K. N. Sreelakshmi She is a consultant fetal medicine in Malnad Diagnostic centre, Shimoga, Karnataka. She has done MBBS from Bangalore Medical College, Karnataka and MS (OBG) from KMC Manipal in the year 2005. She was a former Associate Professor of KMC, Manipal and was working as Professor and Head of Subbaiah Medical College, Shimoga, Karnataka (2012–2018). Her areas of interest are: prenatal diagnosis with special interest in fetal cardiology. Recently she was awarded 3 months (April–July 2018) fellowship from SIAMG (Society of Indian Academy of Medical Genetics) which she has done from KMC, Manipal.
Medical genetics has evolved over a decade, and hence, all investigations are available for clinical practice. Many diseases are diagnosed accurately today because of new investigations. These advanced investigations are affordable, accessible and available in day-to-day practice. Hence, there is a need and it is a time for us to understand these advanced technologies. Karyotyping and rapid aneuploidy tests are basic tests, while chromosomal microarray and next-generation sequencing are advanced technologies. It is time to update the knowledge and utilize them in day-to-day practice. These tests are utilized both in prenatal diagnosis and in some clinical scenarios, which are elaborated in detail. Karyotyping is the basic tool to detect both numerical and structural abnormalities. It is advantageous in that it is accurate with error of 0.001% but has a resolution of up to 5 MB. Rapid aneuploidy detection tests are equally accurate and detect as good as 99%. They are FISH, QF-PCR and MLPA. They have high sensitivity and specificity, and results are available within 3 days of time. Hence, these tests are apt for Indian scenarios, where late detection of anomalies (18–20 weeks) is common. Chromosomal microarray is the hybridization technique which detects aneuploidy of all chromosomes. This is useful for detection of deletion and duplication in chromosomes. This is not available for prenatal diagnosis in India now, whereas this is available for prenatal diagnosis in developed countries. Whole-exome sequencing and whole-genome sequencing are advanced techniques which have been described and discussed at length.
Keywords: Karyotyping · Rapid aneuploidy detection tests · Chromosomal microarray · Whole-exome sequencing · Whole-genome sequencing
There have been tremendous developments in the field of medical genetics in the past decade, especially in deciphering the genome and identifying the phenotypic effects of genomic variations. Diagnosis and management of genetic disorders, namely monogenic and chromosomal disorders, are affected by genomic techniques like next-generation sequencing and microarray.
Here, basic investigations, interpretation of these tests and common clinical conditions needing genetic investigations are discussed at length.
As early as 1882, Walther Flemming, an Austrian cytologist and professor of Anatomy, published the first illustration of human chromosomes. In 1959, Lejeune described extra chromosome in Down syndrome in each cell. The word chromosome is derived from the Greek word “colored body.” Human cells contain 46 chromosomes comprising 22 pairs. The numbers are assigned in descending order of length, size and centromere position of each chromosome pair, with long arm as “q” and short arm as “p.” Karyotype is done by g banding technique with resolution of up to 350–550 MB [1, 2].
Chromosome abnormalities can be either numerical or structural abnormality. Most common abnormalities are aneuploidies, i.e., Down syndrome, Trisomy 21, Trisomy 18, Trisomy 13 and monosomy X (Figs. 1, 2).Common clinical conditions to order karyotype are:
Advantages of karyotyping are:
The various indications for fetal cytogenetic testing include:
In India, many ultrasound abnormalities become apparent during 18–20 weeks of scan; hence, accurate rapid aneuploidy detection techniques are important.
These are primarily targeted for the diagnosis of common autosomal trisomies (13, 18, 21) and sex chromosomal aneuploidies [3–5].
Three methods of diagnostic techniques validated for prenatal diagnosis are:
FISH: Fluorescent In situ Hybridization
It is usually performed on uncultured interphase cells with probes designed specifically for chromosomes 13, 18, 21, X and Y. The number of fluorescent signals per cell gives the number of copies of the targeted chromosome. The technique is known to be almost 100% accurate with an added advantage of ability to detect triploidy also. However, FISH technique is non-automated and time-consuming and necessitates a skilled technician.
QF‑PCR: Quantitative Fluorescent In situ Hybridization
This assay has been widely used for the past 2 decades, which uses fluorescent-labeled primers to amplify specific DNA markers which are polymorphic and unique for chromosomes 13, 18, 21, X and Y. Detection of maternal cell contamination, triploidy and mosaicism as low as 15% is an important advantage of these techniques.
MLPA: Multiplex Ligation‑Dependent Probe Amplification
It is also a PCR-based method, which is cheaper and less labor-intensive than FISH technique. The free ends of the ligated probes are complementary to the primer which enables the amplification of target sites. The technique has a capacity to quantify up to 40–50 different target sequences in one reaction. One of the major drawbacks of this technique is the failure to detect triploidies, especially in a female fetus. It is a completely automated method and is being increasingly used as a method for RAD, especially where large-scale testing of samples is required. Table 1 summarises advantages and disadvantages of RAD techniques.
New Methods of Genetic Testing for Prenatal Diagnosis: Chromosomal Microarray
This is based on the principle of hybridization, and the strength of signals from these probes is interpreted in an automated manner to provide information regarding the number of copies of that region of the genome. In contrast to the commonly used RAD techniques, chromosomal microarray can detect aneuploidies of all 23 chromosomes as well as submicroscopic copy number abnormalities in a genomewide manner [6, 7].
Recurrent pregnancy loss and genetics Recurrent pregnancy losses, also known as recurrent spontaneous abortions, are traditionally defined as 3 or more consecutive pregnancy losses of less than 20 weeks of gestation. A total of 3–5% of couples experience RPL. The cause of RPL is difficult to assess, and in fact, no cause can be determined in half of the cases in spite of a battery of investigations. This suggests the presence of unidentified genetic causes.
In 3–5% of couples, with RPL, one partner is found to carry a balanced chromosomal rearrangement, 50% are balanced reciprocal translocations, and 12% are sex chromosomal mosaicism. Although carriers of balanced translocation are phenotypically normal, their pregnancies are at increased risk miscarriages or live births with congenital abnormalities or intellectual disability. The remainders are chromosomal inversions and other sporadic abnormalities.
In these couples, RPL occurs due to abnormal segregation of gametes at the time of meiosis. In couples, with recurrent miscarriage, chromosomal abnormalities of the embryo account for 30–57% of further miscarriages [8–10].
RCOG recommends karyotyping of products of conception of third and subsequent miscarriages and parental peripheral blood karyotyping in couples with unbalanced structural abnormality [11].
Couples with balanced translocations have a low risk (0.8%) of pregnancies with an unbalanced karyotype surviving into second trimester, and chance of having healthy child is 83%.
Preimplantation genetic diagnosis or fetal karyotyping by amniocentesis is an option for these couples to select fetuses with normal chromosomal content. Some chromosomal variations like pericentric inversion of 9, small or large heterochromatin arm of Y chromosome and inversion Y are seen in many normal individuals and are not known to be associated with poor reproductive outcome.
Premature ovarian aging/failure and genetics
Genetic aberrations comprise one-third of women with premature ovarian aging and also ovarian failure. When FSH is elevated above the age-specific cutoffs, premature ovarian aging is considered. A study by Gleicher et al. titled “Do the etiologies of POF and POA same?” concluded that presumed underlying etiologies of POA follow a similar distribution pattern as reported for POF. They proved the hypotheses that POA is a precursor stage of POF and hence requires similar evaluation [12–14].
Genetic causes comprised approximately 16% of the total in the study conducted by Gleicher et al. Both autosomes and X chromosomal involvement are documented. They are Turner mosaicism, partial X chromosome deletion, X chromosome mosaicism, X chromosome inactivation and FMR 1 (fragile site mental retardation X gene). X chromosome partial deletions are more common, while balanced X chromosome to autosome translocation of Xq13–q26 is rare, but documented. Autosomes involved are at the following gene loci: 3q, 13q, 14q, 17q, 15q and 11p [15, 16]. We had a rare case of triple X syndrome of premature aging in our clinical practice which is reported and Fig. 3 represents karyotype of the same.
Primary amenorrhea and genetics
Clinical features with Turner syndrome like webbed neck, short stature, cubitus valgus with absent menstruation and absent secondary sexual characteristics by the age of 15 years are definite indications for karyotyping. There may be Y element in karyotype, and this is an indication for gonadectomy to prevent gonadoblastoma.
Whole-exome sequencing (WES) and whole-genome sequencing (WGS) [17] are two important new technologies which have become affordable, accessible and available in India. This has enhanced the diagnostic yield in medical genetics. This is not yet used routinely for prenatal diagnosis because of the following reasons:
This is a genomic technique for sequencing of all protein coding genes in genome. This consists of two steps. First step consists of separating the subset of DNA that encodes the proteins called exons, which constitute about 1% of the total genome or approximately 30 million base pairs. The second step is to sequence the exonic DNA using any highthroughput DNA sequencing technology. After sequencing, three kinds of interpretation are obtained:
This is the process of determining the complete DNA sequence of an organism’s genome at a single time. This entails sequencing all of an organism’s chromosomal DNA as well as DNA contained in the mitochondria.
As of now, these diagnostic technologies are used for diagnosis of the following:
Hence, prenatal diagnosis with whole-exome sequencing is now confined to the clinical scenarios wherein there is positive previous targeted gene testing.
Conflicts of interest The author declares that there is no conflict of interest.
Human and Animal Rights Not applicable.
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