A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.     

Whole-Exome Sequencing Efficiently Detects Rare Mutations in Autosomal Recessive Nonsyndromic Hearing Loss

Identification of the pathogenic mutations underlying autosomal recessive nonsyndromic hearing loss (ARNSHL) is difficult, since causative mutations in 39 different genes have so far been reported. After excluding mutations in the most common ARNSHL gene, GJB2, via Sanger sequencing, we performed whole-exome sequencing (WES) in 30 individuals from 20 unrelated multiplex consanguineous families with ARNSHL. Agilent SureSelect Human All Exon 50 Mb kits and an Illumina Hiseq2000 instrument were used. An average of 93%, 84% and 73% of bases were covered to 1X, 10X and 20X within the ARNSHL-related coding RefSeq exons, respectively. Uncovered regions with WES included those that are not targeted by the exome capture kit and regions with high GC content. Twelve homozygous mutations in known deafness genes, of which eight are novel, were identified in 12 families: MYO15A-p.Q1425X, -p.S1481P, -p.A1551D; LOXHD1-p.R1494X, -p.E955X; GIPC3-p.H170N; ILDR1-p.Q274X; MYO7A-p.G2163S; TECTA-p.Y1737C; TMC1-p.S530X; TMPRSS3-p.F13Lfs*10; TRIOBP-p.R785Sfs*50. Each mutation was within a homozygous run documented via WES. Sanger sequencing confirmed co-segregation of the mutation with deafness in each family. Four rare heterozygous variants, predicted to be pathogenic, in known deafness genes were detected in 12 families where homozygous causative variants were already identified. Six heterozygous variants that had similar characteristics to those abovementioned variants were present in 15 ethnically-matched individuals with normal hearing. Our results show that rare causative mutations in known ARNSHL genes can be reliably identified via WES. The excess of heterozygous variants should be considered during search for causative mutations in ARNSHL genes, especially in small-sized families.     

Physiological roles of renal anion transporters NaS1 and Sat1

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.     

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).     

Mutations in myosin light chain kinase cause familial aortic dissections

Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.     

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

Alpha-melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected HEK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent.     

Molecular and cytogenetic analyses of autism in Taiwan

Karyotypic and DNA analyses were both performed on 104 autistic children referred from Taichung Autism Education Academy and Tainan Autism Association in Taiwan. The frequency of fragile sites of the autistic patients did not differ significantly from that of the normal individuals. Of the 12 autistic children with chromosomal abnormalities, 8 had the fragile X, 2 had Down syndrome, and the remaining had other aneuploid constitutions. The results of this study illustrate the contribution of chromosomal abnormalities or variants to the pathogenesis of infantile autism.     

Increased neutralization sensitivity of recently emerged CXCR4-using human immunodeficiency virus type 1 strains compared to coexisting CCR5-using variants from the same patient

CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) variants evolve from CCR5-using (R5) variants relatively late in the natural course of infection in 50% of HIV-1 subtype B-infected individuals and subsequently coexist with R5 HIV-1 variants. This relatively late appearance of X4 HIV-1 variants is poorly understood. Here we tested the neutralization sensitivity for soluble CD4 (sCD4) and the broadly neutralizing antibodies IgG1b12, 2F5, 4E10, and 2G12 of multiple coexisting clonal R5 and (R5)X4 (combined term for monotropic X4 and dualtropic R5X4 viruses) HIV-1 variants that were obtained at two time points after the first appearance of X4 variants in five participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS. Recently emerged (R5)X4 viruses were significantly more sensitive to neutralization by the CD4-binding-site-directed agents sCD4 and IgG1b12 than their coexisting R5 viruses. This difference was less pronounced at the later time point. Early (R5)X4 variants from two out of four patients were also highly sensitive to neutralization by autologous serum (50% inhibition at serum dilutions of >200). Late (R5)X4 viruses from these two patients were neutralized at lower serum dilutions, which suggested escape of X4 variants from humoral immunity. Autologous neutralization of coexisting R5 and (R5)X4 variants was not observed in the other patients. In conclusion, the increased neutralization sensitivity of HIV-1 variants during the transition from CCR5 usage to CXCR4 usage may imply an inhibitory role for humoral immunity in HIV-1 phenotype evolution in some patients, thus potentially contributing to the late emergence of X4 variants.     

Angiotensinogen T174M and M235T Variants and Hypertension in the Hani and Yi Minority Groups of China

A case–control study of 538 individuals investigated whether the angiotensinogen gene (AGT) might be implicated in the pathogenesis of essential hypertension in the Hani and Yi populations of China. Genotypes for two diallelic DNA polymorphisms observed at amino acid residues 174 (T174M) and 235 (M235T) within the coding sequence were determined. M235T and T174M genotyping with PCR-RFLP was performed in 267 normotensive subjects and 271 hypertensive subjects. No significant difference was found between normotensives and hypertensives in genotype distribution and allele frequency for either M235T or T174M in the Hani or the Yi populations (P > 0.05). Relative to carriers of the 235T/235T and 174T/174T combination, the others had a significantly elevated risk of hypertension (OR = 1.62, 95% CI 1.02–2.59; P = 0.043) in the Hani population. The AGT M235T and T174M variants in combination may play a role in the genetic predisposition to develop essential hypertension in the Hani minority of China.     

A392V and R945X mutations cause orofacial clefts via impairing PTCH1 function

The Hedgehog (HH) signaling plays key roles in embryogenesis and organogenesis, and its dysfunction causes a variety of human birth defects. Orofacial cleft (OFC) is one of the most common congenital craniofacial defects, and its etiology is closely related to mutations in multiple components in the HH pathway, including the PTCH1 receptor. A quantity of PTCH1 variants have been associated with OFC, but the pathogenicity and underlying mechanism of these variants have not been functionally validated. In our previous studies, we identified two PTCH1 variants (A392V and R945X) in two families with hereditary OFC. Here we explore the functional consequences of these two variants. In zebrafish embryos, microinjection of wild type PTCH1 mRNA causes curved body axis and craniofacial anomalies. In contrast, microinjection of A392V and R945X PTCH1 mRNAs results in much milder phenotypes, suggesting these two variants are loss-of-function mutations. In mammalian cells, A392V and R945X mutations reverse the inhibitory effect of PTCH1 on HH signaling. Biochemically, the two mutants PTCH1 show lower expression levels and shortened half-life, indicting these mutations decrease the stability of PTCH1. A392V and R945X mutations also appear to cause PTCH1 to localize away from vesicles. Taken together, our findings indicate that A392V and R945X variants are loss-of-function mutations that disrupt the function of PTCH1 and thus cause dysregulation of HH signaling, leading to the pathogenesis of OFC.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Cluster Analysis of Risk Factor Genetic Polymorphisms in Alzheimer’s Disease

Multiple genetic variants may contribute to the risk of developing Alzheimer’s disease. We have analyzed polymorphisms in 9 genes to determine whether particular combinations would contribute to this risk. The genes were APOE, LDLr, CST3, CTSD, TNF, BACE1, MAPT, STH, eNOS, and TFCP2. Three risk groups for the disease were identified. Risk group I was younger, was heterozygous for the CST3 (GA), CTSD2936 (AG), TNF -308 (AG) genetic variants. Risk group II was older, was homozygous for the −427 APOE promoter polymorphism (TT), and heterozygous for the MAPT deletion and for the STH variant (QR). Group III had both the youngest and oldest subjects, were heterozygous for the −863 (AC) and −1031 (CT) TNF promoter polymorphisms. All three groups carried the APOE 4 allele and were heterozygous for both BACE1 polymorphisms. The control groups were carriers of the APOE 3 allele and were homozygous for the BACE1 genetic variants. C. N. Randall, S. N. Morris, A. D. Winkie and G. R. Parker—STAR students. C. N. Randall, D. Strasburger, J. Prozonic, S. N. Morris, A. D. Winkie, G. R. Parker, D. Cheng and E. M. Fennell contributed equally to this study. Special issue article in honor of Dr. George DeVries.     

Molecular analysis of 23 exons of the CFTR gene in Brazilian patients leads to the finding of rare cystic fibrosis mutations

To define mutations present in 23 exons and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 95 patients from Rio de Janeiro, Brazil, we carried out single-strand conformation polymorphism analysis and automated direct sequencing. Mutation detection was achieved in 45% of the alleles presented, and complete genotyping (two mutated alleles) was accomplished in 34.7% of the patients. Twenty patients (21.1%) were found to carry only one mutation, whereas mutated alleles could not be observed in 42 patients (44.2%). Eleven mutations were found, of which four were characterized as rare mutations: P205S (1.05%), Y1092X (0.53%), S549R (0.53%), and S4X (0.53%). The DF508 mutation in this population sample showed a frequency of 28.42%. The low number of individuals (10 of 95; 10.5%) with compound heterozygous (DF508/non-DF508) genotypes could indicate the presence of another severe mutation leading to the premature death of these individuals. In 4 of the aforementioned 10 individuals with compound heterozygous genotypes, the D-7-2-1-2 (XV2c-KM19-IVS6a-TUB9-M470-T854) haplotype was defined.     

Cordyceps sinensis protects against renal ischemia/reperfusion injury in rats

Cordyceps sinensis (CS) is an entomogenous fungus used as a tonic food and Chinese medicine to replenish health. This study investigated the protective effects of CS in rats post-renal ischemia–reperfusion (I/R) sequence by analyzing the influence on stromal cell-derived factor-1α (SDF-1α and chemokine (C-X-C motif) receptor 4 (CXCR4) expressions and senescence during recovery. Chemokine SDF-1 [now called chemokine C-X-C motif ligand 12 (CXCL12)] and its receptor CXCR4 are crucial in kidney repair after ischemic acute renal failure. CS treatment significantly alleviated I/R-induced renal damage assessed by creatinine levels (p < 0.05) and abated renal tubular damages assessed by periodic acid-Schiff with diastase (PASD) staining. CS induced early SDF-1α expression and increased CXCR4 expression 1–6 h post-reperfusion. Histology studies have revealed that CS induced SDF-1α in squamous cells of Bowman’s capsule, mesangial cells, distal convoluted tubules (DCT), and proximal convoluted tubules (PCT). CS also improved renal repair in I/R-induced injury by increasing Ki-67 staining. I/R induced renal senescence after 3 and 6 h of reperfusion. However, CS alleviated I/R-induced senescence at early stage (1 and 3 h). We conclude that CS protects against I/R injury via the SDF-1/CXCR4-signaling axis and alleviates senescence.     

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking,and Persistent Stuttering

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.     

Homozygosity at variant MLH1 can lead to secondary mutation in NF1, neurofibromatosis type I and early onset leukemia

Heterozygous germ-line variants of DNA mismatch repair (MMR) genes predispose individuals to hereditary non-polyposis colorectal cancer. Several independent reports have shown that individuals constitutionally homozygous for MMR allelic variants develop early onset hematological malignancies often associated to features of neurofibromatosis type 1 (NF1) syndrome. The genetic mechanism of NF1 associated to MMR gene deficiency is not fully known. We report here that a child with this form of NF1 displays a heterozygous NF1 gene mutation (c.3721C>T), in addition to a homozygous MLH1 gene mutation (c.676C>T) leading to a truncated MLH1 protein (p.R226X). The parents did not display NF1 features nor the NF1 mutation. This new NF1 gene mutation is recurrent and predicts a truncated neurofibromin (p.R1241X) lacking its GTPase activating function, as well as all C-terminally located functional domains. Our findings suggest that NF1 disease observed in individuals homozygous for deleterious MMR variants may be due to a concomitant NF1 gene mutation. The presence of both homozygous MLH1 and heterozygous NF1 mutation in the child studied here also provides a mechanistic explanation for early onset malignancies that are observed in affected individuals. It also provides a model for cooperation between genetic alterations in human carcinogenesis.     

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.     

A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.     

Validation and fine mapping of lyc12.1, a QTL for increased tomato fruit lycopene content

Lycopene content is a key component of tomato (Solanum lycopersicum L.) fruit quality, and is a focus of many tomato-breeding programs. Two QTLs for increased fruit lycopene content, inherited from a high-lycopene S. pimpinellifolium accession, were previously detected on tomato chromosomes 7 and 12 using a S. lycopersicum × S. pimpinellifolium RIL population, and were identified as potential targets for marker-assisted selection and positional cloning. To validate the phenotypic effect of these two QTLs, a BC₂ population was developed from a cross between a select RIL and the S. lycopersicum recurrent parent. The BC₂ population was field-grown and evaluated for fruit lycopene content using HPLC. Statistical analyses revealed that while lyc7.1 did not significantly increase lycopene content in the heterozygous condition, individuals harboring lyc12.1 in the heterozygous condition contained 70.3 % higher lycopene than the recurrent parent. To eliminate the potential pleiotropic effect of fruit size and minimize the physical size of the lyc12.1 introgression, a marker-assisted backcross program was undertaken and produced a BC₃S₁ NIL population (n = 1,500) segregating for lyc12.1. Lycopene contents from lyc12.1 homozygous and heterozygous recombinants in this population were measured and lyc12.1 was localized to a 1.5 cM region. Furthermore, we determined that lyc12.1 was delimited to a ~1.5 Mb sequence of tomato chromosome 12, and provided some insight into potential candidate genes in the region. The derived sub-NILs will be useful for transferring of lyc12.1 to other tomato genetic backgrounds and for further fine-mapping and cloning of the QTL.