Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis

Abstract

Hereditary cholestasis in childhood and infancy with normal serum gamma-glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low-GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow-up. Liver biopsy specimens revealed giant-cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP-deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated-cholestasis patients (11 of 38 vs. 0 of 13).

Conclusion: MYO5B deficiency may underlie 20% of previously undiagnosed low-GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655-1669).

Figure 1

Histological findings in MYO5B mutant patients (original magnification, all images, ×400). On H&E staining, hepatocellular and canalicular cholestasis, lobular disarray, mild inflammation, and portal‐tract fibrosis were apparent in all specimens. Giant‐cell formation was observed in all patients, with ballooning degeneration of hepatocytes in P4 and P7. CK7 and CK19 immunostaining revealed ductular reaction in all patients but P6, as well as weak heterotopic CK7 expression in some hepatocytes.

Figure 2

MYO5B expression in MYO5B mutant patients (original magnification, all principal images, ×200; insets, ×400). (A) Choledochal cyst control without cholestasis; (B) incidentally resected normal liver control (adjoining excised tumor); (C) biliary atresia control with cholestasis. MYO5B Patients P3‐P5, P6, and P7: Much coarsely granular pigment was observed in every patient specimen (Fig. 3, P3‐P5, P6, and P7), whereas fewer and finer MYO5B‐positive granular deposits were observed in the control individuals, mainly distributed around portal areas (Fig. 3 A,B). The size and number of positive granules in the biliary atresia patient (Fig. 3C) were intermediate between those of the patient group and the control group; the granules in this patient were periportal.

Figure 3

BSEP staining in MYO5B mutant patients (original magnification, all principal images, ×400; insets, 900×). (A) Choledochal cyst control without cholestasis; (B) incidentally resected normal liver control (adjoining excised tumor); (C) biliary atresia control with cholestasis; (D) confirmed PFIC2 patient with biallelic ABCB11 mutations (p.I498T / p.R415X); (E) discarded normal liver control (healthy liver donor). MYO5B patients P3‐P5, P6, and P7: Compared to the control A and B, less expression of BSEP was observed in P3, P4, P6, and P7 (Fig. 3, P3‐P4, P6, and P7), whereas expression was blurred at canaliculi and adjacent cytoplasm in P5 (Fig. 3, P5). Black arrows indicate abnormalities in P5 and P7 (insets).

Figure 4

Bile acids in plasma of MYO5B mutant patients. Bile acid (BA) profiles obtained by UPLC−ESI/MRM‐MS (μM) of plasma from 4 patients carrying MYO5B defects, compared to those from 16 patients confirmed to harbor biallelic ABCB11 mutations and 20 healthy controls. In the MYO5B group, P1, P2, and P4 refer to patients 1, 2, and 4, respectively, and (P5‐1) and (P5‐2) both refer to patient 5, sampled twice, during and after a bout of cholestasis. (A) TBAs based on 62 standards that cover all major bile acids and many rare bile acids (see Supporting Table S4); (B) total conjugated bile acids, including glycol‐CDCA, glyco‐CA, tauro‐DCA,tauro‐CA, and tauro‐CDCA; (C) total free bile acids, including CA, CDCA, DCA and LCA; (D) total primary bile acids, including CA, CDCA, glyco‐CA, glycol‐CDCA, tauro‐CA, and tauro‐CDCA; (E) total secondary bile acids, including DCA, LCA, 7‐KLCA, 12‐KLCA, 67‐diKLCA, and DioxoLCA; (F) total UDCA including free, glycol‐, and tauro‐UDCA and their sulfated forms. The number below each of the MYO5B and ABCB11 groups is the P value of the group versus controls.

Figure 5

Schematic presentations of MYO5B protein and reported homozygous or compound heterozygous mutations. Upper arrows indicate reported mutations in MVID patients.24, 25, 27, 29, 36, 37 Lower arrows show mutations in isolated‐cholestasis patients reported in this article (thicker arrows) and elsewhere.38 Protein data are deduced from UniProt (http://www.uniprot.org/uniprot/Q9ULV0). Severe mutations include truncations and classical splicing mutations. Abbreviation: IQ, Isoleucine‐glutamine (IQ) calmodulin‐binding consensus sequence. ^*Mutations p.I408F and p.L528F were reported in an MVID patient with “atypical” cholestasis.29