Next-Generation Sequencing Advances the Genetic Diagnosis of Cerebral Cavernous Malformation (CCM)

Abstract

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin that predisposes to seizures, focal neurological deficits and fatal intracerebral hemorrhage. It may occur sporadically or in familial forms, segregating as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. Its pathogenesis has been associated with loss-of-function mutations in three genes, namely KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), which are implicated in defense mechanisms against oxidative stress and inflammation. Herein, we screened 21 Italian CCM cases using clinical exome sequencing and found six cases (~29%) with pathogenic variants in CCM genes, including a large 145–256 kb genomic deletion spanning the KRIT1 gene and flanking regions, and the KRIT1 c.1664C>T variant, which we demonstrated to activate a donor splice site in exon 16. The segregation of this cryptic splicing mutation was studied in a large Italian family (five affected and seven unaffected cases), and showed a largely heterogeneous clinical presentation, suggesting the implication of genetic modifiers. Moreover, by extending genetic analysis to a virtual panel of 23 cerebrovascular disease-related genes (Cerebro panel), we found two variants in NOTCH3 and PTEN genes, which could contribute to the abnormal oxidative stress and inflammatory responses to date implicated in CCM disease pathogenesis.

Distant Recurrence of a Cerebral Cavernous Malformation in the Vicinity of a Developmental Venous Anomaly: Case Report of Local Oxy-Inflammatory Events

“…Moreover, additional sources of oxy-inflammatory events already implicated in CCM disease pathogenesis are pathogen-associated molecular patterns (PAMPs), including bacterial PAMPs known to activate TLR4 signaling, such as lipopolysaccharide (LPS) [ 69 ], as well as hypoxia [ 70 ]. Furthermore, consistent with the fact that focal oxy-inflammatory conditions contribute significantly to CCM lesion formation and severity, distinct genetic modifiers of oxy-inflammatory responses have been identified as linked to the incomplete penetrance and highly variable expressivity of CCM disease in large cohorts of fCCM cases [ 8 , 44 , 46 , 51 ]. In addition, new intriguing and significant evidence in animal models suggests that CCM gene mutations can have pathological effects not limited to CCM disease, as they may predispose to the development of other pathological conditions associated with abnormal oxy-inflammatory responses, such as atherosclerosis and hepatic metabolic, antioxidant, and antiglycative dysfunctions [ 77 , 78 ].…”

“…So far, fCCM has been associated with loss-of-function mutations in three genes, KRIT1 / CCM1 , CCM2 and PDCD10 / CCM3 , which have been implicated in a plethora of essential physiological functions, including modulation of cadherin- and integrin-mediated cell adhesion, actin cytoskeleton dynamics, and signal transduction involved in the maintenance of endothelial barrier stability [ 7 ]. However, no mutation in these CCM genes were found in up to 15% of fCCM cases, suggesting the potential existence of additional causative determinants [ 8 , 9 ]. Conversely, mutations in the three known CCM genes were rarely detected in sCCM lesions, which instead resulted associated mainly with somatic mutations in other genes [ 10 , 11 , 12 ], suggesting that fCCM and sCCM lesions may have a distinct genetic origin.…”

“…The pathophysiology of CCM disease remains to be fully elucidated, as there is still some controversy regarding the multiple mechanisms proposed so far. Among others, recent genetic analyses of surgically excised lesions from sCCM patients and distinct studies in conditional knockout mouse models have clearly demonstrated that homozygous loss-of-function mutations in CCM genes are neither necessary nor sufficient to cause the development of CCM lesions, thereby limiting the two-hit model proposed previously [ 42 , 43 ] and suggesting the implication of additional determinants, including local stressful conditions [ 8 , 9 ]. Consistently, whereas the clinical expression of CCM disease is known to be highly variable even among family members carrying the same CCM gene mutation, microenvironmental stressors, including inflammatory and oxidative stress events, and interindividual variability in stress response have progressively emerged as important determinants of CCM disease pathogenesis and severity [ 7 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ].…”

Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes

“…Large deletions have been reported for all three CCM genes [ 2 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. Their size ranges from a few hundred kilobases to 1.9 megabases [ 24 , 30 , 31 ].…”

Heterozygous Loss of KRIT1 in Mice Affects Metabolic Functions of the Liver, Promoting Hepatic Oxidative and Glycative Stress

“…Besides KRIT1 (also known as CCM1), whose mutations account for more than 50% of fCCM cases, CCM disease has been associated with mutations in two other genes, CCM2 and PDCD10 (also known as CCM3), which account for about 20% and 10% of the cases, respectively [ 9 ]. However, accumulated evidence in endothelial-specific conditional knockout (cKO) mouse models has clearly demonstrated that even the homozygous loss of any of the three known CCM genes is not sufficient to cause the formation of CCM lesions, thus confirming the necessary contribution of additional determinants, including microenvironmental risk factors and genetic modifiers of tissue sensitivity to stressful conditions [ 8 , 10 , 11 ]. Accordingly, over the last decade, it has clearly emerged that KRIT1 loss-of-function predisposes the development of CCM lesions by exerting pleiotropic effects on key redox-sensitive mechanisms involved in cellular homeostasis and defenses against oxidative stress and inflammation, leading to enhanced endothelial cell susceptibility to oxy-inflammatory insults [ 8 , 10 , 12 , 13 , 14 ].…”