The benefits and limitations of cell-free DNA screening for 47, XXY (Klinefelter syndrome)

DOI: 10.1002/pd.5044 ORIGINAL ARTICLE The benefits and limitations of cell-free DNA screening for 47, XXY (Klinefelter syndrome) Carole Samango-Sprouse 1,2 *, Colleen Keen 3, Teresa Sadeghin 3 and Andrea Gropman 4 1 Department of Pediatrics, George Washington University, Washington, DC, USA 2 Department of Molecular Genetics, Florida International University, Miami, FL, USA 3 The Focus Foundation, Davidsonville, MD, USA 4 Department of Neurodevelopmental Disorders and Neurogenetics, Children s National Medical Center, Washington, DC, USA *Correspondence to: C. Samango-Sprouse. E-mail: cssprouse@aol.com ABSTRACT Objective The purpose of this paper is to provide an overview of the 47, XXY syndrome, which is the most commonly occurring X and Y chromosomal variation. This paper seeks to review what is currently known of noninvasive prenatal testing (NIPT) and 47, XXY and investigate potential risks and benefits of prenatal identification. Method A literature review of NIPT and 47, XXY was performed to identify limitations of current NIPT techniques. Results As NIPT becomes an increasingly more routine procedure, prenatal findings of 47, XXY may increase. Awareness of this disorder and appropriate genetic counseling is necessary. Conclusion X and Y chromosomal variations will be identified through this screening, and the benefits and limitations to this finding need to be thoughtfully considered. Funding sources: None Conflicts of interest: None declared INTRODUCTION 47, XXY (Klinefelter syndrome) is the most commonly occurring X and Y chromosomal variation (aneuploidies involving the X and/or Y chromosomes) with an estimated incidence of 1 in 450 to 660 live male births. 1 3 X and Y chromosomal variations can arise from either de novo mutations during meiosis of oogenesis, spermatogenesis, or during mitosis after fertilization. 4 There have been studies that have found that the odds of having a child with 47, XXY are statistically higher if paternal and maternal age at the time of conception is advanced (i.e. over the age of 35 years). 5 7 47, XXY has a slightly increased incidence of being maternal rather than paternal in origin, occurring approximately 55% maternally versus 45% paternally. 5 It is still unclear what impact, if any parent-of-origin, also referred to as parental imprinting, can have on the phenotypic presentation and subsequent neurodevelopmental variation that is seen within this disorder. However, it has been postulated that parental imprinting, skewed X inactivation as well as the hetero versus isodisomic nature of the additive X chromosome could contribute to the phenotypic variety of this neurogenetic disorder. 8 10 Despite the relative frequency in which 47, XXY occurs, less than 25% of boys are identified in their lifetime based on a national registry study conducted in Denmark. 11 47, XXY does not present with congenital abnormalities on routine ultrasounds, or with an atypical biochemical profile, thus preventing prenatal identification unless advanced maternal age is present and prenatal testing is offered and accepted. 12,13 Therefore, identification typically occurs postnatally when the individuals with 47, XXY are brought to medical attention for reasons including delayed milestones, behavioral disturbance, incomplete pubertal development, or infertility. 14,15 The majority of research describing the phenotypic profile of boys with 47, XXY has been collected from the prenatal or newborn screening studies more than 40 years ago. These investigations afforded less ascertainment bias than earlier studies which suggested that criminality, psychosexual confusion, and intellectual disability were common. 16 Furthermore, the postnatally identified child and family have often sustained significant frustration from diagnostic odyssey, the delay in diagnosis, and the lack of appropriate services for the neurodevelopmental disturbances. 17,18 Recently, there has been a resurgence of research studies expanding the profile and further characterizing the capabilities of motor, speech and language and executive function domains. 19 22 However, the behavioral and neurodevelopmental phenotypic presentation remain confounded by ascertainment bias,

498 C. Samango-Sprouse et al. variable services, family learning disorders, and absence or presence of testosterone treatment. 23,24 There are salient features that have congealed into a characteristic phenotypic profile that include increased stature relative to what is expected from mean parental height, azoospermia, and hypergonatropic hypogonadism. 25 Due to the hormonal deficiency, testosterone replacement characteristically begins between 11 and 12 years of age, although there is some research to suggest that early testosterone replacement can have cognitive benefits. 26 28 There are also minor dysmorphic features associated with 47, XXY including fifth-finger clinodactyly, pes planus, and increased length of the extremities. 29 The neurodevelopmental phenotype includes enhanced performance intelligence relative to verbal intelligence. Language-based learning dysfunction, motor planning deficits, and an elevated incidence of executive dysfunction have also been reported. In infancy, boys with 47, XXY present with truncal hypotonia and subtle asymmetry of movement patterns. 30 There is an atypical acquisition of sounds and words with increased dependence on consonants b, p, and d. It has been postulated that this is a characteristic pattern of neurodevelopmental disturbance that is more than mere developmental delay but an infantile presentation of developmental dyspraxia (IDD). With maturity, this pattern evolves to motor planning deficits, delayed balance reactions and disturbances in manual coordination and fine motor control. 30,31 Speech and language profiles are characterized by early speech motor disturbances and later language formulation deficits with typically intact receptive vocabulary and delayed expressive vocabulary development. Social cognition and social language deficits in 47, XXY may be common, especially in those untreated with early hormonal treatment in infancy. 27 Social language and social cognition are affected in children with language-based learning disorders who are chromosomally intact. The influence of the additive X on these types of social language deficits warrants further investigation before causality can be assumed. As the use of serum screening and cell free DNA increases, prenatal identification of 47, XXY has become increasingly more common. It is anticipated that noninvasive prenatal testing (NIPT) will become a standard of care within the next five years. 27,32,33 Therefore, the anticipation of early detection of 47, XXY presents many challenges as well as possibilities for genetic counselors, obstetricians, pediatricians, and ancillary health care providers as they interface and guide expectant families. Balanced information should be provided in order to assure an independent and well-informed choice for expectant families. Furthermore, after birth, boys with 47, XXY have significant heterogeneity in neurodevelopmental outcome as well as behavioral and emotional development, making it challenging for health care providers to identify the at-risk child with 47, XXY as early as possible. Conversely, health care providers need to balance addressing the early biomarkers that require therapeutic intervention without creating cases of the vulnerable child syndrome by viewing every nuance of developmental difference as abnormal or delayed. PRENATAL SCREENING Since the discovery of cell-free fetal DNA (cffdna) in maternal serum, medical advancement has pioneered prenatal screening for chromosomal abnormalities. 34 Noninvasive prenatal testing works by analyzing cffdna in maternal blood, and although the test is highly accurate, it is not a diagnostic test and requires cytogenetic confirmation. 35 Most screening tests do not include analysis for sex chromosome aneuploidies unless the patient specifically selects to opt in for these results. The sensitivity and specificity are somewhat lower for sex chromosome abnormalities, in comparison to more common trisomy disorders such as Trisomy 21 or Trisomy 13. 36 38 The American College of Obstetricians and Gynecologists (ACOG) report that NIPT has a sensitivity of 91.0% and specificity of 99.6% to identify sex chromosome abnormalities in patients who receive interpretable results. 36 38 Some studies have reported sensitivity and specificity rates as high as 100% in identifying sex chromosome abnormalities. 36 38 Positive predictive values depend on the particular sex chromosome variant identified, but in general range from 20 to 40%. 38 False positive rates have been reported to be between 0.0 and 0.1%. 39,40 Noninvasive prenatal testing provides a resource for families and physicians to correctly identify 47, XXY prenatally, making it beneficial to the medical community and lay public. However, with the identification of the at-risk pregnancy, the prenatal counseling presents several unique challenges. The significant heterogeneity in the neurodevelopmental profile of 47, XXY, including the impact of familial learning disorders, and the substantive influence of hormonal deficiencies on outcome add another layer of complexity to prenatal counseling. This is uncommon in other neurogenetic syndromes, as well as other sex chromosome aneuploidy (SCA) disorders. CURRENT RECOMMENDATIONS The ACOG, International Society of Prenatal Diagnosis (ISPD), and National Society of Genetic Counselors (NSGC) recommend cffdna screening for common trisomy disorders and only for sex chromosome variants upon patient request. Mild phenotypic presentation, high rates of fetal mosaicism, risk of identifying maternal aneuploidy, and inefficiency of NIPT to properly identify 47, XXY without cytogenetic confirmation are several associated concerns and may contribute to why this screening is not yet offered routinely. 41 Noninvasive prenatal testing is not offered for all pregnancies and should only be completed after proper, comprehensive counseling has been completed. Pretest counseling should identify limitations and benefits of NIPT, as well as the significance of a positive result. Patients should understand before any testing is complete that 47, XXY needs to be confirmed by cytogenetic testing, as well as the risk of identifying a maternal aneuploidy. Mothers should have the right to separately deny maternal sex chromosome aneuploidy results. SCA findings are almost always incidental. Comprehensive information including the variability of the phenotype and response to intervention is often limited. 41 Current counseling for a 47, XXY diagnosis focuses on long-

Benefits and limitations of cell-free DNA screening for XXY 499 term implications involving health, physical development, and intellectual capacity. 42 POTENTIAL BENEFITS OF SCREENING Risk calculations of fetal miscarriage consequential of invasive prenatal testing range from 0.1 and 1.0%. 41,43,44 Despite this relatively small risk, many women are uncomfortable with the idea that any percentage of risk could be wagered against the pregnancy. 45 Therefore, one of the greatest benefits of NIPT is the reduced risk to the fetus and mother. The perspective of eight mothers who had boys affected with 47, XXY (six identified prenatally, and two identified postnatally) were collated. Mothers of prenatally identified boys with 47, XXY thought that the greatest benefit of this early identification was being able to research and prepare for the differences that are associated with 47, XXY. Most of the parents responded that they had enrolled their child in early intervention services sooner than they would have otherwise without a prenatal diagnosis. These mothers also reported increased anxiety during their pregnancy, and expressed feelings of worry and stress, and wished to have a happier pregnancy. Both mothers who had 47, XXY boys postnatally identified were satisfied with the postnatal diagnosis because they had peaceful pregnancies. One family expressed that they wished he could have had early hormonal treatment (testosterone shots) in infancy but restated that they were glad for a postnatal diagnosis because of the happy pregnancy. With the prenatal identification of 47, XXY, parents have more opportunity to receive counseling services prior to delivery that should provide a solid education on subtle developmental differences, fertility procedures, and psychosocial health of males with 47, XXY. If the prenatal identification is confirmed by cytogenetic testing, families will be equipped to identify a multispecialty team of health care providers to assist their child, if necessary. Androgen deficiency has been documented 26,46,47 as one of the hallmarks of 47, XXY syndrome, and this deficiency is highly correlated with benefits on libido, emotional stability, strength, and well-being. Research studies have documented the testosterone deficiency in 47, XXY prenatally, at the newborn period, in adolescence and throughout adulthood. 47 49 In the last five years, there have been several research articles published that reveal beneficial effects on multiple developmental domains of Early Hormonal Treatment (EHT) at 36, 72, and 108 months in boys who are prenatally diagnosed. Improvements have been observed in neurocognition, large and small motor skills, and selected aspects of language-based tasks. 27 Although the administration of testosterone supplementation in infancy has not been established as a standard of care yet, the benefits of EHT and its significant impact on many of these neurodevelopmental differences are becoming more evident. 27,49,50 Some studies have reported that boys prenatally diagnosed with 47, XXY have better neurodevelopmental and behavioral outcomes than postnatally identified 47, XXY boys; however, prenatal diagnosis is not agreed upon as a protective factor at this time. 51,52 This is due in part to the myriad of environmental and biological factors that affect longitudinal outcome. However, with the prenatal diagnosis of 47, XXY, the expectant family has the possibility to seek consultation with appropriate medical providers to develop a treatment care plan that is targeted and syndrome specific that would otherwise be unavailable. Families can assume a potential position of addressing early indicators of developmental disruption as soon as possible, which could be beneficial for a conceivably vulnerable child. LIMITATIONS OF NONINVASIVE PRENATAL TESTING The unique biology of 47, XXY presents different challenges than autosomal trisomy for prenatal detection via cffdna, particularly due to the incidence of mosaicism of both maternal and fetal origin. 53 Presently, NIPT is recommended for expecting mothers at increased risk of chromosomal abnormality, which often includes women of advanced maternal age. A natural consequence of aging involves the loss of the X chromosome, which can result in a baseline rate of mosaicism that would generate maternal cffdna with fewer X chromosome fragments than expected. 53 Approximately 10 to 15% of 47, XXY cases are mosaic. 54 The presence of fetal mosaicism is influential on prenatal counseling because the research studies have suggested that mosaicism may impact outcome in variety of ways including fertility and neurodevelopmental outcome. 55 Confirmatory testing must be completed after NIPT in order to determine if 47, XXY is present. Patients should undergo comprehensive counseling before cytogenetic counseling. In some cases, chorionic villus sampling (CVS) may be more appropriate than amniocentesis, particularly if a result is desired within the first trimester. However, there are several benefits to amniocentesis that CVS does not possess. Amniocentesis can detect neural tube defects and may be more advantageous than CVS because placental mosaicism does not play into effect. The prenatal diagnosis of 47, XXY can be challenging for families because the idea of having a chromosomally typical child has been lost and families must adjust to this new reality. Termination is an option that families may choose, but cytogenetic confirmation and comprehensive nondirective counseling are imperative. Rates of termination following a 47, XXY diagnosis are approximately 70%, which is lower than autosomal trisomies and higher than other sex chromosome disorders such as 47, XYY. 56 Studies have found that the type of medical professional doing the counseling has a significant impact on the outcome of the pregnancy. 57 Health care providers doing the counseling need to have a current understanding of the disorder as well as its longitudinal impact on quality of life. 57 FUTURE CONSIDERATIONS Some studies in adult men postnatally diagnosed with 47, XXY have indicated increased morbidity, reduced quality of life, and increased psychosocial issues when compared to their peers, while others have found no significant differences in quality of life between prenatally and postnatally diagnosed males with 47, XXY. 57,58 Prenatal identification of 47, XXY

500 C. Samango-Sprouse et al. eliminates the risk of diagnostic odyssey many families experience when subtle phenotypic differences present with no known cause. Noninvasive prenatal testing with cytogenetic confirmation provides parents a lifelong awareness and understanding of impacted endocrine, speech, behavioral and motor domains that may otherwise go unnoticed and unaddressed. Parents can be mobilized to create a supportive and enriched environment, which can include early intervention services, for their at-risk child that can further minimize some of the genetic risk. It is well recognized that a prenatal diagnosis affects parental perception and expectations for their child. It is also understood that the type of medical professional providing the post-result counseling, as well as the type of scientific literature presented, can significantly impact whether a termination takes place, and the parent s expectation as their child develops. 57 This altered perception can be optimized if prenatal counseling is sensitive and comprehensive in providing current information on the neuropsychology, fertility, and neurodevelopmental progression associated with this disorder. Rather than waiting to see if improvement is coming, families can be proactive and preventive by developing focused interventional strategies reflective of the vulnerabilities of 47, XXY, which could be longitudinally beneficial for minimizing the developing language-based learning disorders, graphomotor dysfunction, and social anxiety. The risk versus benefit ratio for the prenatal diagnosis of 47, XXY remains hotly debated. Currently, NIPT for 47, XXY is not offered as a standard of care due to its mild phenotypic presentation, risk of identifying a maternal aneuploidy, and the increased rate of false positive results. Furthermore, one study found an increased association between prenatal identification of 47, XXY and preterm delivery, which could be suggestive of maternal anxiety following the result. 42 However, the benefits for early identification and treatment in children with significant neurodevelopmental dysfunction such as Autism Spectrum disorder and Down syndrome have been well demonstrated. 59 61 Furthermore, early identification provides the necessary support as well as enhances the ability of the families to better comprehend the common behavioral and neurodevelopmental manifestations associated with the diagnosis of 47, XXY. WHAT S ALREADY KNOWN ABOUT THIS TOPIC? 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