Vol.2, No.4, 286-290 (2010)Health doi:10.4236/health.2010.24041
Coexistence of Duarte 1 and Duarte 2 variants of galactomia with extrahepatic biliary atresia*
Ramandeep Singh1, Ketan Kulkarni2, Gurjit Kaur3, Babu Ram Thapa2, Rajendra Prasad1
1Department of Biochemistry, Postgraduate Institute of Medical Education and Rearch, Chandigarh, India
2Division of Pediatric Gastroenterology, Postgraduate Institute of Medical Education and Rearch, Chandigarh, India
3Department of Physiology, Government Medical College and Hospital, Chandigarh, India; *******************
Received 3 December 2009; revid 11 January 2010; accepted 15 January 2010.
ABSTRACT
Galactomia is an autosomal recessive disor-der caud by deficient or abnt activities of one of the
three enzymes involved in the ga-lacto metabolic pathway. The predominant form is classic type galactomia caud by vere reduction or abnce of the galacto- 1-phosphate uridyl transfera (GALT) enzyme. Coexistence of extrahepatic biliary atresia (EHBA) with Duarte 1 and 2 variants of galacto-mia has not been described earlier. Here we report a ca of EHBA with concordant Duarte 1 and 2 variants of galactomia in an infant with cholestasis. Genetic analysis of the index pa-tient for galactomia revealed prence of Duarte 1/Duarte 2 variants of galactomia with genotype N314D-L218L/N314D-G1105C-GI391A- G1323A-5’UTR-119delGTCA. Clinical evaluation of the patient showed the prence of EHBA. Henceforth, it may be hypothesized that EHBA may have a genetic basis with simultaneous involvement of the GALT gene.
Keywords:Duarte 1; Duarte 2; Extrahepatic Biliary Atresia; Galactomia; Galato-1-Phosphate
Uridyl Transfera Gene; Los Angles
1. INTRODUCTION
Galactomia is an autosomal recessive disorder of ga-lacto metabolism caud by deficient or abnt activi-ties of one of the three enzymes involved in the galac-to metabolism. The deficiency of galacto-1-phos- phate uridyl transfera (GALT; MIM# 230400) is most common and prents a
s neonatal hepatitis and liver failure. In addition to the predominant classic form, there are two clinical variants: Duarte-1 and Duarte-2 [1]. Al-though coexistence of Duarte 1 and 2 variant of galacto-mia has been reported in literature, synchronous pres-ence of the 2 variants with extrahepatic biliary atresia (EHBA) has not been documented [1,2]. In this commu-nication, we report a ca of EHBA coexisting with Duarte 1 and Duarte 2 variants of galactomia detected at our center during evaluation of cholestatic infants for galactomia.
2. MATERIAL AND METHODS
2.1. Patients
A cohort of 215 infants (age range: 5 days - 10.5 months), admitted in Pediatric Gastroenterology ward of Postgraduate Institute of Medical Education and Re-arch (PGIMER) over a period of 30 months (from January, 2007 to June, 2009) with cholestasis were evaluated for galactomia. This study was approved by Institute’s ethical committee.
2.2. Red Cell (Galacto-1-Phosphate Uridyl
Transfera) GALT Assay
GALT activity was measured using a Perkin-Elmer neo-natal GALT kit (Perkin Elmer Wallac Victor 2D fluoro-meter, Finland) which measured the uridylphosphoglu-co (UDPG) consumption on samples of dried blood eluted from the filter.
2.3. DNA Isolation and Polymera Chain
Reaction (PCR)
Genomic DNA was isolated from whole blood by me- thod of Daly et al. [3].Polymera Chain Reaction (PCR) was performed in order to amplify the required -quences by using specific primers mentioned in Table 1 [4,5].
2.4. Restriction Fragment Length
Polymorphism (RFLP) Analysis
A rapid PCR-bad DNA analysis was performed to confirm or identify the quence variations that create or abolish the given natural and amplification-created re-
*This study was supported by Department of Biotechnology, Ministry
of Science and Technology, Government of India.
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R. Singh et al. / Health 2 (2010) 286-290287
striction sites as mentioned in Table 1 [4,5].
2.5. Ca
The index ca prented as follows. A 3.5 kilogram, term, appropriate for gestational age female child was born to a primigravida mother by lower gment cesar-ean ction. The baby developed jaundice since day 3 of life. She received phototherapy for the same. Jaundice persisted and worned over the next 2 weeks. Baby was passing high colored urine and clay colored stools. Hence, the baby was referred at 3 weeks of age to our institute for further evaluation and management. She was being exclusively breast fed. There was no lethargy, re-fusal to feed, fever, excessive cry, irritability, bleeding, altered nsorium or izures. On enquiry, mother denied any history of rash, joint pains, fever or per vaginal dis-charge during antenatal period. There was no history of previous abortions or significant family history of any illness.
On examination, weight was 3.6 kg, length was 53 cm and occipitofrontal circumference was 35.7 cm. There were no congenital malformations or stigmata of TORCH group of infections. Eye examination did not show any cataracts. The baby had distended abdomen with dilated visible veins and everted umbilicus. There was hepatomegaly (liver was 5 cm below right costal margin with a span of 10 cm) and splenomegaly (4 cm under left costal margin). The liver was firm in consis-tency with well defined margins and coar surface. The rest of the systemic examination was noncontributory and the baby was neurologically appropriate for age.
In view of the history and physical examination, neo-natal cholestasis syndrome was considered and the pa-tient was further evaluated. Hemoglobin was 10.5 gm/dL, total leukocyte count was 15.1 × 109/L with neutrophilic leucocytosis (86% neutrophils). The platelet count was within normal limits (3.7 × 109/L). Liver Function tests showed aspartate-transamina and alanine transamina of 28 U/L and 53 U/L respectively (normal range: 15- 45 U/L). The total rum bilirubin was 8.0 with conju-gated fraction of 4.5 mg/dL. There was hypoalbumine-mia (rum albumin of 1.9 with total rum protein of 5.0 g/dL). The rum alkaline phosphata was 565 IU/L (range: 20-250 IU/L). The coagulation profile revealed a proth- rombin time and activated partial thromboplastin time of 14 (control of 12 conds) and 37 (control of 35 conds) respectively with an international normalized r
atio of 1.05. The C-reactive protein was negative. Blood and urine cultures were sterile. Urine for non-gluco reducing substances was negative. Toxoplasma, retrovi-ral, rubella, cytomegalovirus and herpes virus rologies were negative.
Ultrasonography (along with colour Doppler evalua-tion) of the abdomen showed liver span of 10.5 cm, coarned echotexture, normal outline of liver, normal portal vein and hepatic artery, small and atretic gall bladder, non-visualized common bile duct. The spleen spanned 7 cm. The overall features were suggestive of biliary atresia. The findings of mebrofenin scan were corroborative with the ultrasonography findings. Thus a diagnosis of (EHBA) was established.
In view of cholestasis, as per the study protocol, the patient was evaluated for galactomia. GALT activity of 63% was obrved. Genetic analysis revealed the pres-ence of N314D mutation in exon 10 of the GALT gene. Interestingly, further work-up revealed that N314D mu-tation was prent in two different alleles of the GALT gene: Duarte 1 (D1) and Duarte 2 (D2). D1 allele was
Table 1. Mutations or quence variations, primers ud for PCR and appropriate restriction enzymes ud in detection of normal and mutant alleles.
Detection
Mutations/ Poly-morphisms Primers
Restriction
Enzyme Normal
Allele [bp]
Mutant
allele [bp]教育心得
N314D 5'-GGGTCGACGAGATGCTGGGACTGAGGGTGGAGCA-3'
5'-GGGGTCGACGCCTGCACATACTGCATGTGA-3
Ava II 349 + 81
247 +
102 + 81
L218L 5'-ATGTGGAGGCTTGGAGGTAAA-3'
5'-TTCACCTCTAGCTTTCTCCT-3'
M I 272 147 + 125
IVS4nt-27g→c (G1105C) 5'-ACAGCCAAGCCCTACCTCCCG-3'
5'-ACTCCCTCCTGACCACACCC-3'
Msp I
(ACRS)
171 +
93 + 19
171 + 112
IVS5nt+62g→a (G1323A) 5’-TGGATGGGCAGGGAGGGGGT 3’
5’-TGTCAAGGGGCAAAAGCAGA-3’
Dde I
222 +
120 + 6
186 +
120+ 36 + 6
IVS5nt-24g→a (G1391A) 5'-TGGATGGGCAGGGAGGGGGT -3'
5'-TGTCAAGGGGCAAAAGCAGA -3'
Sac I 239 + 109 348
5’UTR-119delGT
CA 5’-CAGGGCAGCCCAGTCACTCA- 3’
5’-GCGTTGCTGAGGATCGGTTC-3’
Dde I
(ACRS)
145 +
17 + 10
158 + 10
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R. Singh et al. / Health 2 (2010) 286-290
288
found to carry a silent mutation L218L in exon 7 in ad-dition to the N314D in exon 10 of GALT gene (Figure 1). In contrast, D2 allele was found to co-exist with 3 different polymorphisms: IVS4nt-27g →c (G1105C), IVS5nt+62g →a (G1323A) and IVS5nt-24g →a (G1- 391A) and promoter region delet
骊山老母ion of 4 nucleotides (5’UTR-119del GTCA) (Figure 2).
The patient was managed with appropriate supportive and nutritional care along with cessation of breast feed-ing whilst soya-milk formula was initiated. Subquently Kasai’s porto-enterostomy was performed at 3 months of age. Per operatively, the liver was greenish, hypertro-phied; biliary channels were fibrotic although right he-patic duct was visible. Per-operative cholangiogram con-firmed the diagnosis of EHBA.
The patient was discharged on appropriate dietary ad-vice and multivitamin supplementation. At 4 months of follow-up, the infant is passing normal colored stools and is anicteric and gaining weight, although at a slower pace. In view of established biliary cirrhosis the family has been advid regular medical follow-up and has been counled regarding the possible need of liver trans- plantation in future.
3. DISCUSSION
The report prents a peculiar ca of coexistence of 2 variants of galactomia together (Duarte 1 and Duarte 2 together) with EHBA in a solitary ca amongst a cohort
of 215 patients with neonatal cholestasis.
怎么画公主The patient was found to carry the L218L transition along with N314D on one allele which is known as Duarte1 (D1) or Los Angeles variant (LA) of galacto-mia [6,7]. This variant is known to be associated with the incread GALT activity [1]. A molecular mechanism involving an incread rate of translation through codon bias has been propod to be the possible mechanism [8]. In addition, three different transitions were also found: IVS4nt –27 g →c (G1105C) in intron 4 and IVS5nt –24 g →a (G1391A) and IVS5nt +62 g →a (G1323A) in intron 5, all in a heterozygous state. Besides, a deletion of 4 nu-cleotides (5’UTR-119delGTCA) was detected in the promoter region of the GALT gene. This type of geno-type, that is: N314D-G1105C-GI391A-G1323A-5’UTR- 119delGTCA is known to constitute another galacto-mia variant called Duarte 2 (D2) variant which is as-sociated with the decrea GALT activity [6,7]. Investi-gators have suggested that the genetic alterations might be regulatory mutations which may be responsible for the decrea in GALT activity in D2 variants [7]. So the genotype of the patient was N314D-L218L/N314D- G1105C-GI391A-G1323A-5’UTR-119delGTCA or D1/ D2. This unique genotype causing coexistence of D1 and D2 variant galactomia along with EHBA has not been earlier reported in literature.
The coexistence of EHBA alongwith galactomia made us ponder that although EHBA is not believed to be an inherited dia; genetic factors have been re-
个人艺术写真集
A. N314D mutation by Ava II Lane 1: 100 bp DNA ladder Lane 2: Wild type
Lane 3: Homozygous mutant
C. Sequence of GALT gene showing: (a) Normal quence (b) N314D mutation
B. L218L mutation by M I Lane 1: Wild type
Lane 2: Heterozygous mutant Lane 3: 50 bp DNA ladder
D. Sequence of GALT gene showing: (a) Normal quence (b) L218L mutatio
Figure 1. Restriction digestion analysis and electrophoregram of N314D and L218L mutation.
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289乞巧的诗意
172 bp
158 bp 145 bp
1 2 3
171 bp 112 bp 93 bp
1 2 3 4 5 6
222 bp 186 bp 120 bp
1 2 3
348 bp 239 bp
109 bp
A
D
C
B
348 bp十大将
1 2 3 4
A. G1105C using Msp I Lane 1: Wild type
Lane 2 & 3: Heterozygous mutant Lane 4: 50 bp DNA ladder B. G1391A using Sac I
Lane 1: 50 bp DNA ladder Lane 2: Heterozygous mutant Lane 3: Wild type
C. G1323A using Dde I Lane 1: 50 bp Ladder Lane 2: Undigested
Lane 3, 4 & 5: Wild type Lane 6: Heterozygous mutant D. 5’UTR-119delGTCA using Dde I Lane 1: Undigested
Lane 2: 50 bp DNA ladder Lane 3: Heterozygous mutant
Figure 2. Restriction digestion analysis of different polymorphisms associated with Duarte 2.
正切函数ported to be involved in its pathogenesis. Reports of
familial cas provide evidence in favor of this hypothe-sis [9,10]. Worldover, the most widely investigated genes are tho which are related to laterality (inversin) and to the development of bile ducts [11]. In an animal study on mice, authors have obrved a spontaneous mutation in the inversin (inv) gene, on chromosome 4, resulting in situs inversus, obstructive jaundice and death within the first week of life [12]. Analysis of the hepato-biliary system of the inv mice revealed EHBA and intrahepatic ductular proliferation [13]. However, when compared to histopathological findings in infantile EHBA, inconsistent features have been obrved [11]. Further, the human inversin gene has been mapped on chromosome 9 [11], same as that of the gene for the GALT [14]. Despite being a non-syndromic variant of EHBA, our patient might have contemporaneous in-volvement of inversin and GALT genes on chromo-some 9 leading to such peculiar coexistence of disor-ders. Another gene that may play a role in the genesis of EHBA is Jag-1, although its influence on the devel-opment of atresia has not been definitively confirmed [15]. Even though a genetic basis is likely to be con-tributory, the exact mechanism of this co-existence is open to speculation.
The clinical implication of such concordance remains to be conceived. The index patient is currently well and on active follow up. Long term follow up would be able to show the clinical impact of this association on the natural history of EHBA or that of EHBA on the vari-ants of galactomia. More
over, identification of the genetic basis of such a peculiar concordance could have important connotation on prenatal diagnosis of the disorders.
REFERENCES
[1] Langley, S.D., Lai, K., Dembure, P.P., Hjelm, L.N. and
Elsas, L.J. (1997) Molecular basis for Duarte and Los Angeles variant galactomia. American Journal of Hu-man Genetics , 60, 366-372.
[2] Elsas, L.J., Lai, K., Saunders, C.J. and Langley, S.D.
(2001) Functional Analysis of the human galacto- 1-phosphate uridyltransfera promoter in Duarte and LA variant galactomia. Molecular Genetics and Metabo-lism , 72, 297-305.
[3] Daly, A.K., Steen, V .M., Fairbrother, K.S. and Idle, J.R.
(1996) CYP2D6 multiallelism. In Johnson, E.F. and Wateman Eds., Methods in Enzymology , 272, 199-210. [4] Elsas, L.J., Langley, S., Evinger, J., Keil, J.L., Brown, A.,
Singh, R., Fernhoff, P., Hjelm, L.N. and Dembure, P.P.
All Rights Rerved.
R. Singh et al. / Health 2 (2010) 286-290 290
(1995) 7Galactomia: A strategy to identify new bio-
chemical phenotypes and molecular genotypes. American
Journal of Human Genetics, 56, 630-639.
[5]Kozak, L., Francova, H., Fajkusov, L., Pijackova, A.,
Macku, J.M.S., Peskovova, K., Martincova, O., Krijt, J.
and Bzduch, J. (2000) Mutation analysis of the GALT gene in Czech and Slovak galactomia populations: Identification of six novel mutations, including a stop codon mutation (X380R). Human Mutation, 15(2), 206.
崔洪万
[6]Greber, S., Guldberg, P., Scheibenreiter, S. and Strobl, W.
(1995) Mutations in classical and Duarte 2 galactomia.
Pediatric Rearch, 38, 434 (Abstr.).
[7]Podskarbi, T., Kohlmetz, T., Gathof, B.S., Kleinlein, B.,
Bieger, W.P., Grer, U. and Shin, Y.S. (1996) Molecular
characterization of Duarte 1 and Duarte 2 variants of ga-
lacto-1-phosphate uridyl transfera. Journal of Inher-
ited Metabolic Disorders, 19, 638-644.
[8]Kurland, C.G. (1991) Codon bias and gene expression.
FEBS Letters, 285(2), 165-169.
[9]Cunningham, M.L., Sybert, V.P., Opitz, J.M. and Rey-
nolds, J.F. (1988) Idiopathic extrahepatic biliary atresia: Recurrence in sibs in two families. American Journal of Medical Genetics, 31, 421-426.
[10]Danesino, C., Spadoni, E. and Buzzi, A. (1999) Familial
biliary atresia. American Journal of Medical Genetics,85, 195.
[11]Carvalho, E.D., Ivantes, C.A.P. and Bezerra, J.A. (2007)
Extrahepatic biliary atresia: Current concepts and future directions. Jornal de Pediatria, 83(2), 105-120.
[12]Yokoyama, T., Copeland, N.G., Jenkins, N.A., Mont-
gomery, C.A., Elder, F.F. and Overbeek, P.A. (1993) Re-
versal of left-right asymmetry: A situs inversus mutation.
Science, 260, 679-682.
[13]Mazziotti, M.V., Willis, L.K., Heuckeroth, R.O., LaRegi-
na, M.C., Swanson, P.E., Overbeek, P.A. and Perlmutter,
D.H. (1999) Anomalous development of the hepatobil-
iary system in the Inv mou. Hepatology, 30, 372-378. [14]Shih, L.Y., Suslak, L., Rosin, I., Searle, B.M. and Despo-
sito, F. (1984) Gene dosage studies supporting localiza-
tion of the structures genes for galacto-1-phosphate uridyl transfera (GALT) to band 913 of chromosome 9.
American Journal of Medical Genetics, 19, 539-543. [15]Kohsaka, T., Yuan, Z.R., Guo, S.X., Tagawa, M., Naka-
mura, A., Nakano, M., Kawasasaki, H., Inomata, Y., Ta-
naka, K. and Miyauchi, J. (2002) The significance of human jagged 1 mutations detected in vere cas of extrahepatic biliary atresia. Hepatology, 36, 904-912.
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