Non-polyalanine repeat mutation in PHOX2B is detected in autopsy cases of sudden unexpected infant death.

BACKGROUND: Congenital central hypoventilation syndrome (CCHS), which is caused by PHOX2B with phenotypic variations, has a point of controversy: CCHS is putatively involved in autopsy cases of sudden unexpected infant death (SUID) including sudden infant death syndrome.
OBJECTIVE: The relation of CCHS to SUID cases was investigated by extensive genotyping of PHOX2B.
METHODS: We analyzed 93 DNA samples of less than one-year-old SUID cases that were autopsied in our department. Unrelated adult volunteers (n = 942) were used as the control.
RESULTS: No polyalanine tract expansion was detected in the SUID cases. The allelic frequencies of repeat contractions and SNP (rs28647582) in intron 2 were not significantly different from that in those control group. Further extensive sequencing revealed a non-polyalanine repeat mutation (NPARM) of c.905A>C in a sudden death case of a one-month-old male infant. This missense mutation (p.Asn302Thr), registered as rs779068107, was annotated to 'Affected status is unknown', but it might be associated with the sudden death.
CONCLUSION: NPARM was more plausibly related to sudden unexpected death than expansions because of severe clinical complications. This finding indicates possible CCHS involvement in forensic autopsy cases without ante-mortem diagnosis.

Introduction

Congenital central hypoventilation syndrome (CCHS) is characterized by impaired ventilatory response to hypercapnia and hypoxemia during sleep, which usually depend on artificial ventilatory support. This autosomal dominant disorder is caused by the single gene of paired-like homeobox 2B (PHOX2B), which is in turn associated with various phenotypic disorders [1]. Most patients carry expansions of the repetitive alanine tract, ranging from 5 to 13 residues, which are usually generated by de novo mutation in meiosis. The normal allele is 20 times repeated, but a couple of contracted alleles are also prevalent [2]. Furthermore, the minor part carries non-polyalanine repeat mutation (NPARM) of frameshift, nonsense and missense, which more often result in severe complications of Hirschsprung disease and neural crest-derived tumors [3, 4].

The diagnosis of sudden infant death syndrome (SIDS) as a cause of death has decreased in mortality statistics for decades. One reason for the decrease is circumvention by examiners because of indistinguishable disorders such as accidental suffocation in bed. Other broad criteria such as sudden unexpected infant death (SUID) and sudden unexpected death in infancy are preferably chosen to indicate causes for such equivocal unclassifiable infant deaths [5, 6]. The involvement of CCHS in SIDS/SUID has been investigated for forensic autopsy cases because of its similarity of nocturnal onset.

No apparent involvement of CCHS in SIDS cases has been demonstrated in earlier studies [7, 8]. However, Rand et al. demonstrated significant allelic frequency difference of SNP (rs28647582) in intron 2 between SIDS and control groups of the Caucasian population [9]. Liebrechts-Akkerman et al. found significant association of repeat contractions with unclassified sudden infant death in a Dutch population [10]. Ventura et al. reported contraction in an infant autopsy case, but its causality was unclear [11]. Furthermore, a recent study has shown that SNP in the 3′ untranslated region affects the expression of PHOX2B molecules, which potentially impairs respiratory function via the neurological system [12]. In contrast, some negative reports also described that no pathogenic variation of PHOX2B was detected in cases of SIDS [13] or sudden unexpected death in epilepsy cases [14]. The association of CCHS remains controversial.

This report describes a specific investigation of the relation of PHOX2B variation with SIDS/SUID. The distribution of repeat alleles was examined in large groups of Japanese subjects: SUID subjects in the forensic department and healthy control individuals. Then, extensive sequencing of PHOX2B was performed for the SUID samples.

Materials and methods

Subjects

For this study, we analyzed 93 SUID samples from the Department of Forensic Medicine, Tokai University School of Medicine, during 17 years: 2005–2021. The criteria for selection of SUID were fundamentally as described previously [15]. These less than one-year-old 58 male and 35 female subjects had mean age of 3.4 months. Full autopsies were performed including examinations of histopathology, toxicology, clinical chemistry, and virus antibody titers [16]. As the cause of death, SIDS and suspected SIDS cases were 59, undetermined cases were 25, and infections of airways such as bronchitis were 9. Overlay and suffocation during co-sleeping were excluded as possible. As control subjects, unrelated adult volunteers (n = 942) were available [17].

This project was approved by the Ethical Committee of Tokai University Hospital. Written informed consent was obtained from the parents of SUID cases and the control subjects.

Molecular analysis of PHOX2B

DNA was extracted from an oral swab of control subjects and blood at autopsy. An oligonucleotide primer set of 5′-aggtcccaatcccaaccccac-3′ and 5′-gaatccgggatggaggtgatg-3′ was designed to obtain the repetitive tract. Briefly, PCR amplification was performed using Tks Gflex DNA Polymerase (Takara Bio Inc., Kusatsu, Japan) from 10 ng of DNA as the template with 40 thermal cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 10 s. The product was analyzed using a genetic analyzer (3500; Thermo Fisher Scientific, MA, USA).

The GC content of the repeat and its vicinity is very high, around 90%, which often inhibits amplification of sufficient amounts of the expanded alleles [2]. For an earlier study, we developed effective circumvention using bisulfite-treated DNA [18]. In addition to the PCR amplification described above, we applied this modified one to homozygous specimens.

Furthermore, for all SUID cases, the nucleotide sequence of three exons and two introns of PHOX2B was determined for PCR amplicons of 2999 bp using the primer set of 5′-gcgttgagctgtgcacatctc-3′ and 5′-gacgacaatagccttgggcct-3′. Direct sequencing of the products was carried using the Sanger method. The detected substitutions were surveyed on the web site of the National Center for Biotechnology Information Search database (https://www.ncbi.nlm.nih.gov/).

To determine allele frequencies of two substitutions detected in the SUID subjects, the amplified product-length polymorphism (APLP) analysis was employed to the control group [19], using the following primer sets; 5′- tcttcgctccaaaga, 5′- and 5′- catactgctcttcactaaggcg-3′ for rs779068107, and 5′- agtcctggagcctcg, 5′- and 5′- tatttctgatcggccatggggc-3′ for rs28647582, where underline means replaced sites in the oligonucleotides.

Next-generation sequencing (NGS) analysis

DNA libraries were constructed using the Ion AmpliSeq Library kit 2.0 (Thermo Fisher Scientific Inc.), according to the manufacturer’s protocol. Briefly, Ion AmpliSeq Inherited Disease Panel (Thermo Fisher Scientific Inc.) was used as the primer set for all exon regions of 328 disease related genes. After amplification of emulsion PCR in the Ion OneTouch 2 instrument with the Ion PGM Template OT2 400 kit, sequencing was carried out using the Ion 316 Chip kit V2 BC (Thermo Fisher Scientific Inc.). The sequence data were analyzed using Torrent Suite Software ver. 5.2.2. The detected variants were subjected to the predictive in silico tool of Ion Reporter Software ver. 5.1.8 (https://ionreporter.thermofisher.com/ir/) including ClinVar for the causative genes of cardiovascular diseases such as QT prolongation and hypertrophic cardiomyopathy.

Statistical analyses

Categorical variables of Hardy-Weinberg disequilibrium and allelic distribution were compared using either chi-squared tests or Fisher’s exact test when appropriate. A P value of < .05 was inferred as statistically significant.

Results

Repetitive tract analysis

Table 1 summarizes the allele frequency of the polyalanine repeat in two groups. No expansion was detected in the control group (n = 942). Two types of 15-times and 13-times contractions were detected with allele frequencies of 0.038 and 0.004, respectively, of which values were within the Hardy-Weinberg equilibrium (χ2 = 5.48, df = 2).

Table 1

Frequency (number) of genotypes and alleles of the polyalanine repeat in the SUID and control groups.

	Genotype					Allele
	20/20	20/15	20/13	15/15	Expansion	20	15	13	15 +13
SUID (n = 93)	0.946 (88)	0.032 (3)	0.022 (2)	None	None	0.973 (181)	0.016 (3) P = .15	0.011 (2) P = .20	0.027 (5) P = .09
Control (n = 942)	0.920 (867)	0.068 (64)	0.007 (7)	0.004 (4)	None	0.958 (1805)	0.038 (72)	0.004 (7)	0.042 (79)

The high GC content of repetitive tract often inhibits amplification of expanded alleles. To optimize efficacy in raising potential hidden expansions, we applied an alternative analysis using bisulfite-treated DNA to all homozygous samples in the first-round amplification. Nevertheless, no expansion was evident.

A total of 93 samples of SUID cases were analyzed. No expansion was detected from either PCR amplification. The frequencies of 15 and 13 contractions were, respectively, 0.016 and 0.011, which were not significantly different from those in the control group. Moreover, the polyalanine tract comprises four synonymous codons. A polymorphism of synonymous codon was evident for both contraction alleles.

Sequencing of PHOX2B for SUID subjects

For the SUID group, the whole gene were extensively sequenced. Several substitutions were detected in the entire gene (Fig 1). As a characteristic substitution, c.905A>C was detected in heterozygote, which was expected to be a non-synonymous substitution of p.Asn302Thr (Fig 2). The substitution has been registered with an accession number of rs779068107 (NM_003924.4) with an annotation of ‘clinical significance unknown’, for which a comment is accompanied by a potential effect to pathogenesis because of the highly conserved residue of asparagine. Furthermore, the APLP method was developed to detect the substitution of rs779068107 (Fig 3), but any other substitution was detected among the SUID and control groups.

Fig 1

Scheme of detected nucleotide substitutions in PHOX2B in SUID subjects (n = 93).

Fig 2

Electropherogram of heterozygous 905A>C in PHOX2B.

Fig 3

APLP method to detect two substitutions of rs779068107 (left) and rs28647582 (right).

Amplified products were electrophoresed in 12% polyacrylamide gel, followed by ethidium bromide staining. Molecular size markers are in the left lane.

In addition, for SNP (rs28647582) in intron 2, of which detection was performed using the APLP method (Fig 3), no significant allelic difference was obtained in the two groups of SUID and the control (Table 2).

Table 2

Frequency (number) of genotypes and alleles of SNP (rs28647582) in intron 2 in the SUID and control groups.

	Genotype			Allele
	A/A	A/G	G/G	A	G
SUID (n = 93)	0.667 (62)	0.280 (26)	0.054 (5)	0.806 (150)	0.194 (36) P = .92
Control (n = 942)	0.653 (615)	0.314 (296)	0.033 (31)	0.810 (1526)	0.190 (358)

Case

This carrier of rs779068107, a 32-day-old male infant, was found unexpectedly to have no response in bed 3 h after feeding at midnight. At a hospital 30 min later, his death was confirmed. The baby had been delivered at 38 weeks. He weighed 3130 g with Apgar scores of 9 at 1 min and 10 at 5min. No record indicated an apneic event after birth. Potential hearing impairment was found in a screening, but no clinical examination had been done. His parents, both in their twenties, and a 2-year-old sister were healthy. Negative findings were obtained at autopsy, except for general congestion. Furthermore, no critical nucleotide substitutions for cardiovascular diseases, such as KCNQ1, KCNH2 and SCN5A, were found in the comprehensive NGS analysis (supplementary data). SIDS was suspected as the cause of death after the initial examinations. In addition, the polyalanine repeat genotype was 20-times homozygous for the analyses described herein.

Discussion

To arrive at a diagnosis of SIDS, several disorders must be excluded. Molecular analysis has given forensic pathologists opportunities to find out potential physiological disorders of arrhythmic diseases such as long QT syndrome [20]. This approach is now known as molecular autopsy [21]. Actually, CCHS is a genetically detectable disease that presents difficulties for obtaining definitive morphological evidence in routine postmortem examinations [22].

For this study, analyses were performed on a large scale to examine the involvement of repeat expansions and NPARMs in autopsy cases of SUID. No expansion was found in both SUID and control groups. An earlier publication showed positive correlation of repeat contraction with unclassified infant death [10]. By contrast, the present study revealed no significant relation, yielding similar results to that reported by Poetsch et al. [13]. The other report judged the 13-times repeat to be pathogenic [11], but this allele is distributed in healthy individuals even with low frequency, as found in this study. Clinical observations showed that contractions are not pathogenic [2, 7, 8]. The case control study by Bachetti et al. also showed no correlation of SIDS/SUID to the repeat contractions [12]. Therefore, it is reasonable to conclude that contractions are not associated with occurrence of SUID.

SIDS/SUID risk is still likely defined by multifactorial genetic and environmental interactions [23, 24]. In one of such subjects, we detected the missense mutation in PHOX2B. Symptoms such as apnea and cyanosis are typically noticed in the first 48 hr of life, but more patients with NPARMs are known to be evident outside of the newborn period, known as late-onset CCHS [25]. Moreover, as life-threatening events, bradycardia and sinus pauses potentially occur in addition to apnea during sleep [26-28]. Zhou et al. recently demonstrate that phenotypic manifestations of NPARMs are associated with their variant type, location, and effect on transcripts [29]. Although the subject described for the present case seemed to have no episode of respiratory disorder after birth, the degree of severity in the respiratory and cardiovascular defect varies among cases [30, 31]. We infer that SUID cases might have causal NPARMs, rather than expansions.

The majority of CCHS-associated NPARMs are found at the ends of exons 2 and 3 [4]. The rs779068107 variant is also located near the C-terminal of molecules, and this should be the first clinical case. Though it is registered with annotation like ‘clinical significance unknown’, we think that this substitution might be phenotypically pathogenic because of the replacement of highly conserved amino acid residue,

In general, the impaired transfer of abnormal longer protein molecules through the nuclear membrane results in an excess in the cytoplasm to form intracellular aggregates. However, the molecular mechanism of NPARM is distinct from the intracellular localization. Phenotypes of the patients carrying NPARM are variable, and they might depend on mutant protein effects [3, 32]. For instance, a dominant negative effect might be derived from direct interference of the mutated protein with the wild-type activity. Gain-of-function effects might appear by altered expressional control in the original intranuclear distribution [2]. The substituted threonine residue potentially serves as a substrate for phosphorylation and glycosylation, which might exhibit such harmful effects.

Regarding SNP (rs28647582) in intron 2, significant associations with Hirschsprung disease, neuroblastoma and Wilms tumor have been demonstrated in a couple of studies [33, 34]. However, no association with SUID occurrence was obtained in this study. Liang et al. also showed no association of rs28647582 with Hirschsprung disease [35]. This SNP was not linked to polyalanine repeat number and other substitution in this conservative gene. Therefore, we think that this SNP does not have any sufficient effects to function and translated volume of PHOX2B.

In conclusion, our attempts highlight the importance of extensive search for the whole gene including non-polyalanine repeat regions. To date, evidence has indicated arrhythmia diseases such as QT elongation, and metabolic diseases such as fatty acids β-oxidation deficiency as prevalent in forensic autopsy cases of SIDS/SUID. Results of this study indicate the additional possibility of CCHS as a causal disorder for SIDS/SUID.

(XLS)

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15 Feb 2022

1. Line 144-145. Please clarify if the nomenclatures are correct (seems A>C is correct according to Fig 2)(https://www.ncbi.nlm.nih.gov/clinvar?term=((807706[AlleleID])OR(520167[AlleleID]))).

Please place citation(s) for this sentence. If this information is from ClinVar (ID 535771 or 822948), please check the clinical status. "Affected status is unknown" in ClinVar means some individual(s) were tested for certain conditions including Haddad syndrome (e.g., https://www.ncbi.nlm.nih.gov/clinvar/variation/822948/evidence/). This is confusing, but actual clinical conditions (i.e., affected or not) are rarely stated in ClinVar.

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Reviewer #1: The authors analyzed the entire nucleotide sequence of the phox2b locus in Japanese autopsy cases of SIDS/SUID and reported that cchs may be involved in the cause of death in one case with a nonsynonymous substitution. This is a valuable report on the involvement of cchs in sudden infant death syndrome.

Some comments are provided below.

It is desirable to present representative data of the APLP electrophoresis diagram for typing of rs779068107.

The minor allele frequency of rs779068107 in Japanese is required to be described. Compared to the minor allele frequency, do you consider the number of healthy controls to be adequate?

As stated in the manuscript, rs779068107 has been found in patients with Haddad syndrome, but the significance of this mutation in CLINVAR is UNCERTAIN SIGNIFICANCE. As stated in the manuscript, rs779068107 is found in patients with Haddad syndrome, but the significance of this mutation in CLINVAR is UNCERTAIN SIGNIFICANCE. Please discuss this, comparing the mutations around rs779068107 in the phox2b gene may help to solve this problem.

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30 Mar 2022

To the Reviewers' comments

Thank you very much for the detailed review to our submission. The raised comments were helpful to revise the manuscript. The reply is described below. Alterations are indicated in the revised text with track changes in red.

1. Line 144-145. Please clarify if the nomenclatures are correct (seems A>C is correct according to Fig 2) (https://www.ncbi.nlm.nih.gov/clinvar?term=((807706[AlleleID])OR(520167[AlleleID]))).

Reply

Both c.905A>G and c.905A>C are registered in the NCBI web site. As shown in Fig. 2, c.905A>C is correct. However, the authors made a mistake of the amino acid substitution of Asn302Ser in the original manuscript. We have corrected it to Asn302Thr in line 160 of page 11.

2. Line 146-148. "As clinical significance at the site, it was annotated to that observed in a patient with CCHS-Hirschsprung disease (Haddad syndrome)."

Please place citation(s) for this sentence. If this information is from ClinVar (ID 535771 or 822948), please check the clinical status. "Affected status is unknown" in ClinVar means some individual(s) were tested for certain conditions including Haddad syndrome (e.g., https://www.ncbi.nlm.nih.gov/clinvar/variation/822948/evidence/). This is confusing, but actual clinical conditions (i.e., affected or not) are rarely stated in ClinVar.

Reply

As the reviewer pointed out, the web site says "Affected status is unknown" for the missense substitution. The careless description has been rewritten extensively in the revised version, in which the term of Haddad syndrome has been removed thoroughly. Please see lines 31 to 33 in page 2, lines 161 to 163 in page 11, and lines 219 to 220 in page 14. We have extensively rewritten it to the revised version as uncertain significance. Please see lines 33 to 35 in page 2, lines 163 to 165 in page 11, and lines 231 to 234 in page 15.

3. Line 149-151. Regarding rs28647582, the MAF (T>C) is very high in general populations (C=0.382239 in gnomAD). Thus, it is unlikely this SNP is causative. However, some researchers believe this can be a risk factor for SUD (https://www.ncbi.nlm.nih.gov/CBBresearch/Lu/Demo/LitVar/#!?query=rs28647582). This may be discussed (either agree or disagree).

Reply

The authors do not think that the substitution of rs28647582 is related to sudden unexpected infant death. The statement is added into the sections of abstract and discussion of the revised manuscript. Please see lines 28 to 29 in page 2, and lines 245 to 251 in page 16.

In addition, the result of genotyping was separately described as Table 2 with division from the original Table 1. Please see lines 145 to 148 of pages 9 and 10, and lines 182 to 183 of page 12.

As the reviewer found out in the web site, there are a couple of reports that showed positive and negative relations of rs28647582 SNP to Hirschsprung disease, neuroblastoma and Wilms tumor. We also added a new paragraph about these points into the revised text as well. Please see lines 245 to 251 in page 16, and nos. 33-35 of reference list of pages 22 to 23.

4. Line 155-156. Please describe more detail regarding the findings of poly-alanine repeat. Is this condition associated with the phenotype of SIDS? If so, it would be better to add more data and/or discussion.

Reply

According to the advice, the statement regarding poly-alanine repeat to SUID has been added into Discussion of the revised text more clearly. Please see line 208, and lines 214 to 215 in page 14.

5. Please add a statement regarding pathogenic variants of LQTS (LQT1-3) and Brugada syndrome (SCN5A) in the case presentation (page 10). I think excluding variants of congenital arrhythmia will make this study's finding more convincing. If data is unavailable, please state it as a limitation in Discussion section.

Reply

According to the advice from the reviewer, the authors performed the additional genetic testing using next generation sequencing once again. This revealed no critical exchanges in genes associated with the cardiovascular diseases, such as KCNQ1, KCNH2 and SCN5A, in spite of a number of synonymous and missense mutation including common SNPs. The procedure and result were added into the revise version. Please see lines 116 to 126 in page 8, and lines 192 to 195 in page 13.

Further, the raw data is newly added as ‘supplementary data’ with the predictive in silico analysis.

To Reviewer #1

Thank you very much for the detailed review to our submission. The raised comments were helpful to revise the manuscript. The reply is described below. Alterations are indicated in the revised text with track changes in red.

1. It is desirable to present representative data of the APLP electrophoresis diagram for typing of rs779068107.

Reply

According to the advice from the reviewer, the amplified product length polymorphism (APLP) electrophoresis diagram for rs779068107 typing was added into the revised manuscript as Fig. 3. The authors showed the diagram for rs28647582 as well. Please see lines 165 to 167, and lines 174 to 176 in pages 11 to 12, and lines 178 to 179 in page 12.

2. The minor allele frequency of rs779068107 in Japanese is required to be described. Compared to the minor allele frequency, do you consider the number of healthy controls to be adequate?

Reply

This c.905A>C had not been found in the Japanese population as long as referring to the public genome bank. The authors have deposited this substitution to the DDBJ genome bank. Therefore, it was difficult to calculate the frequency in the population. The authors added this point into the revised version. Please see lines 231 to 234 in page 15.

3. As stated in the manuscript, rs779068107 has been found in patients with Haddad syndrome, but the significance of this mutation in CLINVAR is UNCERTAIN SIGNIFICANCE. Please discuss this, comparing the mutations around rs779068107 in the phox2b gene may help to solve this problem.

Reply

As the reviewer pointed out, the substitution was seemingly not confirmed in a clinical patient of Haddad syndrome as long as seeing the web site. It was our fault. The careless description has been rewritten in the revised version, in which the term of Haddad syndrome has been removed thoroughly. Please see lines 31 to 33 in page 2, lines 161 to 163 in page 11, and lines 219 to 220 in page 14. We have extensively rewritten it to the revised version as uncertain significance. Please see lines 33 to 35 in page 2, lines 163 to 165 in page 11, and lines 231 to 234 in page 15.

Submitted filename: Responce to Reviwers final.docx

Click here for additional data file.

18 Apr 2022

Non-polyalanine repeat mutation in PHOX2B is detected in autopsy cases of sudden unexpected infant death

PONE-D-22-00562R1

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Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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Reviewer #1: The manuscript reports on non-polyalanine repeat mutation in PHOX2B in autopsy cases of sudden unexpected infant death. The manuscript has been revised well. The reviewer thinks this manuscript will be acceptable.

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Reviewer #1: No

21 Apr 2022

PONE-D-22-00562R1

Non-polyalanine repeat mutation in PHOX2B is detected in autopsy cases of sudden unexpected infant death

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