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The genetic basis of malignant hyperthermia (MH) is not fully characterised and likely involves more than just the currently classified mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) and the gene encoding the α1 subunit of the dihydropyridine receptor (CACNA1S). In this paper we sequence other genes involved in calcium trafficking within skeletal muscle in patients with positive in vitro contracture tests, searching for alternative genes associated with MH. We identified four rare variants in four different genes (CACNB1, CASQ1, SERCA1 and CASQ2) encoding proteins involved in calcium handling in skeletal muscle in a cohort of 30 Australian MH susceptible probands in whom prior complete sequencing of RYR1 and CACNA1S had yielded no rare variants. These four variants have very low minor allele frequencies and while it is tempting to speculate that they have a role in MH, they remain at present variants of unknown significance. Nevertheless they provide the basis for a new set of functional studies, which may indeed identify novel players in MH.

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Defects in the genes coding for the skeletal muscle ryanodine receptor (RYR1) and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) have been identified as causative for malignant hyperthermia (MH). Sixty-two MH susceptible individuals presenting to the same diagnostic centre had copy deoxyribonucleic acid, derived from muscle ribonucleic acid, sequenced to identify variants with the potential to be responsible for the MH phenotype in both RYR1 and CACNA1S. These genetic findings were combined with clinical episode details and in vitro contracture test results to improve our understanding of the Australian MH cohort. Twelve novel variants were identified in RYR1 and six in CACNA1S. Known RYR1 causative mutations were identified in six persons and novel variants in RYR1 and CACNA1S in a further 17 persons. Trends indicated higher mutation identification in those with more definitive clinical episodes and stronger in vitro contracture test responses.

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A multimodal approach of complementary techniques targeting primarily truncating, deletion and rearrangement mutations provides a robust screening protocol that identifies the vast majority of pathogenic germline APC gene mutations in FAP patients. Patients in whom no mutation is identified through this mutation protocol, may be sub-cohorts representing a different FAP pathogenesis including MYH associated polyposis and somatic cell mosaicism for APC gene mutations.

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Advances in analysis of the RYR1 gene (which encodes the skeletal muscle ryanodine receptor) show that genetic examination is a useful adjunct to the in vitro contracture test in the diagnosis of malignant hyperthermia, as defects in RYR1 have been shown to be responsible for malignant hyperthermia susceptibility. DNA from 34 malignant hyperthermia susceptible individuals and four malignant hyperthermia equivocal subjects was examined using direct sequencing of 'hot-spots' in the RYR1 gene to identify mutations associated with malignant hyperthermia. Seven different causative mutations (as defined by the European Malignant Hyperthermia Group) in nine malignant hyperthermia susceptible individuals were identified. In another six malignant hyperthermia susceptible individuals, five different published but as yet functionally uncharacterised mutations were identified. A further three as yet unpublished and functionally uncharacterised (novel) mutations were identified in three malignant hyperthermia susceptible samples. If the novel and previously published mutations prove to be functionally associated with calcium homeostasis, then this method of analysis achieved a mutation detection rate of 47%. Based on the number of relatives presenting to our unit in the study period, the muscle biopsy rate would have decreased by 25%. That we only identified a genetic defect in RYR1 in 47% of in vitro contracture test positive individuals suggests that there are other areas in RYR1 where pathogenic mutations may occur and that RYR1 may not be the sole gene associated with malignant hyperthermia. It may also reflect a less than 100% specificity of the in vitro contracture test.

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We report our observations in an Australian family with spinocerebellar ataxia type 14 (SCA 14). We describe a novel mutation in exon 5 of the PRKCG gene, altering a highly conserved cysteine to a phenylalanine at codon 150, and record the detailed clinical observations in six affected family members.

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HFE-associated hereditary haemochromatosis is a recessive, iron-overload disorder that affects about one in 200 north Europeans and that can be easily prevented. However, genetic screening for this disease is controversial, and so we assessed whether such screening was suitable for communities. Cheek-brush screening for the Cys282Tyr HFE mutation was offered to individuals in the workplace. Outcomes were assessed by questionnaires before and after testing. 11,307 individuals were screened. We recorded no increase in anxiety in individuals who were homozygous for the Cys282Tyr mutation or non-homozygous. Self-reported tiredness before testing was significantly higher in homozygous participants than in non-homozygous participants (chi2 test, p=0.029). Of the 47 homozygous individuals identified, 46 have taken steps to treat or prevent iron accumulation. Population genetic screening for HFE-associated hereditary haemochromatosis can be practicable and acceptable.

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There is debate as to whether community genetic screening for the mutation(s) causing hereditary hemochromatosis (HH) should be implemented, due to issues including disease penetrance, health economic outcomes, and concerns about community acceptance. Hemochromatosis is a common preventable iron overload disease, due in over 90% of cases to C282Y homozygosity in the HFE gene. We are, therefore, piloting C282Y screening to assess understanding of genetic information and screening acceptability in the workplace setting. In this program, HaemScreen, education was by oral or video presentation in a group setting. C282Y status was assessed by polymerase chain reaction (PCR) and melt-curve analysis on DNA obtained by cheek-brush sampling. Of eligible participants, 5.8% (1.5-15.8%) attended information and screening sessions, of whom 97.7% (5571 individuals) chose to be tested. Twenty-two C282Y (1 : 253) homozygotes were identified and offered clinical follow-up. There were 638 heterozygotes (1 : 8.7). The determinants for participation have been analyzed in terms of the principles outlined in the Health Belief Model. Widespread screening for HH is readily accepted in a workplace setting, and a one-to-many education program is effective. The level of participation varies greatly and the advertizing and session logistics should be adapted to the specific features of each workplace.

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There has been interest in the literature in the possible existence of a gene that predisposes to both breast cancer (BC) and colorectal cancer (CRC). We describe the detailed characterisation of one kindred, MON1080, with 10 cases of BC or CRC invasive cancer among 26 first-, second- or third-degree relatives. Linkage analysis suggested that a mutation was present in BRCA2. DNA sequencing from III: 22 (diagnosed with lobular BC) identified a BRCA2 exon 3 542G>T (L105X) mutation. Her sister (III: 25) had BC and endometrial cancer and carries the same mutation. Following immunohistochemical and microsatellite instability studies, mutation analysis by protein truncation test, cDNA sequencing and quantitative real-time PCR revealed a deletion of MSH2 exon 8 in III: 25, confirming her as a double heterozygote for truncating mutations in both BRCA2 and MSH2. The exon 8 deletion was identified as a 14.9 kb deletion occurring between two Alu sequences. The breakpoint lies within a sequence of 45 bp that is identical in both Alu sequences. In this large BC/CRC kindred, MON1080, disease-causing truncating mutations are present in both MSH2 and BRCA2. There appeared to be no increased susceptibility to the development of colorectal tumours in BRCA2 mutation carriers or to the development of breast tumours in MSH2 mutation carriers. Additionally, two double heterozygotes did not appear to have a different phenotype than would be expected from the presence of a mutation in each gene alone.

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OBJECTIVE: To determine (1) the prevalence and nature of connexin 26 mutations in a cohort of Australian children with non-syndromic hearing loss, and (2) the carrier frequency of the common connexin 26 mutation (35delG) in the general population. DESIGN: A cohort, case-finding study. Mutation analysis was performed on DNA extracted from white blood cells, buccal cells, or Guthrie blood spots. SETTING: A hearing loss investigation clinic and a deafness centre in two Australian capital cities, 1 January 1998 to 31 October 2000. PARTICIPANTS: (1) 243 children (age range, 4 weeks to 16 years; median, 4 years), attending hearing loss clinics in Sydney and Melbourne; (2) 1000 blood samples obtained from anonymous Guthrie card blood spots collected in 1984 [corrected] by the Victorian Clinical Genetics Service as part of the newborn screening program. MAIN OUTCOME MEASURES: (1) The prevalence and types of connexin 26 mutations in a cohort of children with prelingual deafness; (2) the carrier frequency of the common connexin 26 mutation, 35delG, in the general population. RESULTS: Connexin 26 mutations were identified and characterised in 52 (21%) of the 243 children; 14 different mutations, including four previously unreported mutations (135S, C53R, T123N and R127C), were identified. The common 35delG mutation was found in 56 of the 104 alleles (ie, 86 of the connexin 26 alleles in which a mutation was positively identified). The mutations V371 and M34T were also relatively common. The carrier frequency of connexin 26 mutations and of the common 35delG connexin 26 mutation in the Victorian population was estimated to be 1 in 54 and 1 in 100, respectively. CONCLUSIONS: Mutations in the connexin 26 gene (especially the 35delG mutation) are a common cause of prelingual hearing loss in Australia.

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Marker chromosomes containing active human neocentromeres have been described in individuals where the chromosomes are non-mosaic, suggesting that they are mitotically stable, but also in individuals where there is mosaicism, raising the possibility of neocentromere instability. We report two independently ascertained individuals who are mosaic for a supernumerary marker chromosome, shown by reverse chromosome painting to have an 8p origin, resulting in mosaicism for tetrasomy 8p23.1-->pter in the patient. The markers have a primary constriction but show no detectable centromeric alpha-satellite DNA. The marker in Patient 1 demonstrated no centromere protein CENP-B binding, but associated with nine different functionally critical centromere proteins. Investigation of peripheral blood lymphocytes from this patient on five separate occasions over a 13-year period showed 23-46% mosaicism for the marker chromosome with no decrease in incidence. In vitro investigation of primary and secondary sub-clones of a lymphoblast cell line derived from the patient demonstrated 100% stability of the marker chromosome indicating that neocentromere instability is unlikely to be responsible for the mosaicism in the patient. This and other available data support a general model of neocentromerization as a post-zygotic event, irrespective of whether the supernumerary chromosome fragment has arisen during meiosis or post-fertilization at mitosis.

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Hereditary nonpolyposis colorectal cancer (HNPCC) describes the condition of a disparate group of families that have in common a predisposition to colorectal cancer in the absence of a premalignant phenotype. The genetic basis of this disease has been linked to mutations in genes associated with DNA mismatch repair. A large proportion of families harbor changes in one of two genes, hMSH2 and hMLH1. Approximately 35% of families in which the diagnosis is based on the Amsterdam criteria do not appear to harbor mutations in DNA-mismatch-repair genes. In this report we present data from a large series of families with HNPCC and indicate that there are subtle differences between families that harbor germline changes in hMSH2 and families that harbor hMLH1 mutations. Furthermore, there are differences between the mutation-positive group (hMSH2 and hMLH1 combined) of families and the mutation-negative group of families. The major findings identified in this study focus primarily on the extracolonic disease profile observed between the mutation-positive families and the mutation-negative families. Breast cancer was not significantly overrepresented in the hMSH2 mutation-positive group but was overrepresented in the hMLH1 mutation-positive group and in the mutation-negative group. Prostate cancer was not overrepresented in the mutation-positive groups but was overrepresented in the mutation-negative group. In age at diagnosis of colorectal cancer, there was no difference between the hMSH2 mutation-positive group and the hMLH1 mutation-positive group, but there was a significant difference between these two groups and the mutation-negative group.

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The frequencies of various genetically defined spinocerebellar ataxias (SCAs) vary in different populations presumably due to founder effects. No data have been published on the Australian population. Although predominantly of Anglo-Celtic extraction, Australia has also received considerable influx from southeastern Europe and more recently eastern and southeastern Asia. We examined the frequency of mutations for SCA types 1, 2, 3, 6, and 7 in southeastern Australia. Of 88 pedigrees with multiple-affected members, SCA type 1 (SCA1) accounted for 16%, SCA2 for 6%, SCA3 for 12%, SCA6 for 17%, SCA7 for 2%, and 47% (41 pedigrees) were negative for each of SCA1, 2, 3, and 6. Twenty of the 41 negative pedigrees were also negative for dentatorubralpallidoluysian atrophy, and indeed dentatorubralpallidoluysian atrophy has not been reported in Australia. In addition, no pedigree information was available on a further four patients with SCA1, three patients with SCA2, three patients with SCA3, and three patients with SCA6. One SCA1 and two SCA2 patients had no other known affected family members. In total, of 63 pedigrees or individuals with positive tests, 30% were those with SCA1, 15% with SCA2, 22% with SCA3, 30% with SCA6, and 3% with SCA7. Judging by pedigree names, four of the nine SCA2 positive individuals/pedigrees were of Italian extraction, and four of the 14 SCA3 positive individuals/pedigrees were of Chinese descent, whereas only 1 of the 20 SCA1 positive individuals/pedigrees were non-Anglo-Celtic. These results are in accordance with the known ethnic composition of the Australian population and with gene frequencies in these constituent ethnic groups reported by others. The frequency of large-normal alleles for SCA1 and SCA3 in the population reflects the prevalence of these two diseases, supporting the hypothesis that disease alleles arise by expansion of large-normal alleles.

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We recently described a human marker chromosome containing a functional neo-centromere that binds anti-centromere antibodies, but is devoid of centromeric alpha-satellite repeats and derived from a hitherto non-centromeric region of chromosome 10q25. Chromosome walking using cloned single-copy DNA from this region enabled us to identify the antibody-binding domain of this centromere. Extensive restriction mapping indicates that this domain has an identical genomic organization to the corresponding normal chromosomal region, suggesting a mechanism for the origin of this centromere through the activation of a latent centromere that exists within 10q25.

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We have previously reported the identification of a naturally occurring junction between alpha satellite and satellite III DNA on human chromosomes 13, 14 and 21. Direct sequence analysis has shown that the 9 bp alphoid-derived direct repeat sequence (GTGAAAAAG) present at the junction is fully conserved on these chromosomes. A novel protein, pJ alpha, present in HeLa nuclear extracts, binds to the conserved junction sequence. Mutation analysis of the binding site suggests that pJ alpha can recognize one of the two 9 bp repeats and provides some insight into nucleotides that are important for binding. Competition studies support the possibility that this protein binds a significant portion of genomic alpha satellite DNA. Preliminary protein purification experiments have shown that pJ alpha has a molecular weight of 10-15 kDa.

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We present a balanced translocation (X;9) (q28;q21) in which the normal X chromosome is preferentially active. The derivative X chromosome is inactive in 93% of fibroblasts, but the X portion translocated onto chromosome 9 is not inactivated, as apparent from DNA methylation and chromosome replication patterns. Consequently, the patient is functionally disomic for the part of Xq28 distal to the locus LICAM.

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We reviewed 122 cases of balanced X-autosome translocations in females, with respect to the X inactivation pattern, the position of the X break point and the resulting phenotype. In 77% of the patients the translocated X chromosome was early replicating in all cells analysed. The break points in these cases were distributed all along the X chromosome. Most of these patients were either phenotypically normal or had gonadal dysgenesis, some had single gene disorders, and less than 9% had multiple congenital anomalies and/or mental retardation. In the remaining 23% of the cases the translocated X chromosome was late replicating in a proportion of cells. In these cells only one of the translocation products was reported to replicate late, while the remaining portion of the X chromosome showed the same replication pattern as the homologous part of the active, structurally normal X chromosome. The analysis of DNA methylation in one of these cases confirmed noninactivation of the translocated segment. Consequently, these cells were functionally disomic for a part of the X chromosome. The presence of disomic cells was highly prevalent in translocations with break points at Xp22 and Xq28, even though spreading of X inactivation onto the adjacent autosomal segment was noted in most of these cases. This suggests that selection against cells with a late replicating translocated X is driven predominantly by a functional disomy X, and that the efficiency of this process depends primarily on the position of the X break point, and hence the size of the noninactivated region.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25-26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.

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Over 30% of female carriers of the fragile X [fra(X)] syndrome are clinically affected. A nonrandom X chromosome inactivation in these cases could be a plausible explanation. A review of previous studies addressing this question showed inconclusive results; thus, we analysed the X inactivation pattern in fibroblasts of 4 unrelated, mentally retarded fra(X) carriers with a high expression of the fragile site Xq27.3. Using Southern analysis with a highly polymorphic probe M27 beta that recognizes methylation differences between the active and inactive X chromosome we found a 50/50 inactivation pattern in 2 cases and skewed patterns in the other 2. As biased patterns were also observed in control females we conclude that at present no evidence exists for a nonrandom X chromosome inactivation in the fra(X) syndrome in females.

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A de novo interstitial deletion (X)(q27.1q27.3), between the loci DXS 105 and F8, has been found in a mentally retarded female. The deleted X chromosome is preferentially early replicating in fibroblasts, B cells and T cells, suggesting that the missing region plays a role in inactivation of the X chromosome. None of the available DNA probes except DXS 98 maps to the deleted region of about 10,000 kb. The locus FRAXA is either included in the deletion, or located close to the distal break point.

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