Variables of data collection for an Ischemic Stroke Genetics Study:

• Structured interview

• Demographics

• Medications

• Family history

• Past medical history

• Social and behavioural history

• QVSS

• Vital signs

• Medical records

• Blood sample

• Laboratory tests

• Head imaging

• Vascular imaging

• Treatments

• Stroke status

• Subtyping

• Time of stroke onset

• Assessment of the severity of the neurological deficit: NIH Stroke Scale (NIHSS) - http://www.ninds.nih.gov/doctors/NIH_Stroke_Scale.pdf

Definite focal signs - reproduced from the WHO MONICA Manual - http://www.ktl.fi/publications/monica/manual/part4/iv-2.htm#Introduction:

• Unilateral or bilateral motor impairment (including dyscoordination)

• unilateral or bilateral sensory impairment

• aphasis/dysphasis (non-fluent speech)

• hemianopia (half-sided impairment of visual fields)

• Diplopia

• Forced gaze (conjugate deviation)

• Dysphagia of acute onset

• Apraxia of acute onset - http://www.ninds.nih.gov/disorders/apraxia/apraxia.htm

• Ataxia of acute onset - http://www.mdvu.org/library/pediatric/ataxia/

• Perception deficit of acute onset.

Not acceptable as sole evidence of focal dysfunction - although strokes can present in this way, these signs are not specific and cannot therefore be accepted as definite evidence for stroke - reproduced from the WHO MONICA Manual - http://www.ktl.fi/publications/monica/manual/part4/iv-2.htm#Introduction:

• Dizziness, vertigo

• Localized headache

• Blurred vision of both eyes

• Dysarthria (slurred speech)

• Impaired cognitive function (including confusion)

• Impaired consciousness

• Seizures

Diagnostic Categories - reproduced from the WHO MONICA Manual - http://www.ktl.fi/publications/monica/manual/part4/iv-2.htm#Introduction:

There are the following three categories:

(1) = Definite stroke
(4) = Not stroke
(5) = Definite stroke associated with Definite myocardial infarction
(9) = Insufficient data

Insufficient data should be mainly used for fatal cases, especially for cases of sudden death without necropsy.

All patients having insufficient supporting evidence of stroke, but for whom the diagnosis of stroke cannot be entirely excluded, should be classified as insufficient data, e.g. cases with no necropsy, no documented history of focal neurologic deficits and no other diagnosis. Living patients can be classified into this category if:

1. it is impossible to say whether the symptoms were from stroke or from some other disease, e.g. epilepsy, or

2. patients with symptoms and clinical findings otherwise typical for a stroke but the duration remaining uncertain.

• Confirmation of given subject/s being stroke-free: Validating the Questionnaire for Verifying Stroke-Free Status (QVSFS) by Neurological History and Examination - http://stroke.ahajournals.org/cgi/content/abstract/32/10/2232

Measures to asses the severity of neurological deficit in a stroke patient:

NIH Stroke Scale (NIHSS): for assessing the severity of the neurological deficit:

• NIHSS (Original Format) - http://www.ninds.nih.gov/doctors/NIH_Stroke_Scale.pdf

• NIHSS (Booklet) Barthel Index - http://www.ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf

• Oxford Handicap Scale

• Glasgow Outcome Scale - http://www.trauma.org/scores/gos.html

• Trial of Org 10172 in Acute Stroke Treatment (TOAST) - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&list_uids=7678184&dopt=Citation

• Oxfordshire Community Stroke Project (OCSP) - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=1675378&dopt=Abstract

• Baltimore-Washington Young Stroke Study (BWYSS) - http://stroke.ahajournals.org/cgi/content/full/strokeaha;26/1/46

Types of stroke:

Management and prognosis of different stroke subtypes differ. For any stroke study, it is therefore imperative to establish the exact stroke subtype in the studied subjects.

Subarachnoid Haemorrhage ICD 8 or 9 43 - http://www.nlm.nih.gov/medlineplus/ency/article/000701.htm

Symptoms:

Abrupt onset of severe headache or unconsciousness or both. Signs of meningeal irritation (stiff neck, Kernig and Brudzinski signs). Focal neurological deficits are usually not present.

Signs:

At least one of the following must be present additional to typical symptoms.

1. Necropsy - recent subarachnoid haemorrhage and an aneurysm or arteriovenous malformation

2. CT - blood in the Fissura Sylvii or between the frontal lobes or in the basal cistern or in cerebral ventricles

3. Cerebrospinal fluid (liquor) bloody (>2 000rbc per cmm) and an aneurysm or an arteriovenous malformation found on angiography

4. Cerebrospinal fluid (liquor) bloody (>2 000rbc per cmm) and xanthochromic and the possibility of intra-cerebral haemorrhage excluded by necropsy or CT-examination.

Intracerebral haemorrhage ICD 8 or 9 431 - http://www.emedicine.com/emerg/topic557.htm

Symptoms:

Usually sudden onset during activities. Often rapidly developing coma, but small haemorrhage presents no consciousness disturbance.

Signs:

Cerebrospinal fluid often, but not always bloody or xanthochromic. Often severe hypertension present. Haemorrhage must be confirmed by necropsy or by CT-examination.

Brain infarction due to occlusion of precerebral arteries ICD 9 433 ICD 8 432

Symptoms:

May vary.

Signs:

The occlusion must be confirmed by angiography or ultrasound or necropsy.

Brain infarction due to cerebral thrombosis ICD 9 434 ICD 8 433 - http://www.emedicine.com/emerg/topic558.htm

Symptoms:

No severe headache, if at all. Onset acute, sometimes during sleep. Often gradual progression of focal neurologic deficits. Usually, no, or only slight, disturbance of consciousness. TIA can often be detected in history. Often other symptoms of atherosclerosis (CHD, peripheral arterial disease) or underlying diseases (hypertension, diabetes).

Signs:

Brain infarction in the necropsy or in the CT-examination and no evidence for an embolic origin.
 

OR

 

CT scan of satisfactory quality shows no recent brain lesion although clinical criteria of stroke are fulfilled.

Embolic brain infarction ICD 9 434 ICD 8 434 - http://www.emedicine.com/emerg/topic558.htm

Symptoms:

Abrupt onset, usually completion of the neurologic deficits within a few minutes. Disturbance of consciousness absent or only slight at the onset.

Signs:

As in brain infarction due to cerebral thrombosis, but in addition a source of the embolus must be detectable. The most common origins are:

• arrhythmia (atrial flutter and fibrillation)

• valvular heart disease (mitral)

• recent AMI (within previous 3 months).

Hypertension, DM, smoking and hypercholesterolemia are considered as the major risk factors for stroke. It is a well-established fact that conditions like hypertension, DM and hypercholesterolemia have strong genetic basis. Even the effects of exposure to toxins contained in cigarette smoke, and infectious agents are modified by an individual's genetic make-up. In this way, it can be safely said that stroke (atherosclerosis, precisely speaking) has a strong genetic component in its pathobiology.

Infection:

Since host defences are modified by genes, an individual's susceptibility to develop infection is very much governed, at least in part, by his genetic make-up. An example being the strong association of certain human leukocyte antigen (HLA) classes to autoimmune responses. A ubiquitous infectious agent thus may cause an inflammatory response in some but not others.  Hegele et al, (2000) found that some polymorphisms of the mannose-binding lectin gene, which codes for a protein designed to help facilitate phagocytosis, increase the risk of infection in humans. These polymorphisms also were found to correlate with the presence and size of carotid atherosclerotic plaques.

The pro-inflammatory genotype, although helps in wound healing and eradication of infections, may contribute to atherosclerosis and insulin resistance. Many genes coding for molecules involved in inflammatory mechanisms may thus have possible connection to atherosclerotic disease.

Common sporadic strokes - a consequence of polygenic or multifactorial influences:

Most strokes appear to be sporadic. There is considerable evidence that suggests that sporadic stroke is due in part to genetic influences. Rather than being due to a highly penetrant single gene disorder however, common sporadic strokes are thought to arise as a consequence of polygenic or multifactorial influences whereby multiple genes each exert a small influence or risk on phenotype - http://www.acnr.co.uk/pdfs/volume4issue4/v4i4reviewartstroke.pdf

Monogenic stroke - http://brain.oxfordjournals.org/cgi/content/full/123/9/1784

Although, majority of cases of ischaemic stroke are multifactorial in aetiology, mutations in the Notch 3 gene is increasingly appreciated as a cause of familial subcortical stroke. Single-gene defect as a cause of stroke should be considered in the differential diagnosis of any young patient presenting with stroke, or a young or middle-aged adult who lacks the usual risk factors, particularly in the presence of a family history of young-onset stroke.

• Single-gene disorders that cause cardioembolic stroke - http://stroke.ahajournals.org/cgi/content/full/strokeaha;32/12/2762 In cases of embolic stroke, the usual source of embolus is heart. Many cardiac ailments that predispose to thromboembolic phenomenon are single-gene disorders e.g. familial atrial myxomas, hereditary cardiac conduction defects and inherited cardiomyopathies. The latter may present as a primary cardiac disorder (e.g. hypertrophic obstructive cardiomyopathy) or be secondary to a neuromuscular disorder (e.g. Duchenne muscular dystrophy) or one of the inborn errors of metabolism (e.g. Menkes disease).

• Single-gene disorders that cause large artery disease:  Important single-gene disorders include homocysteinuria and dyslipidaemias. The molecular genetic basis of homocysteinuria has been well characterized. The cystathione ß-synthase gene maps to chromosome 21q22.3 (Kraus et al., 1993), and there are several relatively common mutations. The I278T and A114V mutations are widely distributed and frequently found in pyridoxine responders, whilst the G307S and A1224–2C mutations occur more frequently in northern European populations and are associated with a lack of pyridoxine responsiveness (Sebastio et al., 1995). As regards dyslipidaemias, a link has been reported in several disorders, including familial hypoalphalipoproteinaemia, familial hypercholesterolaemia (homozygous form), type II and type IV hyperlipidaemia and Tangier's disease (Brown et al., 1983; Third et al., 1984; Natowicz and Kelley, 1987).

• Single-gene disorders that cause haematological disorders: Haemoglobinopathies, including sickle cell disease and various inherited coagulopathies (deficiency of the natural anticoagulants protein C and protein S) predispose to thrombosis of the cerebral vasculature, in particular in the young. In cases with a positive family history of young-age thrombosis the association is likely to be causal (Kohler et al., 1990; Barinagarrementeria et al., 1994). In elderly individuals, the relationship between the levels of protein C and protein S and stroke appears to be weak (Markus and Hambley, 1998).

• CADASIL - http://ghr.nlm.nih.gov/condition=cadasil: Cases of familial vascular dementia began to appear in the literature in 1977 and since then have been given a number of names. In 1993, the acronym CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) was adopted to designate a form of this disorder as specified by genetic homogeneity (Tournier-Lasserve et al., 1993). The underlying pathology of this condition is non-arteriosclerotic, non-amyloid angiopathy involving the media of the cerebral small vessels.The underlying genetic mutation was identified using a linkage strategy. A sequence of 8 cM (centimorgans) was defined in CADASIL (Joutel et al., 1996; Dichgans et al., 1996), within which the human equivalent of the mouse Notch 3 gene was identified. This was proposed as a candidate gene on the basis of similarity between another gene involved in Notch signalling, Sel 12, and S 182, a gene implicated in Alzheimer's disease (Levitan and Greenwald, 1995). Identification of deleterious mutations confirmed Notch 3 as the causative gene.

• Mitochondrial encephalomyelopathy, lactic acidosis and stroke-like episodes (MELAS - http://www.emedicine.com/ped/topic1406.htm): It is a mitochondrial encephalopathy. Muscle biopsy usually reveals abnormal mitochondria and ragged red fibres. The underlying cause of this disorder is mutations in the mitochondrial DNA. MELAS, like many mitochondrial disorders, demonstrates the phenomenon of heteroplasmy.  MELAS mutations usually lie within the tRNA-leu gene (UUR). Most frequently reported is an AG transition at position 3243 (Enter et al., 1991) and a TC transition at position 3271 (Sakuta et al., 1993). It has been proposed that mutations at position 3243 may result in somatic mutations accumulating in the mitochondrial DNA, leading to progressive mitochondrial dysfunction (Kovalenko et al., 1996).

Genes confer susceptibility to Vascular Cognitive Impairment (VCI):

Genes which confer susceptibility to vascular cognitive impairment can be divided into two categories:

1. Genes that confer susceptibility to cerebrovascular disease.

2. Genes that determine brain tissue responses to cerebrovascular disease (i.e. render parenchymal tissue more or less susceptible to injury or able to repair itself after injury).

Although some progress has been made in identifying genes of the first class, little has been done to explore genes of the second class.

http://content.nejm.org/cgi/content/extract/340/2/115

Atherosclerosis is indeed an inflammatory process. When endothelium is damaged or becomes dysfunctional, a cascade leading to atherogenesis is precipitated, initiating a cycle of injury, immunologic induction, and amplification. Genetics may influence atherosclerosis in numerous ways. For example, genes control the development of inflammatory mediators involved in atherogenesis. In this way genes influence the extent of the pro-inflammatory response or its specificity. Likewise, some other genes are incriminated in influencing the ischemic vulnerability of the tissue and also the ability of the tissue to recover following an ischemic attack. Risk-factors leading to endothelial damage / dysfunction, triggering the inflammatory cascade, include the following:

• Hypertension.
• Diabetes Mellitus (DM).
• Smoking
• Low-density lipoprotein
• Homocysteine
• Infection
  • Chlamydia
  • Herpes

Role of hypertension and Angiotensin II in the pathogenesis of stroke:

Hypertension increases the stroke-risk (risk of atherogenesis) by 3-5 fold  (Benson and Sacco, 2000). Hypertension can result in mechanical injury to endothelium, thereby increasing the number of endothelial adhesion molecules, which attract monocytes and lymphocytes. Angiotensin II, in addition to being a potent vasoconstrictor, can lead to smooth muscle hypertrophy, extracellular matrix production, and induction of cytokines. Expression of angiotensin-converting enzyme (ACE) has been demonstrated in macrophages, lymphocytes, and microvessels neighboring carotid plaques (Fukuhara et al, 2000). Independent of the effects of angiotensin II, hypertension has been shown in animals to increase formation of hydrogen peroxide and free radicals, which in turn can increase leukocyte adhesion (Ross; Swei et al). Thus additional pathophysiologic studies are needed.

American Heart Association Updates Recommendations for Blood Pressure Measurements - http://www.medscape.com/viewarticle/496270

Standard suggestions for measuring blood pressure to use in populations surveys - http://publications.paho.org/english/Vol.14_5_Special.pdf

Diabetes mellitus (DM):

Diabetes mellitus increases stroke-risk by 1.5-3 fold. DM by causing endothelial damage accelerates atherosclerosis. It induces both microangiopathic changes and large-vessel atherosclerosis.

Code of best practice for the measurement of blood glucose and cholesterol - http://www.health.nsw.gov.au/public-health/ehb/general/skinpen/cobp_bg_bc.pdf

Smoking:

Smoking almost doubles the risk of stroke. This is thought to occur by multiple mechanisms. Smoking leads to decreased arterial wall compliance, increased platelet aggregation, increased fibrinogen levels, and decreased HDL cholesterol levels. Smokers show a lower production of endogenous tissue plasminogen activator (tPA) antigen secondary to substance P infusion, suggesting an impairment of endogenous fibrinolysis in smokers (Newby et al, 1999). Physiologically, endogenous t-PA is released by endothelial cells in order to lyse subclinical clots, which develop on denuded areas on the surface of atherosclerotic plaques. An attenuated tPA production thus worsens atherogenesis.

Interestingly, serum t-PA levels have a diurnal pattern with the trough in the morning. This contrasts with levels of the most significant endogenous t-PA inactivator, plasminogen activator inhibitor 1 (PAI-1), which peaks in the morning. Elevated PAI-1 levels can be seen with hypertension, increased age, and insulin resistance. In one study, PAI-1 activity was higher in patients with stroke than in a control group (Lindgren et al, 1996). Nicotine also may increase levels of PAI-1, as shown in endothelial cells in culture, thus inactivating t-PA (Zidovetzki et al, 1999). This may represent another mechanism by which endogenous fibrinolysis is impaired in smokers.

Homocysteine:

Those with genetic enzyme disorders, such as deficiencies in cystathionine beta-synthase, have elevated homocysteine levels and accelerated atherosclerosis associated with the disease homocysteinuria. Interestingly, raised homocysteine levels have been found even in those individuals with no enzymatic defect. In this way, elevated homocysteine level should be considered as an independent risk factor for vascular disease regardless of the presence or absence of an enzymatic defect.

Homocysteine also has been postulated to cause a release of reactive oxygen species. Elevations of both homocysteine and lipid peroxide have been found in patients with acute stroke, while low levels of ascorbic acid (a potent antioxidant) have been detected. This suggests depletion of ascorbic acid in the setting of increased oxidative stress (El Kossi et al, 2000).

An association may exist between elevated homocysteine levels and large- and small-vessel strokes, but not with cardioembolic infarcts, lending support to the contribution of homocysteine in atherogenesis rather than as another mechanism of increased stroke risk (Eikelboom et al, 2000).

Overview of Homocystinuria - http://www.nlm.nih.gov/medlineplus/ency/article/001199.htm

DTT (dithiothreitol) method is a simple and inexpensive assay for homocysteine determination in human plasma for research application - http://medind.nic.in/iaf/t03/i2/iaft03i2p106.pdf

Infection:

Since inflammatory cells play a major role in the pathogenesis of atheroma plaques, a past history of chronic or even acute infection may be regarded as one of the predispositions for vascular disease. The antecedent infection may have very much triggered the inflammatory cascade, which originally initiated the fatty streak formation in the vasculature. Another clue to the involvement of an infectious agent in the pathogenesis of atheromatous plaques comes from the observation of increased mRNA coding for complement proteins in plaques (Yasojima et al, 2001).

Role of C-reactive protein (CRP):

CRP is nowadays considered as a marker for vascular disease. Raised CRP levels in patients of myocardial infarction (MI) and stroke generally means poor prognosis. In addition to the suggestion that CRP is likely a marker for an ongoing inflammatory/infectious process, there is evidence to suggest that CRP actually has a pathogenic role in the progression of atherosclerosis (plaques have a higher expression of CRP mRNA than normal arteries - Yasojima et al, 2001).

Overview of C-reactive protein testing - http://www.nlm.nih.gov/medlineplus/ency/article/003356.htm

Chlamydia:

C pneumoniae has been implicated as a possible causative agent for atherosclerosis more than any other infectious pathogen.

Chlamydia serology - what test to ask?

Chlamydia pneumoniae serology: comparing a commercial enzyme immunoassay and microimmunofluorescence test in patients with cardiovascular disease:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12716394&dopt=Abstract

Importance of Methodology in Determination of Chlamydia pneumoniae Seropositivity in Healthy Subjects and in Patients with Coronary Atherosclerosis:

http://jcm.asm.org/cgi/content/abstract/41/9/4049?ijkey=a4c3b328f6110d35e0a5bfd0a466bfdac825b0a0&keytype2=tf_ipsecsha

Early Carotid Atherosclerosis and Chlamydia pneumoniae Seropositivity: Are There Arguments to Treat With Antibiotics?

http://circ.ahajournals.org/cgi/content/full/110/7/e74

Herpes:

Viruses have been hypothesized to play a pathogenic role in atherosclerosis as well. Although results have been mixed, some prospective studies have failed to find an association between IgG and cytomegalovirus (CMV), herpes simplex virus (HSV), and subsequent stroke (Danesh et al, 1999; Nicholson and Hajjar, 1998; Ridker et al, 1998).

Diagnostics tests for herpes - http://www.herpesalliance.org/diagnostics.htm