Association between platelet endothelial cellular adhesion molecule 1 (PECAM‐1/CD31) polymorphisms and acute myocardial infarction: a study in patients from Sicily

Adhesion of circulating cells to the arterial surface is among the first detectable events in atherogenesis. Cellular adhesion molecules, expressed by the vascular endothelium and by circulating leucocytes, mediate cell recruitment and their transendothelial migration. Platelet endothelial cellular adhesion molecule 1 (PECAM‐1/CD31), involved in this migration, has been associated with the developmental course of atherosclerosis. A few studies have investigated an association between coronary heart disease and single nucleotide polymorphisms (SNPs) located in functionally important domains of the PECAM‐1/CD31 gene. In particular, Ser563Asn and Gly670Arg SNPs have been described as susceptibility factors involved in acute myocardial infarction (AMI) in the Japanese male population. To confirm these observations, we studied 96 male patients (mean age 40 years; age range 20–46) affected by AMI and 118 healthy male controls (mean age 38 years, age range: 20–55), and analysed for the following PECAM‐1/CD31 SNPs: Val125Leu, Asn563Ser and Gly670Arg. The frequency of the Gly670Arg polymorphism was significantly higher in patients with AMI (58.9% vs. 48.3%; P = 0.019), whereas the frequencies of the other two SNPs (Leu125Val and Ser563Asn) were not significantly different between patients and controls. By comparing the observed number of 670Arg/Arg genotypes in the patients with the expected number, calculated from the allele frequency in a healthy population, a significance of P = 0.02 (odds ratio, 2.04; 95% CI: 1.1–3.7) was obtained, supporting a recessive model of inheritance. Hence, the differences between patients and controls are significant, but relatively small. However, as AMI is a multifactorial disease, any single mutation will only provide a small or modest contribution to the risk, which also depends on environmental interaction. All in all, we believe that the results of the present study would add support to the role of pro/anti‐inflammatory genotypes in determining susceptibility or resistance to immune‐inflammatory diseases, including atherosclerosis.     

Polymorphisms of the CYP1B1 gene may be associated with the onset of natural menopause in Chinese women

The Cytochrome P450 1B1 (CYP1B1) is one of the major CYP450 enzymes catalyzing 4-hydroxylation, an important elimination step for estrogens. Relatively little is known, however, about the impact of this gene on the onset and cessation of menstruation, which are significant milestones in a woman's life and predictors of many hormone related diseases. In this report, we described the association of four SNPs in the CYP1B1 gene, Arg48Gly, Ala119Ser, Leu432Val, and Asp449Asp, with the ages of menarche and menopause, years of menstruation and total number of menstrual cycles. Included in the study were 1958 community controls from two recently completed population-based case-control studies of breast cancer and endometrial cancer. No association was observed between the CYP1B1 polymorphisms and the age of menarche among either pre- or post-menopausal women. Among the women who experienced natural menopause, the three non-synonymous SNPs were significantly associated with menopausal age, years of menstruation, and total number of menstrual cycles. The Gly and Ser alleles of Arg48Gly and Ala119Ser were associated with later menopause, more years of menstruation and more menstrual cycles, while women with allele Val at Leu432Val had a 0.9 year earlier menopause, 1.0 year shorter reproductive span, and 12.6 fewer menstrual cycles than those women without this allele. In conclusion, the results from this study suggested that CYP1B1 genetic polymorphisms may be associated with the natural onset of menopause.     

DNA base excision repair gene polymorphisms modulate human cognitive performance and decline during normal life span

To test the hypothesis that single nucleotide polymorphisms (SNPs) in DNA repair genes are associated with cognitive performance during normal aging, the relationship between SNPs in selected exons in DNA base excision repair (BER) genes and cognitive performance was examined in 712 healthy Norwegian individuals aged 20-75 years. SNPs examined included PolB(Pro242Arg), hOGG1(Ser326Cys), MutYH (Met22Val), MutYH(His324Gln), APE1(Gln51His), APE1(Glu148Asp), XRCC1(Lys298Asn), XRCC1(Arg7Leu), NEIL1(Asp252Asn), and NEIL2(Arg257Leu). XRCC1(Arg7Leu) and PolB(Pro242Arg) were characterized by single nucleotide variations (≤0.1% homozygote SNPs). hOGG1(Ser326Cys) (Ser/Cys 40.8%/Cys/Cys 5.7%), MutYH(His324Gln) (His/Gln37%/Gln/Gln 6.0%) and APE1(Glu148Asp) (Glu/Asp 51.3%/Asp/Asp 23.0%) were characterized by higher SNP frequencies. MutYH(Met22Val), APE1(Gln51His) and NEIL2(Arg257Leu) occurred at intermediate SNP frequencies of 11.5, 7.6 and 5.3%, respectively. Interestingly, hOGG1(Ser326Cys) and APE1(Gln51His) had genotype by age interactions with general cognitive function, reasoning, control and speed of processing in cross-sectional analysis and a significant effect on longitudinal decline. Dispersed association effects involving MutYH(His324Gln), MutYH(Met22Val), PolB(Pro242Arg) and NEIL2(Arg257Leu) were also detected when APOE or CHRNA4, were included in the statistical model, a result consistent with proposed involvement of the latter markers in human cognitive decline and/or function. In summary, the results support the notion that polymorphisms in BER genes modulate cognitive performance in healthy elderly individuals.     

Functional analysis of four naturally occurring variants of human constitutive androstane receptor

The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.     

Vacuolating megalencephalic leukoencephalopathy with subcortical cysts: functional studies of novel variants in MLC1

Nine new unrelated patients presenting vacuolating myelinopathy with subcortical cysts were identified and analyzed for variations in the MLC1 gene. We detected 12 mutations (p.Leu37fs, p.Met80Val, p.Leu83Phe, p.Pro92Ser, p.Ser93Leu, p.Ile108fs, p.Gly130Arg, p.Cys171fs, p.Glu202Lys, p.Ser269Tyr, p.Ala275Asn, and p.Leu310_311insLeu) of which nine were novel. In one patient we did not detect mutations. Using a heterologous system, three new missense variants (p.Glu202Lys, p.Ser269Tyr, and p.Ala275Asn) and a single leucine insertion (p.Leu310insLeu)--lying in a stretch of seven leucines--were functionally assayed by determining total protein levels and mutant protein expression at the plasma membrane. No correlation was observed between mutation, clinical features, and plasma membrane expression of mutant protein.     

PARKIN-coding polymorphisms are not associated with Parkinson's disease in a population from northeastern Mexico

Early- and late-onset Parkinson's disease (EOPD and LOPD) have been associated with mutations in the PARKIN gene. Several studies have reported association of Parkinson's disease (PD) with different polymorphisms in different ethnic populations. To study the role of PARKIN polymorphisms as risk factors for PD in a genetically homogeneous northeastern Mexican population, four previously described coding polymorphisms (Ser167Asn, Val380Leu, Arg366Trp, and Asp394Asn) were analyzed by using the PCR-RFLP technique. This case–control study comprised 117 unrelated patients (mean age 59 ± 12 years, range 25–83 years) and 122 healthy unrelated control subjects (mean age 50 ± 15 years, range 25–85 years). The homozygous Trp366 and Asn394 genotypes were not present in our study. The Ser167Asn and Val380Leu polymorphisms were not associated with this disease. For the control group, Ser167Asn and Val380Leu were in Hardy–Weinberg disequilibrium. Given that the main causes of Hardy–Weinberg disequilibrium in controls are selection bias or genotyping error, a competing risk of death associated with the mutant gene could be an explanation of this disequilibrium and lack of association.     

Molecular and biochemical analysis of protective protein/cathepsin A mutations: correlation with clinical severity in galactosialidosis [published erratum appears in Hum Mol Genet 1997 Jan;6(1):146]

Mutations in the gene encoding lysosomal protective protein/cathepsin A (PPCA) are the cause of the lysosomal disorder galactosialidosis (GS). Depending on age of onset and severity of the symptoms, patients present with either an early infantile (EI), a late infantile (LI), or a juvenile/adult (J/A) form of the disease. To study genotype-phenotype correlation in this disorder, we have analyzed the mutations in the PPCA gene of eight clinically different patients. In two EI and one J/A patient, we have identified four novel point mutations (Val104Met, Leu208Pro, Gly411Ser and Ser23Tyr), that prevent phosphorylation and, hence, lysosomal localization and maturation of the mutant precursors. Two amino acid substitutions (Phe412Val and Tyr221Asn) are shared by five LI patients. These mutations appear to be pathognomonic for this phenotype, and determine the clinical outcome depending on whether they are present together or in combination with other mutations. The latter include a single base deletion and a novel amino acid change (Met378Thr), which generates an additional glycosylation site. Within the LI group, patients carrying the Phe412Val mutation are clinically more severe than those with the Tyr221Asn substitution. This is in agreement with the biochemical behavior of the Asn221-mutant protein, that is, like the Phe412Val protein, phosphorylated, routed to lysosomes and proteolytically processed, but its intralysosomal stability is intermediate between that of wild-type PPCA and Val412- PPCA. Overall, these results may explain the clinical heterogeneity observed in GS patients and may help to correlate mutant allelic combinations with specific clinical phenotypes.      

结核分枝杆菌抗原分析及免疫交叉反应研究

目的：筛选和鉴定结核分枝杆菌特异性和保护性抗原，研究结核杆菌的免疫反应特点，以探索结核病诊断和治疗的新途径。方法：采用超声破碎和滤膜抽滤的方法分别得到菌体蛋白和滤液蛋白，通过Western blot试验用结核杆菌的单克隆抗体及结核病人血清来检测蛋白样品，把发生阳性反应的蛋白在BECKMAN LF3200／多肽氨基酸序列测定仪上进行N末端序列分析，并用结核杆菌的单抗对自身抗原组蛋白进行了检测。结果：结核杆菌的31kD和30kD蛋白与结核杆菌的单抗及病人血清反应均呈阳性，但与正常小鼠血清和健康人血清反应呈阴性。31kD和30kD蛋白的N末端序列分别为：Ala Glu Val Asp Trp Leu Val Phe Ala Val和Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr。结核分枝杆菌的单抗与自身抗原组蛋白能发生免疫交叉反应。结论：结核杆菌的31kD和30kD蛋白是免疫保护性抗原，对免疫交叉反应分子基础的进一步研究必将增加对结核免疫机理的了解。     

Identification of novel mutations in the human ornithine transcarbamylase (OTC) gene of Korean patients with OTC deficiency and transient expression of the mutant proteins in vitro

The urea cycle plays key roles to prevent the accumulation of toxic nitrogenous compound and synthesize arginine de novo. Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea cycle, which is inherited in an X-linked manner. This study was undertaken to characterize molecular defects in Korean patients with OTC deficiency. With direct sequence analysis of OTC gene of 26 unrelated Korean patients with OTC deficiency, 23 different mutations were identified. Among these mutations, eleven were novel mutations. The novel mutations were p.Leu9X, p.Arg26Pro, p.Gly100Arg, p.Met205Thr, p.Lys221Asn, p.Asp249Gly, p.Phe281Ser, p.Val323Met, c.571delC, c.853delC, and c.796-805del. All the novel mutations in this study were tested in 100 normal alleles. In vitro expression study of some of novel missense mutations elucidated the correlation of genotype and phenotype of the OTC deficiency.     

Copper-dependent trafficking of Wilson disease mutant ATP7B proteins

We have previously developed a functional assay in yeast for the copper transporter, ATP7B, defective in Wilson disease (WND). Analysis of WND variant ATP7B proteins revealed that several were able to completely, or nearly completely, complement a mutant yeast strain in which the ATP7B ortholog CCC2 was disrupted, indicating that these ATP7B proteins retained copper transport activity. We analyzed the intracellular localization of these active WND ATP7B variant proteins using transient transfection of Chinese hamster ovary cells and triple-label immunofluorescence microscopy, as a second possible aspect of defective function. Two ATP7B variants, Asp765Asn and Leu776Val, which have normal copper transport activity in yeast, retained partial normal Golgi network localization, but were predominantly mislocalized throughout the cell. Asp765Asn and Leu776Val proteins were capable of only partial copper-dependent redistribution. WND variant protein Arg778Leu, which has defective function in yeast, was extensively mislocalized, presumably to the endoplasmic reticulum. ATP7B variant proteins Gly943Ser, which has nearly normal function in yeast, and CysProCys/Ser (mutation of the conserved CysProCys motif to SerProSer), inactive in yeast, were localized normally but were unable to redistribute in response to copper. Localization data from this study, combined with functional data from our yeast studies, provide a biochemical mechanism that can explain in part the variable biochemical features of WND, in particular the normal holo-ceruloplasmin levels observed in some patients. Our data have direct implications for WND diagnosis, indicating that decreased serum ceruloplasmin concentration is not likely to be observed with certain genetic variants of WND.     

Fifteen novel mutations in PKLR associated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

Pyruvate kinase (PK) deficiency is a rare disease but an important cause of hereditary nonspherocytic hemolytic anemia. The disease is caused by mutations in the PKLR gene and shows a marked variability in clinical expression. We report on the molecular characterization of 38 PK‐deficient patients from 35 unrelated families. Twenty‐nine different PKLR mutations were detected, of which 15 are reported here for the first time. Two novel deletions are reported: c.142_159del18 is the largest in‐frame deletion described thus far and predicts the loss of six consecutive amino acids (p.Thr48_Thr53del) in the N‐terminal domain of red blood cell PK. The other deletion removes nearly 1.5 kb of genomic DNA sequence (c.1618+37_2064del1477) and is one of a few large deletional mutants in PKLR. In addition, 13 novel point mutations were identified: one nonsense mutant, p.Arg488X, and 12 missense mutations, predicting the substitution of a single amino acid: p.Arg40Trp, p.Leu73Pro, p.Ile90Asn, p.Gly111Arg, p.Ala154Thr, p.Arg163Leu, p.Gly165Val, p.Leu272Val, p.Ile310Asn, p.Val320Leu, p.Gly358Glu, and p.Leu374Pro. We used the three‐dimensional (3D) structure of recombinant human tetrameric PK to evaluate the protein structural context of the affected residues. In addition, in selected patients red blood cell PK antigen levels were measured by enzyme‐linked immunosorbent assay (ELISA). Collectively, the results provided us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype‐to‐phenotype correlation in PK deficiency as well as the enzyme's structure and function. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Combined in vitro and in silico analyses of FGFR1 variants: genotype-phenotype study in idiopathic hypogonadotropic hypogonadism

Fibroblast growth factor receptor 1 (FGFR1) is an idiopathic hypogonadotropic hypogonadism (IHH)-associated gene, mutated in approximately 10% of the patients with this condition. Through targeted gene sequencing of 153 males with IHH and 100 healthy controls, we identified 10 mutations in FGFR1 from IHH patients with a frequency of 5.9% in the Chinese population of central China. These included nine missense mutations(NM_023110.2, p.Gly687Arg, p.Ala608Asp, p.Gly348Glu, p.Asn296Ser, p.Gly226Asp, p.Arg209Cys, p.Gly97Arg, p.Val71Met, p.Gly70Arg) and a splicing mutation c.1430 + 1G > T. in vitro and in silico analyses of FGFR1 variants were conducted to study the impact of the identified mutations. Our findings indicated that the splicing mutation dramatically affected premRNA processing, causing exon 10 and 6 nucleotides in the 3′ end of exon 9 to be completely skipped. Two variants (p.Gly687Arg and p.Ala608Asp) markedly impaired tyrosine kinase activity, while the other variants had limited impact on the mitogen-activated protein kinase (MAPK) signaling pathway. However, the functional impairment of the mutant receptors was not always consistent with the phenotypes, indicating that FGFR1 mutations might cause IHH in conjunction with other mutant genes. In this study, we expanded the knowledge on the mutation spectrum of FGFR1 in IHH patients and explored the genotype-phenotype relationship.     

Identification of P2X4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin

Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X(4) receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X(4)-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln(36), Leu(40), Val(43), Val(47), Trp(50), Asn(338), Gly(342), Leu(346), Ala(349), and Ile(356) mutants. The substitution-sensitive Arg(33) and Cys(353) mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met(31), Tyr(42), Gly(45), Val(49), Gly(340), Leu(343), Ala(344), Gly(347), Thr(350), Asp(354), and Val(357) map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.     

Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in Russian Charcot-Marie-Tooth neuropathy patients

Charcot-Marie-Tooth disease (CMT) and related inherited peripheral neuropathies, including Dejerine-Sottas syndrome, congenital hypomyelination, and hereditary neuropathy with liability to pressure palsies (HNPP), are caused by mutations in three myelin genes: PMP22, MPZ and Cx32 (GJB1). The most common mutations are the 1.5 Mb CMT1A tandem duplication on chromosome 17p11.2-p12 in CMT1 patients and the reciprocal 1.5 Mb deletion in HNPP patients. We performed a mutation screening in 174 unrelated CMT patients and three HNPP families of Russian origin. The unrelated CMT patients included 108 clinically and electrophysiologically diagnosed CMT1 cases, 32 CMT2 cases, and 34 cases with unspecified CMT. Fifty-nine CMT1A duplications were found, of which 58 belonged to the CMT1 patient group. We found twelve distinct mutations in Cx32, six mutations in MPZ, and two mutations in PMP22. Of these respectively, eight, five, and two lead to a CMT1 phenotype. Eight mutations (Cx32: Ile20Asn/Gly21Ser, Met34Lys, Leu90Val, and Phe193Leu; MPZ: Asp134Gly, Lys138Asn, and Thr139Asn; PMP22: ValSer25-26del) were not reported previously. Phenotype-genotype correlations were based on nerve conduction velocity studies and mutation type.     

Beta1-adrenergic receptor gene polymorphisms in Mexican patients with idiopathic dilated cardiomyopathy

The objective of the study was to evaluate the role of beta1-adrenergic receptor gene polymorphisms (Ser49Gly and Arg389Gly) as susceptibility markers for idiopathic dilated cardiomyopathy (IDC) in Mexican patients. The polymorphisms were analyzed in 47 patients with IDC and 93 ethnically matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The Ser49Gly allele and genotype frequencies were similar in patients and healthy controls. On the other hand, the analysis of the Arg389Gly polymorphism showed an increased frequencies of the *Gly allele (pC = 0.022, OR = 2.16) and *Arg/*Gly genotype (pC = 0.027, OR = 2.70) in the group of IDC patients when compared to healthy controls. The data suggest that Arg389Gly polymorphism could be involved in the genetic susceptibility to develop IDC in Mexicans.     

Functional,structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma‐prone families or patients

Germline mutations of the CDKN2A gene are found in melanoma‐prone families and individuals with multiple sporadic melanomas. The encoded protein, p16^INK4A, comprises four ankyrin‐type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16^INK4a with its physiological targets, the cyclin‐dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16^INK4A structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16^INK4A not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction. Hum Mutat 0, 1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application

Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.     

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.     

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern,North African,and South European countries

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population‐specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.