Fetal exome sequencing for isolated increased nuchal translucency: should we be doing it?

OBJECTIVE: To evaluate the utility of prenatal exome sequencing (ES) for isolated increased nuchal translucency (NT) and to investigate factors that increase diagnostic yield. DESIGN: Retrospective analysis of data from two prospective cohort studies. SETTING: Fetal medicine centres in the UK and USA. POPULATION: Fetuses with increased NT ≥3.5 mm at 11-14 weeks of gestation recruited to the Prenatal Assessment of Genomes and Exomes (PAGE) and Columbia fetal whole exome sequencing studies (n = 213). METHODS: We grouped cases based on (1) the presence of additional structural abnormalities at presentation in the first trimester or later in pregnancy, and (2) NT measurement at presentation. We compared diagnostic rates between groups using Fisher exact test. MAIN OUTCOME MEASURES: Detection of diagnostic genetic variants considered to have caused the observed fetal structural anomaly. RESULTS: Diagnostic variants were detected in 12 (22.2%) of 54 fetuses presenting with non-isolated increased NT, 12 (32.4%) of 37 fetuses with isolated increased NT in the first trimester and additional abnormalities later in pregnancy, and 2 (1.8%) of 111 fetuses with isolated increased NT in the first trimester and no other abnormalities on subsequent scans. Diagnostic rate also increased with increasing size of NT. CONCLUSIONS: The diagnostic yield of prenatal ES is low for fetuses with isolated increased NT but significantly higher where there are additional structural anomalies. Prenatal ES may not be appropriate for truly isolated increased NT but timely, careful ultrasound scanning to identify other anomalies emerging later can direct testing to focus where there is a higher likelihood of diagnosis.

Guidelines for investigating causality of sequence variants in human disease.

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.

Practical considerations in the clinical application of whole-exome sequencing.

Despite the exciting advent of whole-exome sequencing (WES) in medical genetics practices, the optimal interpretation of results requires further actions such as reconsidering clinical information and obtaining further laboratory testing. There are no published data to guide clinicians in this process. In a retrospective study on 93 patients who underwent clinical WES, we set out to assess and resolve these practical challenges. With the laboratories reporting a molecular diagnostic rate of 25.8%, the medical geneticists and the laboratories were 90% concordant in their interpretation of the WES results. Divergence occurred when the medical geneticist reconsidered clinical information and/or additional information regarding pathogenicity of a variant. Variants of uncertain significance were reported in 86% of patients, with 53.7% needing follow-up, such as additional laboratory tests and genotyping of family members. By layering clinical data (e.g. mode of inheritance and phenotypic fit) on to the laboratory results, we developed clinical categories for the WES results. These categories of definite diagnosis (14/93), likely diagnosis (8/93), possible diagnosis (13/93) and no diagnosis (58/93) could be used to convey results to patients uniformly. Our framework for a clinically informed interpretation of the results enhances the utility of WES within medical genetics practices.

The RBMX gene as a candidate for the Shashi X-linked intellectual disability syndrome.

A novel X-linked intellectual disability (XLID) syndrome with moderate intellectual disability and distinguishing craniofacial dysmorphisms had been previously mapped to the Xq26-q27 interval. On whole exome sequencing in the large family originally reported with this disorder, we identified a 23 bp frameshift deletion in the RNA binding motif protein X-linked (RBMX) gene at Xq26 in the affected males (n = 7), one carrier female, absent in unaffected males (n = 2) and in control databases (7800 exomes). The RBMX gene has not been previously causal of human disease. We examined the genic intolerance scores for the coding regions and the non-coding regions of RBMX; the findings were indicative of RBMX being relatively intolerant to loss of function variants, a distinctive pattern seen in a subset of XLID genes. Prior expression and animal modeling studies indicate that loss of function of RBMX results in abnormal brain development. Our finding putatively adds a novel gene to the loci associated with XLID and may enable the identification of other individuals affected with this distinctive syndrome.

SLC1A4 mutations cause a novel disorder of intellectual disability, progressive microcephaly, spasticity and thin corpus callosum.

Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole-exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi-Iraqi patient is compound heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurologic disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.

Islands of linkage disequilibrium.

A detailed knowledge of patterns of linkage disequilibrium in human populations is widely seen as a prerequisite for effective population-based disease gene mapping. New data suggest that linkage disequilibrium is highly structured into discrete blocks of sequence separated by hot spots of recombination.

Population genetic structure of variable drug response.

Geographic patterns of genetic variation, including variation at drug metabolizing enzyme (DME) loci and drug targets, indicate that geographic structuring of inter-individual variation in drug response may occur frequently. This raises two questions: how to represent human population genetic structure in the evaluation of drug safety and efficacy, and how to relate this structure to drug response. We address these by (i) inferring the genetic structure present in a heterogeneous sample and (ii) comparing the distribution of DME variants across the inferred genetic clusters of individuals. We find that commonly used ethnic labels are both insufficient and inaccurate representations of the inferred genetic clusters, and that drug-metabolizing profiles, defined by the distribution of DME variants, differ significantly among the clusters. We note, however, that the complexity of human demographic history means that there is no obvious natural clustering scheme, nor an obvious appropriate degree of resolution. Our comparison of drug-metabolizing profiles across the inferred clusters establishes a framework for assessing the appropriate level of resolution in relating genetic structure to drug response.

Interleukin 28B polymorphisms are the only common genetic variants associated with low-density lipoprotein cholesterol (LDL-C) in genotype-1 chronic hepatitis C and determine the association between LDL-C and treatment response.

Low-density lipoprotein cholesterol (LDL-C) levels and interleukin 28B (IL28B) polymorphism are associated with sustained viral response (SVR) to peginterferon/ribavirin (pegIFN/RBV) for chronic hepatitis C (CHC) infection. IL28B has been linked with LDL-C levels using a candidate gene approach, but it is not known whether other genetic variants are associated with LDL-C, nor how these factors definitively affect SVR. We assessed genetic predictors of serum lipid and triglyceride levels in 1604 patients with genotype 1 (G1) chronic hepatitis C virus (HCV) infection by genome-wide association study and developed multivariable predictive models of SVR. IL28B polymorphisms were the only common genetic variants associated with pretreatment LDL-C level in Caucasians (rs12980275, P = 4.7 × 10(-17), poor response IL28B variants associated with lower LDL-C). The association was dependent on HCV infection, IL28B genotype was no longer associated with LDL-C in SVR patients after treatment, while the association remained significant in non-SVR patients (P < 0.001). LDL-C was significantly associated with SVR for heterozygous IL28B genotype patients (P < 0.001) but not for homozygous genotypes. SVR modelling suggested that IL28B heterozygotes with LDL-C > 130 mg/dL and HCV RNA ≤600 000 IU/mL may anticipate cure rates >80%, while the absence of these two criteria was associated with an SVR rate of <35%. IL28B polymorphisms are the only common genetic variants associated with pretreatment LDL-C in G1-HCV. LDL-C remains significantly associated with SVR for heterozygous IL28B genotype patients, where LDL-C and HCV RNA burden may identify those patients with high or low likelihood of cure with pegIFN/RBV therapy.

Alcohol dependence produced in mice by inhalation of ethanol: grading the withdrawal reaction.

Intoxicating blood levels of ethanol are maintained for several days in mice housed in an atmosphere of ethanol vapor. On removal from the alcohol, all the mice develop withdrawal signs. The signs can be graded to indicate the time course and intensity of the withdrawal reaction.

Genealogical and evolutionary inference with the human Y chromosome.

Population genetics has emerged as a powerful tool for unraveling human history. In addition to the study of mitochondrial and autosomal DNA, attention has recently focused on Y-chromosome variation. Ambiguities and inaccuracies in data analysis, however, pose an important obstacle to further development of the field. Here we review the methods available for genealogical inference using Y-chromosome data. Approaches can be divided into those that do and those that do not use an explicit population model in genealogical inference. We describe the strengths and weaknesses of these model-based and model-free approaches, as well as difficulties associated with the mutation process that affect both methods. In the case of genealogical inference using microsatellite loci, we use coalescent simulations to show that relatively simple generalizations of the mutation process can greatly increase the accuracy of genealogical inference. Because model-free and model-based approaches have different biases and limitations, we conclude that there is considerable benefit in the continued use of both types of approaches.

Drug tolerance in biomembranes: a spin label study of the effects of ethanol.

Ethanol in vitro increased the fluidity of spin-labeled membranes from normal mice. Membranes from mice that had been subjected to long-term ethanol treatment were relatively resistant to this fluidizing effect. The data suggest that the membranes themselves had adapted to the drug, a novel form of drug tolerance.

Population genomics: linkage disequilibrium holds the key.

Current efforts to find disease-causing genes depend on patterns of linkage disequilibrium in human populations. Recent work has shown that linkage disequilibrium can extend over much larger genomic regions than expected, and that the patterns of linkage disequilibrium can differ markedly among populations.

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice.

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanol-treated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.

Heterozygote advantage and the evolution of a dominant diploid phase.

The life cycle of eukaryotic, sexual species is divided into haploid and diploid phases. In multicellular animals and seed plants, the diploid phase is dominant, and the haploid phase is reduced to one, or a very few cells, which are dependent on the diploid form. In other eukaryotic species, however, the haploid phase may dominate or the phases may be equally developed. Even though an alternation between haploid and diploid forms is fundamental to sexual reproduction in eukaryotes, relatively little is known about the evolutionary forces that influence the dominance of haploidy or diploidy. An obvious genetic factor that might result in selection for a dominant diploid phase is heterozygote advantage, since only the diploid phase can be heterozygous. In this paper, I analyze a model designed to determine whether heterozygote advantage could lead to the evolution of a dominant diploid phase. The main result is that heterozygote advantage can lead to an increase in the dominance of the diploid phase, but only if the diploid phase is already sufficiently dominant. Because the diploid phase is unlikely to be increased in organisms that are primarily haploid, I conclude that heterozygote advantage is not a sufficient explanation of the dominance of the diploid phase in higher plants and animals.

Recombination and the evolution of diploidy.

With two copies of every gene, a diploid organism is able to mask recessive deleterious mutations. In this paper we present the analysis of a two-locus model designed to determine when the masking of deleterious alleles favors the evolution of a dominant diploid phase in organisms that alternate between haploid and diploid phases ("alternation of generations"). It is hypothesized that diploidy will be favored whenever masking occurs ("the masking hypothesis"). Using analytical methods, we confirm that this masking hypothesis is essentially correct under free recombination: as long as the heterozygous expression of deleterious alleles is sufficiently masked by the wild-type allele, diploidy is favored over haploidy. When the rate of recombination is lower, however, diploidy is much less likely to be favored over haploidy. In fact, according to our model, the evolution of diploidy is impossible without significant levels of recombination even when masking is fairly strong.

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.

A quantitative genetic analysis of male sexual traits distinguishing the sibling species Drosophila simulans and D. sechellia.

A quantitative trait locus (QTL) genetic analysis of morphological and reproductive traits distinguishing the sibling species Drosophila simulans and D. sechellia was carried out in a backcross design, using 38 markers with an average spacing of 8.4 cM. The direction of QTL effects for the size of the posterior lobe was consistent across the identified QTL, indicating directional selection for this trait. Directional selection also appears to have acted on testis length, indicating that sexual selection may have influenced many reproductive traits, although other forms of directional selection cannot be ruled out. Sex comb tooth number exhibited high levels of variation both within and among isofemale lines and showed no evidence for directional selection and, therefore, may not have been involved in the early speciation process. A database search for genes associated with significant QTL revealed a set of candidate loci for posterior lobe shape and size, sex comb tooth number, testis length, tibia length, and hybrid male fertility. In particular, decapentaplegic (dpp), a gene known to influence the genital arch, was found to be associated with the largest LOD peak for posterior lobe shape and size.

Microsatellite genetic distances with range constraints: analytic description and problems of estimation.

Statistical properties of the symmetric stepwise-mutation model for microsatellite evolution are studied under the assumption that the number of repeats is strictly bounded above and below. An exact analytic expression is found for the expected products of the frequencies of alleles separated by k repeats. This permits characterization of the asymptotic behavior of our distances D1 and (delta mu)2 under range constraints. Based on this characterization we develop transformations that partially restore linearity when allele size is restricted. We show that the appropriate transformation cannot be applied in the case of varying mutation rates (beta) and range constraints (R) because of statistical difficulties. In the special case of no variation in beta and R across loci, however, the transformation simplifies to a usable form and results in a distance much more linear with time than distances developed for an infinite range. Although analytically incorrect in the case of variation in beta and R, the simpler transformation is surprisingly insensitive to variation in these parameters, suggesting that it may have considerable utility in phylogenetic studies.

An evaluation of genetic distances for use with microsatellite loci.

Mutations of alleles at microsatellite loci tend to result in alleles with repeat scores similar to those of the alleles from which they were derived. Therefore the difference in repeat score between alleles carries information about the amount of time that has passed since they shared a common ancestral allele. This information is ignored by genetic distances based on the infinite alleles model. Here we develop a genetic distance based on the stepwise mutation model that includes allelic repeat score. We adapt earlier treatments of the stepwise mutation model to show analytically that the expectation of this distance is a linear function of time. We then use computer simulations to evaluate the overall reliability of this distance and to compare it with allele sharing and Nei's distance. We find that no distance is uniformly superior for all purposes, but that for phylogenetic reconstruction of taxa that are sufficiently diverged, our new distance is preferable.

A low mutation rate for chloroplast microsatellites.

We used chloroplast simple sequence repeats (cpSSRs) to examine whether there is any variation present in the chloroplast genome of Pinus torreyana (Parry ex Carrière) that may previously not have been detected using RFLPs. Analysis of 17 cpSSR loci showed no variation, which is consistent with previous cpRFLP work and confirms that the species is descended from an original, highly monomorphic population following a bottleneck. This lack of biological variation in the chloroplast genome of P. torreyana allowed us to estimate the mutation rates at cpSSR loci as between 3. 2 x 10(-5) and 7.9 x 10(-5). This estimate is lower than published mutation rates at nuclear SSR loci but higher than substitution rates elsewhere in the chloroplast genome.