Identification of a rare LAMB4 variant associated with familial diverticulitis through exome sequencing

Abstract

Diverticulitis is a chronic disease of the colon in which diverticuli, or outpouching through the colonic wall, become inflamed. Although recent observations suggest that genetic factors may play a significant role in diverticulitis, few genes have yet been implicated in disease pathogenesis and familial cases are uncommon. Here, we report results of whole exome sequencing performed on members from a single multi-generational family with early onset diverticulitis in order to identify a genetic component of the disease. We identified a rare single nucleotide variant in the laminin β 4 gene (LAMB4) that segregated with disease in a dominant pattern and causes a damaging missense substitution (D435N). Targeted sequencing of LAMB4 in 148 non-familial and unrelated sporadic diverticulitis patients identified two additional rare variants in the gene. Immunohistochemistry indicated that LAMB4 localizes to the myenteric plexus of colonic tissue and patients harboring LAMB4 variants exhibited reduced LAMB4 protein levels relative to controls. Laminins are constituents of the extracellular matrix and play a major role in regulating the development and function of the enteric nervous system. Reduced LAMB4 levels may therefore alter innervation and morphology of the enteric nervous system, which may contribute to colonic dysmotility associated with diverticulitis.

Figure 1.

Pedigree of the recruited family. A total of five family members were analyzed, including three affected by disease (filled circles and squares, II:2, II:3, and III:4) and two unaffected (open circles and squares, II:4, III:3). Dotted outlines designate those members of the pedigree who were not recruited for the study. The index case (indicated by the arrow) was diagnosed with diverticulitis at age 36. Subjects II:2 and II:3 were diagnosed at the ages of 52 and 50 years, respectively. All three had surgical interventions.

Figure 2.

LAMB4 localization and expression in colonic tissue from control and the index diverticulitis patient. Full thickness cross sections of human colon were immunohistologically stained for LAMB4 protein and imaged at 2X magnification for both control (A) and the index patient (D). 40X images of regions from A and D showing LAMB4 staining in individual myenteric plexus in control (B) and index patient (E). Staining of glial marker S100 as a marker of myenteric plexus on the subsequent cut of tissue for both control (C) and index patient (F).

Figure 3.

Human LAMB4 protein structure and positions of variants identified. LAMB4 consists of 1761 amino acids in three separate domain types: LAMB4 N-Terminal (LAMB4 NT), EGF-Like (E), and coiled coil (CC). Shown are missense variants identified from diverticulitis patients through targeted sequencing of LAMB4. Red: variant in index patient.

Figure 4.

LAMB4 protein levels in myenteric plexus of patients with LAMB4 variants. (A) 40X images of representative myenteric plexus in colonic tissue stained for S100, LAMB4 or DAPI from a non-diverticulitis control patient (1), a patient with sporadic diverticulitis but no damaging variants in LAMB4 (2), a patient with sporadic diverticulitis and heterozygous for the rs2074749 variant in LAMB4 (3) and the index patient (4). (B) Average LAMB4 expression in myenteric plexus in colonic tissue from Control, Sporadic and LAMB4 patients. LAMB4 expression was calculated for each patient sample as the average value of the total level of LAMB4 immunofluorescence in 13 separate myenteric plexus on average for each sample after subtracting the level of fluorescence in the surrounding tissue. Bars show the average of those values for four patients without diverticulitis (Control), eleven patients with diverticulitis but without a variant within the LAMB4 coding region (Sporadic) and six patients with diverticulitis carrying a variant within LAMB4 (LAMB4). Each bar represents the mean (± the standard error of the mean) of the average values from multiple plexus from the sampled tissues. (C) LAMB4 expression levels in colonic sections from individual patients who carried variants in LAMB4. LAMB4 expression was calculated for each patient sample as the average value of the total level of LAMB4 immunofluorescence in at least 13 separate myenteric plexus for each sample after subtracting the level of fluorescence in the surrounding tissue. Any value below zero was set to zero. Patient 1, index patient; patient 2, rs149874137; patient 3, chr7:107746309; patient 4, rs147992634; patient 5, rs2074749; patient 6, chr7:107720090.