Cultural merging invited many inherited disorders in different cultures. Those alleles which remained recessively safer in some population are now becoming lethal and vulnerable in other population. Thalassemia, sickle cell anaemia, Huntington’s disease, cystic fibrosis and other genetic disorders are substantially increased in each and every population every day.
Though genetic counselling and screening are now advanced and accurate, still we cannot terminate genetic disorders spreading in the population. Curing any disease is a prime importance in medical science but as the environment become adversely influencing our health, it is very important to prevent them in the coming years.
In this article, we are going to discuss the newest and most advanced diagnostic and screening technique, called as a prenatal diagnostic technique, PNDT. Also, we will discuss the role of Cell-free DNA is the advancement of prenatal diagnostics.
Essentially, if we understand the status of a genetic condition in the early stage of life, it will be revolutionary. Prenatal genetic screening provides an opportunity to screen genetic conditions in the early stage of development. PCR technology can help in genetic testing. Read some of the interesting articles on PCR,
- The Function of dNTPs in PCR reaction
- Role of DMSO in PCR: DMSO a PCR enhancer
- PCR primer design guidelines
- Role of MgCl2 in PCR reaction
- Function of taq DNA polymerase in PCR
“knowing disease status at an early embryonic stage during the pregnancy”, is called as prenatal genetic screening. PNDT is one of the emerging screening methods in medical science with a lot of opportunities.
Genetic disorders are inherited in one of the two patterns, Dominant or recessive. If it is dominant then the fetus will suffer from vulnerable phenotypes of the disease. But if it remains recessive, the fetus could possibly remain safe.
Depending upon the nature of inheritance, different genetic diseases are characterized as autosomal dominant, autosomal recessive, X linked dominant or X linked recessive and Y linked.
Prenatal diagnostic gives an opportunity to save the fetus if it is absolutely normal or carrier. Amniotic fluid or chorionic villi samples are one of the major sample type routinely used in prenatal screening. DNA is extracted from one of the available samples and it is screened based on the history of parents.
Genetic counselling plays a major role in prenatal diagnosis because a counsellor advice couple whether they can go for prenatal or not. Couples having the previous history of genetic disorders or carrier for any genetic disease can possibly be advised for prenatal diagnosis.
Though Prenatal genetic screening saves many lives and reduces genetic burden globally, it has several limitations. Prenatal genetic screening method deals with an invasive type of sample collection method. The patient may feel pain while collecting the sample.
Invasive sample collection has some serious drawbacks, as we have to collect a sample by holing, niddling or cutting the tissue, there is always remain a chance of infection at the collection site, the patient feels pain and swelling at the site of collection.
However, in the case of prenatal diagnosis, the invasive technique become extremely dangerous because of the chance of miscarriage. Even fetus become infected with some life-threatening disorders.
In the year 1997, Lo YM, Tsui NB, Chiu RW and co-workers developed a safer and non-invasive method for prenatal screening, called as cffDNA. Cell-free fetal DNA is freely floated fetal DNA which is present in maternal blood. During the 10th week of gestation, trophoblastic cells present in the placenta, are the main source of fetal DNA and circulated in the maternal blood.
Cell-free fetal DNAs are small fragments of DNA and are freely floated with mother blood hence we can isolate it by taking serum. Smaller fragments are light weighted than other cell organelles, therefore, it is floated in the serum of maternal blood.
With the help of special kits, we can extract cell-free DNA fragments and can diagnose genetic disease. cell-free fetal DNA technique is highly impactful for single gene disorders such as thalassemia, sickle cell anaemia, cystic fibrosis and Huntington’s disease.
Additionally, it is beneficial in down syndrome, Patau syndrome and Edward syndrome (marker-based screening, not for chromosomal analysis). Since it is non-painful and totally safe, scientists are developing it as a stander prenatal diagnostic tool.
Although it is safer and non-invasive, it has its own drawbacks. The amount of DNA obtained from cell-free DNA is very less if the fetus is less than 10 weeks then the amount is negligible. We cannot extract cell-free DNA with traditional DNA extraction kits and protocols, it requires several major modifications in routine protocols.
Adding formaldehyde in serum may be helpful in increasing the amount of cell-free DNA. The fetus must be older than 10 weeks of gestation for collecting cffDNA.
Moreover, the fragments what we get from cell-free DNAs are always varying. So it is possible that if you want to screen beta-globin gene mutation but could not get beta-globin gene fragments all the time. Perhaps, the copies of the fragment are even very less.
The limited amount of cell-free DNA makes it even sensitive than any other molecular techniques hence we have to proceed sample more carefully because we have no valid reason, why fragment of our interested gene is not extracted.
Further, maternal DNA fragments are present in serum, therefore the chance of a false positive result is higher. What we extracted and confirmed as a recessive, maybe mother’s DNA. Though mother’s DNA fragments are larger than cell-free fetal DNA, it is one of the major limitation of NIPD technique by cffDNA.
Some of the Y bearing and X bearing fragments are frequently found in cell-free DNA hence for fetus sex identification cffDNA is now routinely available. The general procedure for cffDNA extraction are listed here,
- Collect mothers blood sample up to 10 or 12 ml
- Separate serum immediately by centrifugation and proceed immediately.
- Separate cffDNA fragments by standard protocol. QIAGEN virus DNA extraction kit mostly gives good result in all cases.
- Store extracted DNA properly in the cold channel because this piece of DNA is precious as compared to other DNA.
- If the quantity is sufficient, proceed for PCR based screening otherwise sent it directly for sequencing.
If the patient’s pregnancy is at high risk of miscarriage, cffDNA technique is always advisable. For normal pregnancy amniocentesis or chorionic villi sampling gives the best result. Although it is invasive, it is accurate.
In recent time, the cffDNA technique is not ready for routine medical screening but it has potential, it will be the major game changer for prenatal diagnosis in the future.
I personally worked for a year in the prenatal genetic department, and believe me, we are always under pressure of proceeding fetus sample safely because we do not have another chance of sample collection.
Even, we have to cross check all the result all the time because, in the end, it is a matter of someone’s life.
As a geneticist, I recommend to go for genetic screening, if you have any history of genetic disease because congenital genetic diseases are increasing genetic burden globally.
The story covered and written by – Tushar Chauhan
Reviewed by – Debayan Baidya.