| Neonatal hyperbilirubinemia and resultant jaundice are common in 50%-60% of newborn children and to a greater extent in premature infants. Although most cases are benign and do not result in serious consequences, a small number of infants will develop hazardous levels of bilirubin that pose a direct threat of brain damage, and which may result in neuro-developmental abnormalities, such as hearing loss, athetosis, and, rarely, intellectual deficits. Nowadays, phototherapy and blood transfusion are available and effective treatment for neonatal hyperbilirubinemia, however, hazardous hyperbilirubinemia is still prevalent (1%) and problem in the newborn population today, especially in some areas of Asian countries. Thus, understanding the normal and pathological events leading to hyperbilirubinemia is important in clinical practice to prevent the adverse effects of hazardous hyperbilirubinemia.The main feature of neonatal hyperbilirubinemia is an increased bilirubin production that cannot be matched by glucuronidation and the elimination of bilirubin. Bilirubin is the end product of heme catabolism. Heme, produced largely by the breakdown of red blood cells (RBCs), is catabolized by heme oxygenase (HO) to produce an equimolar amount of carbon monoxide (CO) and biliverdin; the latter is reduced to unconjugated bilirubin by biliverdin reductase (BVR). Unconjugated bilirubin is poorly water soluble. In blood, it circulates bound to serum albumin, presumably to prevent the toxicity of free (unbound) bilirubin. Then, unbound bilirubin is rapidly and selectively taken up by hepatocytes and then conjugated to glucuronic acid into bilirubin glucuronides by uridine diphosphate (UDP)-glucuronosyl transferase (UGT1A1) before being secreted into bile. Recent study suggests that transport proteins in the hepatocyte basolateral membrane, particularly among the organic anion transport proteins (OATPs), facilitate the hepatic uptake of unconjugated bilirubin. They belong to the OATP superfamily, which is also called the solute carrier organic anion transporter (SLCO) superfamily. The human SLCO superfamily comprises 11 members grouped into 6 families. Bilirubin is a substrate for OATP1B1 and 0ATP1B3 encoded by SLCO1Bland SLCO1B3 gene。Bilirubin metabolism and its regulation is complex, involving various kinds of enzymes, transporters and regulatory factor. Defective of transporters or enzymes involved in (i) the production of bilirubin from heme; (ii) the metabolism of bilirubin; and (iii) heritable conditions that may reduce red blood cell (RBC) life span and predispose to hemolysis, may contribute to increase levels of unconjugated bilirubin, thereby the increasing the risk of neonatal hyperbilirubinemia, or neonatal jaundice. Due to the physical metabolism characteristics, newborn children can accumulate more bilirubin and were more susceptive to develop hazardous hyperbilirubinemia than adults. The pathogenesis of significant neonatal hyperbilirubinemia is often multifactorial. Both genetic and environmental factors contribute to the development of neonatal hyperbilirubinemia, and the importance of genetic contributions in this disorder has been recently recognized. In clinical, we would found some neonates with unknow reason hyperbilirubinemia, and the genetic factors were always go undetected.Hereditary hyperbilirubinemias can be separated into predominantly unconjugated and predominantly conjugated forms. Unconjugated hyperbilirubinemia is more frequently seen in newborns in the first few weeks after birth. The condition result from the overproduce of bilirubin caused by hereditary hemolytic disorders, or from decreased bilirubin clearance caused by defective in bilirubin uptake, and bilirubin conjugation by hepatocytes. Moreover, kinetic studies has reveal that free (unbound) bilirubin, rather than conjugated bilirubin, was more closely associated with neurologic injury and encephalopathy (kernicterus). Conjugated hyperbilirubin-emias is rare, and the pathogenesis remains unclear. Neonates with conjugated hyperbilirubinemias, jaundice is not apparent. Most cases would not be discovered shortly after birth except the jaundice is apparent. In cases with apparent jaundice, the diagnosis of this disease is usually several months after birth or even in adult. At that time, they often manifested with liver disfunction and cholestasis. Unkown reason of unconjugated hyperbilirubinemias is the focus of our study.Disease associated and suscepbitity genes study is important method to explore the genetic and molecular basis of complex disorder. As the development of DNA detecting techonology, genetic diagnosis has cast a new and fascinating light on the causes of unexplained hyperbilirubinemia. Gene mutations could be classified into three types, point mutations, duplication mutations and deletion mutations. Point mutations and duplication mutations are the main types of mutations in genes involved in bilirubin produce and metabolism. The approach for duplication mutations detecting is still limited, short tandem repeat (STR)-PCR is the most reliable and usually applied genotyping method. As to point mutation, many methods are available, these include PCR-single strand conformational polymorphism analysis, DNA sequencing, amplification refractory mutation system, and the recently developed reverse dot blot assay, probe melting curve, microarray techniques, denaturing high performance liquid chromatography, matrix-assisted laser desorption/ion-ization-time of flight mass spectrometry. However, these approaches are either expensive or technically challenging not suitable for large-scale molecular epidermiology investigation.High-resolution melting (HRM) analysis is recently introduced as a high-through method for variant scanning and genotyping. By precise temperature control and fluorescence monitoring of a saturating dye for observing DNA melting of PCR product, reproducible melting curves were generated by the targeted software for data analysis. Genotyping is based on subtle changes in amplicon Tm and curve shape. Heterozygote detection does not depend on the type of single base substitution or the variant position within the PCR product. PCR and mutation scanning are performed simultaneously in a single procedure within 30 minutes. Sensitivity and specificity for mutation detection are extremely high, and this technique also has the advantages of cost and throughput. Based on its ease of use, simplicity, low cost, rapid, superb sensitivity, and specificity, HRM is quickly becoming the tool of choice being used in both laboratory research and clinical settings to study single nucleotide polymorphisms and small insertions and deletions, and to diagnose associated genetic disorders.To explore the genetic and molecular basis of neonatal hyperbilirubinemia. In this study, we investigated the association of an expanded panel of mutations and polymorphisms across 6 genes that are involved in bilirubin production and metabolism(glucose-6-phosphate dehydrogenase [G6PD], Heme oxygenase-1 [HMOX1], biliverdin reductase A [BLVRA], uridine diphos-phate glucuronosyl transferase 1A1 [UGT1A1], and solute carrier organic anion transporter family member 1B1 [SLCO1B1] and 1B3 [SLCO1B3], and the risk of hyperbilirubinemia in neonates of Chaozhou region of southern China. In addition, we developed a rapid genotype screening assay for a comprehensive analysis of the nine selected SNPs in the 5 bilirubin metabolism genes by using the high-resolution melt analysis (HRM) technology.The study populations were from two resources. The first part of population was from neonates delivered at the Chaozhou Central Hospital, where they received routine newborn screening for the diagnosis of three congenital inherited diseases (G6PD deficiency, congenital hypothyroidism and phenylketonuria), during the period between October 2010 and May 2012. And the second part of studied population consist of term newborns consecutively admitted to the pediatric center of the study hospital from November 2011 to December 2014, with a gestational age of more than 37 weeks, a birth weight≥2500 g and no major birth abnormalities and serious illness. The recorded peak TSB was used to divide the study subjects into case and control subjects. The case subjects included jaundiced infants with a maximum TSB that required phototherapy based on the updated clinical guidelines of the Chinese Medical Association for neonates. Severe neonatal hyperbilirubinemia was defined as a TSB concentration above 20 mg/dL (342μmol/L). Neonates with known clinical risk factors for developing neonatal hyperbilirubinemia, such as hemolysis (a positive Coombs’test), glucose-6-phosphate dehydrogenase deficiency, cephalo-hematoma, infection, perinatal asphyxia, and major organ abnormality, were excluded.From October 2010 to May 2012, a total of 2500 neonates (males:1365, females:1135) delivered in Chaozhou Central Hospital received the routine newborn screening. Of them,67 neonates were suspected to be G6PD deficient based on FST, 44 were males and 23 were females. The overall incidence of G6PD deficiency in Chaozhou was 2.68% by the quantitative assay. Frequency in male population was 3.22%(44/1365) and in female population was 2.03%(23/1135). Comparing to the incidence previous report in other cities of southern China, G6PD deficiceny in Chaozhou population were relatively lower, but still meeting the World Health Organization recommendation for screening all newborns in populations with a prevalence of 3-5% or more in males. Routine neonatal screening of G6PD deficiency in Chaozhou remains necessary and important.Of the 67 deficient infants,58 were detected with G6PD gene mutations by performing HRM analysis and confirmed by directly sequencing.41 males were hemizygotes,17 females were heterozygotes, and no homozygote was found. Seven kinds of G6PD variants (95A>G,392G>T,493A>G,871G>A,1360OT,1376G>C, 1388G>A) were detected.To explore the complex role of multiple genetic modifiers on unconjugated neonatal hyperbilirubinemia. Eleven common mutations and polymorphisms across five bilirubin metabolism genes, namely those encoding UGT1A1, HO-1, BLVRA, SLCO1B1 and SLCO1B3 were determined in our case-control study of Chinese neonates. Most of the selected polymorphisms were located in the promoter or exon region of the genes. In addition, we developed a rapid genotype screening assay for a comprehensive analysis of the nine single base polymorphisms of the eleven selected SNPs using PCR and high-resolution melt analysis (HRM) technology. All selected variants, along with clinical parameters (sex, age and feeding method), were included in our associated evaluation. The allele, genotype and haplotype of the three SNPs in UGT1A1, including rs4148323, rs6742078 and (TA)n were found to have considerable differences between cases and controls. There was also a strong association with neonatal hyperbilirubinemia after being adjusted for known clinical risk factors for neonatal hyperbilirubinemia, including sex, breastfeeding, and age. Specifically, neonates harboring minor alleles of rs4148323 (known as UGT1A1*6, 211G>A) were found to have a significantly increased risk of hyperbilirubinemia (ORadj= 13.02; p=1e-04 for UGT1A1*6 homozygote, ORadj= 2.69; p= 1e-04 for UGT1A1*6 heterozygote), whereas minor alleles at rs6742807 and (TA)n[rs8175347] were observed to have a protective effect on the risk of hyperbilirubinemia (ORadj= 0.16; p= 0.0001 for rs6742807; ORadj= 0.25;p=0.000 for (TA)n). Similarly, haplotype analysis showed that ATA(TA)6 (rs4148323-rs6742078-rs108124-(TA)n) increased the risk (ORadj= 3.00; p= 4e-04), whereas GAT(TA)7 produced a protective effect for hyperbilirubinemia (ORadj= 0.22; p= 0.0018) compared to the most common haplotype GTA(TA)6. Two SNPs in the SLCOs family also also contributed to an increased risk of hyperbilirubinemia. Our data demonstrated that there was a significant gene-environment interaction between the UGT1A1 gene coding region variation and breastfeeding. Neonates carrying heterozygous and homozygous UGTA*6 variants (G/A and A/A genotype) had a substantially higher risk of hyperbilirubinemia than those with the wild phenotype (G/G genotype) in the BF group (breastfed and mixed breastfed) rather than in the SF group (exclusively supplement formula-fed). Neonates with the homozygous UGTA*6 variant that were exclusively fed with breast milk had the highest relative risk of hyperbilirubinemia development (OR=5.33; 95%CI:1.77-16.10, P=0.018).Given the important role of UGT1A1 in bilirubin metabolism,58 neonates with hazardous hyperbilirubinemia (TSB>342umol/L) were further subjected for DNA sequencing of the whole exon of UGT1A1 gene. Common variation of G6PD genes and thalassemia gene were also test and analyzed in those severe jaundiced neonates. Among the 58 term infants with TSB> 20 mg/dL, seven were confirmed with G6PD deficient, four heterozygous for a or β-thalassemia, and forty-four of them detected with at least one heterozygous UGT1A1 functional variant, including nine homozygous for UGT1A1 variation. Aside the predominant c.211 G>A (Gly71Arg) variant, three coding variants of UGT1A1 gene [c.1091C>T (Pro364Leu), C.1352V>T (pro4511eu) and c.1456T>G (Tyr486Asp)] were observed in our case neonates. Moreover, coexpression across these genes has been commonly observed.1) 50% of G6PD deficient neonates co-inherited with at least one variant in UGT1A1 genes.2) All the four thalassemia neonates combined with either G6PD deficiency or UGT1A1 variation. As expected, UGT1A1 variation and G6PD deficiency was more frequent in these neonates with severe hyperbilirubinemia. The results of multivariate logistic regressions, adjusted for covariates, revealed odds ratios for neonates who carried heterozygous, homozygous variation at nucleotide 211 of UGT1A1 gene and G6PD deficiency were 3.47(1.26-9.55),12.46(1.09-142.7) and 12.87(1.32-135.87) compared with those having the wild genotype and normal G6PD activity, respectively.In conclusion, our study revealed the impact of multiple genetic modifiers on neonatal hyperbilirubinemia, reflecting the complex nature of neonatal hyperbilirubinemia. The interaction between multiple gene loci, gene-gene, and gene-nongenetic factors might contribute to the development and severity of neonatal hyperbilirubinemia. Chinese population has a higher risk for severe hyperbilirubinemia, especially in southern China. Besides G6PD deficiency screening, UGTIAI genetic analysis, expecially the UGTlAl*6(c.211G> A, p.Arg71Gly) polymorphism detection, may be taken into consideration for early diagnosis and treatment of severe hyperbilirubinemia newborns in southern China.The establisement of high-resolution melting analysis is a rapid, high-through, effective genotyping method that could be used as a general and rapid method for future large-scale investigation, and a supplement for clinical dignosis of hereditary neonatal hyperbilirubinemias. |
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