New diagnostic workflow improves detection of 5q-spinal muscular atrophy

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5q-spinal muscular atrophy (5q-SMA) is the most common type of spinal muscular atrophy (SMA) affecting approximately one in ten thousand individuals worldwide. Currently, tests are unable to identify all pathogenic genetic variants leading to 5q-SMA. However, researchers using a modified analysis workflow were able to reliably identify patients with 5q-SMA within the diagnostic whole exome analysis, many of whom did not exhibit typical symptoms. They report their results Journal of Neuromuscular Disease.

Recent treatments have shown that early intervention can significantly improve clinical outcomes, so early diagnosis of 5q-SMA is important.”

Angela Abicht, MD, Lead Investigator, Center for Medical Genetics and Department of Neurology, Friedrich-Baur-Institute, LMU Klinikum Munich

96% of cases are caused by homozygous deletion of 5q-SMA SMN1 Gene (loss of both alleles). However, about 4% of patients carry only one SMN1 deletion and a single-nucleotide variant (SNV) in the other allele.

Short-read next-generation sequencing (s-rNGS), commonly applied to whole-exome sequencing, is generally unable to identify individuals with 5q-SMA. The underlying cause is that genes are responsible for the condition, SMN1There is an almost identical but non-functional copy, called SMN2. Thus, common bioinformatics algorithms encounter difficulties when trying to determine the correct sequence reads for any given gene. Ambiguous reads are randomly mapped to a region and then assigned a lower mapping quality, leading to their exclusion by variant callers. Hence, a deleted or altered identification SMN1 Genes are not possible through a standard bioinformatics pipeline.

The investigators retrospectively analyzed two cohorts of patients who underwent diagnostic S-RNGS-based exome analysis over a 12-month period between November 2021 and November 2022. A neuromuscular phenotype was demonstrated in 1,684 patients in cohort 1. Human Phenotype Ontology (HPO) phenotype or its direct descendants: muscle abnormalities or peripheral neuropathy. Cohort 2 consisted of fetal samples from 260 patients who underwent exome sequencing as part of prenatal diagnostics over a one-year period.

In their tailored 5q-SMA variant detection workflow, the investigators used a bioinformatics pipeline that was “SMN2-mukosh, “that is, for SMN1Paralogous regions that comprise SMN2 “hidden” in the reference genome. Consequently, all NGS reads generated were forced to map only SMN1. Later on SMN1/2 In the variable filtering workflow, they used a unique allele frequency SMN1 Its deletion detection variant SMN1 which were homologous and single nucleotide variants SMN1 which were homozygous (consisting of two identical alleles at a given locus), heterozygous (consisting of two different alleles at a locus), or hemizygous (consisting of a single copy of a particular gene).

Application of this new workflow revealed a diagnosis of 5q-SMA in nine patients with a neuromuscular phenotype in cohort 1. One patient with 5q-SMA was identified in cohort 2.

This study demonstrates that it is possible to reliably identify 5q-SMA patients within a diagnostic whole exome analysis using an appropriate 5q-SMA variant detection workflow. This finding is significant because the investigators identified several patients who were not thought to have 5q-SMA because they did not present the typical clinical picture of SMA. In addition, two patients (a 48-year-old woman and a four-month-old child) carried biallelic single nucleotide variants. SMN1.

Co-principal investigator Teresa Neuhann, MD, Medical Genetics Center, Munich, added, “This was an exciting discovery because in all previously described patients, 5q-SMA was caused by either a homozygous deletion. SMN1 (96% of cases) or by one SMN1 deletion and a single-nucleotide variant in the other allele (4% of cases). In both patients, the genetic diagnosis was made after a diagnostic odyssey because the initial clinical diagnosis of 5q-SMA was not confirmed by standard SMA testing with multiplex ligation-dependent probe amplification (MLPA).”

s-rNGS is the current standard test in human genetics. It offers significant diagnostic advantages over the former single gene Sanger sequencing. However, clinicians must be aware of gaps, particularly in the detection of 5q-SMA, an important treatable disorder for which some countries have recently implemented newborn screening.

“To fill the gap, diagnostic laboratories should use adopted pipelines and workflows in short-read sequencing,” commented Dr. Obsession. “On the other hand, physicians can bridge the gap by stepping up SMN1 Sequencing in the case of a hetero- or homozygous absence SMN1 Deletion is indicated by MLPA or real-time PCR methods, but clinical evidence still strongly points to SMA.”

SMA is a disorder that affects motor neurons—the nerve cells that control voluntary muscle movement. These cells are located in the spinal cord. Because muscles cannot respond to signals from nerves, they become weak and shrink from inactivity. 5q-SMA is one of the most common types of SMA with a frequency of approximately one in ten thousand worldwide. SMA patients are divided into three groups: non-sitters, sitters and walkers. Currently approved disease-modifying therapies, including nusinarsen, risdiplam, and onasemnozine abeparvovac, require an early, accurate genetic diagnosis of 5q-SMA to determine and initiate the most appropriate treatment.


Journal Reference:

Kleinle, S., etc. (2023). Closing the gap – detection of 5q-spinal muscular atrophy by short-read next-generation sequencing and unexpected outcomes in a diagnostic patient cohort. Journal of Neuromuscular Disease.

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