Detection of a novel mutation in the CACNA1A gene

Familial hemiplegic migraine (FHM) is a rare autosomal dominant subtype of migraine with aura. It is divided into three subtypes FHM1, FHM2 and FHM3, which are caused by mutations in the CACNA1A, ATP1A2 and SCN1A genes respectively. As part of a regular diagnostic service, we investigated 168 patients with FHM symptoms. Samples were tested for mutations contained within the CACNA1A gene. Some tested samples (4.43%) showed an FHM1 mutation, with five of the mutations found in exon 5, one mutation in exon 16 and one in exon 17. Four polymorphisms were also detected, one of which occurred in a large percentage of samples (14.88%). The exon 16 2094G>A polymorphism, however, has been found to occur in healthy Caucasian control populations up to a frequency of 16% and is not considered to be significantly associated with FHM. A finding of significance, found in a single patient, was the detection of a novel mutation in exon 5 that results in a P225H change. The affected individual was an 8-year-old female. The exact phenotypic effect of this mutation is unknown, and further studies are needed to understand the pathophysiology of this mutation in FHM1. New information will allow for diagnostic procedures to be constantly updated, thus improving accuracy of diagnosis. It is possible that new information will also aid the development of new therapeutic agents for the treatment of FHM.

A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura

Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms. A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia. We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migraine-salient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele. These results therefore support a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target.

Mutations in cardiac T-Box Factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Genetic investigation of methylenetetrahydrofolate reductase (MTHFR) and catechol-O-methyl transferase (COMT) in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease characterized by central nervous system (CNS) inflammation and demyelination. The C677T substitution variant in the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with increased levels of circulating homocysteine and is a mild risk factor for vascular disease. Higher blood levels of homocysteine have also been reported in MS. Thus, the C677T mutation of the MTHFR gene may influence MS susceptibility. Noradrenaline, a neurotransmitter believed to play an immunosupressive role in neuroinflammatory disorders, is catabolized by catechol-O-methyl transferase (COMT). The COMT G158A substitution results in a three- to four-fold decreased activity of the COMT enzyme, which may influence CNS synaptic catecholamine breakdown and could also play a role in MS inflammation. We tested DNA from Australian MS patients and unaffected control subjects, matched for gender, age and ethnicity. Specifically, we genotyped the MTHFR C677T and the COMT G158A mutations. Genotype distributions showed that the homozygous mutant MTHFR genotype (T/T) and the COMT (H/H) genotype were slightly over-represented in the MS group (16% versus 11% and 24% versus 19%, respectively), but both variations failed to reach statistical significance (P=0.15 and P=0.32, respectively). Hence, results from the present study do not support a major role for either functional gene mutation in MS susceptibility.

A genetic analysis of serotonergic biosynthetic and metabolic enzymes in migraine using a DNA pooling approach

Migraine is a common debilitating primary headache disorder with significant mental, physical and social health implications. The brain neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) is involved in nociceptive pathways and has been implicated in the pathophysiology of migraine. With few genetic studies investigating biosynthetic and metabolic enzymes governing the rate of 5-HT activity and their relationship to migraine, it was the objective of this study to assess genetic variants within the human tryptophan hydroxylase (TPH), amino acid decarboxylase (AADC) and monoamine oxidase A (MAOA) genes in migraine susceptibility. This objective was undertaken using a high-throughput DNA pooling experimental design, which proved to be a very accurate, sensitive and specific method of estimating allele frequencies for single nucleotide polymorphism, insertion deletion and variable number tandem repeat loci. Application of DNA pooling to a wide array of genetic loci provides greater scope in the assessment of population-based genetic association study designs. Despite the application of this high-throughput genotyping method, negative results from the two-stage DNA pooling design used to screen loci within the TPH, AADC and MAOA genes did not support their role in migraine susceptibility.

Molecular cytogenetic analysis of basal cell carcinoma DNA using comparative genomic hybridization

In an attempt to define genomic copy number changes associated with the development of basal cell carcinoma, we investigated 15 sporadic tumors by comparative genomic hybridization. With the incorporation of tissue microdissection and degenerate oligonucleotide primed-polymerase chain reaction we were able to isolate, and then universally amplify, DNA from the tumor type. This combined approach allows the investigation of chromosomal imbalances within a histologically distinct region of tissue. Using comparative genomic hybridization we have observed novel and recurrent chromosomal gains at 6p (47%), 6q (20%), 9p (20%), 7 (13%), and X (13%). In addition comparative genomic hybridization revealed regional loss on 9q in 33% of tested tumors encompassing 9q22.3 to which the putative tumor suppressor gene, Patched, has been mapped. The deletion of Patched has been indicated in the development of hereditary and sporadic basal cell carcinomas. The identification of these recurrent genetic aberrations suggests that basal cell carcinomas may not be as genetically stable as previously thought. Further investigation of these regions may lead to the identification of other genes responsible for basal cell carcinoma formation.

TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes : the effect of microorganisms within human follicular fluid collected during IVF cycles

Recently we reported the presence of bacteria within follicular fluid. Previous studies have reported that DNA fragmentation in human spermatozoa after in vivo or in vitro incubation with bacteria results in early embryo demise and a reduced rate of ongoing pregnancy, but the effect of bacteria on oocytes is unknown. This study examined the DNA within mouse oocytes after 12 hours’ incubation within human follicular fluids (n = 5), which were collected from women undergoing in vitro fertilization (IVF) treatment. Each follicular fluid sample was cultured to detect the presence of bacteria. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was used to label DNA fragmentation in ovulated, non-fertilized mouse oocytes following in vitro incubation in human follicular fluid. The bacteria Streptococcus anginosus and Peptoniphilus spp., Lactobacillus gasseri (low-dose), L. gasseri (high-dose), Enterococcus faecalis, or Propionibacterium acnes were detected within the follicular fluids. The most severe DNA fragmentation was observed in oocytes incubated in the follicular fluids containing P. acnes or L. gasseri (high-dose). No DNA fragmentation was observed in the mouse oocytes incubated in the follicular fluid containing low-dose L. gasseri or E. faecalis. Low human oocyte fertilization rates (<29%) were associated with extensive fragmentation in mouse oocytes (80–100%). Bacteria colonizing human follicular fluid in vivo may cause DNA fragmentation in mouse oocytes following 12 h of in vitro incubation. Follicular fluid bacteria may result in poor quality oocytes and/or embryos, leading to poor IVF outcomes.

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the world's major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)- in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells. © 2008 Springer Science+Business Media, LLC.

Analysis of Sdhd and Mmp12 in an affected solar keratosis and control cohort

The incidence of Squamous Cell Carcinoma (SCG) is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 40% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis (SK) lesion and with a small, but significant, chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. More specifically the first aim of this project was to analyse the SDHD and MMP12 genes via Dual-Labelled Probe Real-Time PCR for copy number aberrations in an affected Solar Keratosis and control cohort. It was found that 12 samples had identifiable copy-number aberrations in either the SDHD or MMP12 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The significance of this study is the contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. © 2008 Springer Science+Business Media, LLC.

DNA probes in Charcot-Marie-Tooth neuropathy

Results of Duffy (Fy) linkage confirm genetic heterogeneity in Charcot-Marie-Tooth disease type 1 (CMT1). Of 11 families informative for Fy, four showed probable linkage with CMT1, seven showed probable non-linkage and two showed definite non-linkage. These results suggest that Fy linked CMT1 may be less common than previously thought. These results combined with those of another DNA probe for the antithrombin III gene confirm that there are at least two gene loci for CMT1, termed 1A and 1B.

A linkage and cross-sectional study of hypertension and obesity using a poly(A) Alu-repeat polymorphism at the glucagon receptor gene locus (17q25)

1. Previous glucagon receptor gene (GCGR) studies have shown a Gly40Ser mutation to be more prevalent in essential hypertension and to affect glucagon binding affinity to its receptor. An Alu-repeat poly(A) polymorphism colocalized to GCGR was used in the present study to test for association and linkage in hypertension as well as association in obesity development. 2. Using a cross-sectional approach, 85 hypertensives and 95 normotensives were genotyped using polymerase chain reaction primers flanking the Alu-repeat. Both hypertensive and normotensive populations were subdivided into lean and obese categories based on body mass index (BMI) to determine involvement of this variant in obesity. For the linkage study, 89 Australian Caucasian hypertension affected sibships (174 sibpairs) were genotyped and the results were analysed using GENE-HUNTER, Mapmaker Sibs, ERPA and SPLINK (all freely available from http://linlkage.rockefeller. edu/soft/list.html). 3. Cross-sectional results for both hypertension and obesity were analysed using Chi-squared and Monte Carlo analyses. Results did not show an association of this variant with either hypertension (χ2 = 6.9, P = 0.14; Monte Carlo χ2 = 7.0, P = 0.11; n = 5000) or obesity (χ2 = 3.3, P = 0.35; Monte Carlo χ2 = 3.26, P = 0.34; n = 5000). In addition, results from the linkage study using hypertensive sib-pairs did not indicate linkage of the poly(A) repent with hypertension. Hence, results did not indicate a role far the Alu-repeat in either hypertension or obesity. However, as the heterozygosity of this poly(A) repeat is low (35%), a larger number of hypertensive sib-pairs may be required to draw definitive conclusions.

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives

Obese (BMI ≥ 26 kg/m 2; n = 51) and lean (BMI <26 kg/m 2; n = 61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a HincII RFLP of the low density lipoprotein receptor gene (LDLR). A similar analysis was performed in obese (n = 28) and lean (n = 68) normotensives. A significant association of the C allele of the T→C variant responsible for this RFLP was seen with obesity (χ 2 = 4.6, P = 0.029) in the hypertensive, but not in the normotensive, group (odds ratio = 3.0 for the CC genotype and 2.7 for CT). Furthermore, BMI tracked with genotypes of this allele in the hypertensives (P = 0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the HincII RFLP and an ApaLI RFLP (χ 2 = 33, P<0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for het-erozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.