What Diagnoses Are Missed in Next-Generation Sequencing?
Katta M Girisha Department of Genetics, Sultan Qaboos University, Muscat, Oman. Correspondence to: Katta M GirishaEmail:g.kumar@squ.edu.om
Exome sequencing is quite popular among the clinicians for diagnosis of rare Mendelian disorders but is often
non-diagnostic even when we have a strong clinical suspicion of a monogenic disorder. A proportion of them are solved by
whole genome sequencing. Some families still remain undiagnosed with detection of only one of the two variants necessary
to confirm the diagnosis of an autosomal recessive disorder. Let us look into some of the recent publications that have
solved this problem in some families by identifying the variants that usually escape detection in next-generation
sequencing (NGS).
1 Cryptic second variants in autosomal recessive diseases with one mutation (Moore et al., 2023)
Moore and colleagues investigated definitive autosomal recessive diseases with one hit (only one mutation identified) for
the second hit by whole genome sequencing. They studied 31 patients from the 100,000 Genomes Project who had only
one mutation despite having a strong clinical suspicion of an autosomal recessive disease. Whole genome
sequencing (short read) revealed a diagnosis in eight additional patients by finding the second mutation. These
include a patient with cystic fibrosis harboring a novel exonic LINE1 insertion in CFTR and a patient
with generalized arterial calcification of infancy with complex interlinked duplications involving exons 2–6
of ENPP1. They had to undertake optical genome mapping and RNA analysis for the ENPP1 variant.
2 Retroelements are missed by exome (missing) mutation in ataxia-telangiectasia (Zhao et al., 2023)
Retroelements (retrotransposons) are stretches of DNA that copy and paste themselves into different genomic locations
(transposons). They do this by converting RNA back into DNA through the reverse transcription process. Examples
include Long Interspersed Nuclear Elements-1 (LINE-1 or L1), SINE-VNTR-Alus (SVA) and pseudogene insertions.
Retroelement insertions are already known to cause Mendelian disorders and might be amenable to antisense
oligonucleotide therapy. Zhao and colleagues studied 237 patients with ataxia-telangiectasia who had whole genome
sequencing data and checked for retroelement insertions. They observed 15 individuals harboring one of the five
retroelements. While one was in the coding (exonic) region, the rest were integrated in the non-coding regions. RNA
sequencing, RT-PCR, and/or minigene splicing assays were used to study the functional consequences of these insertions.
Twelve out of 14 intronic insertions led to or contributed to loss of ATM function by exon skipping or activating cryptic
splice sites. Interestingly, these were second (missing) variants in some and third variant in others! They
estimate the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as
~2.1%–5.5%.
3 Deletions and a complex insertion in hereditary hemorrhagic telangiectasia (Xiao et al., 2023)
Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome is caused by loss-of-function (LoF) heterozygous
variants (and a second hit at in the affected tissues) in SMAD4. Xiao and colleagues developed GROFFFY to prioritize
variants in non-coding regions rich in transcribed and critical regulatory sequences. This is an analytic tool that integrates
coordinates for regions with experimental evidence of functionality. They applied GROFFFY to the whole genome
sequence data from solved and unsolved hereditary hemorrhagic telangiectasia recruits to the 100,000 Genomes Project.
They detected three ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor gene SMAD4
which disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein
production. In another individual, a complex insertion was identified. Four undiagnosed cases were thus
solved.
4 DNA methylation signature for unsolved cases of Fanconi anemia (Pagliara et al., 2023)
Fanconi anemia results from inactivating biallelic (predominantly) mutations in one of about 20 genes in the DNA repair
pathway. The wide spectrum of mutations and structural rearrangements make molecular diagnosis of Fanconi anemia
challenging. Assessment of chromosomal fragility is often required to confirm the pathogenicity of the variants and to
firmly establish the diagnosis. Pagliara and colleagues studied the peripheral blood genome-wide DNA
methylation profiles in 25 subjects with molecularly confirmed Fanconi anemia and observed 82 differentially
methylated CpG sites that allow distinguishing subjects with Fanconi anemia from healthy individuals
and patients with other diseases. The episignature was validated using a second cohort of subjects with
Fanconi anemia involving different complementation groups and documented its sensitivity and specificity.
The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating
robustness. They also trained a machine learning tool for identifying DNA methylation signature for this
condition.
To summarize, we need to look for complex rearrangements, retroelements, and alterations in regulatory regions
whenever we do not have a diagnosis for a monogenic disease. Specific DNA methylation signatures might also be a tool
for diagnosis of genetically heterogenous conditions like Fanconi anemia.
References
1. Moore AR, et al. Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases
recruited to the 100 000 Genomes Project. J Med Genet. 2023; 60 (12):1235-1244.
2. Pagliara D, et al. Identification of a robust DNA methylation signature for Fanconi anemia. Am J Hum
Genet. 2023;110 (11):1938-1949.
3. Xiao S, et al. Functional filter for whole-genome sequencing data identifies HHT and stress-associated
non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA. Am J Hum Genet. 2023; 110
(11):1903-1918.
4. Zhao B, et al. Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia.
Am J Hum Genet. 2023;110 (11):1976-1982.