PEDIATRICS Vol. 107 No. 4 April 2001, pp. 690-692

Serotonin Transporter Gene Variation Is a Risk Factor for Sudden Infant Death Syndrome in the Japanese Population

Naoko Narita, MD, PhD^*, , Masaaki Narita, MD, PhD, Sachio Takashima, MD, PhD^§, Masahiro Nakayama, MD, PhD, Toshiro Nagai, MD, PhD^*, and Nobuo Okado, MD, PhD

From the ^* Department of Pediatrics, Dokkyo University, School of Medicine, Koshigaya Hospital, Saitama, Japan;  Neurobiology Laboratory, Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan; the ^§ National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; and  Pathology and Laboratory Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.

	ABSTRACT

Top Abstract Methods Results Discussion References

Objective.  Serotonin (5-HT) in the nervous system is a major factor in facilitation of the brain center for respiration. Variations in the promoter region of the 5-HT transporter (5-HTT) gene have been shown to potentially regulate 5-HT activity in the brain. Therefore, we aimed to identify the possibility that specific allele variants of the 5-HTT gene can be found as a genetic background for sudden infant death syndrome (SIDS).

Methods.  Polymorphisms in the 5' regulatory region of the 5-HTT gene were determined in genomic DNA obtained from 27 SIDS victims and 115 age-matched health control participants.

Results.  There were significant differences in genotype distribution and allele frequency of the 5-HTT promoter gene between SIDS victims and age-matched control participants. The L and XL alleles were more frequently found in SIDS victims than in age-matched control participants.

Conclusion.  Efficiency in the transportation of 5-HTT with the L allele is known to be higher than that with the S allele. The excitatory function by 5-HT is considered to be lower in the respiratory center of individuals with the L allele compared with those with S allele. The XL allele variant has shown another novel biological risk factor for SIDS.  Key words:  sudden infant death syndrome, serotonin, serotonin transporter, promoter, polymorphism, allele variants, respiratory center.

Sudden infant death syndrome (SIDS) is a tragic and common cause of infant death in developed countries. SIDS is defined as "the sudden death of an infant which is unexpected by history, and in which a full postmortem examination fails to demonstrate an adequate cause of death." Possible explanatory mechanisms, such as perinatal and/or postnatal hypoxia, lung dysfunction, and brainstem dysfunction, that might impair ventilatory, circulatory, and arousal responsiveness have been proposed.¹ To date, several risk factors have been identified. These are the prone position,² maternal smoking,³ low birth weight,⁴ low socioeconomic status,⁵ and infection.⁶ Although the precise cause of SIDS is not yet known, attention has been focused recently on malfunction of the respiratory system.⁷

Serotonin (5-HT) exerts potent excitatory effects on the final common pathways in the ventrolateral medulla that controls respiration.⁸ Infants in a risk group for SIDS show a significant increase of rapid eye movement sleep,⁹ suggesting a decreased activity of 5-HT.¹⁰ Given the importance of 5-HT for functional integrity of the respiratory system, changes in 5-HT metabolism have been studied in SIDS victims. Both an increase in the level of a 5-HT metabolite in the cerebrospinal fluid of SIDS victims¹¹ and a decrease in the 5-HT content of the hypothalamus have been documented in SIDS victims.¹²

5-HT modulates diverse brain functions through interactions with 14 different 5-HT receptor subtypes. However, recent evidence has shown that the complex 5-HT neuronal system is under bottleneck control by a single protein, 5-HT transporter (5-HTT).¹³ By controlling reuptake of 5-HT from the extracellular space, 5-HTT regulates the duration and strength of the interactions between 5-HT and its receptors. There is a polymorphism in the promoter region of the 5-HTT gene.^14,15 In humans, the majority of alleles are composed of either 14 (S) or 16 (L) repetitive elements. Although infrequent, there are also 18 and 20 repetitive elements (XL). The activity of the human 5-HTT gene promoter is regulated by these polymorphic repetitive elements, resulting in differences in the efficacy of 5-HTT reuptake among the allelic variants.¹³ Accordingly, the excitatory effect of 5-HT on the respiratory center may differ among 5-HTT alleles. The present study was undertaken to see if specific allelic variations are associated with SIDS victims in the Japanese population.

	METHODS

Top Abstract Methods Results Discussion References

Participants

Twenty-seven SIDS victims 1 to 6 months old (mean ± standard deviation, 2.7 ± 0.5 months old) with a history of sudden and unexpected death were selected for this study. Diagnosis of SIDS was based on the international pathologic criteria for SIDS¹⁶ and an examination of death scene. Human brain tissues (right frontal lobe) of SIDS victims were obtained from National Center of Neurology and Psychiatry in Japan. Blood samples of 115 age-matched (3.1 ± 0.6 months old) control participants were obtained from healthy infants without major disorders in Dokkyo University Koshigaya Hospital. The parents had provided written informed consent. SIDS victims and age-matched control participants were all Japanese.

Determination of Allele Variants

Genomic DNA was extracted from frozen tissue and whole blood using the Genomic DNA Isolation Reagent (Life Technologies, Rockville, MD). The extracted DNA was amplified by polymerase chain reaction (PCR), according to the method of Lesch et al,¹⁵ with minor modifications. Oligonucleotide primers flanking the 5-HTTLPR and corresponding to the nucleotide positions -1416 to -1397 (5'-GGCGTTGCCGCTCTGAATGC) and -910 to -888 (5'-GAGGGACTGAGCTGGACAACCAC) of the 5-HTT gene 5'-flanking regulatory region were used to generate 484- or 528-base pair fragments. PCR amplification was conducted in a final volume of 20 µL consisting of 50 ng of genomic DNA, 2.5 mM deoxyribonucleotides, 20 pmol of forward and reverse primers, 10 mM tris/HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, and 1 U of Taq DNA polymerase. Annealing was conducted at 61°C for 30 seconds, extension at 72°C for 1 minute, and denaturation at 95°C for 30 seconds for 35 cycles. PCR products were visualized by 2% agarose gel electrophoresis followed by ethidium bromide staining.

Statistical Analyses

Genotype distribution and allele frequencies of gene polymorphism were compared between SIDS victims and age-matched control participants by a ² test (2-tailed) and Fisher's exact test.

	RESULTS

Top Abstract Methods Results Discussion References

There were significant differences in genotype distribution (² = 12.49; P = .006, Fisher's exact test; P = .009) and allele frequency (² = 11.36; P = .003, Fisher's exact test; P = .006) between SIDS victims and age-matched control participants (Table 1). The frequency of the L allele was higher in SIDS victims compared with that of the control participants (22.2% vs 13.5%). The XL allele was found in 3 of 54 alleles in SIDS victims (5.6%), whereas there was only 1 XL allele out of 230 alleles in age-matched control participants (0.4%).

	DISCUSSION

Top Abstract Methods Results Discussion References

The present study demonstrates that there are significant differences in genotype distribution and allele frequency of 5-HTT promoter allelic polymorphism between SIDS victims and age-matched control participants. The L and XL alleles in SIDS victims were significantly higher than that of control participants. This finding indicates that having either a L or XL allele is an important risk factor for SIDS. A large scale prospective study is necessary for clinical application of 5-HTT polymorphism to find potential candidates for SIDS. Because 5-HTT promoter L variant was associated with increased expression of the gene in in vitro studies,¹³ our findings suggest that reuptake activity of 5-HTT in the brains of individuals with L allele may be higher and that the 5-HT concentrations in the extracellular space may be maintained lower in individuals with the L allele compared with those with the S allele.

The incidence of SIDS was 0.37 per 1000 infants who survived the neonatal period in Japan during 1997,¹⁷ which was lower than that in the United States0.64 during 1988.¹⁸ It might be possible that the differences may partly be attributable to polymorphic distribution of 5-HTT gene promoter region. The frequencies of L allele shown in the present and previous studies¹⁹ were all <20% in the Japanese population, whereas a nearly 3-time higher frequency of the L allele has been found in whites.¹⁴

The promoter activity of 5-HTT by the allelic variant XL is unknown. However, we found low levels of 5-HT in the blood of individuals with XL allele (unpublished data), which may suggest higher genetic expression of 5-HTT. Additional study on the relationship between 5-HTT promoter genotype and central 5-HT function will also be required to define the relationship between the 5-HTT promoter genotype and 5-HTT function.

The distribution of genotype in our control participants was, in general, consistent with previous reports with Japanese participants.¹² Previous studies showed that XL variant is uniquely present in individuals of African origin¹⁴ and not in the Japanese population,¹⁹ and our data showed 1 XL allele among 115 control Japanese participants and 3 among 27 Japanese SIDS victims. Interestingly, we found a significantly high frequency of XL allele in Japanese individuals with some psychiatric disease compared with control participants (unpublished data). Additional broad study will be required to determine the precise population of XL allele.

The high association of L and XL alleles with SIDS shown in the present study can be used as a biological risk factor for SIDS. The 5-HT hypothesis shows a new understanding on the pathophysiological mechanism for SIDS. Neonatal genetic screening might be applicable to rescue potential candidates for SIDS with proper preventive care including drug administration.

	ACKNOWLEDGMENTS

This study was supported by Grants for Brain Science from the Ministry of Health and Welfare Japan (N.O. and S.T.), and a grant from the Special Research Project on Dynamic Brain Function and Amenity for the Mind, University of Tsukuba (M.N. and N.O.).

We thank Dr Alan L. Schwartz and Dr David B. Wilson (Department of Pediatrics, Washington University School of Medicine) for critical reading of the manuscript.

	FOOTNOTES

The first 2 authors contributed equally to this work.

Received for publication Apr 13, 2000; accepted Jun 22, 2000.

Reprint requests to (N.O.) Neurobiology Laboratory, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail: nokado{at}md.tsukuba.ac.jp

	ABBREVIATIONS

SIDS, sudden infant death syndrome; 5-HT, serotonin; 5-HTT, serotonin transporter; PCR, polymerase chain reaction.

	REFERENCES

Top Abstract Methods Results Discussion References

1. Kinney HC, Filliano JJ, Harper RM The neuropathology of the sudden infant death syndrome. A review. J Neuropathol Exp Neurol 1992; 51:115-126 [Medline]
2. De Jonge GA Cot deaths and sleeping position [letter]. Lancet 1989; 8672:1149-1150
3. Murphy JF, Newcombe RG, Sibert JR The epidemiology of sudden infant syndrome. J Epidemiol Community Health 1982; 36:17-21 [Medline]
4. Lewak N, van der Berg B, Beckwith JB Sudden infant death syndrome risk factors. Prospective data review. Clin Pediatr (Phila) 1979; 18:404-411
5. Marshall TA Sudden infant death syndrome-update. J Sci Med Assoc 1985; 81:605-608
6. Zink P, Drescher J, Veerhagen, et al Serological evidence of recent influenza virus A (H3N2) infections in forensic cases of the sudden infant death syndrome (SIDS). Arch Virol 1987; 93:223-232 [Medline]
7. Poets CF, Meny RG, Chobanian MR, Gasping and other cardiorespiratory patterns during sudden infant deaths. Pediatr Res 1999; 45:350-354 [Medline]
8. Arita H, Sakamoto M, Hirokawa Y, Serotonin innervation patterns differ among the various medullary motoneuronal groups involved in upper airway control. Exp Brain Res 1993; 95:100-110 [Medline]
9. Cornwell AC, Feigenbaum P, Kim A SIDS, abnormal nighttime REM sleep and CNS immaturity. Neuropediatrics 1998; 29:72-79 [Medline]
10. Jacobs BL, Azmitia EC Structure and function of the serotonin system. Physiol Rev 1992; 72:165-229 [Free Full Text]
11. Cramer H, Water J-M, Renaud B, Cerebrospinal fluid adenosine 3',5'-monophosphate, 5-hydroxyindoleacetic acid and homovanillic acid in patients with sleep apnea syndrome. J Neurol Neurosurg Psychiatry 1981; 44:1165-1167 [Medline]
12. Sparks DL, Hunsaker JC III Sudden infant death syndrome: altered aminergic-cholinergic synaptic markers in hypothalamus. J Child Neurol 1991; 6:335-339 [Medline]
13. Lesch K-P, Mosser R Genetically driven variation in serotonin uptake: is there a link to affective spectrum, neurodevelopmental, and neurodegenerative disorders? Biol Psychiatry 1998; 44:179-192 [CrossRef][Medline]
14. Gelernter J, Kranzler H, Coccaro EF, Serotonin transporter protein gene polymorphism and personality measures in African American and European American subjects. Am J Psychiatry 1998; 155:1332-1338 [Abstract/Free Full Text]
15. Lesch K-P, Bengel D, Heils A, Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996; 274:1527-1530 [Abstract/Free Full Text]
16. Krous HF. The international standardized autopsy protocol for sudden infant death. In: Rognum TO, ed. Sudden Infant Death Syndrome. New Trends in the Nineties. Oslo, Norway: Scandinavian University Press; 1995:81-95
17. Statistics and Information Department. Minister's Secretariat, Ministry of Health and Welfare Japan. Vital Statistics of Japan, I. Health Welfare Statistics Association; 1999:331
18. Guyer B, Hoyert DL, Martin JA, Annual summary of vital statistics1998. Pediatrics 1999; 104:1229-1246 [Abstract/Free Full Text]
19. Nakamura T, Muramatsu T, Ono Y, Serotonin transporter gene regulatory region polymorphism and anxiety-related traits in the Japanese. Am J Med Gen 1997; 74:544-545 [CrossRef][Medline]