Citrin plays a role in the transfer of NADH-reducing equivalent from cytosol to mitochondria as part of the malate–aspartate shuttle in liver. Citrin deficiency may cause an impairment of glycolysis due to an increase in the cytosolic NADH/NAD ratio leading to an energy shortage in the liver. Mutations of the SLC25A13 gene are responsible for neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Most patients with NICCD show a resolution of symptoms within the first year of life, but some patients present with severe symptoms and require liver transplantation. We treated four patients including three siblings with NICCD by lactose (galactose)-restricted and medium-chain triglyceride (MCT)-supplemented formula. This formula rapidly improved the clinical condition and laboratory findings. Early treatment was more effective and did not require long-term administration. Lactose (galactose)-restriction can avoid further increase in the cytosolic NADH/NAD ratio in the liver and MCT supplementation can provide energy to hepatic cells by producing an excess of acetyl-CoA in mitochondria. Early treatment with lactose (galactose)-restricted and MCT-supplemented formula is recommended for patients with NICCD and possibly for patients with CTLN2.
Citrin is a liver-type mitochondrial aspartate-glutamate carrier encoded by the SLC25A13 gene, and its deficiency causes adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Here, the authors investigated clinical findings in Korean infants with NICCD and performed mutation analysis on the SLC25A13 gene. Of 47 patients with neonatal cholestasis, three infants had multiple aminoacidemia (involving citrulline, methionine, and arginine) and galactosemia, and thus were diagnosed as having NICCD. Two of these three showed failure to thrive. The laboratory findings showed hypoproteinemia and hyperammonemia, and liver biopsies revealed micro-macrovesicular fatty liver and cholestasis. The three patients each harbored compound heterozygous 1,638-1,660 dup/ S225X mutation, compound heterozygous 851del4/S225X mutation, and heterozygous 1,638-1,660 dup mutation, respectively. With nutritional manipulation, liver functions were normalized and catch-up growth was achieved. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Korean infants.
Cholestasis; Citrin; Citrullinemia; SLC25A13; Mutation
The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), and SLC25A13 mutations cause citrin deficiency (CD), a disease entity that encompasses different age-dependant clinical phenotypes such as Adult-onset Citrullinemia Type II (CTLN2) and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD). The analyses of SLC25A13 gene and its protein/mRNA products remain reliable tools for the definitive diagnoses of CD patients, and so far, the SLC25A13 mutation spectrum in Chinese CD patients has not been well-characterized yet.
Methods and Results
By means of direct DNA sequencing, cDNA cloning and SNP analyses, 16 novel pathogenic mutations, including 9 missense, 4 nonsense, 1 splice-site, 1 deletion and 1 large transposal insertion IVS4ins6kb (GenBank accession number KF425758), were identified in CTLN2 or NICCD patients from China, Japan and Malaysia, respectively, making the SLC25A13 variations worldwide reach the total number of 81. A large NICCD cohort of 116 Chinese cases was also established, and the 4 high-frequency mutations contributed a much larger proportion of the mutated alleles in the patients from south China than in those from the north (χ2 = 14.93, P<0.01), with the latitude of 30°N as the geographic dividing line in mainland China.
This paper further enriched the SLC25A13 variation spectrum worldwide, and formed a substantial contribution to the in-depth understanding of the genotypic feature of Chinese CD patients.
AIM: To explore differences in biochemical indices between neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and that with other etiologies.
METHODS: Patients under 6 mo of age who were referred for investigation of conjugated hyperbilirubinaemia from June 2003 to December 2010 were eligible for this study. After excluding diseases affecting the extrahepatic biliary system, all patients were screened for the two most common SLC25A13 mutations; the coding exons of the entire SLC25A13 gene was sequenced and Western blotting of citrin protein performed in selected cases. Patients in whom homozygous or compound heterozygous SLC25A13 mutation and/or absence of normal citrin protein was detected were defined as having NICCD. Cases in which no specific etiological factor could be ascertained after a comprehensive conjugated hyperbilirubinaemia work-up were defined as idiopathic neonatal cholestasis (INC). Thirty-two NICCD patients, 250 INC patients, and 39 infants with cholangiography-confirmed biliary atresia (BA) were enrolled. Laboratory values at their first visit were abstracted from medical files and compared.
RESULTS: Compared with BA and INC patients, the NICCD patients had significantly higher levels of total bile acid (TBA) [all measures are expressed as median (inter-quartile range): 178.0 (111.2-236.4) μmol/L in NICCD vs 112.0 (84.9-153.9) μmol/L in BA and 103.0 (70.9-135.3) μmol/L in INC, P = 0.0001]. The NICCD patients had significantly lower direct bilirubin [D-Bil 59.6 (43.1-90.9) μmol/L in NICCD vs 134.0 (115.9-151.2) μmol/L in BA and 87.3 (63.0-123.6) μmol/L in INC, P = 0.0001]; alanine aminotransferase [ALT 34.0 (23.0-55.0) U/L in NICCD vs 108.0 (62.0-199.0) U/L in BA and 84.5 (46.0-166.0) U/L in INC, P = 0.0001]; aspartate aminotransferase [AST 74.0 (53.5-150.0) U/L in NICCD vs 153.0 (115.0-239.0) U/L in BA and 130.5 (81.0-223.0) U/L in INC, P = 0.0006]; albumin [34.9 (30.7-38.2) g/L in NICCD vs 38.4 (36.3-42.2) g/L in BA and 39.9 (37.0-42.3) g/L in INC, P = 0.0001]; glucose [3.2 (2.0-4.4) mmol/L in NICCD vs 4.1 (3.4-5.1) mmol/L in BA and 4.0 (3.4-4.6) mmol/L in INC, P = 0.0014] and total cholesterol [TCH 3.33 (2.97-4.00) mmol/L in NICCD vs 4.57 (3.81-5.26) mmol/L in BA and 4.00 (3.24-4.74) mmol/L in INC, P = 0.0155] levels. The D-Bil to total bilirubin (T-Bil) ratio was significantly lower in NICCD patients [all measures are expressed as median (inter-quartile range): 0.54 (0.40-0.74)] than that in BA patients [0.77 (0.72-0.81), P = 0.001] and that in INC patients [0.74 (0.59-0.80), P = 0.0045]. A much higher AST/ALT ratio was found in NICCD patients [2.46 (1.95-3.63)] compared to BA patients [1.38 (0.94-1.97), P = 0.0001] and INC patients [1.48 (1.10-2.26), P = 0.0001]. NICCD patients had significantly higher TBA/D-Bil ratio [3.36 (1.98-4.43) vs 0.85 (0.72-1.09) in BA patients and 1.04 (0.92-1.14) in INC patients, P = 0.0001], and TBA/TCH ratio [60.7 (32.4-70.9) vs 24.7 (19.8-30.2) in BA patients and 24.2 (21.4-26.9) in INC patients, P = 0.0001] compared to the BA and INC groups.
CONCLUSION: NICCD has significantly different biochemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.
Cholestasis; Biliary atresia; Infants; Idiopathic neonatal cholestasis; SLC25A13
Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn−/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn−/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.
One hundred and twenty-four infants admitted to hospitals in Norway between 1955 and 1974 during the first 3 months of life with cholestatic jaundice were studied retrospectively. Sixty-four infants had had extrahepatic atresia of the biliary tree and 60 had had intrahepatic cholestasis. This gives an incidence of about 1:9000 live births for cholestasis. In 4 of the 64 infants with extra-hepatic atresia a bile duct-to-bowel anastomosis had been performed but this was successful in only 2. Sixty of these infants had died by their 2nd birthday. Twenty-six of the infants with intrahepatic cholestasis had died by 1978 and the most common causes of death were cholestasis complicated by infection, bleeding, or hepatoma. The survivors aged between 4 and 23 years were followed up in 1978. In about two-thirds of them aetiological factors--such as alpha-1-antitrypsin deficiency, arteriohepatic dysplasia, cholestasis with lymphoedema--and other familial or genetic factors, or infections were found. Four of the 34 survivors are known to have cirrhosis. Twenty patients had biochemical abnormalities, and 12 had normal liver function tests. Two patients could not be examined. Of the 19 patients with familial or genetic aetiological factors, 4 had cirrhosis, 14 had biochemical abnormalities, and only 5 had normal liver function tests. Of 11 survivors with idiopathic disease or septicaemia, none had cirrhosis and only 4 had abnormal liver function tests.
Between 1960 and 1994 cystic fibrosis was found in nine out of 1474 infants investigated for neonatal cholestasis. Four had delay in passing meconium. In all patients cholestatic jaundice was present during the first 48 hours and in three patients cholestasis was complete, mimicking biliary atresia. Serum cholesterol concentrations were normal in all but two children. Sweat chloride was repeatedly above 95 mmol/l in all instances. Three children had another condition enhancing the risk of cholestasis (alpha1-antitrypsin deficiency, hypopituitarism, perinatal asphyxia, and total parenteral nutrition). Liver histology displayed portal fibrosis and inflammation with bile duct proliferation; mucous plugs in bile ducts were observed in only one patient. Only one child died from cirrhosis. These results indicate that cystic fibrosis is not a major cause of neonatal cholestasis. However early signs of intestinal obstruction and low concentrations of serum cholesterol may indicate cystic fibrosis, regardless of liver histology. Neonatal cholestasis has no prognostic value concerning evolution to cirrhosis.
Progressive familial intrahepatic cholestasis (PFIC) type 2 is caused by mutations in ABCB11, which encodes bile salt export pump (BSEP). We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum γ-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type 2.
ABCB11; Bile salt export pump; Progressive familial intrahepatic cholestasis
Tandem mass spectrometry (MS/MS) has been available in China for 8 years. This technique makes it possible to screen for a wide range of previously unscreened inborn errors of metabolism (IEM) using a single test. This 3-year pilot study investigated the screening, diagnosis, treatment and outcomes of IEM in symptomatic infants and children.
All children encountered in the Newborn Screening Center of Zhejiang Province during a 3-year period with symptoms suspicious for IEM were screened for metabolic diseases. Dried blood spots were collected and analyzed by tandem mass spectrometry. The diagnoses were further confirmed by clinical symptoms and biochemical analysis. Neonatal intrahepatic cholestasis caused by citrin deficiency, ornithine transcarbamylase deficiency and primary carnitine deficiency were confirmed by DNA analysis.
A total of 11,060 symptomatic patients (6,720 boys, 4,340 girls) with a median age of 28.8 months (range: 0.04-168.2 months) were screened. Among these, 62 were diagnosed with IEM, with a detection rate of 0.56%. Thirty-five were males and 27 females and the median age was 3.55 months (range 0.07-143.9 months). Of the 62 patients, 27 (43.5%) had aminoacidemias, 26 (41.9%) had organic acidemias and nine (14.5%) had fatty acid oxidation disorders.
Because most symptomatic patients are diagnosed at an older age, mental retardation and motor delay are difficult to reverse. Additionally, poor medication compliance reduces the efficacy of treatment. More extensive newborn screening is thus imperative for ensuring early diagnosis and enhancing the treatment efficacy of IEM.
Tandem mass spectrometry; Inborn errors of metabolism; Aminoacidemia; Fatty acid oxidation disorders; Organic acidemia
AIM: To investigate the differences in the mutation spectra of the SLC25A13 gene mutations from specific regions of China.
METHODS: Genetic analyses of SLC25A13 mutations were performed in 535 patients with neonatal intrahepatic cholestasis from our center over eight years. Unrelated infants with at least one mutant allele were enrolled to calculate the proportion of SLC25A13 mutations in different regions of China. The boundary between northern and southern China was drawn at the historical border of the Yangtze River.
RESULTS: A total of 63 unrelated patients (about 11% of cases with intrahepatic cholestasis) from 16 provinces or municipalities in China had mutations in the SLC25A13 gene, of these 16 (25%) were homozygotes, 28 (44%) were compound heterozygotes and 19 (30%) were heterozygotes. In addition to four well described common mutations (c.851_854del, c.1638_1660dup23, c.615+5G>A and c.1750+72_1751-4dup17insNM_138459.3:2667 also known as IVS16ins3kb), 13 other mutation types were identified, including three novel mutations: c.985_986insT, c.287T>C and c.1349A>G. According to the geographical division criteria, 60 mutant alleles were identified in patients from the southern areas of China, 43 alleles were identified in patients from the border, and 4 alleles were identified in patients from the northern areas of China. The proportion of four common mutations was higher in south region (56/60, 93%) than that in the border region (34/43, 79%, χ2 = 4.621, P = 0.032) and the northern region (2/4, 50%, χ2 = 8.288, P = 0.041).
CONCLUSION: The SLC25A13 mutation spectra among the three regions of China were different, providing a basis for the improvement of diagnostic strategies and interpretation of genetic diagnosis.
Citrin deficiency; Mutation spectrum; Intrahepatic cholestasis; SLC25A13
Biliary atresia is a rare neonatal disease of unknown etiology, where obstruction of the biliary tree causes severe cholestasis, leading to biliary cirrhosis and death in the first years of life, if the condition is left untreated. Biliary atresia is the most frequent surgical cause of cholestatic jaundice in neonates and should be evoked whenever this clinical sign is associated with pale stools and hepatomegaly. The treatment of biliary atresia is surgical and currently recommended as a sequence of, eventually, two interventions. During the first months of life a hepatoportoenterostomy (a “Kasai,” modifications of which are discussed in this paper) should be performed, in order to restore the biliary flow to the intestine and lessen further damage to the liver. If this fails and/or the disease progresses towards biliary cirrhosis and life-threatening complications, then liver transplantation is indicated, for which biliary atresia represents the most frequent pediatric indication. Of importance, the earlier the Kasai is performed, the later a liver transplantation is usually needed. This warrants a great degree of awareness of biliary atresia, and the implementation of systematic screening for this life-threatening pathology.
Although advances in the management of children with congenital cholestasis have enabled many to survive into adulthood with their native livers, even the most common of these conditions remains rare in adult hepatology practice. Among four congenital cholestatic syndromes (biliary atresia, Alagille syndrome, Caroli disease and congenital hepatic fibrosis, and progressive familial intrahepatic cholestasis), the published data on outcomes of the syndromes into adulthood suggest that a spectrum of severity of liver disease can be expected, from cirrhosis (almost universal in adults with biliary atresia who have not required liver transplantation) to mild and subclinical (eg, in the previously undiagnosed affected parent of an infant with Alagille syndrome). Complications associated with portal hypertension and nutritional deficiencies are common, and other associated features of the cholestatic syndrome may require appropriate attention, such as congenital heart disease in Alagille syndrome. Indications for liver transplantation include synthetic failure, progressive encephalopathy, intractable pruritus, recurrent biliary sepsis and recurrent complications of portal hypertension. Improved understanding of biliary physiology will hopefully translate into improved therapy for children and adults with cholestasis.
Alagille syndrome; Biliary atresia; Caroli syndrome; Children; Cholestasis; PFIC
Radiological studies of bone were performed in infants and children with hepatobiliary disease. Rickets was found in 23 out of 39 patients (59%) with surgically unrepaired biliary atresia, in 4 out of 15 (27%) with surgically repaired biliary atresia, in 11 out of 21 (52%) with neonatal hepatitis, and in 2 out of 4 (50%) with intrahepatic cholestasis. Osteoporosis was found in 23 out of 39 (59%) with unrepaired biliary atresia, in 3 out of 15 (20%) with repaired biliary atresia, in 5 out of 21 (24%) with neonatal hepatitis, and in 1 out of 4 (25%) with intrahepatic cholestasis. 2 girls with Byler disease and 1 infant with choledochal cyst showed no radiological evidence of bone disease.
In unrepaired biliary atresia comparative studies of biochemical data in the groups with and without bone disease showed the following. Serum calcium levels were reduced in the patients with bone disease compared with those in the group without it. Serum magnesium levels were markedly reduced in the groups with and without bone disease. The product of serum calcium and phosphorus was reduced in the group with osteoporosis compared with that in the group without it. The raised levels of serum alkaline phosphatase were unrelated to the presence or absence of bone disease.
Inherited syndromes of intrahepatic cholestasis and biliary atresia are the most common causes of chronic liver disease and the prime indication for liver transplantation in children. Our understanding of the pathogenesis of these diseases has increased substantially by the discovery of genetic mutations in children with intrahepatic cholestasis and the findings that inflammatory circuits are operative at the time of diagnosis of biliary atresia. Building on this solid foundation, recent studies provide new insight into genotype-phenotype relationships and how mutations produce altered bile composition and cholestasis. New evidence exists that although liver transplantation is curative for patients with end-stage liver disease owing to cholestasis, some patients may develop recurrence of cholestasis because of the emergence of autoantibodies that disrupt canalicular function in the new graft. Progress is also evident in biliary atresia, with recent studies identifying candidate modifier genes and directly implicating lymphocytes and inflammatory signals in the pathogenesis of bile duct injury and obstruction.
Cirrhosis; Jaundice; Bilirubin; Hemochromatosis; Biliary atresia; Alagille disease; Transplantation
Infantile cholestatic disorders arise in the context of progressively developing intrahepatic bile ducts. Biliary atresia (BA), a progressive fibroinflammatory disorder of extra- and intrahepatic bile ducts, is the most common identifiable cause of infantile cholestasis and the leading indication for liver transplantation in children. The etiology of BA is unclear, and while there is some evidence for viral, toxic, and complex genetic causes, the exclusive occurrence of BA during a period of biliary growth and remodeling suggests an importance of developmental factors. Interestingly, interferon-γ (IFNγ) signaling is activated in patients and in the frequently utilized Rhesus rotavirus mouse model of BA, and is thought to play a key mechanistic role. Here we demonstrate intrahepatic biliary defects and upregulated hepatic expression of IFNγ pathway genes caused by genetic or pharmacological inhibition of DNA methylation in zebrafish larvae. Biliary defects elicited by inhibition of DNA methylation were reversed by treatment with glucocorticoid, suggesting that the activation of inflammatory pathways was critical. DNA methylation was significantly reduced in bile duct cells from BA patients compared to patients with other infantile cholestatic disorders, thereby establishing a possible etiologic link between decreased DNA methylation, activation of IFNγ signaling, and biliary defects in patients. Conclusion: Inhibition of DNA methylation leads to biliary defects and activation of IFNγ-responsive genes, thus sharing features with BA, which we determine to be associated with DNA hypomethylation. We propose epigenetic activation of IFNγ signaling as a common etiologic mechanism of intrahepatic bile duct defects in BA.
hepatobiliary development; interferon-γ; epigenetics; infantile cholestasis; microarray
One hundred and three infants with prolonged cholestasis beginning before 3 months were classified as having alpha-1-antitrypsin deficiency (17 patients), scanty interlobular bile ducts (16 patients), or "neonatal hepatitis" (70 patients). Twenty-two gradually developed chronic liver disease and the remaining 81 recovered within a few months. Prognosis was found to be poor for infants with alpha-1-antitrypsin deficiency, scanty interlobular bile ducts, and familial "idiopathic" hepatitis. Patients who developed cirrhosis often presented with severe and persistent neonatal cholestasis, mimicking extrahepatic biliary atresia and leading to laparotomy. Thus, a high-risk group of infants-defined by aetiology, family history, and degree of cholestasis-can be recognised in the first months of life.
ABCB4 (MDR3) is a lipid translocator that moves phosphatidylcholine from the inner to outer leaflet of the canalicular membrane for extraction into the lumen by bile salts. Genetic mutations of ABCB4 lead to 3 distinct but related hepatobiliary diseases. Progressive familial intrahepatic cholestasis (PFIC) type 3 is a chronic cholestatic syndrome characterized by a markedly elevated GGTP. Patients present later in life than other types of PFIC with jaundice, pruritis and hepatosplenomegaly. Peri-portal inflammation progresses to biliary cirrhosis and eventually causes portal hypertension. Ursodeoxycholic acid (UDCA), which alters biliary bile composition, normalizes liver function tests in approximately half of treated PFIC type 3 patients. Partial or non-responders will eventually require liver transplantation. Gallstone patients with ABCB4 mutations may have Low Phospholipid Associated Cholelithiasis Syndrome. This is characterized by cholesterol gallstones and intrahepatic microlithiasis, along with recurrent biliary symptoms, despite cholecystectomy. A distinct group of patients with ABCB4 mutations may also develop intrahepatic brown pigment stones. The current treatment for these two diseases is UDCA, which may improve biliary symptoms even before the dissolution of stones occurs. Additional therapies such as Farnesoid X receptor ligands/agonists and benzfibrates show promise as future therapeutic modalities. Intrahepatic cholestasis of pregnancy affects pregnant women with abnormal ABCB4 variants. These women suffer from disabling pruritis and may experience steatorrhea as well. Their fetuses are at high risk for prematurity and still births. The definitive treatment for this condition is delivery of the baby. In the interim, limited fat intake, fat soluble vitamin supplementation and UDCA, with or without S-adenosylmethionine can provide symptomatic relief. In the future, additional hepatobiliary diseases related to ABCB4 mutations are likely to be identified. It is likely that this will result in the discovery of additional therapies for PFIC Type 3, gallstones and intrahepatic cholestasis of pregnancy.
A number of different chronic diseases affect the intrahepatic bile radicles or cholangioles. They include primary and secondary sclerosing cholangitis, primary biliary cirrhosis, chronic cholestatic drug jaundice, atresia, and carcinoma. Aetiological factors include infection, immunological changes, hormones, and congenital defects.
Patients with chronic cholestasis have decreased bile salts in the intestinal contents and suffer from a bile salt deficiency syndrome. Failure to absorb dietary fat is managed by a low-fat diet and by medium-chain trigly-cerides which are absorbed in the absence of intestinal bile salts. Fat-soluble vitamin deficiencies are prevented by parenteral vitamins A, D, and K1. Calcium absorption is defective, and improvement may follow intramuscular vitamin D, medium-chain triglycerides, a low-fat diet, and oral calcium supplements.
In partial intestinal bile salt deficiency the anionic bile-salt-chelating resin cholestyramine controls pruritus though steatorrhoea increases. Pruritus associated with total lack of intestinal bile salts is managed by methyl-testosterone or norethandrolone, though the jaundice increases.
Tandem mass spectrometry (MS/MS) analysis is a powerful tool for newborn screening, and many rare inborn errors of metabolism are currently screened using MS/MS. However, the sensitivity of MS/MS screening for several inborn errors, including citrin deficiency (screened by citrulline level) and carnitine uptake defect (CUD, screened by free carnitine level), is not satisfactory. This study was conducted to determine whether a second-tier molecular test could improve the sensitivity of citrin deficiency and CUD detection without increasing the false-positive rate.
Three mutations in the SLC25A13 gene (for citrin deficiency) and one mutation in the SLC22A5 gene (for CUD) were analyzed in newborns who demonstrated an inconclusive primary screening result (with levels between the screening and diagnostic cutoffs).
The results revealed that 314 of 46 699 newborns received a second-tier test for citrin deficiency, and two patients were identified; 206 of 30 237 newborns received a second-tier testing for CUD, and one patient was identified. No patients were identified using the diagnostic cutoffs. Although the incidences for citrin deficiency (1:23 350) and CUD (1:30 000) detected by screening are still lower than the incidences calculated from the mutation carrier rates, the second-tier molecular test increases the sensitivity of newborn screening for citrin deficiency and CUD without increasing the false-positive rate.
Utilizing a molecular second-tier test for citrin deficiency and carnitine transporter deficiency is feasible.
Newborn screening; Founder mutation; Second-tier molecular test; Citrin deficiency; Carnitine uptake defect
Biliary atresia is a mystifying cause of neonatal cholestasis, manifested by progressive inflammation and fibrosis of both the extrahepatic and intrahepatic bile ducts. It is a devastating disease that leads to cirrhosis and the need for liver transplantation in the majority of children. The etiology is unknown, and one theory is that it may involve a primary perinatal hepatobiliary viral infection and a secondary generation of an autoimmune-mediated bile duct injury. This review will outline the evidence from both human and murine studies supporting a potential cholangiotropic viral infection as the initiator of bile duct injury in biliary atresia and the role of the adaptive immune response and autoimmunity in progression of disease. Delineating the pathways of immune and autoimmune-mediated bile duct injury within biliary atresia could stimulate development of new medical interventions aimed at suppressing the specific immune response, decreasing the inflammatory damage to bile ducts, and delaying or negating the need for liver transplantation.
Reovirus; rotavirus; cholangitis; adaptive immunity; neonatal immunity
To review best practices for early recognition and treatment of conditions resulting in neonatal cholestasis, in order to improve long-term outcomes for affected infants.
QUALITY OF EVIDENCE
Studies, review articles, and meta-analyses pertaining to neonatal-onset cholestasis were sought via electronic databases. Reference lists of studies and review articles supplemented the electronic search. Studies were included if they examined the importance of early diagnosis and intervention for cholestatic jaundice of any cause, and mainly comprised Level II and Level III evidence.
Review of the relevant literature supports the recommendation that infants with jaundice at 2 weeks of age should be tested for cholestasis by quantifying the direct reacting bilirubin levels in their blood. Subsequent rapid investigation using a diagnostic algorithm enables early diagnosis of the specific cause and facilitates timely intervention for conditions whose outcomes are improved by early treatment.
Universal screening for neonatal cholestasis might help with early identification of cases and improve outcomes, although further study is required in the North American setting.
Aetiological factors were sought prospectively in 55 babies with extrahepatic biliary atresia, in 105 with neonatal hepatitis, and in 11 with intrahepatic biliary atresia, seen as a result of nearly complete ascertainment of these conditions in the State of Victoria between 1963 and 1974. In neonatal hepatitis infective causes were shown in 22 babies, galactosaemia in 6 and alpha1-antitrypsin deficiency in 8; familial occurrence was noted in 10 further babies and unrelated second diseases were present in 24 of the remaining 59 babies. The only clues to aetiology in extrahepatic biliary atresia were a suspicion of time-space clusters, a deficiency of affected babies born to young primiparous women, and an unexpected number of spontaneous abortions in the histories given by the mothers. Genetic factors appeared to be important in intrahepatic biliary atresia, but are not reported in detail. Hypotheses for the aetiology of neonatal hepatitis and of extrahepatic biliary atresia are presented. Both are considered syndromes with multiple causes. Recurrence risks in sibs are discussed, and are 1 in 7 for neonatal hepatitis of unknown cause, negligible in extrahepatic biliary atresia, and usually 1 in 2 or 1 in 4 in intrahepatic biliary atresia, depending upon the family history.
Neonatal jaundice persisting beyond 14 days of age is a common clinical scenario. The vast majority of affected children have a benign unconjugated hyperbilirubinemia, but included in this clinical presentation is a group of neonates with conjugated hyperbilirubinemia and liver disease. Early identification of liver disease improves the infant’s outcome, especially for those with extrahepatic biliary atresia. The present paper reviews the approach to the neonate with prolonged jaundice, including clinical presentation, when to proceed with initial investigations, timing of referral, further investigations and management, and provides an overview of the more common causes of neonatal cholestatic liver disease.
Cholestatic liver disease; Neonatal jaundice
Alpha-1-antitrypsin (AAT) deficiency is a rare genetic disorder characterized by hepatitis in neonates, childhood and adulthood (protease inhibitor (PI)*ZZ) and emphysema with or without hepatitis (PI*ZZ)/(PI*SS,SZ or null) in adulthood. We report the case of a female neonate born at 40 weeks of gestation who presented with vitamin K deficiency-related intracranial bleeding and cholestasis of which she died at 28 days of age. At autopsy, the infant was found to have intracranial bleeding, hepatomegaly, and cholestasis with paucity of bile ducts in the liver. Small periodic acid-Schiff diastase positive intrahepatic granules and positive staining with antibodies against AAT protein suggested an AAT deficiency. AAT is a glycoprotein that has a protease inhibitor function. Its deficiency can be the result of various point mutations in Serpin 1 located on chromosome 14. The diagnosis AAT deficiency was confirmed by mutation analysis showing the PI*ZZ genotype in the neonate. In conclusion, AAT deficiency is a rare genetic disorder that can lead to a serious bleeding disorder in the neonatal period if not recognised on time. Pathological diagnosis together with verifying molecular analysis can be used to identify index patients.
Alpha-1-antitrypsin; Death; Deficiency; Neonatal
AIM: To establish the real time fluorescence polymerase chain reaction (RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.
METHODS: Four hundred infants (< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4. Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed. Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes. After a single RT-PCR, results were obtained by analyzing the take-off curves. Twenty-four positive and 14 negative samples were retested by direct sequencing.
RESULTS: Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8% (46/800). Twenty-six and 20 mutant alleles were observed respectively, in 474 and 242 alleles from the intermediate and southern areas of China. No mutant allele was detected in 84 alleles from northern China. Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations, and 14 negative samples were retested by direct sequencing, which confirmed that the accuracy of RT-PCR was 100%.
CONCLUSION: RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.
851del4 mutation; Neonatal intrahepatic cholestasis; Real-time fluorescent polymerase chain reaction; SLC25A13 gene