Pros and Cons of Expanded Carrier Screening

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1		Valeria D'Argenio	--	2108	2022-04-10 11:03:19	\|
2	format corrected.	Beatrix Zheng	+ 1 word(s)	2109	2022-04-11 04:46:40	\|

This entry is adapted from the peer-reviewed paper 10.3390/medicina58030455

Expanded Carrier Screening (ECS) means using molecular techniques to simultaneously analyze multiple DNA mutations or genes responsible for several genetic diseases without a clear risk of inheritance in the individual/couple admitted to the test. In this way, it is possible to avoid biases related to incomplete familial information or difficulties related to the correct estimation of the ethnic risks, also considering the multiethnicity of modern societies. However, more data mean more information, including the so-called incidental findings and the consequent need to finely regulate ECS and the following communication of the obtained results.

genetic carrier screening expanded carrier screening next generation sequencing recessive genetic disease genetic counselling

1. Technical Issues

Expanded Carrier Screening (ECS) has been prompted by recent technological advancements that have made it possible to analyze several genomic loci/genes in a time- and cost-affordable manner ^[1]. Moreover, the current ECS development and diffusion has been driven by the observation that, although ECS screened diseases are individually rare, the cumulative risk of having an affected baby is not so rare, especially if compared to other conditions commonly screened during pregnancy ^[2]. Nevertheless, ECS offering is extremely variable in terms of panels size and used technologies, making it difficult to compare results from different laboratories in addition to confusing patients ^[3].

The first method used to design ECS panels was targeted genotyping ^[2]. Targeted genotyping uses array-based technologies to specifically analyze hundreds or thousands already known DNA variants through specifically designed probes ^[4]. Almost ten years ago, Lazarin et al. reported the use of a mutations panel able to simultaneously analyze more than 400 Mendelian variants associated with more than 100 diseases: by analyzing 23,453 individuals with different ethnicities, they found that 24% carried at least one mutation, supporting the routine use of pan-ethnic ECS ^[5]. However, targeted genotyping is limited to a set of known disease-causing variants for each tested condition; thus, this approach may lack diagnostic sensitivity and accuracy since rare or novel mutations will be undetectable. Moreover, mutations frequency varies across different ethnic groups so that carrier detection may be impaired based on patients’ ancestry. This aspect in turn may make it difficult to estimate the individual residual risk in case of a negative result ^[2].

The recent development of next-generation sequencing (NGS)-based approaches and their validation in routine diagnostic settings ^[6]^[7]^[8] has enhanced the use of NGS-based panels for ECS purposes. Indeed, NGS technologies have been assessed as sensitive and accurate methods for nucleic acids sequence analysis able to detect different kinds of DNA alterations (both known and unknown) and to simultaneously analyze large genomic regions at reduced cost in respect to other techniques ^[9]. Bell et al. reported the evaluation of a preconception, NGS-based ECS to test for 448 severe, recessive diseases in 104 unrelated subjects: they found an average carrier burden of 2.8 and, based on this finding and the test’s lower cost compared to lifetime diseases’ treatments, suggested that NGS-based ECS should be offered to the general population to impact the incidence of rare recessive disorders ^[10]. Hallman et al. carried out a clinical validation of a NGS-based ECS and, by analyzing 11,691 preconception patients, identified 447 carriers and two affected subjects; interestingly, about 25% of the identified mutations were rare mutations not included in traditional tests, thus underlying the advantage of NGS over traditional approaches ^[11]. A study carried out on 805 individuals allowed to identify 352 mutations carriers (43.7%), thus suggesting that all couples who wish to conceive should consider NGS-based ECS irrespective of their ethnicity ^[12]. In this regard, Westemeyer et al. reported the use of NGS-based ECS in the general U.S. population; by analyzing 381,014 individuals, they were able to provide more information compared to traditional screening, thus suggesting the use of NGS to carry out the ECS in all reproductive-age women ^[13].

NGS-based panels for ECS also allow estimating carriers’ frequency in less-studied populations/ethnic groups. Singh et al. used a targeted NGS based approach to analyze 200 unrelated individuals from northern India; they found a carrier frequency of 26% and also highlighted that most of the pathogenic variants identified were different from those commonly found in the West, thus underlying the advantage to use NGS ^[14]. Similarly, Chan et al., using a NGS-based ECS to evaluate 123 infertile Chinese women, found that 58.7% of the tested individuals were carriers of at least a disease and suggested the use of ECS to better estimate carrier frequency in Chinese people ^[15].

Finally, to overcome the already discussed controversy related to different gene panels size, Punj et al. evaluated the possibility to use genome sequencing for ECS purposes: they found that this approach has higher diagnostic sensitivity with respect to the NGS-based targeted ones even if variants interpretation may be challenging ^[16].

The main features of all the studies reviewed herein are summarized in Table 1 to allow their easier comparison.

Table 1. Comparative table reporting the study design and the main findings of the cited ECS studies.

Study	ECS Methodology	DNA Variants/Genes Panel	Studied Population	Main Findings
Lazarin et al. ^[5]	Targeted genotyping	417 DNA variants associated with 108 diseases	23,453 individuals with different ethnicities	24% of individuals carried at least one condition
Bell et al. ^[10]	Targeted NGS	437 genes	104 unrelated subjects	Average carrier burden of 2.8
Hallam et al. ^[11]	Targeted NGS	15 genes	11,691 preconception patients	447 carriers and 2 affected subjects individuated; 25% were rare mutations not included in common preconception tests
Hernandez-Nieto et al. ^[12]	Targeted NGS	283 genes	805 individuals (391 of which are couples)	352 carriers (43.7%)
Westemeyer et al. ^[13]	Targeted NGS	274 genes	381,014 individuals with different ethnicity and age	1 in 44 (2.3%) couples was at risk for genetic disorder
Singh et al. ^[14]	Targeted NGS	88 genes	200 unrelated individuals	52 carriers (26%)
Chan et al. ^[15]	Targeted NGS	104 genes	123 infertile women and 20 of their partners	58.7% were carriers
Punj et al. ^[16]	Genome sequencing	728 genes	202 individuals (131 women and 71 of their partners)	78% were positive carrier for at least one condition; 304 variants found

Overall, the use of NGS has the advantage to allow both pan-ethnic screening and the simultaneous analysis of an increasing number of diseases ^[17]. Indeed, with respect to the mutation-based genotyping panel, often specific for some ethnic groups, NGS can also identify rare and/or novel mutations. However, it has to be mentioned that most of the commonly offered ECS panels do not include intronic regions ^[4]. Moreover, some other limitations of these technologies have to be taken into account, such as the possible lack of mosaicisms due to low sequencing coverage, the need for high data storage, and the identification of a large number of variants of unknown significance (VUSs) ^[17]^[16]. Currently used NGS methodologies are not able to detect all the possible DNA variants, such as triplet repeats, and short reads may be challenging for highly homologous genomic loci ^[17]^[16]. Finally, many inherited diseases are featured by high clinical variability due to variable expression and penetrance, so disease prediction later in life based just on genotype may be very tricky ^[17].

Within the NGS-based approaches, genome sequencing has the advantage to avoid biases that may occur during libraries preparation procedures, such as PCR biases or a not homogeneous representation of the target regions, and may allow a better estimation of structural variants ^[16]; however, the above-mentioned pitfalls related to the high quantity of data generated and their difficult interpretation will be still more represented. It is to be expected that, as has already happened with NGS about ten years ago, the advent of the so-called third-generation sequencers, thanks to long reads implementation, will provide a solution to some of these biases ^[18]. Moreover, novel bioinformatic tools will allow structural variations detection based on sequencing data increasing ECS sensitivity ^[19]. In this context, a very recent publication by Zhao et al. reported the validation of NGS for the detection of SMN1 gene copy numbers by analyzing 478 samples with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR), and NGS; interestingly, they found that NGS performed better than the other two methods and suggested that it can be useful in ECS context to reduce the need for multiple methodologies and, in turn, analysis time and costs ^[20].

These technical innovations will open the way to novel methodologies to improve currently available ECS.

2. Ethical Issues

As mentioned above, ECS allows to simultaneously analyze an increasing number of diseases in individuals and/or couples to identify the “at-risk” subjects, provide them information regarding their risk of transmitting a disease to the offspring, and discuss their consequent reproductive options. As is easy to expect, using this test in a clinical context raises several ethical concerns ^[21].

First of all, pre- and post-test genetic counseling should always be provided, but this is difficult to set up in the case of population-based screening. Accordingly, scientific societies have stated that, in this context, the pre-test counseling is not only not practical, but also unnecessary ^[22], and it has also been proposed that traditional genetic counselling could be reserved just for difficult cases, while in a routine setting, pre-test counseling could be entrusted to other medical professionals ^[2]. This implies a continued medical education to ensure updated knowledge on a rapidly evolving field.

Education programs should also be provided to the general population to minimize anxiety, avoid stigmatization related to genetics, and increase the rate of ECS acceptance. However, it has been already reported many years ago that anxiety related to carrier status seems to be overestimated since it disappears in a few months unless a positive family history and an affected child is already present ^[23]^[24]. Accordingly, based on a survey carried out on 240 women planning a pregnancy who refused ECS, Gilmore et al. found that most of them declined the enrollment for lack of interest/time, while a minority stated they do not want to have this information or are afraid of getting stressed ^[25]. However, further, well-designed studies are required to define the long-term effects of ECS on individuals’ well-being.

While pre-test counseling might be avoided, post-test counseling is crucial to discuss ECS results. Indeed, if the test has been carried out as a preconception screening or during pregnancy, the obtained information could support couples in making informed decisions regarding their reproductive options and gynecologists in properly monitoring pregnancy and delivery. In this context, it has to be mentioned that some of the diseases included in the ECS panel are extremely rare, and their causative mutations, related phenotypes, and long-term outcomes might be largely unknown. This impairs counselling efficacy both in the case of positive results since it does not allow a correct risk estimation and in the case of a negative one since it makes it difficult to evaluate the residual risk.

A particularly critical point is represented by the difficulty to a priori assess the clinical severity of a disease. Considering the enlarged and inclusive design of the currently offered ECSs, really uncommon diseases, whose severity may be not clearly recognized, may be identified, impairing pre-test and, mostly, post-test counseling ^[2]. This situation could be far from rare given that, while diseases prevalence is low, mutations carriers’ rate is high in different populations. To support health care professionals in this task, a disease-severity classification in four tiers has been proposed ^[26]. Even if this kind of classification may somewhat facilitate communication, it does not consider the individual variability and the variable phenotypic expression and penetrance that the same disease, sometimes in the same family, can present ^[17]. Moreover, some disorders may have a mild phenotype that does not justify any intervention but raises questions about their inclusion in the ECS panel. Similar considerations may apply to late-onset diseases, especially if treatments are available.

Another weakness is represented by the potentially high number of VUSs identified in each analyzed subject. Patients should be informed about the possibility of obtaining uncertain results and that variants’ significance interpretation and classification may vary over time ^[27].

Finally, in the case of negative results, it is crucial to underline that this does not mean having any risk. Still, several factors can influence residual risk, including technical factors, current knowledge, or individual factors ^[27].

All the above-mentioned issues have to be carefully taken into account, especially if the researchers consider the potential consequences that the indiscriminate diffusion of enlarged genetic tests, such as ECS, may have at political level and within specific communities. In this context, the Dor Yeshorim project has to be mentioned. It is a premarital carrier testing program implemented in an ultra-orthodox Jewish community with the aim to reduce the number of children affected by genetic diseases; as expected, this program is raising an intense debate on the limitations regarding the personal freedom and on its coercive aspects ^[28]. A similar program is compulsory also for couples before they get married in the United Arab Emirates limited to hemoglobinopathy carrier status assessment and justified by the high prevalence of this disease in that population ^[29]. These experiences, currently limited to a single disease or to restricted ethnic groups, highlight once again the need for clear guidelines to regulate the use of such genomic testing procedures.

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1. Technical Issues

2. Ethical Issues

References