The impact of next-generation sequencing on the diagnosis of pediatric-onset hereditary spastic paraplegias: new genotype-phenotype correlations for rare HSP-related genes

Abstract

Hereditary spastic paraplegias (HSP) are clinical and genetic heterogeneous diseases with more than 80 disease genes identified thus far. Studies on large cohorts of HSP patients showed that, by means of current technologies, the percentage of genetically solved cases is close to 50%. Notably, the percentage of molecularly confirmed diagnoses decreases significantly in sporadic patients. To describe our diagnostic molecular genetic approach on patients with pediatric-onset pure and complex HSP, 47 subjects with HSP underwent molecular screening of 113 known and candidate disease genes by targeted capture and massively parallel sequencing. Negative cases were successively analyzed by multiplex ligation-dependent probe amplification (MLPA) analysis for the SPAST gene and high-resolution SNP array analysis for genome-wide CNV detection. Diagnosis was molecularly confirmed in 29 out of 47 (62%) patients, most of whom had clinical diagnosis of cHSP. Although SPG11 and SPG4 remain the most frequent cause of, respectively, complex and pure HSP, a large number of pathogenic variants were disclosed in POLR3A, FA2H, DDHD2, ATP2B4, ENTPD1, ERLIN2, CAPN1, ALS2, ADAR1, RNASEH2B, TUBB4A, ATL1, and KIF1A. In a subset of these disease genes, phenotypic expansion and novel genotype-phenotype correlations were recognized. Notably, SNP array analysis did not provide any significant contribution in increasing the diagnostic yield. Our findings document the high diagnostic yield of targeted sequencing for patients with pediatric-onset, complex, and pure HSP. MLPA for SPAST and SNP array should be limited to properly selected cases based on clinical suspicion.

Hereditary spastic paraplegia is a novel phenotype for germline de novo ATP1A1 mutation

“…11 The introduction of NGS methodologies has increased both molecular diagnosis and identification of ultra-rare or new genes, but a consistent percentage of patients remain undiagnosed. 5,[12][13][14] We identified a novel de novo ATP1A1 variant in a child with complex HSP, significantly expanding the known phenotypic spectrum. Homology modeling of ATP1A1 allowed us to propose that the p.L337 to proline replacement alters the conformation of the proximal sodium-binding residues impairing their function.…”

“…HSPs are clinically and genetically heterogeneous degenerative disorders of the upper motor neurons with over 90 HSP‐causing genes known . The introduction of NGS methodologies has increased both molecular diagnosis and identification of ultra‐rare or new genes, but a consistent percentage of patients remain undiagnosed . We identified a novel de novo ATP1A1 variant in a child with complex HSP, significantly expanding the known phenotypic spectrum.…”

“…Genomic DNA was extracted from peripheral blood by using NucleoSpin tissue, according to the manufacturer's protocol (Macherey‐Nagel, Germany). Nextera custom enrichment panel (Illumina) was first carried out covering promoters, coding regions, exon‐intron boundaries, and untranslated regions of all known HSP genes as previously described . Successively, a WES was conducted on the proband and his parents by using xGen Exome research panel v1.0 kit (IDT) for exome regions enrichment and Illumina platform for sequencing analysis.…”

“…Nextera custom enrichment panel (Illumina) was first carried out covering promoters, coding regions, exon-intron boundaries, and untranslated regions of all known HSP genes as previously described. 5 Successively, a WES was conducted on the proband and his parents by using xGen Exome research panel v1.0 kit (IDT) for exome regions enrichment and Illumina platform for sequencing analysis. WES data were processed using an in-house implemented pipeline as previously reported, 6 and filtering strategy is summarized in Supporting Information.…”

Expansion of the genetic landscape of ERLIN2‐related disorders

“…The patient with the de novo heterozygous ERLIN2 variant (patient 5) appeared less severely affected compared to the other participants with homozygous ERLIN2 variants, as was shown by a later age of symptom onset, absence of developmental regression, and absence of ID. In fact, among prior reports of ERLIN2 ‐related disorders (Table 2), ID was absent in 5/6 unrelated individuals who had a missense ERLIN2 variant on at least one allele (three families with affected individuals); conversely, ID was present in all individuals with a homozygous truncating variant . This observation suggests that ERLIN2 ‐related disorders can present dosage‐dependent clinical features likely due to reduced protein function.…”