Nanopore Long-Read Sequencing Facilitates Diagnosis of Atypical Ataxia-Telangiectasia

In a case study newly published in Neurology Genetics, Nanopore long-read sequencing confirmed the diagnosis of a 52-year-old Korean woman with atypical late-onset ataxia-telangiectasia (AT) characterized by extrapyramidal symptoms with a very slow progression.¹ This report highlights the utility of Nanopore long-read sequencing in phasing variant haplotypes, which may be beneficial for the diagnosis of autosomal recessive disorders especially for those cases without parental samples.

The patient, the second child of 3 in her family, presented a worsening tremor that initiated 5 years prior. At the age of 7 years, the patient had slower handwriting compared with peers and intermittently had difficulty controlling the left hand when washing her face between 9 and 10 years of age. Later, at the age of 13, intermittent bilateral action tremors started, and the symptoms were maintained into her late teens. In her early 20s, intermittent gait disturbances began while action tremor in the hands remained stable, not significantly impeding daily life until jerky head tremors worsened in her late 40s.

Top Clinical Takeaways

Nanopore sequencing may offer valuable insights into atypical presentations of genetic disorders, aiding accurate diagnosis.
Haplotype phasing through long-read sequencing can resolve complex genetic variations, especially in cases without parental samples.
Advancements like amplification-free sequencing using CRISPR/Cas9 enrichment show promise for more direct and less biased genetic analysis in the future.

“Our case was notable because of the patient exhibiting dystonia and dystonic tremor with slow progression as the primary symptoms rather than ataxia. In addition, the patient maintained an independent gait even in her early 50s. These relatively mild clinical features might result from the residual activity in the hypomorphic missense variant, as previously reported,” senior author Han-Joon Kim, MD, PhD, a neurologist and movement specialist at Seoul National University Hospital, and colleagues wrote.¹

Neurologic examination reported dystonia in the face, hand, and trunk in addition to cervical dystonia at 52 years of age during the patient’s first visit. Researchers reported dystonic postural tremors in both of the patients’ hands and suspected a saccadic pursuit during the extraocular movement examination and noted ocular telangiectasia. Investigators did not observe limb dysmetria in the finger-to-nose and heel-to-shin tests for cerebellar function. The patient had a normal base with no difficulty in walking but showed instability during turning. She performed 4 to 5 steps in the tandem gait test and could walk independently. Notably, cognitive function was revealed as normal, and routine blood tests as well as a brain imaging study produced normal results, but the authors noted an increased level of α-fetoprotein.

All told, the patient had a family history of similar symptoms in her older sister, aged 54 years, who experienced tremor in her right hand beginning in her early 20s and progressively worsened as she got older, but remained significantly less severe than the proband. The sister reported involuntary elevation of the right shoulder and difficulty handwriting because of involuntary flexion of the right wrist since adolescence. During neurological examination of the sister, investigators reported mild dysarthria; head tremor; cervical dystonia; and kinetic, intention, and dystonic postural tremors in both hands, notably worse in the right side. In the cerebellar function tests, the sister showed normal performance and had no definite evidence of telangiectasia. Authors reported a detection of elevated α-fetoprotein level of 108 ng/mL despite other blood tests producing normal results, and a diagnosis of breast cancer was noted from 5 years prior.

In a targeted panel sequencing performed in the proband which included 29 hereditary dystonia–related genes, investigators identified 2 heterozygous variants in the ATM gene: c.6040G>T, p.Glu2014Ter, and c.6154G>A, p.Glu2052Lys. The authors noted difficulty in determining the allelic origin of the variants without parental sampling because of the proband’s decreased father, thus, investigators conducted PCR amplification using primers containing these 2 variants and sequenced them using Nanopore long-read sequencing.

The long-read sequencing aligned with the human reference genome hg38 using minimap2² and provided adequate coverage on both variants, which were set apart by 213 base pairs. Haplotype analysis using WhatsHap³ and visual inspection through the Integrative Genomic Viewer established the allele positions of the variants in trans, which confirmed the molecular diagnosis. Additional familial evaluations using Sanger sequencing in the proband’s siblings showed the same pathogenic variants in the older sister and carrier status of the p.Glu2052Lys variant in the other sibling, the younger brother.

“Although the detection of a single heterozygous allele in siblings would have been a robust indicator of the trans effect of the 2 variants, our approach using Nanopore sequencing provided direct and clear haplotype-resolved information for the 2 ATM gene variants,” Kim et al noted.¹ “In addition, while we conducted PCR amplification before sequencing, a noteworthy development is the potential use of amplification-free sequencing. This method, which involves CRISPR/Cas9 enrichment, could offer a more direct and less biased approach. It is particularly effective for assessing haplotype-resolved single-nucleotide variants, structural variations, and CpG methylation.”