An investigation of polymorphisms in the 4q1 3.3-21.1 CXC chemokine gene cluster for association with multiple sclerosis in Australians.

Susceptibility to multiple sclerosis (MS) is believed to result from the complex interaction of a number of genes, each with modest effect. Vital to the migration of cells to sites of inflammation, including the central nervous system, are chemokines, many of which are implicated in MS pathogenesis. Most of the CXC chemokine genes are encoded in a cluster on chromosome 4q13.3-21.1, which has been identified in several genome-wide screens as being potentially associated with MS. We conducted a two-stage analysis to investigate the chemokine gene cluster for association with MS. Initially, we sequenced the chemokine genes in several DNA pools to identify common polymorphisms, and then genotyped selected SNPs in 373 Australian MS trio families. We found no evidence that the CXC chemokine gene cluster is genetically associated with MS. However, the existence of common variants conferring small risk factors or rare variants with significant risk cannot be excluded.

A genome-wide screen for linkage disequilibrium in Australian HLA-DRB1*1501 positive multiple sclerosis patients.

The association of multiple sclerosis with alleles/haplotypes from the HLA region on chromosome 6p21 is well established although the remainder of the genome remains relatively unexplored. We have completed a genome-wide screen for linkage disequilibrium in a cohort of Australian multiple sclerosis patients positive for HLA-DRB1*1501. A total of 4346 microsatellite markers provided through the "Genetic Analysis of Multiple sclerosis in EuropeanS" (GAMES) collaborative were analysed in DNA separately pooled from cases (n=217) and controls (n=187). Associations were found in four genomic regions (12q15, 16p13, 18p11 and 19q13) previously identified in linkage genome screens. Three additional regions of novel association were also identified (11q12, 11q23 and 14q21). Further analysis of these regions is required to establish whether the associations observed are due to epistatic interaction with the HLA locus.

A genome screen for linkage in Australian sibling-pairs with multiple sclerosis.

The role of genetic factors in determining susceptibility to multiple sclerosis is well established but, despite the global distribution of the disease, systematic efforts to locate susceptibility genes have concentrated exclusively on populations from the Northern Hemisphere. We performed a genome wide screen of linkage in the Australian population using a panel of 397 microsatellite markers in 54 affected sibling-pairs. Multipoint linkage analysis revealed four regions of suggestive linkage (on chromosomes 2p13, 4q26-28, 6q26 and Xp11) and 18 additional regions of potential linkage (at 1q43-44, 3q13-24, 4q24, 4q31-34, 5q11-13, 6q27, 7q33-35, 8p23-21, 9q21, 13q31-32, 16p13, 16p11, 16q23-24, 17p13, 18p11, 20p12-11, Xp21-11 and Xq23-28). Our results contribute to the available data adding new provisional regions of linkage as well as increasing support for areas previously implicated in genetic susceptibility to multiple sclerosis.

Paraoxonase gene cluster is a genetic marker for early microvascular complications in type 1 diabetes.

BACKGROUND: Paraoxonase is a serum enzyme, which prevents oxidation of low-density lipoprotein (LDL) by hydrolyzing lipid peroxides. Two polymorphisms in PON1 gene have been associated with cardiovascular and microvascular diseases in both diabetic and non-diabetic patients. AIMS: The current project was designed to investigate the association between the polymorphisms of two PON genes and diabetes microvascular diseases (retinopathy and microalbuminuria) and any potential linkage between Met54Leu of PON1 and Cys311Ser of PON2 gene. METHODS: Diabetic retinopathy and albumin excretion rate were assessed in 372 adolescents with Type 1 diabetes who were genotyped for the two polymorphisms. RESULTS: We confirmed the increased susceptibility for diabetic retinopathy for the Leu/Leu genotype (odds ratio (OR) 3.34 (confidence interval (CI) 1.95, 5.75), P < 0.0001). The Ser/Ser genotype was significantly more common in those patients with microalbuminuria (albumin excretion rate > or = 20 microg/min) compared with those with albumin excretion rate < 20 microg/min (OR 4.72 (CI 2.65, 8.41), P < 0.0001). The Ser311 of PON2 was in strong linkage disequilibrium with Leu54 of PON1 gene (Delta = 23 x 10(4), P < 0.001). The delta value was higher for those without complications (28 x 104, P < 0.001) compared with those with complications (15.5 x 10(4), P < 0.001). CONCLUSIONS: This study supports the hypothesis that diabetic microangiopathy is genetically heterogeneous. PON1 Leu/Leu increases the risk for retinopathy and PON2 Ser/Ser increases the risk for microalbuminuria.

CCR5 genotyping in an Australian and New Zealand type 1 diabetes cohort.

Infiltration of pancreatic tissue by autoreactive T-cells involves secretion of multiple cytokines and chemokine receptor expression. Genetically determined variation in cell surface expression of the chemokine receptor CCR5 may result in differences in inflammatory cell migration in response to relevant chemokines. Adolescents with type 1 diabetes (T1D) from Australia and New Zealand were genotyped for CCR5-delta32 (n = 626). The allele frequency was compared with that of 253 non-diabetic Australian adolescents and with that of 92 adults with systemic lupus erythematosus. A reduced allele frequency was seen in T1D compared with controls (0.092 vs. 0.123, p = 0.05). This difference was not seen for the cohort of patients with SLE (freq = 0.114). A reduction in the number of CCR5-delta32/delta32 homozygotes, who lack CCR5, in the T1D cohort was also seen and while not statistically significant (2 observed compared to 5.25 expected; p = 0.12) is interesting. These results suggest a partial protection from T1D for CCR5-delta32 homozygous individuals is possible and that CCR5 has a potential role in the pathogenesis of T1D.

T cell receptor beta chain genotyping in Australian relapsing-remitting multiple sclerosis patients.

This study focused on susceptibility to MS within the beta-chain of the T-cell antigen receptor (TCRB locus, 7q35) in a cohort of 122 RR-MS patients compared with 96 normal individuals using biallelic polymorphisms across the bv8s1(Vbeta8.1) to bv11s1 (Vbeta11) TCRB subregion. The markers bv6s5, bv8s1, bv10s1, bv15s1 and bv3s1 were studied for allele and genotype frequencies; haplotypes were assigned with combinations of two of these markers and stratification for HLA-DR15 was also performed. Linkage disequilibrium was found between alleles of the bv8s1, bv10s1/bv15s1 and bv3s1 loci in both patients and controls. An increase among RR-MS patients in the allele frequency of bv8s1*2 (P=0.03) and the haplotype bv8s1*2/bv3s1*1 (P=0.006) was noted and both were found to be statistically significant. In the DR15-positive group, the association between TCRB and MS was seen with the bv8s1*2 allele (Puc=0.05) and the bv8s1*2/bv10s1 haplotypes (Puc=0.048), while the haplotype associations seen among DR15-negative RR-MS patients included the bv3s1*1 allele (bv10s1*1/ bv3s1*1, Puc=0.022; bv8s1*2/bv3s1*1, Puc=0.048). These results support the involvement of the TCRB region in MS susceptibility and encourage further study of the variable gene segments in this region.

Evaluation of the apo-1/Fas promoter mva I polymorphism in multiple sclerosis.

The pathogenesis of multiple sclerosis is under strong genetic control involving several or more genes each of modest effect. Whilst the mechanisms underlying the pathogenesis of MS remain unknown, it has been hypothesised that either decreased apoptosis of autoreactive T cells in the CNS, or increased apoptosis of oligodendrocytes may play an important role. The Apo-1/Fas antigen (CD95), the gene for which is located in a chromosomal region showing linkage in MS genome screens, is a critical inducer of apoptosis and studies have shown aberrant expression of this molecule in MS, correlating with a decrease in T cell apoptosis or increase in CNS tissue damage. This study investigated an Mva I polymorphism in the Apo-1/Fas promoter region in a group of 124 Australian patients with relapsing-remitting MS and in 183 normal controls. Whilst there were increases in the Mva I*2 allele in MS individuals overall (59% vs 52%, P not corrected=0.08), and in HLA-DRB1*1501 negative MS patients (62% vs 55%), these were not significantly different from controls. Interactions were investigated between the Mva I alleles and T cell receptor beta chain variable region (TCRBV) germline polymorphisms, with a trend in MS individuals towards a decrease of the Mva I*1 allele when combined with the TCRBV3S1*2 allele (Relative Risk=0.25, P=0.067), and with the TCRBV8S1*1 allele (Relative Risk=0.44, P=0.12). Overall, the findings of this study indicate a possible effect of the Apo-1/Fas promoter Mva I polymorphism in MS susceptibility, which needs to be confirmed in further studies. Multiple Sclerosis (2000) 6 14 - 18

Lack of restriction of T cell receptor beta variable gene usage in cerebrospinal fluid lymphocytes in acute optic neuritis.

OBJECTIVES: There have been many studies reporting restricted patterns of T cell receptor usage in established multiple sclerosis and these have led to clinical trials of immunomodulation directed at deleting clonal T cell populations. The present study aims to test the hypothesis that highly restricted T cell populations are also present in the CSF in the earliest clinical stages of acute demyelinating disease of the CNS. METHODS: T cell receptor Vbeta (TCRBV) gene expression was studied in CSF and blood in nine patients with acute optic neuritis within 7 days of onset of symptoms, six patients with an acute relapse of multiple sclerosis, and 13 control subjects. RNA was extracted and cDNA synthesised from unstimulated CSF and blood lymphocytes, and TCRBV gene segments were amplified from the cDNA by polymerase chain reaction (PCR) using 21 family specific primers. PCR products were separated by polyacrylamide gel electrophoresis and detected via a labelled oligonucleotide probe. A semiquantitative analysis of band intensity was performed by laser densitometry. RESULTS: TCRBV mRNA was detected in the CSF of eight of nine patients with optic neuritis, six of six patients with multiple sclerosis, and five of 13 controls, and was closely correlated with the presence of oligoclonal IgG. Usage of a single TCRBV family was demonstrated in two of nine patients with optic neuritis and two of six patients with multiple sclerosis. The number of TCRBV families expressed in the other patients ranged between 5 and 15 (optic neuritis) and 4 and 17 (multiple sclerosis). CONCLUSIONS: There is a relative lack of restriction of TCRBV usage by CSF lymphocytes in the very earliest stages (<7 days) of acute optic neuritis. This may imply either that multiple sclerosis is not a monoclonal disease even at onset, or that the autoimmune response has widened before the disease becomes clinically evident. This may have important consequences for the design of immune therapies in multiple sclerosis. Further studies are required to determine whether the CSF T cell repertoire at presentation has prognostic importance. Longitudinal studies are required to follow the CSF T cell repertoire from the time of presentation and to determine whether it may have prognostic significance.

HLA-DMB gene and HLA-DRA promoter region polymorphisms in Australian multiple sclerosis patients.

The MHC region has been shown to contain a susceptibility locus for multiple sclerosis (MS). While the strongest association to date has been between HLA-DRB1*1501 and MS, the exact nature of the MHC association in MS remains unclear. Two candidate polymorphic loci within the MHC class II region, the HLA-DMB gene and the HLA-DRA promoter, which lie close to HLA-DRB1, were therefore examined in an Australian MS population. The HLA-DMB*0103 phenotype was increased in the MS patients (46% vs. 30%) and the frequency of the HLA-DRA promoter A allele was also increased (81% vs. 68%). When the subjects were stratified into HLA-DRB*1501 positive and negative individuals these associations were not significantly different. This is a result of the strong linkage disequilibrium between HLA-DRB*1501 and both HLA-DMB*0103 and the HLA-DRA promoter A allele. The complete linkage between DRB1*1501 and the HLA-DRA promoter A allele indicates that the MS susceptibility haplotype (DRB1*1501-HLA-DQB1*0602-HLA-DQA1* 0102) can be extended out to promoter of the HLA-DRA locus. Interactions between both HLA-DMB and the HLA-DRA promoter and other reported MS susceptibility loci were examined (TCRBV polymorphisms, HLA-DQA1 and HLA-DQB1). Some interactions between specific TCRBV polymorphisms and the HLA-DRA promoter were observed, which is consistent with other published reports suggesting an epistatic interaction between TCRBV and HLA-DRB1.

The DRB1 Val86/Val86 genotype associates with multiple sclerosis in Australian patients.

Genetic susceptibility to multiple sclerosis (MS) has so far been strongly localized to the MHC class II region encoding the alleles of the haplotype HLA-DRB1*1501, -DQA1*0102, -DQB1*0602. However, this haplotype is not carried by approximately 40% of MS patients; a potential explanation could be that they carry other MHC class II alleles with similar function due to the sharing of nucleotide sequences encoding critical amino acid residues. The DRB1 gene is polymorphic at residue 86, encoding valine or glycine. In view of the increasing evidence for a functional role for DRB1 aa86 in the binding and presentation of autoantigenic peptides such as myelin basic protein, this study investigated associations with the residue 86 polymorphism in an Australian MS population. A significant increase in the Val86/Val86 genotype was observed in the MS patients, which was still present in the absence of the DRB1*1501 allele (p = 0.032). This suggest that DRB1 aa86 may have an independent role in contributing to MS susceptibility. The Val86/Val86 genotype was correlated with genotyping for other putative MS susceptibility genes, including T cell receptor beta chain germline polymorphisms, HLA-DMB alleles, and -DQA1 and -DQB1 alleles encoding critical amino acid residues, with a significant interaction only observed with DQB1 Leu26 (p = 0.014). Additional studies of the HLA-DRB1 aa86 polymorphism in MS, and its function, are needed to more fully understand this association.

Increased frequency of CCR-5 delta 32 heterozygotes among long-term non-progressors with HIV-1 infection. The Australian Long-Term Non-Progressor Study Group.

BACKGROUND: The beta-chemokine receptor CCR-5 is used as a coreceptor by macrophage-tropic strains of HIV-1 to gain entry into CD4+ cells. OBJECTIVE: To determine the effect of a common 32 base-pair deletion mutation in the CCR-5 gene (CCR-5 delta 32) on progression of HIV infection to AIDS, and to assess the level of heterozygosity for this mutation in a well-defined group of long-term non-progressors (LTNP). PARTICIPANTS: Sixty-four HIV-1-infected LTNP (CD4+ T lymphocyte count > 500 x 10(6)/l after 8 years) were compared with 95 individuals infected within a similar period (1983-1986) but who had rapidly progressed to AIDS and death, and with a further 120 HIV-positive individuals with CD4+ counts < 500 x 10(6)/l. METHODS: The presence of the CCR-5 delta 32 mutation was assessed using polymerase chain reaction with primers spanning the 32 base-pair deletion. CD4+ and CD8+ counts, plasma HIV-1 RNA, p24 antigen and beta 2-microglobulin levels in LTNP carrying the CCR-5 delta 32 mutation were compared with LTNP lacking the mutation. RESULTS: A marked increase in the frequency of CCR-5 delta 32 heterozygosity was found among LTNP (35.9%) compared with rapid progressors (12.6%; P = 0.0005) and patients selected on the basis of a CD4+ T-cell count < 500 x 10(6)/l (12.5%; P = 0.0004). LTNP heterozygous for CCR-5 delta 32 had a significantly higher CD8+ T-cell count than those without the mutation (1218 versus 972 x 10(6)/l; P = 0.044). No significant correlation was observed between heterozygosity and CD4 count, viral load, p24 antigen or beta 2-microglobulin within the LTNP group. CONCLUSIONS: This study provides the strongest evidence to date for the importance of a single copy of the CCR-5 delta 32 mutation in long-term non-progression of HIV infection, which may involve, in part, CD8+ T lymphocytes.

HLA-DR, -DQA1 and -DQB1 associations in Australian multiple sclerosis patients.

Molecular genotyping for the major histocompatibility complex (MHC) class II loci, HLA-DRB1, -DQB1 and -DQA1, in 100 patients with relapsing/remitting multiple sclerosis (MS) demonstrated an association with the HLA-DR2, DQw6-associated alleles DRB1*1501, DQB1*0602 and DQA1*0102, thereby extending this finding among MS patients in several countries to an Australian population. Analysis by the relative predispositional effect (RPE) method provided no evidence for a second susceptibility allele at either DQA1 or DQB1. However, our data and that of others suggest a negative association with DQA1*0101. Associations were found with DQB1 alleles sharing sequence homology with DQB1*0602, with DQB1 alleles encoding leucine at residue 26 (Leu 26), with DQA1 alleles encoding glutamine at residue 34 (Gln 34) and with Leu 26 plus Gln 34 alleles, but each was shown by two-loci linkage analysis to be secondary to the DRB1*1501, DQB1*0602, DQA1*0102 association. The recently reported negative association with DQA1 alleles encoding phenylalanine at amino acid 25, leucine at amino acid 69 and arginine at amino acid 52 was not found in this study, although there was a trend towards reduced phenylalanine at amino acid 25. The determination at a molecular level of an explanation for the world-wide association with these alleles remains elusive despite major advances in MHC typing.

The CCR5 deletion mutation fails to protect against multiple sclerosis.

Recent advances in the understanding and identification of chemokines and their receptors have provided evidence for their consideration as candidate loci with respect to genetic susceptibility/resistance to MS. Increased levels of the chemokine, macrophage inflammatory protein (MIP)-1 alpha, have been demonstrated in the cerebrospinal fluid of both patients with MS and mice with EAE, and anti-MIP-1 alpha antibodies have been shown to prevent EAE. Recently, a common deletion mutation in the gene for the major receptor for MIP-1 alpha, chemokine receptor 5 (CCR5) has been described. Homozygotes for the mutation fail to express this receptor. Moreover, homozygotes are highly protected against HIV infection this has potential implications for the cell entry of infectious agents in other multifactorial disease where a viral component may be involved. In view of these aspects, a group of 120 unrelated Australian relapsing remitting MS and 168 unrelated control subjects were screened for the CCR5 delta 32 mutation. There was no significant difference in the allele frequency of CCR5 delta 32 gene between the MS patients (0.1125) and the control population (0.0921). The presence of two CCR5 delta 32 homozygotes in the MS patients indicates that the absence of CCR5 is not protective against MS. These data suggest that CCR5 is not an essential component in MS expression, though this may be due to redundancy in the chemokine system where different chemokine receptors may substitute for CCR5 when it is absent.

HLA-DQA1 and -DQB1 genotyping by PCR-RFLP, heteroduplex and homoduplex analysis.

PCR-RFLP typing methods for DQA1 and DQB1 in conjunction with the analysis of heteroduplex and homoduplex patterns have allowed a simple method for typing all of the major DQA1 and DQB1 alleles. This method has advantages over PCR amplification with sequence-specific primers (PCR-SSP), PCR hybridization with sequence-specific oligonucleotide probes (PCR-SSO) and other PCR-RFLP strategies for typing DQ alleles. The analysis of heteroduplex and homoduplex patterns can be used in conjunction with other PCR typing systems such as PCR-SSP as a confirmatory step with little additional work. In addition, a PCR-RFLP strategy was designed for resolving the DQB1*0602 and DQB1*0603 alleles, which involved the use of a primer containing a base mutation, creating a new restriction site which distinguished the two alleles. These techniques have enabled resolution of the major homozygous and heterozygous combinations of these DQA1 and DQB1 alleles. The PCR-RFLP technique does not require the large number of oligonucleotides that are necessary for both the PCR-SSP and PCR-SSO techniques and is thus both time and cost effective for infrequent or small numbers of samples.

The usefulness of a rapid PCR methodology to detect rearranged Ig heavy chain genes in lymphoproliferative disease in a diagnostic setting.

Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the framework 3 region (FR3) of the immunoglobulin heavy (IgH) chain genes, from the tissue of 66 patients with B-lymphoproliferative diseases and 74 patients with other malignant diseases, reactive or normal tissue. The assay performed with 77% sensitivity, 100% specificity and 89% efficacy. In addition, the PCR assay cost less than 25% of the cost performing Southern blot analysis of tumor DNA, which has been the test performed to date, and had a turn around time of 24 hrs rather than the 7-14 days required to obtain a result from Southern blot analysis. These results suggest that PCR analysis of B-cell lymphoproliferative disease is superior to Southern blot analysis, in the setting of a diagnostic laboratory.

TAP2 polymorphisms in Australian multiple sclerosis patients.

Polymorphism of the TAP2 gene locus, situated approximately 150 kb centromeric to the MHC class II loci HLA-DR, DQ was examined in 100 Australian patients with relapsing/remitting multiple sclerosis (MS), in 100 random controls and in 37 selected HLA-DRB1*1501-positive controls. The results were correlated with HLA class I and class II phenotypes. TAP2 encodes a protein involved in the transport and presentation of antigenic peptides by MHC class I molecules and hence is a candidate locus for a putative MS susceptibility gene either through functional interactions with class I alleles or as an explanation, via linkage disequilibrium (LD), for the known association between MS and the alleles DRB1*1501, DQA1*0102, DQB1*0602. Strong LD was found between the allele TAP2*01 and DRB1*1501 in both the MS and control populations. The MS-associated haplotype can therefore be extended to DRB1*1501, DQA1*0102, DQB1*0602, TAP2*01, and the putative gene locus could reside on the centromeric side of DQ. TAP2 typing, however, could not explain the DRB1*1501, DQA1*0102, DQB1*0602-negative patients in whom, interestingly, the frequency of TAP2*01 was decreased compared to controls. The results of this study exclude TAP2 as a locus for a necessary MS/MHC gene but indicate that an MS gene carried by the DRB1*1501, DQA1*0102, DQB1*0602 haplotype could reside centromeric of DQ.

Assessment of HIV status using the polymerase chain reaction in antibody-positive patients and high-risk antibody-negative haemophiliacs.

The polymerase chain reaction (PCR) was used to identify the presence of DNA sequences homologous to HIV-1 in the buffy-coat leukocytes of antibody-positive and antibody-negative individuals in a haemophiliac population. The presence of HIV sequences was demonstrated in all of the antibody-positive haemophiliacs with the exception of one patient who was repeatedly negative. None of the seronegative haemophiliacs gave an overall positive result, although there were clear differences between this population and the negative controls who were examined. We conclude that, in our hands, PCR represents a reliable test which represents a useful diagnostic advance in HIV medicine.

Structure and function of contractile proteins in muscle fibres.

The structural unit of muscle has long been defined as the myofibril, a supramolecular assembly of a dozen or more proteins of which two, actin and myosin, comprise more than 75%. In the past 40 years since Albert Szent-Gyorgyi first described the contractile response from the complex of actin and myosin, knowledge of the structure and function of these contractile proteins has been substantially refined. This paper describes these new discoveries and identifies the problems which remain to be elucidated.

Primary structure of a human glandular kallikrein gene.

To isolate a human glandular kallikrein gene, a human genomic library was screened with a probe made from a mouse kallikrein cDNA (pMK-1). Overlapping clones were obtained and nucleotide sequence determination showed that they together contained a human glandular preprokallikrein gene, hGK-1, of 5.2 kb. The gene encoded a unique preproprotein of 261 amino acids. The sequence of the mature 237-amino-acid protein had 66% homology with the sequence predicted for human kallikrein synthesized in the pancreas, kidney, and salivary gland. Moreover, it had even stronger homology (78%) with human prostate-specific antigen. The latter lacks an aspartic acid residue essential for kallikrein-specific cleavage, whereas the sequence of this new protein had all of the attributes needed to confer kallikrein-like specificity. Southern blotting indicated that the number of glandular kallikrein genes in man could be limited to three, a situation very different from mouse and rat, which each have a large multigene family. Furthermore, unlike kallikrein genes in the mouse, hGK-1 was not closely linked to other human kallikrein genes. In other respects the structure of the human kallikrein gene resembled that in mouse: coding sequences in the five exons were organized similarly, homology was higher with other members of the kallikrein gene family in the same species, and the three key amino acid residues required by serine proteases for their catalytic activity, together with the residue that confers kallikrein-specific cleavage, were conserved and located on different exons. Thus, if hGK-1 is expressed, its product represents a new, and possibly the only other enzyme with true kallikrein-like specificity in man.