UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia

Hui Yang; Huijun Li; Qingyao Xia; Wencheng Dai; Xin Li; Yan Liu; Jie Nie; Fei Yang; Yunfeng Sun; Lei Feng; Liye Yang

doi:10.1371/journal.pone.0279059

. 2022 Dec 15;17(12):e0279059. doi: 10.1371/journal.pone.0279059

UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia

Hui Yang ^1,^*,^#, Huijun Li ^2,^#, Qingyao Xia ³, Wencheng Dai ², Xin Li ¹, Yan Liu ¹, Jie Nie ¹, Fei Yang ¹, Yunfeng Sun ⁴, Lei Feng ⁵, Liye Yang ⁶

Editor: George Vousden⁷

PMCID: PMC9754166 PMID: 36520959

Abstract

To explore the correlation between UGT1A1 variant and neonatal hyperbilirubinemia in Chinese Uighur and Han populations. We conducted this study in Urumqi, China. Umbilical cord blood specimens and clinical information of term infants born in the studied center were collected. Variation status of UGT1A1 was determined by direct sequencing or capillary electrophoresis analysis. 102 Uighur and 99 Han normal term neonates, together with 19 hospitalized term newborns (10 Uighur and 9 Han) due to significant hyperbilirubinemia were enrolled into the final analysis. The incidence of neonates with high-risk transcutaneous bilirubin level (TCB) were much higher in Han newborns than in Uighur newborns(P = 0.01). Also, there was statistically significant difference in (TA) 7 promoter mutation of UGT1A1 between Han and Uighur group(χ2 = 4.675, P = 0.03). Furthermore, exon mutation (c.211 and /or c.1091) in UGT1A1 gene was significantly associated with increased TCB level (OR_adj = 1.41, 95%CI: 0.25–2.51, P = 0.002) and higher risk of hyperbilirubinemia in both Han and Uighur infants after adjusted for covariates (OR_adj = 2.21, 95%CI: 1.09–4.49, P = 0.03). In conclusion, UGT1A1 promoter polymorphism seem to be an important genetic modulator of plasma bilirubin level and neonatal hyperbilirubinemia risk within ethnic groups. Genetic assessment of UGT1A1 coding variants may be useful for clinical diagnosis of neonatal jaundice.

Introduction

Neonatal jaundice or hyperbilirubinemia frequently manifests as a pediatric complex trait or disorder, which is still prevalent in the newborn population today [1]. Although most of them were generally benign, a select number of infants will develop hazardous levels of total serum bilirubin (TSB) that may cause irreversible neurological damage [2–4].

The incidence and severity of neonatal hyperbilirubinemia in Asians and American Indians are much higher, as compared to those in Caucasian and black populations [5, 6]. Both environmental and genetic factors may contribute to this situation. The importance of genetic role in neonatal hyperbilirubinemia has been increasingly recognized [7].

Innate ethnic variation in UGT1A1, which encoded for the key enzyme involved in the conjugation of bilirubin has been reported to contribute to the biologic basis of hyperbilirubinemia risk in Asian. Several polymorphisms in the promoter and coding region of the UGT1A1 gene complex has been described, which was associated with reduction in UGT1A1 enzyme activity and Gilbert syndrome phenotype-an autosomal recessive unconjugated hyperbilirubinemia (UCH) disorder [8–10]. Of these UGT1A1 variants, the coding sequence variants- UGT1A1*6 polymorphism was predominant in East Asian subjects [11, 12]. And UGT1A1 promoter sequence polymorphisms, the most common in the Caucasian population and frequently associated with GS phenotype, were also observed in East Asian subjects, though at lower allele frequencies [13].

China was famous for its large population and was composed of 56 ethnic groups. Recent multi-center epidemiology survey from China revealed that neonatal hyperbilirubinemia was still common in China (34.4% of term neonates in China). Hazardous hyperbilirubinemia remained not rare, especially in some areas of southern China [14]. Our previous studies had confirmed the strong association between the common coding variant G211A (UGT1A1*6) and severe hyperbilirubinemia in Han population of southern China [15–17].

In this study, we aim to further describe the spectrum and prevalence of UGT1A1 variant in newborns of Uighur minority living in northern China. Uighur was one main minority in northern China, with populations of more than 5 million. Growing genetic studies has demonstrated that Uighur living in Xinjiang Uighur Autonomous Region had quite different genetic background which were quite distant from the subpopulations of Chinese Han, Hui and Mongol populations [18, 19]. The study of genetic differentiation across populations would shed insight into the genetic basis of hyperbilirubinemia risk in Asian newborns.

Methods

Study design and sample collection

This study was conducted in the obstetric and newborn ward of Maternal and Child Care Center in Urumqi, Xinjiang Uighur Autonomous Region, China. The study was approved by Ethics Committee of Yangtze University and the hospital.

After informed consent was obtained from the maternal, umbilical cord blood specimens were collected from the newborns born in the obstetric center of Urumqi from March 2018 to December in 2019 by the obstetrician. Clinical records including the birth date, ethnic group, birth weight, delivery method, gestational age, the subsequent transcutaneous bilirubin (TCB) levels in forehead and chest within 1–7 hospital days were collected, Apar score were reviewed. Eligible neonates were those single vaginal delivered term neonates with a gestational age of more than 37 weeks, a birth weight >2500 g and no major birth abnormalities and serious illness, Apar = 10. The ethnic of the newborns was inferred from the self-reported demographic information (name and race) of their parents by reviewing the clinical record. If both their parents were Uighur or Han newborns were included in this study.

Additionally, peripheral blood samples were collected from newborns with continuous hyperbilirubinemia in the pediatric center of the studied hospital between March to September in 2018. Samples were collected and stored at -20°C after completion of the routine blood test and the data were analyzed anonymously. The ethical aboard approved a waiver of written consent.

Hyperbilirubinemia was diagnosed and treated according to the updated clinical guidelines of the Chinese Medical Association for neonates [20]. The recorded peak TCB and/or TSB was used to divide the study subjects into case and control subjects. The case subjects included jaundiced neonates with a maximum TSB /TCB that reached or above P95 percentile value of the hour-specific TSB/TCB nomogram drawn by Chinese Multicenter Study Coordination Group for Neonatal Hyperbilirubinemia [21].

Molecular analysis

The genomic DNA was extracted from surplus EDTA anti-coagulated cord blood specimens using FlexiGene DNA Kit (Qiagen Inc, Valencia, California). The promoter, all five exons, and exon-intron boundaries, of UGT1A1 were tested by polymerase chain reaction (PCR) amplification with 5 pair of primers and direct sequencing as previously described [15–17]. Repeat polymorphism (TA)n in the promoter region was further confirmed by capillary electrophoresis analysis, detailed in our previous studies [15].

Data analysis

Hardy-Weinberg equilibrium (HWE) test and Linkage disequilibrium (LD) analysis for the identified variants in the UGT1A1 locus was performed by using the web tool SNPStats (https://snpstats.net/start.htm), as we have done in our previous study [16, 17].

Differences in the categorical variables within the two groups were compared by Chi-square test or Fisher’s exact test. Independent group t-test or was used to analyze the difference of continuous variables if the dataset was normally distributed; otherwise, the Mann-Whitney test was used.

A linear regression model was used to assess the association between specific polymorphisms or haplotypes of UGT1A1 and the recorded peak TcB values. Logistic regression models were performed to evaluate the association between the specific UGT1A1 variants and the development of neonatal hyperbilirubinemia categorized by the recorded peak TCB levels. Multiple logistic regression analysis was used to evaluate the independence of genetic variants of UGT1A1 with the neonatal hyperbilirubinemia risk after adjusting for known clinical risk factors for neonatal hyperbilirubinemia, including sex, age and ethnic group. The independent variable has been “case” or “control”; a step-down procedure retaining only those variables with P value <0.1 was used. UGT1A1 genotype was forced into the model. Furthermore, the association analysis for each UGT1A1 variant was performed under different genetic model assumptions (co-dominant, dominant or recessive).

All analyses were conducted using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). P< 0.05 was considered statistically significant.

Results

Demographic and clinical characteristics

Finally, a total of 201 term newborns (102 Uighur and 99 Han) from the obstetric ward, together with 19 term newborns (10 Uighur and 9 Han) with significant hyperbilirubinemia (TSB>171umol/L) enrolled in the pediatric center of the studied hospital were taken into the final analysis.

Basic demographic characteristics of 201 term newborns without major birth abnormalities and serious illness were summarized in Table 1. Newborns of Han ethnic group and Uighur ethnic group were analyzed separately. Of the selected factors obtained from chart records, except for the mean TCB levels, the other factors including gestational age, gender, birth weight showed no statistically significant difference between the Han and Uighur newborns. Newborns of Han population has a significantly higher TCB level compared with Uighur newborns. Moreover, the incidence of neonates with high-risk TCB level (according to the hour-specific transcutaneous bilirubin nomogram for neonatal hyperbilirubinemia by the Chinese Multicenter Study Coordination Group) within 1–3 days showed statistically higher in Han normal term newborns (39/73) than in Uighur normal term newborns (25/76) (P = 0.01).

Table 1. Demographic and clinical features of neonates in Han and Uighur group.

	Uighur (n = 102)	Han (n = 99)	P
Sex			NS
male	52(51.0%)	52(52.5%)
female	50(49.0%)	47(47.5%)
Gestational age(week)	39.7±0.11	39.6+0.09	NS
Birth weight(kg)	3.45±0.04	3.27±0.05	NS
Maximum TCB(mg/dl)
Head TCB	10.71±0.29	11.91±0.27	0.003
Chest TCB	11.03±0.29	12.15±0.28	0.006
Neonate jaundice			0.01
Yes (TCB≥12.9mg/dl)	25(32.9%)	39(53.3%)
No (TCB<12.9mg/dl)	51 (67.1%)	34(46.6%)
			-

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NS: No significant

UGT1A1 variant

In addition to the variation in the promoter UGT1A1*28 [A(TA)6TAA/A(TA)7TAA (6/7), rs81753472] and phenobarbital response enhancer module of UGT1A1(-3275 T>G, rs4124874), two variant sites within the coding region of this gene were identified, includingUGT1A1*6(c.211G>A, p.Arg71Gly, rs4148323), *73(c.1091C>T, p.Pro364Leu, rs34946978) (Fig 1). c.211G>A mutation was the predominant exon mutation observed in the study cohort. Specially, the total exon mutation incidence, homozygous mutation rate and heterozygous mutation rate of UGT1A1 were 2.1% (2/96) and 22.9% (22/96) in Uighur newborns, and 2.3% (2/86) and 29.1% (25/86) in Han newborns. As to the (TA)n polymorphism, four kinds of genotype (TA)5/6, 6/6, 6/7 and 7/7 were found (Fig 2), the frequency of TA6/TA7 were 39.2% (40/102) in Uighur newborns and 23.4% (22/94) in Han newborns. Genotype (TA) 7/7 and (TA) 5/6 were observed in one case each in Uighur newborns.

Fig 1 — (A) c. 211 G >A heterozygote (Gly71Arg); (B) c. 211 G >A homozygote (Gly71Arg) (C) c.1091C>T heterozygote (Pro364Leu).

Fig 2 — a(1–2) (TA)6 /(TA)6 homozygote; b(1–2) (TA)6/(TA)7 heterozygote. c(1–2) (TA)7 /(TA)7 homozygote; d(1–2) (TA)5 /(TA)6 homozygote.

None of the polymorphisms showed statistically significant deviations from the HWE in the study subjects except T-3279G polymorphism, which was excluded from the sub-analysis. A strong pairwise LD was observed between the two identified coding SNP (|D|’ = 0.9566, rs4148323 and rs34946978).

There was no statistical difference in c.211G>A mutation frequency between Han and Uighur group, but there was statistically significant difference of (TA)n polymorphism in the promoter region of UGT1A1 gene between Han and Uighur group (P = 0.02) (Table 2).

Table 2. Genotypes frequency of c.-3275T>G in enhance, (TA)_n repeat polymorphism and *6 (c.211G > A, p.Arg71Gly) variant of UGT1A1 gene in Han vs Uighur groups.

	Han		Uighur		P _allele	P _genotype
	n	P _H-W ^a	n	P _H-W ^a	P _allele	P _genotype
(TA)_n		0.35		0.07	0.02	0.02
TA₅/ TA₆	0		1
TA₆/ TA₆	72		60
TA₆/ TA₇	22		40
TA₇/ TA₇	0		1
G211A variation		1		0.68	0.38	0.63
G/G	59		72
G/A	25		22
A/A	2		2
c.-3275T>G		0.32		0.03	0.33
T/T	28		28			0.04
T/G	29		66
G/G	13		16

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a: Hardy-Weinberg Equilibrium test p value.

UGT1A1 variant and neonatal hyperbilirubinemia

Neonates with homozygous and heterozygous UGT1A1 coding mutation (c.211 G>A and/or c.1091 C>T) showed higher TCB level as compared to those with wild UGT1A1 genotype in both Han and Uighur neonate after adjusted for the potential covariance. (OR_adj = 2.36, 95%CI = 1.14–4.88, P = 0.002), while this trend was not observed for the (TA)_n promoter variant (Table 3). Moreover, when analysis of TCB level in neonates clustered according to the UGT1A1 haplotype, it was showed that homozygous mutation including combined heterozygotes variants in the promoter and the exon region of UGT1A1 significantly increased the TCB level of the studied neonates (P<0.05) (Table 4).

Table 3. The associations between TCB level and UGT1A1 mutation adjusted by age, gender and race: Linear regression analysis.

		Han			Uygur			Total
	Mode^#	OR_adj^† (95%CI)	P	Mode^#	OR_adj^† (95%CI)	P	Mode^#	OR_adj^† (95%CI)	P
TATA box	-		NA	Recessive		0.22	Recessive		0.26
TA6/TA6+TA6/TA7		reference			reference			reference
TA7/TA7		-			3.87(-2.21–9.95)			3.85(-2.88–10.57)
c.211 G>A	Dominant		0.046	Recessive		0.02	Dominant		0.03
G/G		reference			reference			reference
G/A		1.78(0.06–3.51)			reference			1.31(0.14–2.49)
A/A		1.78(0.06–3.51)			3.82(0.07–6.93)			1.31(0.14–2.49)
Compound exon mutation	Dominant		0.046	Recessive		0.005	Dominant		0.02
Wildtype		reference			reference			reference
Heterozygote		1.78(0.06–3.51)			reference			1.41(0.25–2.57)
Homozygote ^*		1.78(0.06–3.51)			3.74(1.19–6.29)			1.41(0.25–2.57)

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^# In the linear regression analysis, neonates with wild UGT1A1 genotype (i.e. G/G) was set as reference group under the dominant genetic model assumption, while both those wildtype and the heterozygous UGT1A1 variant carrier (i.e. G/G+G/A) were set as the reference group under the recessive model.

NA: not available

†Adjusted for gender, birth week, race.

*Including compound heterozygous c.211 G>A variant plus heterozygous c.1091 C>T variant.

Table 4. TCB levels of study neonates in the subgroup divided by UGT1A1 genotype.

			Han		Uygur		Total
		n	TCB(mg/dl)	n	TCB(mg/dl)	n	TCB(mg/dl)
Haplotype (TA _n -rs4148323)
①66GG	37		12.17±2.85	33	11.25±3.43	70	11.74±3.15
②67GG	14		13.00±5.05	26	10.56±3.14	40	11.41±4.03
③66GA	23		13.32±3.07	13	10.78±3.68	36	12.40±3.48
④67GA	2		16.20±6.81	4	11.04±2.70	6	12.88±4.56
⑤66AA	4			3	14.16±2.55	7	14.91±5.06
⑥67AA	0			2		2
⑦77AA	0			1		1
Model 1
①	37		12.17±2.85	33	11.25±3.43	70	11.74±3.15
②+③	37		13.20±3.88	39	10.64±3.28	76	11.88±3.79
④+⑤+⑥+⑦	6		16.20±6.81	10	12.91±2.94	16	14.15±4.83
P _a			0.047/0.07/0.016		0.155/0.056/0.169		0.05/0.025/0.018
Model 2
①+②	51		12.4±3.55	59	10.95±3.29	110	11.62±3.48
③+④	25		13.58±3.35	39	10.84±3.40	42	12.47±3.59
⑤+⑥+⑦	4		16.03±8.0	6	14.16±2.55	10	14.91±5.06
P _b			0.114/0.23/0.067		0.072/0.025/0.036		0.017/0.053/0.007

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a: Difference comparison for ① vs ②+③ vs ④+⑤+⑥+⑦ and for ②+③ vs ④+⑤+⑥+⑦ and ① vs④+⑤+⑥+⑦ within the 3 group.

b: Difference comparison for ①+② vs ③+④ vs ⑤+⑥+⑦ and for ③+④ vs ⑤+⑥+⑦ and ①+② vs⑤+⑥+⑦ within the 3 group.

In addition, logistic regression indicated that exon mutation (c.211 and /or c.1091) in UGT1A1 gene was associated with increased risk of hyperbilirubinemia in both Han and Uighur neonates. This strong association of UGT1A1 exon mutation with neonatal hyperbilirubinemia remained statistically significantly after adjusting for known clinical risk factors for neonatal hyperbilirubinemia including gender, age and ethnic group. Specifically, neonates who carried heterozygous or homozygous variation in the exon of UGT1A1 had a 2.21(95%CI:1.09–4.49) fold risk of hyperbilirubinemia as compared with those having the wild genotype (P = 0.03). Moreover, the risk of hyperbilirubinemia was even higher in those newborns with a homozygous exon mutation [9.26 (95%CI: 1.77–48.39), P = 0.008]. As for the (TA)7 promoter variant, homozygous (TA)7 also seemingly associated with increased risk of neonatal hyperbilirubinemia (P = 0.05, Table 5). Similarly, haplotype analysis showed that (TA)6-A ((TA)n -rs4148323) significantly increased the risk of neonatal hyperbilirubinemia (OR_adj = 2.23; P = 0.015) compared to the most common haplotype (TA)6-G.

Table 5. Associations of UGT1A1 variants and neonate hyperbilirubinemia under different inheritage model assumptions: Logistic regression analysis.

	N(%)			Model 1 ^#				Model 2 ^#
	Case	Control		Model	OR _adj (95% CI)^†	P	Model	OR_adj (95% CI)^†	P
(TA) _n				Codominant		0.1	Recessive		0.05
TA6/TA6	55(0.71)	69(0.15)			1			1
TA6/TA7	20(0.26)	36(0.34)			0.73(0.36–1.49)			1
TA7/TA7	2(0.03)	0(0.00)			0 (0.00-NA)			0 (0.00-NA)
c.211G>A				Codominant		0.02	Dominant		0.03
G/G	44(0.60)	67(0.74)			1			1
G/A	22(0.30)	22(0.24)			1.70(0.79–3.63)			2.15(1.05–4.88)
A/A	7(0.10)	2(0.02)			7.98(1.46–43.56)			2.15(1.05–4.88)
Compound exon mutation				Codominant		0.008	Dominant		0.03
Wildtype	43(0.59)	67(0.74)			1			1
Heterozygote	21(0.29)	22(0.24)			1.61(0.75–3.48)			2.21(1.09–4.49)
Homozygote ^*	9(0.12)	2(0.02)			9.26(1.77–48.39)			2.21(1.09–4.49)
Haplotype analysis (TAn-rs4148323)	Frequency
TA6-G	0.60		0.69		1
TA6-A	0.24		0.14		2.01(1.06–3.79)	0.03
TA7-G	0.13		0.17		0.92(0.44–1.96)	0.84
TA7-A	0.03		0.00		4.82(0.38–60.76)	0.22

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# (0, 1,2) set on the three genotype (wildtype, heterozygote and homozygote) for each variant under the codominant genetic model assumption by multiple regression analysis; (0, 1) set on the genotype data (eg: GG+G/A vs AA; GG vs G/A+AA) for each variant under recessive or dominant inheritance model by binary test.

†Adjusted for gender, birth week, race.

* Including compound heterozygous c.211 G>A variant plus heterozygous c.1091 C>T variant.

Discussion and conclusion

Asian neonates were regarded as high-risk population for severe hyperbilirubinemia. The genetic background across population was an important risk factor modulating this disorder. UGT1A1, one of main factors for pathologic hyperbilirubinemia in newborns, varied within and between populations [8, 22]. In this study, we found that the incidence of neonatal hyperbilirubinemia was much higher in full-term Chinese Han newborns than in Uighur newborns of Chinese population. Notably, our data also showed a significant different distribution of promoter variation in UGT1A1- encoding for hepatic bilirubin metabolism enzyme in Han and Uighur. Taken together, we inferred that promoter TATA box variation might be a risk factor that modulated neonatal hyperlipidemia risk in Chinese Han and Uighur newborns. Our results were consistent with Beutler et al. study in African, Europe and Asia population, which has firstly reported the genetic difference of TA7 and serum bilirubin level among populations, they proposed that the (TA)n repeat might be a balanced polymorphism evolutionarily selected to maintain serum bilirubin in an optimal range in the face of largely undefined genetic and environmental pressures [23]. Our study further confirmed this hypothesis. Moreover, considering the difference of TA7 mutation rate and bilirubin level in Asia and Caucasian population, the data obtained in the study added more evidence to previous genetic studies in Uighur ethnic group which has suggested that Uighur ethnic group was a gene admixture of Eastern Asian and European populations, and much closed to European than other ethnics in China [18, 19].

UGT1A1 coding sequence variant c.211 G>A (UGT1A1*6, G71R), common variant predominant in Asia population while not found in Caucasian population, was the main cause of GS in Asia population [11, 12]. In this study, we confirmed the strong association of the UGT1A1 coding sequence variant c.211 G>A (UGT1A1*6, G71R) with neonatal hyperbilirubinemia risk in both Han and Uighur newborns in China. Moreover, our data showed no statistic differences in the frequency of c.211G>A variant among Chinese Han and Uighur newborns. Therefore, on the one hand, the results here demonstrated that Chinese ethic groups shared a clade, though high genetic differentiation exists among Uighur minority and other Chinese ethnic groups. At the same time, it further demonstrated the necessity of screening of this exon mutation to identify high-risk neonates who tend to be suffered from severe neonatal hyperbilirubinemia in all Chinese ethnic groups. On the other hand, our results revealed that c.211 variant could not explain the difference of neonatal hyperbilirubinemia risk in Chinese Han and Uighur newborns, though its predominant role in Chinese neonatal hyperbilirubinemia risk was certain.

As described previously, the (TA)n repeat variant in the UGT1A1 promoter was another extensively studied variant. As for the influence of genetic factors on the UGT1A1 enzyme activity, previous studies believed that the transcription level of UGT1A1 was mainly affected by TATA polymorphisms in the UGT1A1 promoter [23]. Promoters containing seven thymine adenine(ta) repeats have been found to be less active than the wild-type six repeats, and the serum bilirubin levels of persons homozygous or even heterozygous for seven repeats have been found to be higher than those with the wild-type six repeats [24–26]. However, the role of this variant on neonatal hyperbilirubinemia risk was not yet defined. i.e. A(TA)7TAA variations (UGTA*28), was described as the common cause of Gilbert’s syndrome in the Caucasian population, whereas most previous studies in east Asian countries failed to find this association [27, 28]. Recently, one meta-analysis had identified that homozygous while not heterozygous (TA)7 was associated with increased risk of neonatal hyperbilirubinemia in both Asia and European population [29]. In this study, two case of homozygous (TA)7 was observed in Uighur neonate with significant hyperbilirubinemia. Further large-scale population research was necessary to verify the effect of TATA box on the risk of hyperbilirubinemia in the newborns.

We acknowledged that this study has some limitations. First, this study was limited by the small number of neonates enrolled. Some of the trends observed in the study could have reached statistical significance if the study sample had been larger (P = 0.05, Tables 4 and 5). Second, our inability to assess the serum bilirubin for most of the studied neonates, so we used TCB instead of TSB to subgroup our study cohort. Previous studies have shown a linear relationship exists between TcB and TSB, though the correlation significantly decreased when the TCB was high(>229umol/l) [30]. Recently, a system review and meta-analysis by Yu et al also showed that TCB nomogram was as efficient as TSB nomogram for identifying subsequent significant hyperbilirubinemia [31]. Thus, it’s applicable to use TcB nomograms to identify neonatal hyperbilirubinemia in this study.

In conclusion, screening for UGT1A1 coding region 211 G to A variation should be taken into consideration to identify the risk group who tend to be suffered from severe neonatal hyperbilirubinemia in all Chinese populations. What’s more, our results supported previous hypothesis that the importance of UGT1A1 promoter polymorphism in regulation of plasma bilirubin level within ethnic groups. In future studies, it should be necessary to increase the number ethnic groups analyses in order to assess more data about the genetic basis of neonatal hyperbilirubinemia risk from the main ethnic groups of the Chinese population.

Supporting information

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Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was funded by Natural Science Foundation of China (81801509). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0279059.r001

Decision Letter 0

Mingqing Xu

5 Jan 2022

PONE-D-21-33656UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemiaPLOS ONE

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Reviewer #1: In “UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia”, the authors aim to identify genetic variants that are associated with neonatal hyperbilirubinemia in Chinese Uighur and Han populations. Given a higher prevalence of hazardous hyperbilirubinemia in some populations, it is imperative to understand the underlying genetic, and thus, biological mechanisms of this condition. By analyzing populations with varying ancestral backgrounds, the authors aim to identify genetic variants that are associated with hyperbilirubinemia.

There are several major limitations to the current manuscript. One, the datasets are very small and without general population-level information (common population allele frequencies) about the genetic variants , it is difficult to ascertain the strength of the associations presented. Secondly, greater clarity is needed in regards to the statistical analysis methods and presentation of results. As they are currently formatted, the tables (e.g., Tables 3 and 4) are confusing. In their current state it is unclear how many tests were computed (important for multiple comparison correction). It is also currently unclear why some rows appear to be missing p-values or data (more detail, below). If there were count thresholds (e.g. allele counts) that were used to exclude certain comparisons (e.g., in instances of complete separation between cases and controls), then that should be explicitly explained in the methods and table captions.

Details----------

Introduction:

Need an reference for introduction statement “quite distant from the subpopulations of Chinese Han, Hui, and Mongol populations”. Is this based in genetic admixture and ancestral analyses?

Methods:

Given the usage of chi-square or Fisher’s exact test, need to clarify that whether all participants were unrelated, as this is an important methodological consideration. Statistical assumptions of independence would be invalidated if participants were genetically related (e.g., inclusion of siblings or cousins).

Data Analysis: How were the two common variants in the UGT1A1 locus identified? In Reference 15, there are 4 variants within the UGT1A1 locus. Was a specific MAF required? Was this only coding variants?

It would be best to use rsIDs when possible, throughout the paper. This enables readers to more easily connect the presented results to past publications. For instance, it appears that the Arg71Gly corresponds to rs4148323, based on dbSNP and Table 4. However, I’m unable to identifiy the rsID for Pro364Leu, can this be clarified?

Is there a difference between using TCB or TSB for diagnosis of hyperbilirubinemia? Is there a potential source of variation (unintended bias) introduced by using different methods for diagnosis? Can the authors show that there was equal (not-skewed) usage fo TSB/TCB across groups? The usage of two methods is mentioned in the discussion (page 13) but the implication is not explained.

Can the authors clarify how genotype was coded within the logistic regression models? Was this an additive model (e.g., coded 0,1,2) or was it a binary test (0,1) for each genotype (e.g., GG vs G/A+AA; G/A vs GG+AA). In Table 4, it is unclear why some cells appear inconsistently incomplete -please explicitly specify (e.g. in the table caption) which tests were run.

Table 3 appears incomplete. Please better clarify why p-values are presented for some comparisons, but not others. Also, total counts for Compound Exon Mutation are not filled-in for Controls. What does the p=0.015 refer to under Heterozygote for compound exon mutation? Based on the data, it appears the comparison (and statistical test) should be between 20 cases and 20 controls (which presumably does not yield a p-value of 0.015). Please ensure the appropriate data is presented.

Furthermore, the authors should better clarify the exact comparisons for each p-value. What does the p-value of 0.046 assess (under TAn/ 7/7)? From the table, it appears that it compares the 7/7 repeat between cases and controls, but this would not be a statistically feasible comparison with complete separation of counts (0 7/7 copies for controls).

Results:

Given the proximity of the tested variants, what is the linkage disequilibrium among the coding SNPs?

Could the authors include a graph that shows the TSB/TCB levels by genotype and case/control status? It would be helpful to see the effect, respective to the outcome.

Figures:

In figure 1, there is a Typo in parts A and C for heterozygote.

Reviewer #2: In the manuscript entitled “T UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia”, the authors conducted an association test by use of 102 Uighur and 99 Han normal term neonates. The novelty is limited and the scientific questions are not addressed deeply.

(1) I suggest the authors conduct a meta-analysis based on the selected SNPs. to see if the findings are specific to a give ethnic or to all populations, the following papers can be cited and followed for the meta-analytic procedures (if the data is not enough available, at least DISCUSSION should be added as the LIMITATION of this study with enough citation to support the viewpoints): Ref 1: Wu Y, et al. Multi-trait analysis for genome-wide association study of five psychiatric disorders. Transl Psychiatry. 2020 Jun 30;10(1):209. Ref 2: Jiang L, et al. Sex-Specific Association of Circulating Ferritin Level and Risk of Type 2 Diabetes: A Dose-Response Meta-Analysis of Prospective Studies. J Clin Endocrinol Metab. 2019 Oct 1;104(10):4539-4551. Ref 3: Wang X, Wu W, Zheng W, Fang X, Chen L, Rink L, Min J, Wang F. Zinc supplementation improves glycemic control for diabetes prevention and management: a systematic review and meta-analysis of randomized controlled trials. Am J Clin Nutr. 2019 Jul 1;110(1):76-90.

(2) The interactions between environmental and biological factors should also be explored to see specific SNPs are respond to the environmental factors, especially I suggest using mendelian randomization analysis to test if UGT1A1 promoter polymorphism causally trigger the risk of neonatal hyperbilirubinemia through mediating the plasma bilirubin level. If cannot, please discuss the limitations in the Discussion in detail with additional citations to support the viewpoints. For these reasons, the following papers regarding causal inference between genetic variants, inter-mediator phenotype and disease outcome can be cited and followed.

Reference 1: Fuquan Zhang, Ancha Baranova, Chao Zhou, et al. Causal influences of neuroticism on mental health and cardiovascular disease. Human Genetics. 2021 May 1

Reference 2:Fuquan Zhang, et al. Genetic evidence suggests posttraumatic stress disorder as a subtype of major depressive disorder. Journal of Clinical Investigation. 2021 May 30

Reference 3:Xinhui Wang, et al. Genetic support of a causal relationship between iron status and type 2 diabetes: a Mendelian randomization study. The Journal of Clinical Endocrinology & Metabolism. 2021 June 19

(3) In addition, the significantly associated SNPs may be used to predict disease susceptibility, therefore, the authors may explore the possibility to conduct a machine-learning model to predict disease risk based these significant SNPs. For this reason, the authors may cite the following papers to follow these references’ procedure to construct a standard prediction model based on the significant SNPs (probably include the environmental factors). Especially deep learning method is a very promising way to predict disease risk based on clinical information and genetic biomarkers (If deep learning can not be used, please discuss as the LIMITATION of this study with enough citation to support the viewpoints). Ref 4: Yu H, et al. LEPR hypomethylation is significantly associated with gastric cancer in males. Exp Mol Pathol. 2020 Oct;116:104493. Ref 5: Liu M, et al. A multi-model deep convolutional neural network for automatic hippocampus segmentation and classification in Alzheimer's disease. Neuroimage. 2020 Mar;208:116459.

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PLoS One. 2022 Dec 15;17(12):e0279059. doi: 10.1371/journal.pone.0279059.r002

Author response to Decision Letter 0

19 Feb 2022

Journal Requirements:

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This study was funded by Natural Science Foundation of China (81801509).

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Answer: The statement for funder

("The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.") is corrected and not need to amend.

Answer: Thank you for your suggestion. We have removed the phrase that refers to these data as suggested in the revised manuscript.

4,5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Answer: Thank you for your suggestion. We have revised and upload the manuscript as suggested and carefully proof-read the manuscript to minimize the mistakes and typographic, grammatic errors.

#Responds to Review 1

There are several major limitations to the current manuscript.

One, the datasets are very small and without general population-level information (common population allele frequencies) about the genetic variants , it is difficult to ascertain the strength of the associations presented.

Secondly, greater clarity is needed in regards to the statistical analysis methods and presentation of results. As they are currently formatted, the tables (e.g., Tables 3 and 4) are confusing. In their current state it is unclear how many tests were computed (important for multiple comparison correction). It is also currently unclear why some rows appear to be missing p-values or data (more detail, below). If there were count thresholds (e.g. allele counts) that were used to exclude certain comparisons (e.g., in instances of complete separation between cases and controls), then that should be explicitly explained in the methods and table captions.

Answer: Thank you for your suggestion. As the review point that the study is limited by the small size of the study cohort. Our current study is an observation study. However, the outbreak of COVID-19 makes the sample collection really hard. As we described in the method, all the studied sample were collected from 2018.3-2019.12, and stopped when the outbreak of covid-19 in Wuhan in the initial of 2020. Finally, a total of 102 Uighur and 99 Han normal term neonates. We fully acknowledge this limitation. Further large-scale population study is need to identify the association of UGT1A1 mutation and neonatal hyperbilirubinemia in multiple ethnic groups of the Chinese population. We have presented this limitation in the discussion part of the revised manuscript. Also, there are some errors in the table 3 and table 4. We have revised these errors as suggested and marked red in the new version.

Details----------

Introduction:

Need an reference for introduction statement “quite distant from the subpopulations of Chinese Han, Hui, and Mongol populations”. Is this based in genetic admixture and ancestral analyses?

Answer: Thank you for your suggestion. Two references [18, 19] about the genetic admixture analysis of Chinese Uighur and other Chinese population have been cited in the revised version.

[18] Suhua Zhang, Yingnan Bian, Li Li, Kuan Sun, Zheng wang, Qi Zhao, et al. Population genetic study of 34 X-Chromosome markers in 5 main ethnic groups of China. Sci Rep 2015; 5: 17711.

[19] Zhang Z, Wei S, Gui H, Yuan Z, Li, S. The contribution of genetic diversity to subdivide populations living in the silk road of China. PLoS One 2014; 9, e97344

Methods:

Answer: Thank you for your suggestion. All the studied participants (the pregnancy woman and newborn) were recruit from one single hospital from March 2018 to December 2019. And twin gestation was excluded. It’s certain that there are no siblings in our studied neonates. However, whether cousins were included in our studied neonates that was unable to judge according to the information we obtained.

Answer: Thank you for your suggestion. As we described in the manuscript, the promoter, all five exons, and exon-intron boundaries, and a region in the distal promoter (the Phenobarbital response enhancer module) of UGT1A1 were tested by polymerase chain reaction (PCR) amplification with 5 pair of primers and direct sequencing. All the UGT1A1 variants including the two common variants-(TA)n repeat variant(dbSNP rs81753472) and Arg71Gly(rs4148323) were identified by direct sequencing. (TA)n repeat variant was further confirmed by capillary electrophoresis analysis.

In Reference 15, there are 4 variants within the UGT1A1 locus. Was a specific MAF required? Was this only coding variants?

Answer: Thank you for your suggestion. The kind and MAF of UGT1A1 variants were varied in different population. As you see, 4 kinds of coding variants were found in reference 15, while, only 2 kinds of UGT1A1 coding variants (Arg71Gly and Pro364Leu) were identified in this study population.

Answer: Thank you for your suggestion, rsID of the identified UGT1A1 variants were added in the revised manuscript.

Answer: Previous studies have shown a linear relationship exists between TcB and TSB, though the correlation significantly decreased when the TCB was high(>229umol/l) [30]. Recently, a system review and meta-analysis by Yu et al also showed that TCB nomogram was as efficient as TSB nomogram for identifying subsequent significant hyperbilirubinemia. The pooled diagnosis AUC of TCB nomogram was 0.817 which showed no significantly different to the summary TSB nomogram (0.819) [31]. Thus, it’s applicable to use TcB nomograms to identify neonatal hyperbilirubinemia in this study. A system review study by Yu et al. have showed that TcB nomograms had the same predictive value as TSB nomograms for diagnosis of hyperbilirubinemia. we have added explanations for it in the revised version.

[30] Frias C,Kolman KB,Mathieson KM.A comparison of transcutaneous and total serum bilirubin in newborn Hispanic infants at 35 or more weeks of gestation.[J].Journal of the American Board of Family Medicine: JABFM,2007,20(3).

[31] Zhang-Bin Yu, Shu-Ping Han, Chao Chen. Bilirubin nomograms for identification of neonatal hyperbilirubinemia in healthy term and late-preterm infants: a systematic review and meta-analysis. World J Pediatr. 2014,(3).211-218.

Answer: Thank you for your question. As the reviewer pointed that the results presented in Tables (table 3 and 4) is confusing, we have amended and modified the presentation of these results in the new tables (table 3 and table 5).

Results:

Given the proximity of the tested variants, what is the linkage disequilibrium among the coding SNPs?

Answer: Thank you for your suggestion. Linkage disequilibrium analysis was done for the coding SNPs. A strong pairwise LD was observed between the two identified coding SNP (|D|’=0.9566, rs4148323 and rs34946978). We have described this in the revised manuscript and marked red.

Could the authors include a graph that shows the TSB/TCB levels by genotype and case/control status? It would be helpful to see the effect, respective to the outcome.

Answer: Thank you for your suggestion. Tables (Table 3 and 4) have added for showing the TCB levels by genotype and case/control status in the new version.

Figures:

In figure 1, there is a Typo in parts A and C for heterozygote.

Answer: Thank you for your suggestion. we have amended the mistake in the new version.

Responds to Review 2

Reviewer 2: In the manuscript entitled “T UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia”, the authors conducted an association test by use of 102 Uighur and 99 Han normal term neonates. The novelty is limited and the scientific questions are not addressed deeply.

Answer: Thank you for your suggestion. Increasing population study has explore the association between UGT1A1 variant and neonatal hyperbilirubinemia，and confirm the important role of UGT1A1 c.211 variant in Asian neonatal hyperbilirubinemia. However, as to the role of TA promoter in serum bilirubin level and neonatal hyperbilirubinemia risk, the result is controversial. Eg: Meta -analysis study conducted in 2011 doesn’t found its association with neonatal hyperbilirubinemia in Asia, though its significant associated with neonatal hyperbilirubinemia in white population [28]. Recently, another meta-analysis have found that TA promoter was significantly associated in both Asia and causian population. The precise role of TA promoter in serum bilirubin level and neonatal hyperbilirubinemia risk merit further study. That’s the reason why we conduct this study in two main different ethic population in China. Our current study is an observation study. However, the outbreak of COVID-19 makes the sample collection really hard. As we described in the method, all the studied sample were collected from 2018.3-2019.12, and stopped when the outbreak of covid-19 in Wuhan in the initial of 2020. Finally, a total of 102 Uighur and 99 Han normal term neonates. We fully acknowledge that the present study is limited by the small study sample sized. We have presented this limit in the discussion part of the revised manuscript.

Answer: Thank you for your suggestion. As the reviewer point that our current study is an observation study which is limited by the small-scale study sample. As we know, there are two papers by Chinese researchers has reported the meta-analysis study on UGT1A1 variant and neonatal hyperbilirubinemia [28, 29], however, the result is somewhat inconsistent. As to the role of TA promoter in serum bilirubin level and neonatal hyperbilirubinemia risk, the meta -analysis study conducted in 2011 doesn’t found its association with neonatal hyperbilirubinemia in Asia, though its significant associated with neonatal hyperbilirubinemia in white population [28]. That’s the reason why we conduct this study in two main different ethic population in China. Recently, another meta-analysis study (2020) has found that TA promoter was significantly associated in both Asia and European population. We have cited these two meta-analysis studies and added the discussion in my revised manuscript.

[28]Long J, Zhang S, Fang X, Luo Y, Liu J. Association of neonatal hyperbilirubinemia with uridine diphosphate-glucuronosyltransferase 1A1 gene polymorphisms: Meta-analysis. Pediatrics International 2011; 53:530-540.

[29]Jing Wang, Jiansong Yin, Mei Xue, Jun Lyu, Yu Wan. Roles of UGT1A1 Gly71Arg and TATA promoter polymorphisms in neonatal hyperbilirubinemia: A meta-analysis. Gene. 2020;736:144409.

Reference 1: Fuquan Zhang, Ancha Baranova, Chao Zhou, et al. Causal influences of neuroticism on mental health and cardiovascular disease. Human Genetics. 2021 May 1

Reference 2:Fuquan Zhang, et al. Genetic evidence suggests posttraumatic stress disorder as a subtype of major depressive disorder. Journal of Clinical Investigation. 2021 May 30

Answer: Thank you for your suggestion. As the reviewer point that both environment and genetic factors contributed to neonatal hyperbilirubinemia. In this study, all our studied neonates (both Han and Uyghur) all from the same area, and in the case-control association analysis, we have adjusted the potential risk factor like birth week, gender and race. We have detailed this in the method part of this manuscript.

Answer: Thank you for your suggestion. Machine-learning model is a very promising way to predict disease risk based on clinical information and genetic biomarkers. We have fully known that our present study is limited by the small size of the study cohort. we would learn to use machine-learning model to further explore the association of ugt1a1 gene mutation and neonatal hyperbilirubinemia in the further research.

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PLoS One. doi: 10.1371/journal.pone.0279059.r003

Decision Letter 1

George Vousden

18 Jul 2022

PONE-D-21-33656R1UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemiaPLOS ONE

Dear Dr. Yang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Your manuscript has been assessed by the two reviewers who reviewed the original submission. Reviewer has provided some comments; please respond to these in your revised manuscript. Please also respond to the requests I have added below my signature.

Please submit your revised manuscript by Aug 29 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

George Vousden

Deputy Editor in Chief

PLOS ONE

Additional Editor Comments:

A) Please outline how ethnic group was determined in your Methods section. Since any method of identifying an ethnic group suffers from limitations, please discuss the limitations of the method(s) used for this manuscript in your Discussion section.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have worked to address previous concerns. The addition of SNP rsIDs, additional references, and clarifications about the differences between TSB and TCB make the manuscript much clearer. The authors have also better clarified the limitations of the study given the sample size. So while these small numbers provide limits on the types of analyses possible, they do present the relative allele counts which could help inform focus of future studies in larger cohorts.

Only remaining comment is on Table 3. It's unclear why some rows have p-values but not odds ratios (and vice versa). Also, the inclusion of 0.00 odds ratios is confusing, is this meant to indicate there were equal numbers of cases/controls with that particular genotype? It might be clearer to list the counts, and then the p-value next to the Odds ratio. If I'm misunderstanding, perhaps horizontal lines would help clarify how the data is meant to align.

Reviewer #2: The authors have improved their manuscript as suggested, questions are all well addressed. I have not additional concerns.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2022 Dec 15;17(12):e0279059. doi: 10.1371/journal.pone.0279059.r004

Author response to Decision Letter 1

19 Aug 2022

Additional Editor Comments:

Please outline how ethnic group was determined in your Methods section. Since any method of identifying an ethnic group suffers from limitations, please discuss the limitations of the method(s) used for this manuscript in your Discussion section.

Answer：Thank you for your suggestion. We have clarified this question in the revised manuscript. In this study, blood samples (umbilical cord and peripheral blood species) of healthy unrelated newborns from the two main ethnic groups (Uygur and HAN) were collected. The ethnic composition of the newborns was determined according to the ethnic identity of his father and mother. Both their parents were Uygur or Han were included in this study. In fact, intermarriage between Uyghur and Han is rare, though it is allowed by the law of China.

Reviewer #1: Only remaining comment is on Table 3. It's unclear why some rows have p-values but not odds ratios (and vice versa). Also, the inclusion of 0.00 odds ratios is confusing, is this meant to indicate there were equal numbers of cases/controls with that particular genotype? It might be clearer to list the counts, and then the p-value next to the Odds ratio. If I'm misunderstanding, perhaps horizontal lines would help clarify how the data is meant to align.

Answer: Thank you for your question. We have adjusted table 3 and make it easier to see as suggested.

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PLoS One. doi: 10.1371/journal.pone.0279059.r005

Decision Letter 2

George Vousden

21 Oct 2022

PONE-D-21-33656R2UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemiaPLOS ONE

Dear Dr. Yang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. The concerns previously noted by the reviewer have been addressed. However, my previous concerns about how ethnicity was determined have not been addressed satisfactorily. It is indicated that "the ethnic composition of the newborns was determined according to the ethnic identity of his father and mother", but how the ethnic composition was determined remains unclear. Please update your Methods section to provide enough details of how ethnicity was determined, such that another researcher could repeat the procedures with the information provided. In your Discussion section, please discuss whether it was possible that the ethnicity of any of the participants was incorrect.

Please submit your revised manuscript by Dec 05 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

George Vousden

Deputy Editor in Chief

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

**********

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

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Yes, in revisiting Table 3, it is now clearer why certain genotypes (e.g., those that were the reference genotypes) did not have test results.

The samples sizes are small, but the authors highlight this in limitations and list the counts.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

PLoS One. 2022 Dec 15;17(12):e0279059. doi: 10.1371/journal.pone.0279059.r006

Author response to Decision Letter 2

24 Oct 2022

Response to the academic editor and reviewers

1. The concerns previously noted by the reviewer have been addressed. However, my previous concerns about how ethnicity was determined have not been addressed satisfactorily. It is indicated that "the ethnic composition of the newborns was determined according to the ethnic identity of his father and mother", but how the ethnic composition was determined remains unclear. Please update your Methods section to provide enough details of how ethnicity was determined, such that another researcher could repeat the procedures with the information provided. In your Discussion section, please discuss whether it was possible that the ethnicity of any of the participants was incorrect

Answer：Thank you for your suggestion. We have clarified this question more precisely in the revised manuscript. The ethnic of the newborns was inferred from the demographic information (name and race) of their parents by reviewing the clinical record. Both their parents were Uygur or Han were included in this study. In fact, there are so many differences between Uygur and Han, such as their appearance, costume, linguistic, naming way and so on, so that it was not difficult to differential one from the other. Moreover, the demographic information of the study participants was carefully check not less than three times by us, it was not possible that mistaken the ethnicity of any of the participants.

Journal Requirements:

Answer：No change.

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PLoS One. doi: 10.1371/journal.pone.0279059.r007

Decision Letter 3

George Vousden

8 Nov 2022

PONE-D-21-33656R3UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemiaPLOS ONE

Dear Dr. Yang,

Please submit your revised manuscript by Dec 23 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

George Vousden

Deputy Editor in Chief

PLOS ONE

Journal Requirements:

Editor Comments:

Thank you for revising your manuscript in response to my previous comments. Please revise the manuscript to respond to the following minor concerns. Once these concerns are addressed the manuscript will be ready for publication:

1) It is noted that "The ethnic of the newborns was inferred from the demographic information (name and race) of their parents by reviewing the clinical record.". Please indicate how race/ethnicity was originally determined when clinical data was collected - please indicate whether ethnicity was self-reported by patients, or whether this was inferred by clinicians.

2) Suggest to revise the sentence "Both their parents were Uygur or Han were included in this study." to " If both their parents were Uygur or Han newborns were included in this study."

3) Please remove this sentence "In fact, there are so many differences between Uygur and Han, such as their appearance, costume, linguistic, naming way and so on, so that it was not difficult to differential one from the other"

4) Please ensure the spelling of Uyghur is consistent throughout the manuscript, as several spellings are currently used (e.g. Uighur in the title and Uygur in the abstract and other sections of the manuscript)

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2022 Dec 15;17(12):e0279059. doi: 10.1371/journal.pone.0279059.r008

Author response to Decision Letter 3

9 Nov 2022

Response to the editor

Answer：Thank you for your suggestion. Demographic information including the race/ ethnicity was self-reported by the patients on their admission. We have detail this in the revised manuscript and marked red.

2) Suggest to revise the sentence "Both their parents were Uygur or Han were included in this study." to " If both their parents were Uygur or Han newborns were included in this study."

Answer：Thank you for your suggestion. I have done the modification as you suggested in the revised manuscript.

Answer：Thank you for your suggestion. We have done the revision as suggested.

Answer：Thank you for your suggestion. I have done the modification as suggested in the revised manuscript.

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PLoS One. doi: 10.1371/journal.pone.0279059.r009

Decision Letter 4

George Vousden

1 Dec 2022

UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia

PONE-D-21-33656R4

Dear Dr. Yang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

George Vousden

Deputy Editor in Chief

PLOS ONE

Additional Editor Comments:

1) A word is missing from the revised manuscript - I suggested that the sentence "If both their parents were Uygur or Han newborns were included in this study.". However, the word "newborns" is missing from the manuscript. Please revise.

2) We note that you have indicated that “All relevant data are within the manuscript and its Supporting Information files”. However, members of the editorial team have assessed the provided datafiles and are concerned that the data provided do not meet our expectations for minimal datasets. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (see (https://journals.plos.org/plosone/s/data-availability). For example, authors should submit the following data:

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> The points extracted from images for analysis.

Please ensure that you have provided a datafile to meet these requirements with your manuscript or provide relevant accession numbers in your Data Availability Statement.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0279059.r010

Acceptance letter

George Vousden

6 Dec 2022

PONE-D-21-33656R4

UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia

Dear Dr. Yang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

UGT1A1 variants in Chinese Uighur and Han newborns and its correlation with neonatal hyperbilirubinemia

Hui Yang

Huijun Li

Qingyao Xia

Wencheng Dai

Xin Li

Yan Liu

Jie Nie

Fei Yang

Yunfeng Sun

Lei Feng

Liye Yang

Roles

Abstract

Introduction

Methods

Study design and sample collection

Molecular analysis

Data analysis

Results

Demographic and clinical characteristics

Table 1. Demographic and clinical features of neonates in Han and Uighur group.

UGT1A1 variant

Fig 1. Mutations of UGT1A1 found in our study cohort.

Fig 2. A typical chromatograph of capillary electrophoresis of UGT1A1 (TA)n promoter polymorphism followed by direct sequencing.

Table 2. Genotypes frequency of c.-3275T>G in enhance, (TA)n repeat polymorphism and *6 (c.211G > A, p.Arg71Gly) variant of UGT1A1 gene in Han vs Uighur groups.

UGT1A1 variant and neonatal hyperbilirubinemia

Table 3. The associations between TCB level and UGT1A1 mutation adjusted by age, gender and race: Linear regression analysis.

Table 4. TCB levels of study neonates in the subgroup divided by UGT1A1 genotype.

Table 5. Associations of UGT1A1 variants and neonate hyperbilirubinemia under different inheritage model assumptions: Logistic regression analysis.

Discussion and conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Mingqing Xu

Roles

Author response to Decision Letter 0

Decision Letter 1

George Vousden

Roles

Author response to Decision Letter 1

Decision Letter 2

George Vousden

Roles

Author response to Decision Letter 2

Decision Letter 3

George Vousden

Roles

Author response to Decision Letter 3

Decision Letter 4

George Vousden

Roles

Acceptance letter

George Vousden

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Table 2. Genotypes frequency of c.-3275T>G in enhance, (TA)_n repeat polymorphism and *6 (c.211G > A, p.Arg71Gly) variant of UGT1A1 gene in Han vs Uighur groups.