Laboratory of SIDS Research

Hannah C. Kinney, MD, Director, and Robin L. Haynes, PhD, Associate Director,

Research Registry of Robert’s Program

Director, Richard D. Goldstein, MD, Associate Director, Hannah C. Kinney, MD

Boston Children’s Hospital

Harvard Medical School

Boston, MA 02115

December 5 2016

 

  1. Background

 

  • The mission of our SIDS research program is to determine the role of the brain in causing sudden, unexpected, and unexplained death in infants. We are in search of brain abnormalities that contribute to sudden infant death, as well as their causes.  Once the nature of the brain abnormalities and their cause(s) are determined, it will then be possible, we believe, to devise the means to: 1) identify living infants at risk with diagnostic tests, preferably in the newborn period; and 2) to develop interventions to prevent the deaths. Preventing sudden infant death is our over-riding goal.

 

  • We are a group of investigators and trainees with multiple backgrounds and fields of expertise in neuroscience, neuropathology, neurology, biochemistry, pediatrics, pediatric pathology, forensic pathology, genetics, neuroimaging, palliative care, and biostatistics working together in SIDS research. Through various collaborations with each other, we perform the research projects that are ongoing in the laboratory. The research also involves a Research Registry of cases of sudden and unexplained death in early life under the auspices of Robert’s Program on Sudden and Unexpected Death in Pediatrics (SUDP) (see below).  Our work is funded by NIH grants, as well as grants from private SIDS foundations and individual donors.  The funding allows for a breadth and depth of ongoing projects in the laboratory and Robert’s Program, in coordination with each other.  Below we summarize the major ongoing research projects, divided into projects in the basic science laboratory, and those under the clinical translation activities of Robert’s Program.  The following projects are in different stages of progression.

 

  1. Ongoing Laboratory Research Projects in the Laboratory

 

  • The overall hypothesis of our research in the laboratory is that SIDS, or a subset of SIDS, is due to an abnormality in the network of brain circuits that protect against life-threatening stresses during sleep in a critical developmental period (the first year of life). This brain network is called the “central homeostatic network”, and consists of multiple different structures in the brain that are integrated together by interconnecting pathways.  This network orchestrates coordinated responses to dangerous bodily threats during sleep, such as low oxygen (hypoxia), high carbon dioxide (hypercarbia), hypoxia and hypercarbia combined (asphyxia), overheating, and sudden changes in blood pressure.  These threats may complicate unsafe sleep environments, such as face down/prone sleep position, soft bedding, sofas, and adult beds.

 

  • The central homeostatic network acts like an internal
    “alarm system” that is triggered by hypoxia or other stresses during sleep    . When triggered, this alarm system sets in motion protective defenses against the stressors.  Such defenses are arousal from sleep and turning the head to the side for fresh air from the face down position in soft bedding.  If an infant has a defect in this alarm system (that is, in the central homeostatic network), he/she may not be stimulated internally to wake up, but rather, may undergo repeated stresses and ultimately go on to die in his/her sleep from the progressive hypoxia or other stress.

 

  • Over the last two decades, our laboratory has provided compelling evidence of defects in the two major components of the alarm system or central homeostatic network–the lower brainstem and the hippocampus (the latter structure located on each side of the cerebral hemispheres above the brainstem). Our laboratory has provided evidence in four independent datasets of tissue defects indicating dysfunction of the neurotransmitter serotonin in the medulla oblongata (lower brainstem) involved in protective responses to life-threatening challenges during sleep in about 40% of SIDS infants.  These serotonin defects include significantly decreased levels (~26%) of 5-HT itself in SIDS cases compared to age-adjusted controls, as well as abnormalities in serotonin receptors and serotonin’s biosynthetic enzyme. In 2015, we then reported a second new finding, from light microscope studies in a key portion of the hippocampus, called the dentate gyrus.  The dentate gyrus defect was present in a major subgroup of SIDS cases (~40%).  This structural abnormality in the dentate gyrus has been reported in some patients with temporal lobe epilepsy, suggesting the possibility that SIDS deaths may involve seizures originating in the hippocampus and affecting the autonomic nervous system as a major stress. The brainstem is interconnected with the hippocampus, and each structure influences the function and organization of the other, with brainstem influences upon hippocampal development as well.

 

  • The laboratory work is done in a long-standing collaboration with the Office of the Medical Examiner, San Diego, CA, and the program coordinator of SIDS research there, Ms. Elisabeth A. Haas, MPH.

 

  • Project 1: To characterize fully the serotonin defects in the entire brainstem and their relationship to potential serotonin abnormalities in the hippocampus.  A goal is to determine if the serotonergic brainstem abnormalities coexist with defects in the dentate gyrus in the hippocampus in the same infant, or alternatively if these two defects occur in separate SIDS infants, thereby suggesting two separate defects in the central homeostatic network in SIDS.  A variety of modern anatomic and neurochemical techniques are applied to human autopsy tissue analysis in this project, comparing findings in SIDS infants to those in control autopsied infants dying of known causes.  Kinney and Haynes oversee this work.

 

  • Project 2: To determine the gene expression profile of serotonin neurons in the brainstem of SIDS infants compared to control autopsied infants dying of known causes.  Our group has found that not all serotonin neurons are the same, but rather, different serotonin subtypes have different functions important in different aspects of protective responses to stress (homeostasis).  Some serotonin neurons help regulate chemoreception to carbon dioxide, for example, while others regulate responses to temperature changes.  We are attempting to determine what specific types of serotonin neurons are involved in SIDS by determining the different patterns of expression of genes in them, in an effort to determine which serotonin neurons–one or many–are at fault in SIDS.  We hypothesize that abnormal serotonin subtypes will demonstrate different patterns of gene expression irregularities that will help us to identify the molecular pathways that are critical to abnormal cell function, and therefore the molecules to target for drug or other therapies.  State-of-the-art methods in gene expression profiling are applied to this brainstem analysis. The work is done in collaboration with Dr. Benjamin Okaty in the laboratory of Dr. Susan Dymecki, Department of Genetics, Harvard Medical School.  It involves the work of trainees Ms. Emma Giles and Mr. Michael McConville.

 

  • Project 3: To develop a unique platform for the analysis of stem cells (stored as cord blood samples at birth) from human infants who died of SIDS. The underlying premise of this research project is that brainstem serotonin neurons play a direct causal role in SIDS. The different serotonin neuron subtypes are defined by specific gene expression profiles (see above). We postulate in this project that one or more specific 5-HT neuron subtypes are abnormal in SIDS, leading to defective responses to asphyxia and sleep-related sudden death. Our team has generated a functional and molecular map of the different kinds of serotonin neurons in the mouse brain, and is generating data now indicating their likely presence in the infant brainstem. Our specific aim is to convert pluripotent stem cells derived from cord blood from SIDS infants and living controls (collected and stored at birth for potential uses in chronic diseases should they arise in later life) into specific homeostasis-relevant serotonin neuron subtypes, and compare these neurons for differences in gene expression and electrophysiological properties. The general idea is that the stem cells from SIDS infants are transformed in cell culture to become the serotonin neuron subtypes. Physiological methods are applied to the SIDS serotonin neurons in cell culture to determine the abnormal functional properties (electrical discharge patterns) of these neurons; gene expression profiling which is applied to determine aberrant molecular pathways in these neurons.   It is not possible to determine the molecular and cellular features of abnormal serotonin cells in autopsy tissues.  Stem cell methods allow for the unique characterization of the serotonin pathology at the molecular and cellular levels in actual cells from a SIDS infant.   This project involves animal and human work in coordination to establish methodologies and define abnormal findings.  The work is done in collaboration with Dr. Benjamin Okaty in the laboratory of Dr. Susan Dymecki, Department of Genetics, Harvard Medical School.

 

  • Project 4: To characterize in depth the development of the human hippocampus in SIDS infants compared to control infants dying of known causes. Here we seek the cellular, molecular, and structural abnormalities that distinguish the abnormal dentate gyrus in the hippocampus in SIDS in order to define the basic pathways gone awry.  We are focused upon the developmental factors that control the shape and architecture of the dentate gyrus.  We are also attempting to determine if seizure activity has occurred in the dentate gyrus by analyzing tissue markers that change as a result of electrical seizure discharges.  We are also determining if the pattern of gene expression of particular developmental factors is abnormal in the dentate gyrus in SIDS.  The project utilizes techniques in the molecular and neurochemical analysis of human postmortem tissues.  Kinney and Haynes oversee this research. This involves the work of trainees Ms. Emma Giles and Mr. Michael McConville.

 

  • Project 5: To discover potential new abnormalities in the brainstem that, in conjunction with serotonin, contribute to the chain of events that lead to sudden infant death. We want to know the molecular and biochemical basis of the different serotonin abnormalities that we have reported on SIDS. We are currently using “omics” methodologies to simultaneously screen large numbers of proteins (proteomics) and metabolites (metabolomics) for abnormalities in SIDS infants compared to control infants dying of known causes.  Through the use of such screening techniques we will gain insight into the molecules that cause the dysregulation in serotonin factors and into pathological changes beyond what we currently know of SIDS pathology.  This information will provide new clues as to abnormal proteins, metabolites, and/or metabolic pathways that may ultimately serve as new targets for SIDS intervention.  Haynes oversees this research.

 

  • Project 6: To discover a biomarker of infants at risk for SIDS due to brainstem serotonin and/or hippocampal defects. Here, a biomarker, or biological marker, refers to a biochemical or molecular substance whose detection indicates a particular disease state. To be diagnostically useful, biomarkers are derived from readily accessible bodily fluids, such as serum or blood, even if the disease process is centered in another organ site, such as postulated in the brain in SIDS (see above). Work in our laboratory has established important differences in SIDS infants, consistent with an underlying vulnerability in the brainstem and hippocampus that makes certain infants susceptible to SIDS. There is also substantial evidence that death in SIDS is preceded by undetectable dysfunction in respiratory and autonomic control and sleep-wake pattern, including episodic apnea and bradycardia (decrease in heart rate). Currently, there are no biomarkers for SIDS risk in living infants, a major problem given that the death occurs suddenly, without warning, and unwitnessed in seemingly healthy infants. To reach this goal, an association of a candidate biomarker with a pathologic process must first be established. Currently, research in the laboratory is ongoing to determine a biomarker of brainstem serotonin abnormalities. We focused the search for a clinical biomarker in SIDS on readily measurable serum serotonin and found a significant increase in serum levels of serotonin in a subset of SIDS cases (34%). This project is ongoing and is focused on identifying the source or sources of the increased serum serotonin and determining the relationship between serum serotonin levels and brain serotonin abnormalities in SIDS infants. The ultimate goal of the research is to establish serum serotonin as a biomarker of SIDS that could be use for screening of potential infants at risk in the newborn period.  Haynes oversees the biomarker research in the laboratory.

 

  • Project 7: To develop state-of-the-art neuroimaging techniques with the resolution to dentate gyral abnormalities and related hippocampal pathology in living infants at risk for SIDS, as well as in autopsied infants. We are in the process of developing magnetic resonance imaging (MRI) technology that will closely approximate the detailed observations presently observable only under a microscope. We are working with our colleagues, Drs. Sanjay Prabhu and Simon Warfield, in neuroimaging in this research.   Postmortem samples of the temporal lobe with the hippocampus are imaged, and correlations with neuropathology and neuroanatomy are made through serial sections through the tissue specimen with the MRI images.  At this point in our research, we can now distinguish in the postmortem MRI images the microscopic architecture of the hippocampus beyond the resolution of clinically used, standard protocols. The refined technology will allow for a more rapid evaluation of brain regions during the autopsy of children dying suddenly and unexpectedly. This technological progress promises to influence autopsy practice in relevant cases. It will also allow for the detection of those abnormalities in living children found to be at risk, thus opening the door for prevention.

 

 

 

 

 

 

  1. Research in Robert’s Program on Sudden Unexpected Death in Pediatrics

 

  • We have developed a Research Registry for analysis by the clinicians and scientists at Boston Children’s Hospital in the program to evaluate nationally and internationally referred cases of sudden and unexplained death in infants. Detailed information is collected and reviewed by our multidisciplinary team. The Co-Directors of Research in Robert’s Program are Drs. Hannah Kinney and Susan Dymecki.

 

  • Project 1: In the Research Registry in Robert’s Program, we take a different, more broad approach in which we cast a wide net to attempt to define causes of SIDS that may or may not involve the brain.  The major hypothesis in the research in Robert’s Program is that unexplained death in infants is caused by undiagnosed diseases, either completely undiscovered and extreme (lethal) diseases, or known diseases presenting in a previously unrecognized lethal fashion. Research is based on the premise that through careful characterization of individual cases, thoroughly reviewing patient medical records, collecting detailed family pedigrees, and performing pathological examination and genetic testing, underlying biological disorders will be discovered in a large percentage of cases. New rare diseases will be determined, as well as new presentations of known rare diseases. These new diseases likely include the disorders of the central homeostatic network, as defined above, but already involve other important observations found through a careful analysis of larger numbers of cases.   Research in Robert’s Program relies on its case registry, which has an important role in the future testing of new hypotheses, and helps to accelerate discovery, Clinical subtypes discovered by examining human cases of SIDS generate hypotheses about its causes that can be studied in the basic science laboratory. Several different clinical patterns (phenotypes) of sudden unexplained infant death have begun to emerge in this research that are being actively pursued by the entire team of investigators in Robert’s Program.

 

  • Project 2: One ongoing research project in the Research Registry of Robert’s Program is to determine if SIDS infants with hippocampal pathology (SIDS-HP) in the dentate gyrus have variants (mutations, polymorphisms) in genes that are related to epilepsy, including temporal lobe epilepsy, and/or hippocampal maldevelopment using whole exome sequencing. In this project, we are examining the genetic basis of SIDS-HP as an entity potentially related to epilepsy, treating each case as an undiagnosed disease with epilepsy-related neuropathology. Our colleagues in pediatric neurology, Dr. Ann Poduri, and genetics, Dr. Ingrid Holm, are involved in this work.  Genomic DNA samples taken at autopsy from SIDS infants, as well as available living family members, are analyzed.  Known genes linked to sudden death, epilepsy, temporal lobe epilepsy, and dentate gyrus development are analyzed for genetic variants.  Variants deemed pathogenic or likely pathogenic in the SIDS-HP are confirmed using different sequencing techniques.  The opportunity to combine strong clinicopathologic data from the infants in parallel with exome sequencing has future promise of being combined with functional analysis of identified mutations in experimental systems, the latter intended for future work by our group.

 

  • Project 3: We are developing a gene panel to be used in the evaluation of sudden unexpected death in any child, incorporating genes implicated in sudden death due to many causes. This panel will be the most comprehensive gene panel available, and will report critical information of potentially heritable factors involved in sudden death. We are in the process of determining the operating characteristics of the panel. Once achieved, we will make this panel available in the evaluation of all cases of sudden death in children. The panel has continuously been assembled and revised since the opening of Robert’s Program. It will be studied and revised in the future, keeping it in accord with any new discoveries.  This work is a joint effort of the investigative team of Robert’s Program.

 

  • Project 4: Sudden unexpected death carries an enormous burden on the future lives of affected families. Robert’s Program is conducting research to determine which mothers carry an increased risk of more extreme and disabling grief. Once the research accomplishes the means to identify those most at risk, Robert’s Program researchers plan to develop interventions to assist identified families and bring new attention to assisting with coping. Goldstein in Pediatric Palliative Care directs this research.

 

 

 

 

SIDS-Related Publications  Since 2014

 

Original Reports

 

  1. Rognum IJ, Tran H, Haas EA, Hyland K, Paterson DS, Haynes RL, Broadbelt KG, Harty BJ, Mena O, Krous HF, Kinney HC. Serotonin metabolites in the cerebrospinal fluid in the sudden infant death syndrome: In search of a biomarker of risk.  J Neuropathol Exp Neurol 2014; 73: 115-22.
  2. Kinney HC, Cryan, JB, Haynes RL, Paterson DS, Haas EA, Mena OJ, Minter M, Journey KW, Trachtenberg FL, Goldstein RD, Armstrong DD. Dentate gyrus abnormalities in sudden unexplained death in infants: Morphological marker of underlying brain vulnerability.  Acta Neuropathol 2015; 65-80.
  3. Bianciardi M, Toschi N, Edlow BL, Eichner C, Sestompop K, Polimeni JR, Kinney HC, Rosen BR, Wald LL. In vivo neuroimaging template of human brainstem nuclei. Brain Connect 2015 Aug 11 [Epub ahead of print].
  4. Edlow BL, McNab JA, Witzel T, Kinney HC. The structural connectome of the human central homeostatic network. Brain Connect 2016; 6: 187-200.
  5. Hefti MM, Cryan JB, Grafe M, Haas EA, Chadwick AE, Crandall LA, Trachtenberg FL, Kinney HC, Krous HF. Part 1. Sudden unexpected death in early childhood: A series of 151 cases. Forensic Sci Med Pathol 2016; 12; 4-13.
  6. Hefti MM, Cryan JB, Haas EA, Chadwick AE, Crandall LA, Trachtenberg FL, Armstrong DD, Grafe M, Krous F, Kinney HC. Part II. Sudden unexpected death in early childhood: Neuropathologic observations. Forensic Sci Med Pathol 2016; 12; 14-25.
  7. Goldstein RD, Trachtenberg FL, Sens MA, Harty B, Kinney HC. SIDS and infant mortality rates.  Pediatrics 2016; 137: 1-10.
  8. Kinney HC*, Poduri AH* (*, co-first authors), Cryan JB, Haynes RL, Teot L, Sleeper LA, Holm IA, Berry GT, Prabhu, Warfield SK, Brownstein CA, Abram HS, Kruer M, Kemp, WL, Hargitai B, Gastrang J, Mena OJ, Haas EA, Dastjerdi, Armstrong DD, Goldstein, RG. Hippocampal formation maldevelopment and sudden death across the pediatric age spectrum. J Neuropathol Exp Neurol 2016; 75: 984-997.
  9. Haynes RL, Frelinger AL, Kozakewich HW, Tran H, Haas EA, Mena OJ, Trachtenberg FL, Berry GT, Michelson A, Goldstein RD, Kinney, HC. Elevated serum serotonin in a large subset of infants dying of the sudden infant death syndrome.  Under review, 2016.
  10. Goldstein RD, Nields HM, Holm IA, Poduri AH, Morris SE, Brownstein CA, Teot L, Berry GT, Warfield SK, Prabhu SP, Haynes RL, Gilles EK, MacRae CA, Dymecki SM, Kinney HC. A new paradigm for the investigation of sudden unexpected death in young children.  Under review, 2016.
  11. Brownstein CA, Goldstein RD, Kinney HC, Haynes RL, Giles E, Sheidley B, Bainbridge M, Haas EA, Lucas J, Schaber B, Holm IA, Poduri AH. SCN1Aassociated with two cases of sudden infant death syndrome.  Under review, 2016.

 

The Safe Passage Study: Dr. Kinney is  the Principal Investigator of the Developmental Brain and Pathology Center, and member of the Steering Committee of the PASS Network and Safe Passage Study, a NIH-funded consortium to study the effects of prenatal alcohol exposure upon morbidity and mortality of the human fetus and infant.  Dr. Haynes is the Co-Principal Investigator of the DBPC and member of the Steering Committee.

 

  1. Dukes KA, Burd L, Elliott AJ, Fifer WP, Folkerth RD, Hankins GD, Hereld D, Hoffman H, Myers MM, Odendaal HJ, Signore C, Sullivan LM, Willinger M, Wright C, and Kinney HC for the PASS Network. The Safe Passage Study: Design, Methods, Recruitment, and Follow-Up Approach. Pediatr Perinat Epi 2014, Aug 5 [Epub ahead of print].
  2. Human M, Green S, Groenwald C, Goldstein RD, Kinney HC, Odendaal HJ, PASS Network. Psychosocial implications of stillbirth for the mother and her family: A crisis support approach. Social Work (Stellenbosch) 2015; 50: 392.
  3. Boyd TK, Wright CA, Odendall HJ, Sens MA, Folkerth RD, Roberts DJ, Kinney HC, for the PASS Network. The stillbirth classification system for the Safe Passage Study: Incorporating mechanism, etiology, and recurrence. Pediatr Dev Pathol 2016 April 26 [Epub ahead of print].
  4. Dempers JJ, Coldrey J, Burger EH, Thompson V, Wadee SA, Odendaal HJ, Sens MA, Randall BB, Folkerth RD, Kinney HC for the PASS Network. The institution of standardized investigation protocol for sudden infant death in the Eastern Metropole, Cape Town, South Africa. J Forensic Sci 2016, Sept 27 [Epub ahead of print].
  5. Haynes RL, Folkerth RD, Paterson DS, Broadbelt KG, Dan Zaharie S, Hewlett RH, Dempers JJ, Burger E, Wadee S, Schubert P, Wright C, Sens MA, Nelsen L, Randall BB, Tran H, Geldenhuys E, Elliott AJ, Odendaal HJ, Kinney HC; PASS Network. Serotonin receptors in the medulla oblongata of the human fetus and infant: The analytic approach of the international Safe Passage Study. J Neuropathol Exp Neurol. 2016 Sep 15. pii: nlw080.

 

Proceedings of Meetings

 

  1. Goldstein RD, Kinney HC, Willinger MW. Special Article: Sudden unexpected death in fetal life through early childhood: New opportunities (Proceedings of an NICHD Workshop). Pediatrics 2016; 137.

 

Chapter in Book

 

  1. Kinney HC, Heft MM, Goldstein RD, Haynes RL. The Sudden Infant Death Syndrome.  In:  Golden JA, Harding BN (eds). Pathology and Genetics: Acquired and Inherited Diseases of the Developing Nervous System. Basel: ISN Neuropathology Press. 2016, in press.

 

Technical and Other Scientific Interventions

 

  1. Edlow, BL, Folkerth RD, Kinney HC. 2016. The Harvard Ascending Arousal Network (AAN) Atlas. Neuroanatomic atlas of brainstem nuclei of the human AAN in standard MNI152 space for neuroimaging, including diffusion tractography. Available to the academic community

(www.martinos.org/resources/aan-atlas) to enable structural and functional connectivity analyses of the human AAN in healthy subjects and in patients with disorders of consciousness.