Genetic testing for Mendelian stroke due to cerebrovascular anomalies and other syndromes

Ischemic stroke (IS) is a condition caused by complete or partial obstruction of a vessel in the brain, resulting in lack of blood supply and death of brain tissue. Genetic factors play an important role in the development of IS. Studies on twins have demonstrated that stroke prevalence is about five times higher in monozygotic than in dizygotic twins (4). In a large group of stroke patients, the odds ratio of having a family history of stroke was 2.24 for large vessel-disease and 1.93 for small vessel disease (5). For stroke patients under the age of 65, the odds ratio was even higher: 2.93 and 3.15, respectively (5, 6).

The most common causes of IS are atherosclerosis, cardioembolism and small-vessel disease (lacunar stroke) (2). Non-modifiable risk factors (age, African or Asian ethnicity, male sex) and acquired risk factors (hypertension, cigarette smoking, diabetes, atrial fibrillation and obesity) account for much of the risk of ischemic stroke (7). However, the underlying mechanisms of most of these risk factors are genetic.

Incidence rates for IS in the 15 to 45-year age range are ≈10 per 100,000 person years for individuals of predominantly European ancestry, with similar rates for men and women (8).

IS occurs suddenly and may have the following symptoms, depending on the affected area of the brain: unilateral insensitivity or paralysis, confusion and difficulty speaking or following speech, dizziness, loss of balance, coordination, abnormal migraine accompanied by facial pain, sudden reduction or loss of vision, vomiting and altered states of consciousness.

Diagnosis is based on routine physical examination, neurological examination, non-contrast computerized tomography, CT angiography, MR imaging and genetic testing.

Differential diagnosis should consider acute hypoglycemia, brain neoplasms, encephalitis headache, migraine, hypernatremia in emergency medicine, hyperosmolar hyperglycemic nonketotic coma, hypertensive emergencies, hyponatremia, labyrinthitis ossificans, meningitis, hemorrhagic stroke, subarachnoid hemorrhage, subdural hematoma and transient ischemic attack.

Familial and sporadic cases of Mendelian IS are well-documented in the literature and show a greater incidence at young age.

Pathogenic variants may include missense, nonsense, splicing, small insertions and deletions, small indels, gross deletions and duplications.

Hemorrhagic stroke (HS) is an acute manifestation of common progressive cerebrovascular disease among elderly patients. HS is the least treatable and leading cause of acquired disability and mortality. Genetic mechanisms play an important role in its development. It is therefore necessary to develop HS-specific therapeutics and effective primary prevention strategies. Identification of HS-related genes is a promising direction in novel HS prevention (11). Study of genetic mechanisms of HS risk factors, including hypertension, heavy alcohol intake and anticoagulant treatment, is also important (12).

The underlying mechanism of HS is thought to be leakage from small intracerebral arteries damaged by chronic hypertension, bleeding diatheses, iatrogenic anticoagulation, cerebral amyloidosis and cocaine abuse.

HS mostly affects brain sites such as the thalamus, putamen, cerebellum and brainstem. The brain may be damaged by the hemorrhage or by increased intracranial pressure due to a hematoma (13).

According to the World Health Organization, 15 million people suffer stroke worldwide each year. Of these, 5 million die and another 5 million are left permanently disabled (14). The overall incidence of spontaneous HS worldwide is 24.6 per 100 000 person-years. The 30-day mortality rate ranges from 35% to 52% with only 20% of survivors expected to have full functional recovery at 6 months. Approximately half of this mortality occurs within the first 24 hours (15-18).

HS is characterized by a relatively sudden onset of symptoms and neurological deficits, the type and severity of which vary according to lesion type, location and size (19). Findings such as coma, headache, vomiting, seizures, neck stiffness and raised diastolic blood pressure increase the likelihood of HS compared to ischemic stroke, but only neuroimaging can provide a definitive diagnosis (20). Focal neurological deficits present in the following ways: hemiparesis, hemisensory loss, visual field cut on the opposite side to the affected hemisphere, gaze preference on the same side as the affected brain, aphasia, unilateral neglect.

Diagnosis is based on routine physical examination, neurological examination, noncontrast computerized tomography, CT angiography, MR imaging and genetic testing.

Differential diagnosis should consider acute hypoglycemia, brain neoplasms, encephalitis headache, migraine, hypernatremia in emergency medicine, hyperosmolar hyperglycemic nonketotic coma, hypertensive emergencies, hyponatremia, labyrinthitis ossificans, meningitis, stroke, ischemic subarachnoid hemorrhage, subdural hematoma, transient ischemic attack.

HS has autosomal dominant and autosomal recessive inheritance.

Pathogenic variants may include missense, nonsense, splicing, small insertions and deletions, small indels and gross deletions and duplications.

The test is listed in the Orphanet database and is offered by 39 accredited medical genetic laboratories in the EU, and in the GTR database, offered by 17 accredited medical genetic laboratories in the US.

Guidelines for clinical use of the test are described in Genetics Home Reference (ghr.nlm.nih.gov)

Clinically distinguishable syndromes can be analyzed by sequencing only those genes known to be associated with that specific disease using Sanger or Next Generation Sequencing (NGS); if the results are negative, or more generally if clinical signs are ambiguous for diagnosis, a multi-gene NGS panel is used to detect nucleotide variations in coding exons and flanking introns of the above genes.

The test for paradominant genes (KRIT1, CCM2 or PDCD10) is to compare results obtained from germinal lineage (blood or saliva specimens) and affected tissue. For variant selection, a cut-off value (related to biopsy and blood results) is used, and if the variant frequency is higher than the cut-off value it is considered for further analysis. The cut-off depends on tissue quality, extraction method, biocomputing software and other parameters. Potentially causative variants need to be verified by further means (e.g. cloning + Sanger sequencing, Sanger sequencing, minisequencing).

Potentially causative variants and regions with low coverage are Sanger-sequenced. Sanger sequencing is also used for family segregation studies.

Multiplex Ligation Probe Amplification (MLPA) is used to detect duplications and deletions in ABCC6, ACVRL1, APP, CCM2, COL3A1, ENG, FBN1, GLA, KRIT1, MTHFR, NF1, NOTCH3, SAMHD1, SMAD4 and TREX1

To perform molecular diagnosis, a single sample of biological material is normally sufficient. This may be 1 ml peripheral blood in a sterile tube with 0.5 ml K₃EDTA or 1 ml saliva in a sterile tube with 0.5 ml ethanol 95%. Sampling rarely has to be repeated.

A frozen intra-lesional biopsy specimen, in addition to blood or saliva, is necessary to test for somatic variations.

Gene-disease associations and the interpretation of genetic variants are rapidly developing fields. It is therefore possible that the genes mentioned in this note may change as new scientific data is acquired. It is also possible that genetic variants today defined as of “unknown or uncertain significance” may acquire clinical importance.

Identification of pathogenic variants in the above genes confirms the clinical diagnosis and is an indication for family studies.

A pathogenic variant is known to be causative for a given genetic disorder based on previous reports, or predicted to be causative based on loss of protein function or expected significant damage to proteins or protein/protein interactions. In this way it is possible to obtain a molecular diagnosis in new/other subjects, establish the risk of recurrence in family members and plan preventive and/or therapeutic measures.

Detection of a variant of unknown or uncertain significance (VUS): a new variation without any evident pathogenic significance or a known variation with insufficient evidence (or with conflicting evidence) to indicate it is likely benign or likely pathogenic for a given genetic disorder. In these cases, it is advisable to extend testing to the patient’s relatives to assess variant segregation and clarify its contribution. In some cases, it could be necessary to perform further examinations/tests or to do a clinical reassessment of pathological signs.

The absence of variations in the genomic regions investigated does not exclude a clinical diagnosis but suggests the possibility of:

Unexpected results may emerge from the test, for example information regarding consanguinity, absence of family correlation or other genetically-based diseases.