Predictive and diagnostic measures for kernicterus spectrum disorder: a prospective cohort study.

BACKGROUND: Kernicterus spectrum disorder (KSD) resulting from neonatal hyperbilirubinemia remains a common cause of cerebral palsy worldwide. This 12-month prospective cohort study followed neonates with hyperbilirubinemia to determine which clinical measures best predict KSD. METHODS: The study enrolled neonates ≥35 weeks gestation with total serum bilirubin (TSB) ≥ 20 mg/dl admitted to Aminu Kano Hospital, Nigeria. Clinical measures included brain MRI, TSB, modified bilirubin-induced neurologic dysfunction (BIND-M), Barry-Albright Dystonia scale (BAD), auditory brainstem response (ABR), and the modified KSD toolkit. MRI signal alteration of the globus pallidus was scored using the Hyperbilirubinemia Imaging Rating Tool (HIRT). RESULTS: Of 25 neonates enrolled, 13/25 completed 12-month follow-up and six developed KSD. Neonatal BIND-M ≥ 3 was 100% sensitive and 83% specific for KSD. Neonatal ABR was 83% specific and sensitive for KSD. Neonatal HIRT score of 2 was 67% sensitive and 75% specific for KSD; this increased to 100% specificity and sensitivity at 12 months. BAD ≥ 2 was 100% specific for KSD at 3-12 months, with 50-100% sensitivity. CONCLUSIONS: Neonatal MRIs do not reliably predict KSD. BIND-M is an excellent screening tool for KSD, while the BAD or HIRT score at 3 or 12 months can confirm KSD, allowing for early diagnosis and intervention. IMPACT: The first prospective study of children with acute bilirubin encephalopathy evaluating brain MRI findings over the first year of life. Neonatal MRI is not a reliable predictor of kernicterus spectrum disorders (KSD). Brain MRI at 3 or 12 months can confirm KSD. The modified BIND scale obtained at admission for neonatal hyperbilirubinemia is a valuable screening tool to assess risk for developing KSD. The Barry Albright Dystonia scale and brain MRI can be used to establish a diagnosis of KSD in at-risk infants as early as 3 months.

Effect of reduced versus usual lipid emulsion dosing on bilirubin neurotoxicity and neurodevelopmental impairment in extremely preterm infants: study protocol for a randomized controlled trial.

BACKGROUND: Bilirubin neurotoxicity (BN) occurs in premature infants at lower total serum bilirubin levels than term infants and causes neurodevelopmental impairment. Usual dose lipid infusions in preterm infants may increase free fatty acids sufficiently to cause bilirubin displacement from albumin, increasing passage of unbound bilirubin (UB) into the brain leading to BN and neurodevelopmental impairment not reliably identifiable in infancy. These risks may be influenced by whether cycled or continuous phototherapy is used to control bilirubin levels. OBJECTIVE: To assess differences in wave V latency measured by brainstem auditory evoked responses (BAER) at 34-36 weeks gestational age in infants born ≤ 750 g or < 27 weeks' gestational age randomized to receive usual or reduced dose lipid emulsion (half of the usual dose) irrespective of whether cycled or continuous phototherapy is administered. METHODS: Pilot factorial randomized controlled trial (RCT) of lipid dosing (usual and reduced) with treatment groups balanced between cycled or continuous phototherapy assignment. Eligible infants are born at ≤ 750 g or < 27 weeks' gestational age enrolled in the NICHD Neonatal Research Network RCT of cycled or continuous phototherapy. Infants will randomize 1:1 to reduced or usual dose lipid assignment during the first 2 weeks after birth and stratified by phototherapy assignment. Free fatty acids and UB will be measured daily using a novel probe. BAER testing will be performed at 34-36 weeks postmenstrual age or prior to discharge. Blinded neurodevelopmental assessments will be performed at 22-26 months. Intention-to-treat analyses will be performed with generalized linear mixed models with lipid dose and phototherapy assignments as random effects covariates, and assessment for interactions. Bayesian analyses will be performed as a secondary analysis. DISCUSSION: Pragmatic trials are needed to evaluate whether lipid emulsion dosing modifies the effect of phototherapy on BN. This factorial design presents a unique opportunity to evaluate both therapies and their interaction. This study aims to address basic controversial questions about the relationships between lipid administration, free fatty acids, UB, and BN. Findings suggesting a reduced lipid dose can diminish the risk of BN would support the need for a large multicenter RCT of reduced versus usual lipid dosing. TRIAL REGISTRATION: Clinical Trials.gov, NCT04584983, Registered 14 October 2020, https://clinicaltrials.gov/ct2/show/NCT04584983 Protocol version: Version 3.2 (10/5/2022).

Kernicterus Spectrum Disorders Diagnostic Toolkit: validation using retrospective chart review.

BACKGROUND: Kernicterus Spectrum Disorders (KSDs) result from hyperbilirubinemia-induced brain injury. We developed a Toolkit (KSD-TK) to predict the likelihood of KSDs. This study aims to validate the KSD-TK by comparing it to clinical diagnoses made by the Kernicterus Clinic in the Division of Neurology. METHODS: Through retrospective chart review, we completed a KSD-TK for 37 patients evaluated between 2011 and 2019 using highest bilirubin, newborn risk factors, neonatal exam, follow-up exam, auditory testing, tooth enamel, and MRI brain results. KSD-TK results were compared to the clinical diagnoses given by a kernicterus expert (SS). RESULTS: Of 37 patients, 29 were clinically diagnosed with kernicterus, including 14/14 with KSD-TK scored as "definite", 14/15 "probable", and 1/2 with "possible" kernicterus. None of 6 patients with KSD-TK "not kernicterus" were clinically diagnosed with kernicterus. Combining KSD-TK "definite" and "probable", the KSD-TK has 96.6% sensitivity and 87.5% specificity. Each KSD-TK component had high sensitivity, but only three had specificity ≥0.75: auditory neuropathy spectrum disorder, abnormal movements and/or tone on follow-up exam, and abnormal globus pallidus and/or subthalamic nucleus on MRI. CONCLUSION: The KSD-TK is a promising screening tool for patients at risk for kernicterus. IMPACT: This study provides validation of a Kernicterus Spectrum Disorders (KSDs) Toolkit. The toolkit provides screening criteria for predicting KSD diagnosis. Scores of definite or probable have high sensitivity and specificity for KSDs. Abnormal auditory processing, exam, and MRI were most specific for KSDs.

Review of bilirubin neurotoxicity II: preventing and treating acute bilirubin encephalopathy and kernicterus spectrum disorders.

Previously in Part I of this two-part review, we discussed the current and recent advances in the understanding of the molecular biology and neuropathology of bilirubin neurotoxicity (BNTx). Here in Part II, we summarize current treatment options available to treat the severely jaundiced infants to prevent significant brain damage and improve clinical outcomes. In addition, we review potential novel therapies that are in various stages of research and development. We will emphasize treatments for both prevention and treatment of both acute bilirubin encephalopathy (ABE) and kernicterus spectrum disorders (KSDs), highlighting the treatment of the most disabling neurological sequelae of children with mild-to-severe KSDs whose "rare disease" status often means they are overlooked by the clinical research community at large. As with other secondary dystonias, treatment of the dystonic motor symptoms in kernicterus is the greatest clinical challenge.

Review of bilirubin neurotoxicity I: molecular biology and neuropathology of disease.

Despite the availability of successful prevention strategies to prevent excessive hyperbilirubinemia, the neurological sequelae of bilirubin neurotoxicity (BNTx) still occur throughout the world. Kernicterus, encephalopathy due to BNTx, is now understood to be a spectrum of severity and phenotypes known as kernicterus spectrum disorder (KSD). A better understanding of the selective neuropathology and molecular biology of BNTx and using consistent clinical definitions of KSDs as outcome measure can lead to more accurately predicting the risk and causes of BNTx and KSDs. In Part I of our two-part review, we will summarize current and recent advances in the understanding of the selective neuropathology and molecular biology of the disease. Herein we emphasize the role of unbound, free unconjugated bilirubin as well as genetic contributions to the susceptibility BNTx and the development of KSDs. In Part II, we focus on current and possible novel methods to prevent BNTx and ABE and treat ABE and KSDs.

Hyperactivity in the Gunn rat model of neonatal jaundice: age-related attenuation and emergence of gait deficits.

BACKGROUND: Neonatal jaundice resulting from elevated unconjugated bilirubin occurs in 60-80% of newborn infants. Although mild jaundice is generally considered harmless, little is known about its long-term consequences. Recent studies have linked mild bilirubin-induced neurological dysfunction (BIND) with a range of neurological syndromes, including attention-deficit hyperactivity disorder. The goal of this study was to measure BIND across the lifespan in the Gunn rat model of BIND. METHODS: Using a sensitive force plate actometer, we measured locomotor activity and gait in jaundiced (jj) Gunn rats versus their nonjaundiced (Nj) littermates. Data were analyzed for young adult (3-4 mo), early middle-aged (9-10 mo), and late middle-aged (17-20 mo) male rats. RESULTS: jj rats exhibited lower body weights at all ages and a hyperactivity that resolved at 17-20 mo of age. Increased propulsive force and gait velocity accompanied hyperactivity during locomotor bouts at 9-10 mo in jj rats. Stride length did not differ between the two groups at this age. Hyperactivity normalized, and gait deficits, including decreased stride length, propulsive force, and gait velocity, emerged in the 17-20-mo-old jj rats. CONCLUSION: These results demonstrate that, in aging, hyperactivity decreases with the onset of gait deficits in the Gunn rat model of BIND.

Albumin administration protects against bilirubin-induced auditory brainstem dysfunction in Gunn rat pups.

BACKGROUND: Free bilirubin (Bf), the unbound fraction of unconjugated bilirubin (UCB), can induce neurotoxicity, including impairment of the auditory system, which can be assessed by brainstem auditory evoked potentials (BAEPs). We hypothesized that albumin might reduce the risk of neurotoxicity by decreasing Bf and its translocation into the brain. AIM: To determine the effects of albumin on BAEPs and brain bilirubin content in two Gunn rat pup models of acute hyperbilirubinemia. METHODS: We used Gunn rat pups, which have a deficiency of the bilirubin-conjugating enzyme UGT1A1. We induced haemolysis by injection of phenylhydrazine (phz) into 14-days old pups. Subsequently, pups were treated with either i.p. human serum albumin (HSA; 2.5 g/kg; n = 8) or saline (control, n = 8). We induced acute neurotoxicity by injecting 16-days old pups with sulphadimethoxine (sulpha) and treated them with either HSA (n = 9) or saline (control, n = 10). To assess bilirubin neurotoxicity, we used the validated BAEP method and compared relevant parameters; i.e. peak latency values and interwave interval (IWI) between peak I and peak II, a marker of acute neurotoxicity. RESULTS: Phz and sulpha significantly increased IWI I-II by 26% and 29% (P < 0.05) in the haemolysis and the displacement model, respectively. Albumin completely prevented the increase of IWI I-II in either model. The beneficial effect of albumin in the displacement-model by means of normal BAEPs was in line with less bilirubin in the brain (NS). Interestingly, in the haemolysis model the accumulation of total bilirubin in the brain was unaltered, and BAEPs still appeared normal. This might advocate for a role of brain Bf which was calculated and showed that albumin treatment non-significantly reduces Bf concentrations in brain, compared with saline treatment. CONCLUSIONS: Albumin treatment is neuroprotective in acute hyperbilirubinemia in Gunn rat pups. Our present results underline the importance of functional diagnostic test of neurotoxicity above biochemical concentrations.

MGE-Like Neural Progenitor Cell Survival and Expression of Parvalbumin and Proenkephalin in a Jaundiced Rat Model of Kernicterus.

Kernicterus is a permanent condition caused by brain damage from bilirubin toxicity. Dystonia is one of the most debilitating symptoms of kernicterus and results from damage to the globus pallidus (GP). One potential therapeutic strategy to treat dystonia in kernicterus is to replace lost GP neurons and restore basal ganglia circuits through stem cell transplantation. Toward this end, we differentiated human embryonic stem cells (hESCs) into medial ganglion eminence (MGE; the embryological origin of most of the GP neurons)-like neural precursor cells (NPCs). We determined neurochemical phenotype in cell culture and after transplanting into the GP of jaundiced Gunn rats. We also determined grafted cell survival as well as migration, distribution, and morphology after transplantation. As in the GP, most cultured MGE-like NPCs expressed γ-aminobutyric acid (GABA), with some co-expressing markers for parvalbumin (PV) and others expressing markers for pro-enkephalin (PENK). MGE-like NPCs survived in brains at least 7 weeks after transplantation, with most aggregating near the injection site. Grafted cells expressed GABA and PV or PENK as in the normal GP. Although survival was low and the maturity of grafted cells varied, many cells produced neurite outgrowth. While promising, our results suggest the need to further optimize the differentiation protocol for MGE-like NPC for potential use in treating dystonia in kernicterus.

RNA Sequencing of Human Peripheral Nerve in Response to Injury: Distinctive Analysis of the Nerve Repair Pathways.

The development of regenerative therapies for central nervous system diseases can likely benefit from an understanding of the peripheral nervous system repair process, particularly in identifying potential gene pathways involved in human nerve repair. This study employed RNA sequencing (RNA-seq) technology to analyze the whole transcriptome profile of the human peripheral nerve in response to an injury. The distal sural nerve was exposed, completely transected, and a 1 to 2 cm section of nerve fascicles was collected for RNA-seq from six participants with Parkinson's disease, ranging in age between 53 and 70 yr. Two weeks after the initial injury, another section of the nerve fascicles of the distal and pre-degenerated stump of the nerve was dissected and processed for RNA-seq studies. An initial analysis between the pre-lesion status and the postinjury gene expression revealed 3,641 genes that were significantly differentially expressed. In addition, the results support a clear transdifferentiation process that occurred by the end of the 2-wk postinjury. Gene ontology (GO) and hierarchical clustering were used to identify the major signaling pathways affected by the injury. In contrast to previous nonclinical studies, important changes were observed in molecular pathways related to antiapoptotic signaling, neurotrophic factor processes, cell motility, and immune cell chemotactic signaling. The results of our current study provide new insights regarding the essential interactions of different molecular pathways that drive neuronal repair and axonal regeneration in humans.

A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus).

Neonatal hyperbilirubinemia can cause bilirubin encephalopathy (kernicterus). Spontaneously jaundiced (jj) Gunn rats treated with sulfonamide (sulfa) to displace bilirubin from serum albumin, develop bilirubin encephalopathy and abnormal brainstem auditory evoked potentials (BAEPs) comparable with human newborns. We hypothesized phenylhydrazine (PHZ)-induced hemolysis would significantly elevate total plasma bilirubin (TB) in jj Gunn rat pups and produce BAEP abnormalities similar to those observed after sulfa. PHZ 0, 25, 50, or 75 mg/kg was administered intraperitonealy to 15-d-old jjs. An initial TB was recorded in each animal, and a second recorded 1-4 d postinjection to generate a dose-response curve. After PHZ 75 mg/kg, TB peaked at about 30 mg/dL at 48-72 h. A second group of jjs injected with PHZ (0, 25, 50, or 75 mg/kg) and nonjaundiced controls given PHZ 75 mg/kg had HCT and TB at baseline, and HCT, TB, and BAEPs recorded at 48 h. BAEP wave II and III amplitudes decreased, and I-II and I-III interwave intervals increased indicating abnormal central (brainstem) auditory function. PHZ-induced hemolysis in jaundiced Gunn rat pups produces sufficiently elevated TB levels to produce bilirubin encephalopathy. This new model may be a more clinically relevant experimental model of kernicterus- and bilirubin-induced neurologic disorders.

Short Term Development and Fate of MGE-Like Neural Progenitor Cells in Jaundiced and Non-Jaundiced Rat Brain.

Neonatal hyperbilirubinemia targets specific brain regions and can lead to kernicterus. One of the most debilitating symptoms of kernicterus is dystonia, which results from bilirubin toxicity to the globus pallidus (GP). Stem cell transplantation into the GP to replace lost neurons and restore basal ganglia circuits function is a potential therapeutic strategy to treat dystonia in kernicterus. In this study we transplanted human medial ganglionic eminence (MGE)-like neural progenitor cells (NPCs) that we differentiated into a primarily gamma-aminobutyric acid (GABA)ergic phenotype, into the GP of non-immunosuppressed jaundiced (jj) and non-jaundiced (Nj) rats. We assessed the survival and development of graft cells at three time-points post-transplantation. While grafted MGE-like NPCs survived and generated abundant fibers in both jj and Nj brains, NPC survival was greater in the jj brain. These results were consistent with our previous finding that excitatory spinal interneuron-like NPCs exhibited a higher survival rate in the jj brain than in the Nj brain. Our findings further support our hypothesis that slightly elevated bilirubin levels in the jj brain served as an antioxidant and immunosuppressant to protect the transplanted cells. We also identified graft fibers growing toward brain regions that receive projections from the GP, as well as host fibers extending toward the graft. These promising findings suggest that MGE-like NPCs may have the capacity to restore the circuits connecting GP and other nuclei.

Maternal Empowerment - An Underutilized Strategy to Prevent Kernicterus?

BACKGROUND: Kernicterus is a common cause of death and morbidity in many Low- Middle-income Countries (LMICs) and still occurs in affluent nations. In either case, the immediate cause is delayed treatment of severe hyperbilirubinemia. In the West, a provider driven "systems approach" has been widely adopted to identify babies at risk prior to discharge from birthing centers with follow up monitoring based on the serum bilirubin level at time of discharge. The situation is more complicated in regions of the world where kernicterus is endemic, especially in LMICs where Glucose-6-phosphate Dehydrogenase Deficiency (G6PDd) is common and the system of jaundice management is often fragmented. OBJECTIVE: To examine reasons for errors in jaundice management leading to kernicterus and the potential beneficial role of enlisting more parental participation in management decisions. METHOD: We searched world literature related to pitfalls in jaundice management including deficiencies in providers' and parents' knowledge and behavior. Perspectives from mothers of children with kernicterus supplemented the literature review. RESULT: System failures contributing to kernicterus in affluent countries include a lack of follow up planning, bad advice by providers, and a delay in care seeking by parents. In many LMICs, the majority of births occur at home with unskilled attendants. Traditional practices potentiate hemolysis in G6PDd babies. The danger of severe jaundice is frequently underestimated both by parents and care providers, and cultural and economic barriers as well as ineffective therapies delay care seeking. The failure to provide parents information about identifying severe jaundice and knowledge about the risks and treatment of hyperbilirubinemia has contributed to delayed treatment in both affluent and low-middle-income countries. A recent non-randomized clinical trial, supports teaching all parents skills to monitor jaundice, signs of early neurotoxicity, the importance of breast feeding, avoidance of ineffective or dangerous practices, and when/where to seek help. CONCLUSION: Empowering parents allow them to participate more fully in care decisions and to confront obstacles to care when provider services fail.

The Neurological Sequelae of Neonatal Hyperbilirubinemia: Definitions, Diagnosis and Treatment of the Kernicterus Spectrum Disorders (KSDs).

BACKGROUND: Despite its lengthy history, the study of jaundice, hyperbilirubinemia and kernicterus suffers from a lack of clarity and consistency in the key terms used to describe both the clinical and pathophysiological nature of these conditions. For example, the term Bilirubin-induced Neurological Dysfunction (BIND) has been used to refer to all neurological sequelae caused by exposure to high levels of bilirubin, to only mild neurological sequelae, or to scoring systems that quantitate the progressive stages of Acute Bilirubin Encephalopathy (ABE). OBJECTIVE: We seek to clarify and simplify terminology by introducing, defining, and proposing new terms and diagnostic criteria for kernicterus. METHODS: We propose a systematic nomenclature based on pathophysiological and clinical criteria, presenting a logical argument for each term. Acknowledging observations that kernicterus is symptomatically broad and diverse, we propose the use of the overarching term Kernicterus Spectrum Disorders (KSDs) to encompass all the neurological sequelae of bilirubin neurotoxicity including Acute Bilirubin Neurotoxicity (ABE). We further suggest subclassification of KSDs based on the principal disabling features of kernicterus (motor, auditory). Finally, we suggest the term subtle KSD to designate a child with a history of significant bilirubin neurotoxicity with mild or subtle developmental delays. RESULTS AND CONCLUSION: We conclude with a brief description of the limited treatments currently available for KSD, thereby underscoring the importance of further research. We believe that adopting a systematic nomenclature for the spectrum of clinical consequences of hyperbilirubinemia will help unify the field and promote more effective research in both prevention and treatment of KSDs.

Fate of Neural Progenitor Cells Transplanted Into Jaundiced and Nonjaundiced Rat Brains.

High levels of bilirubin in infants can cause kernicterus, which includes basal ganglia damage and dystonia. Stem cell transplantation may be an effective treatment for this disease. In this study, we transplanted human neural progenitor cells differentiated toward propriospinal interneurons into the striatum of 20-day-old spontaneously jaundiced (jj) Gunn rats and nonjaundiced (Nj) littermates. Using immunohistochemical methods, we found that grafted cells survived and grew fibers in jj and Nj brains 3 weeks after transplantation. Grafted cells had a higher survival rate in jj than in Nj brains, suggesting that slightly elevated bilirubin may protect graft survival due to its antioxidative and immunosuppressive effects. Despite their survival, only a small portion of grafted neurons expressed GAD-6 or ChAT, which mark GABAergic and cholinergic neurons, respectively, and are the cells that we are attempting to replace in kernicterus. Thus, NPCs containing large populations of GABAergic and cholinergic neurons should be used for further study in this field.

Biliverdin-induced brainstem auditory evoked potential abnormalities in the jaundiced Gunn rat.

Brainstem auditory evoked potential (BAEP) abnormalities occur in jaundiced Gunn rats given sulfadimethoxine to displace bilirubin bound to serum albumin, releasing it into the tissues. One problem with the model is that after displacement, plasma bilirubin levels drop and do not correlate with neurological dysfunction. In this report, we administered biliverdin, the immediate precursor of bilirubin, in 15- to 17-day-old Gunn rat pups to create an improved model of bilirubin-induced neurological dysfunction. Total plasma bilirubin (TB) levels were measured with a Leica bilirubinometer. Biliverdin (40 mg/kg) or phosphate-buffered saline (PBS) was administered either once and BAEPs recorded 8 h later or twice, 12 h apart, and BAEPs recorded 24 h after the initial injection. A single biliverdin injection produced a significantly decreased amplitude of BAEP wave III, 1.21+/-0.25 vs. 0.49+/-0.27 microV (control vs. biliverdin). The two-injection paradigm resulted in a significantly elevated TB (9.9+/-1.2 vs. 14.9+/-3.1 mg/dl; control vs. biliverdin), significant increases in I-II (1.15+/-0.08 vs. 1.42+/-0.09 ms) and I-III (2.17+/-0.08 vs. 2.5+/-0.13 ms) interwave intervals and a decrease in the amplitude of wave III (1.36+/-0.30 vs. 0.38+/-0.26 microV). Additionally, there were significant correlations between TB and the amplitude of wave III (r2=0.74) and TB and the I-III interwave interval (r2=0.51). In summary, biliverdin administration in jaundiced Gunn rat pups produces BAEP abnormalities consistent with those observed in the sulfadimethoxine model and human newborn hyperbilirubinemia and resulted in increased plasma bilirubin levels that correlate with the degree of neurological dysfunction.

Hyperbilirubinemia and kernicterus.

This article describes new findings concerning the basic science of bilirubin neurotoxicity, new considerations of the definition of clinical kernicterus, and new and useful tools to diagnose kernicterus in older children, and discusses treatments for kernicterus beyond the newborn period and why proper diagnosis is important.

A Novel Perspective on the Biology of Bilirubin in Health and Disease.

Unconjugated bilirubin (UCB) is known to be one of the most potent endogenous antioxidant substances. While hyperbilirubinemia has long been recognized as an ominous sign of liver dysfunction, recent data strongly indicate that mildly elevated bilirubin (BLB) levels can be protective against an array of diseases associated with increased oxidative stress. These clinical observations are supported by new discoveries relating to the role of BLB in immunosuppression and inhibition of protein phosphorylation, resulting in the modulation of intracellular signaling pathways in vascular biology and cancer, among others. Collectively, the evidence suggests that targeting BLB metabolism could be considered a potential therapeutic approach to ameliorate a variety of conditions.

A Hypothesis for Using Pathway Genetic Load Analysis for Understanding Complex Outcomes in Bilirubin Encephalopathy.

Genetic-based susceptibility to bilirubin neurotoxicity and chronic bilirubin encephalopathy (kernicterus) is still poorly understood. Neonatal jaundice affects 60-80% of newborns, and considerable effort goes into preventing this relatively benign condition from escalating into the development of kernicterus making the incidence of this potentially devastating condition very rare in more developed countries. The current understanding of the genetic background of kernicterus is largely comprised of mutations related to alterations of bilirubin production, elimination, or both. Less is known about mutations that may predispose or protect against CNS bilirubin neurotoxicity. The lack of a monogenetic source for this risk of bilirubin neurotoxicity suggests that disease progression is dependent upon an overall decrease in the functionality of one or more essential genetically controlled metabolic pathways. In other words, a "load" is placed on key pathways in the form of multiple genetic variants that combine to create a vulnerable phenotype. The idea of epistatic interactions creating a pathway genetic load (PGL) that affects the response to a specific insult has been previously reported as a PGL score. We hypothesize that the PGL score can be used to investigate whether increased susceptibility to bilirubin-induced CNS damage in neonates is due to a mutational load being placed on key genetic pathways important to the central nervous system's response to bilirubin neurotoxicity. We propose a modification of the PGL score method that replaces the use of a canonical pathway with custom gene lists organized into three tiers with descending levels of evidence combined with the utilization of single nucleotide polymorphism (SNP) causality prediction methods. The PGL score has the potential to explain the genetic background of complex bilirubin induced neurological disorders (BIND) such as kernicterus and could be the key to understanding ranges of outcome severity in complex diseases. We anticipate that this method could be useful for improving the care of jaundiced newborns through its use as an at-risk screen. Importantly, this method would also be useful in uncovering basic knowledge about this and other polygenetic diseases whose genetic source is difficult to discern through traditional means such as a genome-wide association study.