To identify genetic causes for hEDS, a genetic registry was developed at the Medical University of South Carolina (MUSC). Within this registry, a four generation family was identified that presented with autosomal dominant hEDS. Eleven family members were enrolled for genetic analysis, of whom five met the clinical diagnostic criteria for hEDS and three were coded as probable due to age and clinical history at time of analyses. Whole exome sequencing (WES) of the proband (IV-1) and a second cousin (IV-4) was performed. Following variant filtering, four rare and potentially damaging variants were shared between the affected individuals (IV-1 and IV-4). PCR amplification and targeted sequencing of all enrolled family members identified only one of these variants with a perfect phenotype-genotype segregation throughout the pedigree. This variant, located in the Kallikrein serine-protease gene KLK15 (chr19:50825890-C-T), was also found in affected members of a second family. The single nucleotide polymorphism (SNP) results in a missense change (KLK15 p. Gly226Asp), is rare in the population with a minor allele frequency (MAF) of 0.002 (gnomAD v2.1.1), and is predicted to be damaging with CADD and DANN scores of 24 and 0.998, respectively. To determine relevance to connective tissue biology, RT-PCR was performed and confirmed KLK15 mRNA expression in glandular and connective tissues isolated from human and mouse biopsies.
KLK15 is part of a contiguous cluster with 14 other members of the Kallikrein gene family on chromosome 19q13.33. Given the known involvement of Kallikreins in regulating one another through activation cascades, and their shared expression patterns in connective tissues, we evaluated the genetic burden of the entire KLK family of genes in a larger hEDS cohort. WES was performed on 197 clinically diagnosed, unrelated hEDS patients and filtered for KLK variants with MAFs less than 0.01 (<1%) in gnomAD. A total of 76 variants were identified, with 48 being unique in the cohort and 65 patients having at least one rare variant in a KLK gene (32.8%). A gene-based burden test was used to evaluate enrichment of rare variants in individual KLK genes and the entire contiguous gene cluster in hEDS patients. Significant enrichment for qualifying variants was observed in 11 of the 15 KLK genes with p-value <0.05 as well for the entire KLK gene cluster (considered as whole, p = 2.28×10−14); thus supporting a broad role for Kallikrein genes in hEDS.
Exploring the molecular consequences of Kallikrein variants will not only uncover mechanisms of normal connective tissue development and disease but also shed light on the comorbidities commonly associated with hEDS. Notably, Kallikreins are known to interact with substrates in the extracellular matrix, influencing the connective tissue environment in both homeostasis and disease. This class of genes also plays roles in blood pressure regulation and immune cell function, potentially contributing to various comorbidities such as postural orthostatic tachycardia syndrome (POTS) and mast cell activation syndrome, which are frequently observed in hEDS patients. Relatedly, it is curious that there is a tight interaction between Kallikreins and in the innate immune system, specifically the complement system. Kallikreins can cleave complement 3 (C3) and 5 (C5) directly, leading to the generation of C3a and C5a16 , which are potent anaphylatoxins that enhance inflammation upstream of mast cell activation.
Given the autosomal dominant mode of inheritance and the variability of phenotypes associated with hEDS, it is likely that KLK gene variants, such as KLK15 G226D, function primarily in a dominant-negative manner. However, given the known synergistic hierarchy of Kallikreins, where they auto-activate and catalyze the activation of downstream Kallikrein enzymes, even subtle changes in expression levels due to loss-of-function alleles may have damaging effects. While we implicate KLK variants in hEDS, they represent just one aspect of the genetic landscape.
