Prenatal Screening and Prenatal Diagnosis in Genetic diseases
13.1 Difference betweenprenatal screening and prenatal diagnosis
Prenatal testing is available for pregnant women to assess the risk of genetic conditions or congenital disabilities in the fetus This testing can inform various options for managing the pregnancy and delivery, ultimately enhancing the baby's outcome It's essential to differentiate between prenatal screening and prenatal diagnosis, as these two concepts form the foundation of prenatal testing.
According to The American College of Obstetrics and Gynecology, prenatal genetic diagnostic testing aims to accurately identify specific genetic disorders or conditions in the fetus In contrast, prenatal genetic screening evaluates whether a patient has an increased risk of carrying a fetus affected by a genetic disorder.
Initial screening for congenital disabilities was developed in the 1950s with ulfrasound andhasbecome increasinglyprominent inobstetric care Real-tune gray scale imaging became available in the 1970s and improved prenatal diagnosis by
Ultrasonography plays a crucial role in early pregnancy evaluation, focusing on determining gestational age, fetal number, assessing malformations, and ensuring fetal well-being Among the first prenatal diagnostic options, amniocentesis, introduced in the 1950s, has evolved to be safer and is now utilized for genetic screening and infection evaluation Chorionic villus sampling (CVS) offers an earlier diagnostic alternative, while noninvasive methods like serum analyte screening and cell-free DNA screening have also emerged for detecting genetic abnormalities Screening tests can be conducted during both the first and second trimesters, with first-trimester screenings combining ultrasound and maternal blood samples, while second-trimester screenings rely solely on blood tests Results are typically available within a week, and those with positive outcomes are recommended for further diagnostic testing Ultimately, diagnostic tests are the only definitive way to determine if a developing baby has a chromosomal condition.
Diagnostic tests, such as chorionic villus sampling (CVS) and amniocentesis, can accurately identify chromosomal differences in a developing baby with over 99.9% precision Additionally, specific genetic disease tests must be specially requested, and their sensitivity and specificity vary based on the type of test ordered.
Prenatal testing primarily serves as a screening tool to identify pregnancies at high risk for chromosomal abnormalities or congenital conditions Key tests in this category include serum screening, carrier screening, and ultrasound.
Ultrasound can diagnose conditions like open neural tube defects, while serum screening is designed to identify women with pregnancies at an increased risk There are various options for serum screening, each with different test criteria and timing for implementation.
TableJ.3 Overview of current serum screening options /or aneuploidy
(reproduced fromCarlsonand I ora et aL, 201 ~J Ị44J
GeWational Age at Scrwnng (weeks)
MeaswtflttMs Obtained First-tame tier screen 10-13 Nuchâltraniueency Pajp- AhCG
Cell-free DXÃ Any age riier 9-10 week Molecular evaluation of cell-free fetal
Cell-free DNA, commonly known as non-invasive prenatal screening, has been commercially available since 2011 This innovative technology involves the collection of a maternal serum sample to isolate cell-free DNA fragments from the pregnancy, which are primarily derived from placental tissue released by apoptotic trophoblasts The fetal fraction of this DNA increases with gestational age, becoming significantly detectable as early as ten weeks into the pregnancy For accurate analysis, a fetal fraction greater than 4% is essential.
The 2016 screening test for trisomy 21 boasts the highest detection rate among available options, achieving 99% as highlighted in a recent meta-analysis by Gil (2015) However, the American College of Obstetricians and Gynecologists (ACOG) currently recommends cell-free DNA testing for aneuploidy only for women at high pretest risk, including those over 35 years of age at delivery, those with ultrasound findings suggesting increased risk, and women with a history of prior pregnancies affected by trisomy.
Ultrasound has become an essential tool in pregnancy management, with nearly all expectant mothers undergoing at least one ultrasound examination during their routine obstetric care Many women receive multiple scans throughout their pregnancy The primary purposes of ultrasound in obstetric care are to confirm gestational dating and to monitor for potential congenital disabilities.
Diagnostic testing provides patients with crucial information about the potential impact of specific genetic conditions on their pregnancy In the United States, the primary reason for undergoing diagnostic testing is advanced maternal age, particularly for those aged 35 years or older at the estimated delivery date Additional reasons for testing include positive results from aneuploidy screenings, a known family history of genetic disorders, or anomalies detected during ultrasound examinations While the American College of Obstetricians and Gynecologists (ACOG) recommends that all women have access to diagnostic testing, regardless of age, it is essential for patients to receive counseling beforehand due to the associated risks of pregnancy loss.
Chorionic villus sampling (CVS) is the only diagnostic test available during the first trimester, conducted between 11 and 13 weeks of gestation This procedure allows for various diagnostic analyses, including fluorescence in situ hybridization (FISH), karyotype analysis, molecular testing, and gene sequencing CVS can be performed through either a transcervical or transabdominal approach, where chorionic villi are collected for genetic evaluation under ultrasound guidance without entering the amniotic sac The advantage of CVS is that it enables earlier prenatal diagnosis, reducing uncertainty and allowing for safer pregnancy termination if desired However, a drawback is that 1% to 2% of CVS results may indicate confined placental mosaicism instead of true fetal chromosomal abnormalities, which could increase the risk of having a small-for-gestational-age infant Additionally, the estimated risk of pregnancy loss attributed to CVS is approximately 1 in 455.
Amniocentesis is available in the second or third trimesters of pregnancy and may be performed atanygestational age after 15 weeks In this technique, a sterile
Amniocentesis involves the insertion of a 28-gauge needle into the amniotic sac under ultrasound guidance to obtain amniotic fluid for testing This procedure is utilized not only for evaluating genetic disorders but also for detecting intra-amniotic or fetal infections through culture or polymerase chain reaction, as well as identifying neural tube defects by measuring alpha-fetoprotein and acetylcholinesterase levels in the amniotic fluid While complications are more common in earlier gestational ages, the estimated risk of pregnancy loss due to amniocentesis is approximately 1 in 900.
Preimplantation genetic diagnosis (PGD) is increasingly accessible and can identify chromosomal abnormalities earlier in the process This technique is performed after in vitro fertilization (IVF) by either removing a polar body or a single cell from the blastocyst, enabling the detection of abnormalities before embryo transfer, ensuring that only unaffected embryos are implanted It is important to distinguish between PGD and preimplantation genetic screening (PGS); while PGD is diagnostic, PGS serves as a screening method, and its clinical application is still debated PGD is specifically recommended for individuals at high risk of passing on genetic disorders, including all monogenic defects (autosomal recessive, autosomal dominant, and X-linked disorders) and carriers of balanced translocations, who face increased risks of implantation failure and recurrent miscarriages.
SUBJECTS AND METHODS- 24 29
Time and location
The research was conducted at Cenưe for Oene-Protein Research (Hanoi MedicalUniversity)from2019 March to2020 September
Location foramniocentesis NationalObstetrics and Gynecology Hospital 2.2 Subjects
04 patients with suspects of IGHMBP-2 related neuromuscular disorders and family members (parents, siblings)
- Clinical presentations with muscle weaknessand or respi rat on’ symptoms
- Age ofonset: frombirth to adolescence
• Respiratory symptoms appealabruptly and progress rapidly to respiratory distress without a historyof congenitalheart diseaseorrespiratory disease
- Muscle weakness beganatthedistalpart of the limbs (hands or legs)
- No familyhistoryof similar diseases was recorded.
Laboratory ’ equipment and apparatus
- Eppendorf tube 1,5 ml: 0,5 ml: 02 nil
- Falcon tube 15 ml and 50 ml
- Gene Amp PCR System9700(USA).
- w Transillununator Cbenudoc EQ-Bio-Rad (USA).
- Centrifuge and filtration Beckman (USA) Eppendorf (Germany)
Sanger Sequencinginstrument aBI Prism3 IOC Genetic Analyzer (USA)
• Next Generation Sequencing instrument Illumina HiSeq (USA)
Chemicals
♦ Chemicals for DX.i sequencing: Sequencing kỉ: se:for ỉBI 3000 and IlluminaHiSeq
- Step I: Four patients were carefully examined and ruled out muscle weakness and respiratory distress due to prior medical condition orabnormalities (congenital diabetes, vitaminB12deficiency, autoimmune disease !
- Step 2 Blood samples were collectedfromfour patients and family members for DNA extraction purification andamplification(by PCR)
In Step 3, targeted panel sequencing using Next Generation Sequencing and Sanger Sequencing was conducted to identify IGHMBP2 variants in four patients The pathogenicity of these IGHMBP2 variants was assessed following the guidelines established by the American College of Medical Genetics.
- Step 4 Blood sample horn family members were collected and used for Sanger Sequencingto identifyĨGHMBP2 path?genic variants
- Step $: Prenatal diagnosis was performed forthe mother of patient 3 using amniocentesis to detect chromosomaland geneticabnocnalities
- Step 6 Basedon genetic anah sii genetic counseling was given to thefamily for thedecision onthe pregnancy
Patients and family members provided 2 ml to 5 ml of peripheral blood for DNA extraction using the Phenol Chloroform method at the Center for Gen-Protein Research DNA concentration was measured with a Nano Drop 1000, and only samples with a purity range of 1.8 to 2.0 were deemed suitable for detecting genetic variants.
Step I Red blood cell hsis
- Eppendorf 1.5ml tube 1ml of RBC lysis (Lysis buffer - RBC) * 0.5ml whole bloodEDTA coated.
- Lightlyshake -• Voltcx — case ill room temperature for 5 minutes
- Centrifuge at4000rpm X 5 minutesat 4*c collectprecipĩtíion
- Redo the abovementioned steps 2-3 times until detecting white precipitation (white blood cell)
Step 2 White blood cells wash
- Add 1 nil Phosphate-buffered saline (PBS) for WBC lysis to the Eppendorf tube containing theprecipitation.
- Lightly shake -• Voile* -* case ill mom temperature tor5 minutes
• Cenuifuge at4000rpm X 5 minutes,at 4*c collect precipitation
Step3 White blood cell lysis
- Add C (p.His445Pro), c.1574T>C (p.Leu525Pro), and c.2362OT (p.Arg788Tô) Among the patients, only one was homozygous for an IGHMBP2 variant, while the remaining three exhibited compound heterozygous variants To confirm these findings, Sanger sequencing was performed on each patient's blood sample.
Identification of carriers among family members and application on prenatal
4.1 C linical manifestations and genetic testing of patients and family members
The clinical course of four patients showed significant heterogeneity Patients 2 and 3 experienced severe hypotonia before the age of one and suffered from respiratory distress, which ultimately led to their deaths In contrast, patient 4 did not exhibit respiratory symptoms but faced a gradual loss of ambulation Muscle weakness in patient 4 began distally and progressed proximally, presenting as largely asymptomatic, a characteristic that can be observed in various peripheral neuropathies.
Early-onset respiratory distress due to severe pneumonia and muscle tone loss was observed in patients I, 2, and 3, prompting a comprehensive literature review on this combination of symptoms The abnormalities primarily affected the peripheral muscles, particularly with distal onset in the limbs, leading us to focus on conditions related to the peripheral nervous system or neuromuscular diseases.
Neuromuscular diseases lack a unified classification but can be broadly categorized into three types based on the anatomy of the peripheral nervous system: motor neuron disease, peripheral neuropathy, and end-plate disorders In our analysis, we ruled out neuromuscular junction diseases, as these typically present in middle-aged individuals, with myasthenia gravis and Lambert-Eaton syndrome being the most prevalent Both conditions involve antibodies targeting the acetylcholine receptor, muscle-specific kinase (MUSK), and lipoprotein-related protein 4 (LRP4), leading to proximal muscle weakness Notably, our patients exhibited distal-onset muscle weakness, which differed from the characteristics of the end-plate group, and we found no respiratory system involvement in these cases.
DISCUSSION _ 45 4.1 Clinical manifestations and genetic testing of patients and family members 45
Options for prenatal diagnosis for hereditary disease
Genetic analysis revealed that the parents of four patients are carriers of the IGHXÍBP2 gene As SMARD1 and CMT2S are autosomal recessive disorders, there is a 25% chance that carrier parents may have an affected child Therefore, genetic counseling options like prenatal diagnosis or pre-implantation genetic diagnosis can help parents reduce the likelihood of having a homozygous child.
During the research, the mother of patient family 3 was 17 weeks pregnant Following the diagnosis of SMARD1 with compound heterozygous variants in patient three, the family was recommended to undergo amniocentesis for genetic diagnostic testing The genetic analysis of the fetus confirmed the presence of compound heterozygous variants.
Prenatal genetic testing is crucial for identifying potential genetic disorders in a fetus, allowing parents to make informed decisions about their pregnancy In a case where a 5Ổ genotype indicated a risk of SXCARDi, the parents opted to terminate the pregnancy after comprehensive genetic counseling This testing provides numerous benefits, such as reassuring parents with normal results, identifying conditions that may require prenatal treatment, optimizing neonatal care by ensuring proper delivery settings, and facilitating the possibility of pregnancy termination when necessary Techniques like chorionic villus sampling (CVS), amniocentesis, and preimplantation genetic diagnosis (PGD) are essential tools for effective intrauterine genetic diagnosis.
Chorionic villus sampling (CVS) is typically conducted between 10 to 13 weeks of gestation, while amniocentesis is performed after 15 weeks The key benefit of CVS is its earlier timing, which allows for quicker analysis of viable cells, with results available in 5 to 7 days compared to 7 to 10 days for amniocentesis This expedited process provides crucial management options following an abnormal first-trimester ultrasound or screening test, although amniocentesis remains a viable diagnostic alternative.
The prevalence of pregnancy loss associated with chorionic villus sampling (CVS) has significantly declined over time A recent meta-analysis involving 8,899 women who underwent CVS and 37,388 who did not found a procedure-related loss rate of just 0.22% (1 in 455) [Akolekar 2015, Bulletin 162] While some studies have suggested a link between CVS and limb reduction defects, the associated risk appears to be very low, particularly for procedures conducted after ten weeks of gestation Additionally, vaginal spotting or bleeding is a potential complication, occurring in up to 32% of patients following transcervical CVS, while the incidence after transabdominal CVS is lower.
-w •* CN ôG lower than that The incidenceof culture failure amniotic fluidleakage or infection after CVS is less than 0 5% (68) (69)
Amniocentesis for genetic diagnosis is typically performed between 15 and 20 weeks of gestation, but it can be conducted at later stages as well Extensive multicenter studies have confirmed the safety and cytogenetic diagnostic accuracy of this procedure To minimize risks, especially in cases of alloimmunization, transplacental needle passage is generally avoided when technically feasible However, data indicates that the procedure-related loss rate is similar for both transplacental and non-transplacental approaches Additionally, if the amnion and chorion have not fused, the procedure may be delayed due to an increased likelihood of failing to obtain amniotic fluid or needing a second puncture.
Amniocentesis carries a significant risk of pregnancy loss, with the procedure-related loss rate during mid-trimester amniocentesis decreasing over time due to advancements in technique and increased provider experience Accurate data on miscarriage rates following amniocentesis is difficult to obtain due to the rarity of such outcomes and challenges in establishing appropriate control groups Estimates suggest that the risk of procedure-related pregnancy loss is approximately 0.1% to 0.3% when performed by experienced healthcare providers, though findings can vary based on medical settings and techniques It is essential for healthcare providers to contextualize the risk of miscarriage associated with amniocentesis within the patient's overall background risk Minor complications, such as transient vaginal spotting or amniotic fluid leakage, occur in about 1-2% of cases Historically, early amniocentesis, conducted between 10 and 13 weeks of gestation, has been associated with significantly higher rates of pregnancy loss compared to mid-trimester procedures.
58 complications than mid trimester amniocentesis Therefore early amniocentesis (before 14 weeks of gestation)isnot recommended
Preimplantation genetic testing (PGT) is a procedure that evaluates embryos for specific genetic disorders prior to implantation This testing can be conducted on polar bodies from the oocyte, a single blastomere from a cleavage-stage embryo, or a group of cells from the trophectoderm at the blastocyst stage Utilizing cytogenetic or molecular techniques, preimplantation genetic diagnosis (PGD) is applicable to early embryos created through in vitro fertilization and can screen for most genetic conditions with identified mutations in the family However, since PGD typically analyzes only one or a few cells, it is advisable to confirm results through chorionic villus sampling (CVS) or amniocentesis.
In this research we reachedtwo conclusions
Firstly 14 patient was homozygous for an ỈGHMBP2 variant and 3 4 had compound heterozygous variants Four variants found included: C.1235-3AX3 (intron 8) c 1334A>C (p Hts445Pro) (exon 9) C.1574T>C (pLeu525Pro) (exon
The C2362OT (p.Arg788Ter) variant, along with the novel C.1574T>G (p.Leu525Pro), was identified in patients who exhibited muscle weakness in the lower limbs Among the affected individuals, three out of four experienced respiratory distress, with two tragically passing away before reaching the age of two.
Secondly, parents and siblings of four patients were all earners and genetic testing was successfully applied onprenataldiagnosis for the familyofpatient 3
HubbardJ ed (1974), 77ằ PeripheralNervous System springer US
< 1078) A clinician** view ofneuromusculardiseases, by Michael II Brooke.225 pp illustrated $18 95 the ^ĩllianis & Wilkins Company, Baltimore 197'