Redefining tissue specificity of genetic regulation of gene expression in the presence of allelic heterogeneity

Understanding the mechanisms that underlie genetic regulation of gene expression is crucial to explaining the diversity that governs complex traits. Large scale expression quantitative trait locus (eQTL) studies have been instrumental in identifying genetic variants that influence the expression of target genes. However, a large fraction of disease-associated genetic variants have not been clearly explained by current eQTL data, frustrating attempts to use these data to comprehensively characterize disease loci. One notable observation from recent studies is that cis-eQTL effects are often shared across different cell types and tissues. This would suggest that common genetic variants impacting steady-state, adult gene expression are largely tolerated, shared across tissues, and less relevant to disease. However, allelic heterogeneity and complex patterns of linkage disequilibrium (LD) within each locus may skew the quantification of sharing of genetic effects between tissues, impede our ability to identify causal variants, and hinder the identification of regulatory effects for disease-associated genetic variants. Indeed, recent research suggests that multiple causal variants are often present in many eQTL and complex trait associated loci. Here, we re-analyze tissue-specificity of genetic effects in the presence of LD and allelic heterogeneity, proposing a novel method, CAFEH, that improves the identification of causal regulatory variants across tissues and their relationship to disease loci.

Extensive pleiotropism and allelic heterogeneity mediate metabolic effects of IRX3 and IRX5

Whereas coding variants often have pleiotropic effects across multiple tissues, noncoding variants are thought to mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. Here, we investigated the genetic and functional architecture of a genomic region within the FTO gene that is strongly associated with obesity risk. We show that multiple variants on a common haplotype modify the regulatory properties of several enhancers targeting IRX3 and IRX5 from megabase distances. We demonstrate that these enhancers affect gene expression in multiple tissues, including adipose and brain, and impart regulatory effects during a restricted temporal window. Our data indicate that the genetic architecture of disease-associated loci may involve extensive pleiotropy, allelic heterogeneity, shared allelic effects across tissues, and temporally restricted effects.

MO045THE APPLICATION OF A NGS KIDNEY PANEL REVEALED KEY CHALLENGES OF PKD1-2 ANALYSIS: INTERPRETATION OF MISSENSE VARIANTS, SIGNIFICANCE OF VARIANTS IN DUPLICATED REGIONS AND HIGH ALLELIC HETEROGENEITY

Background and Aims Genetic testing has changed the clinical management of inherited kidney diseases patients, improving prognosis, surveillance and therapy. On the other hand, it has put geneticists and clinicians in front of new challenges, as the heterogeneity of these disorders and the high number of variants, with no clear genotype-phenotype correlation. Method 108 patients underwent genetic analysis through a kidney focused NGS panel, named Nephroplex, containing 119 genetic loci associated with inherited kidney disorders. The study aimed to addressed the genetic landscape of cystic individuals and to analyze PKD1 and PKD2 variants in non-cystic individuals. Results Following diagnostic criteria, patients were divided as cystic kidney diseases (n=36) and non-cystic kidney diseases (n=72). Among the group of cystic patients, a causative mutation was detected in 51% of cases. We found thirty-seven PKD1 and PKD2 variants in 26 out of 35 individuals. In particular, 12 variants were shown to be damaging and nine of that were reported in public database, as CLINVAR and Mayo Clinic databases. Among pathogenic variants, twelve were truncating and the remaining were missense variants. Of note, 7 out of 12 damaging PKD1 mutations were located in duplicated regions. Moreover, in three cystic patients, we found a (i) a frameshift hemizygote OFD1 mutation (ii) compound heterozygote PKHD1 variants and (iii) a frameshift MUC1 variant, framing the diagnosis of oro-facio-digital type 1, autosomal recessive polycystic kidney disease and autosomal dominant tubulointerstitial disease, respectively. Interestingly, we detected 28 PKD1-2 rare variants in 21 out of 75 adult non cystic patients (28%). The most were observed in PKD1 genes (82% vs 18% in PKD2). Eighteen of 28 variants were described in the literature as likely benign or as mutations of uncertain significance, while we found 10 novel variants. In silico analysis revealed as pathogenic a frameshift mutation located in exon 15. Of note, the great part of these variations reside into the duplicated PKD1 regions. Conclusion Our data showed that genetic analysis of ADPKD retains unique challenges, given the high degree of homology of PKD1 with his pseudogenes and the high allelic heterogeneity in non-cystic individuals.

Hypophosphatasia: A Unique Disorder of Bone Mineralization

Hypophosphatasia (HPP) is a rare genetic disease characterized by a decrease in the activity of tissue non-specific alkaline phosphatase (TNSALP). TNSALP is encoded by the ALPL gene, which is abundantly expressed in the skeleton, liver, kidney, and developing teeth. HPP exhibits high clinical variability largely due to the high allelic heterogeneity of the ALPL gene. HPP is characterized by multisystemic complications, although the most common clinical manifestations are those that occur in the skeleton, muscles, and teeth. These complications are mainly due to the accumulation of inorganic pyrophosphate (PPi) and pyridoxal-5′-phosphate (PLP). It has been observed that the prevalence of mild forms of the disease is more than 40 times the prevalence of severe forms. Patients with HPP present at least one mutation in the ALPL gene. However, it is known that there are other causes that lead to decreased alkaline phosphatase (ALP) levels without mutations in the ALPL gene. Although the phenotype can be correlated with the genotype in HPP, the prediction of the phenotype from the genotype cannot be made with complete certainty. The availability of a specific enzyme replacement therapy for HPP undoubtedly represents an advance in therapeutic strategy, especially in severe forms of the disease in pediatric patients.

MRLocus: Identifying causal genes mediating a trait through Bayesian estimation of allelic heterogeneity

Expression quantitative trait loci (eQTL) studies are used to understand the regulatory function of non-coding genome-wide association study (GWAS) risk loci, but colocalization alone does not demonstrate a causal relationship of gene expression affecting a trait. Evidence for mediation, that perturbation of gene expression in a given tissue or developmental context will induce a change in the downstream GWAS trait, can be provided by two-sample Mendelian Randomization (MR). Here, we introduce a new statistical method, MRLocus, for Bayesian estimation of the gene-to-trait effect from eQTL and GWAS summary data for loci with evidence of allelic heterogeneity, that is, containing multiple causal variants. MRLocus makes use of a colocalization step applied to each nearly-LD-independent eQTL, followed by an MR analysis step across eQTLs. Additionally, our method involves estimation of the extent of allelic heterogeneity through a dispersion parameter, indicating variable mediation effects from each individual eQTL on the downstream trait. Our method is evaluated against other state-of-the-art methods for estimation of the gene-to-trait mediation effect, using an existing simulation framework. In simulation, MRLocus often has the highest accuracy among competing methods, and in each case provides more accurate estimation of uncertainty as assessed through interval coverage. MRLocus is then applied to five candidate causal genes for mediation of particular GWAS traits, where gene-to-trait effects are concordant with those previously reported. We find that MRLocus’s estimation of the causal effect across eQTLs within a locus provides useful information for determining how perturbation of gene expression or individual regulatory elements will affect downstream traits. The MRLocus method is implemented as an R package available at https://mikelove.github.io/mrlocus.

Giant axonal neuropathy: The first Iranian case with a variation in the gigaxonin gene and a glance to the other cases

Background: Giant axonal neuropathy (GAN) is a very rare fatal neurodegenerative disorder with clinical and allelic heterogeneity. The disease is caused by mutations in the GAN (gigaxonin) gene. Herein, we reported the clinical presentations and results of genetic analysis of the first Iranian GAN case. Methods: Phenotypic data were obtained by neurologic examination, brain magnetic resonance imaging (MRI), electromyography (EMG), electroencephalography (EEG), and sonography from the proband. Deoxyribonucleic acid (DNA) was isolated from peripheral blood leucocytes and whole exome sequencing (WES) was performed. The candidate variant was screened by Sanger sequencing in the proband and her family members. Results: The proband was a 7-year-old girl who was admitted with a chief complaint of ataxia, muscle weakness, delayed developmental milestones, and history of psychiatric disorders. She was very moody and had clumsy gait, decreased deep tendon reflexes (DTRs) of lower limbs, and kinky hair. The brain MRI revealed white matter abnormality. The EMG revealed that her disease was compatible with the chronic axonal type of sensorimotor polyneuropathy; however, her EEG was normal. Results of the WES revealed a homozygous variant; c.G778T:p.E260* in the GAN gene, indicating the GAN disorder. Conclusion: The present study affirmed GAN allelic heterogeneity and resulted in the expansion of the phenotypic spectrum of GAN pathogenic variants. Identification of more families with mutations in GAN gene helps to further understand the molecular basis of the disease and provides an opportunity for genetic counseling especially in the populations with a high degree of consanguineous marriage such as the Iranian population.

Cystic fibrosis in Tuscany: evolution of newborn screening strategies over time to the present

Background Cystic fibrosis (CF) is a life-threatening disease affecting about 1:3000 newborns in Caucasian populations. The introduction of newborn screening for cystic fibrosis (CF NBS) has improved the clinical outcomes of individuals with CF through early diagnosis and early treatment. NBS strategies have been implemented over time. CF NBS was introduced extensively in 1984 in Tuscany, a region with 3.7 million people, characterized by a high allelic heterogeneity of CFTR gene. Aim and methods The aim of the study is to present the results from 34 years (1984–2018) of CF NBS, retrospectively evaluating the sensitivity, specificity and predictive values of the tests. In particular, we studied the impact of the introduction of DNA molecular analysis in NBS in a region with high allelic heterogeneity, such as Tuscany. Results Over these 34 years, 919,520 neonates were screened, using four different NBS strategies. From 1984 to 1991, CF NBS was performed by the determination of albumin on dried meconium (sensitivity 68.75%; specificity 99.82%). Subsequently, the analysis of immunoreactive trypsinogen on a blood spot was adopted as CF NBS protocol (sensitivity 83.33%; specificity 99.77%). From 1992 to 2010, this strategy was associated with lactase meconium dosage: IRT1/IRT2 + LACT protocol (sensitivity 87.50%; specificity 99.82%). From 2011, when the existing algorithm was integrated by analysis of CF causing variants of the CFTR gene (IRT1/IRT2 + LACT + IRT1/DNA protocol), a substantial improvement in sensitivity was seen (senisitivity 96.15%; specificity 99.75%). Other improved parameters with DNA analysis in the NBS programme, compared with the previous method, were the diagnosis time (52 days vs. 38 days) and the recall rate (0.58 to 0.38%). Conclusion The inclusion of DNA analysis in the NBS was a fundamental step in improving sensitivity, even in a region with high allelic variability.