neutropenia, anaemia, intermittent lymphopaenia and thrombocytopenia. Through whole-exome sequencing analysis, a compound heterozygous genotype consisting of 2 single-nucleotide deletions in exons 1 and 4 of G6PC3 was identified (c.210delC, p. Ile70HisfsTer46; c.421del, p. Trp141GlyfsTer2); this second frameshift variant has not been previously described and is not found in gnomAD. The patient is alive under treatment with G-CSF 3 mcg/ kg/day and prophylaxis with TMP-SMZ and fluconazole.

| Ethics approval
This case series study was granted exemption by the National Institute of Pediatrics Research and Ethics Committee due to its retrospective design. All authors subscribe the 1964 Declaration of Helsinki by the World Medical Organization and its later amendments regarding human experimentation. All patients or their guardians consented to genetic diagnostic research and publication.

| Review of the literature
We searched the PubMed/Medline database for the terms ('G6PC3 deficiency' OR 'Dursun syndrome' OR 'Severe congenital neutropenia type 4'), and selected articles published in English from 2009 to 2020. We found 89 patients reported from at least 14 countries in 4 continents (See Tables 1-3. Not all reports included the country of origin). Table 1 describes their origins, demographic, clinical and laboratory features, treatment and outcome. Table 2 collects their genetic variants. In Table 3, we summarize the most common features and findings.
Ninety-four patients were included in this review (whenever the denominator is less than 94, that attribute was not available for some patients). The distribution of the disease was similar between males (52/92, 56%) and females. Most cases were from Middle East countries (47.8%). None of the patients had adverse reactions to live vaccines such as BCG. All patients presented at least 1 severe infection prior to 2 years of age; however, only 28.5% (26/91) were diagnosed before the age of 2.
Immunological analysis was not performed homogeneously in all patients; flow cytometry, immunoglobulins or lymphoproliferation was available only for 21/94. Although they all had severe neutropenia, some showed an intermittent increase in neutrophil counts. Intermittent thrombocytopenia was reported in 37/94 patients (39.3%), and 17 of 94 (18%) had lymphocyte counts below 1500/mcL. Nineteen patients had their serum immunoglobulin levels measured, of which 9 (47.3%) presented with hypergammaglobulinaemia and 2 hypogammaglobulinaemia (10.5%).
Treatment was described in 76 patients; 1 patient did not require any pharmacological intervention, while 63/76 (82.8%) were given G-CSF, 2.6% received pegfilgrastim and 3.9% received prophylactic co-trimoxazole as only treatment; IVIG was administered in 5/76 (6.5%) patients. Only 3 patients (4%) received haematopoietic stem cell transplant (HSCT). Patient 69 underwent HSCT due to severe IBD refractory to medical treatment and showed complete resolution of gastrointestinal symptoms; whereas patient 70 was transplanted because of the severe presentation of the disease and died from complications associated with the procedure. Long-term Among the 94 reported patients, homozygous missense was the most frequent variant type (Table 2). Homozygous frameshift insertions, deletions, splice site or nonsense was also reported. Although the transition c.130C>T was associated with non-syndromic neutropenia in Pakistani patients, a genotype-phenotype correlation has not been confirmed.

| DISCUSSION
Including our 5 patients, we describe a total of 94 patients with G6PC3. They frequently present with recurrent and severe bacterial infections during the first year of life, such as otitis media, skin abscesses, urinary tract infections, sino-pulmonary infections and sepsis. The diagnosis is suspected based on microbes (bacterial and fungal), infection sites (mouth, skin, bone, blood, lymph nodes, umbilical stump, respiratory and gastrointestinal tracts) and haematological findings: severe neutropenia was found in all reported patients; 37 patients had intermittent thrombocytopenia without clinical bleeding. Bone marrow aspirates of patients with G6PC3 deficiency showed a great diversity of findings, including: normocellular or hypercellular bone marrow, maturation arrest and even myelokathexis. 7 So far, there is no evidence that G6PC3 deficiency is a preleukaemic condition. In this review, we found only 1 case of leukaemia (P70) 8 ; no other reports of malignant transformation has been described in other SCN syndromes. 5,9 We have been able to include here most of the patients reported in the medical literature, expanding the phenotype and describing the typical haematological and nonhaematological features of G6PC3-deficient patients. The main limitations are the descriptive retrospective nature of the study, the fact that the information we rely upon is provided by different sources and authors, and lastly, the possibility of important data being lost throughout. Additionally, this is not a systematic review, and we are only including cases reported in English.
The activity of glucose-6-phosphatase is regulated by 3 genes: G6PC1, expressed in liver, small intestine and kidney, is related to the glycogenolytic and gluconeogenic pathways; G6PC2, expressed only in the pancreatic cells, is related to glucose level control; and G6PC3, ubiquitously expressed, hydrolyses glucose-6-phosphate to glucose in the final step of gluconeogenesis and glycogenolysis 10 in the endoplasmic reticulum. G6PC3 is essential to control neutrophil viability 3 ; its loss of function is associated with neutropenia due to an increase in endoplasmic reticulum stress and abnormal glucose homeostasis that leads to an increased susceptibility to apoptosis of neutrophils, skin fibroblasts and myeloid cells. 11,12 The above findings suggest that G6PC3 deficiency is a quantitative and qualitative neutrophil disease. 13 The immunological features are diverse and include T cell lymphopaenia, thymic hypoplasia and dysgammaglobulinaemia. Some articles suggest that the T cell lymphopaenia found in patients with G6PC3 deficiency may be associated with thymic hypoplasia. Nevertheless, the mechanisms leading to this thymic alteration are unclear. 14 Some G6PC3-deficient patients may have a more profound immunological defect, and might require a deeper approach, including T cell flow cytometry and lymphoproliferation assays. When abnormalities in the function of T and B cells are demonstrated, it is mandatory to consider immunoglobulin replacement. 9 Progressive lymphopaenia has also been reported; therefore, immunological long-term follow-up is required.
Non-haematological features are pivotal in differentiating G6PC3 deficiency from other causes of SCN: F I G U R E 2 Patient 2 also had prominent superficial veins in limbs, digital clubbing, brachymetatarsia of the third toe and bilateral hypoacusia structural heart defects, prominent superficial veins, urogenital malformations, growth retardation, pubertal and developmental delay. 15 The phenotypic spectrum of the disease is expanding, as it might be syndromic or non-syndromic, the latter group being harder to diagnose given the absence of non-haematological features.. 16 In this review, 23/94 (24.4%) of the patients did not have any syndromic association. Thus, G6PC3 deficiency should be considered in any SCN of unknown aetiology.
The most frequent non-haematological features are cardiovascular malformations, 17 atrial septal defect being the most common among those (55.3%). A wide range of cardiac abnormalities has been described in the literature, including heart valve abnormalities (mitral insufficiency, pulmonary valve stenosis, mitral and tricuspid insufficiency), followed by patent ductus arteriosus, coronary aneurysm, hypoplastic left ventricle and foramen ovale. The prominent superficial venous pattern was present in 58/94 (61.7%) of the patients reported, making it a frequent non-haematological feature. This alteration is less evident in childhood but becomes more prominent with age. These vascular changes have been attributed to increased cell apoptosis in myeloid cells, neutrophils and skin fibroblasts secondary to G6PC3 deficiency; as a consequence they can develop into varicose veins and ulcers during adulthood. 11 IBD has been reported in 10/94 reported patients with G6PC3 deficiency. Some authors suggest that autoinflammation through inflammasome activation may aggravate the IBD activity in G6PC3 deficiency. 12 IBD is a common finding in phagocyte defects, such as chronic granulomatous disease and leucocyte adhesion deficiency; these patients show dysregulated and poorly controlled inflammation perpetuated by a breakdown in the mucosal homeostasis and defective bacterial recognition and clearance. [18][19][20][21] Faecal calprotectin is not a good marker of inflammation in these patients since the neutropenia may give false-negative results; stool α1 antitrypsin may be a more reliable marker. 22 Haematopoietic stem cell transplant is a reasonable alternative for severe gastrointestinal manifestations resistant to conventional treatments. 17 The treatment most frequently used was filgrastim (G-CSF), leading to an increase in the number of neutrophils, together with an improvement in the patient's quality of life due to a decrease in the infection rate. In a murine G6PC3 −/− model, G-CSF delayed, but did not prevent, neutrophil apoptosis. In that study, a 5-day G-CSF treatment regime corrected neutropenia, stimulated glucose uptake and improved neutrophil function. 23 Depending on the severity of the defect, some patients received prophylactic antibiotics; mild phenotypes were treated with co-trimoxazole alone. On the other hand, more severe phenotypes may require G-CSF, antibiotics and gamma globulin replacement. In general, the reported survival is high, with a good quality of life as long as patients use filgrastim (G-CSF) and their malformations are surgically corrected. In recent years, therapeutic options other than G-CSF have emerged, such as SGLT2 inhibitor empagliflozin, a drug that decreases the concentration of 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a toxic metabolite that accumulates in the plasma of G6PC3 patients 24 ; empagliflozin reduces the concentration of toxic metabolites allowing a recovery in neutrophil function. 25 This novel treatment appears promising, although further studies are needed. So far, there is no recommendation to endorse or promote HSCT as a definitive treatment for the neutropenia. In this review, only 3 patients underwent HSCT, of which 1 died due to complications associated with the procedure.
We recommend the use of filgrastim (G-CSF) in patients with recurrent or severe infections, as it is considered safe and improves neutrophil counts, prevents the recurrence of infections and it subjectively improved the quality of life in all reported cases. The dose and frequency of administration vary with the needs of each patient. We also recommend the use of oral ambulatory prophylactic antibiotics; co-trimoxazole is a cheap and safe choice to diminish the rate and severity of infections, particularly in countries where the access to G-CSF is limited. A small group of patients has a higher immune compromise, so we suggest that all patients who either continue to suffer recurrent infections despite having normal levels of neutrophils or who have persistent lymphopaenia should be further evaluated with immunoglobulin levels testing, flow cytometry and lymphoproliferation assays. So far, HSCT is only suggested in patients with IBD refractory to conventional pharmacological treatment and in patients with G-CSF refractory disease (G-CSF dose >50 μg/kg/ day or myelodysplastic syndrome). HSCT is not routinely recommended, with most reports showing a good evolution with the exclusive use of filgrastim (G-CSF) and surgery to treat malformations.
Although G6PC3 deficiency is a rare disease, there is an increasing number of case reports around the world. This allows us to have a clearer idea of the clinical picture, the associated syndromic features and the best treatment for these patients. Given a clinical suspicion of G6PC3, it is appropriate to carry out a complete medical history that includes consanguinity, type and number of infections and microbiological isolates. An exhaustive physical examination is mandatory, searching for a triangular face, depressed nasal bridge, redundant neck skin, cutis laxa, prominent veins, cardiac murmur, overlapping toes and urogenital malformations, as they may be present in the syndromic phenotype.
All patients with suspected G6PC3 deficiency should have an echocardiogram and renal ultrasound. They must also have a close monitoring of weight, height and growth velocity, with any alteration prompting measurements of growth hormone levels and an evaluation by an endocrinologist. Although it is an infrequent manifestation, the presence of loose stools or bloody diarrhoea, tenesmus or abdominal pain suggests IBD. These patients should be evaluated with a complete blood count, erythrocyte sedimentation rate or C-reactive protein, albumin, faecal calprotectin and upper endoscopy/colonoscopy with biopsies.
The c.210delC variant was present in all five Mexican patients from this report and in most Hispanic patients reported to date. Banka and Newman 17 pointed out there might be some founder effects: Arg253His is frequent in the Middle East, Gly260Arg is more frequent in Caucasians from Europe and Phe71SerfsTer46 (c.210delC) is common among unrelated individuals of Hispanic descent. With a frequency of 0.00011 in gnomAD (gnomad.broad insti tute. org/varia nt/17-42148 542-TC-T?datas et=gnomad_r2_1), all 28 existing alleles (16 women and 12 men, all heterozygous) are from Latino/Admixed American individuals. Our 5 patients came from different regions of the country, mostly from Central and North Mexico. Although there was no known history of consanguinity, endogamy at small, geographically isolated communities might explain the homozygosity in three of the families.
There are multiple unanswered questions about G6PC3-deficient patients. The cause for the heterogeneity of the bone marrow findings is currently unknown, as is the reason why some patients present with thymic hypoplasia, lymphopaenia and hypogammaglobulinaemia. Whether the neurodevelopmental delay and hearing loss described in some patients are caused by prolonged hospitalizations and recurrent infections, or they are part of the disease phenotype, is also unclear. Other exceptional clinical features, such as oculocutaneous albinism, might result from different autosomal recessive gene defects in highly consanguineous populations.
In conclusion, we described five and reviewed 89 more cases of G6PC3 deficiency reported in the literature during the last 10 years, including clinical features, treatment, prognosis and mutational analysis. It is a disease with a high heterogeneity, with syndromic and non-syndromic phenotypes. A possible G6PC3 deficiency should be considered in every patient with severe congenital neutropenia. The follow-up should include growth and development evaluation, as well as an assessment of complete blood count, echocardiogram and renal ultrasound, while individualizing the needs of each patient, as clinical penetrance is variable, and no genotype-phenotype has been described. The ongoing management of the disease should be conducted within a multidisciplinary team. We recommend treating with G-CSF and antibiotic prophylaxis, as they seem to improve the frequency of infections and quality of life. An increasing number of cases of G6PC3 deficiency in the world is likely to continue to be identified due to easier access to diagnostic methods, expanding our understanding of this disease.