The challenge of personalized cell biology: The example of microvillus inclusion disease

Abstract

Whole exome sequencing now provides a tool for rapid analysis of patients manifesting congenital diseases. Congenital diarrheal diseases provide a critical example of the challenges of combining identification of genetic mutations responsible for disease with characterization of the cell biological and cell physiological deficits observed in patients. Recent studies exploring the cellular events associated with loss of functional Myosin 5B (MYO5B) have demonstrated the importance of cell biological and physiological analyses to provide a greater understanding of the implications of pathological mutations. Development of enteroids derived from biopsies of patients with complex congenital diarrheal diseases provides a critical resource for evaluation of the cell biological impact of specific monogenic mutations on enterocyte function. The ability to identify putative causative mutations for congenital disease now provides an opportunity to coordinate the efforts of physicians and cell biologists in an effort to provide patients with personalized cell biology analysis to improve patient diagnosis and treatment.

The development of personalized or precision medicine strategies has led to increasing utilization of approaches to tailor treatments to the individual characteristics of patients. Much of this process of specification of treatments relies on genetic analysis of patients or their tumors. While analysis of the genetics of cancers had led to new strategies for more targeted therapies for cancers, genetic analysis of more benign conditions has often lagged. Publications in Traffic have often highlighted the impact of mutations in trafficking proteins in human diseases.^1,2 The identification of genetic mutations responsible for pediatric congenital diarrhea syndromes represents a notable example of how genetic analysis is impacting the diagnosis and eventual treatment of monogenic pathology. In recent years, examination of genetics is radically changing the diagnostic algorithms applied to the presentation of congenital diarrhea syndromes.³ Just as importantly, whole exome and whole genome sequencing of patients with undiagnosed congenital diarrhea syndromes is ushering in a new age of personalized cell biology that allows genetic mutations to be connected more rapidly to manifestations of altered cell biology and cell physiology.

Recent studies have identified mutations in key regulatory proteins that are responsible for congenital diarrhea syndromes. A major class of proteins affected by these mutations are proteins involved in vesicle trafficking in enterocytes, including MYO5B, DGAT1, STX3, Munc18-2, and TTC7A.³ These mutations seem to alter the trafficking of key transporters to the apical membrane to establish appropriate absorption of sodium and water from the gut lumen. Perhaps the most studied congenital diarrhea syndrome is microvillus inclusion disease (MVID), which is caused by truncation or mis-sense mutations in MYO5B that lead to loss of MYO5B function.^4,5 A less severe related congenital diarrhea syndrome can also be caused by mutations in either syntaxin 3 or MUNC-18-2.^6,7 Recent studies have shown that loss of MYO5B induces deficits in apical trafficking of NHE3 and SGLT1, while CFTR is still trafficked to the apical membrane.⁸ While the disease was first described by the presence of large inclusions lined by microvilli in enterocytes, studies in MYO5B KO mouse models have suggested that microvillus inclusions are not the cause of the apical trafficking defect.^8,9 Indeed, analysis of the microvillus inclusions in MYO5B KO mice has revealed that an unrecognized apical bulk endocytosis pathway in enterocytes from these mutant mice is responsible for inclusion formation.¹⁰ These findings demonstrate how analysis of genetic mutations can lead to recognition of basic cell biological insights. Thus, it is clear from a number of studies that there must be multiple vesicle trafficking pathways to the apical membrane in enterocytes and likely multiple paradigms for endocytosis and recycling. Indeed, the devastating impact of MYO5B loss or inactivation may accrue from the range of Rab proteins that can interact with MYO5B, including Rab11a, Rab11b, Rab25, Rab8a, Rab10, and Rab6.^11–15

The example of MYO5B and MVID also highlights the remaining problems in making definitive diagnoses for patients with congenital diarrhea. Essentially all patients with MVID display biallelic MYO5B mutations in either homozygous or compound-heterozygous form. Individuals with single heterozygous allele mutations are asymptomatic. The presence of two damaged alleles can be confirmed through biallelic inheritance of mutations from the parents. The demonstration of bi-allelic inheritance highlights why the parents of probands should also be examined with “trio”-whole exome sequencing (ie, sequencing of the patient and both parents) as the most efficient and comprehensive mode for genetic analysis. When one of the parents is not available for sequencing or when one of the alleles might come from a de novo mutation, more sophisticated methods are necessary to demonstrate that both alleles in the proband are damaged,¹⁶ since as noted, MYO5B mutant heterozygotes tend to be asymptomatic. Also, the number of newly-identified mutations in MYO5B continues to grow. While some of these can be traced to particular critical regions in the motor domain or in the motor lever arm, the question of actual impact can be of importance in making the diagnosis. While morphological criteria are sometimes helpful, the presence of microvillus inclusions is often highly variable. In normal enterocytes, MYO5B is distributed underneath the apical brush border, so an absence of MYO5B immunostaining or a mislocalization of MYO5B may support an alteration in functional motor activity. Few of the motor mutations have been studied directly, but altered function in expressed mutated protein can be demonstrated in the P660L mutation present in Navajo MVID patients.¹⁷ While complete loss of MYO5B leading to MVID has been replicated in mice,^8,9,18,19 the impact of particular mutations on specific aspects of disease remains unclear. Thus, it is unclear whether particular mutations can induce a range of disease manifestations through variations in the severity of deficits in trafficking. In addition, while most MVID patients show both severe intestinal disease as well as more moderate cholestasis, it is now clear that certain patients may manifest only liver disease.^20,21 The molecular impact of these mutations has not been defined in detail, so it is not clear how these MYO5B mutations elicit a predominantly liver-specific phenotype. These findings underscore the need to define the impact of mutations on MYO5B function.

Mouse models have provided important systems for the interpretation of gene deletions and mutations. Analyses of mouse models of MYO5B loss have been critical in gaining an understanding of trafficking defects.^8,18 Furthermore, streamlined methods for CRISPR production of mouse point mutants have now made development and utilization of directed mutant mice a reasonable approach in understanding new genetic mutations along a 4-6 months timeline. Nevertheless, perhaps the methods with the greatest utility lie in the preparation and characterization of cell biological and physiological deficits in enteroids derived from patient biopsies or induced pluripotent stem cells (iPSCs) from patients. In particular, enteroids represent an in vitro cell culture system derived from stem cells grown in three-dimensional matrices, that can maintain all intestinal cell types and recapitulate many physiological cell functions. These cultured enteroid can be rapidly developed from patient biopsies within 4-6 weeks and studied through a number of cell biological systems, including comparison of enteroid trafficking patterns by immunostaining between patient biopsies and enteroids. Human enteroids also can polarize into monolayers, and the utilization of air-liquid interfaces can promote terminal differentiation with both construction of a fully developed brush border and appropriate trafficking of transporters and apical enzymes into the brush border. iPSC-derived enterocytes can also be of great use and can be derived from patients without endoscopy, but the processes for lineage differentiation are complex and true terminal differentiation characteristics are less well established. All of these patient derived organoid systems model the epithelial compartment, which is the focus for most congenital diarrhea syndromes, rather than the stromal or immune elements that contribute to other entities such as early onset inflammatory bowel disease.

The question remains of how detailed analysis of the cell biological and cell physiological alterations that ensue from mutations in trafficking proteins can be integrated into clinical care to facilitate more rapid diagnosis and improved treatment. In the case of congenital diarrheal diseases, a recent publication by the COngenital Diarrhea and Enteropathy (PediCODE) consortium has promoted the pathway of enteroid/organoid development from biopsies at the time of initial diagnostic endoscopy, coordinated with detailed immunostaining phenotyping of biopsy tissues and whole exome sequencing of the proband and their parents.³ This pathway certainly provides the most efficient approach to these diseases, but in the case of identification of novel mutations, a rapid coordinated effort to define the molecular impact of putative mutations is also needed. These efforts can often define a candidate pathogenic mutation and guide therapeutic interventions, as has been seen recently for patients with mutations that lead to loss of DGAT1.^22–24 Still, facilitating genetic diagnosis cannot guarantee that clear treatment options are possible, and present gene therapy approaches are not suited for restoration of function across the 20 ft of small intestine. It should also be recognized that for some congenital diarrheal syndromes that result from monogenic mutations, even life-threatening ones, symptoms may resolve spontaneously if the children can be sustained through early life by total parenteral nutrition. Thus, the intestinal mucosa may find ways around specific mutations and heal itself, given enough time.

In the final analysis, it is clear that we have entered a new age where characterization of the cell biological and cell physiological function of patient derived enteroids/organoids will have an increasingly important role in defining patient diagnoses as well as in deriving appropriate possible treatments. To establish this personalized cell biology, physicians must build strong relationships with basic cell biologists to integrate enteroid preparation and characterization as well as direct molecular analyses into the normal clinical paradigm for taking care of patients with congenital diarrheal disease in a timely fashion. This means that cell biologists will need to be mobilized as part of a total care team integrated with geneticists and neonatologists.