Bp Indel Research Articles

Fast and accurate read alignment for resequencing

Next-generation sequence analysis has become an important task both in laboratory and clinical settings. A key stage in the majority sequence analysis workflows, such as resequencing, is the alignment of genomic reads to a reference genome. The accurate alignment of reads with large indels is a computationally challenging task for researchers. We introduce SeqAlto as a new algorithm for read alignment. For reads longer than or equal to 100 bp, SeqAlto is up to 10 × faster than existing algorithms, while retaining high accuracy and the ability to align reads with large (up to 50 bp) indels. This improvement in efficiency is particularly important in the analysis of future sequencing data where the number of reads approaches many billions. Furthermore, SeqAlto uses less than 8 GB of memory to align against the human genome. SeqAlto is benchmarked against several existing tools with both real and simulated data. Linux and Mac OS X binaries free for academic use are available at http://www.stanford.edu/group/wonglab/seqalto whwong@stanford.edu.

Fine mapping of Rf3 and Rf4 fertility restorer loci of WA-CMS of rice (Oryza sativa L.) and validation of the developed marker system for identification of restorer lines

The Wild Abortive (WA) system is the major cytoplasmic male sterility (CMS) source for hybrid rice production in indica rice and its fertility restoration is reported to be controlled by two major loci viz. Rf3 on chromosome 1 and Rf4 on chromosome 10. With the availability of the rice genome sequence, an attempt was made to fine map, develop candidate gene based markers for Rf3 and Rf4 and validate the developed marker system in a set of known restorer lines. Using polymorphic markers developed from microsatellite markers and candidate gene based markers from Rf3 and Rf4 loci, local linkage maps were constructed in two mapping populations of ~1,500 F2 progeny from KRH2 (IR58025A/KMR3R) and DRRH2 (IR68897A/DR714-1-2R) hybrids. QTLs and their interactions for fertility restoration in Rf3 and Rf4 loci were identified. The identified QTL in both mapping populations together explained 66–72 % of the phenotypic variance of the trait suggesting their utility in developing a marker system for identification of fertility restorers for WA-CMS. Sequence comparison of the two candidate genes from the Rf3 and Rf4 regions in male sterile (A) and restorer (R) lines showed 2–3 bp indels and a few substitutions in the Rf3 region and indels of 327 and 106 bp in the Rf4 region respectively. The marker system identified in the present study was validated in 212 restorers and 34 maintainers along with earlier reported markers for fertility restoration of WA-CMS. Together DRCG-RF4-14 and DRCG-RF4-8 for the Rf4 locus and DRRM-RF3-5/DRRM-RF3-10 for the Rf3 locus showed a maximum efficiency of 92 % for identification of restorers.

Mutation in promoter region of a serine protease inhibitor confers Perkinsus marinus resistance in the eastern oyster (Crassostrea virginica)

Protease inhibitors from the host may inhibit proteases from invading pathogens and confer resistance. We have previously shown that a single-nucleotide polymorphism (SNP198C) in a serine protease inhibitor gene (cvSI-1) is associated with Perkinsus marinus resistance in the eastern oyster. As SNP198 is synonymous, we studied whether its linkage to polymorphism at the promoter region could explain the resistance. A 631 bp fragment of the promoter region was cloned by genome-walking and resequenced, revealing 22 SNPs and 3 insertion/deletions (indels). A 25 bp indel at position −404 was genotyped along with SNP198 for association analysis using before- and after-mortality samples. After mortalities that were primarily caused by P. marinus, the frequency of deletion allele at −404indel increased by 15.6% (p = 0.0437), while that of SNP198C increased by only 3.4% (p = 0.5756). The resistance alleles at the two loci were coupled in 79.6% of the oysters. Oysters with the deletion allele at −404indel showed significant (p = 0.0189) up-regulation of cvSI-1 expression under P. marinus challenge. Our results suggest that mutation at the promoter region causes increased transcription of cvSI-1, which in turn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenic proteases from the parasite.

PRNP gene variation in Pakistani cattle and buffaloes

Bovine spongiform encephalopathy (BSE) is a neurodegenerative prion protein misfolding disorder of cattle. BSE is of two types, classical BSE and atypical BSE which in turn is of two types, H-type BSE and L-type BSE. Both H-type BSE and L-type BSE are primarily sporadic prion disorders. However, one case of H-type BSE has recently been associated with E211K polymorphism in the prion protein gene (PRNP). Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23bp insertion/deletion (indel) in the PRNP promoter region and a 12bp indel in the first intron. No information regarding BSE susceptibility in Pakistani cattle is available. The present study aimed at achieving this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23bp and 12bp indels employing triplex PCR. The E211K polymorphism was not detected in any of the animals studied. The 23bp insertion allele was underrepresented in studied cattle breeds while the 12bp insertion allele was overrepresented. Both 23bp and 12bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23bp and 12bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle in terms of allele, genotype and haplotype frequencies of 23bp and 12bp indels. The higher observed frequency of 12bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE than European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.

Overexpression of the sterol 14α‐demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype

The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14α-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ΔY459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola.

Discrimination ofTrichophyton tonsuransandTrichophyton equinumby PCR-RFLP and by β-tubulin and Translation Elongation Factor 1-α sequencing

Trichophyton tonsurans and T. equinum are two closely related sister species of dermatophytes, but differ in their preferred hosts, i.e., humans or horses, respectively. Routine procedures for their identification depend on studies of their phenotypic, physiological and biochemical characteristics, which are laborious and may yield ambiguous results. Molecular methods using rDNA ITS also had been judged to be insufficiently discriminatory. In the present study two genetic markers were sequenced in addition to the ITS region, i.e., partial β-tubulin (BT2) and translation elongation factor 1-α (TEF1). The TEF1 locus revealed a consistent differences in a 13 bp indel and an additional SNP between the two species, along with a single base substitution in BT2 and one ITS1, enabling unambiguous distinction of the two taxa. RFLP targeting the ITS region was evaluated as a potential tool for routine screening of suspected isolates of T. tonsurans and T. equinum.

Genetic characterization of Red Junglefowl (Gallus gallus), Thai indigenous chicken (Gallus domesticus), and two commercial lines using selective functional genes compared to microsatellite markers

Genetic characterization among Red Junglefowl (GS, Gallus gallus spadiceus), Thai indigenous chicken (TIC, Gallus domesticus) and commercial lines has been widely used for studies of genealogical origin, genetic diversity, and effects of selection. We compared the efficiency of genetic characterization of chicken populations that had been under different intensities of selection using selective functional gene versus microsatellite marker analyses. We genotyped 151 chickens from five populations: Red Junglefowl, TIC and commercial lines (BR, broiler and WL, White Leghorn). Genetic structure analyses using six loci of five functional genes - corresponding to heat tolerance (heat shock protein 70, HSP70/C, HSP70/M), broodiness (vasoactive intestinal peptide receptor-1, VIPR-1), egg production-[24-bp indel (insertion or deletion) prolactin, 24bpPRL], ovulation rate (growth hormone receptor, GHR), and growth (insulin-like growth factor-1, IGF-1) - were compared with 18 microsatellite markers. PCR-RFLP and allele specific PCR were used for functional gene typing. A neighbor-joining tree from Nei's genetic distance was constructed to show genetic relationships. A similar pattern was found with both functional genes and microsatellites. Three groups consisting of BR, WL and TIC-GS-GG were formed. A principal component plot based on individual similarity using Dice's coefficient was also constructed to confirm the relationship. Different patterns were found when using functional genes versus microsatellites. A principal component plot with functional genes also gave three clusters consisting of BR, WL and TIC-GS-GG. A principal component plot using microsatellites gave four clusters, consisting of WL, GG, TIC, and BR-GS. Characterization of BR and GS differs from previous studies. We concluded that genetic characterization with appropriate functional genes is more accurate when differences in genetic make-up among populations are known. Genetic characterization using functional gene data was consistent in neighbor joining and principal component plot analyses, while genetic characterization using microsatellite data gave varied results depending on the analysis methodology.

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.

New evidence for balancing selection at the HLA-G locus in South Amerindians

HLA-G is a non-classical HLA (Human Leukocyte Antigen) molecule characterized by limited tissue distribution under normal physiological conditions and low variability at both DNA and protein levels. Several studies suggest that HLA-G could play a role, as an immunoregulatory molecule, in situations as diverse as transplantation, cancer, viral infections and inflammatory diseases. A total of 237 individuals from 21 South American tribes speaking nine different linguistic families were studied in relation to the 14 bp insertion/deletion polymorphism at the HLA-G gene. A consistent (seven in nine) excess of heterozygosity in samples classified by language was obtained. Our data supply evidences for balancing selection acting at the HLA-G 14 bp INDEL region. Enhanced fetal survival in a pathogen-rich environment may account for these findings.

Polymorphism of the prion protein gene (PRNP) in Polish cattle affected by classical bovine spongiform encephalopathy

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of two insertion/deletion (indel) polymorphisms, located within the promoter and intron 1 of the prion protein gene PRNP, showing a significant association with the occurrence of classical form of the disease. Because the effect of the polymorphisms was studied only in few populations, in this study we investigated whether previously described association of PRNP indel polymorphisms with BSE susceptibility in cattle is also present in Polish cattle population. We found a significant relation between the investigated PRNP indel polymorphisms (23 and 12bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P<0.05). The deletion variants of both polymorphisms were related to increased susceptibility, whereas insertion variants were protective against BSE.

Systematics of the Eupatorium album Complex (Asteraceae) from Eastern North America

Abstract Analysis of ITS sequence data showed unexpected complexity for Eupatorium album and E. petaloideum, a closely related species pair from eastern North America that has been considered to form a single species. The two species consistently differed in ITS sequence by seven to eight bp as well as a one bp indel, and detailed analysis revealed little intraspecific variation. The ITS sequences of other samples that had similar but slightly differing morphology and were pollen sterile gave several patterns involving bp or indel polymorphisms that indicated that they were of hybrid derivation. Based on differences in leaf and phyllary shape and glandular trichome distribution it was possible to delimit the individual hybrid combinations from one another and from their progenitor species. Material previously called E. album from the western part of its range is of hybrid origin from E. album and E. lancifolium, and is described here as Eupatorium sullivaniae. Material from the northeastern part of the ra...

Bovine spongiform encephalopathy associated insertion/deletion polymorphisms of the prion protein gene in the four beef cattle breeds from North China

Two insertion/deletion (indel) polymorphisms of the prion protein gene (PRNP), a 23-bp indel in the putative promoter region and a 12-bp indel within intron I, are associated with the susceptibility to bovine spongiform encephalopathy (BSE) in cattle. In the present study, the polymorphism frequencies of the two indels in four main beef cattle breeds (Hereford, Simmental, Black Angus, and Mongolian) from North China were studied. The results showed that the frequencies of deletion genotypes and alleles of 23- and 12-bp indels were lower, whereas the frequencies of insertion genotypes and alleles of the two indels were higher in Mongolian cattle than in the other three cattle breeds. In Mongolian cattle, the 23-bp insertion / 12-bp insertion was the major haplotype, whereas in Hereford, Simmental, and Black Angus cattle, the 23-bp deletion / 12-bp deletion was the major haplotype. These results demonstrated that Mongolian cattle could be more resistant to BSE, compared with the other three cattle breeds, because of its relatively low frequencies of deletion genotypes and alleles of 23- and 12-bp indel polymorphisms. Thus, this race could be important for selective breeding to improve resistance against BSE in this area.

A 14 bp indel variation in the NCX1 gene modulates the age at onset in late-onset Alzheimer’s disease

Calcium homeostasis is critical to amyloid beta precursor protein (APP) processing. Na(+)/Ca(2+) exchanger (NCX) proteins play an important role in maintaining intracellular Na(+) and Ca(2+) homeostasis in the brain under physiological and pathological conditions. We sequenced a hyper-variable region in intron 2 of the Na(+)/Ca(2+) exchanger 1 gene (NCX1), and investigated whether insertion/deletion variations in this region are associated with the occurrence for Alzheimer's disease (AD). Examining 413 AD patients and 361 healthy controls, we identified 3 insertion/deletion polymorphisms. No significant differences of the allele and genotype frequencies were observed between the AD cases and the controls for any of the three polymorphisms. However, among the AD patients whose age at onset (AAO) was 65years or older (n=299), carriers of a 14bp insertion showed a lower average AAO (ins/ins and ins/del vs. del/del, 72.49±5.17 vs. 74.28±5.79, p=0.016). It suggested that this 14bp insertion/deletion polymorphism might modulate AAO in late-onset AD patients.

Role of Dopaminergic Gene Polymorphisms (DBH 19 bp Indel and DRD2Nco I) in Genetic Susceptibility to Migraine in North Indian Population: Table 1

The dopaminergic system plays a major role in migraine. We aimed to look for association of polymorphisms in dopaminergic genes in genetic susceptibility to migraine in North Indian population. In the present study, two polymorphisms, DBH 19 bp indel (rs no. 72393728) and DRD2 Nco I (rs no. 6275), were selected. Using case-control design, 301 migraine patients (202 migraine without aura and 99 migraine with aura) and 202 healthy controls were recruited in the study. Genotyping was done using polymerase chain reaction (PCR) (DBH 19 bp indel) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (DRD2 Nco I). Logistic regression analysis was used to find the contribution of these polymorphisms in migraine. Bonferroni's correction was applied for multiple comparisons. In DBH 19 bp indel polymorphism, a significant association was observed at allelic level (P = 0.027) especially in females (P = 0.016) on comparing migraine patients with healthy controls. In DRD2 Nco I polymorphism, no significant association was observed in migraine patients or any subgroups. Our study suggests a potentially significant role of DBH 19 bp indel polymorphism in migraine susceptibility.

Polymorphisms of myostatin gene (MSTN) in four goat breeds and their effects on Boer goat growth performance

Polymorphisms of myostatin (MSTN) gene were investigated as a candidate marker for goat growth in 687 individuals by gene sequencing and polymerase chain reaction restriction fragment length polymorphism methods. Three potential genotypes (AA, AB and BB) of 5 bp indel (1,256 TTTTA/-) in 5'UTR were detected in four breeds. The polymorphism (CC, CD and DD) of substitution (1,388 T/A) in exon 1 was only segregating in Boer. Genotype AB resulted in significant increases in body weights at birth (BW0), 90 (BW90) and 300 (BW300) days of age, and birth body length compared to BB (P < 0.05). Whilst genotype CD contributed to heavier BW0 and BW90, and larger birth body height (BH) compared to CC (P < 0.05). Individuals either with AB or CD genotype also had greater values in other body sizes, although no significant differences appeared (P > 0.05). When in combination, the combined genotype ABCD displayed positive impacts on better growth traits in BW0, BW90, BW300, BH and BCG (P < 0.05). These results indicate that these markers in myostatin (MSTN) are associated with Boer growth and may be useful for marker assisted selection.

Molecular characterization and identification of a novel polymorphism of 200 bp indel associated with age at first egg of the promoter region in chicken follicle-stimulating hormone receptor (FSHR) gene

Follicle-stimulating hormone receptor (FSHR) plays an important role in animal follicular development. Polymorphisms in FSHR promoter region likely impact transcription and follicle growth and maturation. In this study, a fragment of ~1.9 kb of cFSHR promoter for Zang, Xianju, Lohmann Brown, Jining Bairi and Wenchang breeds (line) was obtained. Totally 49 variations were revealed, of which 39 are single nucleotide substitutions, one is nucleotide substitution of (TTG) to (CAC) and nine are indels. Polymorphism at -874 site (a 200 bp indel mutation) was identified, and their effects on egg production traits as well as gene expression were analyzed. At this site, allele I(+) was dominant in Lohmann Brown and Xinyang Brown (a synthetic egg-laying line) lines, but very rare in three Chinese indigenous chicken breeds, namely Jining Bairi, Wenchang, Zang and one synthetic boiler line (Luqin). In Xinyang Brown population, the polymorphism was associated with age at first egg (AFE) (P < 0.05) and its effect on egg number at 37 weeks of age (E37) and egg number at 57 weeks of age (E57) was not significantly different (P > 0.05). The cFSHR mRNA level was not significantly different between three genotypes in small white and small yellow follicles of Xinyang Brown hens, however, allele I(+) tends to increase cFSHR transcription.

Association ofHLA-G3′ UTR 14-bp Insertion/Deletion Polymorphism with Hepatocellular Carcinoma Susceptibility in a Chinese Population

The 14-bp insertion/deletion (indel) polymorphism located in the 3' UTR of the human leukocyte antigen-G (HLA-G) gene plays a role in several autoimmune and chronic inflammatory diseases. HLA-G expression is associated with hepatocellular carcinoma (HCC) prognosis, especially in early stage, with high expression independently associated with shortened overall survival and increased tumor recurrence. In the present study, we carried out a case-control study in a Chinese population (318 cases and 599 controls) to estimate the susceptibility to HCC associated with the 14-bp indel polymorphism. Logistic regression analysis showed that the heterozygote and the homozygote 14-bp ins/ins confer a lower risk of HCC (adjusted OR = 0.75, 95% CI: 0.57-1.01, p = 0.061; OR = 0.54, 95% CI: 0.30-0.98, p = 0.031, respectively). Hepatitis B virus (HBV) stratification analysis showed that the associations were stronger in the HBV-positive population. Immunohistochemical analysis further showed that HLA-G expression in HCC tissues with 14-bp del/del genotype was more prominent than for heterozygous and 14-bp ins/ins genotype (p < 0.01). Taken together, our results suggested that the HLA-G 14-bp indel polymorphism may be a marker for genetic susceptibility to HCC in Chinese populations. Further studies from different populations with larger sample size are warranted to validate our findings.

CDNA Cloning and Characterization of Adipose Triglyceride Lipase Gene in Zebra Finch (Taeniopygia guttata) and Java Sparrow (Padda oryzivora)

Adipose triglyceride lipase (ATGL) is a crucial lipase that catalyzes triglyceride hydrolysis. In this study, reverse transcription (RT)-polymerase chain reaction, rapid amplification of cDNA ends, and mRNA real-time analysis were performed to characterize the ATGL gene in zebra finch and Java sparrow. Two cDNA of the zebra finch ATGL (zfATGL) gene were 1877 and 1626 bp were obtained; both encoded the same 481 amino acid protein. A 251 bp indel in the 3' UTR led to two variant transcripts. The zfATGL gene at chromosome 5 spanned over 28 Kb in length and comprised 9 exons. As in chicken, both the zfATGL gene and its intron 1 were much longer than mammals, which seemed to be one characteristic of ATGL gene in birds. The zfATGL gene was predominantly expressed in abdominal fat, subcutaneous fat, breast muscle, and leg muscle in both male and female. Two cDNA (1938 and 1885 bp) of the Java sparrow ATGL gene were also obtained, both of which encoded the same 481 amino acid protein. A 53 bp indel in 3' UTR led to two variant transcripts. The zfATGL gene was predominantly expressed in breast muscle, subcutaneous fat, abdominal fat, heart, and leg muscle. ATGL homology analysis showed that GASAG and GCGFLG motifs in the patatin region were conserved among 16 species. It was further indicated that the 251- and 53 bp indels at the 3' UTR of ATGL were potential binding sites for miRNA tgu-miR-2987. In conclusion, our study identified the full length cDNA and their variant transcripts of zfATGL and Java sparrow ATGL, and they were predominantly expressed in muscle, fat, and heart tissues.

Populationwide Investigation of Two Indel Polymorphisms at the Prion Protein Gene in Polish Holstein–Friesian Cattle

The allele, genotype, and haplotype frequencies among 837 Polish Holstein-Friesian cattle were determined at two regulatory indel polymorphisms of the PRNP gene. Allele frequencies at the 23 bp indel promoter polymorphism were 0.622 (del) and 0.378 (ins), with 0.613 and 0.387 in sires and 0.633 and 0.366 in dams. Allele frequencies at the 12 bp indel intron polymorphism were 0.527 (del) and 0.473 (ins), with 0.529 and 0.471 in sires and 0.543 and 0.456 in dams. Four haplotypes were identified in this population (23-12del, 23-12ins, 23del-12ins, and 23ins-12del). Haplotype 23-12del occurred most frequently in both sire and dam groups. Comparative analysis of Polish Holstein-Friesian and German Holstein populations revealed a similar genetic structure for the 23 bp indel polymorphism and a significantly different one for the 12 bp indel polymorphism. In allele and haplotype analysis, significant differences were observed between the Polish Holstein-Friesian population and a BSE-free German Holstein population.

Role of 14‐bp insertion/deletion polymorphism in HLA‐G among Indian women with recurrent spontaneous abortions

Human leukocyte antigen (HLA)-G is predominantly expressed on the extravillous cytotrophoblasts at the fetal-maternal interface. The 14-bp polymorphism in exon 8 is associated with HLA-G messenger ribonucleic acid (mRNA) stability and isoform alternative splicing patterns, thereby influencing the functionality of HLA-G in pregnancy. We analysed the 14-bp indel polymorphism in 143 recurrent spontaneous abortions (RSAs) and 150 control couples. We did not find any significant difference in the 14-bp insertion/deletion allele frequencies among the RSA and control couples. Analysis for increased sharing of the polymorphism in the RSA and the control couples also did not show any significant difference. However, we found an increase in the frequency of the 14-bp deletion homozygotes in the RSA women, which could lead to extremely high levels of soluble HLA-G (sHLA-G).