- Email: [email protected]
Medico-legal investigation in an explicable case of congenital central hypoventilation syndrome due to a rare variant of the PHOX2B gene
Journal of Forensic and Legal Medicine 58 (2018) 1–5
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine journal homepage: www.elsevier.com/locate/yjflm
Research Paper
Medico-legal investigation in an explicable case of congenital central hypoventilation syndrome due to a rare variant of the PHOX2B gene
T
Francesco Venturaa,∗, Rosario Barrancoa, Tiziana Bachettib, Paolo Nozzac, Ezio Fulcherid,e, Antonella Palmierif, Isabella Ceccherinib a
Department of Forensic and Legal Medicine, University of Genova, Via De’ Toni 12, 16132 Genova, Italy U.O.C. Medical Genetics, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy c U.O.C. Clinical Pathology, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy d U.O.S.D. Fetal and Perinatal Pathology, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy e School of Medicine, University of Genova, Italy f Department of Pediatric Emergency - Sudden Infant Death Syndrome Liguria Centre, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Congenital central hypoventilation syndrome PHOX2B gene polyAlanine contractions
The heterozygous PHOX2B gene mutation is related to congenital central hypoventilation syndrome (CCHS). It is characterized by defective autonomous nervous system development leading to inadequate breathing response to hypoxia and hypercapnia, leading to hypoventilation especially during non-REM sleep, but also during waking in the more severe cases. Herein we report a case of sudden death in a 28-day-old child. The mother reported the infant was found lying on her own bed in the prone position. The infant was wearing a romper and lying in her crib without any blanket or other objects. At autopsy no significant pathological findings were detected. Histologically, sparse aspirated milk residues were present in some lung fields. Toxicological and microbiological examinations were within the norm. The initial postmortem investigation ruled out any readily identifiable cause of death. However, genetic analysis revealed a rare heterozygous 21bp in-frame deletion of the polyalanine coding sequences of the PHOX2B gene. In-frame contractions of the poly-Ala tract of the PHOX2B gene have already been reported in patients with symptoms suggestive of sporadic hypoventilation, apparent life-threatening events or neonatal respiratory distress.
1. Introduction Congenital central hypoventilation syndrome (CCHS) is a potentially fatal condition compromising the regulation of respiratory and autonomic nervous system functions. It presents in new-borns and, rarely, as a milder delayed-onset form in toddlers, children, and even adults.1,2 It has been classified within a distinct and burgeoning nosological category of somewhat rare disorders labelled respiratory and autonomic disorders of infancy, childhood, and adulthood.3 The incidence is estimated to be at 1 of 200 000 livebirths4 and in fatal cases children usually succumb at night, only to be discovered dead the following morning. The forensic investigation of sudden death of infants is extremely challenging. According to respected authors, at least three different scenarios may present after thorough investigation: a first, lacking any prior reported symptoms or significant pathological findings, ∗
constitutes a death of unknown aetiology; a second (symptomatic) group, with the presence of symptoms and/or an underlying disease state, whose severity, nonetheless, by no means appears of such a degree as to justify death. Between these extremes there is a another “borderline” group with only minor or intermediate pathological findings, however deemed unlikely to suffice as the sole cause of death.5 CCHS is a diagnosis of exclusion and is mainly associated with mutations in the paired-like homeobox (PHOX2B) gene, which plays a role in controlling the autonomic regulation of breathing. Herein we report a presumed case of central hypoventilation characterized by a rare PHOX2B gene mutation, confirming the genetic predisposition to the disease as well as highlighting the value of genetic analysis in the post-mortem differential diagnosis of sudden infant death.
Corresponding author. E-mail address: [email protected] (F. Ventura).
https://doi.org/10.1016/j.jflm.2018.04.009 Received 23 June 2017; Received in revised form 9 April 2018; Accepted 13 April 2018 Available online 17 April 2018 1752-928X/ © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Journal of Forensic and Legal Medicine 58 (2018) 1–5
F. Ventura et al.
2. Materials and methods In 2010, the Liguria Region (Italy) officially sanctioned the institution of the Unexpected Sudden Infant Death Liguria Centre, Pediatric Emergency Department, Giannina Gaslini Children's Hospital in Genoa, Italy. As part of its research activities, all cases of death within the first year of life are directed to the regional centre for postmortem study aimed at identifying the causes of death. The Centre is coordinated by an Emergency/Urgency unit, comprising a team of experts (pediatrician, clinical pathologist, forensic pathologist, geneticist, cardiologist, specialist in metabolic disorders, microbiologists). Within the centre, an interdisciplinary commission was established to create a post-mortem pathway for the patient with suspected sudden infant death syndrome, as recognized by international experiences and literature data, coordinated by the Director of the centre. Multicentre analysis provides a comprehensive autopsy, histological, toxicological, microbiological and genetic evaluation.
Fig. 1. Histological Findings: lung specimens with a few foreign material (milk) associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. H&E x10, x40.
3. Case history A.K.V. (female, 28 days old, of Ecuadorian descent) was found lifeless after spending the night with her family at the home of friends. The mother reported the infant was found lying in her own crib in a prone position, wearing a romper and without any blanket or other objects. After prolonged and unsuccessful cardiopulmonary resuscitation (CPR), the infant was declared dead. Third-born of healthy parents, she was born at term after an uneventful pregnancy and delivery. The neonatal period was also routine with no noteworthy episodes of infection, rhinitis or cough, anomalous bowel discharge and/or vomiting. Moreover, parents reported normal growth and efficient suckling, with breast milk added to feeding formula. In the family history, a maternal ancestor had died from stroke at age 38 and a maternal aunt had a history of ill-described cardiac surgery at age 51. The siblings, of 2 and 5 years of age, were reportedly healthy.
Fig. 2. Histological Findings: lung specimens with a few foreign material (milk) associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. H&E x10, x40.
4. Autopsy, toxicological and microbiological findings
this or another variant in strict Linkage Disequilibrium (LD) may play a predisposing role in the occurrence of life threatening events. According to our Institute's protocol, to assess the involvement of PHOX2B defects in sudden intra-uterine and infant deaths, direct DNA sequencing of both strands of the amplification products corresponding to coding exons and flanking intronic regions of the PHOX2B gene (GenBank NM_003924.3) was applied to the victim's DNA specimens. A heterozygous 21bp in-frame deletion of the poly-Alanine stretch of the PHOX2B exon 3 (c.754-774del21), corresponding to a contraction of 7 alanine residues in the protein, was detected. Moreover, by coupling exon 3 amplification with FAM-tagged primers and capillary electrophoresis, the -7Ala contraction was confirmed in the victim's and excluded in mother's DNA (Fig. 3). Unfortunately, the father and his older brothers had returned to their native country, so their DNA was not available for analysis.
At autopsy the following findings were revealed: development of visceral organs compatible with age and absence of any abnormalities or malformations. The stomach and the proximal small bowel contained curdled milky contents. No noteworthy pathological findings or malformations of its parenchyma. Macroscopically, the organs revealed no malformations or abnormalities, and no further pathological findings were observed. Histopathological examination (Figs. 1–2) revealed foreign material (milk) present in some lung fields associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. The central nervous system showed normal development and myelination. There was no evidence of malformation nor signs of hypoxia/ischemia or haemorrhage. Toxicological investigations ruled out the presence of any drug or substance. Microbiological examinations were normal and excluded existing infectious disease processes.
6. Discussion
5. Genetic findings
CCHS was first described by Mellins et al.6 and is characterized by defective autonomous nervous system development leading to inadequate breathing response to hypoxia and hypercapnia, responsible for hypoventilation, especially during non-REM sleep, but also during waking hours in more severe cases.7 In 20% of cases this disease is associated with Hirschsprung disease and in another 5–10% there is also a tumour of autonomic neural crest derivatives, such as a ganglioneuroma or neuroblastoma.8 Patients with congenital central hypoventilation syndrome present a
The protocol involving the mutation screening of the PHOX2B gene in cases with sudden unexpected infant death has been applied for a few years in our Institute. Seven DNA samples have been tested so far and in addition to the present one, showing a PHOX2B polyAla contraction, two others have resulted to carry the variant allele of a rare synonymous polymorphism S184S (rs17885216, MAF = 0.01; https://www. ncbi.nlm.nih.gov/projects/SNP). This raises the question of whether 2
Journal of Forensic and Legal Medicine 58 (2018) 1–5
F. Ventura et al.
gene promoter target transcription are more closely associated with increased poly-Alanine repeats.12 In a smaller subset of cases (less than 10%), instead of exhibiting poly-Alanine expansions, other mutations in the PHOX2B gene are present, such as frameshift, missense, nonsense, and stop codon mutations, are present.10 Deletions have also been identified as causes of CCHS in a small subgroup of patients.3,13 In all these latter cases the phenotypic spectrum is extremely variable and sometimes even far worse.3 On the other hand, subjects with poly-Alanine tracts of decreased repeat lengths may survive into adulthood without manifesting the early acute respiratory failure typically associated with the CCHS phenotype. Indeed, poly-Alanine contractions represent a rare mutation that can also be found in seemingly healthy subjects.14,15 In CCHS, a functional analysis using Magnetic Resonance Imaging,16 reported alterations of signal intensity in the thalamus, mesencephalus, medial pons as well as the region spanning from the insular cortex to the hippocampus. This pathology involves a disorder of the autonomic nervous system. An abnormality of central integration of neuronal signals on the part of the central chemokines is the current hypothesis. Symptoms include apnoea and cyanosis, in the absence of cardiac, pulmonary, neuromuscular, or brainstem malformations.1 Affected infants show blunted physiological increases in rate or effort of breathing and, without a timely diagnosis, may develop right heart enlargement and pulmonary hypertension in response to chronic hypercapnia.13 In children with CCHS there is an increase in the incidence of arrhythmic episodes, mainly sinus bradycardia, and blood pressure values are lower during the waking and higher during sleep, indicating an attenuation of the physiological drop in sleeping blood pressures.17 Moreover, perception of asphyxia is absent.18 The diagnosis of CCHS is clinical, confirmed by molecular genetics. In most cases it is diagnosed within 48 h of birth, following an episode of cyanosis, hypercapnia and O2 desaturation during sleep. Sometimes, diagnosis is made in the wake of asphyxiation resulting in irreversible brain damage or death.13 There is evidence supporting the involvement of PHOX2B defects in a wider spectrum of disease phenotypes, all sharing an impaired autonomous nervous system functioning.11 In one of these studies, the entire PHOX2B gene was investigated in 23 Japanese and 91 AfricanAmerican and Caucasian sudden infant death cases, finding no PHOX2B mutation but only polymorphic alleles, whose overall frequency however turned out to be statistically higher than in matched controls.9,19 Furthermore, a molecular analysis of genes, previously implicated in the serotonin household or in CCHS, carried out in 195 Dutch USID (Unclassified Sudden Infant Death)/SIDS cases and 846 healthy controls confirmed a role of common PHOX2B polyalanine contraction alleles, resulted to be more frequent in cases than controls.20 In-frame contractions, carrying only 7, 13, 14 or 15 Ala-residues at the poly-Ala tract of the PHOX2B gene, have already been reported i) in CCHS patients, in association with additional causative mutations, ii) in about 3% of the general population, iii) in a small proportion of CCHS patients' parents and, iv) in patients with symptoms suggestive of sporadic hypoventilation, apparent life-threatening events or neonatal respiratory distress.21,22 These latter observations support a possible different effect of the PHOX2B contractions, which may manifest under specific circumstances and/or with certain genetic backgrounds. Consequently, the PHOX2B variant thus found, has had, with all likelihood, a decisive role in the alteration of the ventilation of the child and in originating the hypoventilation mechanism. Therefore, the PHOX2B variant in the present case could have had a role in worsening the effects of some other predisposing environmental factors. Despite only one parent being available for the analysis, the absence of the PHOX2B contractions from mother's DNA is consistent with, and
Fig. 3. Genetic analysis: peak scanner profiles obtained after capillary electrophoresis of FAM-conjugated PCR products corresponding to the polyAla stretch region of the PHOX2B exon 3. From top to bottom, the following peak scanner profiles are reported: (1) a standard control coming from a healthy individual, (2) the mother, turned out to carry wild type PHOX2B allele, (3) the victim, heterozygous for the 7Ala contracted PHOX2B allele (c.754-774del21), and (4) a control without DNA template (blank).
mutation of the PHOX2B gene.9 This maps onto the 4p12 chromosome and encodes a transcriptional factor of 314 amino acids with two short and stable poly-Alanine repeat expansions of 9 and 20 residues.8 Its mutation entails either autosomal dominant transmission or de novo occurrence in the first generation of polyalanine expansion mutations (PARM) and other less frequent non-polyalanine mutations (NPARM).8 In addition, in about 10% of cases of CCHS, an asymptomatic parent shows mosaicism for the mutation found in the child.10 In particular, heterozygous nucleotide in-frame duplications of the PHOX2B gene (MIM #603851), occurring in a gene stretch coding for twenty alanine residues, range from 15 to 39 nucleotide insertions, leading to an increase in normal 20-polyalanine tract up to 25–33 repeats.10,11 Increased poly-Alanine expansion (genotypes 20/26 to 20/33) was associated with greater severity of autonomic dysfunction with increased R-R segments on Holter recordings and increased ventilation dysfunction. In addition, further anomalies such as PHOX2B mislocalizations to cellular cytoplasm (normally present only in the nucleus), a misfolding tendency of the PHOX2B protein, and alterations in 3
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F. Ventura et al.
more, SIDS cases are presently decreasing also thanks to a higher diagnostic yield from targeted screening procedures. Since family history of SIDS is a well-known risk factor for sudden infant death, the genetic analysis is of the utmost significance for family counselling and potential prevention of sudden death in siblings. The case herein presented corroborates the fact that contractions in poly-Alanine repeats may, in the presence of accompanying predisposing conditions, lead to severe alveolar hypoventilation that may, in turn, result in death. Finally, from a clinical perspective, cases of infant hypoventilation during sleep warrant genetic investigation so as to exclude PHOX2B gene mutations.
does not exclude a possible de novo occurrence of the variant. Our findings are also in line with results of the above mentioned retrospective study of 195 Dutch USID cases,20 whereas the results of a successive German study excluded such an association, although based only to very short polyAla contractions.14 Nonetheless, we contribute further evidence to the correlation between predisposition to infant sudden death and common and apparently neutral PHOX2B variants. Indeed, in several functional studies making use of in vitro gene reporter assays, PHOX2B contractions have been reported to reduce the transactivation of PHOX2B target promoters,23,24 thus suggesting that, besides poly-Ala expansions, also poly-Ala tracts shorter than 20 residues can impair, though to a lesser extent, the ability of PHOX2B to express its target genes and therefore to correctly contribute to development and function of specific autonomous nervous system cell lineages. Therefore, effects of poly-Ala contractions may be described in terms of loss-of-function of the transcription factor, a view consistent with the already reported association between patients affected with Hirschsprung Disease (HSCR), another developmental defect of the autonomous nervous system, and poly-Ala contractions as well as interstitial deletions of the whole PHOX2B locus.24–26 Developmental alterations of the respiratory human retrotrapezoid nucleus (hRTN) were reported in sudden unexplained foetal and infant death.15 In particular, authors observed a frequency of structural and/ or PHOX2B-expression abnormalities of the hRTN that were higher in USID cases than in controls and concluded that such developmental abnormalities may seriously compromise chemoreception control. Consistent with such an observation, PHOX2B-expressing retrotrapezoid neurons of transgenic mice have been demonstrated to be intrinsically responsive to ventilation stimuli, as expected for a respiratory chemoreceptor.27 Based on the observations collected at the scene, followed by supporting post-mortem test findings, the sudden death of the infant was attributed to the underlying genetic predisposition to congenital central hypoventilation (i.e. low-penetrance PHOX2B gene), manifesting clinically as transient hypoventilation, correlated to prone position during sleep, whose combined effects likely resulted in a critical impairment of autonomic control of respiration at a susceptible stage of development. In such cases we can refer to the triple-risk model, whereby three separate factors simultaneously interact in the pathogenesis of death. The first factor is an underlying vulnerability of the infant (PHOX2B gene mutation); the second, a critical developmental period; and lastly, an exogenous stressor (prone position).28,29 Nevertheless, the plausible role of terminal airway involvement resulting from the prone position assumed at night, should be considered for its possible contribution to the sudden death. It would seem that position played a marginal role since postmortem analysis failed to detect clear and apparent signs of asphyxic distress (absence of acute pulmonary emphysema or ruptured septa), consistent with CCHS cases. In light of the triple risk model, the CCHS condition hindered pulmonary gas exchanges with ensuing hypoventilation and hypercapnia. It may, therefore, be asserted that the observed mutation played a significant role in the onset of a hypoventilation process. In this case, our in-depth multidisciplinary approach effectively elucidated the cause of death. We, therefore, underline the need for a careful and comprehensive multidisciplinary approach in cases of sudden infant death. An integrated analysis can in fact allow to trace the exact pathological mechanism causing the death and assure an adequate differential diagnosis. Although early autopsy investigations led to the classification of the case as sudden infant death syndrome due to the absence of specific pathological findings, advanced genetic study has allowed to define the mode and cause of death. Due to the advances in genetics and other ancillary investigations, the underlying cause of death could be identified in many cases of sudden unexpected death in infancy. All the
Conflicts of interest The authors declare that they have no conflict of interest. References 1. Nirupam N, Sharma R, Chhapola V, Kanwal SK, Berry-Kravis EM, Kumar V. Congenital central hypoventilation syndrome with PHOX2B gene mutation: are we missing the diagnosis? Indian J Pediatr. 2013;80(8):688–690. 2. Weese-Mayer DE, Marazita ML, Rand CM, et al. Congenital central hypoventilation syndrome. In: Adam MP, Ardinger HH, Pagon RA, eds. GeneReviews. Seattle (WA). Seattle: University of Washington; 2004:1993–2017. 3. Rand CM, Patwari PP, Carroll MS, Weese-Mayer DE. Congenital central hypoventilation syndrome and sudden infant death syndrome: disorders of autonomic regulation. Semin Pediatr Neurol. 2013;20(1):44–55. 4. Trang H, Dehan M, Beaufils F, Zaccaria I, Amiel J, Gaultier C. French CCHS Working Group. The French congenital central hypoventilation syndrome registry: general data, phenotype, and genotype. Chest. 2005;127:72–79. 5. Bajanowski T, Vennemann M, Bohnert M, Rauch E, Brinkmann B, Mitchell EA. GeSID Group. Unnatural causes of sudden unexpected deaths initially thought to be sudden infant death syndrome. Int J Legal Med. 2005;119(4):213–216. 6. Mellins RB, Balfour Jr HH, Turino GM, Winters RW. Failure of automatic control of ventilation (Ondine's curse). Report of an infant born with this syndrome and review of the literature. Medicine (Baltim). 1970;49:487–504. 7. Szymońska I, Borgenvik TL, Karlsvik TM, Halsen A, Malecki BK, Saetre SE, Jagła M, Kruczek P, Talowska AM, Drabik G, Zasada M, Malecki M. Novel mutation-deletion in the PHOX2B gene of the patient diagnosed with neuroblastoma, Hirschsprung's disease, and congenital central hypoventilation syndrome (NB-HSCR-CCHS) cluster. J Genet Syndr Gene Ther. 2015;6(3):269. 8. Trochet D, O'Brien LM, Gozal D, et al. PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome. Am J Hum Genet. 2005;76(3):421–426. 9. Kijima K, Sasaki A, Niki T, et al. Sudden infant death syndrome is not associated with the mutation of PHOX2B gene, a major causative gene of congenital central hypoventilation syndrome. Tohoku J Exp Med. 2004;203(1):65–68. 10. Berry-Kravis EM, Zhou L, Rand CM, Weese-Mayer DE. Congenital central hypoventilation syndrome: PHOX2B mutations and phenotype. Am J Respir Crit Care Med. 2006;174(10):1139–1144. 11. Weese-Mayer DE, Berry-Kravis EM, Ceccherini I, Keens TG, Loghmanee DA, Trang H. An official ATS clinical policy statement: congenital central hypoventilation syndrome. Genetic basis, diagnosis, and management. Am J Respir Crit Care Med. 2010;181:626–644. 12. Antic NA, Malow BA, Lange N, et al. PHOX2B mutation-confirmed congenital central hypoventilation syndrome: presentation in adulthood. Am J Respir Crit Care Med. 2006;174(8):923–927. 13. Marion TL, Bradshaw WT. Congenital central hypoventilation syndrome and the PHOX2B gene mutation. Neonatal Netw. 2011;30(6):397–401. 14. Poetsch M, Todt R, Vennemann M, Bajanowski T. That's not it, either-neither polymorphisms in PHOX2B nor in MIF are involved in sudden infant death syndrome (SIDS). Int J Legal Med. 2015;129(5):985–989. 15. Lavezzi AM, Weese-Mayer DE, Yu MY, et al. Developmental alterations of the respiratory human retrotrapezoid nucleus in sudden unexplained fetal and infant death. Auton Neurosci. 2012;170:12–19. 16. Patwari PP, Carroll MS, Rand CM, Kumar R, Harper R, Weese-Mayer DE. Congenital central hypoventilation syndrome and the PHOX2B gene: a model of respiratory and autonomic dysregulation. Respir Physiol Neurobiol. 2010;173(3):322–335. 17. Gronli JO, Santucci BA, Leurgans SE, Berry-Kravis EM, Weese-Mayer DE. Congenital central hypoventilation syndrome: PHOX2B genotype determines risk for sudden death. Pediatr Pulmonol. 2008;43(1):77–86. 18. Dejhalla M, Parton P, Golombek SG. Case report of Haddad syndrome in a newborn: congenital central hypoventilation syndrome and Hirschsprung's disease. J Perinatol. 2006;26(4):259–260. 19. Rand CM, Weese-Mayer DE, Zhou L, et al. Sudden infant death syndrome: casecontrol frequency differences in paired like homeobox (PHOX) 2B gene. Am J Med Genet A. 2006;140:1687–1691. 20. Liebrechts-Akkerman G, Liu F, Lao O, et al. PHOX2B polyalanine repeat length is associated with sudden infant death syndrome and unclassified sudden infant death
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in the Dutch population. Int J Legal Med. 2014;128:621–629. 21. Matera I, Bachetti T, Puppo F, et al. PHOX2B mutations and polyalanine expansions correlate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Central Hypoventilation Syndrome. J Med Genet. 2004;41:373–380. 22. Bachetti T, Parodi S, Di Duca M, Santamaria G, Ravazzolo R, Ceccherini I. Low amounts of PHOX2B expanded alleles in asymptomatic parents suggest unsuspected recurrence risk in congenital central hypoventilation syndrome. J Mol Med. 2011;89:505–513. 23. Toyota T, Yoshitsugu K, Ebihara M, et al. Association between schizophrenia with ocular misalignment and polyalanine length variation in PMX2B. Hum Mol Genet. 2004;13:551–561. 24. Di Zanni E, Adamo A, Belligni E, et al. Ceccherini I. Common PHOX2B poly-alanine
25. 26. 27. 28.
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contractions impair RET gene transcription, predisposing to Hirschsprung disease. Biochim Biophys Acta. 2017;1863:1770–1777. Benailly HK, Lapierre JM, Laudier B, et al. PMX2B, a new candidate gene for Hirschsprung's disease. Clin Genet. 2003;64:204–209. Fernández RM, Mathieu Y, Luzón-Toro B, et al. Contributions of PHOX2B in the pathogenesis of Hirschsprung disease. PLoS One. 2013;8:e54043. Wang S, Shi Y, Shu S, Guyenet PG, Bayliss DA. Phox2b-expressing retrotrapezoid neurons are intrinsically responsive to H+ and CO2. J Neurosci. 2013;33:7756–7761. Filiano JJ, Kinney HC. A perspective on neuropathologic findings in victims of the sudden infant death syndrome: the triple-risk model. Biol Neonate. 1994;65(34):194–197. Kinney HC, Thach BT. The sudden infant death syndrome. N Engl J Med. 2009 Aug 20;361(8):795–805.
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine journal homepage: www.elsevier.com/locate/yjflm
Research Paper
Medico-legal investigation in an explicable case of congenital central hypoventilation syndrome due to a rare variant of the PHOX2B gene
T
Francesco Venturaa,∗, Rosario Barrancoa, Tiziana Bachettib, Paolo Nozzac, Ezio Fulcherid,e, Antonella Palmierif, Isabella Ceccherinib a
Department of Forensic and Legal Medicine, University of Genova, Via De’ Toni 12, 16132 Genova, Italy U.O.C. Medical Genetics, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy c U.O.C. Clinical Pathology, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy d U.O.S.D. Fetal and Perinatal Pathology, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy e School of Medicine, University of Genova, Italy f Department of Pediatric Emergency - Sudden Infant Death Syndrome Liguria Centre, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16148 Genova, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Congenital central hypoventilation syndrome PHOX2B gene polyAlanine contractions
The heterozygous PHOX2B gene mutation is related to congenital central hypoventilation syndrome (CCHS). It is characterized by defective autonomous nervous system development leading to inadequate breathing response to hypoxia and hypercapnia, leading to hypoventilation especially during non-REM sleep, but also during waking in the more severe cases. Herein we report a case of sudden death in a 28-day-old child. The mother reported the infant was found lying on her own bed in the prone position. The infant was wearing a romper and lying in her crib without any blanket or other objects. At autopsy no significant pathological findings were detected. Histologically, sparse aspirated milk residues were present in some lung fields. Toxicological and microbiological examinations were within the norm. The initial postmortem investigation ruled out any readily identifiable cause of death. However, genetic analysis revealed a rare heterozygous 21bp in-frame deletion of the polyalanine coding sequences of the PHOX2B gene. In-frame contractions of the poly-Ala tract of the PHOX2B gene have already been reported in patients with symptoms suggestive of sporadic hypoventilation, apparent life-threatening events or neonatal respiratory distress.
1. Introduction Congenital central hypoventilation syndrome (CCHS) is a potentially fatal condition compromising the regulation of respiratory and autonomic nervous system functions. It presents in new-borns and, rarely, as a milder delayed-onset form in toddlers, children, and even adults.1,2 It has been classified within a distinct and burgeoning nosological category of somewhat rare disorders labelled respiratory and autonomic disorders of infancy, childhood, and adulthood.3 The incidence is estimated to be at 1 of 200 000 livebirths4 and in fatal cases children usually succumb at night, only to be discovered dead the following morning. The forensic investigation of sudden death of infants is extremely challenging. According to respected authors, at least three different scenarios may present after thorough investigation: a first, lacking any prior reported symptoms or significant pathological findings, ∗
constitutes a death of unknown aetiology; a second (symptomatic) group, with the presence of symptoms and/or an underlying disease state, whose severity, nonetheless, by no means appears of such a degree as to justify death. Between these extremes there is a another “borderline” group with only minor or intermediate pathological findings, however deemed unlikely to suffice as the sole cause of death.5 CCHS is a diagnosis of exclusion and is mainly associated with mutations in the paired-like homeobox (PHOX2B) gene, which plays a role in controlling the autonomic regulation of breathing. Herein we report a presumed case of central hypoventilation characterized by a rare PHOX2B gene mutation, confirming the genetic predisposition to the disease as well as highlighting the value of genetic analysis in the post-mortem differential diagnosis of sudden infant death.
Corresponding author. E-mail address: [email protected] (F. Ventura).
https://doi.org/10.1016/j.jflm.2018.04.009 Received 23 June 2017; Received in revised form 9 April 2018; Accepted 13 April 2018 Available online 17 April 2018 1752-928X/ © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Journal of Forensic and Legal Medicine 58 (2018) 1–5
F. Ventura et al.
2. Materials and methods In 2010, the Liguria Region (Italy) officially sanctioned the institution of the Unexpected Sudden Infant Death Liguria Centre, Pediatric Emergency Department, Giannina Gaslini Children's Hospital in Genoa, Italy. As part of its research activities, all cases of death within the first year of life are directed to the regional centre for postmortem study aimed at identifying the causes of death. The Centre is coordinated by an Emergency/Urgency unit, comprising a team of experts (pediatrician, clinical pathologist, forensic pathologist, geneticist, cardiologist, specialist in metabolic disorders, microbiologists). Within the centre, an interdisciplinary commission was established to create a post-mortem pathway for the patient with suspected sudden infant death syndrome, as recognized by international experiences and literature data, coordinated by the Director of the centre. Multicentre analysis provides a comprehensive autopsy, histological, toxicological, microbiological and genetic evaluation.
Fig. 1. Histological Findings: lung specimens with a few foreign material (milk) associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. H&E x10, x40.
3. Case history A.K.V. (female, 28 days old, of Ecuadorian descent) was found lifeless after spending the night with her family at the home of friends. The mother reported the infant was found lying in her own crib in a prone position, wearing a romper and without any blanket or other objects. After prolonged and unsuccessful cardiopulmonary resuscitation (CPR), the infant was declared dead. Third-born of healthy parents, she was born at term after an uneventful pregnancy and delivery. The neonatal period was also routine with no noteworthy episodes of infection, rhinitis or cough, anomalous bowel discharge and/or vomiting. Moreover, parents reported normal growth and efficient suckling, with breast milk added to feeding formula. In the family history, a maternal ancestor had died from stroke at age 38 and a maternal aunt had a history of ill-described cardiac surgery at age 51. The siblings, of 2 and 5 years of age, were reportedly healthy.
Fig. 2. Histological Findings: lung specimens with a few foreign material (milk) associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. H&E x10, x40.
4. Autopsy, toxicological and microbiological findings
this or another variant in strict Linkage Disequilibrium (LD) may play a predisposing role in the occurrence of life threatening events. According to our Institute's protocol, to assess the involvement of PHOX2B defects in sudden intra-uterine and infant deaths, direct DNA sequencing of both strands of the amplification products corresponding to coding exons and flanking intronic regions of the PHOX2B gene (GenBank NM_003924.3) was applied to the victim's DNA specimens. A heterozygous 21bp in-frame deletion of the poly-Alanine stretch of the PHOX2B exon 3 (c.754-774del21), corresponding to a contraction of 7 alanine residues in the protein, was detected. Moreover, by coupling exon 3 amplification with FAM-tagged primers and capillary electrophoresis, the -7Ala contraction was confirmed in the victim's and excluded in mother's DNA (Fig. 3). Unfortunately, the father and his older brothers had returned to their native country, so their DNA was not available for analysis.
At autopsy the following findings were revealed: development of visceral organs compatible with age and absence of any abnormalities or malformations. The stomach and the proximal small bowel contained curdled milky contents. No noteworthy pathological findings or malformations of its parenchyma. Macroscopically, the organs revealed no malformations or abnormalities, and no further pathological findings were observed. Histopathological examination (Figs. 1–2) revealed foreign material (milk) present in some lung fields associated with acute pulmonary stasis. There was, however, no tissue reaction to the presence of the foreign material. The central nervous system showed normal development and myelination. There was no evidence of malformation nor signs of hypoxia/ischemia or haemorrhage. Toxicological investigations ruled out the presence of any drug or substance. Microbiological examinations were normal and excluded existing infectious disease processes.
6. Discussion
5. Genetic findings
CCHS was first described by Mellins et al.6 and is characterized by defective autonomous nervous system development leading to inadequate breathing response to hypoxia and hypercapnia, responsible for hypoventilation, especially during non-REM sleep, but also during waking hours in more severe cases.7 In 20% of cases this disease is associated with Hirschsprung disease and in another 5–10% there is also a tumour of autonomic neural crest derivatives, such as a ganglioneuroma or neuroblastoma.8 Patients with congenital central hypoventilation syndrome present a
The protocol involving the mutation screening of the PHOX2B gene in cases with sudden unexpected infant death has been applied for a few years in our Institute. Seven DNA samples have been tested so far and in addition to the present one, showing a PHOX2B polyAla contraction, two others have resulted to carry the variant allele of a rare synonymous polymorphism S184S (rs17885216, MAF = 0.01; https://www. ncbi.nlm.nih.gov/projects/SNP). This raises the question of whether 2
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gene promoter target transcription are more closely associated with increased poly-Alanine repeats.12 In a smaller subset of cases (less than 10%), instead of exhibiting poly-Alanine expansions, other mutations in the PHOX2B gene are present, such as frameshift, missense, nonsense, and stop codon mutations, are present.10 Deletions have also been identified as causes of CCHS in a small subgroup of patients.3,13 In all these latter cases the phenotypic spectrum is extremely variable and sometimes even far worse.3 On the other hand, subjects with poly-Alanine tracts of decreased repeat lengths may survive into adulthood without manifesting the early acute respiratory failure typically associated with the CCHS phenotype. Indeed, poly-Alanine contractions represent a rare mutation that can also be found in seemingly healthy subjects.14,15 In CCHS, a functional analysis using Magnetic Resonance Imaging,16 reported alterations of signal intensity in the thalamus, mesencephalus, medial pons as well as the region spanning from the insular cortex to the hippocampus. This pathology involves a disorder of the autonomic nervous system. An abnormality of central integration of neuronal signals on the part of the central chemokines is the current hypothesis. Symptoms include apnoea and cyanosis, in the absence of cardiac, pulmonary, neuromuscular, or brainstem malformations.1 Affected infants show blunted physiological increases in rate or effort of breathing and, without a timely diagnosis, may develop right heart enlargement and pulmonary hypertension in response to chronic hypercapnia.13 In children with CCHS there is an increase in the incidence of arrhythmic episodes, mainly sinus bradycardia, and blood pressure values are lower during the waking and higher during sleep, indicating an attenuation of the physiological drop in sleeping blood pressures.17 Moreover, perception of asphyxia is absent.18 The diagnosis of CCHS is clinical, confirmed by molecular genetics. In most cases it is diagnosed within 48 h of birth, following an episode of cyanosis, hypercapnia and O2 desaturation during sleep. Sometimes, diagnosis is made in the wake of asphyxiation resulting in irreversible brain damage or death.13 There is evidence supporting the involvement of PHOX2B defects in a wider spectrum of disease phenotypes, all sharing an impaired autonomous nervous system functioning.11 In one of these studies, the entire PHOX2B gene was investigated in 23 Japanese and 91 AfricanAmerican and Caucasian sudden infant death cases, finding no PHOX2B mutation but only polymorphic alleles, whose overall frequency however turned out to be statistically higher than in matched controls.9,19 Furthermore, a molecular analysis of genes, previously implicated in the serotonin household or in CCHS, carried out in 195 Dutch USID (Unclassified Sudden Infant Death)/SIDS cases and 846 healthy controls confirmed a role of common PHOX2B polyalanine contraction alleles, resulted to be more frequent in cases than controls.20 In-frame contractions, carrying only 7, 13, 14 or 15 Ala-residues at the poly-Ala tract of the PHOX2B gene, have already been reported i) in CCHS patients, in association with additional causative mutations, ii) in about 3% of the general population, iii) in a small proportion of CCHS patients' parents and, iv) in patients with symptoms suggestive of sporadic hypoventilation, apparent life-threatening events or neonatal respiratory distress.21,22 These latter observations support a possible different effect of the PHOX2B contractions, which may manifest under specific circumstances and/or with certain genetic backgrounds. Consequently, the PHOX2B variant thus found, has had, with all likelihood, a decisive role in the alteration of the ventilation of the child and in originating the hypoventilation mechanism. Therefore, the PHOX2B variant in the present case could have had a role in worsening the effects of some other predisposing environmental factors. Despite only one parent being available for the analysis, the absence of the PHOX2B contractions from mother's DNA is consistent with, and
Fig. 3. Genetic analysis: peak scanner profiles obtained after capillary electrophoresis of FAM-conjugated PCR products corresponding to the polyAla stretch region of the PHOX2B exon 3. From top to bottom, the following peak scanner profiles are reported: (1) a standard control coming from a healthy individual, (2) the mother, turned out to carry wild type PHOX2B allele, (3) the victim, heterozygous for the 7Ala contracted PHOX2B allele (c.754-774del21), and (4) a control without DNA template (blank).
mutation of the PHOX2B gene.9 This maps onto the 4p12 chromosome and encodes a transcriptional factor of 314 amino acids with two short and stable poly-Alanine repeat expansions of 9 and 20 residues.8 Its mutation entails either autosomal dominant transmission or de novo occurrence in the first generation of polyalanine expansion mutations (PARM) and other less frequent non-polyalanine mutations (NPARM).8 In addition, in about 10% of cases of CCHS, an asymptomatic parent shows mosaicism for the mutation found in the child.10 In particular, heterozygous nucleotide in-frame duplications of the PHOX2B gene (MIM #603851), occurring in a gene stretch coding for twenty alanine residues, range from 15 to 39 nucleotide insertions, leading to an increase in normal 20-polyalanine tract up to 25–33 repeats.10,11 Increased poly-Alanine expansion (genotypes 20/26 to 20/33) was associated with greater severity of autonomic dysfunction with increased R-R segments on Holter recordings and increased ventilation dysfunction. In addition, further anomalies such as PHOX2B mislocalizations to cellular cytoplasm (normally present only in the nucleus), a misfolding tendency of the PHOX2B protein, and alterations in 3
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more, SIDS cases are presently decreasing also thanks to a higher diagnostic yield from targeted screening procedures. Since family history of SIDS is a well-known risk factor for sudden infant death, the genetic analysis is of the utmost significance for family counselling and potential prevention of sudden death in siblings. The case herein presented corroborates the fact that contractions in poly-Alanine repeats may, in the presence of accompanying predisposing conditions, lead to severe alveolar hypoventilation that may, in turn, result in death. Finally, from a clinical perspective, cases of infant hypoventilation during sleep warrant genetic investigation so as to exclude PHOX2B gene mutations.
does not exclude a possible de novo occurrence of the variant. Our findings are also in line with results of the above mentioned retrospective study of 195 Dutch USID cases,20 whereas the results of a successive German study excluded such an association, although based only to very short polyAla contractions.14 Nonetheless, we contribute further evidence to the correlation between predisposition to infant sudden death and common and apparently neutral PHOX2B variants. Indeed, in several functional studies making use of in vitro gene reporter assays, PHOX2B contractions have been reported to reduce the transactivation of PHOX2B target promoters,23,24 thus suggesting that, besides poly-Ala expansions, also poly-Ala tracts shorter than 20 residues can impair, though to a lesser extent, the ability of PHOX2B to express its target genes and therefore to correctly contribute to development and function of specific autonomous nervous system cell lineages. Therefore, effects of poly-Ala contractions may be described in terms of loss-of-function of the transcription factor, a view consistent with the already reported association between patients affected with Hirschsprung Disease (HSCR), another developmental defect of the autonomous nervous system, and poly-Ala contractions as well as interstitial deletions of the whole PHOX2B locus.24–26 Developmental alterations of the respiratory human retrotrapezoid nucleus (hRTN) were reported in sudden unexplained foetal and infant death.15 In particular, authors observed a frequency of structural and/ or PHOX2B-expression abnormalities of the hRTN that were higher in USID cases than in controls and concluded that such developmental abnormalities may seriously compromise chemoreception control. Consistent with such an observation, PHOX2B-expressing retrotrapezoid neurons of transgenic mice have been demonstrated to be intrinsically responsive to ventilation stimuli, as expected for a respiratory chemoreceptor.27 Based on the observations collected at the scene, followed by supporting post-mortem test findings, the sudden death of the infant was attributed to the underlying genetic predisposition to congenital central hypoventilation (i.e. low-penetrance PHOX2B gene), manifesting clinically as transient hypoventilation, correlated to prone position during sleep, whose combined effects likely resulted in a critical impairment of autonomic control of respiration at a susceptible stage of development. In such cases we can refer to the triple-risk model, whereby three separate factors simultaneously interact in the pathogenesis of death. The first factor is an underlying vulnerability of the infant (PHOX2B gene mutation); the second, a critical developmental period; and lastly, an exogenous stressor (prone position).28,29 Nevertheless, the plausible role of terminal airway involvement resulting from the prone position assumed at night, should be considered for its possible contribution to the sudden death. It would seem that position played a marginal role since postmortem analysis failed to detect clear and apparent signs of asphyxic distress (absence of acute pulmonary emphysema or ruptured septa), consistent with CCHS cases. In light of the triple risk model, the CCHS condition hindered pulmonary gas exchanges with ensuing hypoventilation and hypercapnia. It may, therefore, be asserted that the observed mutation played a significant role in the onset of a hypoventilation process. In this case, our in-depth multidisciplinary approach effectively elucidated the cause of death. We, therefore, underline the need for a careful and comprehensive multidisciplinary approach in cases of sudden infant death. An integrated analysis can in fact allow to trace the exact pathological mechanism causing the death and assure an adequate differential diagnosis. Although early autopsy investigations led to the classification of the case as sudden infant death syndrome due to the absence of specific pathological findings, advanced genetic study has allowed to define the mode and cause of death. Due to the advances in genetics and other ancillary investigations, the underlying cause of death could be identified in many cases of sudden unexpected death in infancy. All the
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