Fig. 1: Phenotypic and dysmorphic features of patient 1 (A and B),...
Could Down Syndrome Be Eliminated? Scientists Say Cutting-edge Gene Editing Tool Could Cut Out Extra Chromosome
Cutting-edge gene editing technology could eradicate Down syndrome, according to Japanese scientists.
Down syndrome, which causes a range of developmental differences and affects 1 in 700 newborns in the United States, is caused by the presence of an extra copy of chromosome 21.
The extra chromosome, also known as trisomy 21, causes cellular overactivity, compromises a range of processes within the body, and can manifest in distinctive physical traits, learning difficulties and health concerns.
Now new research out of Mie University in Japan suggests that by using the DNA-modifying tech CRISPR, it is possible to remove the surplus chromosome in affected cells and bring cellular behavior closer to typical function.
Down syndrome, which causes a range of developmental differences and affects 1 in 700 newborns in the United States, is caused by the presence of an extra copy of chromosome 21. Mongkolchon – stock.Adobe.ComCRISPR-Cas9 is a gene-editing system that utilizes an enzyme to identify specific DNA sequences. Once the enzyme locates a matching site, it snips through the DNA strands.
Ryotaro Hashizume and his colleagues designed CRISPR guides to target only the trisomy 21 chromosome, a process called allele-specific editing, which directs the cutting enzyme to the desired spot.
When they used it on lab-grown cells, removing the extra copy of the gene normalized the way the genes expressed themselves in the body — suggesting that the genetic burden had been removed.
They also found that after the extra chromosome was removed, genes tied to nervous system development were more active and those related to metabolism were less active. This backs up previous research that found extra copies of chromosome 21 disrupt brain development during early fetal growth.
Researchers also tested their CRISPR guides on skin fibroblasts, which are mature, non-stem cells taken from people with Down syndrome.
In these fully developed cells, the editing method successfully removed the extra chromosome in a number of cases.
Hashizume and his team designed CRISPR guides to target only the trisomy 21 chromosome, a process called allele-specific editing, which directs the cutting enzyme to the desired spot. Gorodenkoff – stock.Adobe.ComAfter removal, these corrected cells grew faster and had a shorter doubling time than untreated cells, suggesting that removing the extra chromosome may help with the biological strain that slows down cell growth.
But the CRISPR can affect healthy chromosomes, too, and researchers are refining their program so that it only attaches to the extra copy of chromosome 21.
This work proves that, rather than making small fixes, CRISPR can eliminate an entire chromosome.
The scientists published their findings in PNAS Nexus.
Hashizume and his team are hopeful that their work may be used to design regenerative therapies and treatments that address genetic surplus at its source.
Researchers will continue to analyze the risks of DNA changes and monitor how modified cells function over time and their viability in real-world settings.
Researchers also tested their CRISPR guides on skin fibroblasts, mature, non-stem cells taken from people with Down syndrome. Yakobchuk Olena – stock.Adobe.ComPeople with Down syndrome often deal with a range of health concerns, including developmental delays and intellectual disabilities.
Roughly half of people with Down syndrome are born with a congenital heart defect — most commonly atrioventricular septal defect, a hole in the center of the heart.
They're also more likely to have problems with digestion, immunity, weight, sleep apnea, seizures, hearing, vision and teeth. They're at higher risk for leukemia and spinal problems, as well as thyroid conditions.
Additionally, they face a much higher risk of developing Alzheimer's-related dementia as they age — an estimated three to five times higher than the general population.
Scientists are still working to pinpoint the exact cause, but it's believed that the extra copy of chromosome 21 drives the overproduction of amyloid precursor protein. This excess production leads to the buildup of amyloid beta plaques in the brain, a hallmark of Alzheimer's disease.
Yet eliminating Down syndrome has proven to be a controversial topic. Iceland has made headlines over the years for nearly eradicating it in its population of just under 400,000, largely because nearly all women there choose to abort when their prenatal screening tests are positive for it.
"When we start listening to what people with Down syndrome themselves have to say about this … they find it disturbing … and we hear the same stories from their families," Ástríður Stefánsdóttir, a medical doctor and a professor in applied ethics at the University of Iceland, told ABC News Australia.
Down Syndrome Research Should Look At The Whole Cell Not Just The Extra Chromosome, Scientists SayScienceDaily
Research on understanding the effect of extra chromosomes for conditions like Down syndrome typically involves examining what genes play a role in the symptoms of these conditions. However, researchers from Germany and the US propose a new way of looking at these conditions, suggesting that when an extra chromosome is present, the impact on the cell depends less on which chromosome is duplicated and more on the presence of extra DNA. This work appears in a review publishing December 1in the American Journal of Human Genetics.
"Understanding the complexity and general nature of disease phenotypes allows us to see a bigger picture and not get stuck focusing on a single gene, due to its presence on the extra chromosome," says lead author Maria Krivega, developmental biologist at Heidelberg University.
Every cell starts out with extra chromosomes during early embryogenesis; however, this DNA gets sorted into pairs after about a week of growth. When this process goes awry, it often leads to death of the embryo, with only a few being able to survive with the extra DNA, like in the case of Down syndrome.
By taking a step back and looking at the entire cell, researchers were able to create a new understanding of these syndromes. Krivega and her collaborators took a critical look at recent evidence suggesting that Down syndrome phenotypes arise not only because of increased dosage of genes on chromosome 21 but also because of global effects of chromosome gain.
The researchers sifted through published datasets of proteins and RNA of individuals with Down syndrome and compared these to laboratory made cells with trisomies of chromosomes 3, 5, 12, and 21. What they found from this comparison was that it didn't matter which chromosome was in excess, the cells all had decreased ability to replicate, survive, and maintain their DNA.
"We were interested to find out why cells with imbalanced chromosomal content -- in other words, aneuploid -- are capable of surviving," says Krivega. "It was particularly exciting to me to learn if viable aneuploid embryonic cells have similarities with aneuploid cancer cells or cell lines, derived in the laboratory."
Additionally, they found that the adaptive T cell immune system was underdeveloped in all cells, while the innate immune system seemed to be overactive. The authors suggest that this is a consequence of general chromosome gain. This research can be expanded into autoimmune diseases, such as Alzheimer disease or acute leukemias in trisomy chr. 8 or 21, that also exist without any connection to aneuploidy.
"We hope that our work elucidating a complex trisomy phenotype should help to improve such kids' development," says Krivega.
Mapping The Down Syndrome Chromosome RegionEuropean Journal Of Human Genetics - Nature
Genomic Map of the D21S55-ETS2 RegionA partial physical map of this region in lymphocyte DNA has already been published [37] showing a physical linkage between D21S55 and ERG. We describe here a physical linkage between D21S55, ERG and ETS2, established by complete and partial digestion of fibroblast DNA [15].
Fragment sizes, detected after hybridization with these three probes, for complete or partial digestion with the enzymes NotI, NruI, SfiI, KspI, BssHII and MluI, were analyzed. No common fragment was found with SfiI, KspI, and BssHII with the three probes. With MluI, we identified two MluI fragments for ETS2 (360 and 800 kb), and one fragment of 1,650 kb for D21S55 and ERG. With NotI, we identified a 700-kb ETS2 fragment, a 1,450-kb ERG-D21S55 fragment and a 2,100-kb partial NotI fragment common to the three probes (data not shown). With NruI, these three probes hybridized to different complete restriction fragments: 600 kb ETS2, < 1,000 kb ERG and 1,200 kb D21S55. Figure 1 shows the partial NruI restriction fragments for these three probes. Two fragments from partial digests were common to ERG and D21S55 (1,900 and 2,400 kb), whereas no fragments from partial digests and carrying ETS2 contained ERG or D21S55. These fragments presumably contained contiguous NruI fragments distal to ETS2.
Fig. 1Sequential hybridizations of a PFGE blot with D21S55, ERG cDNA5′, and ETS2 cDNA. Fibroblast DNA was digested with NruI. 1 = 10 mM Mg2+; 2 = 0.5 mM Mg2+.
These data established the physical linkage between ERG, D21S55 and ETS2 but are insufficient to map the ERG gene with respect to D21S55 and ETS2.
YAC ContigThe YAC libraries were screened by PCR for ETS2 and ERG. The characteristics of the positive clones are summarized in table 1.
Table 1 Characterization of YAC clonesAluPCR. The AluPCR method allows the identification of unique sequences localized between two Alu sequences in human DNA.
The PCR conditions were determined for the amplification of a few fragments. By varying the Mg2+ concentration, the number of visible bands could be increased but with a higher background signal as well [data not shown, and 37]. As PCR fragments were cut out of the gels and used as probes with no further purification, we performed AluPCR in conditions giving the lowest background.
With the primer AluIV (fig. 2), we obtained fragments of the same size in different YACs. To determine their position on chromosome 21, their identity and the overlap between the different YAC clones, these fragments were used as probes on Southern blots of YAC (data not shown), human, and hybrid DNAs. Figure 3 shows an example of such an analysis: the two PCR products n1, namely AluIV: 1 from 259H11 and AluIV:1 from 448D5 hybridized with the same genomic fragment and are therefore identical, indicating that these two YACs overlap in a region containing this AluPCR product. YACs B19C12, 259H11 and A125B12 overlap as do 259H11, A125B12 and 448D5. As 448D5 and A196B6 both hybridize with the ETS2 cDNA probe, they are also overlapping. These data allow the construction of a contig between the ERG and the ETS2 YACs. More bands were amplified with the primer PDJ33 than with the primer AluIV. A similar analysis of the results with the primer PDJ33 confirmed the YAC overlaps identified with AluIV (data not shown).
Fig. 2Ethidium-bromide-stained gel of inter-AluIV PCR products. 1 = 259H11; 2 = A125B12; 3 = 448D5; 4 = A196B6; 5 = B19C12; 6 = yeast Saccharomyces cerevisiae AB972; nl indicates the inter-Alu IV fragments used as AluIV: 1 probes (fig. 3, 4).
Fig. 3Sequential hybridizations of EcoRI restricted DNAs. 1 = Human fibroblast; 2 = hybrid Acem; 3 = hybrid GA9–3; 4 = hybrid 1881c–13b; 5 = hybrid 9542c–5a; 6 = Chinese hamster; 7 = WA17, and 8 = mouse with AluIV: 1 probes from 448D5 and 259H11 identifying the same 15-kb fragment.
YAC Ends. YAC ends were identified by vector-AluPCR and the results are summarized in table 2. Only 50% of the ends could be isolated by this method. The amplification products were used as probes on Southern blots to determine their localizations on chromosome 21 and the overlap between YAC clones (table 2). The right arm of 259H11 and that of A196B6 did not hybridize with any other YACs, indicating that these ends map at each extremity of the contig. A125B12 (left arm) hybridized with A125B12 and 448D5, and 448D5 (left arm) with A125B12, 259H11 and 448D5. Thus these three YACs overlap and can be oriented with respect to each other. The left arm of B19C12 hybridized with 259H11, but not with the other YACs. This confirms the overlap between B19C12 and 259H11, as determined by AluPCR, and orients B19C12 with respect to 259H11 and A125B12.
Table 2 Isolation and characterization of YAC endsProbing YAC Southern blots with the left YAC end from 448D5 (probe LE448) visualized an EcoRI fragment of 2.4 kb in the two clones from the CEPH library (259H11 and 448D5) and an EcoRI fragment of 2.7 kb in the clone from the CHR21JYE library (A125B12) (fig. 4A). This size difference may be due to polymorphism. To test this hypothesis, we tested this probe on hybrid DNAs (data not shown) and on 9 different human DNAs (fig. 4B). In hybrid DNAs, we found either the 2.4- or the 2.7-kb fragment. Similarly, using human DNAs we found the 2.4-kb fragment in 5 individuals, the 2.7-kb fragment in 1 individual and both fragments (2.4 kb + 2.7 kb) in 3 individuals. These results confirm that probe LE448 detects an EcoRI polymorphism.
Fig. 4EcoRI polymorphism detected by LE448 probe. A YAC DNAs from 259H11 (1), A125B12 (2), 448D5 (3), and A196B6 (4). B Nine different human DNAs.
Restriction Map of the YACs. The five YACs were completely digested each with BssHII, MluI, SalI, NruI, NotI, SfiI and KspI. 448D5 and 259H11 (fig. 5) were partially digested by NruI, KspI, SfiI and BssHII and by NruI, SalI, BssHII and KspI, respectively. The resulting blots were hybridized with both YAC vector arms (pBR2.7 and pBR1.6) and the internal probes 5′ERG, 3′ERG, ETS2 promoter and ETS2 cDNA. The resulting data allowed the construction of the restriction map for the five YACs (fig. 6).
Fig. 5Sequential hybridizations of a PFGE blot with pBR1.6, ERG cDNA5′ and pBR2.7 probes. 259H11 YAC DNA was partially digested with 5 U SalI (0), 1 U SalI (1), 5 U NruI (2), 1 U NruI (3), 5 U BssHII (4), 1 U BssHII (5), 5 U KspI (6), and 1 U KspI (7).
Fig. 6Contig map of overlapping YAC clones. Right arm (R), left arm (L) and enzymes used for the restriction map are indicated on the figure. Dashed lines correspond to maximum size of the regions hybridizing with the ERG and the ETS2 probes. (?) = Restriction site not found.
The probes 5′ and 3′ERG overlap for 300 bp. The orientation of the ERG gene in the contig was deduced from the following data (shown in fig. 6): in 259H11 and A125B12, these two probes mapped on the same fragment (15 kb NruI, 30 kb SalI and 90 kb partial SalI fragments). The 5′ERG probe mapped on a 60-kb SalI and on a 170-kb NruI fragment. In A125B12, pBR1.6 mapped on the same 30-kb SalI fragment as the two ERG probes, whereas the 5′ERG 60-kb SalI fragment was not visualized. In B19C12, the two ERG probes and the pBR1.6 probe mapped on a 20-kb SalI and on a < 10-kb NruI fragment. The 5′ERG 60-kb SalI and 170-kb NruI fragments are not found in this YAC. Thus, the use of two ERG probes allows the orientation of this gene in the YACs 259H11 and A125B12, and indicates that the YAC B19C12 does not contain the entire gene.
To orient the ETS2 gene in the YACs 448D5 and A196B6, we used two internal probes: the ETS2 cDNA probe hybridized with the two YACs and the ETS2 promoter probe hybridized only with 448D5 (shown in fig. 6). In 448D5, the probes pBR1.6, ETS2 cDNA and ETS2 promoter were on different NruI fragments (40, 48 and 42 kb, respectively), but on single SalI (150 kb) and MluI (120 kb) fragments. Probes pBR1.6 and ETS2 cDNA were on the same 90 kb KspI, 90 kb BssHII and 88 kb partial NruI fragments. Probes ETS2 promoter and ETS2 cDNA were on the same 65 kb SfiI fragment. In A196B6, pBR2.7 and ETS2 cDNA mapped on 45 kb SfiI, 50 kb NruI, 130 kb SalI, 140 kb KspI, 140 kb BssHII and 180 kb MluI fragments. Thus the probes for the ETS2 gene can be ordered as follows in 448D5: right arm — ETS2 cDNA — ETS2 promoter; and in A196B6: left arm — ETS2 cDNA. This shows that the overlapping of these two YACs involves the coding region of the ETS2 gene.
An overlapping contig map (fig. 6) was constructed by comparing the restriction maps of the five YACs. There were a few differences between the maps of these YACs: one MluI site and one SalI site, which were not digested to completion in 259H11, were not found in A125B12. Similarly one NruI site which was only partially cleaved in 259H11 was not found in B19C12. This contig contains the two protooncogenes ERG and ETS2 in a head-to-head orientation. Based on the genomic map, ERG is localized between D21S55 and ETS2.
The presence of HMG14 in this contig was tested by hybridization on YAC Southern blots. The human fragment specific for HGM14 was not found in the YACs of the contig.
In situ Hybridization. All the five YACs were used for in situ hybridization assays on metaphasic lymphocyte chromosomes of normal individuals, to check the absence of coligation artifacts in these five YACs, and their localization on chromosome 21. All the YACs gave a hybridization signal in the same region, the 21q22.2–21q22.3 junction, and no signal on other chromosomes. Thus there are no coligation artifacts in these YACs.
The 3 ERG YACs (259H11, B19C12 and A125B12) were hybridized with the metaphasic lymphocyte chromosomes of patient FG. The karyotype of this patient is 46 XY, dir dup (21) (pter→q22.300:ql 1.205→qter). Previous data obtained by in situ hybridization with probes close to the borders of the duplicated segments were consistent with a true tandem duplication of chromosome 21 [13]. Only 259H11 gave 2 signals on the duplicated chromosome, showing that a large part of this YAC is duplicated in this patient (fig. 7). These data indicate that part of the 259H11 YAC is within the DCR.
Fig. 7In situ hybridization on metaphasic chromosomes of the patient FG with the YAC 259H11. Spots of hybridization on the normal and rearranged chromosomes are indicated by white arrows.
Localization of the ERG Gene with Respect to the DCR. To test if the ERG gene is within the DCR, we estimated the copy number of the 5′ERG sequence in blood DNA from patient FG by the slot blot method [18]. No duplication of this gene in patient FG was found (data not shown).
Previous PFGE analysis of the patient FG, with D21S55 and D21S16, indicated an abnormal NotI restriction fragment which contains the distal boundary of the DCR [13]. PFGE analysis with the ERG probes did not reveal this abnormal NotI restriction fragment (data not shown). This confirms that the ERG gene is distal to D21S55 and shows that it is outside the DCR.
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