Treatment with plasma exchange of a pregnant woman with anti-PP1Pk alloimmunization: A case report.

The histo-blood group antigens P, P1 and Pk are a closely related set of glycosphingolipid structures expressed by red blood cells and other tissues. None of these three characters is expressed on p cells, a null phenotype that arises in the context of homozygous mutation of the A4GALT gene. Subjects with p phenotype spontaneously develop a natural alloantibody named anti-PP1Pk, which is a mixture of IgG and IgM against P1, P and Pk. While anti-P1 is a weak cold antibody with poor clinical significance, anti-P and anti-Pk antibodies are potent haemolysins responsible for severe hemolytic transfusion reactions. The rare anti-PP1Pk alloantibodies are associated with recurrent spontaneous abortion in the first trimester of gestation. P and Pk antigens are expressed at high levels on the placenta and antibodies directed against both these structures are deleterious to placental trophoblasts. Here we describe the use of plasma exchange (PEX) in a nulliparous 39-year-old woman with anti-PP1Pk antibodies and a history of repeated spontaneous early abortions and hypofertility. The patient underwent apheresis starting from the third week throughout the pregnancy and a healthy child was delivered by cesarean section at 35 WG. The newborn required only phototherapy within a few days of life. We can state that an early treatment with the only PEX has proven to be effective and safe in the management of a fetomaternal P-incompatibility caused by a high anti-PP1Pk titer (256).

[Application of hyaluronic acid microneedles in insulin intelligent delivery system for the treatment of diabetes].

In this study, insulin (insulin, INS)/Ca3PO4 complex and glucose oxidase (glucose oxidase, GOx)/Cu3(PO4)2 complex were prepared by coprecipitation method. The mineralized insulin (mineralized insulin, m-INS) showed irregular crystalline clusters, and the mineralized glucose oxidase (m-GOx) showed flower spherical morphology, with a diameter of about 1-2 µm. In vitro simulated release experiment showed that m-INS released INS as the pH value of the medium decreased. When the pH value was 4.5, the release amount reached 96.68%. The enzyme activity detection experiment showed that the enzyme activity stability of m-GOx was higher than that of free GOx. It still maintained high activity after 10 days at room temperature, while the activity of GOx was less than 60%. The glucose solution was prepared to simulate the state of normal blood glucose (5.6 mmol/L) and hyperglycemia (22.2 mmol/L). When m-INS and m-GOx were added to the glucose solution, the release amount of INS showed a significant glucose concentration dependence. The higher the glucose concentration, the greater the release amount and release rate of INS. Finally, m-INS, m-GOx and hyaluronic acid (HA) solution were mixed to prepare HA microneedle arrays loaded with m-INS and m-GOx. Type 1 diabetes mice were constructed to evaluate the effect of drug-loaded HA microarray on blood glucose control in diabetic rats. The results show that the HA microneedles loaded with m-INS/m-GOx could deliver drugs effectively. The average blood glucose concentration in diabetic rats dropped to about 7 mmol/L within 1 h, normal blood glucose concentration could be maintained for 10 h, and the overall blood glucose concentration was lower than the level before administration for 36 hours. Compared with HA microneedles loaded with INS only, m-ins microneedles showed better glucose tolerance, longer-lasting glucose control effect and less risk of hypoglycemia. Compared with other sustained-release systems, the preparation process of the core components in this study is simple, efficient, safe and effective, and has great commercial potential.

Multidisciplinary management of anti-PP1Pk or anti-P alloimmunization during pregnancy: A new case with anti-P and a literature review.

BACKGROUND: Red blood cell alloimmunization is the first cause of fetal and neonatal anemia. Alloimmunizations with anti-PP1Pk or anti-P can cause recurrent miscarriages and hemolytic disease of the fetus and newborn in the 2nd and 3rd trimesters of pregnancy. We report on a pregnant patient immunized with anti-P and a history of recurrent miscarriages. CASE REPORT: This P2k (GLOB:-1; P1PK:-1,3) patient had a first pregnancy marked by a caesarean at 38 weeks of gestation (WG) for non-reassuring fetal heart rate. Then, she had three early spontaneous miscarriages. The fifth pregnancy began with a high titer of anti-P at 128. Early initiation of treatment with Intravenous Immunoglobulins (IVIg) and plasma exchanges (PE) starting at 5 WG permitted us to reduce the titer of anti-P below 32. A healthy infant was delivered by caesarean at 38 WG without anemia at birth and no exchange transfusion was required. DISCUSSION AND REVIEW OF THE LITERATURE: The P and Pk antigens are expressed on placental, trophoblastic, and embryonic cells. This explains why P1k (GLOB:-1; P1PK:1,3), P2k (GLOB:-1; P1PK:-1,3), or Tj(a-)/p (GLOB:-1; P1PK:-1,-3) patients are prone to recurrent abortions in the first trimester of pregnancy. A literature review demonstrated 87% (68/78) of miscarriages in p patients. However, publication biases are possible with the most severe cases being reported. CONCLUSION: Immunizations to P and PP1Pk antigens differ from others in their physiopathology and precocity. The association of PE and IVIg seems to be an effective treatment in the management of anti-PP1Pk or anti-P fetomaternal incompatibilities.

Anti-PP₁P(k) (Tj(a)) Antibody in a Korean Female Patient with p Phenotype Confirmed by Genotyping

Second Tunisian case of Anti -Tja mediated recurrent abortions.

The diagnosis and the treatment of rare phenotypes remain a problematic situation in many countries especially in Tunisia. Individuals with rare phenotype may develop clinically significant red cell antibodies directed against the high incidence Antigens they lack. A 35 years old patient was referred to our laboratory to explain a high incidence (twelve) of recurrent miscarriage during the first and second terms of pregnancy. This patient was grouped as O Rhesus: 1, -2, -3, 4, 5 K:-1. In her plasma we identified a pan-reactive anti-PP1PK antibody (anti-Tja) recognized to be responsible of spontaneous recurrent abortions. The red cell phenotype was P1 and Tja negative. More investigations concluded to the absence of auto and other allo-antibodies association. Therapeutic plasmapheresis from early stages was suggested for the future pregnancy to remove anti-public antibodies in order to maintain normal placenta functions. The Anti-Tja antibody, naturally occurring in patients with rare p phenotype, has the ability to induce recurrent spontaneous miscarriages and to cause immediate hemolytic transfusion reactions. Despite the absence of compatible donors in her family, this patient is not in an impasse situation because two donors with the same phenotype were identified when investigating a first case in 2013.

The P1 histo-blood group antigen is present on human red blood cell glycoproteins.

BACKGROUND: The P1 antigen was first described in 1927 and belongs to the P1PK histo-blood group system, together with Pk and NOR. The A4GALT-encoded 4-α-galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS: RBCs of known P1 /P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α-galactosidases, papain, and/or peptide-N-glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblot. A new P 1 /P 2 -allelic discrimination assay based on rs5751348 was validated. RESULTS: P1 occurs on various glycoproteins, seen as smearlike patterns in anti-P1-stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P 1 -homozygous and P 1 -heterozygous RBCs (P 1 P 1 > P 1 P 2 ), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α-galactosidase of broad Galα-1,3/4/6-specificity. Peptide-N-glycosidase F treatment reduced the P1 signal, while papain or α-1,3-specific galactosidase did not. P 1 /P 2 status was confirmed by a new rs5751348 assay. CONCLUSION: Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N-glycans.

A hemolytic anti-LKE associated with a rare LKE-negative, "weak P" red blood cell phenotype: alloanti-LKE and alloanti-P recognize galactosylgloboside and monosialogalactosylgloboside (LKE) antigens.

BACKGROUND: "Weak P" is a rare red blood cell (RBC) phenotype, characterized by a global decrease in P(k) and P antigens. We now describe a second weak P individual who also typed LKE-negative (LKE-N) and possessed a clinically significant anti-LKE. STUDY DESIGN AND METHODS: Patient RBCs and plasma were examined by standard serology and flow cytometry. Glycosphingolipids (GSLs) from patient, P(k) , and LKE-strong (LKE-S) RBCs were isolated and analyzed by high-performance thin-layer chromatography (HPTLC). To confirm antibody specificity, patient serum and 30 human polyclonal controls, including alloanti-P and anti-PP1 P(k) , were tested against a panel of GSLs by HPTLC immunostaining. RESULTS: The patient typed P1 +, P+, and LKE-N and possessed a "P-like" panagglutinin. In a two-stage indirect antiglobulin test, the patient's plasma caused hemolysis of LKE-S cells but not p, P(k) , or LKE-N cells. Clinically, transfusion of P+ RBCs compatible by a prewarmed technique had shortened RBC survival with laboratory evidence of hemolysis. Analysis of the patient's isolated RBC GSLs showed a 30% relative decrease in Gb3 (P(k) ) and Gb4 (P) and a 90% decrease in monosialogalactosylgloboside (MSGG, LKE), accompanied by increased lactosylceramide (CDH), paragloboside, and GM3. On HPTLC immunostaining, the patient's plasma strongly bound MSSG with weak binding to galactosylgloboside (Gb5). Binding to MSGG, Gb5, and Gb4 was also observed with some examples of alloanti-P from P(k) individuals, but not anti-PP1 P(k) , autoanti-P, or normal controls. CONCLUSIONS: We describe the first example of a clinically significant anti-LKE in the setting of a rare weak P background. Human alloanti-LKE and some alloanti-P recognized Gb5 and MSGG.

A systematic study of single-nucleotide polymorphisms in the A4GALT gene suggests a molecular genetic basis for the P1/P2 blood groups.

BACKGROUND: The molecular mechanism for the formation of the P1/P2 blood groups remains unsolved. It has been shown that the P1/P2 polymorphism is connected to the different A4GALT gene expression levels in P1 and P2 red blood cells. STUDY DESIGN AND METHODS: The present investigation conducted a pilot investigation that involved the detailed and stepwise screening of single-nucleotide polymorphisms (SNPs) in the A4GALT gene, followed by a larger-scale association study. The transcription-inducing activity by the different genotypes of SNPs was analyzed using reporter assays. RESULTS: A total of 416 different SNP sites in the A4GALT genes from four P1 and four P2 individuals were analyzed in the pilot investigation, and 11 SNP sites, distributed in the A4GALT Intron 1 region, exhibited an association with the P1/P2 phenotypes. In the follow-up association study, the genotypes at the 11 SNPs of a total of 338 individuals across four different ethnic populations were determined, and the results show that two SNPs, rs2143918 and rs5751348, are consistently associated with the P1/P2 phenotypes. Reporter assays demonstrated significantly higher transcription-inducing activity by the SNPs bearing the P(1)-allele genotype than by the SNPs bearing the P(2)-allele genotype and that the difference in transcriptional activity was determined by the different genotypes at SNP rs5751348. CONCLUSION: The results of this investigation demonstrate a consistent association of A4GALT SNPs rs2143918 and rs5751348 with the P1/P2 phenotypes and suggest that SNP rs5751348 may lead to allelic variations in A4GALT gene expression and consequently leads to the formation of the P1/P2 phenotypes.

P1PK, GLOB, and FORS blood group systems and GLOB collection: biochemical and clinical aspects. Do we understand it all yet?

Antigens belonging to the P1PK, GLOB, and FORS blood group systems and the GLOB blood group collection represent a closely related set of 13 glycosphingolipids (GSLs). They are synthesized by the coordinated action of glycosyltransferases, encoded by at least 7 different loci. Three of these enzymes show either different activity or a different mRNA expression profile due to genetic polymorphisms, resulting in blood group diversity. In recent years, significant progress has been made in understanding the molecular background and biological functions of these GSLs. Their medical significance is often related to the existence of natural antibodies, as they may cause complications after transfusions and during pregnancies. In addition, GSLs belonging to these blood group systems are receptors for several pathogens. This review summarizes the present knowledge about the complicated network of enzymatic interactions leading to synthesis of these GSLs, as well as their clinical implications.

An update on the GLOB blood group system and collection.

The P blood group antigen of the GLOB system is a glycolipid structure, also known as globoside, on the red blood cells (RBCs) of almost all individuals worldwide. The P antigen is intimately related to the Pk and NOR antigens discussed in the review about the P1PK blood group system. Naturally occurring anti-P is present in the serum of individuals with the rare globoside-deficient phenotypes p, P1k, and P2k and has been implicated in hemolytic transfusion reactions as well as unfavorable outcomes of pregnancy. The molecular genetic basis of globoside deficiency is absence of functional P synthase as a result of mutations at the B3GALNT1 locus. Other related glycolipid structures, the LKE and PX2 antigens, remain in the GLOB blood group collection pending further evidence about the genes and gene products responsible for their synthesis.

P1PK: the blood group system that changed its name and expanded.

The antigens in the P1PK blood group system are carried on glycosphingolipids. The system currently includes three different antigens, P1, Pk, and NOR. The P1 antigen was disovered in 1927 by Landsteiner and Levine, and Pk and NOR were described in 1951 and 1982, respectively. As in the ABO system, naturally occurring antibodies of the immunoglobulin (Ig) M or IgG class, against the missing carbohydrate structures, can be present in the sera of people lacking the corresponding antigen. Anti-P1 is generally a weak and cold-reactive antibody not implicated in hemolytic transfusion reaction (HTR) or hemolytic disease of the fetus and newborn while Pk antibodies can cause HTR, and anti-NOR is regarded as a polyagglutinin. A higher frequency of miscarriage is seen in women with the rare phenotypes p, P1k, and P2k. Furthermore, the Pk and P1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. Why p individuals lack not only Pk and P expression but also P1 has been a longstanding enigma. Recently, it was shown that the same A4GALT-encoded galactosyltransferase synthesizes both the P1 and Pk antigens and that a polymorphism in a new exon in this gene predicts the P1 and P2 phenotypes.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo-blood group systems.

BACKGROUND: The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. STUDY DESIGN AND METHODS: The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. RESULTS: The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. CONCLUSION: For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

[Serological and genetic study of a pedigree featuring a rare p phenotype].

OBJECTIVE: To explore genetic background of a pedigree with a rare p phenotype from Guangdong province. METHODS: The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing. RESULTS: The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34. CONCLUSION: The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.

Serological and genetic study of a pedigree featuring a rare p phenotype / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore genetic background of a pedigree with a rare p phenotype from Guangdong province.</p><p><b>METHODS</b>The rare p phenotype was identified by a conventional serologic method. With genomic DNA of proband and family members extracted, exon 3 of alpha-(1,4)galactosyltransferase (A4GALT) gene was amplified with PCR and analyzed by direct sequencing. The mutation found in the pedigree was screened in a normal population using direct sequencing.</p><p><b>RESULTS</b>The proband and 4 family members with the rare p phenotype have all carried a point mutation c.100G>A (p.Val34Ile) in combination with a deletion-insertional mutation c.418_428del11ins34(p.Gln139Trpfs*72), which renders a compound mutation of A4GALT gene. One family member with P2 phenotype has carried a same heterozygous mutation. Of the 100 healthy donors, 5 have carried a heterozygous point mutation c.100G>A, and none carried the deletion-insertional mutation c.418_428del11ins34.</p><p><b>CONCLUSION</b>The rare p phenotype of the pedigree has resulted from a compound mutation of the A4GALT gene, which is in keeping with a recessive inheritance pattern of the p phenotype.</p>

Functional characterisation of a complex mutation in the α(1,4)galactosyltransferase gene in Taiwanese individuals with p phenotype.

BACKGROUND AND OBJECTIVE: Individuals with p phenotype lack P1, P(k) and P antigens on red blood cells, presumably as a result of deficiency in the enzyme α(1,4)galactosyltransferase (A4GALT). The aim of this study was to explore the molecular background of a Taiwanese family with p phenotype. MATERIALS AND METHODS: Blood samples from two p siblings and seven family members were investigated. The coding region of the A4GALT gene was analysed by polymerase chain reaction and direct sequencing. The wild- and mutant-complementary DNAs (cDNAs) of A4GALT were cloned into an expression vector and transfected to Chinese hamster ovary (CHO) cells. P(k) expression on the transfected cells was analysed by flow cytometry and the activities of A4GALT were measured by high-performance liquid chromatography. RESULTS: The two individuals with p phenotype were homozygous for the complex mutation, which was caused by a combined deletion and insertion between nt 418 and 428. No expression of P(k) and no enzyme activity were observed in cells transfected with the mutant construct. CONCLUSION: The first case of p phenotype in Taiwan was caused by a non-functional allele resulting from a homozygous complex mutation of A4GALT gene.

Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups.

The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Galα1-4Gal of the P(k) (Gb3/CD77) glycosphingolipid, important in transfusion medicine, obstetrics, and pathogen susceptibility. Critical nucleotide changes in A4GALT not only abolish P(k) formation but also another Galα1-4Gal-defined antigen, P1, which belongs to the only blood group system for which the responsible locus remains undefined. Since known A4GALT polymorphisms do not explain the P1-P(k)+ phenotype, P(2), we set out to elucidate the genetic basis of P(1)/P(2). Despite marked differences (P(1) > P(2)) in A4GALT transcript levels in blood, luciferase experiments showed no difference between P(1)/P(2)-related promoter sequences. Investigation of A4GALT mRNA in cultured human bone marrow cells revealed novel transcripts containing only the noncoding exon 1 and a sequence (here termed exon 2a) from intron 1. These 5'-capped transcripts include poly-A tails and 3 polymorphic sites, one of which was P(1)/P(2)-specific among > 200 donors and opens a short reading frame in P(2) alleles. We exploited these data to devise the first genotyping assays to predict P1 status. P(1)/P(2) genotypes correlated with both transcript levels and P1/P(k) expression on red cells. Thus, P(1) zygosity partially explains the well-known interindividual variation in P1 strength. Future investigations need to focus on regulatory mechanisms underlying P1 synthesis.