Maternal and paternal carriage of the annexin A5 M2 haplotype: a possible risk factor for recurrent implantation failure (RIF).

OBJECTIVE: This study was carried out to determine the potential role of the M2/ANXA5 haplotype as a risk factor for recurrent implantation failure (RIF). Carriage of the M2/ANXA5 haplotype that induces prothrombotic changes has been implicated in failure of early pregnancies and placenta-mediated complications (preeclampsia, IUGR, preterm birth). MATERIAL AND METHODS: In the present case control study, 63 couples (females and males) with RIF presenting for IVF/ICSI to the Fertility Center of [masked] were analyzed. RIF was defined as ≥ 4 consecutive failed ART-transfers of ≥ 4 blastocysts or ≥ 8 cleavage-stage embryos of optimal quality and maternal age ≤ 41. Fertile female controls (n = 90) were recruited from the same center. Population controls (n = 533) were drafted from the PopGen biobank, UKSH Kiel. RESULTS: Couples carrying the M2/ANXA5 haplotype turned out to have a significantly increased relative risk (RR) for RIF. Compared with female fertile controls, RR was 1.81 with p = 0.037 (OR 2.1, 95%CI 1.0-4.3) and RR was 1.70, with p = 0.004 (OR 2.0, 95%CI 1.2-3.1) compared with population controls (15.4% M2 carriers). Male partners were comparable with RIF females for M2/ANXA5 haplotypes (28.6% vs. 23.8%, p = 0.54). RIF females compared with population controls had a RR of 1.55 (p = 0.09) and RIF males compared with population controls had a RR of 1.9 (p = 0.01). Couples with ≥ 7 failed transfers showed a RR of 1.82 (p = 0.02) compared with population controls. CONCLUSION: Our findings suggest that maternal as well as paternal M2/ANXA5 haplotype carriages are risk factors for RIF. These results allow new insights into the pathogenesis of RIF and might help to identify relevant risk groups.

The relevance of ANXA5 genetic variants on male fertility.

PURPOSE: To investigate the effect of the anticoagulation factor annexin A5 on male fertility and to provide perspective on the influence of members of the coagulation cascade on fertility. METHODS: Patients with normozoospermia and with unexplained severe oligozoospermia were retrospectively selected and their genomic DNA sequenced for the promoter region of ANXA5. The genotypes proportions and the odds ratio for carriership of the haplotype M2 were compared between the groups and population control. The clinical data used were gathered from parameters determined during routine clinical assessment and were compared between carriers and non-carriers within the patient groups. RESULTS: The carrier rates for the haplotype M2/ANXA5 were of 25.73%, 20.81%, and 15.3% in the severe oligozoospermic, the normozoospermic, and the general population control groups, respectively. The OR between patients groups was of 1.31 (95% CI 0.88 to 1.96 p = 0.176). Oligozoospermic and normozoospermic patients compared with the control group had an OR of 1.9 (95% CI 1.33 to 2.73 p < 0.001) and 1.45 (95% CI 0.99 to 2.10 p = 0.054) respectively. The clinical parameters that differed between the carriers and non-carriers of the haplotype M2/ANXA5 were prolactin, α-glucosidase, and fructose. The differences were only statistically significant in the normozoospermic group. CONCLUSIONS: Athough the infertile patient groups had a higher prevalence of promoter variants, we could not demonstrate any biologically relevant effect of lower levels of annexin A5 on most male fertility parameters. A deficiency in an anticoagulation factor does not seem to impact male fertility.

Assessment of M2/ANXA5 haplotype as a risk factor in couples with placenta-mediated pregnancy complications.

PURPOSE: The aim of this study was to confirm the associated M2/ANXA5 carrier risk in women with placenta-mediated pregnancy complications (PMPC) and to test their male partners for such association. Further analysis evaluated the influence of maternal vs. paternal M2 alleles on miscarriage. METHODS: Two hundred eighty-eight couples with preeclampsia (PE), intrauterine growth restriction (IUGR), or premature birth (PB) were recruited (n = 96 of each phenotype). The prevalence of the M2 haplotype was compared to two control cohorts. They included a group of women with a history of normal pregnancy without gestational pathology (Munich controls, n = 94) and a random population sample (PopGen controls, n = 533). RESULTS: Significant association of M2 haplotype and pregnancy complications was confirmed for women and for couples, where prevalence was elevated from 15.4 to 23.8% (p < 0.001). Post hoc analyses demonstrated an association for IUGR and PB individually. A strong link between previous miscarriages and M2 carrier status was identified which may explain the predisposition to placental pregnancy complication. M2/ANXA5 appears to be a risk factor for adverse pregnancy outcomes related, but not limited to miscarriages, with similar prevalence in women and their male partners. CONCLUSION: These findings support the proposed physiological function of ANXA5 as an embryonic anticoagulant that appears deficient in contiguous specter of thrombophilia-related pregnancy complications culminating more frequently in miscarriage in a maternal M2 carrier background.

Maternal carriers of the ANXA5 M2 haplotype are exposed to a greater risk for placenta-mediated pregnancy complications.

PURPOSE: Annexin A5 (ANXA5) is a protein abundantly expressed in normal placenta where it contributes to the healthy outcome of a pregnancy. Lower ANXA5 levels have been observed in M2/ANXA5 haplotype carrying chorion. Consequently, this study aimed to assess the potential association of M2 maternal carrier status with the risk of recurrent pregnancy loss (RPL), the timing of miscarriages, and other obstetric complications, for the first time in a population from Latin America. METHODS: This study was designed as a prospective recruitment of RPL patients with post hoc analysis. The distribution of the M2/ANXA5 haplotype was compared between a group of 229 Argentine women with RPL and 100 parous controls, and was further analyzed in subgroups of patients stratified according to the timing of miscarriages and in relation to other obstetric complications. RESULTS: No significant differences were found in the distribution of M2 haplotype among either RPL patients or the subgroups with embryonic, early fetal, or late fetal losses compared to parous controls. Notwithstanding, maternal M2/ANXA5 was found to be independently associated with a higher risk of suffering intrauterine growth restriction (IUGR) and/or preeclampsia (PE). Simultaneously, the presence of inherited and/or acquired thrombophilia also proved to be an independent risk factor for these. CONCLUSIONS: The association found between the maternal carriage of the M2/ANXA5 haplotype and an elevated risk of IUGR and/or PE supports the hypothesis that carrier status of this haplotype and the consequently reduced placental ANXA5 expression might be responsible, at least partially, for the onset of these gestational vascular complications.

Genetic analysis of the M2/ANXA5 haplotype as recurrent pregnancy loss predisposition in the Malay population.

PURPOSE: The aim of this study was to evaluate a new predisposition factor, M2/ANXA5 (RPRGL3), in recurrent pregnancy loss (RPL) patients of Malay origin, since it was previously known that the prevalence of this condition is relatively high among the Malay population of Malaysia, where conventional hereditary thrombophilia factors have been generally ruled out. METHODS: A total of 232 women who had experienced ≥2 unexplained RPL and 141 available male partners were recruited, with 360 healthy Malay and 166 parous female controls. Prevalence of M2 carriage and RPL odds ratios were calculated in (a) control and patient groups; (b) clinically defined subgroups in categories of pregnancy loss, primary, secondary, and tertiary; and (c) timing of pregnancy loss in early, ≤15th gestation week and "late" fetal losses, and >15th gestation week subgroups. RESULTS: Both male and female subjects had similar M2/ANXA5 allele frequencies. The carrier rate of M2/ANXA5 for the general Malay population was 42.2 and 34.9% for parous controls. These carrier rates compared to Malay RPL subjects (52% M2 carriers) resulted in elevated odds ratios (95% confidence interval) of 1.53 (1.1 to 2.1) and 1.97 (1.3 to 3.1) accordingly for early fetal losses. Moreover, exceeding copy numbers of M2/ANXA5 alleles seemed to afflict a greater chance of RPL in couples, especially when both partners were M2 carriers. CONCLUSION: This study confirmed the proposed role of M2/ANXA5 as embryonic, genetically associated thrombophilia predisposition factor for early RPL among ethnic Malay of Malaysia.

Annexin A5 haplotype M2 is not a risk factor for recurrent spontaneous abortion in Northern Europe: is there sufficient evidence?

The M2 haplotype of the annexin A5 gene is a well-recognized predisposition factor for recurrent spontaneous abortion (RSA). A recent publication by Nagirnaja et al. (2015) in PLoS One discusses the risk role of the M2 haplotype for RSA in cases compared with controls of North European extraction and arrives at a negative result. As a number of previous and fairly recent studies have supported the proposed involvement of the M2 haplotype in the cause of idiopathic RSA, this commentary aims to highlight problematic issues in the above publication. It is the opinion of the authors that the study by Nagirnaja et al. (2015) does not generate adequate proof of the absence of RSA risk, attributable to carriage of the M2 haplotype.

Functional characterisation of alpha-galactosidase a mutations as a basis for a new classification system in fabry disease.

Fabry disease (FD) is an X-linked hereditary defect of glycosphingolipid storage caused by mutations in the gene encoding the lysosomal hydrolase α-galactosidase A (GLA, α-gal A). To date, over 400 mutations causing amino acid substitutions have been described. Most of these mutations are related to the classical Fabry phenotype. Generally in lysosomal storage disorders a reliable genotype/phenotype correlation is difficult to achieve, especially in FD with its X-linked mode of inheritance. In order to predict the metabolic consequence of a given mutation, we combined in vitro enzyme activity with in vivo biomarker data. Furthermore, we used the pharmacological chaperone (PC) 1-deoxygalactonojirimycin (DGJ) as a tool to analyse the influence of individual mutations on subcellular organelle-trafficking and stability. We analysed a significant number of mutations and correlated the obtained properties to the clinical manifestation related to the mutation in order to improve our knowledge of the identity of functional relevant amino acids. Additionally, we illustrate the consequences of different mutations on plasma lyso-globotriaosylsphingosine (lyso-Gb3) accumulation in the patients' plasma, a biomarker proven to reflect the impaired substrate clearance caused by specific mutations. The established system enables us to provide information for the clinical relevance of PC therapy for a given mutant. Finally, in order to generate reliable predictions of mutant GLA defects we compared the different data sets to reveal the most coherent system to reflect the clinical situation.

Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion.

Annexin A5 is a placental anti-coagulant protein that contains four nucleotide substitutions (M2 haplotype) in its promoter. This haplotype is a risk factor for recurrent spontaneous abortion (RSA). The influence of the M2 haplotype in the gestational timing of spontaneous abortions, paternal risk and relationships with known risk factors were investigated. European couples (n = 500) who had experienced three or more consecutive spontaneous abortions, and two fertile control groups, were selected for this study. The allele frequency of M2 was significantly higher among patients who had experienced early RSA than among controls (P = 0.002). No difference was found between controls and patients who had undergone late spontaneous abortions. No difference was found between patients who had experienced RSA who had a live birth or no live births, or between patients who were positive or negative for known risk factors. Male and female partners in each group had similar allele frequencies of M2. The M2 haplotype is a risk factor for early spontaneous abortions, before the 12th week of gestation, and confers about the same relative risk to carriers of both sexes. Having one or more M2 allele(s) in combination with other risk factors further increases the RSA risk.

M2/ANXA5 haplotype as a predisposition factor in Malay women and couples experiencing recurrent spontaneous abortion: a pilot study.

Recurrent spontaneous abortion (RSA) is a prevalent condition among the Malay population of Malaysia, where carriage risk of conventional hereditary thrombophilia factors has been generally ruled out. The contribution of M2/ANXA5, a common haplotype in the annexin A5 gene promoter, was evalauted for RSA in Malay. Seventy-seven women who had experienced two or more unexplained RSA and 41 available male partners were selected for study, with 360 population controls recruited from healthy Malay individuals. Incidence of M2 carriage and odds ratios were calculated between control and patient groups, and clinically defined subgroups and RSA risk was evaluated. M2/ANXA5, found in 42.2% of the general Malay population, was associated with greater risks for women with primary and secondary RSA with early (gestational week 5-15) losses. The risk was somewhat higher in Malay couples when both partners were carriers and a trend of higher prevalence was seen for the male partners patients who had experienced RSA. M2 carriage seems to be a risk factor with unusually high incidence in Malay women and couples with primary and secondary RSA with 'early' spontaneous abortions. The associated male partner risk confirms the proposed role of M2/ANXA5 as a genetic trait impeding embryonic anticoagulation.

The haplotype M2 of the ANXA5 gene is not associated with antitrophoblast antibodies.

PURPOSE: The M2 haplotype in ANXA5 as well as antitrophoblast antibodies predispose to recurrent pregnancy loss (RPL). Since M2/ANXA5 can be a factor for development of antiphospholipid antibodies (aPL), this study aimed to trace a possible association of M2 with antitrophoblast antibodies. METHODS: One hundred patients with two or more consecutive, idiopathic RPLs were divided in two subgroups, JEG-3(+) (n = 42) and JEG-3(-) (n = 58), according to the anti-JEG-3 reactivity measured in subjects' sera. Both subgroups were genotyped for ANXA5 promoter haplotypes and genetic frequencies were compared to available fertile and control populations, as well as within the subgroups. RESULTS: M2/ANXA5 was generally enriched in the JEG-3 screened cohort of RPL patients in comparison to fertile and population controls. Despite the relatively higher abundance of the haplotype in the JEG-3(-) sample as compared to JEG-3(+) patients and in the JEG-3(-) primary RPL subset in particular, compared to the rest of patients, there was no statistically significant difference between both, JEG-3(-) and JEG-3(+) subgroups. CONCLUSION: It appears that the haplotype M2/ANXA5 is not associated with the presence of anti-trophoblast antibodies. Our finding indicates that anti-trophoblast antibodies are a class of molecules that differ from aPL and from anti-b2-GPI antibodies, apparently not directed to same or similar epitopes that aPL and anti-b2-GPI would recognize.

Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

Polymorphisms in the annexin A5 gene promoter in Japanese women with recurrent pregnancy loss.

Recent findings have raised the possibility that polymorphisms within the annexin A5 gene (ANXA5) promoter contribute to the etiology of recurrent pregnancy loss (RPL). In our present study, 243 Japanese women who had suffered more than three fetal losses and a group of 119 fertile controls were genotyped for four ANXA5 gene promoter single-nucleotide polymorphisms (SNPs; SNP1-4: g.-467G >A, g.-448A>C, g.-422T>C, g.-373G>A) previously reported to be associated with this disorder. An additional two SNPs located within the 5'-untranslated region of the ANXA5 (SNP5 and 6: g.-302T>G, g.-1C>T) were also evaluated. Our case--control study revealed that the minor allele was significantly more frequent in the RPL group than controls for all six of these SNPs, among which SNP5 showed the highest significance (P= 0.002). As with the M2 haplotype for SNP1-4 (A-C-C-A) for a western population in previous reports, a haplotype comprising all of the minor alleles for SNP1-6 (A-C-C-A-G-T), the third major haplotype in the Japanese population, showed a significantly higher frequency in our current RPL subjects than in controls (P= 0.025). In addition, the second major haplotype (G-A-T-G-G-C) was found to confer a significant risk of RPL (P= 0.036), implicating SNP5 as a major risk determinant for this disease. Our present findings support the hypothesis that genomic variations within the ANXA5 gene upstream region impact upon the disease susceptibility to RPL. Our data indicate that SNP5 is a novel risk factor for this disease in the Japanese population.

Incompletely penetrant PKD1 alleles mimic the renal manifestations of ARPKD.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutation in PKD1 or PKD2, is usually an adult-onset disorder but can rarely manifest as a neonatal disease within a family characterized by otherwise typical ADPKD. Coinheritance of a hypomorphic PKD1 allele in trans with an inactivating PKD1 allele is one mechanism that can cause early onset ADPKD. Here, we describe two pedigrees without a history of cystic kidney disease that each contain two patients with onset of massive PKD in utero. The presentations were typical of autosomal recessive PKD (ARPKD) but they were not linked to the known ARPKD gene, PKHD1. Mutation analysis of the ADPKD genes provided strong evidence that both families inherited, in trans, two incompletely penetrant PKD1 alleles. These patients illustrate that PKD1 mutations can manifest as a phenocopy of ARPKD with respect to renal involvement and highlight the perils of linkage-based diagnostics in ARPKD without positive PKHD1 mutation data. Furthermore, the phenotypic overlap between ARPKD and these patients resulting from incomplete penetrant PKD1 alleles support a common pathogenesis for these diseases.

Micromolar Zinc in Annexin A5 Anticoagulation as a Potential Remedy for RPRGL3-Associated Recurrent Pregnancy Loss.

Deficient expression of the placental anticoagulant annexin A5 (ANXA5) has been associated with thrombophilia-related pregnancy complications and ultimately with recurrent pregnancy loss (RPL). Carrier status of M2/ANXA5 ( RPRGL3), common ANXA5 promoter variant, has been identified as genetic cause of reduced ANXA5 levels and proposed as biomarker for successful anticoagulant treatment of RPL women. A murine model of AnxA5 loss of function displayed characteristic placental pathology and fetal loss that was alleviated through anticoagulant intervention. This study identified an alternative means of supplementing anticoagulation, through elevated ANXA5 expression. Physiological micromolar Zn2+ stimulated ANXA5 transcription, raising ANXA5 protein expression and surface abundance on BeWo and human umbilical vein endothelial cells (HUVEC), thus resulting in prolonged coagulation times. Zn2-fed AnxA5 functionally deficient pregnant mice showed a trend to increase litter size when primiparous that grew comparable to wild-type progeny in subsequent pregnancies. Elevated AnxA5 signal upon Zn2+ treatment was confirmed in murine placentae. Micromolar Zn2+ stimulated ANXA5 expression in cell culture directly and alleviated RPL in AnxA5 genetically deficient mice, without notable toxicity effects.

Association between M2/ANXA5 haplotype and repeated pregnancy loss: a meta-analysis.

OBJECTIVE: To ascertain the magnitude and precision of the association between M2/ANXA5 haplotype and repeated pregnancy loss (RPL). DESIGN: Meta-analysis of odds ratios. SETTING: Not applicable. PATIENT(S): Subjects were women with RPL and their partners. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): The association between M2/ANXA5 haplotype and RPL was evaluated in a meta-analysis of odds ratios. We further scrutinized this association according to [1] the sequence of miscarriages, [2] the number of consecutive losses, [3] the extent of excluding other pathologies of RPL, and [4] the timing of fetal loss. RESULT(S): Fourteen individual studies (n = 4,664 subjects) were included in this meta-analysis. The results show that women with the M2/ANXA5 haplotype have 1.54 times (95% confidence interval, 1.08-2.20) the odds of having associated RPL compared with women with the normal haplotype, regardless of consecutive or nonconsecutive pregnancy losses. Acknowledging the clinical heterogeneity among the studies, this significant association comes with a caveat that the lower bound of the confidence interval is close to unity. In couple populations (n = 2,449), M2/ANXA5 haplotype subjects have an odds ratio of 1.48 (95% confidence interval, 1.14-1.91) of experiencing RPL, which suggests contributions from paternal M2/ANXA5 carriers in RPL. CONCLUSION(S): This meta-analysis ascertains that women with the M2/ANXA5 haplotype have a higher risk of experiencing RPL, especially consecutive early idiopathic RPL. Male partners with the M2/ANXA5 haplotype partly contribute to this risk. Hence, screening for the M2/ANXA5 haplotype as a panel of laboratory investigations for RPL is recommended.

Chromosomal evolution of the PKD1 gene family in primates.

BACKGROUND: The autosomal dominant polycystic kidney disease (ADPKD) is mostly caused by mutations in the PKD1 (polycystic kidney disease 1) gene located in 16p13.3. Moreover, there are six pseudogenes of PKD1 that are located proximal to the master gene in 16p13.1. In contrast, no pseudogene could be detected in the mouse genome, only a single copy gene on chromosome 17. The question arises how the human situation originated phylogenetically. To address this question we applied comparative FISH-mapping of a human PKD1-containing genomic BAC clone and a PKD1-cDNA clone to chromosomes of a variety of primate species and the dog as a non-primate outgroup species. RESULTS: Comparative FISH with the PKD1-cDNA clone clearly shows that in all primate species studied distinct single signals map in subtelomeric chromosomal positions orthologous to the short arm of human chromosome 16 harbouring the master PKD1 gene. Only in human and African great apes, but not in orangutan, FISH with both BAC and cDNA clones reveals additional signal clusters located proximal of and clearly separated from the PKD1 master genes indicating the chromosomal position of PKD1 pseudogenes in 16p of these species, respectively. Indeed, this is in accordance with sequencing data in human, chimpanzee and orangutan. Apart from the master PKD1 gene, six pseudogenes are identified in both, human and chimpanzee, while only a single-copy gene is present in the whole-genome sequence of orangutan. The phylogenetic reconstruction of the PKD1-tree reveals that all human pseudogenes are closely related to the human PKD1 gene, and all chimpanzee pseudogenes are closely related to the chimpanzee PKD1 gene. However, our statistical analyses provide strong indication that gene conversion events may have occurred within the PKD1 family members of human and chimpanzee, respectively. CONCLUSION: PKD1 must have undergone amplification very recently in hominid evolution. Duplicative transposition of the PKD1 gene and further amplification and evolution of the PKD1 pseudogenes may have arisen in a common ancestor of Homo, Pan and Gorilla approximately 8 MYA. Reticulate evolutionary processes such as gene conversion and non-allelic homologous recombination (NAHR) may have resulted in concerted evolution of PKD1 family members in human and chimpanzee and, thus, simulate an independent evolution of the PKD1 pseudogenes from their master PKD1 genes in human and chimpanzee.

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions.

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.

Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A.

The amount of residual F8 (FVIII:C) determines the clinical severity of hemophilia A. Recently, we showed that the mutation detection rate in severely affected male patients (FVIII:C<1% of normal) is virtually 100% when testing for the common intron 22-/intron 1- inversions and big deletions, followed by genomic sequencing of the F8 gene. Here we report on the spectrum of mutations and their distribution throughout the F8 gene sequence in 135 patients with moderate (n=23) or mild (n=112) hemophilia A. In contrast to the severe form of the disorder, analysis on the genomic level failed to detect the molecular defect in approximately 4% of the moderately and in approximately 12% of the mildly affected patients. A total of 36 of the mutations identified in this study are novel. The vast majority of the detected changes were missense. The newly detected amino acid substitutions were scored for potential distant or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling. Two molecular changes in the promoter region of the factor VIII gene (c.-112G>A and -219C>T), affecting the core segment (minimal promoter) were detected in two patients with mild hemophilia A. To our knowledge this is the first report on promoter mutations in the F8 gene.

Lessons From the EThIGII Trial: Proper Putative Benefit Assessment of Low-Molecular-Weight Heparin Treatment in M2/ANXA5 Haplotype Carriers.

This study presents sample size considerations derived from the Efficacy of Thromboprophylaxis as an Intervention during Gravidity (EThIGII) trial (ClinicalTrials.gov: NCT00400387) to address the question of low-molecular-weight heparin (LMWH) treatment in women with recurrent pregnancy loss (RPL) depending on the M2/ANXA5 haplotype. To evaluate the possible influence of such treatment on miscarriage rates of trial participants, a post hoc analysis of ANXA5 promoter genotypes in the light of M2/ANXA5 (RPRGL3) distribution was performed using logistic models. DNA for genotyping was available from 129 LMWH and 95 control patients, 44 (19.6%) of whom were M2/ANXA5 carriers. Miscarriages occurred in 1 (4.0%) of 25 M2/ANXA5 carriers from the LMWH group compared to 4 (21.1%) of 19 in the control group, resulting in an odds ratio (95% confidence interval) for miscarriage of 0.16 (0.016-1.5) for women treated with LMWH. In noncarriers, miscarriage rates were 6 (5.8%) of 104 versus 7 (9.2%) of 76 for the LMWH and the control groups, respectively, corresponding to an odds ratio for miscarriage of 0.60 (0.19-1.9). The apparent beneficial effects of miscarriage rate reduction in M2/ANXA5 carriers with RPL concur with biological considerations about improvement in reduced ANXA5 function through LMWH treatment in an adequate murine model. The data obtained were instrumental to design proper assessment of the existence and magnitude of this effect.