Intellectual disability genomics: current state, pitfalls and future challenges

Intellectual disability (ID) can be caused by non-genetic and genetic factors, the latter being responsible for more than 1700 ID-related disorders. The broad ID phenotypic and genetic heterogeneity, as well as the difficulty in the establishment of the inheritance pattern, often result in a delay in the diagnosis. It has become apparent that massive parallel sequencing can overcome these difficulties. In this review we address: (i) ID genetic aetiology, (ii) clinical/medical settings testing, (iii) massive parallel sequencing, (iv) variant filtering and prioritization, (v) variant classification guidelines and functional studies, and (vi) ID diagnostic yield. Furthermore, the need for a constant update of the methodologies and functional tests, is essential. Thus, international collaborations, to gather expertise, data and resources through multidisciplinary contributions, are fundamental to keep track of the fast progress in ID gene discovery.

Assessment of the gene mosaicism burden in blood and its implications for immune disorders

There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results.

Analysis of age-dependent DNA methylation changes in plucked hair samples using massive parallel sequencing

The analysis of age-dependent DNA methylation changes is a valuable tool in epigenetic research and forensic genetics. With some exceptions, most studies in the past concentrated on the analysis of blood, buccal, and saliva samples. Another important sample type in forensic investigations is hair, where age-dependent DNA methylation has not been investigated so far. In this pilot study a deeper look was taken at the possibilities and challenges of DNA methylation analysis in hair. The DNA methylation of selected age-dependent 5’-C-phosphate-G‑3’ (CpG) sites were characterized for their potential use as a biomarker for age prediction using plucked hair samples and massive parallel sequencing. Plucked hair roots of 49 individuals were included in the study. The DNA methylation of 31 hairs was successfully analyzed. The DNA methylation pattern of 10 loci, including ELOVL2, F5, KLF14, and TRIM59, was determined by amplicon-based massive parallel sequencing. Age-dependent changes were found for several markers. The results demonstrate the possible use of already established age-dependent markers but at the same time they have tissue/cell type-specific characteristics. Special challenges such as low amounts of DNA and degraded DNA as well as the possible heterogeneous cellular composition of plucked hair samples, have to be considered.

Massive parallel sequencing in a family with rectal cancer

Background We have previously reported a family with a suspected autosomal dominant rectal and gastric cancer syndrome without any obvious causative genetic variant. Here, we focused the study on a potentially isolated rectal cancer syndrome in this family. Methods We included seven family members (six obligate carriers). Whole-exome sequencing and whole-genome sequencing data were analyzed and filtered for shared coding and splicing sequence and structural variants among the affected individuals. Results When considering family members with rectal cancer or advanced adenomas as affected, we found six new potentially cancer-associated variants in the genes CENPB, ZBTB20, CLINK, LRRC26, TRPM1, and NPEPL1. All variants were missense variants and none of the genes have previously been linked to inherited rectal cancer. No structural variant was found. Conclusion By massive parallel sequencing in a family suspected of carrying a highly penetrant rectal cancer predisposing genetic variant, we found six genetic missense variants with a potential connection to the rectal cancer in this family. One of them could be a high-risk genetic variant, or one or more of them could be low risk variants. The p.(Glu438Lys) variant in the CENPB gene was found to be of particular interest. The CENPB protein binds DNA and helps form centromeres during mitosis. It is involved in the WNT signaling pathway, which is critical for colorectal cancer development and its role in inherited rectal cancer needs to be further examined.