Multiple effects of SERCA2b mutations associated with Darier's disease

Darier's disease (DD) is an autosomal dominant disorder caused by mutations in the ATP2A2 gene, encoding sarco/endoplasmic reticulum Ca2+-ATPase pump type 2b isoform (SERCA2b). Although >100 mutations in the ATP2A2 gene were identified, no apparent relation between genotype/phenotype emerged. In this work, we analyzed 12 DD-associated mutations from all of the regions of SERCA2b to study the underlying pathologic mechanism of DD and to elucidate the role of dimerization in SERCA2b activity. Most mutations markedly affected protein expression, partially because of enhanced proteasome-mediated degradation. All of the mutants showed lower activity than the wild type pump. Notably, several mutants that cause relatively severe phenotype of DD inhibited the activity of the endogenous and the co-expressed wild type SERCA2b. Importantly, these effects were not attributed to changes in passive Ca2+ leak, inositol 1,4,5-trisphosphate receptor activity, or sensitivity to inositol 1,4,5-trisphosphate. Rather, co-immunoprecipitation experiments showed that SERCA2b monomers interact to influence the activity of each other. These findings reveal multiple molecular mechanisms to account for the plethora of pathologic states observed in DD and provide the first evidence for the importance of SERCA2b dimerization in pump function in vivo.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

Secretory pathway stress responses as possible mechanisms of disease involving Golgi Ca2+ pump dysfunction

In mammalian tissues, uptake of Ca(2+) and Mn(2+) by Golgi membranes is mediated by the secretory pathway Ca(2+) -ATPases, SPCA1 and SPCA2, encoded by the ATP2C1 and ATP2C2 genes. Loss of one copy of the ATP2C1 gene, which causes SPCA1 haploinsufficiency, leads to squamous cell tumors of keratinized epithelia in mice and to Hailey-Hailey disease, an acantholytic skin disease, in humans. Although the disease phenotypes resulting from SPCA1 haploinsufficiency in mice and humans are quite different, each species-specific phenotype is remarkably similar to those arising as a result of null mutations in one copy of the ATP2A2 gene, encoding SERCA2, the endoplasmic reticulum (ER) Ca(2+) pump. SERCA2 haploinsufficiency, like SPCA1 haploinsufficiency, causes squamous cell tumors in mice and Darier's disease, also an acantholytic skin disease, in humans. The phenotypic similarities between SPCA1 and SERCA2 haploinsufficiency in the two species, and the general functions of the two pumps in consecutive compartments of the secretory pathway, suggest that the underlying disease mechanisms are similar. In this review, we discuss evidence supporting the view that chronic Golgi stress and/or ER stress resulting from Ca(2+) pump haploinsufficiencies leads to activation of cellular stress responses in keratinocytes, with the predominance of proapoptotic pathways (although not necessarily apoptosis itself) leading to acantholytic skin disease in humans and the predominance of prosurvival pathways leading to tumors in mice.     

Cloning and characterization of the human GFRA2 locus and investigation of the gene in Hirschsprung disease

The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha (GFRalpha) are cell surface bound glycoproteins that mediate interactions of the GDNF ligand family with the RET receptor. These interactions are crucial to the development of the kidney and some peripheral nerve lineages. In humans, mutations of RET or RET ligands are associated with the congenital abnormality Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are absent. As the GFRalpha family are required for normal activation of the RET receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is required for the development of the myenteric nerve plexus, is an excellent candidate gene for HSCR. In this study, we cloned the human GFRA2 locus, characterized the gene structure, and compared it with other GFRA family members. We further investigated the GFRA2 gene for mutations in a panel of HSCR patients. GFRA2 has nine coding exons that are similar in size and organization to those of other GFRA family genes. We identified six sequence variants of GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2 protein. Two further changes that resulted in amino acid substitutions were found in exon 9 and were predicted to lie in the amino acid sequence encoding the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no difference in frequency of any of the sequence variants between control and HSCR populations. Our data indicate that members of the GFRA gene family are closely related in intron/exon structure and in sequence. We have not detected any correlation between sequence variants of GFRA2 and the HSCR phenotype.     

KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.     

A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene

Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. The axonal form of the disease is designated as "CMT type 2" (CMT2). Although four loci known to be implicated in autosomal dominant CMT2 have been mapped thus far (on 1p35-p36, 3q13. 1, 3q13-q22, and 7p14), no one causative gene is yet known. A large Russian family with CMT2 was found in the Mordovian Republic (Russia). Affected members had the typical CMT2 phenotype. Additionally, several patients suffered from hyperkeratosis, although the association, if any, between the two disorders is not clear. Linkage with the CMT loci already known (CMT1A, CMT1B, CMT2A, CMT2B, CMT2D, and a number of other CMT-related loci) was excluded. Genomewide screening pinpointed the disease locus in this family to chromosome 8p21, within a 16-cM interval between markers D8S136 and D8S1769. A maximum two-point LOD score of 5.93 was yielded by a microsatellite from the 5' region of the neurofilament-light gene (NF-L). Neurofilament proteins play an important role in axonal structure and are implicated in several neuronal disorders. Screening of affected family members for mutations in the NF-L gene and in the tightly linked neurofilament-medium gene (NF-M) revealed the only DNA alteration linked with the disease: a A998C transversion in the first exon of NF-L, which converts a conserved Gln333 amino acid to proline. This alteration was not found in 180 normal chromosomes. Twenty unrelated CMT2 patients, as well as 26 others with an undetermined form of CMT, also were screened for mutations in NF-L, but no additional mutations were found. It is suggested that Gln333Pro represents a rare disease-causing mutation, which results in the CMT2 phenotype.     

Effects of the mutant von Willebrand factor gene in von Willebrand disease

Von Willebrand disease (vWD) is a common inherited bleeding disorder in humans, and can be divided into a mild (type 1) and severe (type 3) form. Previous linkage studies identified one subject with vWD type 1 who transmitted different alleles of the von Willebrand factor (vWF) gene to his two affected children, one having vWD type 3 and the other having type 1. By screening the promoter and coding sequence (52 exons) of the vWF gene, three missense mutations were detected in this family. The type 1 individual who transmitted different alleles of the gene to his two sick children carries two substitutions, one in exon 5 and the other in exon 18 on the respective alleles. The relationship between the genotype (mutations) and the phenotype in this family is complex. In order further to correlate the relationship in vWD type 1 individuals, fifty-five subjects who carry one null allele of the vWF gene were collected. All these subjects are from vWD type 3 families with known mutations. Biochemical data of these 55 subjects indicate that gene dosage and other factors, such as blood group, age, and environment factors, play a critical role in the development of the phenotype of the disease.     

Mutational and haplotype analyses of families with familial partial lipodystrophy (Dunnigan variety) reveal recurrent missense mutations in the globular C-terminal domain of lamin A/C

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.     

Further characterization of ATP6V0A2-related autosomal recessive cutis laxa

Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H⁺-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients’ dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-β signalling and increased TGF-β1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.     

Germline mutations in the 3′ part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5′ end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3′ part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3′ part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype. Received: 18 June 1996 / Revised: 8 July 1996     

APC mutation in the alternatively spliced region of exon 9 associated with late onset familial adenomatous polyposis

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.     

Truncating and Missense Mutations in IGHMBP2 Cause Charcot-Marie Tooth Disease Type 2

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-μ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5′ region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.     

A novel splicing mutation in the ABCA1 gene,causing Tangier disease and familial HDL deficiency in a large family

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene.ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G?>?A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86?±?0.16?mmol/L and 92.2?±?10.9?mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G?>?A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency.Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.     

Detection of a novel missense mutation and second recurrent mutation in the CACNA1A gene in individuals with EA-2 and FHM

Mutations in the brain specific P/Q type Ca2+ channel alpha1 subunit gene, CACNA1A, have been identified in three clinically distinct disorders, viz. episodic ataxia type 2 (EA-2), familial hemiplegic migraine (FHM) and spinocerebellar ataxia 6 (SCA6). For individuals with EA-2, the mutations described thus far are presumed to result in a truncated protein product. Several different missense mutations have been identified in patients with FHM. At least two of these mutations have been identified on two different chromosome 19p13 haplotypes and thus represent recurrent mutations. In the present study, we have screened several individuals for mutations in all 47 exons in the CACNA1A gene by single-strand conformation analysis. We have characterised a novel missense mutation, G5260A, in exon 32 in a family segregating for EA-2. The consequence of this mutation is an amino acid substitution at a highly conserved position within the CACNA1A gene. This represents the first point mutation not resulting in a proposed truncated protein. Furthermore, this mutation has been detected in a family member with mild clinical signs including only migraine. Additionally, a second previously identified recurrent muta tion, C2272T, in exon 16 has been discovered in a patient with FHM.     

Altered apoptosis regulation in Kufor-Rakeb syndrome patients with mutations in the ATP13A2 gene

ATP13A2 gene encodes for a protein of the group 5 P-type ATPase family. ATP13A2 mutations are responsible for Kufor-Rakeb syndrome (KRS), a rare autosomal recessive juvenile parkinsonism characterized by the subacute onset of extrapyramidal, pyramidal and cognitive dysfunction with secondary nonresponsiveness to levodopa. FBXO7 protein is an F-box-containing protein. Recessive FBXO7 mutations are responsible for PARK15, a rare juvenile parkinsonism characterized by progressive neurodegeneration with extrapyramidal and pyramidal system involvement. Our aim was to evaluate apoptosis in cells from two KRS siblings carrying a homozygous ATP13A2 mutation and a heterozygous FBXO7 mutation. We also analysed apoptosis in the patients' healthy parents. Peripheral blood lymphocytes from the KRS patients and parents were exposed to 2-deoxy-D-ribose; apoptosis was analysed by flow cytometry and fluorescence microscopy. Apoptosis was much higher in lymphocytes from the KRS patients and parents than in controls, both in standard conditions and after induction with a pro-apoptotic stimulus. The lack of correlation between increased apoptosis and the presence of the mutated FBXO7 gene rules out the involvement of FBXO7 in apoptosis regulation. The altered apoptotic pattern of subjects with mutated ATP13A2 suggests a correlation between apoptosis alteration and the mutated ATP13A2 protein. We hypothesize that ATP13A2 mutations may compromise protein function, disrupting cell cation balance and rendering cells prone to apoptosis. However, the deregulation of apoptosis in KRS patients displaying different disease severity suggested that the altered apoptotic pathway probably does not have a pathogenetic role in KRS by itself.     

Analysis of the coding regions of <Emphasis Type="Italic">VEGFR3</Emphasis> and <Emphasis Type="Italic">VEGFC</Emphasis> in Milroy disease and other primary lymphoedemas

Milroy disease (hereditary lymphoedema type I, MIM 153100) is a congenital onset primary lymphoedema with autosomal dominant inheritance. Mutations in the gene, vascular endothelial growth factor receptor 3, VEGFR3 (FLT4), are known to cause Milroy disease, but there is uncertainty about the prevalence of VEGFR3 mutations in patients with primary lymphoedema and more specifically in those with a phenotype that resembles Milroy disease. This study aims to address this issue and thereby delineate the Milroy disease phenotype. Fifty-two patients with primary lymphoedema were analysed for mutations in the coding regions of VEGFR3. Patients were divided into four groups: Typical Milroy disease with family history (group I), typical Milroy disease with no family history (group II), atypical Milroy disease (group III), and complex primary lymphoedema (group IV). Results demonstrated that with rigorous phenotyping the likelihood of detecting VEGFR3 mutations is optimised. Mutation prevalence is 75% in typical Milroy patients with a family history (group I) and 68% if positive family history is not a diagnostic criterion. A positive family history is not essential in Milroy disease. The likelihood of detecting VEGFR3 mutations in patients who have a phenotype which is not typical of Milroy disease is very small (<5%). For the 22 mutation positive patients, 14 novel VEGFR3 mutations were identified, two of which were in exon 22 and one in exon 17, confirming that these exons should be included in VEGFR3 analysis. No mutations were found outside the kinase domains, showing that analysis of this part of the gene is not useful for Milroy disease patients. VEGFC, which encodes the ligand for VEGFR3, was sequenced in all patients with typical Milroy disease (groups I and II) and no mutations were identified. F. C. Connell and P. Ostergaard contributed equally to this work. An erratum to this article can be found at     

Molecular characterization of the human delta-aminolevulinate dehydratase 2 (ALAD2) allele: implications for molecular screening of individuals for genetic susceptibility to lead poisoning.

Reports of families with members affected with both von Willebrand disease (vWD) and hereditary hemorrhagic telangiectasia (HHT) suggest a possible relationship between these two disorders. vWD, the most common inherited bleeding disorder in humans, is due to either a quantitative or qualitative defect in von Willebrand factor (vWF). The gene for vWF has been cloned and mapped to chromosome 12 (12p12----12pter). HHT, an uncommon inherited bleeding disorder, is characterized by malformed, dilated, fragile blood vessels. The chromosomal location of the gene for HHT is unknown. We studied two families by RFLP analysis to determine whether there is a molecular basis for the association of vWD and HHT. Family A is affected with both type IIA vWD and HHT; family B is affected with HHT alone. Linkage of HHT to the vWF gene was not detected, and vWF was ruled out as a candidate gene for HHT. The vWF gene was found to be tightly linked to type IIA vWD in family A (lod score 3.61 at recombination fraction .00). By PCR and DNA sequence analysis of vWF exon 28, a single T----C transition resulting in the substitution of Thr for Ile865 was identified. This substitution is located immediately adjacent to two previously identified type IIA vWD mutations.     

Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.