MR imaging in sports-related glenohumeral instability

Sports-related shoulder pain and injuries represent a common problem. In this context, glenohumeral instability is currently believed to play a central role either as a recognized or as an unrecognized condition. Shoulder instabilities can roughly be divided into traumatic, atraumatic, and microtraumatic glenohumeral instabilities. In athletes, atraumatic and microtraumatic instabilities can lead to secondary impingement syndromes and chronic damage to intraarticular structures. Magnetic resonance (MR) arthrography is superior to conventional MR imaging in the diagnosis of labro-ligamentous injuries, intrinsic impingement, and SLAP (superior labral anteroposterior) lesions, and thus represents the most informative imaging modality in the overall assessment of glenohumeral instability. This article reviews the imaging criteria for the detection and classification of instability-related injuries in athletes with special emphasis on the influence of MR findings on therapeutic decisions.
Introduction
In the last decade, understanding of the biomechanics and pathophysiology of the athletic shoulder has significantly improved. Especially in overhead athletes and throwers several pathologic mechanisms have been identified which could not be explained by the traditional concepts of instability and impingement. With an expanded definition, glenohumeral instability is now believed to play the crucial role in the etiology of the painful athletic shoulder. Instability related to sports is not infrequently unrecognized and initiates a vicious circle of secondary impingement, muscular dysfunction, and damage to intraarticular structures that can be devastating and may end the athlete’s career [1–5].
The term “instability” constitutes a spectrum of disorders which includes hyperlaxity, subluxation, and dislocation. Principally, glenohumeral instability can be classified according to its etiology, degree, frequency, and direction. The classic categorization of affected individuals into two groups with traumatic and atraumatic instability represented by the mnemonics TUBS and AMBRII has been supplemented by a further grouping that is mainly comprised of overhead athletes with so-called “microinstability” (microtraumatic instability) and that has been labelled with the acronym AIOS (Table 1). However, it should be emphasized that congenital or acquired hyperlaxity, microinstability, and traumatic instability can overlap particularly in athletes engaged in overhead sports [2, 6].
Table 1Mnemonics for classification of glenohumeral instabilityTUBSAMBRIIAIOSTraumaticAtraumaticAcquiredUnidirectionalMultidirectionalInstabilityBankart lesionBilateralOverstressSurgeryRehabilitationSurgeryInferior capsular shift(Rotator) Interval closure
With reference to the complex relationships beween instability and impingement in overhead athletes, Jobe and co-workers developed a classification system (Table 2) with four groups of pathologic conditions [1]. Whereas groups 1 and 4 represent clearly defined entities with either impingement or instability, groups 2 and 3 include pathologies in which both mechanisms coexist. Group 1 corresponds to classic subacromial impingement, which appears to be relatively rare in athletes, especially in younger individuals. Group 2 refers to microinstability caused by repetitive microtraumatization of the capsulo-labral structures as the main pathologic process, leading to secondary impingement syndromes such as posterosuperior impingement. Group 3 equals the AMBRII group, with athletes that have congenital hyperlaxity of both shoulders as well as of other joints. Group 4 finally includes pure traumatic instabilities without secondary impingement as seen in the TUBS group. In the majority of athletes, traumatic instability occurs in an anterior direction; sports-related posterior “macroinstability” is very rare [1, 2, 4, 7, 8].
Table 2Modified classification of instability and impingement in the athletic shoulder [1, 2, 4]GroupPathologyEtiologyPattern of Lesions1Primary impingementSubacromial stenosisRotator cuff lesionsNo instability2Primary instabilityRepetitive microtraumaLabro-ligamentous lesionsSecondary impingementRotator cuff lesionsSLAP lesionsPosterosuperior impingement3Primary instabilityGeneralized hyperlaxityLarge capsular volumeSecondary impingementRotator cuff lesionsLabral abnormalities4Primary instabilityAcute trauma (dislocation)Labro-ligamentous injuriesNo impingementSLAP lesions
Magnetic resonance (MR) imaging plays an important role in the diagnostic work-up of the painful athletic shoulder and with advanced techniques has in many instances already replaced diagnostic arthroscopy. This article discusses the role of MR imaging and MR arthrography in the context of the most common forms of sports-related glenohumeral instability and presents the typical MR arthrographic appearance of their corresponding intraarticular lesions and patterns of injury.
Technical considerations
Particularly in athletes, MR arthrography has been advocated for accurate depiction of labro-ligamentous injuries as well as rotator cuff pathology [5, 7–11]. With the exception of examinations following acute shoulder dislocation, where joint effusion or hemorrhage serve as “natural” contrast media, at our institution MR arthrography is performed on a routine basis in individuals with clinical evidence of shoulder instability and in athletes with unclear shoulder pain. For intraarticular injection of contrast media, several different techniques have been described that can all successfully be employed in MR arthrography [9]. We inject 12–20 ml of a 2.5 mmol/l solution of a gadolinium chelate following fluoroscopically guided puncture of the glenohumeral joint via a classic anterior approach and verification of the intraarticular position of the needle tip by application of a small amount of iodinated contrast media. With the use of a dedicated shoulder coil transverse, coronal oblique, and sagittal oblique T1-weighted spin-echo (SE) sequences are then obtained. Fat-suppression is routinely applied in the coronal oblique plane, but not necessarily in the transverse and sagittal oblique planes. The examination is supplemented by acquisition of a coronal oblique intermediate weighted (echo time=33–45 ms) turbo spin-echo (TSE) sequence with spectral fat-saturation. In patients with anterior instability or suspected posterosuperior glenoid impingement we generally add a T1-weighted SE and/or an intermediate weighted TSE sequence in the ABER (abduction and external rotation) position, achieved by transverse oblique orientation of the images along the long axis of the humerus after flexing the elbow and placing the patient’s hand behind his head or neck (Fig. 1) [5, 9, 12]. Geometric parameters are identical for all pulse sequences with a section thickness of 3 mm and an in plane resolution of 0.4×0.3–0.5 mm.
Fig. 1a, bABER position: normal findings. a Coronal localizer image of the shoulder obtained in abduction and external rotation shows correct orientation of transverse oblique sections aligned with the humeral shaft. b Resulting transverse oblique T1-weighted MR arthrogram demonstrates normal appearance of anteroinferior labrum (white arrow) with taut IGHL (arrowheads), posterosuperior labrum (black arrow), and supraspinatus tendon (**) (T greater tuberosity of humeral head)
The non-enhanced MR imaging protocol performed after acute shoulder dislocation includes a coronal oblique T1-weighted SE sequence, coronal oblique and transverse intermediate weighted TSE sequences with fat-saturation, and a sagittal oblique T2-weighted TSE sequence with the same geometric parameters as mentioned above.
Traumatic glenohumeral instability
Traumatic glenohumeral instability is typically initiated by a specific traumatic event, followed by other episodes of dislocation or subluxation with a unidirectional pattern [2, 7]. In the vast majority of athletes traumatic instability occurs in an anteroinferior direction when a sudden force overwhelms the anterior capsular structures while the athlete’s arm is in an abducted, externally rotated, and extended position [13]. The resulting combination of injuries represents the source of chronic instability, particularly those involving the inferior glenohumeral ligament (IGHL), which, according to the current opinion, is the most important passive stabilizer of the shoulder joint. The IGHL is formed by an anterior and a posterior band, which represent condensations of the capsule running from the inferior labrum to the humeral neck. The anteroinferior labrum and the anterior band of the IGHL together form the anteroinferior labro-ligamentous complex. The labrum is thought to serve more as an insertion site for the IGHL than to provide stability to the glenohumeral joint by deepening the glenoid fossa [13–15]. Although generally common in sports, this type of instability is rarely observed in throwers or overhead athletes, but, if present, can cause secondary damage to the rotator cuff and the superior and posterior labrum [2].
Labro-ligamentous injuries
The high diagnostic accuracy of MR arthrography in the detection of labro-ligamentous lesions has been demonstrated in several studies. MR arthrography identifies labral tears with a sensitivity of 88–96% and a specificity of 91–98% [14, 16–19]. For the detection of lesions of the superior, middle, and inferior glenohumeral ligaments, Chandnani and co-workers reported sensitivities and specificities of 88–100% [20]. The sensitivities and specificities of unenhanced MR imaging for the diagnosis of labro-ligamentous injuries vary widely in the literature. Although a direct comparison with MR arthrography has not yet been performed in a larger series, we regard the role of standard MR imaging in the diagnostic work-up of shoulder instability as questionable, particularly with a view to chronic cases and the depiction of associated pathology [7, 9, 14, 21, 22]. The advantages of MR arthrography are obvious and result from capsular distension with separation of anatomic structures and improved delineation of tears following entrance of contrast media [8, 9]. MR arthrography thereby allows more confident distinction of pathologic conditions from the common anatomic variations of labral morphology as well as from congenital variants of the glenohumeral ligaments and the labro-ligamentous unit, such as the Buford complex [7, 9, 14, 23–26]. Furthermore, the imaging appearance of Bankart variant lesions has been described only with MR arthrography [7, 14, 27, 28].
Few studies have directly compared MR arthrography and computed tomography (CT) arthrography in the diagnosis of labro-ligamentous injuries. The results suggest that, with the exception of bony changes, CT arthrography is less sensitive than MR arthrography [5, 16]. However, CT arthrography with the use of new multidetector techniques has not yet been fully evaluated in the shoulder. First results indicate that due to its capability of isotropic data acquisition it will probably perform equivalent to MR arthrography [8, 26].
Since modern surgical stabilization procedures aim at restoration of normal anatomy, a more differentiated view at labro-ligamentous injuries has developed in the recent years, and several variants of traumatic lesions of the anteroinferior labrum and/or capsular structures have been described. The following types of lesions should be distinguished (Fig. 2).
Fig. 2a–fClassification of Bankart and Bankart variant lesions. a Bankart lesion, b bony Bankart lesion, c Perthes lesion, d ALPSA (anterior labro-ligamentous periosteal sleeve avulsion) lesion, e GLAD (glenolabral articular disruption) lesion, f HAGL (humeral avulsion of glenohumeral ligaments) lesion. (LLC anteroinferior labro-ligamentous complex, P scapular periosteum, HH humeral head, AC articular cartilage of glenoid, IGHL inferior glenohumeral ligament)
Bankart lesion
The classic labral injury described by Bankart (Fig. 2a) is a complete detachment of the anteroinferior labro-ligamentous complex from the glenoid associated with a rupture of the scapular periosteum [29, 30]. The Bankart lesion represents the most common form of labro-ligamentous injury in patients with first-time traumatic dislocations of the shoulder [31, 32]. Due to the lost contact with the periosteum the lesion shows no tendency to heal. Surgical treatment is by reattachment of the labro-ligamentous complex to the glenoid either arthroscopically or during an open procedure (Bankart repair) [31, 32]. MR arthrography (Fig. 3) typically shows a deformed anteroinferior labrum which is completely separated from the glenoid and therefore is “floating” in the anterior capsular recess adherent to the anterior band of the IGHL [7]. In many cases the torn periosteum can be visualized by transverse MR arthrograms.
Fig. 3a–cBankart lesion. a Transverse and b coronal oblique T1-weighted MR arthrograms show complete detachment of the anteroinferior labrum (arrows) which “floats” within the anterior capsular recess but remains attached to the IGHL (arrowheads). c Fat-suppressed T1-weighted MR arthrogram obtained in ABER position reveals separation of the anteroinferior labrum from the glenoid edge as well as disruption of the periosteum (arrow). Note taut IGHL adherent to the labrum (arrowhead)
Bony Bankart lesion
In a bony Bankart lesion (Fig. 2b) an osseous fragment of variable size is avulsed from the anteroinferior glenoid together with the labro-ligamentous complex. The size of the fragment influences the therapeutic concept, since larger glenoidal defects can give rise to recurrent instability after stabilizing soft tissue procedures such as the Bankart procedure, particularly in contact athletes [33, 34]. In a study with cadaveric specimens and experimentally created defects of the glenoid rim the critical fragment size was >7 mm in average width [34]. Burkhardt and De Beer coined the term “inverted peer” to describe the visual impression of a significant bone defect of the glenoid at arthroscopy. The “inverted peer” geometry is the reverse of the normal peer-shaped glenoid, which means that its anteroposterior diameter below the midglenoid notch is smaller than that above it [33]. This configuration usually precludes arthroscopic repair and represents an indication for fragment refixation or bone grafting. MR arthrography is less accurate in depiction of bony Bankart lesions compared with CT or CT arthrography [35]. Smaller osseous fragments can easily be overlooked, particularly if fat suppression is used, whereas glenoid fractures with larger fragments can usually be diagnosed with sufficient confidence. Sagittal oblique images are useful to estimate the geometry of the glenoid (Fig. 4).
Fig. 4a–dBony Bankart lesion. a Coronal oblique and b transverse T1-weighted MR arthrograms show avulsion of a large osseous fragment (arrowheads) from the glenoid together with the attaching anteroinferior labrum and IGHL (arrow). c Sagittal oblique T1-weighted MR arthrogram demonstrates “inverted peer” geometry of glenoid in presence of a “relevant” bony Bankart lesion (arrowhead). Note smaller anteroposterior diameter of glenoid below (**) midglenoid notch than above (++) it. For comparison, normal peer-shaped configuration of glenoid is shown on d sagittal oblique T1-weighted MR arthrogram from individual with stable shoulder (C coracoid process)
Perthes lesion
The Perthes lesion (Fig. 2c) is a variant of the Bankart lesion, which also occurs in patients with acute anterior instability. In the Perthes lesion the anteroinferior labro-ligamentous complex is detached from the glenoid, but unlike in the injury described by Bankart, the periosteum remains intact and is stripped anteromedially [27, 31, 36]. Therefore, unless only loosely attached, the labrum may remain in its normal anatomic position. The integrity of the periosteum allows partial “healing” of the labrum, which might also become resynovialized and thus, although incompetent, can look normal on arthroscopic inspection. Because scar tissue may prevent contrast media from entering the labral tear, nondisplaced Perthes lesions can be difficult to detect on MR arthrography as well [14, 27]. MR arthrograms obtained in the ABER position have been reported to be more sensitive in the diagnosis of Perthes lesions compared with conventional transverse MR arthrograms, although these results are based on a relatively small series of patients [12, 27]. In our experience, the ABER position can at times help to visualize nondisplaced Perthes lesions by separating the base of the anteroinferior labrum from the glenoid and consecutively allowing the entrance of contrast media. On the other hand, displaced or slightly displaced lesions can be realigned with the glenoid and therefore be less conspicuous on ABER images than on transverse images obtained in neutral position (Fig. 5).
Fig. 5a, bPerthes lesion. a Transverse fat-suppressed T1-weighted MR arthrogram shows detachment and slight displacement of the anteroinferior labro-ligamentous complex (arrow) and stripping of the intact scapular periosteum (arrowhead). On b corresponding T1-weighted MR arthrogram obtained in ABER position the anteroinferior labrum is realigned with the glenoid edge by the taut IGHL (arrowhead). Although the tear is still demarcated by contrast media (arrow), it is less conspicuous than on the conventional transverse section in this case. (M middle glenohumeral ligament
ALPSA lesion
The ALPSA (anterior labro-ligamentous periosteal sleeve avulsion) lesion (Fig. 2d) has also been termed “medialized Bankart lesion” and is more common in patients with recurrent than with first-time traumatic dislocations of the shoulder [14, 30]. It represents an anteroinferior labral avulsion that leads to medial displacement and inferior rotation of the labro-ligamentous complex together with the intact periosteum in a sleeve-like fashion, thereby causing incompetence of the IGHL and consecutively anterior instability. The labrum may eventually heal and be resynovialized in this abnormal position [7, 30]. Transformation of the ALPSA lesion into a Bankart lesion by dissection of the complex from the glenoid followed by anatomic refixation (Bankart repair) has been recommended as the treatment of choice [30]. MR arthrography (Fig. 6) shows the typical medial and inferior displacement of the deformed labro-ligamentous complex which is best seen on transverse and coronal oblique images. Contrast medium often outlines a crease or cleft between the glenoid and the nodular shaped fibrous tissue on the glenoid neck, whereas the glenoid edge typically lacks a normal labral structure [7, 14, 30]. Familiarity with these signs can prevent misinterpretation of the ALPSA lesion as an anatomic variant in cases with only slight displacement of the labrum.
Fig. 6a, bALPSA lesion. a Coronal oblique and b transverse T1-weighted MR arthrograms demonstrate a nodular shaped anteroinferior labroligamentous complex (arrows) that is displaced medially and inferiorly on the scapular neck. Note cleft (arrowheads) between fibrous tissue and glenoid
GLAD lesion
The GLAD (glenolabral articular disruption) lesion (Fig. 2e), as described by Neviaser is a superficial tear of the anteroinferior labrum in combination with an articular cartilage lesion of the anterior inferior quadrant of the glenoid. The injury is thought to result from glenohumeral impaction while the arm is abducted and externally rotated. Persistent anterior shoulder pain after a fall onto an outstretched arm is the most common clinical presentation, and arthroscopic debridement has been proposed as the treatment of choice [37].
The labral component of the abnormality typically represents an inferior based flap tear without evidence of capsulo-periosteal stripping. Since the anterior fibers of the IGHL remain strongly attached to the labrum and glenoid, the GLAD lesion is usually not associated with anterior instability. The degree of articular cartilage damage is variable and ranges from softening and fibrillation to deep surface defects with loose fragments of cartilage which might remain attached to the anterior labrum and can be undermined by contrast media on MR arthrography (Fig. 7) [7, 14, 28, 37]. Particularly if the chondral lesion is small and therefore hardly visible on MR imaging, the GLAD lesion can look similar to instability-related labral injuries. It is therefore important to note that the labrum is nondisplaced and both the IGHL as well as the periosteum are intact.
Fig. 7GLAD lesion. Transverse T1-weighted MR arthrogram shows a nondisplaced tear of the anterior labrum (arrow) associated with a full-thickness chondral defect of the anterior glenoid (arrowhead). A small fragment of hyaline cartilage adheres to the torn labrum
HAGL lesion
In contrast to the Bankart lesion and its variants, the HAGL (humeral avulsion of glenohumeral ligaments) lesion (Fig. 2f) does not involve the labro-ligamentous complex at the glenoid, but represents an isolated tear of the IGHL at its humeral insertion following vigorous shoulder dislocation [38, 39]. The injury is associated with anterior instability and represents a pitfall at arthroscopy as well as open shoulder surgery, since it can be overlooked if the area of the humeral neck is not specifically searched for this finding [39, 40]. The majority of patients in whom a HAGL lesion is detected are male and are involved in contact sports, such as rugby, (American) football, or ice hockey [33, 40]. The injury therefore is thought to be relatively rare in countries where these kinds of sport are unpopular. Treatment is by surgical reattachment of the IGHL to its humeral insertion [38, 40]. Particularly in subacute cases with absence of joint effusion, the HAGL lesion can only be diagnosed with confidence if MR arthrography is used. As a sequel of humeral avulsion the IGHL is seen as a J-shaped rather than a U-shaped structure on coronal oblique arthrograms, and contrast extravasation can occur at its insertion site (Fig. 8). Acute injuries are usually associated with edematous changes of the soft tissues anterior to the humeral neck [39]. Approximately 20% of HAGL lesions occur with avulsion of a bony fragment from the humerus (bony HAGL=BHAGL) [41]. On rare occasions the HAGL lesion can be associated with a classic Bankart lesion. Due to the complete isolation of the anterior band of the IGHL which results from this combination of injuries, the lesion has been described as the “floating AIGHL” [42]. Other associated injuries include subscapularis tears and Hill-Sachs defects as well as other osteochondral lesions [38, 40].
Fig. 8a, bHAGL lesion. a Coronal oblique fat-suppressed T1-weighted MR arthrogram and b corresponding intermediate weighted TSE image demonstrate avulsion of the IGHL at its humeral insertion causing a J-shaped configuration of the axillary recess (arrowheads). Contrast extravasation can be seen at the site of the tear (arrows). Bone marrow edema within humeral head (*) was caused by acute Hill-Sachs defect (not shown)
Particularly in athletes, the decision whether an arthroscopic or an open procedure should be performed to achieve the most favourable outcome, can be very difficult and is subject to a controverse discussion. Criteria in favour of open rather than arthroscopic surgery include engagement in contact sports, presence of a relevant glenoid defect and/or Hill-Sachs defect, a HAGL lesion, and poor quality of the labro-ligamentous complex [14, 33, 38, 43]. A recent study could demonstrate the potential of MR arthrography in classification of anteroinferior labro-ligamentous injuries with a view to therapeutic decisions [14]. With arthroscopy as the “gold standard,” Bankart and Bankart variant lesions could be correctly classified with a sensitivity of 77%, a specificity of 91%, and an accuracy of 84%. Furthermore, MR arthrography was capable of identification of injuries with poor quality of the anterior labrum and IGHL, which in high concordance with arthroscopy could not be assigned to one of the specific types of lesions mentioned above. All of these “nonclassifiable” lesions with degenerative changes and scar tissue formation were observed in patients with chronic anterior instability. On MR arthrography (Fig. 9) these injuries are characterized by a swollen inferior glenohumeral ligament complex without distinct depiction of the labrum, IGHL, and scapular periosteum [14].
Fig. 9Nonclassifiable anteroinferior labro-ligamentous injury. Transverse T1-weighted MR arthrogram shows markedly deformed and enlarged labro-ligamentous complex without differentiability of labrum, IGHL, and scapular periosteum. The lesion was associated with chronic anteroinferior instability
Hill-Sachs defect
The Hill-Sachs defect represents a compression injury of the posterolateral humeral head which develops during anteroinferior dislocation as the relatively large and soft humeral head impacts against the comparatively small and hard bony glenoid [13]. The incidence of the lesion at arthroscopy varies considerably from 47 to 100% among different series of patients with first-time traumatic dislocations [31, 32, 44]. The depth and size of a Hill-Sachs lesion, ranging from shallow chondral defects to deep osteochondral impaction fractures, is thought to reflect the tightness of the joint capsule [44]. In athletes with hyperlaxity at the time of the first shoulder dislocation, the humeral injury therefore typically is small or absent. Osteochondral Hill-Sachs defects that involve less than one third of the circumference of the humeral head are usually regarded as prognostically irrelevant. Larger lesions, especially if oriented with their long axis parallel to the glenoid, can engage the anterior corner of the glenoid in abduction and external rotation and thus lead to repeated subluxations or redislocations (“engaging” Hill-Sachs defect) [33].
On transverse MR images, the Hill-Sachs defect is typically seen at or slightly above the level of the coracoid process (Fig. 10). False positive diagnoses can occur due to misinterpretation of a normal anatomic groove at the posterolateral humerus, which can often be found caudad to that position [45]. In acute or subacute cases with edema of adjacent bone marrow, the impaction injury can easily be detected on fat-suppressed intermediate/T2-weighted or STIR images. Chronic lesions, particularly if small, are often better visualized by CT [35]. However, clinically significant osteochondral defects are sufficiently demonstrable by MR and rarely necessitate further imaging.
Fig. 10a–cHill-Sachs defect: MR appearance. a Acute lesion: transverse fat-suppressed intermediate weighted TSE image shows impaction of the posterolateral humeral head (arrowhead) with adjacent bone marrow edema following acute anteroinferior dislocation. b, c Chronic lesions: b transverse fat-suppressed T1-weighted and c transverse T1-weighted MR arthrograms obtained in two different patients several months after anteroinferior dislocation depict circumscribed (b) and extensive (c) osteochondral defects (arrowheads) of the humeral head (C tip of coracoid process)
Atraumatic glenohumeral instability
Atraumatic glenohumeral instability is typically multidirectional and usually evident in individuals with congenital hypermobility syndrome. The diagnosis is predominantly based on the clinical examination, which uncovers bilateral affection of the shoulders in association with generalized hyperlaxity of ligaments and joints [1, 2, 35, 46]. The increased baseline laxity of the shoulder seen in athletes with generalized hypermobility is advantageous for several types of sports, but at the same time harbors a high risk of long-term injury with damage of intra- and periarticular structures [1, 2]. If conservative management fails, multidirectional instability is usually treated surgically by inferior capsular shift and closure of the rotator interval [6].
There are no specific findings to diagnose atraumatic glenohumeral instability by MR imaging or MR arthrography [46]. In most non-athletic AMBRII patients, MR arthrography shows an increased capsular volume but no or little substantial alterations of the intraarticular structures. In athletes, laxity of the capsule is frequently associated with secondary damage to the labrum and the rotator cuff [2]. The labrum may be hypoplastic or torn, but can as well appear swollen and increased in signal intensity due to more or less extensive degenerative change (Fig. 11). Further morphologic alterations can involve the biceps anchor and the articular surface of the rotator cuff [2]. MR arthrography can help with therapeutic decisions by identification or exclusion of significant intraarticular pathology that might represent an indication for surgical repair in addition to capsular reduction.
Fig. 11a, bAtraumatic glenohumeral instability: labral degeneration. a Transverse fat-suppressed T1-weighted and b corresponding sagittal oblique T1-weighted MR arthrograms reveal markedly swollen and enhanced but nondisplaced anterior glenoid labrum in an athlete with multidirectional shoulder instability. Extensive labral degeneration was verified by arthroscopy (not shown)
Microtraumatic glenohumeral instability
Microtraumatic glenohumeral instability is thought to result from chronic traumatization of the capsular structures in throwers and overhead athletes, such as tennis players, swimmers, and handball players. In contrast to atraumatic glenohumeral instability, microinstability is typically seen unilaterally only in the dominant shoulder of the athlete. Whereas repetitive abduction and external rotation cause microtrauma of the anterior capsule and thus can provoke anterior instability, repetitive overhead activity with abduction, flexion, and internal rotation might give rise to posterior microinstability as a result of injury to the posterior labro-ligamentous elements [4, 7, 9, 35, 46].
Structural abnormalities consecutive to microtraumatic glenohumeral instability can be depicted by MR arthrography and include laxity of the anterior or posterior capsule, labral injuries ranging from degeneration and fraying to tearing and detachment, SLAP (superior labral anteroposterior) lesions, and tears of the rotator cuff caused by secondary impingement [2].
Posterosuperior glenoid impingement (PSI)
PSI is a form of internal impingement which represents a common problem in throwers and overhead athletes, presenting with acute or chronic posterior shoulder pain [4, 5, 47, 48]. The basic observation was reported by Walch, who described impingement between the articular side of the supraspinatus tendon and the posterosuperior edge of the glenoid evident in abduction and external rotation [49]. In throwers and overhead athletes, PSI can lead to a typical pattern of injuries, the so-called “kissing lesions”, which includes corresponding lesions of the undersurface of the rotator cuff, the posterosuperior labrum, the greater tuberosity, and the superior bony glenoid [47, 48, 50–52]. The development of PSI has been attributed to chronic injury (repetitive stretching) of the anterior capsular structures, particularly the IGHL, with consecutive anterior microinstability, which causes anterior subluxation of the humeral head in abduction and external rotation during overhead movements, and thus allows excessive contact between the rotator cuff and the posterosuperior glenoid [47, 48]. However, this theory is not generally accepted. Other authors described a contracture of the posteroinferior capsule and a posterior SLAP lesion as the essential lesions for the development of PSI in throwers [3]. Although the basic mechanism is still subject to discussion, the high coincidence of PSI and SLAP lesions is undoubted. Furthermore, there is apparently a huge overlap of clinical symptoms in athletes with SLAP lesions, PSI, or both. It is important to stress that contact between the undersurface of the rotator cuff and the posterosuperior glenoid, as seen on arthroscopy, is not pathologic per se. Posterosuperior glenoid impingement should only be diagnosed if this contact is associated with clinical symptoms and corresponding lesions of the involved anatomic structures [2, 13, 51, 52]. Conservative treatment is usually suited in athletes with minor structural abnormalities, whereas surgical debridement and repair (eventually in combination with capsular plication) are indicated in the presence of relevant rotator cuff and labral lesions.
MR imaging can be used to confirm the diagnosis of PSI and to help with therapeutic decisions by demonstrating the extent of articular damage. Since MR arthrography has been shown to be more accurate than conventional MR imaging in the detection of partial rotator cuff tears, labral tears, and SLAP lesions, it is also regarded as the method of choice in athletes with suspected PSI, which is accompanied by a combination of these abnormalities [50, 53–55]. MR arthrograms (Fig. 12) typically show articular-sided partial tearing of the supraspinatus and/or infraspinatus tendons. The supraspinatus lesion, unlike in patients with subacromial impingement, usually involves the posterior part of the tendon. Damage to the posterosuperior labrum ranges from degenerative change and fraying to tears and detachment and might be associated with ganglia and SLAP lesions. Alterations of bone at the greater tuberosity and the superior glenoid are best demonstrated by fat-suppressed intermediate or T2-weighted images and include erosions, bone marrow edema, cyst formation, and sclerosis [50–52]. MR arthrograms obtained in the ABER position (Fig. 12d,e) usually show contact between the lesions of the rotator cuff and the superior labrum/glenoid and might even reveal interposition of the supraspinatus tendon pinched between the glenoid and the greater tuberosity. Furthermore, the ABER position is useful in demonstration of subluxation of the humeral head and depiction of abnormalities of the anterior capsule. The anterior band of the IGHL can appear attenuated and elongated or might even be torn [50].
Fig. 12a–ePSI: spectrum of findings on MR arthrography. a Transverse fat-suppressed T1-weighted MR arthrogram and b, c corresponding coronal oblique images obtained in a tennis player with posterior shoulder pain show a posterosuperior labral tear (arrow in a) associated with an articular-sided partial tear of the posterior aspect of the supraspinatus tendon (arrowhead in b) and a SLAP type 2 lesion (arrow in c). Note large volume of anterior capsule (* in a). d, e T1-weighted MR arthrograms obtained in ABER position from two athletes with PSI: d Arthrogram demonstrates the “kissing lesion” pattern with corresponding tears of the posterosuperior labrum (white arrow) and the undersurface of the supraspinatus tendon (black arrowhead) accompanied by sclerosis of the greater tuberosity of the humeral head at the contact zone with the glenoid (black arrow). Note elongation of the IGHL (white arrowhead). e Arthrogram shows interposition of the supraspinatus tendon (*) between the greater tuberosity and the superior glenoid as well as a tear of the IGHL (arrowhead) with consecutive contrast extravasation
SLAP lesions
SLAP lesions of the shoulder are common injuries in athletes which represent tears of the superior glenoid labrum that extend in an anterior to posterior direction [55, 56]. Four different types of SLAP lesions have been described in the original classification by Snyder:
degenerative fraying of the superior labrum;avulsion of the superior labrum and biceps anchor from the glenoid;bucket-handle tear of the superior labrum with preserved biceps anchor;bucket-handle tear of the superior labrum involving the long head of biceps tendon [56].
This classification has been expanded with several further types of lesions, which mainly represent combinations of the most common form, the SLAP type 2 lesion, with other injuries of the labrum, medial glenohumeral ligament, or rotator cuff. However, only the Snyder classifications have found wide acceptance, and most institutions categorize these injuries as type 1–4 with or without asscociated lesions. Whereas type 1 and 2 lesions are typically caused by repetitive torsion of the biceps anchor (the so-called “peelback mechanism”) in throwers and overhead athletes, types 3 and 4 lesions are thought to occurr more often after a fall onto an outstretched arm or onto a flexed elbow [3, 55, 56]. Furthermore, type 2 lesions are frequently observed in athletes with traumatic anterior shoulder instability, where they develop in association with lesions of the anteroinferior labro-ligamentous complex (most often a classic Bankart lesion). In these cases, the labro-ligamentous avulsion can be considered as the main lesion, with the SLAP lesion being an associated injury [55]. Surgical treatment is indicated in all types of SLAP lesions except type 1 lesions which are usually of no clinical relevance. Since type 2 and 4 lesions impair the stabilizing function of the biceps insertion and therefore can provoke glenohumeral instability, secondary impingement, and rotator cuff lesions, they are commonly treated by refixation. Type 3 bucket-handle tears are usually not surgically reattached but treated by simple debridement [55, 56].
MR arthrography has proved to be superior to conventional MR imaging with regard to the identification of SLAP lesions and has been shown to be reliable in assessment of the stability of the biceps anchor and the detection of associated injuries [55, 57–59]. However, its ability to correctly classify the different types of lesions is limited [55].
Most SLAP lesions are best depicted on MR arthrograms oriented in the coronal oblique plane (Fig. 13). Increased signal intensity and/or surface irregularity of the superior labrum can be seen in type 1 lesions which, however, can only infrequently be diagnosed by MR imaging. Type 2 lesions are characterized by linear extension of contrast media into the superior labrum and the biceps anchor. This most common type of SLAP lesion also represents the most problematic in terms of differential diagnosis. False positive and false negative diagnoses of a SLAP type 2 lesion can be caused by misinterpretation of a sublabral recess as a tear and vice versa [55]. The sublabral recess (Fig. 14) is a very common anatomic variant with an incidence of >70% in cadaveric studies [26, 60]. It represents a variably sized synovialized cleft between the superior labrum and the bony glenoid which is typically oriented medially and points at the supraglenoidal tubercule [26, 55, 60]. The most important criterion for the presence of a SLAP type 2 lesion rather than a sublabral recess therefore is lateral or superior extension of the tear. If the tear extends medially (like a sublabral recess) it often shows irregular margins and/or a relatively wide separation between the labrum and the glenoid. In individuals with traumatic anterior instability the type 2 lesion often originates from an anteroinferior tear of the labro-ligamentous complex that extends cranially up into the superior labrum and the biceps anchor (Fig. 15) [55]. Type 3 lesions are characterized by a vertical and an additional horizontal contrast interface which separate the avulsed superior labrum as a triangular-shaped fragment from the intact biceps tendon. In Type 4 lesions the horizontal component of the tear additionally extends into the long head of biceps tendon. The bucket-handle fragment therefore is composed of the superior labrum as well as a portion of the tendon and might be more or less displaced from the superior glenoid [55, 59].
Fig. 13a–dSLAP lesions: classification according to Snyder [56] on coronal oblique MR arthrograms (from [35]; modified). a SLAP type 1 lesion, b SLAP type 2 lesion, c SLAP type 3 lesion, d SLAP type 4 lesion (LBC labral-bicipital complex, HH humeral head, G glenoid)Fig. 14Sublabral recess. Coronal oblique T1-weighted MR arthrogram demonstrates medially oriented cleft (arrowhead) between the superior labrum and the glenoid outlined by contrast media which was also diagnosed as a sublabral recess with a stable biceps insertion on arthroscopy (not shown). Note meniscoid shape of superior labrum (*) and integrity of the biceps anchor (arrow)Fig. 15a–dSLAP type 2 lesion in association with a Bankart lesion in traumatic anterior glenohumeral instability. a Coronal oblique fat-suppressed T1-weighted MR arthrogram shows superior extension of contrast media into the superior labrum and biceps anchor (arrowhead). b Corresponding sagittal oblique MR arthrogram reveals tearing of the entire anterior labrum (arrowheads) extending from inferior to superior. c, d Corresponding transverse MR arthrograms demonstrate detachment of the anterior labrum that continues as a classic Bankart lesion anteroinferiorly (arrows)
Conclusion
Sports-related glenohumeral instability is a complex and in many aspects still controversial subject. MR imaging can play an important role in evaluation of the athlete’s shoulder if it aims at providing essential information for therapeutic decisions at eye level with diagnostic arthroscopy. Therefore, the radiologist should be familiar with the mechanisms and classification of injuries as well as the advantages and limitations of his examination techniques. At present, MR arthrography represents the best evaluated and most informative imaging technique for the assessment of shoulder pathology in many aspects and thus, appears to be most valuable for the diagnostic work-up of glenohumeral instability and unclear shoulder pain in athletes.

magnetic resonance
mr arthrography
shoulder dislocation
shoulder mr
shoulder injuries


Genetic polymorphisms of the RAS-cytokine pathway and chronic kidney disease

Chronic kidney disease (CKD) in children is irreversible. It is associated with renal failure progression and atherosclerotic cardiovascular (CV) abnormalities. Nearly 60% of children with CKD are affected since birth with congenital or inherited kidney disorders. Preliminary evidence primarily from adult CKD studies indicates common genetic risk factors for CKD and atherosclerotic CV disease. Although multiple physiologic pathways share common genes for CKD and CV disease, substantial evidence supports our attention to the renin angiotensin system (RAS) and the interlinked inflammatory cascade because they modulate the progressions of renal and CV disease. Gene polymorphisms in the RAS-cytokine pathway, through altered gene expression of inflammatory cytokines, are potential factors that modulate the rate of CKD progression and CV abnormalities in patients with CKD. For studying such hypotheses, the cooperative efforts among scientific groups and the availability of robust and affordable technologies to genotype thousands of single nucleotide polymorphisms (SNPs) across the genome make genome-wide association studies an attractive paradigm for studying polygenic diseases such as CKD. Although attractive, such studies should be interpreted carefully, with a fundamental understanding of their potential weaknesses. Nevertheless, whole-genome association studies for diabetic nephropathy and future studies pertaining to other types of CKD will offer further insight for the development of targeted interventions to treat CKD and associated atherosclerotic CV abnormalities in the pediatric CKD population.
Introduction
Chronic kidney disease (CKD) is irreversible and progressive [1]. In children, CKD is underappreciated, understudied, and an important cause of morbidity and mortality [1, 2]. Furthermore, adjusted mortality rates since 1991 among the pediatric end-stage renal disease (ESRD) population increased by 5% to 26.6 per million general population in 2005; and cardiovascular (CV) mortality among pediatric ESRD patients has increased from 17.7 deaths per 1,000 patient years at risk in 1991 to 23.4 in 2005 [3]. Children with CKD live with the consequences of abnormal renal function for their entire lives, with nearly 60% affected since birth with congenital or inherited kidney disorders [2]. Diabetic nephropathy and hypertension, which are the dominant causes of CKD in adults, are rare causes of CKD in childhood. CKD in children is the result of heterogeneous diseases of the kidney and urinary tract that range from common congenital malformations of the urinary tract to rare inborn errors of metabolism. Although some patients have stable kidney function for years, others have a rapid decline in function. The factors associated with an accelerated decline in kidney function include: the cause of CKD, proteinuria, hypertension, anemia, hyperphosphatemia, and metabolic acidosis [4–9]. In common with children and adults with CKD, progression to kidney failure occurs via a final common pathway characterized by progressive interstitial fibrosis, peritubular capillary loss with hypoxia, and destruction of functioning nephrons because of tubular atrophy [10]. Despite the diverse initiating and secondary factors noted above, CKD progression is strongly influenced by common inflammatory mechanisms [11].
CKD is a well-known risk factor for atherosclerotic CV disease [12–16]. Children with kidney failure receiving chronic dialysis have a cardiac death rate 1,000-fold higher compared with children in the general population [15]. Children with mild to moderate CKD have a high prevalence of traditional risk factors for atherosclerotic CV disease, including hypertension, hyperlipidemia, and elevated homocysteine levels [17–19]. Left ventricular hypertrophy (LVH), a pathophysiologic adaptation of the myocardium, is viewed as a marker for early CV disease in pediatric patients with CKD [20, 21]. The long-standing and progressive atherosclerotic CV abnormalities that begin in childhood CKD contribute to the increased CV morbidity in adulthood [22]. CKD promotes maladaptive interactions between the heart and kidneys, which in turn amplifies the progressive failure of these organs [23]. The emerging evidence suggests that cytokines may play a vital regulatory role in initiation and progression of both renal and CV disease in patients with CKD [24].
Preliminary evidence primarily from adult CKD studies [24] indicates common genetic risk factors for CKD and atherosclerotic CV disease. Although multiple physiologic pathways share common genes for CKD and CV disease, substantial evidence [10, 25–28] supports our attention to the renin angiotensin system (RAS) and the interlinked inflammatory cascade because they modulate the progression of renal and CV disease. Current research suggests that the natural variations of the genes involving the RAS-cytokine pathway influence the rate of progressions for renal and CV disease in CKD patients [24]. Insights gained by understanding how variations in this pathway influence the progressions of renal and CV disease will lead to hypotheses for targeted interventions to treat CKD and associated atherosclerotic CV abnormalities in the pediatric CKD population. This article reviews clinically relevant candidate genes of the RAS-cytokine pathway and the fundamentals of genotype–phenotype association studies.
CKD–CV disease link: RAS-cytokine pathway
Intervention trials in adults with CKD have demonstrated that blockade of the RAS slow progression of renal disease via antihypertensive and anti-inflammatory mechanisms [26–28]. The RAS generates circulating angiotensin II (AT2), which regulates blood pressure and intravascular volume. In contrast to its endocrine function, tissue RAS produces AT2 that is involved in autocrine and paracrine signaling within all bodily organs, including the heart, blood vessels, and kidneys [29]. Tissue RAS exerts a pivotal role in the regulation of cytokine signaling, potentially modulating the inflammatory response associated with renal disease progression and susceptibility for CV dysfunction.
Tissue RAS via AT2 regulates the cytokine pathway responsible for progressive injury in the kidney and heart [25, 30–32]. As depicted in Fig. 1, activation of tissue RAS increases the local production of AT2. After AT2 stimulates the AT2 receptor, a number of signaling systems are triggered, including that of nuclear factor kappa B (NF-κB), which is responsible for upregulation of proinflammatory cytokines [33]. The cytokine signaling modulates endothelial dysfunction, adhesion and migration of circulating immune cells (monocyte, leukocytes, or neutrophils) into the interstitium, and activation of resident fibroblasts [10, 11]. Cytokines are soluble polypeptides that act as important humoral modulators in immunoregulation, hematopoiesis, and inflammation. Cytokines act in a highly complex coordinated network with considerable overlap and redundancy between the function of individual cytokines. Being pleiotropic in their actions, these molecules can induce or repress their own synthesis as well as that of other cytokines and cytokine receptors [24, 34].
Fig. 1Activation of the renin angiotensin system (RAS) and an increase in the local production of angiotensin II (AT2) triggers the inflammatory host response
In the kidney, the inflammatory host response leads to renal interstitial fibrosis and progression [10]. These actions within the kidney are mediated by proinflammatory [tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6] and profibrotic cytokines [TGF-β and plasminogen activator inhibitor (PAI)-1] [10, 35]. Proteinuria stimulates interstitial inflammation and fibrosis in the kidney; it also is a risk factor for future decline in kidney function [36]. NF-κB activity is stimulated by albumin [37] and is the pathway that links proteinuria and tubulointerstitial inflammation and fibrosis in the kidney [38, 39]. In the heart, the AT2-stimulated inflammatory response leads to LVH [40, 41]. The myocardial hypertrophy is caused by an increase in cell size and accompanied by changes in gene expression in response to AT2 [42, 43]. In addition to AT2, proinflammatory cytokines IL-1, IL-6, TNF-α, and TGF-β are responsible for myocyte hypertrophy and interstitial fibrosis [44, 45]. In the blood vessel, the AT2 pathway is the molecular mechanism leading to atherosclerosis [25, 32]. There is preliminary evidence indicating impaired flow-mediated dilation among hypertensive patients with mutations in the promoter region of the NF-κB gene [46]. Hence the upregulation of RAS-cytokine pathway activity is associated with renal progression, LVH, and atherosclerosis. The magnitude of this response may depend on genetic polymorphisms, which may either increase or decrease expression of these genes [24, 47].
Genetic polymorphisms
Different versions of a gene at a specific chromosomal location, or loci, that encode a trait are called alleles (see Table 1 for a glossary of common terms). Complementary alleles are inherited from each parent. A change in one nucleotide (base pair) within a gene is called a single nucleotide polymorphism (SNP). About 11 million SNPs with minor allele frequencies (MAF) of at least 1% are estimated to exist in the human genome. SNPs that affect native protein function, i.e. functional SNPs, can occur in gene promoter regions, coding regions, splice junctions, and 3’-untranslated regions (UTR) and may be causally involved in the etiology of human disease. Other types of polymorphisms include insertion/deletion (indel) polymorphisms and mini- and microsatellites (di-, tri-, and tetranucleotide repeats) [48]. Variation in phenotypic expression of a gene may be affected by epigenetic factors, where gene expression is affected by mechanisms other than alterations in the nucleotide sequence, but this is beyond the scope of this review.
Table 1Glossary of common genetic termsAlleles: Alternate sequences of the same gene, one inherited from each parent.Biological pathway: A set of proteins that interact to produce normal and abnormal physiology.Candidate gene: A gene in which variants could plausibly explain a given phenotype, such as severity of disease or variable response to drug. Methods to identify candidate genes include basic science studies, identifying DNA sequences conserved across species, human genetics, epidemiologic association studies, or genome-wide analyses.Epigenetics: Heritable change in the pattern of gene expression mediated by mechanism other than alterations in the primary nucleotide sequence of gene.Genome: The collection of all DNA in an organism. Only a small proportion (probably <3%) of human genomes encodes proteins.Genotype: The genetic makeup of an individual, which may refer to the whole genome or to specific genes or regions of genes.Haplotype: A set of genetic variants that are inherited together. Polymorphisms that are coinherited more often than by chance alone are in linkage disequilibrium (LD). Haplotype blocks may include many individual polymorphisms in high LD; as a result, establishing genotype at any single polymorphic site with such a block may establish genotypes at linked sites within the block. Individual single-nucleotide polymorphisms (SNPs) that can be used to establish genotype within a haplotype block are termed tag SNPs.Heterozygous: Having different alleles in a specific region of DNA.Homozygous: Having the same alleles in a specific region of DNAPhenotype: Measurable characteristics of an organism. These may derive from genotype, environment, or the combination. Organisms with the same phenotype can have different genotypes.Polymorphisms: DNA variants that are common, often defined as >1% in a given population. Polymorphisms can be in coding regions (where they may be synonymous or nonsynonymous) or, more commonly, in noncoding regions, and often vary by ethnicity. The most common type of polymorphism is a change in one nucleotide (base pair) in a DNA sequence, referred to as an SNP. Other polymorphisms are insertion and deletion of multiple sequential nucleotides (indels); variable numbers of repeats, such as doublets or triplets; or large-scale duplications or deletions. Although some genetic variants are known to alter protein abundance or function, the functional consequences of most polymorphisms are unknown.Tag SNPs: These are maximally informative SNPs that characterize common haplotypes.
The study of individual SNPs has yielded exciting insight into the factors involved in CKD progression. Although candidate-gene-based approaches are a logical first step, they are unlikely to provide a complete answer. The progressions to renal and CV disease are complex traits involving multiple genes. As discussed later, the cooperative efforts among scientific groups and the availability of robust and affordable technologies that can identify thousands of SNPs across the genome make genome-wide association studies an attractive paradigm for studying polygenic diseases such as CKD. Whole-genome association studies are being used to identify the genetic basis for CKD, with large consortiums investigating the genetic predisposition to diabetic nephropathy in Europe and North America [49, 50].
Although a complete overview of all known genetic polymorphisms of the RAS-cytokine pathway is beyond the scope of this review, a complete listing can be found at the National Center for Biotechnology Information (NCBI) SNP database (dbSNP) (www.ncbi.nlm.nih.gove/projects/SNP/). There are polymorphisms of the RAS-cytokine genes that have been reported to be associated with renal progression and/or CV morbidity and are summarized below (Table 2). Despite some of the potential weaknesses of the studies included, these candidate-gene association studies offer some preliminary information worthy of further investigation.Table 2Candidate-gene polymorphisms and associations with renal and cardiovascular (CV) diseases among subjects with chronic kidney disease (CKD)AuthorGene and genotypeaStudy population/study typebSample size (n)Clinical significanceRenin Angiotensin System (RAS)Boright A [53]ACE haplotypesUS diabetic nephropathy CKD (DCCT-EDIC)/cohort studyRestricted to Caucasian cohort = 1,365The genetic variation of the ACE gene is associated with microalbuminuria and diabetic nephropathy.Hadjadj S [49]ACE insertion/deletion polymorphismEuropean diabetic nephropathy CKD/case-control studyAdult diabetic nephropathy cases = 1,057, controls = 1,127The haplotype including the ACE deletion allele was associated with diabetic nephropathy.Haszon I [57]ACE insertion/deletion polymorphismEuropean Vesicoureteral reflux CKD/case-control studyPediatric VUR cases = 77, controls = 80A deletion at both alleles is linked to renal scarring in VUR.Hohenfellner K [55]ACE insertion/deletion polymorphismEuropean CKD (Nutritional Treatment of Chronic Renal Failure in Childhood Study)/cohort studyPediatric cohort = 95A deletion at both alleles is linked to a higher risk of renal progression among children with congenital renal malformation.Lovati E [58]ACE insertion/deletion polymorphismEuropean ESRD/case-control studyAdult ESRD cases = 260, controls = 327A deletion at both alleles is linked to a higher risk of renal progression among adults.Ng D [54]ACE insertion/deletion haplotypeUS diabetic nephropathy CKD/case-control studyAdult Caucasians type 2 with diabetic nephropathy cases = 291, controls = 167The deletion allele haplotype is associated with diabetic nephropathy.Papp F [56]ACE insertion/deletion polymorphismEuropean ESRD/case-control studyPediatric ESRD cases = 20, controls = 150A deletion at both alleles is linked to ESRD.Hsu C [64]AGT-6 G>AUS CKD (ARIC Study)/ cohort studyRestricted to Adult African American CKD cohort = 3,381 cGenotype A/A is linked to a higher risk of renal progression among African American CKD patients.Reis K [111]AGT M235TEuropean kidney transplant/case-control studyAdult kidney transplant cases = 82, controls = 100Genotype Thr/Thr linked to chronic allograft dysfunction.Buraczynska M [66]AT1R A>CEuropean ESRD/case-control studyAdult ESRD cases = 745, controls = 520Genotype C/C or A/C is linked to higher risk of renal progression in adults.Interleukin (IL)-1Wetmore JB [73]IL-1α g.-889 C>TUS ESRD/case-control studyAdult ESRD cases = 239, controls = 252Genotype T/T is linked with risk for ESRD.Amoli M [82]IL-1β g.-511 C>TEuropean Henoch-Schonlein purpura disease (HSP)/case-control studyAdult and pediatric HSP cases = 49, controls = 146Carriage of the T allele linked to severity of renal involvement with Henoch-Schonlein purpura.Interleukin-1 Receptor AntagonistBuraczynska M [83]IL-2RN*2European ESRD/case-control studyAdult ESRD cases = 602, controls = 433Homozygous for the IL2RN*2 allele linked with more rapid progression in patients with glomerulonephritis and diabetic nephropathy and risk for ESRD.Wetmore JB [73]IL-1RN*2US ESRD/case-control studyAdult ESRD cases = 239, controls = 252Homozygous for the IL2RN*2 allele is linked to risk for ESRD.Watanabe M [81]IL-12N*2Japanese IgA nephropathy CKD /case-control studyAdult IgA Nephropathy cases = 106, controls = 74Carriage of the IL2RN*2 allele is linked to severe proteinuria and increased creatinine in IgA nephropathy.Interleukin-6Balakrishnan V [72]g.-174G>CUS ESRD (HEMO Study)/cross-sectional studyAdult ESRD cohort = 187Genotype G/G or G/C is linked to increased comorbid conditions and decreased functional status among dialysis patients.Losito A [86]g.-174G>CEuropean ESRD/case-control studyAdult ESRD cases = 161, controls = 169Carriage of the C allele linked to LVH in hemodialysis patients, especially those with diabetes.Muller-Steinhardt M [87]g.-174G>CEuropean Kidney transplant/cohort studyAdult kidney transplant cohort = 158Carriage of the C allele linked to decreased kidney allograft survival.Interleukin-10Girndt M [88]g.-1082 G>AEuropean ESRD/cohort studyAdult ESRD cohort = 300The A/A genotype is linked to a lower production of IL-10 and increased CV morbidityTumor Necrosis Factor (TNF)-αBalakrishnan V [72]g.-308 G>AUS ESRD (HEMO Study)/cross-sectional studyAdult ESRD cohort = 187Genotype A/A or A/G is linked to low serum albumin, increased comorbid conditions, and decreased functional status among dialysis patients.Transforming Growth Factor-βSato F [106]g.-509C>T and g.+869T>CJapanese IgA Nephropathy CKD/cross-sectional studyAdult IgA nephropathy cases = 329, controls = 297The -509C/C and 869C/C genotypes are linked with heavy proteinuria and mesangial cell proliferation in patients with IgA nephropathy.Rao M [107]g.+915G>CUS ESRD (HEMO Study)/cross-sectional, and cohort studiesAdult ESRD cohort = 187Genotype G/C vs G/G was linked with risk for prevalent vascular disease, new onset cardiac morbidity and cardiac mortality in HD patients.Plasminogen Activator Inhibitor (PAI)-1Aucella F [113]4G/5GEuropean ESRD/cohort studyAdult ESRD cohort = 417Genotype 4G/4G is linked to increased risk for fatal MI among HD patients.Reis K [111]4G/5GEuropean Kidney transplant/case-control studyAdult kidney transplant cases = 82, controls = 100Carriage of the 4G allele linked to chronic allograft dysfunction.Wong A [112]4G/5GChinese Systemic lupus erythematosus CKD/case-control studyAdult diabetic nephropathy cases = 95, controls = 46Genotype of the 4G/4G linked to increased severity lupus nephritis among SLE patients.ACE angiotensin converting enzyme, AGT angiotensin, AT1R angiotensin II type 1 receptor, DCCT Diabetes Control and Complications Trial, EDIC Epidemiology of Diabetes Interventions and Complications, ESRD end-stage renal disease, ARIC Arthrosclerosis Risk in Communities, HEMO hemodialysis, VUR vesicoureteral reflux, HD hemodyalysis,  SLE systemic lupus erythematosus, MI myocardial infarctiona The gene and gene polymorphism of interestb Study population is given to indicate potential for population stratification and type of CKD population (name of cohort study)/study type by: cross-sectional, cohort, or case controlc In the study by Hsu et al., 3,449 subjects had AGT genotyping, whereas 3,381 subjects had both AGT and AT1R genotyping
The renin-angiotensin system
Polymorphisms in the RAS system are associated with clinically significant renal and CV disease morbidity and thought to occur through a proinflammatory mechanism. The inflammatory response is activated by the RAS through the recruitment of proinflammatory cells to the site of injury [51, 52] and the upregulation of adhesion molecules on vascular endothelial cells and smooth muscle cells [30].
A naturally occurring variant in the angiotensin-converting enzyme (ACE) gene, located on 17q23, is a 250-base pair deletion in intron 16. The D/D genotype is associated with a high ACE level, whereas the I/I genotype is associated with a low ACE level. In subjects with type 1 and type 2 diabetes, the haplotype insertion allele of the ACE gene has been associated with lower risk of diabetic nephropathy compared with the haplotypes including the deletion allele [49, 53, 54]. The D/D genotype has been associated with renal progression in children and adults with CKD [55–58]. The D/D polymorphism has been associated with LVH and QTc interval prolongation in patients with ESRD [59, 60].
Polymorphisms in angiotensinogen (AGT), located on 1q42-q43, are associated with increase in risk for renal progression and CV disease. Reported frequently in the literature, the M235T SNP is a methionine (Met) to threonine (Thr) amino acid substitution at codon 235. The Thr/Thr genotype is associated with an increased risk for hypertension in the general population [61, 62] and in kidney transplant patients with chronic allograft dysfunction. Investigators have recently recommended a name change of the SNP M268T for the substitution at amino acid 268, to be consistent with accepted human gene mutation nomenclature [63]. Another SNP is the AGT-6 G/A promoter variant, which is associated with a higher risk for renal progression in the African American CKD population [64].
A polymorphism in the AT2 type 1 receptor (AT1R) gene polymorphism, located on chromosome 3 (3q21-q25) [65], has also been associated with risk of renal progression and CV disease. The polymorphism of interest is a nucleotide change from an adenine (A) to cytosine (C) in the 3′-UTR at nucleotide 1166. The C/C genotype has been associated with a more rapid onset of renal failure compared with those with the A/A genotype [66]. Furthermore, the C/C genotype also is associated with the development of hypertension and coronary artery disease [67, 68].
Interleukin-1 and IL-1 receptor antagonist (IL-1Ra)
The IL-1 family consists of two proinflammatory cytokines, IL-1α and IL-1β, and a naturally occurring anti-inflammatory agent, the IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases [69, 70]. Plasma IL-1 and IL-1Ra have been shown to predict cardiovascular outcome [71] and mortality in ESRD [72]. The genes of the IL-1 complex map to the 430-kb region on the long arm of chromosome 2. The IL-1α gene has an SNP g.-889C>T, which is a base pair change from a cytosine to a thymine (C→T) [73]. The IL-1β gene has an SNP g.-511C>T, which has a base pair change exchange (C→T) [73] and g.+3953 [74]. The IL-1Ra gene contains a variable number of tandem repeat (VNTR) polymorphisms in intron 2 (IL-1RN) [75]. The IL-1RN allele 2 is related to increased production of IL-1β [76]. Polymorphisms in IL-1α have been associated with ESRD [73]. Gene polymorphisms of IL-1β and IL-1RN have been associated with hypertension [77], atherosclerosis [78–80], CAD [78–80], and progression of renal disease [81–83].
Interleukin-6 (IL-6)
IL-6 is a proinflammatory cytokine that stimulates the production of C-reactive protein (CRP) and fibrinogen. There is a promoter polymorphism at position –174 of the IL-6 gene (g.-174G>C). Carriage of the C allele is associated with higher levels of IL-6 production in response to pathologic stimuli [84]. The polymorphism is associated with a higher risk of CV disease in the general population [84, 85]. In dialysis patients, the carriage of the C allele is associated with high blood pressure, LVH, and decrease in functional status [47, 86]; in kidney transplant recipients, it is associated with decreased graft survival [87].
Interleukin-10 (IL-10)
IL-10 attenuates the inflammatory response [47]. Decreased production of IL-10 is associated with increased CRP and higher cardiovascular mortality [88]. The IL-10 gene is located on 1q31-32 and is composed of five exons and has SNPs at positions g.-592 C>A, g.-819 C>T, and g.-1082 G>A [89, 90]. The low producer genotype A/A of the g.-1082G>A SNP is associated with increased CV mortality in ESRD patients [88].
Tumor necrosis factor-α (TNF-α)
TNF-α production is stimulated by AT2 and associated with tubulointerstitial fibrosis [91]. Furthermore, elevated TNF-α levels are associated with CV disease comorbidities: coronary artery disease [92], LVH [93], and congestive heart failure [94]. The TNF-α gene is located on chromosome 6 and is highly polymorphic. Numerous promoter-region SNPs exist and are located at the upstream positions -1031, -863, -857, -851, -419, -376, -308, -238, -163, and -49 relative to the transcription start site; another SNP is at +488 in the intron [95, 96]. In hemodialysis patients, the polymorphism g.-308 G>A in the promoter region of the TNF-α gene has been associated with significant comorbidity; the carriage of the A allele is associated with a low serum albumin, higher burden of comorbid conditions, and a low Karnofsky score [47].
Transforming growth factor-β (TGF-β)
TGF-β is a cytokine that regulates cell growth, differentiation, and extracellular matrix production [97]. TGF-β transmits the profibrotic signaling of AT2 that promotes interstitial fibrosis in the kidney [98]. Blockade of AT2 by ACE inhibitors and AT2 receptor blocker (ARB) drugs reduces intrarenal TGF-β [99]. Furthermore, TGF-β is responsible for the production of additional fibrosis-promoting molecules such as connective tissue growth factor (CTGF) and PAI-1 [10]. Overproduction of TGF-β1 is associated with renal progression [10, 100], hypertension [101], and LVH [102]. Several polymorphisms have been identified in the TGF-β gene [103]. There are two polymorphisms in the signal peptide sequence Leu10->Pro (g.+869T>C), Arg25→Pro (g.+915G>C) associated with higher production of TGF-β1 [104, 105]. Gene polymorphisms in TGF-β1 have been associated with an increase in proteinuria and mesangial cell hypertrophy in patient with IgA nephropathy [106]. Furthermore, the +915G/C genotype at codon 25 (Arg/Pro) may be a genetic susceptibility factor for the development of atherosclerosis due to the genotype’s association with an increased risk for cardiac morbidity and cardiac-specific mortality in hemodialysis (HD) patients [107].
Plasminogen activator inhibitor-1 (PAI-1)
Upregulation of PAI-1 favors extracellular matrix accumulation and fibrosis by inhibiting fibrinolysis [108]. AT2 signaling via the type 1 receptor increases the production of PAI-1 [108]. Furthermore, regression of sclerosis is associated with blockade AT2 and a reduction in PAI-1 [109]. The gene polymorphism of interest is a 4G/5G insertion/deletion 675 base pairs from the start of the promoter. The polymorphism affects the binding of nuclear proteins involved in the regulation of PAI-1 gene transcription, leading to higher rate of synthesis with the 4G/4G genotype [110]. The genotype is associated with chronic kidney allograft nephropathy [111] and increased activity of lupus nephritis [112], The PAI-1 4G/5G polymorphism is associated with fatal and nonfatal myocardial infarction in dialysis patients [113]. In summary, much of the data supporting the link between RAS-cytokine gene polymorphisms and the progression of renal and CV disease stem from adult studies that involve a significantly larger prevalent population. However, there are preliminary data supporting efforts for genotype–phenotype association studies in children [55–57]. Although there is heterogeneity for the causes of CKD between the adult and pediatric populations, progression for renal and CV abnormalities shares the same final common pathway, as discussed above.
Finding the link between genotype and phenotype
As stated previously, the natural variations of the genes involving the RAS–cytokine pathway potentially influence the rate of progressions for renal and CV disease in CKD patients. For example, a study by Balakrishnan et al. demonstrates the relationship between cytokine gene polymorphism and cytokine secretion from peripheral blood monocytes (PBMC) in hemodialysis patients [47]. Genotyping was performed for SNPs in the promoter region of IL-6 (-174 G>C), TNF-α (-308G>A), and IL-10 (-1082 G>A) in 183 ESRD patients. Plasma cytokine levels by endotoxin-stimulated PBMCs were measured by enzyme-linked immunosorbent assay (ELISA). Plasma IL-6 levels were higher in the circulating blood from patients having -174G/C or C/C genotype; and IL-10 secretion was increased in -1082 G/G genotype (Table 3). The inflammatory response to the uremic milieu is variable and associated with cytokine gene polymorphisms in CKD patients.Table 3Relationship between genotype and plasma cytokine levels [72]CytokineGenotypeTranscription/secretion level (expected)Plasma level in pg/ml mean ± SDInterleukin (IL)-6-174 C/CLow12.2 ± 5.1-174 G/G, G/CHigh15.01 ± 17.4aTumor necrosis factor (TNF)-α-308 G/GLow998.8 ± 1156.2-308 G/A, A/AHigh1131 ± 1616.2IL-10-1082 A/ALow344.8 ± 356.3-1082 G/AIntermediate391.0 ± 440.5-1082 G/GHigh627.4 ± 506.2baLevene’s test for unequal variance, p=0.05, bKruskall–Wallis test, p = 0.01
By analyzing differences in DNA sequences in large cohort studies, the disease phenotype can be mapped according to known genetic markers (i.e. known locations of SNPs) by linkage analysis. Unlike monogenic disorders, polygenic disorders as in CKD and CV abnormalities are the result of complex interactions of intra- and intercellular systems that are governed by multiple gene loci (polygenic), often modified by gene–environment and gene–gene interactions (epistasis) [24]. The genetic determinants of renal and CV disease progression are cumulative variations of gene transcription and function in these interdependent pathways.
A consistent picture of genotype–phenotype relationships in CKD and CV disease are lacking, probably because: (1) other unknown functional loci may be present; (2) polymorphisms that are known but which have not yet been recognized to be functional may exist; (3) inconsistent definitions of CKD; (4) heterogeneity of diseases that cause CKD; (5) limitations due to sample size, especially in the pediatric CKD population. Polymorphisms do not exist in isolation, and it may be the combination of base changes at several proximal sites along the allele, i.e. the haplotype that influences the function. Haplotype methods may capture a large proportion of the genetic variation across sizable regions using a minimal number of tag SNPs [114, 115], as discussed further below.
Haplotype and HapMap
Alleles that are in close proximity along a DNA strand tend to cross over together during recombination and comprise a haplotype. In a population, common haplotypes can be inherited among many individuals from a common ancestor, with complementary haplotypes being given by each parent. The characterization haplotype variants among human populations (http://www.hapmap.org) offers new opportunities in the genetic analysis of CKD through whole-genome association studies.
At this time, the International Haplotype Map (HapMap) Consortium is characterizing where and how frequently sequence variants occur in the human genome in four different ethnic groups worldwide [116]. Here, SNPs along a chromosome that tend to be inherited together can define a haplotype. Although a chromosomal region may contain many SNPs, there only a few tag SNPs that offer the most information about genetic variation in that region. Within candidate genes, the number of common polymorphisms is finite [117], but direct assay of all existing common polymorphism is inefficient, because genotypes at many of these sites are strongly correlated. Selection of the maximally informative set of a common tag SNP set can comprehensively interrogate for main effects of the haplotype [118, 119].
The HapMap project has opened the way for whole-genome association studies. Association refers to the statistical dependence between two variables, which is a measure of the degree to which the frequency of a risk factor, in this case a genetic marker, is different between persons with disease compared with those without disease. Hence, studies to determine the genes that influence CKD progression will compare CKD patients with a more rapid decline in glomerular filtration rate (GFR) to those with slower progression. Regions where the two groups differ in their haplotype frequencies might contain genes associated with renal progression. However, the dawning of new technology for rapid sequencing of DNA has implications of advancing this field of research beyond the HapMap [120].
Potential limitations of gene association studies
The presence of an association does not imply that the observed relationship is one of cause and effect. A judgment of causation from epidemiologic data relies on assessing the validity of the observed statistical association and the cumulative evidence from a variety of sources in order to support a causal inference regarding the genetic marker of interest. An association between a gene and disease may be indicative of a true relationship between the gene and the disease; however, the association may not be valid due to chance (type I error), limited sample size (type II error), bias, or confounding. Ideally, a gene association study will have sufficient cases and controls (on the order of thousands) to identify a gene locus or region that is truly associated with the disease given a threshold of significance on the order of P value < 10−6 [121]. However, an investigation of this nature is not likely to be achieved immediately in the pediatric CKD population without preliminary evidence from studies of modest sizes varying in degrees of quality. Furthermore, no single center alone will have a sufficient number of pediatric CKD subjects to power such an analysis and would likely need a cooperative effort among a number of pediatric CKD centers.
Type I and type II error
Typically, investigators may submit their data sets for genotyping with a standard, very large, set of SNPs, which will include markers located in the candidate gene along with a few thousand which are not. Spurious associations where the genetic marker of interest is correlated with the disease by chance may occur, especially in the presence of multiple comparisons [122]. To illustrate the multiple comparisons problem, we use a hypothetical case-control study of 10,000 unrelated SNPs to be tested for association with the disease of interest, with the threshold for statistical significance set to a P value of 0.01. If 10,000 statistical tests are performed to assess the association between each of the SNPs and the disease, by chance alone, we would expect 100 SNPs at random to be statistically associated with the disease, even though there is no true relationship; this is a type I error. With increasing number of genetic markers being typed and multiple intermediate phenotypes being tested, strict guidelines for publication of gene association studies have been proposed [123–125].
In studies of modest size, SNP markers in the candidate gene may produce relatively modest evidence in favor of association with a level of P < 0.01, even with hundreds of subjects [126]. Thus, a marker having a real but modest effect is not expected to produce an odds ratio (OR) with a smaller P value than markers producing apparently significant results by chance [126]. In addressing this limitation, setting a stringent P value (P < 10−6) is a generally accepted convention for large studies with markers of low prior probability of true association [121]. Applicable to studies of smaller magnitude, alternative methods relying on Bayesian strategies have been proposed [126, 127]. Under these approaches, the prior probability of association based on preliminary evidence is used to weight the P value obtained. This supports the general recommendation for having biologic plausibility of the observed association such that the genetic variant of interest is involved the pathogenesis of disease [122, 124, 128]. It is worth considering whether the known function of the gene is linked to the observed phenotype. Furthermore, the association between gene and disease is less likely to be spurious if the relationship is observed in other independent studies. Although mildly controversial [129], there is general agreement that the initial findings of a gene–disease association study be replicated by other studies [130, 131].
Many gene association studies are not replicated for a number of reasons. A small sample size may limit a study’s ability to detect an association if one truly exists. Concluding that there is no association when one truly exists defines a type II error. The magnitude of the contribution of single gene variants to polygenic disorders is small, with a typical effect sizes corresponding to ORs of 1.2–1.6 [132]. Compared with the smaller sample sizes needed to detect a larger effect (e.g. OR ≥ 2.0), detecting associations of smaller magnitude requires a much larger sample size, which may dramatically increase the recruitment costs and make some studies unfeasible [132].
Furthermore, the lack of consistency in proposed gene–disease association across studies may also be reflective in the inherent complexity and heterogeneity of common diseases, including CKD [131], which are beyond the scope of this review. A proposed solution for studies with insufficient power are to: (1) emphasize replication and obtain data to determine biologic plausibility; (2) synthesize results of individual studies for meta-analysis; or (3) obtain data on individual subjects from several studies to perform a pooled analysis [125].
Limitations in study design
Biased estimates for an observed association between genotype and phenotype may originate from a flaw in the design or conduct of the study that has introduced systematic error or bias into the result. There are numerous considerations to this point, but a few topics deserve some mention in this general overview. A more complete discussion of these considerations can be found elsewhere [122, 125, 133, 134]. The adequacy of any epidemiologic study design depends on the scientific question [135]. A study that is designed to detect an association may “overselect” cases in order to detect an association. However, the measures of the association from studies designed to detect genotype–phenotype associations are not valid or generalizable measures of association for the population [125].
The case–control study is a common study paradigm for genome association studies because it is economically efficient, allows for the evaluation of diseases with long latent periods, and can examine multiple etiologic factors for one disease; however compared with other study paradigms, case–control studies are particularly prone to bias if controls are not properly selected [136]. Genetic association studies may be biased or confounded by population stratification [133] and genotyping errors [134]. Population stratification can create the appearance of a SNP–disease association and arises when race and/or ethnicity is related both to the SNP under investigation (e.g. differences in allele frequencies within distinct ethnic groups) and to the disease of interest. Similar to epidemiologic studies needing to address race and ethnicity as potential confounders, gene polymorphism studies should assess the potential for bias and confounding due to population stratification [137].
Of additional concern, genotyping errors can be significant, leading to null results or erroneous conclusions [134]. Genotyping errors can stem from a number of causes, including: (1) inadequate sample quality; (2) artifacts due to biochemical or equipment problems; (3) errors from the DNA amplification process; (4) human factors. Genotyping errors in phenotype–genotype association studies will tend to bias estimates of association toward the null assuming that errors occur at equal frequency across case status [134]. However, if cases and controls are genotyped using different assays or run separately in distinct batches, differential errors may occur resulting in either over- or underestimation of the true association [134].
Conclusions
Genetic association studies have the potential to provide new insights into the factors responsible for CKD renal and CV progression. These investigations provide hope for new drug targets to treat or modify individual disease risk. In the case of CKD, genetic polymorphisms in the RAS–cytokine pathway may be responsible for the intraindividual variation in renal and cardiac progression in patients with CKD and may offer new targets for drug therapy.
The Human Genome and HapMap projects have made it possible to evaluate a multitude of candidate genes that might be linked to CKD progression. The enthusiasm for these investigations must be tempered by acknowledging the limitations of gene association studies. Attention to biologic plausibility and appropriate study design will help the interpretability of published results. Independent investigations replicating initial findings are needed to support an inference of a causal association between the gene polymorphism of interest and the disease phenotype.
Whole-genome association studies are becoming widely available and are being performed to investigate the genetic predisposition to diabetic nephropathy [49, 50]. In an effort to understand the risk factors for progression of CKD and CV disease, the ongoing CKD cohort studies in adult and pediatric patients [138, 139] are evaluating known risk factors for CKD progression, including etiology of CKD, proteinuria, and hypertension. These cohort studies are collecting biologic and genetic samples for future studies of cytokines or their genetic polymorphisms, which may yield scientific insight into the pathophysiologic mechanisms of CKD progression in both adults and children.

genetic
polymorphism
cytokine
chronic kidney disease
progression
pediatric
cardiovascular disease