Center for Molecular Medicine Cologne

Spinal Muscular Atrophy: specific DNA sequence protects against symptoms


Plastin 3 escapes X inactivation in some women: on the one hand this protects against spinal muscular atrophy, on the other hand it is a biomarker for cancer

"I assume that in the future, the length of the DXZ4 DNA sequence that alters the X inactivation can be used specifically for diagnosis as well as for prediction using special techniques (long-read next generation sequencing) and improved bioinformatic tools", comments Prof. Dr. Brunhilde Wirth.

Long-drawn DNA strands from a patient were applied to a very thin glass plate (molecular combing) and stained with fluorescent probes. The green bands indicate the copy number of the macrosatellite DXZ4 Photo: Eike Strathmann

Females and males differ genetically in their sex chromosomes. Although females have two X chromosomes, one of them is randomly inactivated early in embryonic development. As a result, approximately 85% of all X-linked genes are transcribed in similar amount in males and females.

The research group led by Professor Brunhilde Wirth from the Institute of Human Genetics at the University Hospital Cologne and the Center for Molecular Medicine Cologne has now discovered that plastin 3, a gene located on the X chromosome, escapes X-inactivation in some tissues. The reason is the DXZ4 macrosatellite, a highly repetitive segment of DNA that plays an important role in X inactivation. When DXZ4 is particularly rich in repetitive units, X-inactivation is partially disrupted and plastin 3, which is in close proximity, produces RNA and protein from both of a cell's X chromosomes. Elevated level of plastin-3 protects against life-threatening spinal muscular atrophy (SMA), but is also a biomarker for many types of cancer. These results were published on February 21, 2023 in the internationally renowned American Journal of Human Genetics with following title Epigenetic regulation of plastin-3 expression by the macrosatellite DXZ4 and the transcriptional regulator CHD4 -

Females have two X chromosomes, males only one and the extra Y chromosome. This genetic difference is crucial for the formation of the respective sex during embryonic development. One of the two X chromosomes is randomly inactivated early in embryonic development, a process known as X-chromosomal inactivation. In every female cell, either the paternal or the maternal X chromosome is inactivated. This X-linked inactivation ensures that the X-linked genes are expressed equally in both sexes. But that's not always the case. About 15% of all female X-linked genes escape tissue-specific X-inactivation. In other words, some X-linked genes produce up to twice as much RNA and protein. This can either be an advantage or sometimes a disadvantage for the function of the cell.

The plastin 3 (PLS3) gene is located on the X chromosome. PLS3, a protein of the cytoskeleton, is very important for many processes in the cell. The migration of cells or the formation of long neuronal processes such as axons, the endocytosis, i.e. the absorption of cell components or pathogens, depends on the amount of PLS3. As early as 2013, Wirth's group showed in a publication in the New England Journal of Medicine that mutations in the PLS3 gene lead to osteoporosis with bone fractures in men and women in menopause are more likely to get osteoporosis. Elevated PLS3 levels, on the other hand, protect against spinal muscular atrophy (SMA). Wirth published these groundbreaking results in Science back in 2008. SMA is a common, fatal neuromuscular disease caused by loss of the SMN1 gene. However, all individuals with SMA have one to four copies of the SMN2 gene, although these only produce about 10% functional protein per copy. Wirth’s research group was able to show in seven SMA families that some female siblings of SMA-affected brothers remain asymptomatic despite carrying the same SMN1 deletion and identical SMN2 copies as their affected brothers. They are all protected by overexpression of PLS3.

However, PLS3 overexpression is not always beneficial. Elevated PLS3 levels are associated with a worse prognosis in the most common types of cancer and are used there as so-called biomarkers. Elevated PLS3 levels also lead to osteoarthritis. But what is the reason for the increased PLS3 expression?

Only now, 15 years after the Science publication, has the research team led by Professor Dr. Brunhilde Wirth uncovered the cause of the increased PLS3 amount. A so-called macrosatellite DXZ4, which is located in the immediate vicinity of PLS3, is largely responsible for this. DXZ4 consists of 3.4 kilobase units, which occur in varying numbers in humans. DXZ4 is partially responsible for X-inactivation in females.

Dr Eike Strathmann, a doctoral student in Wirth's working group, was able to use a special method in cooperation with the Cologne Center for Genomics (CCG) to precisely determine the number of DXZ4 copies. So-called molecular combing with special fluorescent DNA samples was used. Linearized DNA strands from a large number of subjects with and without PLS3 expression in the blood, from asymptomatic SMA patients and control persons were applied to glass plates and stained with four different fluorescent markers: two probes mark the beginning and end of each repeat unit and two further probes the adjacent regions of the entire DXZ4 macrosatellite. All females that showed elevated PLS3 levels showed a particularly long DXZ4 on one X chromosome. If DXZ4 contains particularly many 3.5 kb-units, PLS3 can escape X-inactivation; the PLS3 protein can then be produced by both copies of the gene in a cell.

Furthermore, motoneurons—the cells most affected by SMA—were differentiated from induced pluripotent stem cells of two SMA families with asymptomatic and SMA siblings, and the entire transcriptome analyzed. PLS3 was twice as highly expressed in asymptomatic females as it was expressed from both X chromosomes in a cell. In addition, Strathmann has identified the CHD4/NuRD protein complex that epigenetically regulates the expression of PLS3 in both females and males.

Wirth is confident that the length of the DXZ4 macrosatellite can be determined and used diagnostically and predictively in the future with long-read next-generation sequencing, improved bioinformatic methods and with cheaper prices.

Eike A. Strathmann, Irmgard Hölker, Nikolai Tschernoster, Seyyedmohsen Hosseinibarkooie, Julien Come, Cecile Martinat, Janine Altmüller and Brunhilde Wirth
Epigenetic regulation of plastin-3 expression by the macrosatellite DXZ4 and the transcriptional regulator CHD4
American Journal of Human Genetics

Prof. Dr. Brunhilde Wirth

Institute of Human Genetics and Center for Molecular Medicine Cologne
Medical Faculty, University of Cologne and University Hospital

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