Concurrent IVF and spontaneous conception resulting in a quadruplet pregnancy.

Blastocyst transfer of just one or two embryos has been used to help limit the number of high-order gestations. In this case report we describe the occurrence of a quadruplet pregnancy after the transfer of only two blastocysts during IVF. Sonographic examination showed four fetuses and what appeared to be quadriamniotic/quadrichorionic sacs, suggesting that a concomitant spontaneous conception had occurred. Definite confirmation of zygosity was obtained by genetic testing using DNA microsatellite polymorphism determinations after the birth of one boy and three girls at 32 weeks gestation. Although this event has not been reported previously, the possibility of its occurrence should be kept in mind. IVF patients with patent Fallopian tubes should be cautioned against intercourse late in their controlled ovarian stimulation, especially if they would decline multifetal reduction.

CD31 mismatching affects marrow transplantation outcome.

Graft-versus-host disease (GVHD) complicating allogeneic bone marrow transplantation (BMT) is often attributed to mismatched minor histocompatibility antigens (mHags), which are poorly defined in humans. CD31 is a candidate human mHag relevant to acute GVHD, but reports disagree about its level of significance, the role of HLA restriction, and the relative importance of different polymorphic codons within the molecule. We therefore examined in greater detail the impact of CD31-matching on BMT outcome in a prospective study from a single institution. Samples of recipient and donor DNA were collected pretransplantation for all patients receiving unmanipulated bone marrow from an HLA-identical sibling over a 45-month period at our institution. CD31 DNA typing of alleles at the 3 polymorphic codons 125 (L or V), 563 (N or S), and 670 (R or G) was performed for 118 patient-donor pairs plus 2 additional pairs who had codon 125 typing only. Donor-recipient CD31 nonidentity was tested for correlation with BMT clinical outcome measures of severe acute GVHD, chronic GVHD, relapse, and survival. Gene frequencies of approximately 0.5 for each allele at all 3 codons were comparable to previous reports. Because complete association was seen for 563N with 670G and for 563S with 670R, nonidentity for those codons was analyzed as a single genetic marker designated codon 563/670. Donor-recipient CD31 nonidentity was a significant risk factor for overall survival, both at codon 563/670 (hazard ratio [hr] = 2.58, P = .005) and at codon 125 (hr = 1.07, P = .036). Similar results held for disease-free survival. Nonidentity at codon 563/670 was also a significant risk factor (odds ratio [OR] = 11.15, P = .011) for severe (grades III, IV) versus no (grade 0) acute GVHD. Nonidentity at codon 125 posed less but still significant risk (OR = 9.30, P = .030). When the comparison group without severe acute GVHD was expanded to include grade I as well as grade 0 patients, the risk from CD31 nonidentity increased for both codon 563/670 (OR = 12.31, P = .010) and codon 125 (OR = 11.24, P = .011). CD31 nonidentity remained a significant independent risk factor for survival and for severe acute GVHD when tested in multivariate analysis with the covariates of adulthood, recipient-donor sex difference, ethnic group, disease, pretransplantation risk category, HLA-A2 type, B44-like types, and GVHD prophylactic regimen. CD31 nonidentity showed a trend but failed to achieve statistical significance as a risk factor for relapse and for chronic GVHD. In conclusion, donor-recipient CD31 nonidentity is a significant risk factor for survival and for severe acute GVHD in HLA-identical sibling BMT. The stronger associations with codon 563/670 suggest that polymorphism may be more important than the linked polymorphism at codon 125.

Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects.

Toxicities have limited the use of allogeneic hematopoietic cell transplantation (HCT) to younger, medically fit patients. In a canine HCT model, a combination of postgrafting mycophenolate mofetil (MMF) and cyclosporine (CSP) allowed stable allogeneic engraftment after minimally toxic conditioning with low-dose (200 cGy) total-body irradiation (TBI). These findings, together with the known antitumor effects of donor leukocyte infusions (DLIs), led to the design of this trial. Forty-five patients (median age 56 years) with hematologic malignancies, HLA-identical sibling donors, and relative contraindications to conventional HCT were treated. Immunosuppression involved TBI of 200 cGy before and CSP/MMF after HCT. DLIs were given after HCT for persistent malignancy, mixed chimerism, or both. Regimen toxicities and myelosuppression were mild, allowing 53% of eligible patients to have entirely outpatient transplantations. Nonfatal graft rejection occurred in 20% of patients. Grades II to III acute graft-versus-host disease (GVHD) occurred in 47% of patients with sustained engraftment. With median follow-up of 417 days, survival was 66.7%, nonrelapse mortality 6.7%, and relapse mortality 26.7%. Fifty-three percent of patients with sustained engraftment were in complete remission, including 8 with molecular remissions. This novel allografting approach, based on the use of postgrafting immunosuppression to control graft rejection and GVHD, has dramatically reduced the acute toxicities of allografting. HCT with the induction of potent graft-versus-tumor effects can be performed in previously ineligible patients, largely in an outpatient setting. Future protocol modifications should reduce rejection and GVHD, thereby facilitating studies of allogeneic immunotherapy for a variety of malignancies. (Blood. 2001;97:3390-3400)

Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.

BACKGROUND: Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans. METHODS: A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens. RESULTS: The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies. CONCLUSIONS: Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

HLA-class II genes modify outcome of Toxoplasma gondii infection.

Associations between Human Leukocyte Antigen (HLA) (i.e. human major histocompatibility complex [MHC]) genes and susceptibility to infections and inflammatory processes have been described, but causal relationships have not been proven. We characterized effects of HLA-DQ alleles on outcome of congenital toxoplasma infection and found that among Caucasians, the DQ3 gene frequency was significantly higher in infected infants with hydrocephalus (0.783) than infected infants without hydrocephalus (0.444) or published normal controls (0.487). We then developed a novel animal model to definitively determine the effect of these HLA DQ molecules on the severity of toxoplasmosis. Human MHC-Class II transgenes reduced parasite burden and necrosis in brains of mice infected with Toxoplasma gondii. Consistent with the observed association between DQ3 and hydrocephalus in human infants, in the murine model the DQ3(DQ8; DQB1*0302) gene protected less than DQ1 (DQ6; DQB1*0601). Our findings definitively prove a cause and effect relationship between human MHC genes and resistance to infection, provide novel means to characterise human immune responses that are protective or pathogenic in infections, and are important for vaccine development.

Familial coagulation factor V deficiency caused by a novel 4 base pair insertion in the factor V gene: factor V Stanford.

An index patient with pseudohomozygosity for factor V Leiden was identified. Each of his two children inherited a different paternal factor V allele; a daughter was heterozygous for factor V Leiden, with 100% factor V activity, and a son was heterozygous for factor V deficiency, with 50% factor V activity. Genomic DNA was obtained from family members, and the 25 factor V exons and flanking intronic regions were sequenced in the proband and confirmed in the children. Within exon 13 of factor V, a 4 base insertion was found at NT 2856 in the proband and son. but not the daughter. This mutation, here designated factor V Stanford, results in a frameshift with loss of a thrombin activation site (R1545V) and premature termination of translation at amino acid 1560.

Specific sHLA in healthy donors and donor-specific sHLA in renal transplant patients.

We studied cadaver kidney transplant recipients to determine if their serum levels of donor-specific class I sHLA correlated with graft outcome. Testing of sHLA was performed by an ELISA sandwich assay using allospecific monoclonal trapping antibodies and anti-beta2-mu detecting antibody. Sufficient sHLA sensitivity (<1 ng/ml) was achieved by using two synergistic trapping antibodies. Suitable antibodies were available for A2 and B7, and data were collected for these two antigens. Stability of these sHLA was determined in plasma and serum as were ranges of normal and background levels. Background levels varied substantially. Five A2- recipients of A2+ grafts and 5 B7- recipients of B7+ grafts were studied with appropriate sHLA levels measured pre-transplant and at intervals post-transplant. Graft outcome was assessed by serum creatinines, renal biopsies and/or therapy for rejection. In the 5 patients (3 A2- and 2 B7-) whose post-transplant donor-specific sHLA never exceeded immunological complications (e.g., post-operative ATN, ureteral obstruction) did not affect the correlation. In the 5 patients with post-transplant levels exceeding pre-transplant levels, subsequent evidence of rejection was observed. Periodic measurement of donor-specific sHLA should be a useful instrument for monitoring renal allograft rejection.

HLA-DQB1*0602 homozygosity increases relative risk for narcolepsy but not disease severity in two ethnic groups. US Modafinil in Narcolepsy Multicenter Study Group.

Narcolepsy is a neurological disorder known to be tightly associated with HLA-DQA1*0102 and DQB1*0602. In this study, we have examined if homozygosity for DQB1*0602 increases disease susceptibility and/or severity. Patients diagnosed at Stanford University (n=160) or enrolled in a multicenter clinical trial (n=509) were included in this analysis. In both African-Americans and Caucasian-Americans with or without cataplexy, a significantly higher than expected number of subjects were DQB1*0602 homozygotes. Relative risks were 2-4 times higher in DQB1*0602 homozygotes vs heterozygotes across all patient groups. In contrast, symptom severity did not differ between DQB1*0602 homozygous and heterozygous subjects. These results indicate that HLA-DQB1*0602 homozygosity increases susceptibility to narcolepsy but does not appear to influence disease severity.

HLA DR15 (DR2) and DQB1*0602 typing studies in 188 narcoleptic patients with cataplexy.

Narcolepsy is considered a homogeneous clinical entity when excessive daytime sleepiness and cataplexy are present. Cataplexy is a polymorphic symptom that can be very mild and is thus subjectively defined. The Multiple Sleep Latency Test (MSLT) is widely used as a diagnostic test for narcolepsy. A short mean sleep latency and multiple sleep onset REM periods (SOREMPs) are typically observed in narcoleptic patients. The discovery of a tight association of narcolepsy with HLA class II antigens offers a unique opportunity to explore the respective value of the MSLT or of the presence of clear-cut cataplexy in defining an etiologically homogeneous group of narcoleptic patients. In this study, we carried out HLA typing for DR15(DR2) and DQB1*0602 in 188 narcoleptic patients with cataplexy in three ethnic groups (24 Asians, 61 Blacks, and 103 Caucasians). These results confirm the importance of DQB1*0602 typing rather than DR15 (DR2) typing in Black narcoleptic patients and demonstrate that the presence of clear-cut cataplexy is a better predictor for DQB1*0602 positivity than the presence of abnormal MSLT results.

Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy.

Narcolepsy is a sleep disorder that has been shown to be tightly associated with HLA DR15 (DR2). In this study, 58 non-DR15 patients with narcolepsy-cataplexy were typed at the HLA DRB1, DQA1 and DQB1 loci. Subjects included both sporadic cases and narcoleptic probands from multiplex families. Additional markers studied in the class II region were the promoters of the DQA1 and DQB1 genes, two CA repeat polymorphisms (DQCAR and DQCARII) located between the DQA1 and DQB1 genes, three CA repeat markers (G51152, T16CAR and G411624R) located between DQB1 and DQB3 and polymorphisms at the DQB2 locus. Twenty-one (36%) of these 58 non-DR15 narcoleptic patients were DQA1*0102 and DQB1*0602, a DQ1 subtype normally associated with DRB1*15 in DR2-positive narcoleptic subjects. Additional microsatellite and DQA1 promoter diversity was found in some of these non-DR15 but DQB1*0602-positive haplotypes but the known allele specific codons of DQA1*0102 and DQB1*0602 were maintained in all 21 cases. The 37 non-DQA1*0102/DQB1*0602 subjects did not share any particular HLA DR or DQ alleles. We conclude that HLA DQA1*0102 and DQB1*0602 are the most likely primary candidate susceptibility genes for narcolepsy in the HLA class II region.

HLA DQB1*0602 is associated with cataplexy in 509 narcoleptic patients.

Narcolepsy is a sleep disorder associated with HLA DR15 (DR2) and DQB1*0602. We HLA typed 509 patients enrolled in a clinical trial for the drug modafinil and analyzed the results in relation to cataplexy, a symptom of narcolepsy characterized by muscle weakness triggered by emotions. The patients were either subjects with cataplexy who had a mean sleep latency (SL) of less than 8 minutes and two or more sleep onset rapid eye movement (REM) periods (SOREMPs) during a multiple sleep latency test, or narcoleptic patients without cataplexy but with a mean SL shorter than 5 minutes and two or more SOREMPs. The respective values of DRB1*15 (DR2) and DQB1*0602 as markers for narcolepsy were first compared in different ethnic groups and in patients with and without cataplexy. DQB1*0602 was found to be a more sensitive marker for narcolepsy than DRB1*15 across all ethnic groups. DQB1*0602 frequency was strikingly higher in patients with cataplexy versus patients without cataplexy (76.1% in 421 patients versus 40.9% in 88 patients). Positivity was highest in patients with severe cataplexy (94.8%) and progressively decreased to 54.2% in patients with the mildest cataplexy. A voluntary 50-item questionnaire focusing on cataplexy was also analyzed in 212 of the 509 HLA-typed patients. Subjects with definite cataplexy as observed by an experienced clinician were more frequently HLA DQB1*0602-positive than those with doubtful cataplexy, and the manifestations of cataplexy were clinically more typical in DQB1*0602-positive patients. These results show that the HLA association is as tight as previously reported (85-95%) when cataplexy is clinically typical or severe. We also found that patients with mild, atypical, or no cataplexy have a significantly increased DQB1*0602 frequency (40-60%) in comparison with ethnically matched controls (24%). These results could be explained by increased disease heterogeneity in the noncataplexy group or by a direct effect of the HLA DQB1*0602 genotype on the clinical expression of narcolepsy.

Polymorphism of adhesion molecule CD31 and its role in acute graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GVHD) caused by poorly defined minor (i.e., other than HLA) histocompatibility antigens remains a serious problem in recipients of bone marrow transplants. We sought to determine whether the CD31 adhesion molecule is a minor alloantigen. METHODS: We directly sequenced samples of complementary DNA (cDNA) encoding CD31 molecules from 21 unrelated normal subjects. Sequence-specific primers were then designed to amplify alleles by the polymerase chain reaction, thereby permitting CD31 typing of genomic DNA from additional normal subjects. To assess the relevance of CD31 matching to bone marrow transplantation, we performed CD31 typing of 46 recipients of bone marrow (32 without GVHD and 14 with severe [grade III or IV] acute GVHD) and their HLA-identical sibling donors. The immunoreactivity of CD31 phenotypes with anti-CD31 monoclonal antibodies was compared by flow cytometry. RESULTS: Direct sequencing of cDNA for CD31 from the 21 normal subjects identified a single polymorphism, CTG-->GTG (Leu-->Val), at codon 125; we designated the resulting alleles CD31.L and CD31.V, respectively. The CD31 genotypes of these and 142 other unrelated subjects were of the expected frequencies. Among the transplant recipients, 71 percent of those with acute GVHD had CD31 genotypes that were not identical to the donor's genotype, as compared with 22 percent of the recipients without GVHD (P = 0.004). The binding of anti-CD31 monoclonal antibodies as measured by fluorescence-activated cell sorting correlated with the CD31 types of homozygous cell lines. CONCLUSIONS: The adhesion molecule CD31 is polymorphic. When donor and recipient genotypes are not identical, the risk of GVHD increases. Prospective CD31 typing may reduce the risk of acute GVHD.

Evidence for genetic regulation of susceptibility to toxoplasmic encephalitis in AIDS patients.

The frequency of HLA-DQ antigens in AIDS patients with toxoplasmic encephalitis (TE) were examined. HLA-DQ3 was significantly more frequent in white North American AIDS patients with TE (85.0%) than in the general white population (51.8%; P = .007, corrected P = .028) or randomly selected control AIDS patients who had not developed TE (40.0%; P = .016). In contrast, the frequency of HLA-DQ1 was lower in TE patients than in healthy controls (40.0% vs. 66.5%, P = .027), but this difference did not reach statistical significance when corrected for the number of variables tested (corrected P = .108 for the general white population). HLA-DQ3 thus appears to be a genetic marker of susceptibility to development of TE in AIDS patients, and DQ1 may be a resistance marker. These HLA associations with disease indicate that development of TE in AIDS patients is affected by a gene or genes in the HLA complex and that HLA-DQ typing may help in decisions regarding TE prophylaxis.

Extensive polymorphism of a (CA)n microsatellite located in the HLA-DQA1/DQB1 class II region.

A highly polymorphic (CA)n microsatellite marker (DQCAR), located between the DQA1 and the DQB1 genes, was characterized in four ethnic groups. Based on length polymorphism, 12 alleles could be defined. The marker is located 1- to 2-kb telomeric to the DQB1 gene and 10 kb centromeric to the DQA1 gene and was shown to be in tight linkage disequilibrium with HLA-DQ. Analysis of the linkage disequilibrium pattern revealed little additional diversity in DQ1-associated haplotypes. Almost all DQ1 subjects examined were DQCAR 103 or DQCAR 107 (13 and 15 CA repeats, respectively). In contrast, significant haplotypic diversity was observed for most DQ2-, DQ3-, and DQ4-associated haplotypes. These haplotypes often had longer allele sizes (DQCAR > 111, more than 17 CA repeats) and more DQCAR alleles per haplotype. These haplotypes also carried DQCAR alleles of different sizes, even though they bore the same DQA1 and DQB1 alleles, and sometimes the same DRB1 allele as well. These results indicate that DQCAR could be a useful marker to better define disease associations with HLA. Our results are also consistent with the hypothesis that CAR alleles with higher numbers of repeats have higher mutation rates and that recombination within the HLA-DR/DQ region is haplotype dependent.

Narcolepsy and immunity.

Narcolepsy is a neurological disorder known to be associated with human leukocyte antigen (HLA)-DQB1*0602 in humans. In a canine model, the disorder is also genetically linked to a gene of high homology with the human mu-switch-like immunoglobulin (Ig) gene (current LOD score 13.6 at 0% recombination). Since association with HLA or other immune function polymorphic genes (T cell receptor of Ig, mainly) is a hallmark of most autoimmune diseases, it is proposed that autoimmunity may also play a role in the development of narcolepsy. Arguments for and against this hypothesis are reviewed. It is shown that both on the basis of the most recent molecular studies, and because of some of its clinical features, narcolepsy may be an autoimmune disorder. However, neither systemic nor central nervous system (CNS) evidence of any autoimmune abnormality have ever been found. To reconcile this discrepancy, it is suggested that the pathological immune process involved in narcolepsy could be difficult to detect because it is restricted to a very small region of the brain or targets a low abundance neuroeffector. Alternatively, it is possible that a more fundamental relationship is involved between sleep generation and immune regulation. The pathophysiology of narcolepsy may then involve new CNS-immune mechanisms that may shed new light on the sleep process itself.

An immunoglobulin switchlike sequence is linked with canine narcolepsy.

Canine narcolepsy is an animal model of the human disorder that is transmitted as a single autosomal recessive gene with full penetrance (canarc-1) in Dobermans and Labradors. In previous experiments, we have identified a very tight linkage marker for canarc-1. This marker, a 0.85-kb band cross reacting with a human mu-switch Heavy-Chain Immunoglobulin probe (maximum logarithm of odds [LOD] score Zmax = 10.8 at 0% recombination), has now been cloned and sequenced. The gene, composed of GC rich repeats, is 75% homologous to the human mu-switch gene and is similar in organization to immunoglobulin switch genes. Curiously, however, this mu-switchlike segment appears to be unlinked with other switchlike polymorphisms detected at high stringency with the human mu-switch probe. Because in most animal species all switch genes are located within 300-500 kb and show tight linkage in families, this result suggests two possible hypotheses: 1) Our 0.85 kb is a true immunoglobulin switch segment, but the map of the canine Variable Heavy-Chain loci is organized in unlinked clusters, or 2) our 0.85-kb segment is not an immunoglobulin switch segment and is located elsewhere in the genome in all species. We are now using chromosome walking and Yeast Artificial Chromosome Cloning techniques, together with corresponding studies in humans to identify the pathological gene.

DQB1*0602 and DQA1*0102 (DQ1) are better markers than DR2 for narcolepsy in Caucasian and black Americans.

In the present study, we tested 19 Caucasian and 28 Black American narcoleptics for the presence of the human leucocyte antigen (HLA) DQB1*0602 and DQA1*0102 (DQ1) genes using a specific polymerase chain reaction (PCR)-oligotyping technique. A similar technique was also used to identify DRB1*1501 and DRB1*1503 (DR2). Results indicate that all but one Caucasian patient (previously identified) were DRB1*1501 (DR2) and DQB1*0602/DQA1*102 (DQ1) positive. In Black Americans, however, DRB1*1501 (DR2) was a poor marker for narcolepsy. Only 75% of patients were DR2 positive, most of them being DRB1*1503, but not DRB1*1501 positive. DQB1*0602 was found in all but one Black narcoleptic patient. The clinical and polygraphic results for this patient were typical, thus confirming the existence of a rare, but genuine form of DQB1*0602 negative narcolepsy. These results demonstrate that DQB1*0602/DQA1*0102 is the best marker for narcolepsy across all ethnic groups.