Here we report the identification of a family with 3β-HSD deficiency in which affected individuals show striking phenotypic variability. The proband had chronic liver disease since childhood, but survived without medical care into her early 20s and then died at age 24. Her paternal first cousin (III.1) died at age 6 years of liver disease. The sister of III.1 (III.5) had an apparently self-limited liver disorder in childhood that was severe enough to require multiple hospitalizations and yet she has been asymptomatic for the last 22 years. We confirmed that she was homozygous for a null allele of HSD3B7
, yet her liver function tests were normal at age 32 years. The lack of 3β-HSD activity was biochemically confirmed by FAB-MS analysis of the serum. This 32 year old family member represents the oldest patient yet diagnosed with 3β-HSD deficiency and the only adult with the disorder who does not have symptoms. The disorder can present in older children and teenagers, typically with mild elevations in transaminases, fat-soluble vitamin malabsorption and sometimes with rickets that resolves with vitamin supplementation; the disease then presents later with hepatosplenomegaly (2
). To our knowledge, only one other patient with 3β-HSD deficiency was diagnosed as an adult; he had neonatal cholestasis and rickets in childhood and presented again at age 26 with cholestasis (23
The clinical features of 3β-HSD deficiency are not easy to distinguish from those of other inherited disorders of bile acid synthesis or transport (2
).. Currently, the diagnosis is dependent on the measurement of bile acids in the urine using mass spectrometry (21
). Patients with 3β-HSD deficiency accumulate 3β-hydroxy-Δ5
bile acids that are reduced in mass by two Daltons from normal saturated bile acids, indicating the presence of a double-bond. Most of these abnormal bile acids are preferentially sulfated at the 3β-hydroxy group and are conjugated in the side-chain with glycine, but not with taurine (5
). In the absence of a urine sample from individual III.5, we confirmed the deficiency in 3β-HSD activity by analyzing an extract of her serum. It is usually difficult to detect bile acids by FAB-MS of serum unless a patient has significant cholestasis (24
), so the presence of atypical 3β-hydroxy-Δ5
- bile acids in serum not only established definitively this genetic defect in bile acid synthesis but also the presence of cholestasis, despite the patient having normal serum liver function tests. These atypical bile acids are cholestatic and hepatotoxic (25
) and in the absence of normal primary bile acids their continued synthesis leads to progressive cholestatic liver disease (2
Liver injury in 3β-HSD deficiency is the result of a lack of normal primary bile acids that are required to stimulate bile flow, combined with the presence of increased production of 3β-hydroxy-Δ5
- bile acids that accumulate due to the enzyme defect (26
). Replacement therapy with oral administration of the primary bile acid, cholic acid reduces the levels of 3β-hydroxy-Δ5
- bile acids through negative feedback inhibition on endogenous bile acid synthesis and this leads to a normalization of the clinical symptoms, liver function tests, and liver histology if initiated prior to development of significant cirrhosis (3
). Prolonged treatment with cholic acid (>15 years) is both safe and efficacious (7
3β-HSD deficiency shows variable expressivity and pleiotropy, but the absence of symptoms in a 32 year old is remarkable. Other clinically asymptomatic individuals have been identified in the course of screening families of patients with 3β-HSD deficiency, but all were significantly younger than this patient. In one family, mass spectrometry of urinary bile acids revealed asymptomatic 3β-HSD deficiency in two of the probands siblings, ages 2 months and 3 years; both siblings had normal serum levels of liver enzymes, although one was vitamin D deficient (15
Patients with 3β-HSD deficiency differ in presentation. Some patients present with signs of liver disease (jaundice, hepatosplenomegaly), others with fat soluble vitamin deficiencies (hypocalcemia, rickets, coagulopathy) or fat malabsorption as a result of cholestasis, or a combination of these features (2
). The proband in our family did not have clinical evidence of cholestasis at presentation, although her bilirubin level was mildly elevated. Although she did not report symptoms consistent with fat malabsorption, she had a history of recurrent mucocutaneous bleeding since childhood which was likely caused by vitamin K deficiency due to cholestasis.
The mechanism responsible for the phenotypic variability in 3β-HSD deficiency remains unknown. One possibility is functional redundancy, such that another enzyme compensates for the loss of 3β-HSD activity. Differences in the ability to metabolize the hepatotoxic and cholestatic bile acids, possibly by intestinal bacterial flora or by other endogenous pathways, could also contribute to the wide variability in expression of this disorder. Finally, individuals may differ in the rate of excretion of the toxic bile acids due to differences in the rate of secretion or efficiency of reabsorption of bile acids that enter the biliary enterohepatic circulation. None of these possibilities explain the mild phenotype in our patient since she had no detectable primary bile acids and the levels of abnormal 3β-hydroxy-Δ5 bile acids in her serum were comparable to those seen in other patients with clinically severe disease.
The c.45–46del AG mutation in HSD3B7
identified in this family was previously found in two unrelated families of British and Canadian origin (3
) and in a French-Senegalese patient with 3β-HSD deficiency (7
). No haplotype data are available to determine if the mutation is a new or recurrent mutation, but the presence of the same mutation in patients of diverse ethnicities implies that this may be a mutational hot spot. Patients carrying this mutation do not show any distinguishing phenotypic features and the age at presentation varies from a few months to 13.5 years. Genotype-phenotype correlation has not been demonstrated for any of the other 20 mutations reported in HSD3B7
It is essential to establish the diagnosis of 3β-HSD deficiency since this is a treatable disorder. Patient III.5 is an ideal candidate for oral cholic acid therapy, which can be expected to lead to a resolution of cholestasis, a suppression of the atypical bile acids by feedback inhibition on hepatic bile acids synthesis, and a concomitant clinical improvement; initiation of oral cholic acid therapy in most cases results in a striking reversal of the histological hallmarks of the disease, even at relatively advanced stages (7
). Dramatic responses to treatment were seen even among patients with severe cholestasis and marked lobular and septal portal fibrosis on liver biopsy (7
Here we used homozygosity mapping with single nucleotide polymorphism (SNP) microarray genotyping as an initial genetic test to pinpoint the causative mutation in this family. With the increasing use of SNP microarrays for whole genome scanning, homozygosity mapping has become easy and rapid. This approach is particularly powerful in situations where there is an increased likelihood of inheriting two alleles identical-by-descent, such as consanguinity or inbreeding. Although the likelihood of homozygosity is smaller in outbred populations, homozygosity mapping has become easy and rapid and widely available through the use of SNP microarrays for routine cytogenetic analysis.
The extent of homozygosity found in this family confirmed a high degree of consanguinity. Offspring of first cousins are expected to be homozygous for ~ 6% (or 1/16th) of their genome. However, in populations with a history of consanguineous matings, the proportion of the genome that is homozygous can reach 11% when considering only homozygous regions that are associated with recessive disease, that is, greater than 3 cM (27
). In this family, individuals III.5, III.6 and III.14, whose DNA was used for homozygosity mapping, were offspring of first cousins. Their genome homozygosity associated with recessive disease was estimated to be 21%, 9.5% and 10%, respectively, indicating that consanguinity was practiced for generations in this family.
An alternative genetic approach that could have been used to identify the causative gene in this family is whole exome (or whole genome) sequencing (28
). This approach has the potential added advantage of revealing modifier genes that contribute to the phenotypic variability of this disorder. Unfortunately, it is unlikely that exome sequencing would have been helpful in identifying modifier genes contributing to the phenotypic variability in this family, given the small number of affected individuals (n=2) available for study and the large number of sequence variations found in genomes. A large scale sequencing study that includes large numbers of carefully phenotyped patients with 3β-HSD deficiency may provide the opportunity to identify modifier genes for this disorder.
In conclusion, we present here a highly consanguineous Arab-Iranian pedigree with four individuals suffering from 3β-HSD deficiency, caused by a recurrent mutation. The clinical presentation was extremely variable with both prolonged asymptomatic and fatal course occurring in the different affected family members. Increased awareness of possible 3β-HSD deficiency in clinical evaluation of cirrhosis in young adults, as well as in children, is essential, since this condition has an excellent prognosis with primary bile acid treatment.