Exome sequencing identifies a mutation in the ACTN2 gene in a family with idiopathic ventricular fibrillation, left ventricular noncompaction, and sudden death.

BACKGROUND: Potentially lethal and heritable cardiomyopathies and cardiac channelopathies are caused by heterogeneous autosomal dominant mutations in over 50 distinct genes, and multiple genes are responsible for a given disease. Clinical genetic tests are available for several of the inherited cardiac diseases and clinical investigations guide which test to order. This study describes a family with cardiac disease in which marked clinical diversity exists. In the absence of a unified clinical diagnosis, we used exome sequencing to identify a causal mutation. METHODS: Clinical evaluation of family members was performed, including physical examination, electrocardiography, 2D transthoracic echocardiography and review of autopsy records. Exome sequencing was performed on a clinically affected individual and co-segregation studies and haplotype analysis were performed to further confirm pathogenicity. RESULTS: Clinically affected members showed marked cardiac phenotype heterogeneity. While some individuals were asymptomatic, other presentations included left ventricular non-compaction, a resuscitated cardiac arrest due to idiopathic ventricular fibrillation, dilated cardiomyopathy, and sudden unexplained death. Whole exome sequencing identified an Ala119Thr mutation in the alpha-actinin-2 (ACTN2) gene that segregated with disease. Haplotype analysis showed that this mutation segregated with an identical haplotype in a second, previously described family with clinically diverse cardiac disease, and is likely inherited from a common ancestor. CONCLUSIONS: Mutations in the ACTN2 gene can be responsible for marked cardiac phenotype heterogeneity in families. The diverse mechanistic roles of ACTN2 in the cardiac Z-disc may explain this heterogeneous clinical presentation. Exome sequencing is a useful adjunct to cardiac genetic testing in families with mixed clinical presentations.

Diagnostic validation of a familial hypercholesterolaemia cohort provides a model for using targeted next generation DNA sequencing in the clinical setting.

Our aim was to assess the sensitivity and specificity of a next generation DNA sequencing (NGS) platform using a capture based DNA library preparation method. Data and experience gained from this diagnostic validation can be used to progress the applications of NGS in the wider molecular diagnostic setting. A technical cross-validation comparing the current molecular diagnostic gold standard methods of Sanger DNA sequencing and multiplex ligation-dependant probe amplification (MLPA) versus a customised capture based targeted re-sequencing method on a SOLiD 5500 sequencing platform was carried out using a cohort of 96 familial hypercholesterolaemia (FH) samples. We compared a total of 595 DNA variations (488 common single nucleotide polymorphisms, 73 missense mutations, 9 nonsense mutations, 3 splice site point mutations, 13 small indels, 2 multi-exonic duplications and 7 multi-exonic deletions) found previously in the 96 FH samples. DNA variation detection sensitivity and specificity were both 100% for the SOLiD 5500 NGS platform compared with Sanger sequencing and MLPA only when both LifeScope and Integrative Genomics Viewer softwares were utilised. The methods described here offer a high-quality strategy for the detection of a wide range of DNA mutations in diseases with a moderate number of well described causative genes. However, there are important issues related to the bioinformatic algorithms employed to detect small indels.

DNA methylation: bisulphite modification and analysis.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs. This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing. DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al. and Clark et al., methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

Estimation of effective lens position using a method independent of preoperative keratometry readings.

PURPOSE: To evaluate the validity of a keratometry (K)-independent method of estimating effective lens position (ELP) before phacoemulsification cataract surgery. SETTING: Institute of Eye Surgery, Whitfield Clinic, Waterford, Ireland. DESIGN: Evaluation of diagnostic test or technology. METHODS: The anterior chamber diameter and corneal height in eyes scheduled for cataract surgery were measured with a rotating Scheimpflug camera. Corneal height and anterior chamber diameter were used to estimate the ELP in a K-independent method (using the SRK/T [ELP(rs)] and Holladay 1 [ELP(rh)] formulas). RESULTS: The mean ELP was calculated using the traditional (mean ELP(s) 5.59 mm ± 0.52 mm [SD]; mean ELP(h) 5.63 ± 0.42 mm) and K-independent (mean ELP(rs) 5.55 ± 0.42 mm; mean ELP(rh) ± SD 5.60 ± 0.36 mm) methods. Agreement between ELP(s) and ELP(rs) and between ELP(h) and ELP(rh) were represented by Bland-Altman plots, with mean differences (± 1.96 SD) of 0.06 ± 0.65 mm (range -0.59 to +0.71 mm; P=.08) in association with ELP(rs) and -0.04 ± 0.39 mm (range -0.43 to +0.35 mm; P=.08) in association with ELP(rh). The mean absolute error for ELP(s) versus ELP(rs) estimation and for ELP(h) versus ELP(rh) estimation was 0.242 ± 0.222 mm (range 0.001 to 1.272 mm) and 0.152 ± 0.137 mm (range 0.001 to 0.814 mm), respectively. CONCLUSION: This study confirms that the K-independent ELP estimation method is comparable to traditional K-dependent methods and may be useful in post-refractive surgery patients.

Value of dual biometry in the detection and investigation of error in the preoperative prediction of refractive status following cataract surgery.

PURPOSE: To report the value of dual biometry in the detection of biometry errors. METHODS: Study 1: retrospective study of 224 consecutive cataract operations. The intraocular lens power calculation was based on immersion biometry. Study 2: immersion biometry was compared with optical coherence biometry (OCB) in terms of axial length, anterior chamber depth, keratometry readings and the recommended lens power to achieve emmetropia. Study 3: prospective study of 61 consecutive cataract operations. Both immersion and OCB were performed, but lens power calculation was based on the latter. RESULTS: Study 1: 115 (86%), 101 (75.4%), 90 (67.2%) and 50 (37.3%) of postoperative spherical equivalents were within +/-1.5 dioptres (D), +/-1.25 D, +/-1 D and +/-0.5 D of the target, respectively. Study 2: excellent agreement between axial length readings, anterior chamber depth readings and keratometry readings by immersion biometry and OCB was observed (reflected in a mean bias of -0.065 mm, -0.048 mm and +0.1803 D, respectively, in association with OCB). Agreement between the lens power recommended by each technique to achieve emmetropia was poor (mean bias of +1.16 D in association with OCB), but improved following appropriate modification of lens constants in the Accutome A-scan software (mean bias with OCB = -0.4 D). Study 3: 37 (92.5%) and 23 (57.5%) of operated eyes achieved a postoperative refraction within +/-1 D and +/-0.5 D of target, respectively. CONCLUSION: Systematic errors in biometry can exist, in the presence of acceptable postoperative refractive results. Dual biometry allows each biometric parameter to be scrutinized in isolation, and identify sources of error that may otherwise go undetected.

Periconceptional undernutrition in normal and overweight ewes leads to increased adrenal growth and epigenetic changes in adrenal IGF2/H19 gene in offspring.

Adverse conditions in early life result in increased activation of the hypothalamo-pituitary-adrenal axis and in stress responsiveness in offspring. We have developed a model in which "donor" ewes are either normally nourished or overnourished prior to a period of dietary restriction, before transfer of the embryo at 6-7 d after conception to a ewe of normal weight and nutritional history. A moderate restriction of energy intake during the periconceptional period in both normal weight and overweight ewes resulted in increased adrenal mass in male and female lambs and an increased cortisol response to stress in female lambs. The increase in adrenal weight in lambs exposed to periconceptional undernutrition was associated with a decrease in the adrenal mRNA expression of IGF2 and decreased methylation in the proximal CTCF-binding site in the differentially methylated region of the IGF2/H19 gene. Thus, weight loss in both normal and overweight mothers during the periconceptional period results in epigenetic modification of IGF2 in the adrenal gland, adrenal overgrowth, and increased vulnerability to stress in offspring. Determining the appropriate approach to weight loss in the periconceptional period may therefore be important in overweight or obese women seeking to become pregnant.

Surgically induced astigmatism after phacoemulsification with and without correction for posture-related ocular cyclotorsion: randomized controlled study.

PURPOSE: To report the impact of posture-related ocular cyclotorsion on one surgeon's surgically induced astigmatism (SIA) results and the variance in SIA. SETTING: Institute of Eye Surgery, Whitfield Clinic, Waterford, Ireland. METHODS: This prospective randomized controlled study included eyes that had phacoemulsification with intraocular lens implantation. Eyes were randomly assigned to have (intervention group) or not have (control group) correction for posture-related ocular cyclotorsion. In the intervention group, the clear corneal incision was placed precisely at the 120-degree meridian with instruments designed to correct posture-related ocular cyclotorsion. In the control group, the surgeon endeavored to place the incision at the 120-degree meridian, but without markings. RESULTS: The intervention group comprised 41 eyes and the control group, 61 eyes. The mean absolute SIA was 0.74 diopters (D) in the intervention group and 0.78 D in the control group; the difference between groups was not statistically significant (P>.5, unpaired 2-tailed Student t test). The variance in SIA was 0.29 D(2) and 0.31 D(2), respectively; the difference between groups was not statistically significant (P>.5, unpaired F test). CONCLUSIONS: Attempts to correct for posture-related ocular cyclotorsion did not influence SIA or its variance in a single-surgeon series. These results should be interpreted with full appreciation of the limitations of currently available techniques to correct for posture-related ocular cyclotorsion in the clinical setting.