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.

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.

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.

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.

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.

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.

No major role of common SV2A variation for predisposition or levetiracetam response in epilepsy.

Levetiracetam (LEV), a newer antiepileptic drug (AED) useful for several epilepsy syndromes, binds to SV2A. Identifying genetic variants that influence response to LEV may allow more tailored use of LEV. Obvious candidate genes are SV2A, SV2B and SV2C, which encode the only known binding site, synaptic vesicle protein 2 (SV2), with LEV binding to the SV2A isoform. SV2A is an essential protein as homozygous SV2A knockout mice appear normal at birth but fail to grow, experience severe seizures and die by 3 weeks. We addressed characterising AED response issues in pharmacogenetics and whether variation in these genes associates with response to LEV in two independent cohorts with epilepsy. We also investigated whether variation in these three genes associated with epilepsy predisposition in two larger cohorts of patients with various epilepsy phenotypes. Common genetic variation in SV2A, encoding the actual binding site of LEV, was fully represented in this study whereas SV2B and SV2C were not fully covered. None of the polymorphisms tested in SV2A, SV2B or SV2C influence LEV response or predisposition to epilepsy. We found no association between genetic variation in SV2A, SV2B or SV2C and response to LEV or epilepsy predisposition. We suggest this study design may be used in future pharmacogenetic work examining AED or LEV efficacy. However, different study designs would be needed to examine common variation with minor effect sizes, or rare variation, influencing AED or LEV response or epilepsy predisposition.

Obesity is associated with genetic variants that alter dopamine availability.

Human and animal studies have implicated dopamine in appetite regulation, and family studies have shown that BMI has a strong genetic component. Dopamine availability is controlled largely by three enzymes: COMT, MAOA and MAOB, and by the dopamine transporter SLC6A3, and each gene has a well-characterized functional variant. Here we look at these four functional polymorphisms together, to investigate how heritable variation in dopamine levels influences the risk of obesity in a cohort of 1150, including 240 defined as obese (BMI > or = 30). The COMT and SLC6A3 polymorphisms showed no association with either weight, BMI or obesity risk. We found, however, that both MAOA and MAOB show an excess of the low-activity genotypes in obese individuals (MAOA:chi2= 15.45, p = 0.004; MAOB:chi2= 8.05, p = 0.018). Additionally, the MAOA genotype was significantly associated with both weight (p = 0.0005) and BMI (p = 0.001). When considered together, the 'at risk genotype'--low activity genotypes at both the MAOA and MAOB loci--shows a relative risk for obesity of 5.01. These results have not been replicated and, given the experience of complex trait genetics, warrant caution in interpretation. In implicating both the MAOA and MOAB variants, however, this study provides the first indication that dopamine availability (as opposed to other effects of MAOA) is involved in human obesity. It is therefore a priority to assess the associations in replication datasets.

Population structure in the Mediterranean basin: a Y chromosome perspective.

The Mediterranean region has been characterised by a number of pre-historical and historical demographic events whose legacy on the current genetic landscape is still a matter of debate. In order to investigate the degree of population structure across the Mediterranean, we have investigated Y chromosome variation in a large dataset of Mediterranean populations, 11 of which are first described here. Our analyses identify four main clusters in the Mediterranean that can be labelled as North Africa, Arab, Central-East and West Mediterranean. In particular, Near Eastern samples tend to separate according to the presence of Arab Y chromosome lineages, suggesting that the Arab expansion played a major role in shaping the current genetic structuring within the Fertile Crescent.

The alpha-synuclein gene in multiple system atrophy.

BACKGROUND: The formation of alpha-synuclein aggregates may be a critical event in the pathogenesis of multiple system atrophy (MSA). However, the role of this gene in the aetiology of MSA is unknown and untested. METHOD: The linkage disequilibrium (LD) structure of the alpha-synuclein gene was established and LD patterns were used to identify a set of tagging single nucleotide polymorphisms (SNPs) that represent 95% of the haplotype diversity across the entire gene. The effect of polymorphisms on the pathological expression of MSA in pathologically confirmed cases was also evaluated. RESULTS AND CONCLUSION: In 253 Gilman probable or definite MSA patients, 457 possible, probable, and definite MSA cases and 1472 controls, a frequency difference for the individual tagging SNPs or tag-defined haplotypes was not detected. No effect was observed of polymorphisms on the pathological expression of MSA in pathologically confirmed cases.

ABCB1/MDR1 gene determines susceptibility and phenotype in ulcerative colitis: discrimination of critical variants using a gene-wide haplotype tagging approach.

Several lines of evidence suggest a role for the multidrug resistance gene (ABCB1/MDR1) and its product, P-glycoprotein 170, in the pathogenesis of inflammatory bowel disease (IBD). In addition, P-glycoprotein activity determines bioavailability of many drugs used regularly in many medical specialties, and ABCB/MDR1 variation appears to be a critical pharmacogenetic determinant. We have utilized a gene-wide haplotype tagging approach to further define the identity of germ-line variations in the ABCB1/MDR1 gene contributing to IBD susceptibility. Six haplotype tagging single nucleotide polymorphisms (tSNPs) representing the haplotypic variations of the ABCB1/MDR1 gene were identified initially following the characterization of the haplotype structure of this gene in 24 Centre d'Etude du Polymorphisme Humain Caucasian trios. Genotyping was performed in 249 ulcerative colitis (UC) and 179 Crohn's disease (CD) patients and 260 healthy controls. Using log-likelihood analysis, we observed a highly significant association between the common haplotypes and UC (P=4.22 x 10(-7)) but not CD (P=0.22). This significant association was critically dependent on one tSNP, intronic variant rs3789243. All haplotypes with this variant retained a highly significant association (P=3.2 x 10(-7)-3.6 x 10(-12)), whereas significance was lost when rs3789243 was dropped in systematic haplotypic analysis. The effect of this tSNP was independent of C3435T SNP, previously suggested to be the critical variant in disease susceptibility and drug transport. The association with UC was shown to be strongest with the phenotype of extensive disease (P=1.7 x 10(-7)). This 'candidate gene' approach provides compelling evidence to support the contribution of the ABCB1/MDR1 gene in determining risk to UC but not to CD and provides new insights into the localization of the critical susceptibility determinants within the gene. In addition, these findings have potentially important implications in the application of pharmacogenetics across a range of common diseases, including HIV, epilepsy and colorectal cancer.

Population genetic approaches to neurological disease: Parkinson's disease as an example.

Parkinson's disease (PD) is a common, progressive, incurable disabling condition. The cause is unknown but over the past few years tremendous progress in our understanding of the genetic bases of this condition has been made. To date, this has almost exclusively come from the study of relatively rare Mendelian forms of the disease and there are no currently, widely accepted common variants known to increase susceptibility. The role that the "Mendelian" genes play in common sporadic forms of PD is unknown. Moreover, most studies in PD can really be described as candidate polymorphism studies rather than true and complete assessments of the genes themselves. We provide a model of how one might tackle some of these issues using Parkinson's disease as an illustration. One of the emerging hypotheses of gene environment interaction in Parkinson's disease is based on drug metabolizing (or xenobiotic) enzymes and their interaction with putative environmental toxins. This motivated us to describe a tagging approach for an extensive but not exhaustive list of 55 drug metabolizing enzyme genes. We use these data to illustrate the power, and some of the limitations of a haplotype tagging approach. We show that haplotype tagging is extremely efficient and works well with only a modest increase in effort through different populations. The tagging approach works much less well if the minor allele frequency is below 5%. However, it will now be possible using these tags to evaluate these genes comprehensively in PD and other neurodegenerative conditions.

The -1021C->T DBH gene variant is not associated with epilepsy or antiepileptic drug response.

Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine (NE). Animal studies show that genes in the NE pathway are candidates for susceptibility to epilepsy and antiepileptic drug (AED) response. The authors genotyped the -1021C-->T major functional polymorphism in the DBH gene in 675 patients with epilepsy and 1,087 controls. The authors found no association with epilepsy, several epilepsy subtypes, or AED response. The results suggest that the -1021C-->T variant does not contribute to epilepsy.

Quantitative genetic analysis of natural variation in body size in Drosophila melanogaster.

Latitudinal, genetic variation in body size is a commonly observed phenomenon in many invertebrate species and is shaped by natural selection. In this study, we use a chromosome substitution and a quantitative trait locus (QTL) mapping approach to identify chromosomes and genomic regions associated with adaptive variation in body size in natural populations of Drosophila melanogaster from the extreme ends of clines in South America and Australia. Chromosome substitution revealed the largest effects on chromosome three in both continents, and minor effects on the X and second chromosome. Similarly, QTL analysis of the Australian cline identified QTL with largest effects on the third chromosome, with smaller effects on the second. However, no QTL were found on the X chromosome. We also compared the coincidence of locations of QTL with the locations of five microsatellite loci previously shown to vary clinally in Australia. Permutation tests using both the sum of the LOD scores and the sum distance to nearest QTL peak revealed there were no significant associations between locations of clinal markers and QTL's. The lack of significance may, in part, be due to broad QTL peaks identified in this study. Future studies using higher resolution QTL maps should reveal whether the degree of clinality in microsatellite allele frequencies can be used to identify QTL in traits that vary along an environmental gradient.

The genetic bases of divergence in desiccation and starvation resistance among tropical and temperate populations of Drosophila melanogaster.

Desiccation and starvation resistance are two stress-related traits which vary geographically with climate in Drosophila melanogaster. To investigate the contribution of epistasis to population divergence for these traits, we crossed tropical and temperate populations from two different geographical regions to produce F1, F2 and first backcross generations. Line-cross analysis of generation means revealed that genetic bases of divergence for both traits were complex and remarkably similar in a number of respects. Strong additive and dominance effects were present in most of the models, whereas epistatic and maternal effects were less common. The presence of epistatic effects in approximately half of the models presented in this study is consistent with line-cross studies of diverged traits in other animals, and does not support the view that epistasis is the predominant means by which populations diverge. In addition, evidence of maternal effects in both traits adds to a growing body of recent evidence that suggests maternal contributions to population differentiation are more widespread than previously thought. This finding undermines the accuracy of studies inferring epistasis directly from the magnitude of F2 breakdown. More line-cross analysis studies of naturally diverged populations that take into account maternal effects will shed further light on the true incidence of epistasis and its importance in the evolutionary process.

Genetic sampling error of distance (delta(mu))2 and variation in mutation rate among microsatellite loci.

An expression is obtained for the time-dependent variance of the microsatellite genetic distance (delta(mu))2 when the mutation rate is allowed to vary randomly among loci. An estimator is presented for the coefficient of variation, C(w), in the mutation rate. Estimated values of C(w) from genetic distances between African and non-African populations were less than 100%. Caveats to this conclusion are discussed.

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.

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.