The diagnosis of inherited disorders in the early years of life and later in life can be very complex. The increasing knowledge of the genetic basis for many of the inherited disorders affecting the blood, together with the power of genomic approaches, has opened the way for the relatively simple screening for such disorders. The optimum approach for this is not fully established as yet but can be done by either the identification of single gene variants or multiple variants in a specific disease area, or by sequencing the entire genome. Sequencing the entire genome is the most comprehensive approach but at this stage brings with it issues of data handling, analysis, and ethics associated with the potential to sequence everybody in their early life. However, it is likely that such approaches will come into widespread use over time.

 Some examples of the potential approaches in hematologic disorders are given as follows:

 The Hemoglobinopathies

 The genetic basis for the hemoglobinopathies and thalassemias is well known, and many causative genetic variants can be detected using simple polymerase chain reaction (PCR) approaches. Some uncommon mutations (Hb Q-India, HbNedlands, Hb Queens Park) require specific primers, but the approach to detecting such disorders is readily applied during prenatal screening. Deletions and mutations can readily be detected using allele-specific PCR for mutations or Gap-PCR for deletions, which use primers that bind to both sides of a deletion and can be used to successfully diagnose α-thalassemias, resulting from variable-sized deletions of α-globin gene.

Clotting Disorders

 Genomic techniques can be helpful to refine thrombotic risk prediction. Current approaches focus on five common genetic risk factors for venous thromboembolism, including antithrombin, protein C, and protein S deficiency; factor V Leiden; and the G20210A pro thrombin gene variant. Although the diagnosis of these thrombophilias is routinely based on functional assays of the coagulation cascades, the use of genetic testing to augment this approach can be useful. To date, genotyping has not replaced plasma-based assays for diagnostic purposes, with the exception of the prothrombin gene variant. Testing for activated protein C resistance remains controversial, even with the second-generation plasma assays using factor V–deficient plasmas. Some institutions simply do factor V Leiden DNA testing, whereas others use a less expensive plasma-based PCR assay and do DNA testing only for validation.

Disease with Rare Penetrant Variants Involving Multiple Loci

The ability of NGS to capture and analyze multiple gene loci has given it the ability to screen multiple loci in a single test. This approach relies upon a knowledge of the genetic basis of the disorder and the development of a specific testing panel. Thus genome-wide targeted exon capture followed by high-throughput DNA sequencing can provide an unbiased analysis of coding exons and is applicable to dis eases associated with significant genotypic variability caused by mutations in numerous genes that result in the same clinical phenotype. One example of such a disease is Fanconi anemia, a heterogeneous bone marrow failure syndrome associated with defective DNA repair associated with cancer predisposition and congenital anomalies. It is inherited primarily as an autosomal recessive fashion, with more than a dozen Fanconi genes having been described. Application of exome sequencing to Fanconi patients has identified a variety of mutations in Fanconi-associated genes, several of which are novel, such as the XRCC2, one of five RAD51 paralogs that act nonredundantly in the pathway of homologous recombination repair.[1] The increasing knowledge of the genetic basis for such disorders will allow the design and application of increasingly refined panels in a clinical setting. Currently the approach is readily applicable and easier to apply than sequencing the entire genome; however, as technology improves, it is likely that whole genome sequencing will replace looking for variants already described.

 Common Low Penetrance Risk Variants

 Inherited variants can modify disease response by the inheritance of common genetic variants with low penetrance. These inherited variants have been investigated by genome-wide association studies (GWASs), which often require thousands of patient samples to have sufficient power to detect statistically significant associations. Many GWASs have been performed, attempting to identify common variants contributing to complex disease. An example is the sequencing of candidate genes near loci implicated in fetal hemoglobin (HbF) level variation, which showed that rare variants in MYB to be associated with HbF levels.[2]

 The approach of identifying common variants that modify responses of specific pathways has been extensively explored in the coagulation cascades. Numerous clinical studies have addressed genetic variation at VCORC1 and CYP2C9 to identify risk in the use of vitamin K antagonists for anticoagulation.[3] These approaches have been extended further to define genetic risk scores associated with venous thromboembolism (VTE) bAe with the goal of personalizing anticoagulation therapy for prevention of recurrent VTE, but much more development is required before such approaches are clinically useful. Similar GWAS approaches have been evaluated for antiplatelet agents. Clopidogrel, a P2Y12 inhibitor, is activated by the cytochrome P450 system. Patients carrying the CYP2C19*2 allele metabolize clopidogrel poorly and are good candidates for alternative P2Y12 inhibitors due to their higher risk of arterial thrombosis.[4]

References

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1- Shamseldin HE, Elfaki M, Alkuraya FS. Exome sequencing reveals a novel Fanconi group defined by XRCC2 mutation. J Med Genet. 2012;49(3):184 186. https://doi.org/10.1136/jmedgenet-2011-100585.

2- Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine-mapping at three loci known to affect fetal hemoglobin levels explains additional genetic variation. Nat Genet. 2010;42(12):1049–1051. https://doi.org/10.1038/ng.707.

3- Misasi S, Martini G, Paoletti O, et al. VKORC1 and CYP2C9 polymorphisms related to adverse events in case-control cohort of anticoagulated patients. Medicine (Baltimore). 2016;95(52):e5451. https:// doi.org/10.1097/MD.0000000000005451.

4- Dean L. Clopidogrel therapy and CYP2C19 genotype. In: Pratt VM, McLeod HL, Rubinstein WS, eds. Medical Genetics Summaries. National Center for Biotechnology Information (US); 2012. Accessed March 18, 2020. http://www.ncbi.nlm.nih.gov/books/NBK84114/.