Genetic Defects in Bile Acid Conjugation Cause Fat-soluble Vitamin Deficiency

Kenneth D. R. Setchell; James E. Heubi; Sohela Shah; Joel E. Lavine; David Suskind; Mohammed Al–Edreesi; Carol Potter; David W. Russell; Nancy C. O'Connell; Brian Wolfe; Pinky Jha; Wujuan Zhang; Kevin E. Bove; Alex S. Knisely; Alan F. Hofmann; Philip Rosenthal; Laura N. Bull

Disclosures

Gastroenterology. 2013;144(5):945-955. 

In This Article

Patients and Methods

Clinical Descriptions of Patients

The demographic information and presentations of 10 patients from 7 families are summarized in the following text and in Table 1, with more detail provided in Supplementary Patients and Methods.

Evaluation of jaundice and anemia at age 40 days in patient 1, a male infant born at term (2.6 kg) to parents not identified as consanguine, showed α-thalassemia. A prolonged prothrombin time (PT) at age 5 months responded to fresh-frozen plasma and vitamin K. Severe anemia, congestive heart failure, pulmonary edema, and rickets with a fracture of the proximal fibula were recorded at 1 year. Marked growth retardation and hepatosplenomegaly at age 14 years prompted reevaluation, with bile acid analysis by mass spectrometry.

On evaluation of jaundice with acholic stools at age 28 days in patient 2, a male infant born at term (3.3 kg) to first-cousin parents with 2 nominally well children, sonographic examination showed no gallbladder and cholescintigraphy showed no contrast excretion; an intraoperative cholangiogram was interpreted as consonant with biliary atresia. Jaundice did not resolve with portoenterostomy. At 7 months, rickets was diagnosed, as well as a fracture of the right humerus and low serum vitamin D and E levels. Bile acid analysis of a urine sample was performed at 4 years, with follow-up liver biopsy.

Urine and serum samples from patient 3, the 8-year-old full sister of patient 2, were screened for a bile acid synthetic defect by mass spectrometry, although family members considered her healthy. She was later found to have had rickets at age 6 months and fractures at age 3 years. At age 12 years, serum vitamin E level was low without hypocoagulability, other hypovitaminosis, or other abnormal clinical/biochemistry test results.

Immunizations at age 4 months in patient 4, a female infant born at 36 weeks' gestation (2.3 kg) to parents not identified as consanguine and with one well child, immediately produced large ecchymoses. A prolonged PT responded to fresh-frozen plasma and vitamin K. Serum vitamin D and E levels were low, and rickets was seen on imaging. Serum vitamin A level was normal, without other abnormal clinical/biochemistry test results. Liver biopsy was performed at 15 months, with mass spectrometry screening for a bile acid synthetic defect.

Hydrocephalus ascribed to aqueductal stenosis, respiratory distress, and hypoglycemia suspect for sepsis led to hospital admission at age 3 days for patient 5, a male neonate born at term (4.58 kg) to consanguine parents. Liver failure developed and was successfully treated with a liver transplant. Evaluation before transplant included liver biopsy and screening for a bile acid synthesis defect.

Patient 6, the full sister of patient 5, had never been ill and was clinically well when, at age 9 years, a urine sample was screened by mass spectrometry for abnormal bile acid levels. The only abnormal clinical/laboratory test result was a low total serum tocopherol concentration; hypocoagulability, other hypovitaminoses, or other abnormal clinical/biochemistry test results were not found.

Patient 7, the full sister of patients 5 and 6, had severe jaundice as a neonate. Hepatitis was diagnosed. Ursodeoxycholic acid and vitamin A were given for several years. At age 10 years, a urine sample was screened for a bile acid synthesis defect by mass spectroscopy, and serum vitamin A and D and total tocopherol levels were low, without hypocoagulability or other abnormal clinical/biochemistry test results.

Growth failure at age 4 months prompted evaluation of patient 8, a female infant born at term (3.63 kg) to parents not identified as consanguine and with one well child. A prolonged PT responded to parenteral vitamin K; serum vitamin A, D, and E levels were low and serum alkaline phosphatase activity was high, without other abnormal clinical/biochemistry test results. A urine sample was screened by mass spectroscopy for a bile acid synthesis defect.

On evaluation at age 5 months of growth retardation, jaundice, and rickets, patient 9, a male infant born at term (2.5 kg), exhibited mild hepatomegaly without splenomegaly. A prolonged PT responded to parenteral vitamin K; serum vitamins D and E levels were low, without hypovitaminosis A. Conjugated and nonconjugated hyperbilirubinemia accompanied elevations in serum transaminase and alkaline phosphatase activities. Liver biopsy was performed, as well as bile acid analysis by mass spectroscopy.

Poor weight gain led to evaluation of patient 10, a girl; a urine sample was screened by mass spectroscopy at age 8 years, when duodenal stenosis was surgically palliated, and earlier clinical details are lacking. A urine sample was again screened at age 10 years.

Analytical Techniques

The bile acid composition of urine, serum, bile, and feces was examined in detail using a combination of methodologies previously reported, including liquid-solid extraction, lipophilic anion exchange chromatography to isolate bile acids based on conjugate classes, and analysis of these fractions by gas chromatography–mass spectrometry (GC-MS) after derivatization to methyl ester–trimethylsilyl ethers.[8] The initial screening procedure for diagnosis of a bile acid synthetic defect was performed by direct analysis of the urine sample using fast atom bombardment ionization–mass spectrometry (FAB-MS) and GC-MS.[8,9]

Molecular Genetic Analysis of BAAT and SLC27A5

Human genomic DNA was isolated from white blood cells using Puregene DNA Isolation Kits (Qiagen, Valencia, CA). The 3 coding exons of BAAT and the 10 coding exons of SLC27A5 were amplified by polymerase chain reaction. The polymerase chain reaction products were purified and sequenced using standard approaches. Sequences were aligned to a reference gene sequence. Absence of candidate mutations from publically (dbSNP) and locally available control sequence data was confirmed. Predicted functional consequences of missense changes were evaluated using Polyphen2 (Polymorphism Phenotyping v2; https://genetics.bwh.harvard.edu/pph2/).

Control Samples. For the mutation in patients 2 and 3, 80 control chromosomes from individuals of Arab ancestry were assayed. For the other mutations, 113 control chromosomes from HapMap families of Northern and Western European ancestry were assayed.[10]

Histologic Analysis

Sections of formalin-fixed paraffin-embedded liver tissue from patients 1, 2, 4, and 5 were stained with H&E, periodic acid–Schiff/diastase, reticulin, and Masson trichrome methods. Patients 1, 2, and 5 had second liver samples obtained at ages 14 years, 4.5 years, and 6 months, respectively. Tissue samples from the second biopsy specimen from patient 2, the only specimen from patient 4, and the first specimen from patient 5 were processed for ultrastructural study (glutaraldehyde fixed, osmium tetroxide postfixed, resin embedded). Ultrathin sections of resin-embedded liver were stained with uranyl oxide/lead citrate and examined using a transmission electron microscope. In patients 2, 4, and 5, expression of BACL and BAAT was assessed immunohistochemically using antibodies against BACL (HPA007292; Sigma, St Louis, MO) and BAAT (ab97455; Abcam, Cambridge, England) with EnVision reaction development (Dako UK, Ely, England) and hematoxylin counterstaining as described elsewhere.[11]

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