Congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS): Kindred disorders of autonomic regulation

Respiratory Physiology & Neurobiology 164 (2008) 38–48

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Congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS): Kindred disorders of autonomic regulation Debra E. Weese-Mayer a,∗ , Elizabeth M. Berry-Kravis b , Isabella Ceccherini d , Casey M. Rand c a Northwestern University Feinberg School of Medicine, Center for Autonomic Medicine in Pediatrics, Children’s Memorial Hospital, 2300 Children’s Plaza, Chicago, IL 60614, United States b Departments of Pediatrics, Neurological Sciences, and Biochemistry, Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, United States c Department of Pediatrics, Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, United States d Laboratory of Molecular Genetics, Istituto Giannina Gaslini, 16148 Genova, Italy

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Article history: Accepted 13 May 2008 Keywords: SIDS CCHS ANS dysregulation 5-HTT FEV PHOX2B SLC6A4

a b s t r a c t Congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS) were long considered rare disorders of respiratory control and more recently have been highlighted as part of a growing spectrum of disorders within the rubric of autonomic nervous system (ANS) dysregulation (ANSD). CCHS typically presents in the newborn period with a phenotype including alveolar hypoventilation, symptoms of ANSD and, in a subset of cases, Hirschsprung disease and later tumors of neural crest origin. Study of genes related to autonomic dysregulation and the embryologic origin of the neural crest led to the discovery of PHOX2B as the disease-defining gene for CCHS. Like CCHS, SIDS is thought to result from central deficits in control of breathing and ANSD, although SIDS risk is most likely defined by complex multifactorial genetic and environmental interactions. Some early genetic and neuropathological evidence is emerging to implicate serotonin systems in SIDS risk. The purpose of this article is to review the current understanding of the genetic basis for CCHS and SIDS, and discuss the impact of this information on clinical practice and future research directions. © 2008 Elsevier B.V. All rights reserved.

1. Introduction A comprehensive description of the genetic basis of respiratory control disorders was recently published, thus providing a thorough account of the field (Gaultier, editor, 2008). The aim of this publication is to focus the clinician and basic scientist on two disorders, typically considered to be pediatric in nature, and long identified primarily as “control of breathing” disorders: congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS). Though both CCHS and SIDS have been considered rare orphan diseases due to their relatively low incidence, more recently they have been highlighted as part of a growing spectrum of disorders within the rubric of autonomic nervous system (ANS) dysregulation (Axelrod et al., 2006). Study of genes related to physiologic and anatomic ANS dysregulation (ANSD) has led to successful identification of the genetic basis for CCHS. In contrast to CCHS, the genetic profile for the infant at risk for SIDS remains ill

∗ Corresponding author. Tel.: +1 773 880 8188; fax: +1 773 880 8100. E-mail addresses: [email protected] (D.E. Weese-Mayer), Elizabeth M [email protected] (E.M. Berry-Kravis), [email protected] (I. Ceccherini), Casey M [email protected] (C.M. Rand). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.05.011

defined and primarily at the stage of analysis of risk associated with candidate genes. The purpose of this article is to provide a comprehensive review of current research into the genetic basis for CCHS and SIDS. 2. Congenital central hypoventilation syndrome (CCHS) CCHS is a disorder of respiratory control with diffuse autonomic dysregulation (Weese-Mayer et al., 1999, 2001) that was first described in 1970 (Mellins et al., 1970), and in association with Hirschsprung disease and tumors of neural crest origin in 1978 (Haddad et al., 1978). The symptoms of ANSD include decreased heart rate variability (Woo et al., 1992; Ogawa et al., 1993; Silvestri et al., 2000; Trang et al., 2005a,b), an attenuated heart rate response to exercise (Silvestri et al., 1995), transient abrupt asystoles (Gronli et al., 2008), altered blood pressure homeostasis (Trang et al., 2003, 2005b), severe constipation (Weese-Mayer et al., 1992), esophageal dysmotility/dysphagia (Faure et al., 2002), decreased perception of discomfort, pupillary abnormalities (Weese-Mayer et al., 1992; Goldberg and Ludwig, 1996), decreased perception of anxiety (Pine et al., 1994), sporadic profuse sweating, and decreased basal body temperature among others. A characteristic boxy shaped facies has been described in children and young adults with CCHS, with faces

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that are generally shorter relative to the width and flatter, and with an inferior inflection of the lateral segment of vermillion border on the upper lip (Todd et al., 2006). Though it is now widely accepted that the paired-like homeobox protein (PHOX)2B is the disease-defining gene for CCHS, it is educational, particularly in terms of application to other less well understood disorders, to understand the steps that led to determining the relationship between PHOX2B and CCHS. 2.1. Familial recurrence data and rationale for genetic basis for CCHS A genetic component to CCHS was initially postulated based on familial recurrence identified in monozygotic female twins (Khalifa et al., 1988), sisters (Haddad et al., 1978), male–female sibs (WeeseMayer et al., 1993), and male–female half sibs (Hamilton and Bodurtha, 1989) with CCHS. Subsequently multiple cases of women diagnosed with CCHS in their own childhoods giving birth to infants with CCHS were reported (Silvestri et al., 2002; Sritippayawan et al., 2002). The association of CCHS with Hirschsprung disease and tumors of neural crest origin further indicated a genetic basis for the disease. Report of a child with CCHS born to a woman who had neuroblastoma as an infant (Devriendt et al., 2000) provided support for a transmitted genetic component affecting the phenotypic spectrum of ANSD and CCHS. 2.2. Genetic studies in CCHS Pursuit of the genetic basis for CCHS was initially limited due to the perceived rarity of the disease. Early research focused on genes studied in Hirschsprung disease. These studies identified twenty CCHS patients with unique protein-altering mutations in specific genes including: rearranged during transfection factor (RET) (Amiel et al., 1998; Sakai et al., 2001; Fitze et al., 2003; Sasaki et al., 2003), glial-derived neurotrophic factor (GDNF) (Amiel et al., 1998), endothelin-3 (EDN3) (Bolk et al., 1996b), brain-derived neurotrophic factor (BDNF) (Weese-Mayer et al., 2002), human aschaete-scute homolog-1 (HASH1) (de Pontual et al., 2003; Sasaki et al., 2003), paired-like homeobox 2A (PHOX2A) (Sasaki et al., 2003), GFRA1 (Sasaki et al., 2003), bone morphogenic protein-2 (BMP2) (Weese-Mayer et al., 2003a), and endothelin converting enzyme-1 (ECE1) (Weese-Mayer et al., 2003a). However, other reports indicated an absence of RET (Bolk et al., 1996a) and RNX mutations in CCHS (Matera et al., 2002; Amiel et al., 2003b) and none of these mutations could account for the majority of CCHS cases. 2.2.1. Association of PHOX2B and CCHS PHOX2B, located on chromosome 4p12, encodes a highly conserved homeobox domain transcription factor with 2 stable polyalanine (PA) repeats of 9 and 20 residues and is a key gene in ANS development with a role in early embryologic development as a transcriptional activator in promotion of pan-neuronal differentiation including upregulation of proneural genes, mammalian aschaete-scute homologue-1 (MASH1) expression and motoneural differentiation (Lo et al., 1998). PHOX2B has a separate role by a different pathway wherein it represses expression of inhibitors of neurogenesis (Lo et al., 1999). Further, PHOX2B is required to express tyrosine hydroxylase, dopamine beta hydroxylase (Hirsch et al., 1998), and RET, and to maintain MASH1, thereby regulating noradrenergic neuronal specification in vertebrates (Pattyn et al., 1999). These roles of PHOX2B early in the embryologic origin of the ANS with a role in determining the fate of early neuronal cells coupled with the known ANS dysfunction in CCHS led researchers to investigate PHOX2B in CCHS.

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Amiel et al. (2003a) identified an expansion of the 20 alanine repeat located in exon 3 of PHOX2B in 18 of 29 (62%) French CCHS cases. Expanded alleles identified in the study ranged from 25 to 29 repeats. They concluded that each of the expansions must have occurred de novo since the expansions varied in length and were not present on mutation screening in the eight sets of parents of the CCHS cases. In addition to the PA repeat expansions, they identified frameshift mutations in PHOX2B in 2 of 29 (7%) CCHS cases. None of these PHOX2B mutations were present in controls. In the same publication, Amiel et al. (2003a) demonstrated the presence of PHOX2B expression in early developmental human embryos in both central autonomic neuron circuits and in peripheral neural crest derivatives. Amiel and colleagues concluded that PHOX2B plays a central role in the development and/or function of the neuronal network involved in autonomic regulation of respiration and that mutations in PHOX2B may result in CCHS in a subset of cases. 2.2.2. PHOX2B is the disease-defining gene for CCHS Concurrent to studies by the Amiel group, Weese-Mayer et al. (2003a) selected a set of candidate genes involved in ANS development for mutation screening, including MASH1, BMP2, EN1, TLX3, ECE1, EDN1, and PHOX2A via direct DNA sequencing in a cohort of 67 CCHS and 67 matched controls. No novel disease-causing mutations were found in any of these genes; however, Weese-Mayer et al. (2003a) identified PHOX2B exon 3 PA repeat expansions in 65 of 67 (97%) primarily American CCHS probands. Of the two remaining cases a nonsense mutation (premature stop codon) in PHOX2B was identified in one patient and a PA expansion was later identified in the other case after repeat testing (Berry-Kravis et al., 2006). Collectively, Weese-Mayer et al. (2003a) identified mutations in exon 3 of the PHOX2B gene in 100% of 67 children with the CCHS phenotype, indicating that PHOX2B is the disease-defining gene in CCHS. They also identified a broader range for PA repeat mutation length in CCHS with expansions ranging from 25 to 33 repeats. To date more than 350 cases of CCHS have been confirmed with genetic testing in the U.S. using the clinical PHOX2B assay (Weese-Mayer et al., 2003a; Berry-Kravis et al., 2006; Weese-Mayer, Berry-Kravis, Zhou, Rand, personal communication) (Fig. 1). Subsequently, Matera et al. (2004) identified PHOX2B PA expansion mutations in 21 of 24 CCHS cases (88%) from Italy, Germany and the Netherlands with an expansion range of 25–33 repeats. They also identified two CCHS cases with heterozygous frameshift mutations (8% of cases). PHOX2B mutations were therefore identified in 96% of cases of CCHS. This study also reported that the technique described by Amiel et al. (2003a) failed to PCR amplify some alleles in the GC rich PA region of PHOX2B, resulting in underidentification of the number of CCHS cases with a PHOX2B mutation. Subsequently, the Amiel group (Trang et al., 2005a) (re)analyzed 34 CCHS patients, and identified a PHOX2B mutation in 91% of the cases and later (Trochet et al., 2005b) in 174 cases (92.6% identification). The PA expansion mutations were not found in any of 302 controls from the above-cited publications. These results further implicated PHOX2B as disease-defining in cases of CCHS. 2.3. PHOX2B PA expansion genotype-CCHS phenotype correlations The range for number of repeats in the PA expansion on the affected allele is 24–33 (Weese-Mayer et al., 2003a; Matera et al., 2004; Repetto et al., 2008). An association between increasing PHOX2B PA repeat expansion mutation length and increased severity of respiratory phenotype and autonomic dysfunction (number of ANSD symptoms) (Weese-Mayer et al., 2003a; Matera et al., 2004), length of longest R–R interval on Holter monitoring (Gronli et al., 2008), and the severity of the facial phenotype characteristic of

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Fig. 1. PHOX2B mutations in congenital central hypoventilation syndrome (CCHS) and sudden infant death syndrome (SIDS). Individuals with CCHS are heterozygous for mutations in the PHOX2B gene. CCHS-related mutations include polyalanine repeat expansion mutations in exon 3 (more than 90% of the cases) as well as non-polyalanine expansion mutations (missense, nonsense, and frameshift mutations) (less than 10% of the cases). Mutations shown are inclusive of all mutations reported in the literature as of early 2008. SIDS mutations include an intron 2 polymorphism and 8 discrete mutations in exon 3. The SIDS mutations have not been identified in CCHS subjects. However, it is anticipated that a subset of CCHS cases with the least number of repeats might die in infancy and will be misidentified as SIDS.

CCHS (Todd et al., 2006) has been established. Deletion variants with only 14 or 15 repeats in the PA repeat tract have been reported in three children with CCHS (Amiel et al., 2003a; Weese-Mayer et al., 2003a). 2.4. PHOX2B non-polyalanine repeat mutations in PHOX2B and relationship to CCHS phenotype Non-polyalanine repeat mutations (NPARMs) (Loghmanee et al., 2008) with significant effect on the PHOX2B protein have been reported in association with CCHS by groups in the U.S. (WeeseMayer et al., 2003a; Berry-Kravis et al., 2006; Raabe et al., 2008), Italy (Matera et al., 2004; Bachetti et al., 2005; Parodi et al., 2008), Japan (Sasaki et al., 2003), France (Amiel et al., 2003a; Trochet et al., 2005b), Germany (Hennewig et al., 2008), and Australia (Bajaj et al., 2005). Thus far, 43 individuals with CCHS and NPARMs in PHOX2B have been described worldwide, and mutations include mostly frameshift mutations (30/43, 70%), but also nonsense (2/43, 5%), missense (10/43, 23%), and missense with stop codon alteration (1/43, 2%). Recurrent 38 and 35 bp deletions causing frameshift beginning in the first codon of the PA repeat have been identified by several groups in different countries, suggesting a specific mutational mechanism. Most CCHS-associated mutations are found at the end of exon 2 or in exon 3. While most CCHS-associated frameshift mutations in PHOX2B are de novo, a few similarly located mutations (618delC, 577delG) have been inherited and are variably penetrant in families (Matera et al., 2004; Berry-Kravis et al., 2006), suggesting that −1 frameshifts in this area may produce a milder cellular deficit than other described frameshift mutations. Review of clinical information from subjects with NPARMs revealed a more severe phenotype with a much higher rate of Hirschsprung disease, higher frequency of 24 h ventilation and more frequent neural crest tumors, than observed in subject cohorts with the more common PA repeat mutations (Amiel et al., 2003a; Weese-Mayer et al., 2003a; Sasaki et al., 2003; Matera et al.,

2004; Trochet et al., 2005b; Berry-Kravis et al., 2006). This would suggest that, as a group, NPARMs produce more severe dysfunction of PHOX2B. Patients with a CCHS phenotype and no PA repeat expansion mutation, especially when there is severe Hirschsprung disease, are likely to be heterozygous for an alternative mutation in PHOX2B. 2.5. Inheritance of PHOX2B mutations and genetic counseling for CCHS families Most parents of affected children with CCHS do not carry a PHOX2B mutation at all, indicating a high de novo mutation rate in affected individuals, 5–10% of cases inherited mutations from an unaffected parent (Weese-Mayer et al., 2003a; Trochet et al., 2005b). Inherited mutations fall into two categories: germline mutations, inherited and present in all cells of the body may exhibit incomplete penetrance depending on their nature, and somatic mutations, occurring de novo in somatic cells after formation of the embryo and therefore present in only a fraction of cells in the body. These latter mutations result in mosaicism in the parent of the proband but are always fully penetrant when inherited in the germline. The 24 and 25 repeat expansions of the PA tract mutations and a few NPARMs can be found in the germline of asymptomatic parents and family members of children with CCHS, suggesting these mutations are inherited as dominant with incomplete penetrance (Matera et al., 2004; Weese-Mayer et al., 2005; Antic et al., 2006; Berry-Kravis et al., 2006; Repetto et al., 2008). These alleles are characterized by somewhat milder, although variable, phenotypic effects in the CCHS-affected children or other family members. Carriers of these alleles who do not have CCHS may show other ANSD phenotypes, including Hirschsprung disease or neuroblastoma (Devriendt et al., 2000; Berry-Kravis et al., 2006), or may present in adulthood or later in childhood with symptoms of obstructive and/or central sleep apnea, ventilatory insufficiency following a respiratory illness, or central apnea and

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prolonged recovery of alertness following sedation or anesthesia (Weese-Mayer et al., 2005; Diedrich et al., 2007). PHOX2B mutations in CCHS proband parents with somatic mosaicism include PA alleles larger than 25 repeats and non-PA mutations (Weese-Mayer et al., 2003a; Trochet et al., 2005b; BerryKravis et al., 2006; Parodi et al., 2008). Somatic mosaicism for an expanded PA PHOX2B allele was first reported by Weese-Mayer et al. (2003a) in 4 parents, whose children carried their same mutations, out of 54 available families (7.4%). Trochet et al. (2005b) subsequently identified somatic mosaicism in one parent of each of 10 CCHS patients, confirming that roughly 10% of probands will inherit the mutation from a mosaic parent. The percent of cells with the somatic mutation has been found to range from 9% to 35% in a quantitative assay in a subset of mosaic parents (Trochet et al., 2008). Further, no asymptomatic carrier of a 25 repeat allele was found to be a mosaic, confirming that in these cases lack of the disease phenotype can be ascribed to reduced penetrance of a germline mutation (Parodi et al., 2008; Trochet et al., 2008). Taken together these data allow the hypothesis that germline PA expansions larger than 25 repeats are fully penetrant and by extension, asymptomatic carriers of these alleles will always have significant degrees of somatic mosaicism. Further, neither CCHS nor the later presentation CCHS probands have shown so far any degree of somatic mosaicism, an observation which suggests a germline origin for expansion mutations in most of these patients, although it is possible that individuals mosaic for PA expansions may show symptoms of hypoventilation with more rigorous study. Genetic counseling is crucial for individuals diagnosed with CCHS, their parents and, in some cases, specific family members. For all affected individuals with CCHS, there is a 50% chance of transmitting the mutation and therefore the disease phenotype, to each child. If an unaffected parent is found to be mosaic for a PHOX2B mutation (usually identified because of an affected child), there will be up to a 50% chance of recurrence in any subsequent children. Mosaic individuals always can be assumed to have a new mutation (the mutation cannot be inherited in mosaic fashion) and therefore, there would not be a concern about family members other than children with CCHS being carriers of the mutation. On the other hand, if unaffected parents do carry a germline mutation (i.e. a 25 PA mutant allele) there may be numerous other family members who can carry the same mutation without having obvious symptoms. In this light, ALL parents of a CCHS proband should have genetic testing done to rule out mosaicism (PA repeat mutations of 26 or more, non-PA repeat mutations) or a non-penetrant carrier state (24 and 25 PA repeat mutations and non-PA repeat mutations). This will allow exclusion of recurrence risk in the family and also will alert the asymptomatic carrier parent of his or her own potential respiratory and anesthesia risks. Moreover, in the situation when an asymptomatic parent and a proband with CCHS carry a 24 or 25 repeat expanded allele (or a non-PA repeat mutation), the proband’s siblings, the parent’s siblings, and the grandparents on the side of the carrier parent should be tested, as these family members may be mutation carriers and have risk for hypoventilation and/or offspring with CCHS. Despite negative testing of the parents of a proband with CCHS, germline mosaicism cannot be ruled out and prenatal testing for subsequent pregnancies should be considered. Currently, the patented assay developed by Weese-Mayer et al. (2003a) is clinically available for the diagnosis of CCHS using a simple and accurate method for sizing the repeat sequence associated with the PA tract expansion (www.genetests.org) (assay patented; proceeds support CCHS research). This assay has applicability for the diagnosis of probands with CCHS and mosaic parents as well as prenatal diagnosis of subsequent siblings, offspring of mosaic parents, and offspring of CCHS probands. The assay also has appli-

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cability in diagnosing CCHS in adults with unexplained hypercarbia or control of breathing deficits (Weese-Mayer et al., 2005; Antic et al., 2006). 2.6. Pathogenetic mechanisms of PHOX2B mutations and the CCHS phenotype The finding that PHOX2B binds directly to the regulatory regions of the dopamine-␤-hydroxylase (D␤H), PHOX2A and TLX-2 genes (Borghini et al., 2006), has suggested an efficient functional approach to assay the molecular effects of CCHS-associated PHOX2B mutations. In particular, PHOX2B mutations have been tested so far for potential disruption of the normal function of the protein with respect to transactivation of different target promoters, DNA binding, aggregate formation, and subcellular localization. Distinct CCHS pathogenetic mechanisms for PHOX2B PA expansions and frameshift mutations have thus been postulated. In addition, the cellular response to PHOX2B PA expansions has been investigated to determine whether there exist cellular mechanisms that could be targeted to limit the cytotoxicity of these mutations. 2.6.1. PHOX2B PA repeat expansions The ability of expression constructs containing PA mutations to regulate the transcription of known target genes (DˇH and PHOX2A) has been compared, in two different laboratories, to a wild type PHOX2B construct, demonstrating a strict inverse correlation between the transactivating ability of PHOX2B constructs and the length of the PA tract (Bachetti et al., 2005; Trochet et al., 2005a). On the other hand, the same mutant PHOX2B versions had only a weak effect on the TLX-2 promoter (Borghini et al., 2006). Finally, a significant reduction of the transactivating activity of PHOX2B constructs bearing common PA contractions on the DˇH promoter has also been observed in a similar assay (Toyota et al., 2004). Unfortunately, this observation could not be replicated in a different cell system (Trochet et al., 2005a) suggesting the need for additional investigation before concluding that PA contractions may result in some disruption of PHOX2B function. Fluorescence microscopy has shown that the wild type PHOX2B protein is present almost exclusively in the nucleus. On the other hand, increasing proportions of cells characterized by a complete or partial cytoplasmic mislocalization of the protein and by the presence of large aggregates have been correlated with increasing PA expansion lengths. This suggests that impaired subcellular PHOX2B localization might result from the aggregation-prone effect of the PA expansion (Bachetti et al., 2005). Similar experiments performed in HeLa cells confirmed formation of PHOX2B mutant PA aggregates (Trochet et al., 2005a), providing further evidence that mislocalization of the mutant protein is a common pathogenetic mechanism leading to impaired transcriptional activity of mutant PHOX2B containing expanded alanine tracts. PHOX2B DNA binding is affected for PHOX2B proteins containing expansions of 29 alanines and above, which become unavailable for DNA binding due to spontaneous formation of oligomers (Trochet et al., 2005a). Finally, in addition to functional haploinsufficiency, PA mutations have been demonstrated to exert a partial dominant negative effect, blocking the normal protein from performing its usual function because of abnormal aggregation with the mutant protein (Bachetti et al., 2005; Trochet et al., 2005a). In the attempt to assess the fate of cells expressing PA expanded PHOX2B, in vitro experiments have demonstrated that activation of the heat shock response by the drug geldanamycin, a naturally occurring antibiotic, is efficient both in preventing formation and in inducing clearance of PHOX2B pre-formed PA aggregates and, ultimately, also in rescuing the PHOX2B ability to transactivate the D␤H promoter (Bachetti et al., 2005; Trochet et al., 2005a). In

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addition, elimination of PHOX2B mutant proteins by two cellular mechanisms, the proteasome and autophagy, already known to be involved in the clearance of poly-Q and poly-A aggregates, has been demonstrated (Bachetti et al., 2007).

be a weak paternal expansion bias which would require analysis of a larger sample of parent–child trios.

2.6.2. PHOX2B non-PA repeat mutations (NPARMs) Mutant PHOX2B proteins carrying either frameshift, missense or nonsense mutations have shown severely compromised transcriptional activation activity for the D␤H and TLX2 promoters, with more severe disruption of activity for frameshift mutations correlated with length of the disrupted C-terminal sequence (Bachetti et al., 2005; Trochet et al., 2005a; Borghini et al., 2006). Surprisingly, PHOX2B frameshift mutations have induced a 10–30% increased activation of the PHOX2A regulatory region (Bachetti et al., 2005). Moreover, similar to PA expansions larger than 9 residues, frameshift and missense mutations have mainly shown a complete loss of DNA binding, despite correct localization in the nucleus (Bachetti et al., 2005; Trochet et al., 2005a). Finally, a recent study has not only confirmed these findings but also shown that NPARM mutant PHOX2B constructs retained the ability to suppress cellular proliferation, without being able to promote differentiation, suggesting an explanation for the association of frameshift and missense PHOX2B mutations with risk of neuroblastoma, and therefore a mechanism which might promote development of neural crest tumors (Raabe et al., 2008).

SIDS is defined as the sudden and unexpected death of an infant less than one year of age, whose death remains unexplained despite a thorough autopsy, death scene investigation, and review of clinical history (Willinger et al., 1991). The 1992 “Back to Sleep” campaign identified modifiable environmental risk factors for SIDS and led to a decrease in SIDS incidence from 1.2/1000 live births (Kochanek, 1995) to 0.529/1000 live births in 2003 (Hoyert et al., 2006) in the U.S. Despite the success of the Back to Sleep campaign in the U.S., SIDS remains one of the most prevalent causes of infant mortality, with 2162 reported cases of SIDS in 2003 in the U.S. alone (Hoyert et al., 2006). Despite the decline in SIDS rates in the U.S., African American infants have a 2.7 fold higher SIDS rate than Caucasian infants (Hoyert et al., 2006). This ethnic disparity, coupled with SIDS deaths despite improved compliance with modifiable risk factors, led investigators to consider a genetic basis for SIDS. A candidate gene-based approach to identifying genetic factors responsible for SIDS susceptibility, similar to that used to establish PHOX2B as the disease-defining gene in CCHS, is underway. Genetic studies to date have been motivated by clinical, epidemiological, and/or neuropathological observations in SIDS victims, with subsequent pursuit of candidate genes in five categories: (1) genes for the serotonergic system based on decreased receptor binding in brainstems of SIDS victims, (2) genes pertinent to the early embryology of the ANS (linked to the 5-HT system) based on reports of ANS dysregulation in SIDS victims, (3) genes for nicotine metabolizing enzymes based on evidence of cigarette smoking as a modifiable risk factor for SIDS, (4) genes for ion channel proteins based on electrocardiographic evidence of prolonged QT intervals in SIDS victims, and (5) genes regulating inflammation, energy production, hypoglycemia, and thermal regulation based on reports of postnatal infection, low birth weight, and/or overheating in SIDS victims. In the following sections we will review the evidence for candidate genes pertinent to the first three of these categories, encompassing the serotonin system, the early embryology of the ANS, and nicotine metabolizing genes. For a more comprehensive review of the other genes studied in SIDS the reader is referred to a recent review article (Weese-Mayer et al., 2007).

2.7. Mechanism of PA expansion Poly-A tracts, predicted in roughly 500 human proteins, particularly transcription factor genes like PHOX2B, have already been found to be expanded in association with at least nine different congenital disorders, including mental retardation and malformations of the brain, digits and midline structures (Amiel et al., 2004). Thus PA expansions are members of a broader category of trinucleotide repeat-associated disorders that also includes polyglutamine (poly-Q) expansions. Unlike poly-Q tracts, poly-A stretches are generally stable, are usually coded by imperfect trinucleotide repeats and, with the exception of rare contractions, are not present as polymorphic tracts in the human population, thus suggesting an unequal allelic homologous recombination (cross-over) during meiosis and/or mitosis as the most attractive diseasecausing mechanism for poly-A tract expansions (Amiel et al., 2004). However, since in mosaic individuals only two alleles (wild type and expanded alleles), instead of the three alleles (wild type, contracted and expanded alleles) expected after occurrence of a somatic event of unequal crossing-over, have been reported, an alternative mutational mechanism must be considered to explain the origin of these trinucleotide repeat expansions (Parodi et al., 2008; Trochet et al., 2008). Indeed, by reasoning that imperfect trinucleotide repeat sequences, typical of poly-A tracts, would reduce the ability of the repeats to form misaligned structures and strengthen their stability, replication slippage has been proposed as a more plausible mechanism than unequal crossing-over for generation of poly-A tract expansions (Chen et al., 2005). More recently, segregation analysis of PHOX2B markers in four informative CCHS families has suggested the occurrence of unequal sister chromatid exchange at the base of PA expansions, either during gametogenesis or in post-zygotic somatic cells (Arai et al., 2007). In this latter study, a paternal origin of the gametes transmitting expansions has also been reported in six informative de novo CCHS trios, while in a larger cohort of 20 trios no significant parental expansion bias has been demonstrated, with 13 mutations occurring on the paternal and 7 on the maternal chromosome (Parodi et al., 2008). Thus, occurrence of poly-A repeats expansions may be independent from processes specific to sperm or oocyte development, or there may

3. Sudden infant death syndrome

3.1. Serotonergic system genes in SIDS 3.1.1. Rationale Panigrahy et al. (2000) demonstrated that SIDS infants displayed decreased serotonergic (5-HT) receptor binding in both the arcuate nucleus and the nucleus raphe´ obscurus, an area especially important for carbon dioxide chemoreception (Nattie and Li, 2001), of the medulla. Kinney et al. (2003) provided corroborating data in a group of Native American infants, a population at increased risk for SIDS. Ozawa and Okado (2002) demonstrated reduced expression of 5-HT receptor 1A (HTR1A) in medullary regions responsible for cardiorespiratory control among SIDS cases relative to controls. Paterson et al. (2006) subsequently reported quantitative discrepancies between several serotonergic system components in SIDS cases vs. controls, including an increased density and number of serotonergic neurons in the medulla of SIDS cases, specifically in areas important for homeostatic control. These results indicate a larger involvement of the 5-HT network, strengthen the hypothesis of serotonergic involvement in the pathogenesis of SIDS, and have motivated studies focused on genes involved in the serotonergic system.

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3.1.2. Serotonin transporter gene (SLC6A4, 5-HTT) Located at 17q11.1-q12 (Ramamoorthy et al., 1993), the serotonin transporter gene controls the duration and strength of interactions between 5-HT and its receptors by regulating membrane re-uptake of 5-HT from the extracellular space (Lesch et al., 1994; Heils et al., 1996; Fiskerstrand et al., 1999; MacKenzie and Quinn, 1999). Two polymorphisms in the 5 regulatory region of the 5-HTT gene differentially modulate gene expression: (1) an insertion–deletion in a repeat sequence in the promoter region and (2) a variable number tandem repeat (VNTR) sequence in intron 2. The most common alleles of the promoter polymorphism include the short allele (S) (14 copies of the 20–23 bp repeat unit) and the long allele (L) (16 copies). The L allele is a more effective promoter within cell transfection models (Heils et al., 1996). Subjects with the L/L genotype have an increased availability of raphe´ 5-HT transporters on in vivo neuroimaging studies (Heinz et al., 2000), as well as increased midbrain 5-HTT binding and 5-HTT mRNA levels in human postmortem brain (Little et al., 1998) (vs. individuals with at least one S allele). Intron 2 VNTR alleles contain 9, 10, or 12 copies of a 16–17 bp repeat sequence (Ogilvie et al., 1996), with the 12 repeat allele resulting in highest levels of expression of 5HTT in in vitro assays (Fiskerstrand et al., 1999) and transgenic mice (MacKenzie and Quinn, 1999). The 12 repeat construct was a stronger enhancer in differentiating embryonic stem cells, suggesting an effect on distribution and rate of transcriptional control (Fiskerstrand et al., 1999). Specific sequence variants within individual VNTR repeats also contribute to variability in transcriptional efficiency (Lovejoy et al., 2003), suggesting that both repeat copy number and the primary sequence of the repeat units play a role in tissue-specific 5-HTT expression. Other polymorphisms have been described, such as a 3 untranslated region (UTR) single nucleotide polymorphism (SNP) within a putative polyadenylation signal for 5-HTT mRNA, which may also contribute to variation in expression of this transporter in the CNS.

3.1.2.1. 5-HTT promoter region polymorphism in SIDS. Narita et al. (2001) demonstrated significant differences in genotype distribution and allele frequency of the 5-HTT promoter polymorphism between 27 Japanese SIDS cases and 115 age-matched controls, with an excess of the L/L genotype and L allele in the SIDS group relative to controls (7.4% vs. 1.7% for L/L; 22.2% vs. 13.5% for L allele). Weese-Mayer et al. (2003b) replicated this finding in an independent sample of 87 SIDS cases (43 African American, 44 Caucasian) and 87 gender/ethnicity-matched controls from the U.S., with significant differences in both genotype distribution and allele frequency in the combined dataset and for allele frequency in the Caucasian dataset. Specifically, there was an excess of the L/L genotype and the L allele in the SIDS group relative to controls (54.0% vs. 39.1% for L/L; 73.0% vs. 58.6% for L allele). Further, significantly fewer SIDS cases vs. controls with no L allele (S/S genotype) were reported in the entire cohort (8.0% vs. 21.8%) and within the Caucasian subgroup (13.6% vs. 34.1%). In addition to the case–control results, Weese-Mayer et al. (2003b) examined allele and genotype frequency differences by ethnicity in an additional set of 334 control subjects. The frequency of the long allele was increased in African Americans (73.9%) vs. Caucasians (53%). Weese-Mayer et al. concluded that the promoter polymorphism in 5-HTT may play an important role in SIDS risk and may explain, in part, the ethnic differences in SIDS risk. Specifically, SIDS rates are high among African Americans and low among Japanese, and the 5-HTT L allele frequency is high among African Americans (Heils et al., 1996; Du et al., 2000; Gelernter et al., 1998, 1999) and low among Japanese controls (13.5% in Narita et al. study).

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3.1.2.2. 5-HTT intron 2 VNTR in SIDS. Weese-Mayer et al. (2003c) studied the 5-HTT intron 2 VNTR genotype in a cohort of 90 pairs of SIDS cases and gender/ethnicity-matched controls (46 Caucasian, 44 African American). Genotype distribution, allele frequency for the 12 repeat allele, and frequency of the 12/12 genotype differed significantly between African American SIDS cases and controls. Similar to the promoter variant, there was a higher frequency of the 12-allele in the African American population, in both cases and controls. Significant associations between SIDS and the combined “promoter L/L or L/S and the intron 2 VNTR 12/12” genotype were identified in the total dataset and the African American subgroup, but not the Caucasian subgroup (Weese-Mayer et al., 2003c). Finally, the L/12 haplotype (on the same chromosome) was significantly more frequent in SIDS cases in the overall cohort, but this was driven by the significant association of this haplotype with SIDS in the African American group, while the haplotype was not increased in Caucasian SIDS cases vs. controls. These studies established an association between SIDS and the 12 repeat allele of the intron 2 VNTR and the L-12 haplotype in the African American subgroup. 3.1.2.3. 5-HTT 3 untranslated region in SIDS. The 3 UTR SNP in a putative polyadenylation site (Battersby et al., 1999) was not found to be associated with SIDS (Maher et al., 2006) in 92 pairs of gender/ethnicity-matched SIDS cases and controls, nor in the Caucasian or African American subgroups. This SNP has not clearly been associated with functional changes in 5-HTT expressions, suggesting that associations between 5-HTT and SIDS may only be found for polymorphisms associated with increased expression of 5-HTT. 3.1.3. 5-HT FEV gene Located on chromosome 2, the Fifth Ewing Variant (FEV) gene is the human homologue of the mouse Pet1 gene. FEV and Pet1 (Hendricks et al., 1999, 2003; Iyo et al., 2005; Maurer et al., 2004; Peter et al., 1997; Pfaar et al., 2002) were identified among the Sonic hedgehog (Shh)-regulated series of transcription factors working in concert to specify a 5-HT neuronal phenotype, with a necessary role for terminal induction of 5-HT neurons. Pet1 is expressed in the central 5-HT system, its onset of expression consistently precedes the appearance of 5-HT by 0.5 days (Hendricks et al., 1999), it establishes and maintains the 5-HT phenotype, it is the only known transcription factor whose brain expression is limited to developing and adult 5-HT neurons, and in the Pet-1 knockout mouse nearly all 5-HT neurons fail to differentiate and the remaining neurons exhibit deficient expression of genes required for 5-HT synthesis, uptake and vesicular storage (Hendricks et al., 2003). Pet1 interacts with the regulatory regions of genes whose expression is characteristic of the 5-HT phenotype including the 5-HTT, 5-HT receptor 1A (HTR1A), tryptophan hydroxylase and aromatic l-amino acid decarboxylase genes (Hendricks et al., 1999). Based on its sequence similarity to the predicted mouse Pet1 gene (Peter et al., 1997; Pfaar et al., 2002) and its similar restricted expression pattern in the 5-HT system (Maurer et al., 2004; Iyo et al., 2005), a similar function for the human FEV gene in the differentiation and development of the human 5-HT neuronal phenotype is anticipated. Recently a 1.8 kb 5 region immediately upstream of the Pet1 coding region was demonstrated to be necessary and sufficient to cause 5-HT neuron-specific expression of Pet1 (Scott et al., 2005). This region shows 70–90% sequence identity with the human FEV gene and is therefore expected to play a similar role of specifying FEV expression to 5-HT neurons in the human brain. Because FEV specifically interacts with the 5-HTT promoter, already implicated in SIDS risk, and has been predicted to play an integral role in 5-HT neuronal differentiation, FEV was

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hypothesized to be a likely candidate gene for contribution to SIDS etiology. 3.1.3.1. FEV intron 2 mutation in SIDS. Rand et al. (2007) reported a previously unidentified heterozygous insertion mutation (IVS2191 190insA) in intron 2 of the FEV gene, 190 bp upstream of the 5 exon 3 splice site, in 6 of 96 SIDS cases, while the mutation was not found in 96 gender- and ethnicity-matched controls. All individuals with the mutation were African Americans. No other variants in FEV identified in this cohort were specifically associated with SIDS although African Americans had generally more polymorphism at all sites. Though the IVS2-191 190insA SIDS-specific mutation is located outside of the amino acid coding region and does not appear to be involved in splicing, it may have a regulatory role alone or in combination with variations identified in 5-HTT or other, yet unidentified, variations in genes involved in 5-HT system development. Studies of this mutation in a larger cohort of SIDS cases and matched controls to assess the effect of interaction between variants in serotonergic system genes on SIDS risk are indicated, as are functional analyses to ascertain the possible pathogenetic mechanism associated with the mutation. 3.1.4. 5-HT receptor 1A (HTR1A) gene Variation of the HTR1A receptor density in SIDS cases was first identified when decreased 5-HT receptor 1A and 2A immunoreactivity in the dorsal nucleus of the vagus, the solitary nucleus, and the ventrolateral medulla of the medulla oblongata was observed in SIDS cases compared to controls (Ozawa and Okado, 2002). Recently, expression of the HTR1A receptor was found to be significantly decreased in SIDS cases, especially in male infants (Paterson et al., 2006). The HTR1A receptor is key to the autonomic response to homeostatic stress, and acts as an inhibitory somatodendritic autoreceptor densely distributed in medullary regions pertinent to cardiorespiratory regulation (Thor et al., 1992). Stimulation of the HTR1A receptor in these regions has been found to reduce the ventilatory response to hypercarbia, cause fragmented sleep characterized by a decrease in body temperature, heart rate, body movements, and rapid eye movements, and diminish the normal cardiovascular response brought on by acute psychological and inflammatory stress (Taylor et al., 2005; Darnall et al., 2005; Nalivaiko et al., 2005; Messier et al., 2004). Six variations within the coding region of HTR1A were identified in a cohort of 96 SIDS cases and 96 gender- and ethnicity-matched controls (Morley et al., 2008). There was no association of SIDS with allele frequency or genotype distribution for any of these variants, although variation was noted to be greater in males in both African American and Caucasian subgroups, for both SIDS subjects and controls. 3.2. Autonomic nervous system (ANS) genes in SIDS 3.2.1. Rationale Symptoms compatible with ANSD reported in SIDS include profuse sweating, elevated body temperature, tachycardia then bradycardia preceding the terminal event, reduced heart rate variability, drenching sweats and facial pallor, and decreased responses to obstructive sleep events (Kahn et al., 1992; Fleming et al., 1990; Ponsonby et al., 1992; Meny et al., 1994; Schechtman et al., 1988; Ledwidge et al., 1998; Taylor et al., 1996; Franco et al., 1999). Accordingly, genes pertinent to the early embryology of the ANS were considered to potentially confer SIDS risk. PHOX2B, the disease-defining gene for CCHS (Amiel et al., 2003a; Weese-Mayer et al., 2003a; Sasaki et al., 2003; Matera et al., 2004; Trochet et al., 2005b; Berry-Kravis et al., 2006), is a key gene in ANS development with a role in early embryologic development (see CCHS section above). Recent studies indicate that PHOX2B plays

a regulatory role in the selection between motor neuron or serotonergic neuronal fate in the development of the central nervous system (Pattyn et al., 2003, 2004). Loss of function experiments in mice have shown that for the transition from motor neuron production to 5-HT neuron production to commence, downregulation of PHOX2B is required (Pattyn et al., 2003). 3.2.2. ANS genes in SIDS DNA from 92 SIDS cases and from 26 of the 92 matched controls was sequenced for exon and splice site mutations in bone morphogenic protein-2 (BMP2), mammalian achaete-scute homolog-1 (MASH1), PHOX2A, RET, ECE1, endothelin-1 (EDN1), T-cell leukemia homeobox protein (TLX3), and engrailed-1 (EN1) (Weese-Mayer et al., 2004). Any base change expected to affect a splice site or result in modification of the protein sequence identified in SIDS subjects or controls was further screened in all 92 controls. Sequence data from PHOX2A, RET, ECE1, TLX3, and EN1 revealed 11 rare proteinchanging polymorphisms in 14 SIDS cases (15.2% of SIDS cases) and subsequent genotyping for these polymorphisms in controls identified 1 polymorphism in 2 controls (2.2% of controls). Each mutation occurred in 1 SIDS case with the exception of the TLX3 base change that occurred in 4 SIDS cases and 2 controls. African American infants accounted for 10 of the SIDS cases and the 2 controls with protein-changing mutations. Four common protein-changing polymorphisms were identified in BMP2, RET, ECE1, and EDN1, though allele frequencies did not differ between SIDS cases and controls. However, allele frequencies for the BMP2 common polymorphism differed significantly between Caucasian and African American infants. Among controls the allele frequencies for the BMP2 and ECE1 polymorphisms differed significantly between Caucasian and African American infants. 3.2.3. PHOX2B gene in SIDS Based on the established relationship between SIDS, 5-HTT, and ANS dysregulation coupled with the recognized role of PHOX2B in ANS and 5-HT system development, Weese-Mayer et al. (2004) studied a cohort of 91 SIDS cases and 91 matched controls for the PHOX2B PA expansion mutation characteristic of CCHS. None of the study subjects demonstrated the PHOX2B PA mutation. Subsequently, Rand et al. (2006a) sequenced the coding regions and intron–exon boundaries of PHOX2B in the same SIDS/control cohort, and identified a common polymorphism (IVS2+101A>G; g.1364A>G) in intron 2 of the PHOX2B gene located 100 bp downstream of the exon 2 splice site. The frequency of subjects carrying the variant G allele (genotype GG or GA) of this polymorphism was significantly higher in the SIDS group than in the matched control group, and also higher in Caucasian SIDS cases than in matched control subjects. Eight polymorphisms located in the third exon of the PHOX2B gene occurred significantly more frequently among SIDS cases (34 occurrences) than controls (19 occurrences). Frequency of these variants was higher in both Caucasian and African American SIDS subgroups. Two of the 8 polymorphisms identified were protein-altering missense mutations (F153L and S176T), occurring in 9 SIDS cases and 4 controls (10% and 4%, respectively) (Fig. 1). Kijima et al. (2004) also sequenced the PHOX2B gene in 23 Japanese SIDS cases and 50 controls and identified 1 polymorphism in exon 2 of PHOX2B and 2 intron 2 polymorphisms, none of which were identified in the Rand et al. (2006a) study. These polymorphisms were identified in 1%, 1%, and 9% of subjects, respectively, but the authors do not clarify if these were identified in SIDS cases or controls. Conversely, none of the PHOX2B exon 3 polymorphisms that Rand et al. (2006a) described in the Caucasian and African Americans were reported in the Japanese cases.

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3.3. Nicotine metabolizing genes in SIDS 3.3.1. Rationale Exposure to tobacco, both prenatal as well as postnatal, has been identified as a key risk factor in the etiology of SIDS (Anderson and Cook, 1997; Blair et al., 1996; Brooke et al., 1997; MacDorman et al., 1997; Mitchell et al., 1997). A relationship between tobacco exposure and altered ANS function has long been recognized for adults with both chronic and acute (Kotamaki, 1995; Lucini et al., 1996; Niedermaier et al., 1993; Piha, 1994; Pope et al., 2001) exposure and more recently for infants exposed to smoke prenatally (Franco et al., 2000). Based on these relationships between SIDS, tobacco exposure, and ANS dysregulation, genes involved in nicotine metabolism were identified as possible candidate genes for further study of the genetic basis for SIDS. The ability to convert toxic metabolites in cigarette smoke to less harmful compounds is key to minimizing the adverse health effects of exposure to tobacco. Polycyclic aromatic hydrocarbons (PAHs), some of the most important carcinogens in cigarette smoke, are metabolized through a two-stage process. Cytochrome P-450 1A1 (CYP1A1) encodes aryl hydrocarbon hydroxylase, a major enzyme responsible for phase 1 metabolism of PAHs. GST theta 1 GSTT1 is encoded by the GSTT1 gene, and is a major enzyme in phase 2 of cigarette smoke metabolism (Hayashi et al., 1992; Nakachi et al., 1993; Bartsch et al., 2000). Polymorphisms in both the CYP1A1 and GSTT1 genes (Ishibe et al., 1997; Bartsch et al., 2000), have been reported to impact the metabolic detoxification process for cigarette smoke. Thus, expression of polymorphisms in these genes have been associated with low birth weight (Wang et al., 2002), and may account for the varying susceptibility to other adverse health consequences of cigarette smoke exposure, including SIDS. 3.3.2. CYP1A1 and GSTT1 in SIDS Rand et al. (2006b) reported on frequency of known CYP1A1 and GSTT1 polymorphisms in 106 SIDS cases and 106 gender- and ethnicity-matched control subjects. The frequency of the GSTT1 homozygous deletion genotype did not differ between SIDS cases (22/106; 20%) and matched controls (32/106; 30%) in either the complete sample or the Caucasian or African American subgroups. Likewise, no association between SIDS and CYP1A1 was observed for genotype distribution or allele frequencies at any of three CYP1A1 polymorphisms, or when multiple CYP1A1 polymorphisms were considered in combination, nor was there any association of SIDS with containing both the GSTT1 deletion genotype and a CYP1A1 polymorphism. 3.4. Genetics of SIDS summary and significance The 5-HTT promoter variant L allele and VNTR 12 repeat alleles present the most robust genetic association with SIDS to date. As these alleles both constitute the more effective promoters (Fiskerstrand et al., 1999; Heils et al., 1996) and both are associated with increased expression of 5-HTT transporters in various brain regions, synaptic serotonin levels would be expected to be lower in those infants with a long or 12 repeat allele, and perhaps lowest in those with both variants. Increased prevalence of the more effective promoter alleles in SIDS cases would suggest that lower synaptic serotonin levels are associated with SIDS risk, perhaps resulting in alterations in serotonergic receptor binding observed in SIDS brain through a developmental effect on serotonergic neurons or networks in the raphe´ and elsewhere. The serotonin transporter is expressed early in ontogenesis in the mouse and the rat (Zhou et al., 2000), and may influence serotonin synapse formation, ¨ serotonin-dependent patterning of neuronal networks (Bruning et al., 1997), and serotonin-dependent cranial neural crest migration

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(Moiseiwitsch and Lauder, 1995). The 5-HTT short allele has been associated with anxiety, phobias, and an increased fear response (Lesch et al., 1996; Du et al., 2000; Hariri et al., 2002; Hu et al., 2000; Katsuragi et al., 1999), and one might also postulate that SIDS would be less likely in infants with the S/S genotype due to an exaggerated fear response and increased arousability. In any case it appears that SIDS risk is specifically related to functional variants that increase expression of 5-HT transporter protein and future investigations on the influence of 5-HTT on SIDS risk should focus on polymorphisms which directly impact regulation of transporter protein expression or function. The finding of higher frequency of protein-changing mutations in conserved residues of genes that regulate ANS development (Weese-Mayer et al., 2004) including PHOX2A, RET, ECE1, TLX3, and EN1 suggests that polymorphisms in these genes may confer some SIDS risk. The greatest number of rare mutations was identified in the RET gene. This is of particular interest because of the relationship of RET to Hirschsprung disease and to CCHS (both diseases of neural crest origin), and because of the RET knock-out model with a depressed ventilatory response to inhaled carbon dioxide with decreased frequency and tidal volume (Burton et al., 1997). The observation that none of the SIDS cases demonstrated the PHOX2B PA expansion mutation previously identified in CCHS indicates less specific overlap between the two diseases than previously considered. However, as families of CCHS probands have a higher incidence of SIDS history in a family member (Weese-Mayer et al., 1993), and as the anticipated incidence of this PHOX2B mutation is low in the general population, a very large sample size would be needed to detect a case. Therefore, it is still appropriate to evaluate infants with SIDS for the CCHS PHOX2B mutation in order to ascertain that CCHS was not the cause of death. However, polymorphisms in PHOX2B that do not cause CCHS are more common in SIDS cases (Rand et al., 2006a) (Fig. 1) and may confer SIDS risk independently or when present in combination with other mutations of genes in the pathway for specification of ANS neurons (e.g. RET), consistent with the finding of a possible interaction between polymorphisms in PHOX2B and RET in mediating SIDS risk, and suggesting that genetic changes at multiple points in the pathway could combine to amplify risk. Although PHOX2B plays a key role in the differentiation of central 5-HT neurons (Pattyn et al., 2003, 2004), the absence of significant interactions between PHOX2B and 5-HTT polymorphisms suggests the two genes exert independent effects on SIDS risk, potentially by acting on different aspects of 5-HT system function. The identification of polymorphisms in genes pertinent to the embryologic origin of the ANS in SIDS cases lends support to the overriding hypothesis that infants who succumb to SIDS have an underlying genetic predisposition. The low rate of occurrence of mutations in ANS genes studied suggests that there are yet unidentified genes that are responsible for the SIDS phenotype, either directly or in conjunction with the polymorphisms identified in PHOX2B, RET, 5-HTT, FEV and/or other genes involved in ANS or 5HT system development. Sequencing of additional genes involved in ANS or 5-HT development in a larger group of SIDS cases will be expected to yield insight into the relationship between PHOX2B, additional candidate genes, SIDS, and CCHS.

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