GCAT|Panel, a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing.

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.

Internal validation and improvement of mitochondrial genome sequencing using the Precision ID mtDNA Whole Genome Panel.

With the recent advances in next-generation sequencing (NGS), mitochondrial whole-genome sequencing has begun to be applied to the field of the forensic biology as an alternative to the traditional Sanger-type sequencing (STS). However, experimental workflows, commercial solutions, and output data analysis must be strictly validated before being implemented into the forensic laboratory. In this study, we performed an internal validation for an NGS-based typing of the entire mitochondrial genome using the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) on the Ion S5 sequencer (Thermo Fisher Scientific). Concordance, repeatability, reproducibility, sensitivity, and heteroplasmy detection analyses were assessed using the 2800 M and 9947A standard control DNA as well as typical casework specimens, and results were compared with conventional Sanger sequencing and another NGS sequencer in a different laboratory. We discuss the strengths and limitations of this approach, highlighting some issues regarding noise thresholds and heteroplasmy detection, and suggesting solutions to mitigate these effects and improve overall data interpretation. Results confirmed that the Precision ID Whole mtDNA Genome Panel is highly reproducible and sensitive, yielding useful full mitochondrial DNA sequences also from challenging DNA specimens, thus providing further support for its use in forensic practice.

The impact of non-additive genetic associations on age-related complex diseases.

Genome-wide association studies (GWAS) are not fully comprehensive, as current strategies typically test only the additive model, exclude the X chromosome, and use only one reference panel for genotype imputation. We implement an extensive GWAS strategy, GUIDANCE, which improves genotype imputation by using multiple reference panels and includes the analysis of the X chromosome and non-additive models to test for association. We apply this methodology to 62,281 subjects across 22 age-related diseases and identify 94 genome-wide associated loci, including 26 previously unreported. Moreover, we observe that 27.7% of the 94 loci are missed if we use standard imputation strategies with a single reference panel, such as HRC, and only test the additive model. Among the new findings, we identify three novel low-frequency recessive variants with odds ratios larger than 4, which need at least a three-fold larger sample size to be detected under the additive model. This study highlights the benefits of applying innovative strategies to better uncover the genetic architecture of complex diseases.

Performance of four models for eye color prediction in an Italian population sample.

Forensic DNA phenotyping (FDP) has recently provided important advancements in forensic investigations, by predicting the physical appearance of a subject from a biological sample, using SNP markers. The majority of operable prediction models have been developed for iris color; however, replication studies to understand their applicability on a worldwide scale are still limited for many of them. In this work, 4 models for eye color prediction (IrisPlex, Ruiz, Allwood and Hart models) were systematically evaluated in a sample of 296 subjects of Italian origin. Genotypes were determined by a custom NGS-based panel targeting all the predictive SNPs included in the 4 tested models. Overall, 60-69% of the Italian sample could be correctly predicted with the IrisPlex, Ruiz and Allwood models, applying the recommended threshold. The IrisPlex model showed the lowest frequency of errors (17%), but also the highest number of inconclusive results (18%). In the absence of the threshold, the highest proportion of correct predictions was again obtained with the IrisPlex model (76%), followed by the Allwood (73%) and the Ruiz (65%) models. Lastly, the Hart predictive algorithm had the lowest error rate (2%), but the majority of predictions (87%) were restricted to the less informative categories of "not-blue" and "not-brown", and correct color predictions were obtained only for 11% of the sample. As observed in previous studies, the majority of incorrect and undefined predictions were ascribable to the intermediate category, which represented 25% of the Italian sample. An adjustment of the IrisPlex (multinomial logistic regression) and Ruiz models (Snipper Bayesian classifier) with Italian allele frequencies gave only minor improvements in predicting intermediate eye color and no remarkable overall changes in performance. This suggests an incomplete knowledge underlying the intermediate colors. Considering the impact of this phenotype in the Italian sample as well as in other admixed populations, future improvements of eye color prediction methods should include a better genetic and phenotypic characterization of this category.

Clinical and genetic characterization of an Italian family with slow-channel syndrome.

INTRODUCTION: The slow-channel congenital myasthenic syndrome (SCCMS) is a postsynaptic form of congenital myasthenic syndromes (CMSs), a clinically heterogeneous group of disorders caused by genetic defects leading to an abnormal signal transmission at the endplate. METHODS: We report clinical and molecular data of a multigenerational family in which the presentation of a progressive proximal-distal weakness with ocular involvement led to a number of different clinical diagnoses. RESULTS: A comprehensive genetic study which included whole-genome linkage analysis and whole-exome sequencing identified a heterozygous missense substitution (c.721C>T, p.L241F) in the ε subunit of the acetylcholine receptor (CHRNE) that was consistent with clinical weakness in all patients. DISCUSSION: SCCMS is characterized by a broad and heterogeneous clinical phenotype in which disease onset, symptoms, severity, and progression can be highly variable even between family members. The identification of a CHRNE mutation allowed to make the definitive diagnosis of CMS in this family and contributed to define the clinical spectrum of this disease.

Diagnosis of Primary Ciliary Dyskinesia by a Targeted Next-Generation Sequencing Panel: Molecular and Clinical Findings in Italian Patients.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder that alters mucociliary clearance, with consequent chronic disease of upper and lower airways. Diagnosis of PCD is challenging, and genetic testing is hampered by the high heterogeneity of the disease, because autosomal recessive causative mutations were found in 34 different genes. In this study, we clinically and molecularly characterized a cohort of 51 Italian patients with clinical signs of PCD. A custom next-generation sequencing panel that enables the affordable and simultaneous screening of 24 PCD genes was developed for genetic analysis. After variant filtering and prioritization, the molecular diagnosis of PCD was achieved in 43% of the patients. Overall, 5 homozygous and 27 compound heterozygous mutations, 21 of which were never reported before, were identified in 11 PCD genes. The DNAH5 and DNAH11 genes were the most common cause of PCD in Italy, but some population specificities were identified. In addition, the number of unsolved cases and the identification of only a single mutation in six patients suggest further genetic heterogeneity and invoke the need of novel strategies to detect unconventional pathogenic DNA variants. Finally, despite the availability of mutation databases and in silico prediction tools helping the interpretation of variants in next-generation sequencing screenings, a comprehensive segregation analysis is required to establish the in trans inheritance and support the pathogenic role of mutations.

Improving the knowledge of amyotrophic lateral sclerosis genetics: novel SOD1 and FUS variants.

Amyotrophic lateral sclerosis (ALS) is as an adult-onset neurodegenerative disorder involving both upper and lower motor neurons. About 5% of all cases exhibit signs of frontotemporal degeneration (FTD). We established the mutation frequency of C9ORF72, SOD1, TARDBP, and FUS genes in 307 patients with sporadic ALS, 46 patients with familial ALS (FALS), and 73 patients affected with FTD, all originating from the northeastern part of Italy. C9ORF72 pathogenic expansion was found on 22% of familial ALS, 5% of sporadic ALS, and 14% of FTD patients, resulting the most frequently genetic determinant in our cohort. Sequence analysis of ALS cohort identified 2 novel variants on SOD1 (p.Glu41Gly) and FUS (p.Gly496Glyfs*31). Interestingly, the single base deletion on FUS was observed in an homozygous state, suggesting a recessive pattern of inheritance. No point mutations were identified on FTD cohort. Although useful to direct genetic testing, this study results expand the current knowledge of ALS genetics.