lNTRODUCTlON

Paroxysmal nocturnal hemoglobinuria(PNH)is an acquired hematopoietic stem cell(HSC)disorder.Deficient HSCs give rise to a clonal population of blood cells deficient in proteins anchored with glycosylphosphatidylinositol(GPI-anchored),a glycolipid moiety that secures 100 different proteins to the cell surface[1].In 2019,4 patients with typical PNH and autoinflammatory symptoms,including recurrent aseptic meningitis,were found to have a germline point mutation in one

allele,with the other

allele being removed by somatic deletion of a 20q region implicated in hematological malignancies.Analyses of patient leukocytes revealed free GPI expressed on the cell surface,triggering autoinflammation through increased IL-1β secretion,activation of the lectin pathway of complement and production of C5b-9 complexes[2].Therefore,eculizumab treatment abrogates not only intravascular hemolysis but also autoinflammation.We report the fifth case of PIGT-PNH and the first time that allogeneic hematopoietic stem cell transplantation has been applied as treatment.This procedure was readily feasible with no excess alloreactivity or toxicity.

CASE PRESENTATlON

Chief complaints

A 47-year-old man was referred to our hospital for pancytopenia with PNH cloning and meningitidis and urticaria.

And in it was spread an enormous repast, while the table so glittered with golden cups and plates, flagons and dishes, candlesticks and a hundred other things beside, that it was hardly possible to look steadily46 at it

The king watched her till she was quite out of sight, so as to make sure that she was not spying upon him; then he took the golden lion into his room and lifted some loose boards from the floor

History of present illness

Upon admission to our hospital,he presented with fever,sudden brownish urine and altered consciousness with mild pancytopenia(hemoglobin 93 g/L,platelets 137.109/L and leukocytes 2.109/L).

History of past illness

C reactive protein levels were mildly elevated at 20 mg/L.Examination of lumbar cerebrospinal fluid showed 307 polymorphonuclear leukocytes/mm

.No bacteria,fungi,viruses or mycobacteria were identified,nor were autoantibodies.A biopsy from one urticarial lesion revealed mixed inflammatory(neutrophils and monocytes)infiltrate.Flow cytometric analysis of both erythrocytes and granulocytes indicated deficiency of GPI-anchored proteins(Figure 1);complement system dosing showed a normal CH50 Level.Factor H and Factor I plasma concentrations and anti-Factor H antibodies were also normal.Examination of cellular morphology based on bone marrow aspiration revealed multilineage dysplasia with no excess blasts(＜ 2%).Medullar cytogenetic analysis detected a 20q deletion in the karyotype,and Sanger sequencing highlighted a deletion of 4 nucleotides(NM_015937.6:c.766_769del)in exon 6(p.Lys256ThrfsTer38)leading to a frameshift and a premature stop codon.This mutation was found in the heterozygous state in both T lymphocytes and in the negative cellular fraction,suggesting a constitutional anomaly.These results were confirmed using another sample consisting of DNA extracted from fibroblast culture cells collected after skin biopsy.This finding is reported only once in the ClinVar database(RCV000735856.1).According to the CGH array,we detected a large somatic deletion of 18 Mb from 20q11.21 to 20q13.13,an area including the entire PIGT gene.This 20q deletion associated with heterozygous constitutional mutation of PIGT leads to biallelic inactivation of the gene(Figure 2).

I wasn t prepared for a reply, but with resignation, he said, I really am homeless and I really am hungry! You can come with me and watch me eat! But I kept on walking.

Personal and family history

Allogeneic stem cell transplantation has not been reported for treating PIGT-PNH,yet this therapy addresses the concern regarding a high risk of alloreactivity and toxicity in patients with activated NLRP3 inflammasomes in mononuclear cells.Our case is the first to be successfully treated with allo-SCT,and no toxicity(especially GvHD)was observed.

Physical examination

Examination revealed urticaria and symptoms of meningitis including headache and stiff neck.His meningitis symptoms were resolved at 3 d after initiation of meropenem.During hospitalization,he experienced 4 episodes of aseptic meningitis and general fatigue,arthralgia and urticaria preceded each episode.He recovered quickly within 3 d from the last episode of meningitis with corticosteroids and without antibiotics.The patient developed severe chronic hemolysis after the first meningitidis episode.

PNH is a clonal disorder involving blood cells deficient in glycosylphosphatidylinositol-anchored(GPIanchored)proteins[1,3],which is often caused by a deficient initial step in GPI anchor synthesis,as catalyzed by the GPI-GlcNAc transferase encoded by the X-chromosomal gene

[4-6].However,22

genes participate in the biosynthesis and protein attachment of GPI[7,8].The

gene on chromosome 20,at position 20q13.12 with 12 exons,encodes phosphatidylinositol-glycan biosynthesis class T protein(PIG-T),a subunit of the heteropentameric GPI transamidase complex that facilitates attachment of GPI anchors to proteins[9].Four cases of PHN with recurrent inflammatory symptoms have been reported[2]with PIGT defects and successfully treated with eculizumab.In 2013,PNH due to 2 mutation events was reported: a germline splice site mutation and a somatic deletion in

(c.1401-2A＞G),as identified by next-generation sequencing[10].In 2018,a second patient with long-term severe urticaria and joint pain before developing PNH harbored similar mutations in

(c.250G＞T)and exhibited recurrent aseptic meningitis in addition to inflammatory symptoms[11].Both cases clearly improved with eculizumab treatment.In 2019,2 additional patients with PHN and inflammatory symptoms were reported: one had chronic lymphocytic leukemia,and the other carried the

-V617F mutation.Both patients harbored germline mutations in one

allele(one patient with c.761_764delGAAA and the other with c.197delA)associated with somatic deletions,including the entire

gene in the second allele without

gene abnormalities[2].In known cases of PHN with

disruption,one of the

alleles is removed due to a somatic deletion of varying size,including the common deleted region(CDR)of 1.9 Mb,which is close to the centromeric region,often described in myeloid malignancies with 20q deletion.Based on a family segregation study,

haploinsufficiency is not sufficient for the development of autoinflammatory symptoms.In our case,the development of MDS with 20q deletion was an indispensable additional abnormality resulting in biallelic inactivation of

explaining the PNH.If mutations in both

and

can induce PNH,recurrent inflammatory symptoms,including meningitis,are in particular found with

mutations.Therefore,some authors have proposed creating a new entity named PIGT-PHN[2].In PNH-PIGT syndrome,cytokine dosing suggests that increased free GPI might over activate NLRP3 inflammasomes in mononuclear cells with strong IL-1β and IL-18 responses.IL-18 is produced by activated inflammasomes[12,13]and is also produced during clinical GvHD.NLRP3 is known to play a role in enhancing GvHD[14].Our case report is the fifth published case of PIGT-PNH.Among 4 patients previously described,3 patients were partially controlled with corticosteroids,colchicine,diphenhydramine,cromoglycin,azathioprine,mycophenolate mofetil,dapsone,anakinra and canakinumab.Only eculizumab treatment abrogates autoinflammation for one patient.We confirm that eculizumab is the best treatment to abrogate intravascular hemolysis and autoinflammation.Because we know that complement activation and inflammatory dysregulation before allo-SCT might be associated to a higher incidence of severe acute GvHD in patients,our main concern was about the toxicity of this procedure.We report fort the first time that allogeneic hematopoietic stem cell transplantation is a readily feasible procedure with no excess alloreactivity or toxicity.

Laboratory examinations

The patient reported a history of recurrent urticaria and arthralgia since he was 30-years-old.Three months and one month prior,he was hospitalized for acute dermohypodermitis with pancytopenia and no documented microbiologic agent.He was successfully treated with piperacillin and tazobactam for 14 d.

We observed rapid myeloid engraftment,with a time for neutrophils ＞ 0.5 × 10

/L and platelet recovery(＞ 20.10

/L)of 15 d and 16 d,respectively.Chimerism was complete donor at 1,3,12 and 18 mo posttransplant.No acute GvHD was observed.Six months after transplantation,he developed moderate chronic hepatic and skin GvHD that improved by enhancing the calcineurin inhibitor and starting 1 mg/kg/d corticosteroid therapy.At the time of writing,at 24 mo after transplantation,chronic GvHD is in complete remission with no immunosuppressant.

Imaging examinations

Cerebral magnetic resonance imaging results were normal.

FlNAL DlAGNOSlS

PIGT-PNH.

TREATMENT

All the patient's symptoms,including urticaria,arthralgia,headache/meningitidis and hemolysis,completely disappeared after eculizumab was administered regularly.Finally,after 8 mo on eculizumab treatment,the pancytopenia worsened(hemoglobin 90 g/L,platelets 67.10

/L and leukocytes 1.10

/L),and the patient presented a sepsis secondary to a catheter-related bacteriemia of staphylococcus epidermidis resistant to methicillin.Bone marrow tests revealed 8% blast.We decided to transplant the patient because of the episode of severe infection and bone marrow smear results.The decision of transplantation was difficult,because in common PNH caused by mutation of PIGA,there is a high risk of developing GVHD,especially in patients older than 40-years-old with no sibling donors.No data were available about transplantation in PNH caused by mutation of PIGT,and our patient had no sibling or matched unrelated donors.However,recent retrospective studies demonstrated promising results with HLA-mismatched/haploidentical hematopoietic stem cell transplantation after reduced intensity conditioning and GVHD prophylaxis with post-Transplant cyclophosphamide in refractory severe aplastic anemia patients.Moreover,inflammatory symptoms in our patient were totally controlled by eculizumab.We hypothesized that it could be a good time for transplantation.Therefore,allogeneic hematopoietic stem cell transplantation with peripheral blood stem cells from an HLAmismatched unrelated donor was carried out after a reduced-intensity conditioning regimen consisting of thiotepa(5 mg/m

at day -7),a total fludarabine dose of 150 mg/m

(30 mg/m

from day -5 to day -1),and total intravenous(i.v.)busulfan 6.4 mg/kg(3.2 mg/kg/d on days -4 and -3).Graft

host disease(GvHD)prophylaxis consisted of posttransplant cyclophosphamide(50 mg/kg/j on days+3 and+4),cyclosporine A(starting on day+5 at 3 mg/kg/day)as a continuous i.v.infusion,and i.v.MMF(starting on day+5 at 15 mg/kg every 12 h).A dose of 6×10

CD34

/kg body weight was infused.

OUTCOME AND FOLLOW-UP

The King s son declared that he would fulfil his promise, and when his parents mildly remarked that the girl was only a keeper of sheep, and a very ugly one too, the maiden boldly said that she was born a princess, and that, if they would only give her some water and leave her alone in a room for a few minutes, she would show that she could look as well as anyone in fine clothes

DlSCUSSlON

In a moment the Princess Hyacinthia had changed herself, the Prince, and his charger into a thick wood where a thousand paths and roads crossed each other

CONCLUSlON

No special family history was reported.

ACKNOWLEDGEMENTS

We thank the patient for his full consent.

FOOTNOTES

D'Aveni M provided the concept and design and reviewed and revised the manuscript;Schenone L wrote the manuscript;Schenone L,Notarantonio AB,Latger-Cannard V,Fremeaux-Bacchi V,De Carvalho-Bittencourt M and Muller M performed the analysis;Detrait M,Rubio MT,D’Aveni M took care of the patient;Rubio MT and Muller M revised the manuscript.

Informed written consent was obtained from the patient.

4.Meanest work of the house:The meanest work would be the filthiest51, harshest and most demeaning work in the household, such as scrubbing floors and emptying chamber pots.Return to place in story.

The authors declare no competing financial interests.

The authors have read the CARE Checklist(2016),and the manuscript was prepared and revised according to the CARE Checklist(2016).

Thereupon she made the Princess sit down beside her, and began fingering her silken robe, while she muttered Lace on top, lace underneath49! This must have cost you a pretty penny! It would have been better to save enough to feed yourself, and not come begging to those who want all they have for themselves

This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial(CC BYNC 4.0)license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is noncommercial.See: https://creativecommons.org/Licenses/by-nc/4.0/

France

Laurence Schenone 0000-0002-6006-4917;Anne-Béatrice Notarantonio 0000-0002-7939-9477;Véronique Latger-Cannard 0000-0002-2831-1850;Veronique Fremeaux-Bacchi 0000-0002-4865-8528;Marcelo De Carvalho-Bittencourt 0000-0002-2698-2458;Marie-Thérèse Rubio 0000-0003-3732-5442;Marc Muller 0000-0002-7190-7684;Maud D'Aveni 0000-0002-5909-2742.

Wu YXJ

Filipodia

Wu YXJ

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