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Bundock EA, Corey TS, Andrew TA, et al., editors. Unexplained Pediatric Deaths: Investigation, Certification, and Family Needs [Internet]. San Diego (CA): Academic Forensic Pathology International; 2019.
JACK
“Jack was 2.5 years old and a wonderful big brother to his 6-month-old sister at the time of his sudden death. Jack’s cause of death was ruled undetermined by the medical examiner. It was so frustrating and frightening to not know why he died. We pursued research that included genetic analysis from specimens the medical examiner had saved. Research was able to identify a mutation in the CALM1 gene, which likely caused Jack’s heart to stop. This information allowed us to test our daughter and ourselves to ensure we didn’t have the same mutation. When we received the assurance that this was a unique finding in Jack, it gave us such peace of mind. Explaining his death doesn’t bring Jack back or change how much we miss him, but it does give us reassurance that the same thing won’t happen to our daughter.”
– Jack’s Mom and Dad
OUTLINE.
Sudden pediatric deaths may result from unsuspected genetic conditions, including metabolic disorders. Currently recognized etiologies of sudden pediatric death that have a genetic basis are predominantly structural (congenital anomalies, structural cardiomyopathies) or functional cardiac conditions (channelopathies), with epileptogenic mutations and metabolic disorders becoming increasingly recognized. Cardiomyopathies may be primary disorders due to specific genetic mutations, secondary to metabolic disease, multifactorial, or of unknown etiology. Genes associated with channelopathies, such as long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) are the focus of intense research.
While genetic associations are being identified rapidly, the field is still limited by a lack of genotype-phenotype correlations. Rapidly advancing technologies and libraries of known mutations make postmortem genetic testing desirable, yet established guidelines for sample collection, testing algorithms, definitions of variants, interpretation of significance, and resource allocation are needed to successfully adjudicate cause of death and counsel survivors. This chapter provides recommendations for sample collection, DNA storage, and genetic testing algorithms (including phenotype-driven and whole exome approaches) in autopsy-negative sudden deaths in the pediatric population. Further research is needed to identify the mechanisms through which these mutations exert their lethal effect, so we can better understand and counsel families on cause of death and disease variability and severity.
While most explained sudden deaths in the young (“young” used in the literature to indicate children and adults generally <50 years of age) are due to pathologies identifiable at postmortem examination, it is estimated that 3–30% have no identifiable cause at autopsy (autopsy-negative sudden unexplained death) (1–6). Numerous molecular defects in genes encoding proteins for sodium and potassium ion channels of the sarcolemma or encoding receptors regulating intracellular calcium release in the sarcoplasmic reticulum have been implicated in sudden deaths (Table 9.1, modified from [7]), potentially explaining up to one third of autopsy-negative cases (8–10). Several postmortem genetic studies of unexplained sudden infant deaths indicate that up to 20% may be due to channelopathies (11, 12), but a more conservative estimate is less than 15% (11). Mutations in genes associated with structural cardiomyopathies (e.g., hypertrophic cardiomyopathy) have also been identified in sudden unexplained deaths in infancy with normal hearts (13), and variants of genes involved in autonomic function, neurotransmission, and energy metabolism suggest non-cardiac mechanisms of death may explain other autopsy-negative cases (14–18). Very few population-based genetic studies focus on children older than one year (19).
Major Cardiac Channelopathy-Susceptibility Genes.
Genetic risk factors contributing to post-infancy and prepubescent sudden unexpected deaths have not undergone the same focused research attention as those contributing to the sudden unexpected deaths of infants and young adults (11, 19–21). When children, aged 1–10 years, have been included in published series, their limited numbers prevent generalized conclusions for this age group (19, 22, 23). Most studies including older children have focused on cardiac genetic disease specifically, ignoring some of the genetic risks associated with sudden infant death related to the autonomic central nervous system, immune dysfunction, metabolic disease, and nicotine response (20). In addition, sudden deaths in toddlers, unlike infants, teens and young adults, are associated with an eight-fold increase in febrile seizures (24) suggesting this cohort requires specific research into seizure-related and other central nervous system factors.
In 1998, Schwartz and colleagues published descriptions of 12 cases of sudden death in infants with previously diagnosed QTc prolongation (diagnosis by neonatal electrocardiogram) (25). This article provided real evidence for a causal link between QT interval prolongation and some cases of sudden unexplained infant death, a hypothesis that had been discussed in the literature for over ten years (26, 27). Shortly after Schwartz’s 1998 article was published, reports appeared in the literature documenting LQTS-causing mutations of the sodium channel gene SCN5A in an infant who was successfully resuscitated from an out-of-hospital cardiac arrest (28) and in two cases of sudden unexplained infant death (29) and a mutation of the potassium channel gene KVLQT1 in a 17-year-old boy found dead in bed (30). While gene variants associated with channelopathies were first reported at a trickle, there is now a rapidly expanding list (Table 9.1). The difficulty is no longer in identifying associated variants, but in determining which can be classified as “pathogenic” and integrating identified genetic variants into the final determination of cause of death. While research will continue to identify pathogenic variants and improve diagnostic yield, the practical problem for the forensic pathologist remains unchanged; which cases should have genetic testing and what test panel should be used?
The investigative phase of a sudden pediatric death is an opportune time to gather family and medical history that may point toward a causative genetic disorder. A careful review of signs and symptoms and history is the best indicator for targeted genetic or metabolic testing, but a negative investigation does not exclude a genetic etiology. Furthermore, even when the death scene or autopsy offers an apparent cause of death (e.g., unsafe sleep environment or signs of infection), a genetic disorder may have made the child more likely to die than a child without a disorder in the same circumstance. Generally, once unnatural etiologies and obvious infectious, syndromic, and congenital defects are ruled out by autopsy, metabolic disorders and channelopathies should be considered, even in cases with some extrinsic risk factors, such as unsafe sleeping conditions.
Inborn errors of metabolism, such as fatty acid oxidation defects, organic acidurias, congenital adrenal hypoplasia, galactosemia, congenital hypothyroidism and biotin deficiency, are rare and typically associated with premortem illness such as anorexia or viral type symptoms, metabolic acidosis, hypoglycemia, vomiting, lethargy, hypotonia or coma. Children with congenital adrenal hypoplasia may experience excessive urination and thirst as well as salt craving. However, these symptoms may not be apparent or well described by caregivers, especially during the acute shock and grieving process. If state-mandated newborn screening was performed prior to the death, the results of the screening should be obtained (either as an indicator for postmortem testing or to help confirm postmortem test results). While most states test for the 34 core conditions (including amino acid, endocrine, fatty acid oxidation, hemoglobin, and organic acid disorders) specified by the Health Resources and Services Administration, some states include tests for secondary conditions. Pathologists should be familiar with the panel performed in their jurisdiction (31, 32). Nonspecific findings at autopsy such as hepatic steatosis, cystic dysplasia of the brain or kidneys, or nonspecific intracerebral hemorrhage should prompt consideration of a metabolic disorder. Although many medical examiners routinely screen for metabolic disorders in an autopsy-negative sudden death, this practice has a low diagnostic yield and has therefore been criticized as a poor use of resources (33). Although newborn screening methodology is targeted to early detection of disease, analyte levels may be higher after the neonatal period and repeat testing at autopsy may result in diagnosis when neonatal screening was negative. In summary, metabolic testing is relatively inexpensive and although the positive yield may be small, this testing can be useful even with previously normal neonatal testing. Positive results not only explain the death but often have significant implications for other family members and family planning.
There are many other historical clues for a potential genetic or metabolic cause of death. A family history of premature death at a young age (<50 years), drowning in shallow water or drowning of an accomplished unimpaired swimmer, congenital heart disease (including premature heart failure and/or cardiomyopathy), or a personal or family history of syncope, arrhythmia, heart failure and/or seizures, can all be important clues to an underlying condition. A careful description of the scene, activity at time of death, and witness statements are also important (34). Since de novo mutations in cardiac and epilepsy syndromes continue to be identified, absence of a concerning family or personal history does not exclude a genetic etiology, although it does reduce diagnostic yield of testing. Recent studies have shown that de novo mutations are predominantly of paternal origin and increase with advanced paternal age (35).
Medicolegal death investigation of sudden pediatric deaths can be frustrating when a clear cause of death is not identified. The preservation and availability of viable genetic specimens can maintain opportunities for genetic analysis that may provide clarity on cause of the sudden death, identify at-risk relatives, as well as confirm familial risk of an explained cause of death. While confirming familial risk is not imperative for the mandate of a medicolegal death investigation system to determine cause and manner of death, it is of vital importance for surviving family members and their efficient and appropriate use of medical screening.
The National Association of Medical Examiners (NAME) Autopsy Performance standards and Inspection and Accreditation Checklist include routine collection and preservation of blood, urine, and vitreous specimens (36). Until recently, there were no uniform requirements for retention of a high-quality blood sample specifically for DNA extraction. In an attempt to address this gap, NAME issued a position paper regarding postmortem samples for genetic testing (37). These guidelines discuss the need for uniform collection with recommendations for case selection, type of sample, storage, and retention. Sample(s) should be collected for possible genetic testing in all pediatric cases of sudden unexpected death, drowning of an experienced swimmer, and suspected seizure deaths. Blood and heart tissue are the preferred samples, with highest-quality material being (in order of preference): 1) extracted soon after collection from fresh sample (e.g., sent immediately to testing laboratory or for DNA banking), 2) stabilized in an intermediary preservative and stored, or 3) snap-frozen and stored frozen. Blood stored in potassium ethylene-diaminetetraacetic acid (EDTA), a calcium chelating agent that prevents clotting and preserves blood cells, usually yields good results. While blood spot cards are economical and can be useful for paternity testing, often only small amounts of variable quality DNA can be extracted. This limits detection of deletions and duplications and some commercial laboratories will not accept blood spot cards for gene panels.
The sample preferred by most commercial laboratories is 10 mL of fresh blood in potassium EDTA (pink or purple tubes). Freezing blood may rupture cells and release enzymes that degrade DNA. However, in the forensic setting a decision to initiate genetic testing may be delayed by many weeks or months. A long-term storage solution is, therefore, necessary. Blood DNA yield is affected by long-term (>1yr) storage, but DNA integrity will remain (38). For medical examiner/coroner offices that cannot manage storage (long-term or short-term), sending a fresh sample to a commercial laboratory at the time of autopsy for extraction and banking of DNA, with next of kin consent, can be considered in the case of a suspected genetic etiology.
Fresh tissue (heart, liver, or spleen, in order of preference), trimmed to less than or equal to 5 mm wide, 10 mm high, and 20 mm long, submerged in 5 volumes of RNA stabilization solution (e.g., a 0.5 g sample requires about 2.5 mL of RNA stabilizing solution) are the next best choice. Tissue samples can be stored at 25°C for one week, at 4°C (refrigerator) for one month, or at −20°C (freezer) or −70°C (ultra-low temp freezer) indefinitely (39, 40).
When blood in EDTA and fresh or frozen tissues are not available, formalin-fixed paraffin-embedded tissues, sample residuals remaining after toxicology testing, or even blood cards remaining after state-mandated newborn screening may be a last resort source for genetic testing. As genetic testing technology advances, testing of these samples may become more routine.
Once the DNA sample is collected and stored, the medical examiner/coroner, pediatrician, genetic counselor and family have time to consider death circumstances and autopsy findings in the context of the decedent’s medical and family history and choose an appropriate testing strategy. Ideally, the family should consult with a genetic counselor early in the process. Issues of timing of testing and cost will usually arise. Phenotype-driven genetic testing, which uses family/personal history, signs/symptoms, and/or autopsy findings to select relevant genes based on known phenotype-gene associations should be considered first. Phenotype-driven genetic testing is recommended when pathognomonic signs of a condition, for example hypertrophic cardiomyopathy (HCM), are present at autopsy. There are two genes that cause approximately 40% of HCM cases (41), so this will be a small and affordable test. If that is negative, then a larger HCM panel (~30 genes), cardiomyopathy panel (~45 genes), or comprehensive arrhythmia panel (~90 genes) containing cardiomyopathy and channelopathy genes can be ordered.
If the decedent has a history of febrile seizures, and/or a relative with epilepsy, an epilepsy panel is a targeted test that may yield positive results. However, it is important to note that a history of seizures may indicate arrhythmias presenting as syncope. Testing for a combination of epilepsy and arrhythmia gene variants may be warranted.
In the absence of physical autopsy findings or a significant personal or family history, a common situation in pediatric sudden death, the possibility of targeted testing yielding results is reduced and more comprehensive testing may be needed. Testing for channelopathies (LQTS, SQTS, BrS, CPVT) is indicated in autopsy-negative cases, especially in young people. A review of the literature showed that about 25–35% of autopsy-negative sudden deaths and approximately 10% of SIDS cases yield variants in genes associated with these conditions (42). Thus, a reasonable approach would be to order a targeted arrhythmia panel first, with subsequent whole exome sequencing (cardiac and epilepsy genes) if the panel is negative. Whole exome sequencing is more expensive and has a higher likelihood of revealing variants of uncertain significance. In addition, whole exome sequencing is only informative when performed for a trio (decedent and both biological parents), which may not always be possible. If the care team and family feel comfortable with these risks, comprehensive testing may be an appropriate first step in an autopsy-negative case.
It is important to note that the yield of cardiac gene testing varies by age, with infants having a yield that does not exceed 10% in large cohort studies, especially when other risk factors for sudden death are present (11, 43). The yield for children over one year is not well studied. As such, expanding testing beyond cardiac genes (e.g., including epilepsy genes), the possibility of uncertain results, criteria for cases to be tested, and resource allocation are important discussions. Glengarry et al. recommend testing a subset of sudden infant deaths which meet criteria that will increase diagnostic yield (43). Criteria used by Glengarry et al. may be expanded to include autopsy-negative cases, those with a known personal or family history of previous sudden death, syncope, seizures, or proven cardiac arrhythmias, those without strong evidence of asphyxia (e.g., absence of clear overlay or covering of the nose and mouth), and sudden collapse while awake. It is important to note that, in people who have passed away due to complication of long QT syndrome, the first sign of the condition is death in 10–15% of cases (44). Guidelines for pursuing genetic testing that are more stringent than those proposed by Glengarry may miss these cases.
The cost of sequencing continues to decrease (45) and large numbers of private, disease-inducing mutations exist (46). While it is somewhat outdated to target specific (“common”) mutations, such panels are now offered at a cost approaching practicality for use by medical examiner/coroner offices. Sequencing entire coding regions of genes of interest is the current state of clinical testing. The cost to generate a high-quality, whole, human genome sequence is near $1000, as compared to $4000 in 2015. This showcases the rate at which cost is decreasing (47).
In most areas of the country, the cost of genetic testing largely falls on family members of the decedent. A recent study showed that 42% of testing was self-pay (48). Fourteen percent of testing was financed by medical examiner/coroner offices when budget allowed or grant funding was available. Insurance rarely covers this testing (6%), even when living insured individuals are part of a trio being tested (decedent and survivors). In 2019, the out-of-pocket cost for an individual or medical examiner/coroner office to self-pay can be as low as $250. The top commercial laboratories are generally competitive, reliable, and offer similar test panels for cardiomyopathies, channelopathies, and epilepsy. Choosing a laboratory is a part of the conversation between the care team and the decedent’s family, with aspects panel customization, turn-around-time, and cost considered. The Genetic Testing Registry can be utilized to compare tests and laboratories (49).
Procedural guidance for genetic and metabolic testing is summarized in Table 9.2.
Procedural Guidance and Key Considerations for Evaluation of Genetic and Metabolic Disorders.
Due to precipitous postmortem deterioration in specimen quality (and thus test utility), successful identification of inborn errors of metabolism mandates urgent specimen collection, one of the only true “emergencies” in forensic pathology. Specimen degradation becomes apparent within 30 minutes of death, and false negative tests may occur after four hours postmortem. Therefore, each death investigation system should consider ways to facilitate rapid specimen collection after death (see Recommended Strategies below). Regardless of specimen collection timing, pathologists should be prepared for storage of a variety of samples (Table 9.3).
Specimen Type and Collection/Storage Standards (33).
Specimen collection will be easier if the pathologist has access to proper tools for pediatric autopsies. Small bore needles (23GA to 25GA) with low volume syringes (5 mL or less) will make fluid collection easier. Furthermore, using small bore, “butterfly needles” with small volume syringes may facilitate the low, continuous suction needed to withdraw sufficient blood from the smallest infants.
Many medical examiner/coroner offices will not have a −70°C ultra-low freezer or proper storage solutions such as RPMI (Roswell Park Memorial Institute media for cell/tissue culture), glutaraldehyde (fixative for electron microscopy), and RNA stabilization solutions (for preservation of specimens for genetic testing). The freezer is a significant investment that will likely be underutilized in all but the larger death investigation centers. However, partnering in advance with local academic or clinical institutions that have such equipment (e.g., a university pathology department or a children’s hospital) may fulfill the need. Academic departments are frequently keen to assist when there is the potential for collaboration on interesting/challenging cases or when residents or fellows can participate in the work-up of apparent pediatric cases of inborn errors of metabolism. When such collaboration is not possible because of geographic or other considerations, the specimen containers may be purchased, and small quantities of storage solutions can be kept in ready supply, mindful of expiration dates.
The unexpected death of an infant or young child – alone – is not a valid reason to expend resources on metabolic testing. Similarly, previously normal newborn metabolic screening test results should not exclude further testing for metabolic derangements when such testing is suggested by investigative history or abnormal autopsy results.
As the major barrier to the proper identification and classification of inborn errors of metabolism is specimen adequacy, it is recommended that death investigation systems develop standard operating guidelines for all pediatric deaths. Such operating guidelines should standardize expeditious gathering of a thorough history (which includes relevant information about apparent hypoglycemic episodes, fever/sepsis, unexplained sweats, virilism, etc.) and communication of the information gathered to the on-call forensic pathologist. This will give the pathologist a sound basis for decisions concerning the urgency of autopsy (or of specimen collection ahead of autopsy).
It is recommended that pathologists perform autopsies on infants and young children as soon as possible. A metabolic blood/bile spot card should be prepared at the time of autopsy and held. When history, signs and symptoms, and/or pathologic findings suggest the potential for an inborn error of metabolism, sending the metabolic blood/bile spot card for screening is an appropriate first step, but may be insufficient for final diagnosis. Therefore, storage of fresh frozen tissues at −70°C (heart, brain, liver, kidney, skeletal muscle), glutaraldehyde-fixed tissues (brain, kidney, skeletal muscle), and tissue for fibroblast culture should be considered in such cases. Appropriate samples for various studies are detailed in Table 9.3. Directed testing of stored samples should be requested when 1) suggested by abnormal historical markers, 2) indicated by pathologic findings, or 3) some combination of the two. Given the rarity and complexity of such cases, broad consultations may be helpful to maximize appropriate use of limited resources.
Procedural guidance for genetic and metabolic testing is summarized in Table 9.2.
Compounding the family’s overwhelming grief is a common concern for underlying medical conditions that may impact other relatives and require evaluation and counseling. The NAME position paper on retaining postmortem samples for genetic testing discusses interactions between the medical examiner and family, including obtaining informed consent, how and when to communicate the need for genetic testing, and discussing results (37). As soon as practicable after the death, the medical examiner/coroner should inform the family, as well as the primary care physician/pediatrician, if a genetic or metabolic condition is suspected. This conversation may determine sample storage methods and therefore DNA quality. If a metabolic condition is suspected, early involvement of a genetic counselor, and pediatric pathologist is recommended. Surviving families have indicated that they appreciate a more direct approach from medical professionals (50) because the mourning process hampers their own search for information and decision-making.
Upon completion of the autopsy, regardless of final cause of death, communication with the family and pediatrician should be frequent and ideally both in writing and by phone. Depending on local regulations and state statutes, some medical examiner/coroner offices may require written consent from next of kin before release of information to healthcare providers. Clinical cardiac or genetic evaluation of family members should be timed with the release of the decedent’s genetic testing results. In at-risk relatives, testing should be targeted to the identified familial variant, as this mitigates cost and uncertainty.
Relationships between the medical examiner/coroner, genetic counselors, cardiologists, other healthcare providers, and testing laboratories should be well established in advance. This advanced preparation simplifies the process for involved professionals and assists families with decision making and testing of survivors (when results from testing of the deceased or clinical concerns indicate). Genetic counselors are well equipped to assist with informed consent for testing and educating families about the process and pitfalls and ramifications of testing. While many people feel compelled to obtain more information, genetic testing can cause harm. Children diagnosed with a cardiac condition after the death of a sibling can be faced with significant limitations and restrictions in life, requiring ongoing support and professional follow-up. Genetic testing may reveal mutations that are unrelated to sudden death yet are significant for survivor health and cause additional follow-up and stress. Genetic testing of a deceased child may also reveal misattributed paternity.
The National Society of Genetic Counselors’ postmortem working group has online resources and an on-call email address (gro.cgsn@metromtsop) that can be utilized to assist in the genetic testing process (51). Additionally, the Sudden Death in the Young Case Registry provides tools for providers, resources for families, and opportunities for research (52). Advocacy organizations such as the SADS Foundation is also a good resource for medical professionals, researchers, and families (53).
Many barriers to testing can be remedied by discussion, education, and outreach within and between specialties; by creating a regional network of professionals for coordinated, quality care (54); and by establishing standard procedures in advance. Medical examiner/coroner offices have limited staff and resources and, therefore, need access to colleagues who can provide technical services for sample storage, help evaluate clinical circumstances, recommend tests, obtain family consent, provide result interpretation, and counsel families.
Technological limitations are no longer paramount but cost of genetic testing remains a significant barrier, particularly for medical examiner/coroner offices with high case volume. Currently many insurance companies do not reimburse families for postmortem genetic testing. If the family has insurance, testing of living relatives may be covered. Many medical examiner/coroner offices struggle to comfortably shoulder the burden of investigating unnatural deaths in their jurisdiction, making genetic testing for natural diseases a distant consideration. Aside from the cost of testing itself, the medical examiner/coroner facility incurs costs for sample collection and storage (e.g., freezers, RNA stabilization solutions, liquid nitrogen for snap freezing, dry ice, storage and mailing containers) and shipping fees. Financial aspects of both postmortem and survivor genetic testing need to be addressed by our medicolegal death investigation, insurance, and commercial laboratory systems at local and national levels.
The scope of evaluations among surviving first-degree relatives is not standardized, and the response to an unexplained sudden death of a child by the survivor’s physicians may range from simple inquiry as to symptoms to invasive testing. Testing strategies and result interpretation remain unsettled, as emergence of new genetic technology and an increasingly refined understanding of mutation significance continue to change. Future genetic testing algorithms will be influenced by decreasing cost of sequencing and increased knowledge of the genetics of human health and disease. A targeted approach with subsequent expansion to more comprehensive testing is the current strategy, but this may become outmoded as the quick, accurate, and affordable sequencing of entire genomes becomes available.
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