Magnetic resonance imaging protocols for paediatric neuroradiology Increasingly , radiologists are encouraged to have protocols for all imaging studies and to include imaging guidelines in care pathways set up by the referring clinicians . This is particularly advantageous in MRI where magnet time is limited and a radiologist 's review of each patient 's images often results in additional sequences and longer scanning times without the advantage of improvement in diagnostic ability . The difficulties of imaging small children and the challenges presented to the radiologist as the brain develops are discussed . We present our protocols for imaging the brain and spine of children based on 20 years experience of paediatric neurological MRI . The protocols are adapted to suit children under the age of 2 years , small body parts and paediatric clinical scenarios . Introduction The use of imaging protocols allows the standardization of procedures and workflow as well as consistency of image quality . With appropriate training , radiographers can select and implement imaging protocols with relatively little requirement for radiological involvement . This use of skill mix provides radiographers with an expanded role and greater responsibility that increases job satisfaction and staff retention . Imaging protocols can also provide guidance for radiologists and radiographers for patients in shared care between secondary and tertiary care institutions . The use of protocols set up by the referring centre will prevent repeat MR scanning with the additional burden on MR scanner time . These advantages are offset by the minor disadvantage that radiologists ' personal preferences and the intricacies of individual scanners are not taken into consideration . At Great Ormond Street Hospital -LRB- GOSH -RRB- , MRI protocols have been implemented for nearly 20 years and have been modified as scanners are replaced and new sequences developed . Our success is measured by the minimal number of patients recalled -LRB- less than 1 % -RRB- . Increasingly , we are asked about our MR protocols by departments throughout the United Kingdom and have set them out here to make them freely available to those who may find them beneficial to patient care . The protocols provided are confined to conventional imaging techniques and diffusion-weighted imaging -LRB- DWI -RRB- , and do not include the advanced imaging techniques of perfusion imaging , diffusion tensor imaging and MR spectroscopy that remain largely within the research domain . Practical issues of MRI in children One of the biggest challenges of paediatric neuroimaging is the acquisition of high-quality diagnostic images , as it requires the infant or child to keep still for a long period of time -LRB- sometimes nearly 1 h -RRB- . Children over the age of 7 years are scanned , where possible , without sedation and with recourse to play specialists and videos . Neonates under 2 months old -LRB- corrected age , if premature -RRB- are scanned during natural sleep induced by food , comfort and warmth -LRB- feed and wrap -RRB- , often best after a period of sleep deprivation . If this fails then light oral sedation , e.g. chloral hydrate , may be used . The majority of our patients -LRB- approximately 70 % -RRB- require sedation or a general anaesthetic . When sedation is required , it is usually heavy and heavier than that required for CT or nuclear medicine studies . Sedation is applied by specially trained nurses who are trained to decide when and how much to administer and monitor its effects , and who are skilled in the recovery of the child -LSB- 1 -RSB- . Details can be found in a review of the management of painless imaging in children -LSB- 2 -RSB- . All MR sequences may disturb the sleeping infant or child and ear protection such as earplugs and baby earmuffs should be used . Some motion can be avoided by swaddling infants , keeping them warm , and by placing moulded foam or airbags around the baby 's or child 's head . Cardiorespiratory monitoring with MR-compatible equipment is essential in all infants , whether sedated or not , and all sedated or anaesthetized children -LSB- 2 -RSB- . Commercial MR machines are designed for adult practice and few manufacturers make provision for the issues encountered in paediatric practice . Currently , most children are imaged in units that have a wider adult practice with only a handful of institutions throughout the world having dedicated paediatric MR facilities . The structures we need to examine in children are generally small and we aim for a maximum slice thickness of 5 mm in the brain and 3 mm in the spine . The slice thickness is reduced to 3 mm when acquiring images of the pituitary gland and orbits . We do not recommend ultrafast ` breath-hold ' - type T2-weighted -LRB- T2-W -RRB- sequences that have reduced contrast to noise as an alternative to sedating a child , as achieving high contrast to noise is of utmost importance given the small size of the paediatric head . Advances in coil design have improved signal-to-noise and we now use multichannel head and spine coils . Practical consideration of the changing brain with age Changes in normal values of measured parameters in very young children occur as the brain undergoes very rapid development during the first 2 -- 3 years of life . This necessitates the creative modification of pulse sequence parameters in order to optimize the soft-tissue contrast between anatomical and pathological structures . The two most important physiological changes to take place within the first 2 -- 3 years are reduction in the total brain water content and T1 and T2 shortening largely as a consequence of the maturation of myelin -LSB- 3 -RSB- . From birth to 6 months of age the maturation of myelin is best assessed using T1-weighted -LRB- T1-W -RRB- images . From 6 months until the completion of myelination at around 2 years of age , T2-W images are more useful . During the process of myelination on T1-W images the signal from white matter goes from dark to bright . For a variable period the subcortical white matter becomes isointense with grey matter . This loss of grey/white matter differentiation obscures structural detail and makes identification of subtle abnormalities such as polymicrogyria difficult to detect within the first 6 months of life . The white matter shows maturity later , between 6 months and 2 years , on T2-W images and at this stage T1-W images are essential to evaluate structural abnormalities -LSB- 4 -RSB- . To overcome the difficulty of increased brain water in those under 2 years of age , we use a dual-echo short-tau inversion recovery -LRB- STIR -RRB- sequence that has improved contrast resolution in this age group -LRB- Fig. 1 -RRB- -LSB- 5 -RSB- . Typical values for a dual-echo STIR sequence are TE 30/128 ms , TR 5,400 ms and TI 130 ms. We prefer this sequence to the more conventional T2-W fast -LRB- or turbo -RRB- spin-echo sequences , for reasons of increased lesion conspicuity . However , we recognize that similar tissue contrast may be achieved if such fast spin-echo sequences are modified using increased TR and TE to compensate for the increased water content in this age group . The additional brain water is difficult to suppress and we do not find the fluid-attenuated inversion recovery -LRB- FLAIR -RRB- technique useful in this age group as lesion conspicuity is poor . Fig. 1Axial images acquired through the bodies of the lateral ventricles of a 6-month-old child . First -LRB- a -RRB- and second -LRB- b -RRB- echo -LRB- TE 30/120 ms , TR 5,500 ms , TI 130 ms -RRB- of a dual-echo short-tau sequence shows the increased contrast between grey and white matter on the second echo compared to -LRB- c -RRB- the T2-W -LRB- TE 90 ms , TR 3,500 ms -RRB- sequence that would be used in those over 2 years of age . The slice thickness -LRB- 5 mm -RRB- and matrix size -LRB- 512 × 192 -RRB- were the same for both sequences Diffusion measurements in the brain of newborns and infants show that there is more movement of water -LRB- high apparent diffusion coefficient , ADC -RRB- with less directionality -LRB- low anisotropy -RRB- within the immature and unmyelinated brain than in the mature and myelinated adult brain -LSB- 6 , 7 -RSB- . The most dramatic decrease in ADC values occurs within the first few months of life and the greatest changes are seen in the frontal and parietal white matter -LSB- 8 -RSB- . Failure to appreciate the normal changes with age of the ADC will result in the erroneous reporting of white matter abnormality particularly in the neonatal period . Factors influencing sequence choice For any imaging protocol , it is the specific combination of sequences that determines the diagnostic efficacy of the examination . It is clear that imaging protocols vary between institutions . This is largely because rapid advances in imaging technology and variations between manufacturers , applied in the context of investigating relatively rare disorders , precludes an effective evidence-based approach to sequence choice . The main advantages that MRI offers over alternative imaging modalities is the ability to demonstrate different tissue contrasts -LRB- principally T1-W , T2-W and spin density , but also flow and diffusion -RRB- in multiple imaging planes -LRB- principally sagittal , coronal and axial -RRB- . The disadvantage of MRI is the artefacts that it generates in almost every image . The choice of sequence combination should reflect the multicontrast and multiplanar capabilities of MRI . We have found that applying the generic principle of combining T2-W images in two planes , supported by T1-W imaging in two planes , as the basis of our imaging protocols , serves to optimize the benefits of MRI whilst minimizing the impact of artefacts . Standard MRI studies Brain Our standard brain sequence -LRB- > 2 years of age -RRB- is composed of an axial T2-W , coronal FLAIR , and coronal and sagittal T1-W images . Sagittal T1-W images allow the assessment of the midline structures , particularly the corpus callosum and cerebellum , which are frequently abnormal in congenital abnormalities . Coronal T1-W images are valuable in the diagnosis of abnormalities of the anterior visual pathway , schizencephaly and holoprosencephaly . Axial T2-W and coronal FLAIR images provide T2-W images in two different planes and have been shown to be complementary in children -LSB- 9 -RSB- . DWI is acquired in all children unless artefacts from , for example , dental braces or a ventriculoperitoneal shunt , preclude it , and an ADC is calculated using automated computer software and provided for reporting . In those under 2 years of age , the T2-W sequence is replaced by a dual-echo axial STIR sequence -LRB- see above -RRB- . In some cases , a T2 * - W gradient-echo -LRB- GE -RRB- sequence -LRB- `` susceptibility-weighted '' sequence -RRB- , sensitive to changes in local field inhomogeneity caused by the breakdown products of haemoglobin , is added . The sequence is particularly useful in trauma and vascular malformations such as multiple cavernomas . Spine MRI is the modality of choice for imaging the intraspinal components of the paediatric spine . Prior to ossification of the posterior elements of the spinal column , US has been shown to be a valuable screening tool ; however , infants with an abnormal sonogram or who have a neurological abnormality in the context of a normal sonogram , still require MR imaging -LSB- 10 -RSB- . Our standard spine imaging includes sagittal , fast spin-echo T1 - and T2-W sequences -LRB- 3-mm-thick slices -RRB- . Both axial T1-W and T2-W images are acquired through any abnormality . Unlike most adult spine imaging protocols , groups of axial images through disc levels are not applied because degenerative disc disease is rare . Children with scoliosis and/or suspected spinal dysraphism routinely have axial T1-W images through the conus and filum terminale to detect lipomas of the filum terminale that may not be visible on sagittal imaging . Contrast medium The use of contrast medium is a personal choice and in the majority of units is requested on a case-by-case basis . At GOSH the radiographers or sedation nurses administer MR contrast medium according to guidelines set up by the neuroradiologists -LRB- Table 1 -RRB- . In the event of an unexpected finding the radiographers are encouraged to make a decision on contrast administration without discussion with a radiologist . Table 1Indications for contrast medium administrationVarious indicationsAcute inflammation Acute disseminated encephalomyelitis -LRB- ADEM -RRB- Optic neuritisAcute infection Abscess Cerebritis Discitis Empyema Encephalitis Meningitis Transverse myelitisNeurocutaneous disorders Congenital melanocytic naevus Neurofibromatosis type IITumours Benign and malignant Intracranial IntraspinalWhite matter disordersaVascular anomalies Cavernomas Developmental venous anomaliesVascular disorders Intraparenchymal haemorrhage Sturge-Weber syndrome VasculitisaAt presentation and at follow-up if necessary MRI protocols for specific areas The strategies applied to the brain are also applied to small parts such as the orbits and pituitary gland . The orbit is scanned using a STIR sequence to benefit from the fat saturation properties of the sequence and improve the conspicuity of lesions within the orbit . The slice thickness is reduced to 3 mm and , to increase the signal-to-noise , the matrix size is reduced , thereby slightly reducing the in-plane resolution . A heavily T2-W 3-D volume sequence of the petrous temporal bones is used to image the membranous labyrinth and is reconstructed in the coronal plane of the petrous temporal bone , and the axial and sagittal plane of the internal auditory meati -LRB- IAM -RRB- . The sagittal plane is used to view the vestibulocochlear nerve in cross section . A constructive interference steady state sequence -LRB- CISS -RRB- , available on Siemens scanners , is acquired and reconstructed at a slice thickness of 0.7 mm . The GE equivalent is the FIESTA sequence and the Philips equivalent is DRIVE . MRI protocols for specific neurological or neurosurgical presentations The majority of children undergoing an MRI brain scan will have epilepsy , stroke or a brain tumour and are often referred to specialists with an interest in these areas . Tumours of the brain and spine Brain Imaging of brain tumours is performed using our MRI brain with contrast enhancement protocol . We add contrast-enhanced T1-W imaging of the spine to establish the presence of any distant metastases in accordance with the United Kingdom Children 's Cancer Study Group -LRB- UKCCSG -RRB- guidelines for the imaging of brain tumours -LSB- 11 -RSB- . One of the commonest reasons for repeat scanning of a child with a brain tumour is the acquisition of contrast-enhanced imaging of the spine in a child who has had only a brain scan at the referring centre . The purpose of preoperative imaging of brain tumours is to assess tumour location and type , establish whether there are single or multiple lesions , define its relationship with vital structures and look for any complications such as hydrocephalus . Contrast-enhanced T1-W volumetric acquisitions are also acquired for image-guided surgery . Imaging of the spine is performed in all children with intracranial tumours -LRB- not only posterior fossa tumours -RRB- and although this requires a change of coil and an additional sequence , we find it beneficial as tumour histology is often not certain at the time of the initial imaging . Some tumours unexpectedly metastasize to the spine and assessment of the postoperative spine is made more difficult in the presence of blood products . Postoperative MRI is acquired using our MRI brain with contrast enhancement protocol to assess the degree of tumour clearance . We aim to do this within 72 h of surgery to reduce enhancement due to postoperative changes that can complicate the assessment of tumour clearance , in accordance with guidelines proposed by the UKCCSG -LSB- 11 -RSB- . If preoperative spinal imaging has not been acquired , the spine is imaged with both unenhanced and contrast-enhanced T1-W imaging to enable differentiation between haematoma and tumour metastases . Surveillance imaging -LRB- SI -RRB- during adjuvant treatment and follow-up is a major part of the management of childhood tumours and scanning frequency has important implications to anaesthetic and radiology service provision . In children with ependymoma and medulloblastoma , studies at GOSH have shown that SI reveals a substantial number of asymptomatic recurrences and survival appears to be improved in these patients compared with those patients in whom the recurrence is identified by symptoms -LSB- 12 , 13 -RSB- . Benefits of SI following incomplete resection of posterior fossa low-grade pilocytic astrocytoma have also been shown -LSB- 14 , 15 -RSB- . SI schedules have been written based on this work -LRB- Table 2 -RRB- . Table 2Proposed protocols for surveillance imaging in children with ependymoma , medulloblastoma and pilocytic astrocytomaTumourTiming of postoperative imagingFrequency of studyMacroscopically complete excisionIncomplete excisionCranial studySpinal studyCranial studySpinal studyEpendymoma24 -- 48 hFirstNoneFirstNone1st year3 -- 6 months6 months3 months3 -- 6 months2nd -- 5th years6 months6 -- 12 months6 months6 -- 12 monthsMedulloblastomaa24 -- 48 hFirstNoneFirstNone1st year3 -- 4 months3 -- 4 months3 -- 4 months3 -- 4 months2nd -- 6th year6 -- 8 months6 -- 8 months6 -- 8 months6 -- 8 monthsCerebellar low-grade astrocytomab24 -- 48 hFirstNoneFirstNone1st year6 monthsNone6 monthsNone2nd yearAt 24 monthsNone6 monthsNone3rd yearAt 3.5 yearsNone6 monthsNone4 -- 5th yearAt 5 yearsNone1 yearNone6th year onwardsNoneNone2 yearsNoneaAll imaging studies in children with medulloblastoma should include the entire neuroaxis.bThe surveillance imaging protocol following complete resection should be applied to children who have radiotherapy with the postoperative scan omitted Spine Imaging of spinal tumours is performed using our MRI spine with contrast enhancement protocol -LRB- Table 3 -RRB- with dedicated axial imaging through the tumour . We add a standard brain scan with contrast enhancement to the initial or immediate postoperative scan to stage the tumour . If the brain scan is normal , further brain imaging is not acquired as part of SI and acquired only when clinically indicated -LRB- Table 4 -RRB- . Table 3Protocols for specific areasSpecific areasProtocolsOrbits -LRB- 3-mm slices -RRB- Coronal and axial dual-echo STIR Coronal and axial T1-W spin-echoOrbits with contrast enhancement -LRB- 3-mm slices -RRB- Coronal dual-echo STIR Coronal and axial T1-W spin-echo Contrast-enhanced coronal and axial T1-W images with fat saturationPituitary -LRB- 3-mm slices -RRB- Sagittal and coronal T1-W spin-echo Coronal T2-W spin-echoPituitary with contrast enhancement -LRB- 3-mm slices -RRB- Pituitary protocol Contrast-enhanced coronal and sagittal T1-W spin-echoInternal auditory meati 3-D volume axial CISS Brain MRIFace and neck MRI Coronal and axial dual-echo STIR Coronal and axial T1-W spin-echo Fat-saturated contrast-enhanced coronal and axial T1-W spin-echoMidline facial lesions Axial dual-echo STIR from floor of anterior cranial fossa to hard palate Sagittal T1-W and T2-W spin-echo -LRB- 3-mm slices -RRB- Coronal T1-W spin-echo from nose to brainstemTable 4Standard MRI brain and spine protocolsTypes of brain and spine MRIProtocolsMRI brain -LRB- under 2 years old -RRB- Axial and coronal dual echo STIR Coronal and sagittal T1-W spin-echo DWI in three planes and calculated ADC map Axial `` susceptibility-weighted '' GE sequenceaMRI brain -LRB- over 2 years old -RRB- Axial T2-W fast spin-echo Coronal FLAIR Coronal and sagittal T1-W spin-echo DWI in three planes and calculated ADC map Axial `` susceptibility-weighted '' GE sequenceaMRI brain with contrast enhancement -LRB- under 2 years old -RRB- Axial and coronal dual-echo STIR Coronal T1-W spin-echo DWI in three planes and calculated ADC map Contrast-enhanced axial , coronal and sagittal T1-W spin-echo with magnetization transfer Axial `` susceptibility-weighted '' GE sequenceaMRI brain with contrast enhancement -LRB- over 2 years old -RRB- Axial T2-W fast spin-echo Coronal FLAIR Coronal T1-W spin-echo DWI in three planes and calculated ADC map Contrast-enhanced axial , coronal and sagittal T1-W spin-echo with magnetization transfer Axial `` susceptibility-weighted '' GE sequenceaMRI spine Sagittal T1-W and T2-W fast spin-echo Axial T1-W and T2-W fast spin-echo through target area and conus -LRB- Coronal T1-W spin-echo for scoliosis , if patient compliant -RRB- MRI spine with contrast enhancement Sagittal T1-W and T2-W fast spin-echo Contrast-enhanced sagittal T1-W fast spin-echo Axial T1-W spin-echo through target areaaOptional sequence Epilepsy UK guidelines for the imaging of epilepsy include children who have either multiple or focal seizures -LRB- NICE guidelines -RRB- -LSB- 16 -RSB- . In children with epilepsy we are aiming to detect focal cortical abnormalities ; the majority are extratemporal and in a smaller proportion they are mesial temporal in origin . The majority of cortical abnormalities are easily diagnosed using conventional brain imaging and our standard brain protocol is used to investigate these children . The acquisition of a coronal T2-W sequence -LRB- either STIR , T2-W or FLAIR -RRB- allows the visualization of the mesial temporal lobe , and is a sequence often not acquired in referring centres . There are a group of children with intractable epilepsy , i.e. seizures not controlled by three drugs , who are considered for epilepsy surgery . In this subset of children a more rigorous epilepsy protocol is carried out that includes a 3-D volume T1-W acquisition and hippocampal T2 relaxometry -LRB- Table 5 -RRB- . To identify mesial temporal abnormalities the coronal studies are planned on a parasagittal scout image to lie perpendicular to the hippocampi . The 3-D , T1-W gradient-echo dataset is acquired isometrically in the sagittal plane -LRB- this saves time -RRB- and reconstructed in all three planes with the axial and coronal images tilted to follow the plane of the hippocampus . More recently a 3-D FLAIR sequence reconstructed along the same lines has been added . A sequence is acquired that measures the true T2 values of the hippocampi -LRB- T2 relaxometry -RRB- , which is only of value in suspected mesial temporal sclerosis -LRB- MTS -RRB- . It has been shown that T2 values are more sensitive to the presence of MTS than visual inspection and T2 relaxometry is of particular value in bilateral disease -LSB- 17 -RSB- . Normal values will vary between scanners , so normative data should be acquired . The radiographers carry out the postprocessing of the 3-D dataset and the T2 relaxometry . Table 5Protocols for particular clinical indicationsTypes of clinical indicationProtocolsBrain tumours MRI brain with contrast enhancement Contrast-enhanced sagittal T1-W images of whole spine Contrast-enhanced image-guided images when requiredStrokeAcute Axial T2-W fast spin-echoa Coronal FLAIRb Sagittal T1-W spin-echoDWI in three planes and calculated ADC mapIntracerebral 3-D TOF MRAAxial dual-echo STIR and T1-W spin-echo through the neckExtracerebral 2-D TOF MRA of the neck down to the aortic rootNon-acuteAcute stroke protocol without imaging of the neckEpilepsy Axial T2-W fast spin-echoa 3-D T1-W volume acquisition reconstructed in three planes Coronal T2-W fast spin-echob Coronal FLAIR -LRB- or 3-D FLAIR if available -RRB- Hippocampal T2-relaxometry -LRB- see text -RRB- Intraparenchymal haemorrhage MRI brain with contrast enhancement Intracerebral 3-D TOF MRA MRVNon-accidental head injury Standard MRI brain Axial GE `` susceptibility-weighted '' sequence Sagittal T2-W spin-echo and GE `` susceptibility-weighted '' sequence of the cervical spineMPRAGE magnetization prepared rapid acquisition gradient echo , MRV magnetic resonance venographyaReplaced by dual echo STIR sequence in the under 2 age groupbReplaced by a dual echo STIR coronal in the under 2 year age group . Non-traumatic intraparenchymal haemorrhage The aim of imaging children with intraparenchymal haemorrhage -LRB- IPH -RRB- is to detect the underlying cause and we advocate the use of CT angiography as a matter of urgency in those children who have a depressed level of consciousness or whose clinical condition is deteriorating . MRI is used to investigate clinically stable children , and we perform brain MRI with contrast enhancement and a 3-D , time-of-flight -LRB- TOF -RRB- intracranial MR angiography -LRB- MRA -RRB- . In our experience nearly two-thirds of children presenting with nontraumatic IPH have an underlying cause diagnosed by MRI and MRA , with aetiologies including tumour , arteriovenous malformation -LRB- AVM -RRB- , venous sinus thrombosis and cavernous angioma -LSB- 18 -RSB- . An attempt is made to perform MRI within 72 h of the ictus to overcome the problem of the visibility of blood products -LRB- T1 shortening due to methaemoglobin -RRB- on 3-D TOF sequences that may obscure the underlying pathology . The value of intravenous contrast agents in identifying underlying vascular causes has not been proven . We have a largely anecdotal view that it is helpful in identifying small AVMs and venous anomalies . Stroke The aims of conventional MRI are not only to detect the infarct , but also to provide information to establish the cause of the stroke and to exclude other causes -LRB- such as tumour or infection -RRB- . The majority of children with stroke have a combination of risk factors including sickle cell disease , congenital heart disease , anaemia , prothrombotic disorders and infections such as varicella-zoster -LSB- 19 -RSB- . The rationale for the inclusion of intracranial MRA in all children with stroke is that cerebral arteriopathy is found in up to 80 % of these children and most commonly affects focal areas of large intracranial arteries -LSB- 19 -RSB- . Specific entities such as moyamoya disease may also be diagnosed . The commonest abnormality identified is occlusion or stenosis , of unknown aetiology , affecting the terminal internal carotid artery -LRB- ICA -RRB- or proximal middle cerebral artery -LRB- MCA -RRB- . Extracranial MRA and , either a dual-echo STIR or fat-saturated axial T1-W imaging , through the neck can detect arterial dissection , particularly in children with posterior circulation infarcts -LSB- 20 -RSB- . The inversion time of the STIR sequence is selected to suppress the fat within the neck . The fat saturation provided by both these sequences improves the conspicuity of the haematoma within the extracranial vessel wall . A 3-D TOF MRA sequence is used for the imaging of the intracranial vessels and a 2-D TOF MRA sequence for the extracranial vessels . The TOF scan times are shorter than phase-contrast -LRB- PC -RRB- MRA and there is lack of dependence on the choice of correct velocity encoding with obvious advantages when scanning ill children . Intracranial 3-D TOF MRA is also included in the investigation of children with IPH , although its sensitivity to T1 shortening may obscure the underlying abnormality -LRB- see above -RRB- . As CT is often the first-line investigation in children with stroke , the potential of DWI to detect hyperacute cerebral infarction prior to changes on T2-W MRI is not realized . However , inpatients -LRB- e.g. cardiac patients , patients with recent-onset stroke -RRB- can be imaged early , and in these children DWI can be used to detect infarcts of different ages . Non-accidental head injury Although CT is the primary imaging tool in the evaluation of nonaccidental head injury -LRB- NAHI -RRB- , we perform MRI in children with an abnormal CT or neurological symptoms and signs on day 3 -- 4 in accordance with the protocol proposed by Jaspan et al. -LSB- 21 -RSB- . The rationale for performing MRI is that it is more sensitive than CT to both hypoxic -- ischaemic injury , particularly when DWI is used , and subdural haematomas adjacent to the calvarium or in the posterior fossa . MRI is repeated at 2 -- 3 months in those children with parenchymal brain injury or a persistent neurological abnormality . Children with suspected NAHI are imaged using our standard brain protocol . In addition axial GE -LRB- susceptibility-weighted -RRB- sequences are acquired , as they are more sensitive to blood products than standard sequences . Sagittal T2-W and GE images are acquired of the cervical cord , including the brainstem , particularly to identify shear injuries within the brainstem that have been proposed as a mechanism of hypoxic -- ischaemic injury in children who have been shaken as the mechanism of injury -LSB- 22 , 23 -RSB- . The aim of the sagittal T2-W sequence of the cervical cord is to look for any evidence of trauma as a result of violent shaking in an attempt to establish the true , albeit rare , incidence of pathology in the upper cervical spine -LSB- 21 -RSB- . Neonatal imaging Conventional imaging can detect patterns of regional brain injury in the neonatal period that can help time the injury , determine underlying mechanisms and ultimately provide some prognostic information . MRI is useful in detecting hypoxic -- ischaemic injury , and germinal matrix and intraventricular haemorrhage , and can be useful to distinguish other pathologies that may mimic hypoxic -- ischaemic encephalopathy in the neonatal period such as venous infarction , metabolic disease , infection , and congenital developmental abnormalities . Our neonatal imaging protocol is the same as the under-2-year brain protocol . Achieving high signal-to-noise is of utmost importance given the small size of the infant head and is improved by decreasing the slice thickness to 4 mm whilst decreasing the matrix size from 512 × 512 to 256 × 256 in a field-of-view of 180 mm . Although specific neonatal head coils have been developed , most nonpaediatric centres are unlikely to have access to them and improved results can be obtained by using an adult knee coil . Developmental delay Developmental delay , without other clinical features such as epilepsy or dysmorphic features , is not considered an indication for an MRI scan at our institution . This decision was made in conjunction with our neurologists as it was recognized that there is an extremely low yield of clinically relevant abnormalities seen in patients with developmental delay alone . The future The future of paediatric neuroimaging lies in the incorporation of research techniques , such as perfusion imaging and diffusion tensor imaging , into standard imaging protocols once they have been demonstrated to be of value within the clinical arena . The use of higher field strength magnets will have the advantage of increasing signal-to-noise and reducing scanning time , which will be of benefit to the child who is both small and liable to move .

O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


A

genome

search

for

primary

vesicoureteral

reflux

shows

further

evidence

for

genetic

heterogeneity

Vesicoureteral

reflux

-LRB-

VUR

-RRB-

is

the

most

common

disease

of

the

urinary

tract

in

children

In

order

to

identify

gene

-LRB-

s

-RRB-

involved

in

this

complex

disorder

we

performed

a

genome-wide

search

in

a

selected

sample

of

patients

with

primary

VUR

from

eight

families

originating

from

southern

Italy

Sixteen

additional

families

with

patients

were

included

in

a

second

stage

Nonparametric

affected-only

linkage

analysis

identified

four

genomic

areas

on

chromosomes

and

-LRB-

p

-RRB-

the

best

result

corresponded

to

the

interval

on

chromosome

-LRB-

nonparametric

linkage

score

NPL

p

-RRB-

This

region

was

then

saturated

with

additional

markers

tested

in

the

complete

group

of

patients

from

families

-LRB-

NPL

p

-RRB-

We

identified

a

genomic

area

on

where

patients

from

six

multiplex

families

shared

overlapping

haplotypes

However

we

did

not

find

evidence

for

a

common

ancestral

haplotype

The

region

on

chromosome

was

delimited

to

-LRB-

max

NPL

p

-RRB-

after

additional

fine

typing

Furthermore

on

chromosome

patients

from

a

single

family

showed

excess

allele

sharing

-LRB-

NPL

p

-RRB-

Only

the

chromosome

region

has

been

previously

reported

in

the

single

genome-wide

screening

available

for

primary

VUR

Our

results

suggest

the

presence

of

several

novel

loci

for

primary

VUR

giving

further

evidence

for

the

genetic

heterogeneity

of

this

disorder

Introduction

Vesicoureteral

reflux

-LRB-

VUR

-RRB-

-LRB-

OMIM

-RRB-

is

the

most

common

disease

of

the

urinary

tract

in

children

and

affects

of

the

Caucasian

population

-LSB-

-RSB-

VUR

may

be

associated

with

both

acquired

postinfectious

and

congenital

parenchymal

damage

currently

known

as

reflux

nephropathy

-LRB-

RN

-RRB-

-LSB-

-RSB-

The

most

serious

consequence

of

RN

is

chronic

renal

insufficiency

-LRB-

CRI

-RRB-

leading

to

end-stage

renal

failure

-LRB-

ESRF

-RRB-

dialysis

and/or

renal

transplantation

of

children

affected

with

CRI

have

RN

-LSB-

-RSB-

As

a

consequence

the

impact

of

VUR

on

public

health

is

considerable

and

despite

medical

and

surgical

interventions

for

the

past

decades

the

incidence

of

VUR-related

renal

failure

has

not

decreased

-LSB-

-RSB-

The

reflux

may

occur

isolated

or

in

association

with

other

congenital

abnormalities

of

kidney/urinary

tract

-LRB-

CAKUT

-RRB-

or

as

part

of

syndromic

entities

such

as

renal-coloboma

or

branchio-oto-renal

syndromes

-LSB-

-RSB-

The

exact

etiology

of

primary

VUR

is

not

known

but

it

is

probably

related

to

an

abnormal

morphogenesis

of

the

ureteral

bud

leading

to

a

defect

of

the

ureterovesical

junction

-LSB-

-RSB-

The

initial

evidence

suggesting

a

genetic

origin

of

primary

VUR

came

from

twin

studies

showing

an

concordance

for

VUR

in

monozygotic

twins

vs.

a

concordance

in

dizygotic

twins

-LSB-

-RSB-

Subsequent

evidence

included

familial

clustering

of

VUR

-LSB-

-RSB-

ethnic

differences

between

affected

and

nonaffected

individuals

-LSB-

-RSB-

and

an

increased

risk

-LRB-

-RRB-

of

developing

VUR

in

first-degree

relatives

of

an

index

case

-LSB-

-RSB-

From

family

studies

a

range

of

inheritance

patterns

was

reported

including

autosomal

dominant

with

incomplete

penetrance

-LSB-

-RSB-

autosomal

recessive

-LSB-

-RSB-

polygenic

-LSB-

-RSB-

and

even

X-linked

-LSB-

-RSB-

Previous

studies

suggest

a

urinary

tract

malformation

locus

on

chromosome

-LSB-

-RSB-

Studies

of

humans

with

chromosomal

abnormalities

also

suggest

candidate

loci

or

genes

on

chromosomes

-LSB-

-RSB-

-LRB-

gene

-RRB-

-LSB-

-RSB-

and

-LSB-

-RSB-

Because

mutations

in

on

cause

renal-coloboma

syndrome

a

rare

autosomal

dominant

disease

with

kidney

anomalies

that

include

VUR

this

gene

was

also

proposed

as

a

candidate

-LSB-

-RSB-

However

of

these

loci

or

genes

has

been

shown

causally

related

to

primary

VUR

-LSB-

-RSB-

Recently

Lu

et

al.

-LSB-

-RSB-

showed

that

mutations

in

the

gene

contribute

to

the

pathogenesis

of

VUR/CAKUT

in

a

small

proportion

of

families

In

the

only

genome-wide

linkage

study

reported

to

date

Feather

et

al.

-LSB-

-RSB-

demonstrated

linkage

to

chromosome

for

primary

VUR

under

a

model

of

autosomal

dominant

inheritance

with

reduced

penetrance

Here

we

describe

the

results

of

the

second

genome-wide

scan

for

primary

VUR

Differently

from

previous

studies

and

aiming

to

collect

a

homogeneous

sample

set

our

patients

were

ascertained

in

a

single

geographic

region

Our

results

suggest

the

presence

of

several

novel

loci

for

primary

VUR

giving

further

evidence

for

the

genetic

heterogeneity

of

this

disorder

Methods

Patients

and

families

Fifty-one

pedigrees

with

multiple

patients

with

VUR

coming

from

Campania

-LRB-

southern

Italy

-RRB-

were

enrolled

in

the

study

-LRB-

Fig.

-RRB-

All

families

were

ascertained

through

an

index

case

with

VUR

documented

by

voiding

cystourethrography

-LRB-

VCUG

-RRB-

in

males

and

direct

radionuclide

cystography

-LRB-

RNC

-RRB-

in

females

and

family

members

Three

pediatric

nephrologists

and

one

radiologist

assessed

the

patients

RN

was

diagnosed

by

DMSA

scintigraphy

-LRB-

dimercaptosuccinic

acid

labeled

with

m

-RRB-

and

defined

as

focal

defects

of

radionuclide

uptake

and/or

by

one-kidney

differential

uptake

below

-LSB-

-RSB-

VUR

grading

was

made

according

to

the

International

Grading

System

of

Vesicoureteral

Reflux

-LSB-

-RSB-

Fig.

and

distribution

of

families

and

patients

included

in

the

study

VUR

vesicoureteral

reflux

Additional

family

members

were

considered

as

affected

based

on

the

presence

of

reflux

documented

by

VCUG/RNC

and/or

the

diagnosis

of

RN

or

the

detection

of

ESRF/renal

replacement

in

absence

of

other

known

causes

As

VUR

may

spontaneously

disappear

during

childhood

and

adolescence

-LSB-

-RSB-

the

finding

of

scintigraphic

signs

of

RN

in

relatives

of

VUR

patients

strongly

suggests

the

previous

occurrence

of

reflux

-LSB-

-RSB-

Individuals

with

renal

symptoms

indicative

of

VUR

such

as

previous

urinary

tract

infections

and/or

hypertension

and/or

proteinuria

not

supported

by

additional

findings

were

classified

as

diagnosis

unknown

Patients

with

secondary

VUR

i.e.

neurogenic

bladder

and

posterior

urethral

valves

or

other

urinary

tract

abnormalities

i.e.

ureterocele

and

obstructive

hydronephrosis

were

excluded

The

study

focused

on

primary

familial

VUR

Eight

families

with

patients

with

VUR

were

selected

for

study

phase

-LRB-

genome

scan

-RRB-

according

to

the

following

criteria

diagnosis

of

primary

VUR

in

absence

of

any

other

malformation

two

or

more

affected

individuals

per

family

and

a

pattern

of

inheritance

compatible

with

an

autosomal

dominant

model

The

second

sample

-LRB-

follow-up

-RRB-

consisted

of

five

affected

relative

pairs

-LRB-

parent

child

trios

ten

patients

-RRB-

and

small

families

-LRB-

patients

-RRB-

fulfilling

the

same

criteria

-LRB-

Fig.

-RRB-

Informed

consent

from

patients

and

family

members

-LRB-

parents

for

their

children

-RRB-

and

approval

from

the

Ethic

Committee

at

Second

University

of

Naples

were

obtained

previously

Laboratory

analysis

Genomic

DNA

was

isolated

from

peripheral

blood

leukocytes

by

standard

techniques

and

was

sent

from

the

Paediatrics

Department

of

Second

University

of

Naples

to

the

Department

of

Clinical

Genetics

Erasmus

Medical

Centre

in

Rotterdam

A

systematic

genome

scan

was

performed

using

the

ABI

Prism

set

-LRB-

Applied

Biosystems

-RRB-

consisting

of

short-tandem-repeat

polymorphisms

markers

-LRB-

STRPs

-RRB-

average

spaced

cM

Additional

markers

for

further

characterization

of

candidate

regions

were

selected

from

the

gender-average

Marshfield

genetic

map

Information

about

marker

order

and

distances

were

obtained

from

the

National

Center

for

Biotechnology

Information

-LRB-

NCBI

-RRB-

physical

map

and

Marshfield

integrated

genetic

map

Polymerase

chain

reaction

-LRB-

PCR

-RRB-

products

were

resolved

on

an

automated

sequencer

and

genotypes

were

analyzed

using

the

GeneMapper

software

v.

-LRB-

Applied

Biosystems

-RRB-

Linkage

analysis

One

thousand

simulations

were

performed

-LRB-

SLINK

MSIM

-RRB-

-LSB-

-RSB-

to

investigate

the

statistical

power

of

our

sample

set

An

autosomal

dominant

mode

of

inheritance

was

assumed

with

a

penetrance

of

a

mutant

allele

frequency

of

and

a

phenocopy

rate

of

The

calculations

were

done

under

the

assumption

of

genetic

heterogeneity

Assuming

of

unlinked

families

a

maximum

total

log

of

odds

-LRB-

LOD

-RRB-

score

of

was

obtained

with

a

low

average

LOD

of

-LSB-

standard

deviation

-LRB-

SD

-RRB-

-RSB-

after

analyzing

all

eight

pedigrees

selected

previously

Increasing

the

percentage

of

unlinked

families

to

a

maximum

LOD

score

of

was

observed

-LRB-

average

SD

-RRB-

Families

and

had

the

largest

contribution

to

the

total

LOD

score

-LRB-

LOD

and

respectively

-RRB-

followed

by

families

and

-LRB-

LOD

-RRB-

Due

to

their

small

size

families

and

had

less

contribution

to

the

final

LOD

-LRB-

LOD

-RRB-

Simulations

were

calculated

in

the

replication

group

-LRB-

families

-RRB-

after

exclusion

of

five

parent

child

trios

giving

a

maximum

LOD

of

-LRB-

average

SD

-RRB-

and

a

maximum

LOD

of

-LRB-

average

SD

-RRB-

under

assumption

of

and

unlinked

families

respectively

Family

with

seven

patients

was

the

main

contributor

to

the

total

LOD

score

-LRB-

LOD

-RRB-

Finally

simulations

were

performed

in

all

families

-LRB-

genome

scan

group

and

replication

group

-RRB-

reaching

a

maximum

LOD

of

-LRB-

average

of

SD

-RRB-

and

a

maximum

LOD

of

-LRB-

average

of

SD

-RRB-

under

assumption

of

and

unlinked

families

respectively

-LSB-

-RSB-

was

used

to

process

the

genetic

data

whereas

the

accuracy

of

allele

segregation

within

the

families

was

confirmed

with

Pedcheck

The

program

GENEHUNTER

v.

-LSB-

-RSB-

was

used

to

compute

multipoint

parametric

and

nonparametric

-LRB-

or

model-free

-RRB-

linkage

analysis

As

VUR

may

often

disappear

with

age

all

individuals

in

whom

VUR

RN

or

both

were

not

clinically

proven

were

classified

-LRB-

and

analyzed

-RRB-

as

diagnosis

unknown

Thus

a

conservative

affected-only

analysis

was

performed

based

on

these

criteria

Due

to

the

uncertain

pattern

of

inheritance

nonparametric

analysis

was

computed

The

rationale

of

a

nonparametric

analysis

is

that

among

affected

relatives

excess

sharing

of

haplotypes

identical

by

descent

would

be

expected

irrespective

of

the

mode

of

inheritance

The

nonparametric

linkage

statistics

examining

all

individuals

simultaneously

-LRB-

NPL-all

-RRB-

is

reported

A

parametric

analysis

was

performed

as

well

An

autosomal

dominant

mode

of

inheritance

with

reduced

penetrance

as

described

above

-LRB-

power

calculations

-RRB-

was

used

Marker

allele

frequencies

were

calculated

using

all

spouses

-LRB-

unrelated

individuals

-RRB-

coming

from

the

same

geographic

area

Due

to

the

genetic

heterogeneity

of

the

disease

the

genome

scan

data

was

analyzed

maximizing

the

heterogeneity

LOD

score

-LRB-

HLOD

-RRB-

with

respect

to

the

proportion

of

linked

families

-LRB-

α

-RRB-

Nonparametric

-LRB-

NPL

-RRB-

and

parametric

-LRB-

LOD

-RRB-

scores

were

calculated

for

each

of

the

families

and

then

total

NPL

and

HLOD

scores

were

obtained

Results

Patients

Fifty-one

pedigrees

with

multiple

patients

with

VUR

originating

from

the

same

region

of

southern

Italy

-LRB-

Campania

-RRB-

were

available

for

the

study

All

persons

in

the

study

were

ascertained

through

an

index

case

documented

by

VCUG/RNC

A

total

of

patients

were

detected

According

to

the

phenotype

three

groups

of

families

were

identified

primary

VUR

VUR

associated

with

additional

abnormalities

of

kidney/urinary

tract

such

as

duplicated

collecting

system

renal

agenesis

or

hypospadia

and

VUR

occurring

in

syndromes

such

as

renal-coloboma

branchio-oto-renal

syndromes

and

reflux

associated

with

congenital

ichthyosis

-LRB-

Fig.

-RRB-

A

total

of

primary

VUR

patients

belonging

to

pedigrees

were

identified

-LRB-

based

on

a

positive

VCUG/RNC

based

on

detection

of

RN

diagnosed

as

having

both

reflux

and

RN

and

six

with

ESRF

-RRB-

During

the

course

of

the

study

a

new

patient

affected

with

RN

was

detected

in

family

and

included

in

the

second

stage

Twenty-eight

patients

were

males

and

were

females

-LRB-

ratio

M/F

-RRB-

The

median

age

at

diagnosis

was

years

-LRB-

range

month

years

-RRB-

Among

the

patients

were

treated

surgically

and

four

out

of

six

who

developed

ESRF

underwent

renal

transplantation

The

most

informative

pedigrees

are

shown

in

Fig.

b

The

pedigrees

showed

patients

in

several

generations

and

male-to-male

transmission

was

observed

in

some

families

supporting

an

autosomal

dominant

mode

of

inheritance

As

reported

before

for

primary

VUR

we

observed

several

obligate

carriers

in

our

families

i.e.

families

and

Family

is

especially

interesting

when

assessing

the

parents

-LRB-

asymptomatic

-RRB-

of

the

four

symptomatic

children

both

individuals

were

found

to

have

RN

No

consanguinity

was

reported

but

history

of

ESRF

in

both

branches

was

described

We

are

currently

recruiting

clinical

information

of

those

patients

-LRB-

family

second

generation

Fig.

-RRB-

Due

to

this

bilinear

transmission

this

family

was

excluded

from

the

linkage

analysis

Fig.

selection

of

pedigrees

enrolled

in

the

study

is

shown

Squares

indicate

males

and

circles

indicate

females

Family

members

with

unknown

phenotype

are

in

grey

whereas

those

unaffected

-LRB-

normal

voiding

cystourethrography

or

direct

radionuclide

cystography

before

age

of

years

and

unrelated

spouses

-RRB-

are

in

white

An

asterisk

highlights

the

individuals

genotyped

in

the

study

A

number

following

the

asterisk

indicates

the

patients/obligate

carriers

reported

in

Fig.

Individuals

with

urinary

tract

infections

by

history

and

obligate

carriers

are

considered

as

unknown

in

all

analyses

a

Nine

multiplex

pedigrees

included

in

the

genome-wide

scan

are

shown

An

additional

five

parent

child

trios

are

not

displayed

Family

was

excluded

from

the

linkage

analysis

due

to

the

bilineal

inheritance

b

All

multiplex

pedigrees

included

in

the

fine-typing

stage

are

shown

VUR

vesicoureteral

reflux

RN

reflux

nephropathy

ESRF

end-stage

renal

failure

UTIs

urinary

tract

infections

Genome

search

We

performed

a

systematic

genome-wide

scan

using

microsatellite

markers

Results

from

both

parametric

and

nonparametric

linkage

analysis

excluded

most

of

the

genome

-LRB-

data

not

shown

-RRB-

Furthermore

negative

scores

were

found

on

both

VUR

loci

-LSB-

-RSB-

-LRB-

NPL

p

-RRB-

and

-LSB-

-RSB-

-LRB-

NPL

p

-RRB-

previously

reported

We

used

a

nominal

p

value

of

to

decide

whether

a

region

was

promising

for

further

study

Table

summarizes

the

genome-wide

scan

results

showing

all

regions

that

yielded

a

total

NPL

corresponding

to

p

These

regions

were

located

on

chromosome

-LRB-

-RRB-

chromosome

-LRB-

-RRB-

and

chromosome

-LRB-

-RRB-

Furthermore

on

chromosome

patients

from

a

single

family

showed

excess

allele

sharing

-LRB-

-RRB-

The

best

evidence

of

linkage

was

observed

on

chromosome

which

yielded

the

highest

NPL

score

-LRB-

p

HLOD

α

-RRB-

and

on

chromosome

-LRB-

NPL

p

HLOD

α

-RRB-

Recently

mutations

in

were

described

in

two

VUR/CAKUT

patients

-LSB-

-RSB-

which

is

an

ideal

functional

candidate

gene

for

VUR/CAKUT

maps

on

chromosome

at

the

border

of

our

region

Thus

we

retrospectively

sequenced

all

gene

exons

in

our

index

patients

and

no

mutations

were

found

Table

of

the

genome-wide

scan

results

Total

nonparametric

linkage

-LRB-

NPL

-RRB-

scores

with

p

values

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Total

NPL

-LRB-

p

-RRB-

-LRB-

p

-RRB-

-LRB-

p

-RRB-

-LRB-

p

-RRB-

-LRB-

p

-RRB-

Total

HLOD

-LRB-

α

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

NPL

and

-LRB-

LOD

-RRB-

scores

per

family

are

shown

at

each

locus

Total

NPL

-LRB-

p

value

-RRB-

and

HLOD

-LRB-

α

-RRB-

obtained

from

the

analysis

of

all

eight

pedigrees

are

displayed

HLOD

was

obtained

maximizing

the

heterogeneity

LOD

score

with

respect

to

the

proportion

of

linked

families

-LRB-

α

-RRB-

Negative

NPL

and

LOD

scores

indicate

no

linkage

or

inconclusive

resultNPL

nonparametric

LOD

score

HLOD

heterogeneity

LOD

score

α

proportion

of

linked

families

The

NPL

score

of

-LRB-

p

-RRB-

on

chromosome

was

mainly

due

to

family

-LRB-

NPL

p

LOD

-RRB-

Yet

allele

sharing

among

the

patients

of

this

family

was

observed

also

on

chromosome

-LRB-

NPL

p

LOD

-RRB-

The

only

region

in

which

patients

from

family

-LRB-

Fig.

-RRB-

showed

excess

allele

sharing

was

on

chromosome

-LRB-

NPL

p

LOD

-RRB-

We

then

closely

inspected

the

haplotypes

from

the

positive

regions

in

all

genotyped

families

Families

and

displayed

haplotype

sharing

among

each

family

's

patients

on

chromosome

supporting

the

linkage

to

this

area

Thus

in

six

out

of

eight

informative

families

we

observed

allele/haplotype

sharing

in

an

overlapping

area

on

chromosome

We

noted

also

on

chromosome

haplotype

sharing

in

four

out

of

eight

multiplex

families

-LRB-

families

and

-RRB-

extending

from

until

Therefore

we

decided

to

explore

these

genomic

areas

Refinement

of

the

chromosome

locus

The

chromosome

region

between

and

was

saturated

with

additional

microsatellites

markers

-LRB-

average

spaced

approximately

cM

-RRB-

A

maximum

HLOD

-LRB-

α

-RRB-

was

reached

with

an

NPL

p

In

a

second

stage

the

fine

mapping

was

extended

with

additional

families

with

patients

The

expansion

of

pedigrees

introduced

additional

unlinked

families

which

lowered

the

maximum

HLOD

to

-LRB-

α

-RRB-

giving

a

total

NPL

-LRB-

p

-RRB-

-LRB-

Table

-RRB-

Pedigree

was

the

main

contributor

to

the

total

score

with

an

NPL

p

-LRB-

LOD

-RRB-

We

then

performed

a

detailed

haplotype

analysis

in

all

families

Patients

from

families

and

shared

a

common

region

delimited

by

markers

and

containing

genes

according

with

the

NCBI

map

built

Instead

families

and

were

sharing

a

more

centromeric

area

not

overlapping

with

the

other

families

-LRB-

Fig.

-RRB-

We

did

not

observe

a

common

ancestral

haplotype

shared

across

the

families

Fig.

analysis

in

families

supporting

the

chromosome

region

after

fine

typing

A

selection

of

microsatellite

markers

is

shown

The

shared

genomic

region

is

shown

in

grey

recombinants

are

shown

in

white

blocksTable

of

fine-typing

results

in

chromosome

and

-LRB-

total

NPL

scores

with

p

values

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Total

NPL

-LRB-

p

-RRB-

-LRB-

p

-RRB-

-LRB-

p

-RRB-

Total

HLOD

-LRB-

α

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

NPL

and

-LRB-

LOD

-RRB-

scores

per

family

are

shown

at

each

locus

Total

NPL

-LRB-

p

value

-RRB-

and

HLOD

-LRB-

α

-RRB-

obtained

from

the

analysis

of

all

eight

pedigrees

are

displayed

HLOD

was

obtained

maximizing

the

heterogeneity

LOD

score

with

respect

to

the

proportion

of

linked

families

-LRB-

α

-RRB-

Negative

NPL

and

LOD

scores

indicate

no

linkage

or

inconclusive

resultNPL

nonparametric

LOD

score

HLOD

heterogeneity

LOD

score

α

proportion

of

linked

families

Refinement

of

chromosome

locus

The

region

between

-LRB-

cM

-RRB-

and

-LRB-

cM

-RRB-

was

saturated

with

seven

extra

microsatellite

markers

-LRB-

total

spaced

cM

-RRB-

in

the

complete

group

of

families

-LRB-

Table

-RRB-

We

observed

a

maximum

HLOD

of

-LRB-

α

-RRB-

and

NPL

of

-LRB-

p

-RRB-

The

haplotype

analysis

showed

allele

sharing

in

families

and

between

-LRB-

cM

-RRB-

and

-LRB-

cM

-RRB-

Patients

from

families

and

displayed

haplotype

sharing

but

it

was

limited

to

the

upper

or

lower

part

of

the

region

thus

showing

no

overlapping

with

the

rest

of

the

families

Candidate

genes

There

were

several

interesting

genes

map

on

the

chromosome

region

The

gene

encodes

the

protein

phosphatase

one

of

four

major

protein

phosphatases

identified

in

eukaryotic

cells

implicated

in

the

regulation

of

most

major

metabolic

pathways

It

has

been

shown

to

be

expressed

ubiquitously

in

kidneys

regulating

the

mitogenic

activity

in

the

early

embryonic

kidney

-LSB-

-RSB-

The

gene

encodes

a

type

of

fibronectin

expressed

mainly

in

fetal

kidney

-LSB-

-RSB-

implicated

in

the

development

of

renal

basement

membrane

-LSB-

-RSB-

and

genes

-LRB-

Retinol-binding-proteins

type

and

-RRB-

are

involved

in

the

metabolism

of

vitamin

A

which

in

its

active

form

of

retinoic

acid

plays

a

critical

role

during

kidney

development

even

connecting

ureters

to

bladder

-LSB-

-RSB-

The

gene

encodes

the

angiotensin

receptor

II

type

-LRB-

-RRB-

shown

to

be

related

to

gross

abnormalities

in

renal

morphogenesis

in

mutant

mice

-LRB-

hydronephrosis

-RRB-

as

well

as

a

poor

proliferation

of

ureteral

smooth

cells

-LSB-

-RSB-

Discussion

This

study

reports

the

second

genome-wide

search

for

primary

VUR

Our

results

suggest

the

existence

of

several

loci

mapping

to

chromosomes

and

further

supporting

the

hypothesis

that

primary

VUR

is

genetically

heterogeneous

We

as

others

encountered

several

pitfalls

when

studying

VUR

We

observed

clinical

variability

among

and

within

families

and

the

presence

of

obligate

carriers

-LRB-

individuals

who

carry

and

transmit

the

disease

allele

but

do

not

manifest

any

disease

sign

or

symptom

-RRB-

Although

the

appropriate

clinical

investigations

were

performed

in

several

apparently

healthy

individuals

-LRB-

carriers

-RRB-

no

evidence

of

disease

was

found

indicating

a

reduced

or

age-dependent

penetrance

As

most

of

these

individuals

were

recruited

during

their

adulthood

we

could

not

exclude

an

earlier

disease

condition

that

evolved

to

a

spontaneous

resolution

To

overcome

these

problems

we

first

performed

a

careful

clinical

evaluation

of

patients

and

available

relatives

All

individuals

older

than

years

of

age

with

insufficient

or

no

evidence

of

VUR

were

classified

as

diagnosis

unknown

despite

the

consequent

loss

of

power

for

the

statistical

analysis

We

performed

the

first

genome

search

in

VUR

that

includes

families

with

the

same

ethnic

origin

In

order

to

strengthen

the

genetic

homogeneity

of

the

patients

all

families

included

in

this

study

originated

from

the

same

geographic

area

in

the

southern

part

of

Italy

Yet

our

results

strongly

support

that

primary

VUR

is

a

-LRB-

highly

-RRB-

genetic

heterogeneous

condition

Whereas

a

large

number

of

our

patients

and

families

supported

the

chromosome

locus

one

relatively

large

pedigree

-LRB-

family

-RRB-

showed

evidence

of

linkage

on

chromosome

Six

of

our

families

supported

a

locus

on

chromosome

-LRB-

cM

-RRB-

This

region

is

fully

overlapping

with

one

of

the

loci

reported

by

Feather

et

al.

-LSB-

-RSB-

In

their

study

the

interval

on

chromosome

-LRB-

from

to

cM

-RRB-

showed

a

high

α

-LRB-

-RRB-

with

an

NPL

p

This

region

was

supported

by

one

of

their

largest

pedigrees

-LRB-

with

seven

patients

-RRB-

that

was

clearly

not

linked

to

the

chromosome

locus

It

is

interesting

that

two

independent

studies

have

found

the

same

genomic

region

in

distinct

groups

of

patients

Besides

the

number

of

patients

included

in

our

genome

scan

the

results

of

the

statistical

analysis

are

quite

modest

and

do

not

reach

the

criteria

suggested

by

Lander

and

Kruglyak

-LSB-

-RSB-

to

declare

significant

linkage

Therefore

some

of

the

identified

loci

may

represent

false

positive

hits

and

should

be

interpreted

with

caution

The

complex

disease

etiology

of

primary

VUR

has

shown

to

be

difficult

to

disentangle

Genetic

heterogeneity

and

lack

of

knowledge

of

the

true

genetic

model

for

VUR

are

probably

the

main

difficulties

in

the

identification

of

the

genetic

etiology

of

VUR

Although

genetic

studies

in

VUR

are

still

in

an

early

phase

we

can

presume

that

primary

VUR

is

likely

a

complex

disorder

with

a

number

of

not

fully

penetrant

genes

causing

most

of

the

familiar

cases

Finally

primary

VUR

could

be

caused

by

simultaneous

gene-environment

interactions

In

conclusion

our

results

show

further

evidence

for

the

genetic

heterogeneity

in

primary

VUR

We

will

next

focus

on

the

refinement

of

the

identified

genomic

regions

and

the

sequence

analysis

of

the

candidate

genes

according

to

their

tissutal

expression

and

biological

function

Replication

of

the

results

in

additional

families

will

be

essential

first

to

confirm

and

eventually

to

evaluate

the

contribution

of

these

loci

to

the

pathogenesis

of

primary

nonsyndromic

VUR

O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O

primary familial vesicoureteral reflux
genetic linkage