Integration Of Microarray Technology Into Prenatal Diagnosis: Counseling Issues Generated During The NICHD Clinical Trial

Abstract

Cytogenetic microarray analysis (CMA) in prenatal testing detects chromosome abnormalities and new genetic syndromes that would be missed by conventional cytogenetics and has the potential to significantly enhance prenatal genetic evaluation. A large NICHD-sponsored multi-centered trial to assess the role of CMA as a primary prenatal diagnostic tool has been completed, and results will soon be available. Integration of this technology into clinical care will require thoughtful changes in patient counseling. Here we examine four cases, all ascertained in the NICHD prenatal microarray study, to illustrate the challenges and subtleties of genetic counseling required with prenatal CMA testing. While the specifics of each case are distinct, the underlying genetic principles of uncertainty, variable expressivity, and lack of precise genotype-phenotype correlation are well known and already part of prenatal counseling. Counselor and practitioner education will need to include both the science of interpreting array findings as well as development of improved approaches to uncertainty. A team approach to interpretation will need to be developed, as will standardized guidelines by professional organizations and laboratories. Of equal import is additional research into patient attitudes and desires, and a better understanding of the full phenotypic spectrum of copy number variants discovered in-utero.

Microarray copy number analysis has proven to be a powerful tool in the evaluation of infants and children with congenital anomalies, dysmorphic features, and neurocognitive disabilities including developmental delay and autism spectrum disorder, with clinically significant findings reported in up to 15% of cases with normal conventional karyotypes ^(1–3). Based on this experience, the use of cytogenetic microarray analysis (CMA) in prenatal testing is being utilized in some centers, especially for the evaluation of fetal structural anomalies identified by ultrasound ^(4–13). Further, a large NICHD-sponsored multi-centered trial to assess the role of CMA as a primary prenatal diagnostic tool has been completed, and results will be available shortly. Reports to date suggest that CMA is a more sensitive diagnostic test and adds incremental value to conventional karyotyping with the potential to replace karyotyping in the initial cytogenetic evaluation of the fetus. As the technical aspects of CMA testing continue to evolve and prospective studies are completed to evaluate the clinical validity and utility of CMA in the prenatal setting, integration of this information into clinical care will require thoughtful changes in patient counseling.

Although the paradigm for genetic testing in the postnatal setting has shifted to CMA as the first-line test in many situations, there are distinct differences in the prenatal use of genetic information.. Whereas infants and children are identified for testing because of structural or developmental anomalies allowing correlation of the CMA results with the clinical features, prenatal results frequently require counseling with incomplete or absent knowledge of the phenotype because of the inherent limitations of ultrasound investigations. This is further complicated by the present literature describing the clinical effects of microdeletions and duplications based on postnatal recognition of an abnormal phenotype, and likely biased toward the more severe end of the phenotypic spectrum, as those children are more likely to come to clinical attention and be referred for diagnostic testing.

One of the unique aspects of prenatal testing is the option to use the information to make difficult decisions about continuation of a pregnancy whereas postnatal results provide parents with an explanation for the child’s phenotype and are used to plan future management of the child and future reproductive planning. Parents choosing prenatal testing are frequently using the information to assure the birth of an unaffected child.

In most cases, determination of the clinical significance of a copy number variant (CNV) discovered during prenatal testing is straightforward. Many of these have been previously described in the literature and/or curated databases. Some CNVs are benign variants that have no significant clinical impact. Others have been confirmed as being pathogenic but the penetrance and expressivity are variable. If insufficient evidence is available to unequivocally determine if a CNV is of either benign or pathogenic clinical significance, then the CNV is reported by the lab as a CNV of uncertain clinical significance. Although with more experience uncertain findings are becoming less frequent, they still occur in up to 2% of prenatal cases (14), with the frequency varying depending on the content and resolution of the array. Although high-resolution whole genome arrays are likely to discover more clinically significant findings, they are also more apt to uncover findings of uncertain significance (9). Some arrays are designed to emphasize detection of known microdeletion/microduplication syndromes, such as the 22q11.2 deletion or Smith-Magenis syndrome, as well as other known loci of inherited Mendelian disorders. These targeted arrays may also provide less dense coverage throughout the genome to detect large changes in the “non-targeted” (backbone) regions. This can minimize the chance of finding a variant of uncertain significance (VOUS), but also may fail to detect a rare/novel genomic imbalance. However, regardless of which array design is used, the need to counsel patients about uncertainty will be necessary. When a VOUS is found, predictive counseling will require determination of the likelihood that a CNV is pathogenic and likely to result in a clinically significant phenotype based on size, gene content, review of the literature and information in databases. While clinical information such as ultrasound findings, family history, and inheritance of the copy number variant can be taken into consideration, none of these will allow definitive prediction of the phenotype. Though sonographic anomalies may be suggestive of an association, it is also possible that they are coincidental. Likewise, whereas inheritance from a normal parent could be considered reassuring and a de novo event more suspicious, incomplete penetrance and variable expressivity must be considered as a possibility even for inherited CNVs.

Early experience with the interpretation of array data in prenatal diagnosis has already demonstrated some of the challenges that practitioners and counselors will face as CMA is incorporated into clinical practice. Here, we outline some of these areas with clinical examples, and discuss potential counseling dilemmas and options. We also suggest topics which should be reviewed with patients during a pretest counseling session. Although CMA raises new questions and will require the use of new tools and knowledge, the prenatal genetic counseling approach and issues are similar to those we address for any type of genetic testing. The following four cases, all ascertained in the NICHD prenatal microarray study, illustrate the challenges and subtleties of genetic counseling in this new era of genetic testing.

Case 1: Invasive testing performed for ultrasound finding of a unilateral club foot. Array results: a de-novo 1.60 Mb gain (microduplication) arr7q11.23 (72,404,248-73,976,981)x1

Review of the literature reveals a well characterized 7q11.2 microduplication syndrome, the reciprocal duplication of the Williams–Beuren syndrome critical region ⁽¹⁵⁾. The probands were identified in a series of patients with idiopathic intellectual disability. An association with autism is also present. Both familial and de novo cases have been described. Variable speech delay is a constant finding and cognitive abilities range from normal to moderate mental retardation. Hypotonia and congenital anomalies, including club feet, were noted in some probands. Further confirmation of the pathogenic potential of this microduplication comes from review of two large databases. Of the 17 cases of this microduplication reported in the experience of the ISCA consortium, 16 were discovered in patients studied because of developmental delay and only one was seen in a large series of “normal” individuals⁽¹⁶⁾. This results in an odds ratio of 10.3 for a pathogenic phenotype (p =0.0046) (17). Cooper, et al have reported similar findings. Based on this information, the couple can be counseled that the array finding is pathogenic.

Predicting the impact on future development is less certain. While all probands had significant developmental delay leading to their testing, many of the parents having the same microduplication were employed and functioning well, albeit with a history of learning difficulties, language delay or both. This raises the question of whether some patients outgrow some of their childhood cognitive problems, or whether the phenotype in the specific adult patient group studied represents the mild end of the phenotypic spectrum. If it were not for the more severely affected children, parents would never have been diagnosed. It thus remains to be determined how frequently the duplication is present in cohorts of unaffected or mildly affected controls. Long term follow-up studies of pregnancies with this microduplication but without in utero abnormalities are required to address this question before one can definitively counsel patients.

Case 2:CVS done for previous child with developmental delay secondary to dup 5q31.2-31.3 (5.7Mb). CMA analysis of this case: Normal 5q but a de-novo 450kb duplication arr 1q22 (154,348,185-154,799,248) x3

Review of the literature and DECIPHER database ⁽¹⁸⁾ reveals no similar cases. The UCSC genome browser ⁽¹⁹⁾ shows 18 genes in the duplicated segment, two of which are potentially disease-causing. Neuronal acetylcholine receptor subunit beta-2 is a protein that in humans is encoded by the CHRNB2 gene which resides in the duplicated segment. Mutations in this gene are associated with autosomal dominant nocturnal frontal lobe epilepsy. The RNA-specific adenosine deaminase gene is also located in this segment and in both humans and animals mutations of this gene are associated with dyschromatosis symmetrica hereditaria (OMIM DSH; 127400), a benign autosomal-dominant skin disorder. Based on these findings, the patient was informed that the finding was of uncertain clinical significance.

When a result is uncertain, the clinician should assist the patient in understanding the relative likelihood of the CNV being pathogenic or benign. Any such speculation must be well supported, but the uncertainty of this classification must still be communicated. In this case, the small size of the CNV, the fact that similar cases have not been reported, and the much lower likelihood that a duplication will be pathogenic compared to a deletion are all reassuring. However, there is biologic plausibility of a potential phenotypic consequence based on the gene function. All of these factors make counseling this patient difficult. Cases such as this one demonstrate the value of a multidisciplinary team approach composed of laboratory personnel familiar with CMA technology, a clinical geneticist, and genetic counselors. Each team member should be well educated in the unique aspects of CMA interpretation, and if such a resource is not locally available, referral is appropriate. Recall, however, that uncertain results are not unique to CMA technology and arise in interpretation of all forms of genetic testing, including evaluation of a karyotype.

As whole genome arrays with higher resolution are designed, more findings of uncertain clinical significance will be identified ⁽⁹⁾. This has led to the suggestion that these not be reported to the patient⁽²⁰⁾. Others have found this approach too paternalistic, and believe that with counseling, patients are capable of understanding uncertain or inconclusive results. Alternatively, as part of the pre-test counseling and consent process, patients choose whether they wish to be made aware of uncertain laboratory results. At present, there is no consensus on the best approach or practice guidelines; hence, it is recommended that laboratories discuss this with referring physicians to develop local policies.

Case 3: Patient has an amniocentesis for positive Down syndrome screening. CMA analysis: a de-novo 751 kb deletion in 22q11.2. arr22 q11.2 (19,084,421-19,835,417)x1. A fetal echocardiogram is normal

Another difficult area of interpretation results when a CNV is discovered in a known pathogenic region, but the size or location is different than previously described cases. In this case, the deletion was within the well characterized DiGeorge syndrome region but is smaller than the 3 Mb deletion found in 90% of patients with features of DiGeorge and velocardiofacial syndromes. CMA results indicate that the deletion is located within the distal end of the 3 Mb common deletion, between two of the eight low-copy repeat sequences in this region, which predispose to nonallelic homologous recombination and result in deletions. Review of the literature revealed that about 7% of patients with DiGeorge/VCFS Syndrome have a smaller, nested deletion of about 1.5Mb within the common deletion, and the remaining patients have smaller “atypical” deletions within the region or distal to the common deletion region ^{(21)(22, 23)}.

The size and location of deletions within this region do not correlate with the phenotype, which is highly variable ⁽²⁴⁾. In this case, a detailed anatomic survey and fetal echocardiogram are indicated to look for evidence of congenital anomalies associated with the deletion. The absence of a heart defect on fetal echocardiogram does not exclude the potential for this CNV to be pathogenic. Cardiac defects occur in only 75% of patients with 22q11.2 deletions, and subtle cardiac lesions could be evident later in pregnancy or postnatally⁽²⁵⁾.

Although this is an atypical 22q11 deletion, and does not include the major candidate gene (TBX 1). the most prudent approach would be to counsel the parents about the potential for any of the features of the 22q11.2 deletion syndrome including neuro-cognitive difficulties, speech delay, and psychiatric and behavioral problems, as well as recommend a post-natal evaluation by a geneticist or individual with expertise with the deletion syndrome.

Case 4: CVS performed for advanced maternal age. CMA analysis: a 203 kb maternally inherited deletion arr3p26.2. (4,395,412 – 4,598,515)x1mat

The majority of cytogenetic disorders discovered by prenatal karyotype result in significant fetal and neonatal morbidities which commence at birth and result in severe lifetime morbidity and neurocognitive impairment. This is not always the case with CMA and some microdeletions and duplications may result in adult-onset diseases.

Review of the literature in Case 4 revealed that this deletion is associated with spinocerebellar ataxia 15, an adult onset disorder with significant variable expressivity ⁽²⁶⁾. Onset is between ages 7 and 66 years, usually with gait ataxia but sometimes with tremor. Affected individuals remain ambulatory for 10 to 54 years after onset.

Appropriate counseling for the fetus and mother in this case is uncertain. Some guidance comes from The American College of Medical Genetics ⁽²⁷⁾ which recommends that when CNVs associated with pre-symptomatic conditions are discovered postnatally, the CNV should be reported to facilitate early access to medical care. This may not always be appropriate or desirable with prenatal testing, and may result in heightened parental anxiety. One may query parents during the pretest consent process as to whether they wish to be informed and include their decision in the consent form which will provide referring physicians and genetic counselors some guidance when such findings are discovered. However, fully informing parents of the implications of such findings during a pre-procedure counseling session is impractical and counselors will still have to use their discretion of how to manage each specific situation. For example, when CNVs associated with a treatable or modifiable condition such as neoplasia are discovered and information on penetrance, lifetime risk, disease spectrum, and clinical management are known, these findings should be reported to the parents to maximize future care and prognosis.

Whether to inform the parent of their pre-symptomatic status for a non-modifiable adult onset diseases is less clear. In the present case, maternal testing was not performed for the purpose of diagnosing asymptomatic maternal SCA15 and such findings were not anticipated by the mother. Experience with the prenatal diagnosis of Huntington disease, in which some parents go to great strides, including assisted reproductive technology, to avoid knowing their disease status, suggests that not all individuals are prepared for or desire this information. In many labs, discovery of a fetal CNV results in reflex CMA of the parents to determine whether the finding is de-novo or inherited, For expedience, this is frequently done without re-contacting the family. We suggest however that when the CNV, if inherited, can potentially impact parental health, ascertainment of the parents’ preference prior to testing represents the best approach.

DISCUSSION

CMA detects chromosome abnormalities and new genetic syndromes that would be missed by conventional cytogenetics and has the potential to significantly enhance prenatal genetic evaluation. While some may suggest that its use be limited to pregnancies with fetal structural anomalies, it appears that its improved diagnostic ability to detect clinically relevant abnormalities is likely to lead to its adaptation as a primary prenatal diagnostic tool. At first this will lead to frequent counseling dilemmas, as does the translation of any new technology into clinical care, but with time, education, and the development of appropriate care guidelines, these should become infrequent.

Counselor and practitioner education will be important in the implementation of CMA testing, and will need to include both an introduction to the science of interpreting array findings as well as development of improved approaches to uncertainty. While most copy number variants can be interpreted based on gene content, size, inheritance, clinical findings, and review of the literature and databases, many practitioners and laboratories will initially require training and experience before becoming comfortable in their interpretation. This transition period will require close collaboration between laboratories performing arrays, clinical geneticists with expert knowledge and expertise in the area, and counselors skilled in relating results to patients. In many centers a team approach to interpretation will need to be developed, as will local guidelines as to the reporting and management of specific array findings. Professional organizations and laboratories will need to develop standardized guidelines for testing and reporting of results to minimize the uncertainty and ensure that patients and practitioners are provided with accurate information and appropriate counseling tools.

Presently the tools available to assist in interpreting prenatal CMA findings remain incomplete. Most critical is the development of large databases of CNVs discovered during prenatal testing, thus obviating the known ascertainment bias unavoidable in postnatal cases studied only because of existing abnormalities. Only through such data can the full phenotype be sufficiently well defined to provide accurate predictive counseling. Of equal necessity is research into patient attitudes and desires, and an understanding of in which populations the use of CMA technology will be most beneficial.

While each of the examples above may result in heightened patient anxiety and confusion, fortunately they will be infrequent if the prenatal diagnosis program insists on thorough pre-test counseling and implements a formal consent process. While the specifics of each case are distinct, the underlying genetic principles of uncertainty, variable expressivity, and lack of precise genotype-phenotype correlation are well known and should be a standard part of routine prenatal counseling. To minimize the impact of these findings, pre-test counseling for CMA should cover each of these possibilities. A discussion of the couples’ tolerance for uncertainty and whether they want to be alerted to these findings may be appropriate, but additional research is required to understand the implications of this approach.

STATEMENT.

• It is known from post natal evaluation of infants and children with dysmorphic features, neurocognitive difficulties or structural anomalies that microarray analysis will provide important clinically relevant information but also can result in findings of uncertain clinical significance.

• This study gives an early insight into the counseling quandaries that prenatal array use can generate.