Manini et al. 2025 — Whole Genome Sequencing in PLS Cohorts
The genetic landscape of adult sporadic PLS has remained poorly defined compared to ALS, partly because PLS is rare enough that assembling large cohorts is difficult, and partly because earlier targeted gene panels consistently returned negative results. The Manini et al. 2025 study applied whole genome sequencing to a PLS cohort — capturing variants that panel testing misses — and produced the most comprehensive picture yet of what the genetics of adult PLS actually looks like.
Why whole genome sequencing matters for PLS
Genetic testing in clinical practice has historically used targeted approaches: sequencing a defined panel of genes known to be associated with a condition. For PLS, panels have typically included ALS-associated genes (SOD1, C9orf72, TARDBP, FUS, and others) plus hereditary spastic paraplegia genes (SPG loci). These panels are useful — they identify the subset of PLS patients who turn out to carry a known pathogenic variant — but they are inherently limited by what is known at the time the panel is designed.
Whole genome sequencing (WGS) has no predefined gene list. It sequences all three billion base pairs of the genome, capturing not just coding variants in known disease genes but also variants in regions previously considered non-causal: promoters, enhancers, untranslated regions, deep intronic sequences, and structural variants. The tradeoff is interpretive complexity — WGS produces vastly more variants than targeted panels, and distinguishing disease-causing changes from the enormous background of normal genetic variation requires careful bioinformatics and functional validation. The reward, when a study is well designed, is the possibility of discovering genetic causes that panel testing would never find.
For PLS specifically, WGS also addresses a fundamental question: is the lack of genetic findings in adult PLS a real biological feature (most adult PLS is genuinely non-genetic), or an artifact of the limited scope of panel testing? WGS can distinguish between these possibilities in a way that targeted approaches cannot.
What they did
Manini and colleagues at the University of Milan applied whole genome sequencing to a cohort of PLS patients. The study analyzed both coding and non-coding variants systematically, using established bioinformatics pipelines to identify variants of potential relevance to upper motor neuron degeneration. Variants were assessed for predicted pathogenicity, population frequency, and consistency with known mechanisms of motor neuron disease.
The cohort consisted of adult-onset PLS patients meeting clinical diagnostic criteria — not juvenile cases, which are biologically distinct and caused by ALS2 mutations. This distinction is important: the study was designed to characterize the genetics of the sporadic adult form that represents the majority of PLS patients.
The analysis specifically looked for variants in genes associated with hereditary spastic paraplegia (SPG loci), ALS-associated genes, and novel candidate loci suggested by the WGS data. The combination of a clinically well-characterized cohort and a comprehensive sequencing approach made this study the most thorough genetic characterization of adult PLS published to date.
What they found
The predominant finding was consistent with prior smaller studies: the overwhelming majority of adult sporadic PLS patients had no identifiable causative genetic variant even under comprehensive WGS analysis. This is a meaningful negative finding — it strengthens the conclusion that adult sporadic PLS is genuinely non-Mendelian in its genetic architecture, not simply under-tested.
Within the cohort, a minority of patients carried rare variants in genes associated with hereditary spastic paraplegia — SPG loci. These findings mirror what has been reported in smaller case series: some patients who present clinically with apparent PLS turn out to carry SPG gene mutations that, properly interpreted, reclassify their diagnosis as late-onset HSP. WGS is sensitive enough to capture these variants even when they are in non-coding regions that panel testing might miss.
The study also identified novel variants in genes not previously associated with PLS — candidates that require further functional validation before they can be considered causal. Identifying candidate variants through WGS is a first step; confirming that those variants cause disease requires additional work in cell or animal models. These candidates represent the leading edge of PLS genetics research rather than established findings.
No single dominant genetic cause of sporadic adult PLS was identified. The picture that emerges is of a genetically heterogeneous condition where rare variants across multiple genes may contribute to disease susceptibility in individual patients, rather than one or two high-frequency causal genes comparable to C9orf72 in ALS.
Why it matters
This study is important for several reasons that extend beyond the specific variants it identified. It establishes a credible baseline: with WGS-level sensitivity, most adult PLS is still genetically clean. That has direct implications for genetic counseling — it supports the clinical message that adult sporadic PLS is not typically inherited, and that children of affected individuals are not at substantially elevated genetic risk. This is a meaningful statement to be able to make to patients and families, and it now rests on more comprehensive genetic evidence than before.
The study also establishes the research infrastructure and analytical precedent for future larger WGS efforts. PLS is rare enough that single-institution cohorts are limited in size. Multi-center international WGS initiatives — pooling patients across clinical centers — are the natural next step, and the Manini analysis demonstrates the methodology and confirms the feasibility of the approach.
For the subset of patients where SPG variants were found, the clinical implication is direct: reclassification from PLS to HSP, with associated changes in genetic counseling and family risk assessment. This is the clearest argument for comprehensive genetic testing — including WGS where available — in adult PLS: not because a PLS-causing gene will usually be found, but because an HSP-causing gene occasionally will be, and that finding changes the clinical picture.
Caution is warranted in interpreting the novel candidates the study identified. Variants of uncertain significance are an inherent output of WGS, and without functional validation, they cannot be treated as established causes of disease. The most appropriate reading of the Manini data is as a source of hypotheses for further study, not a list of confirmed PLS genes.
Limitations
Cohort size is the primary limitation of any WGS study of a rare disease. PLS is uncommon enough that even a dedicated single-institution recruitment effort over years will produce a cohort far smaller than those available for common diseases. Small sample sizes limit statistical power to detect genuine genetic associations, particularly for rare variants. This is not a criticism of the study — it reflects the fundamental challenge of rare disease genetics — but it means the candidate variants identified require replication in independent cohorts before confidence in them is warranted.
Post-translational modifications, epigenetic factors, and somatic mutations — all outside the scope of standard WGS analysis — remain unexplored potential contributors to PLS susceptibility. WGS characterizes inherited germline variation; it cannot assess everything that contributes to why a given motor neuron degenerates.
How this connects
The Manini WGS study is the empirical basis for the statement that adult sporadic PLS is predominantly genetically clean even under comprehensive testing. The Genetics Research hub provides broader context for how this fits into the overall PLS genetics landscape. For patients who come to genetic testing expecting an answer, the Genetics patient guide addresses how to frame a negative result and what it does and does not mean. The SPG gene hits found in a minority of Manini patients are the same phenomenon discussed in detail at PLS and HSP — The Genetic Boundary. For the contrast with juvenile PLS, which does have a defined genetic cause, see ALS2 and Juvenile PLS.
Citation
Manini A, et al. Whole genome sequencing in primary lateral sclerosis cohorts. University of Milan (UNIMI). 2025. Available at: air.unimi.it.