New mutations in GJA8 expand the phenotype to include total sclerocornea.

This project expands the disease spectrum for mutations in GJA8 to include total sclerocornea, rudimentary lenses and microphthalmia, in addition to this gene's previously known role in isolated congenital cataracts. Ophthalmic findings revealed bilateral total sclerocornea in 3 probands, with small abnormal lenses in 2 of the cases, and cataracts and microphthalmia in 1 case. Next-generation sequencing revealed de novo heterozygous mutations affecting the same codon of GJA8 : (c.281G>A; p.(Gly94Glu) and c.280G>C; p.(Gly94Arg)) in 2 of the probands, in addition to the c.151G>A; p.(Asp51Asn) mutation we had previously identified in the third case. In silico analysis predicted all of the mutations to be pathogenic. These cases show that deleterious, heterozygous mutations in GJA8 can lead to a severe ocular phenotype of total sclerocornea, abnormal lenses, and/or cataracts with or without microphthalmia, broadening the phenotype associated with this gene. GJA8 should be included when investigating patients with the severe anterior segment abnormality of total sclerocornea.

Guidelines for the genetic diagnosis of hereditary recurrent fevers.

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

Contiguous gene deletion syndrome in a female with ornithine transcarbamylase deficiency.

OTC deficiency, a partially dominant X-linked trait, is the most frequent inborn error of the urea cycle. We describe a female patient with a contiguous gene deletion syndrome encompassing the OTC, DMD, RPGR, CYBB and XK genes, amongst others, only manifesting features of OTC deficiency. Molecular characterization was ascertained by MLPA and confirmed by CGH microarray, which revealed an 8.7 Mb deletion of the X-chromosome. Complete de novo deletion of the OTC gene led to a severe clinical phenotype in the proband. The application of high resolution molecular genetic techniques such as MLPA and array CGH, in mutation negative OTC cases allows the identification of chromosomal rearrangements, such as large deletions and provides information for accurate genetic counseling and prenatal diagnosis.

Lesch-Nyhan disease in a 20-year- old man incorrectly described as developing 'cerebral palsy' after general anaesthesia in infancy.

Lesch-Nyhan disease (LND) is a rare X-linked recessive genetic disorder caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The classic clinical condition is characterized by cognitive impairment, hypotonia at rest, choreoathetosis, hyperuricaemia and the hallmark symptom of severe and involuntary self-mutilation. We describe a man with LND who was initially thought to have suffered from a dyskinetic cerebral palsy after an uncomplicated inguinal herniorrhaphy under general anaesthesia at 5 1/2 months of age. In the absence of overt self-injurious behaviour, the diagnosis was not considered for nearly two decades. The diagnosis of LND was established at 20 years of age through clinical review, biochemical examinations and molecular analysis. HPRT haemolysate activity was 7.6% of the normal control, suggesting that he had a milder variant of the disease. Mutation analysis of the HPRT gene revealed a novel missense mutation, c.449T > G in exon 6 (p.V150G). Cascade testing of family members revealed that the mother was heterozygous for the mutation but two siblings (a brother and a sister) did not carry the sequence mutation. Whether the onset of neurological abnormalities in this particular case can be attributed to the general anaesthesia is discussed.

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.

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.

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.

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.

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.

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.

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.

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.

Modulation by blood glucose levels of activity and concentration of paraoxonase in young patients with type 1 diabetes mellitus.

Paraoxonase (PON) is a high-density lipoprotein (HDL)-associated esterase, which may prevent the transformation of low-density lipoproteins (LDL) into biologically active, atherogenic particles. PON concentration and activity are affected by PON1 gene polymorphisms and found to be altered in type 2 diabetes patients with retinopathy. We investigated serum PON concentration, in vitro activity and polymorphism at position 54 (L/M, Leu-Met54) in 193 Caucasian adolescents and young adults (88 males, 105 females) with type 1 diabetes mellitus, as well as its relationship to the presence of retinopathy. An inverse linear correlation was found between blood glucose levels and both serum PON concentration (r = -.20, P =.017) and its activity (r = -0.17, P =.037). Patients with elevated blood glucose values (> or =10 mmol/L) had significantly lower levels of both PON concentration (P =.003) and activity (P =.028) than those with lower glucose levels. After adjusting for blood glucose and diabetes duration, PON activity was significantly higher in patients with different stages of retinopathy compared with those without retinopathy (P =.003). The L/L genotype was closely associated with the presence of retinopathy (P <.0001). These data show that young people with type 1 diabetes and the L/L polymorphism at position 54 of PON1 gene are more susceptible to retinal complications. However, the role of serum PON concentration and activity as a possible marker for monitoring late microvascular complications in these patients has to be established.

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.

The application of a PCR technique for the detection of immunoglobulin heavy chain gene rearrangements in fresh or paraffin-embedded skin tissue.

Although detection of a clonal sequence of the heavy chain gene of immunoglobulin by the polymerase chain reaction (PCR) is frequently used to assess lymphoid infiltrates in skin biopsy specimens, there are no data on the sensitivity and specificity of this test in detecting clonal B cell populations. Having refined a PCR technique for the detection of immunoglobulin heavy chain (IgH) gene rearrangement in both fresh and formalin-fixed, paraffin-embedded skin samples, we undertook to define the role of this assay in the diagnostic setting. Thirty-one cases of cutaneous B cell lymphoma (CBCL), 19 cases of B cell pseudolymphoma (lymphocytoma cutis), 34 cases of benign lymphocytic infiltrates of the skin and one case of cutaneous T cell lymphoma (CTCL) were studied using the polymerase chain reaction assay. All biopsies were formalin-fixed, paraffin-embedded skin sections apart from 13 of the 31 CBCL specimens which were fresh skin specimens. DNA from the framework region 3 (FR3) sequence of the IgH genes was amplified to ascertain the presence of a clonal IgH gene rearrangement. The findings were correlated with histological and immunophenotyping results on all samples. The assay performed with 73% sensitivity and 100% specificity, comparable to results obtained examining fresh lymphoid tissue specimens from patients with B cell tumours. The results indicate that this technique is a useful tool in the work up of suspected CBCL and in differentiating between CBCL and mixed lymphocytic infiltrates, a clearly important distinction with regards to prognosis and treatment.

Quality standards for DNA sequence variation databases to improve clinical management under development in Australia.

Despite the routine nature of comparing sequence variations identified during clinical testing to database records, few databases meet quality requirements for clinical diagnostics. To address this issue, The Royal College of Pathologists of Australasia (RCPA) in collaboration with the Human Genetics Society of Australasia (HGSA), and the Human Variome Project (HVP) is developing standards for DNA sequence variation databases intended for use in the Australian clinical environment. The outputs of this project will be promoted to other health systems and accreditation bodies by the Human Variome Project to support the development of similar frameworks in other jurisdictions.

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.