Judy Savige

Array-Based Gene Discovery with Three Unrelated Subjects Shows SCARB2/LIMP-2 Deficiency Causes Myoclonus Epilepsy and Glomerulosclerosis

Action myoclonus-renal failure syndrome (AMRF) is an autosomal-recessive disorder with the remarkable combination of focal glomerulosclerosis, frequently with glomerular collapse, and progressive myoclonus epilepsy associated with storage material in the brain. Here, we employed a novel combination of molecular strategies to find the responsible gene and show its effects in an animal model. Utilizing only three unrelated affected individuals and their relatives, we used homozygosity mapping with single-nucleotide polymorphism chips to localize AMRF. We then used microarray-expression analysis to prioritize candidates prior to sequencing. The disorder was mapped to 4q13-21, and microarray-expression analysis identified SCARB2/Limp2, which encodes a lysosomal-membrane protein, as the likely candidate. Mutations in SCARB2/Limp2 were found in all three families used for mapping and subsequently confirmed in two other unrelated AMRF families. The mutations were associated with lack of SCARB2 protein. Reanalysis of an existing Limp2 knockout mouse showed intracellular inclusions in cerebral and cerebellar cortex, and the kidneys showed subtle glomerular changes. This study highlights that recessive genes can be identified with a very small number of subjects. The ancestral lysosomal-membrane protein SCARB2/LIMP-2 is responsible for AMRF. The heterogeneous pathology in the kidney and brain suggests that SCARB2/Limp2 has pleiotropic effects that may be relevant to understanding the pathogenesis of other forms of glomerulosclerosis or collapse and myoclonic epilepsies.

A review of the ocular manifestations

Alport syndrome has a prevalence of 1/5000, and 85% of patients have the X-linked form, where affected males develop renal failure and usually have a high-tone sensorineural deafness by the age of 20. The typical ocular associations are a dot-and-fleck retinopathy which occurs in about 85% of affected adult males, anterior lenticonus which occurs in about 25%, and the rare posterior polymorphous corneal dystrophy. The retinopathy and anterior lenticonus are not usually demonstrated in childhood but worsen with time so that the retinal lesion is often present at the onset of renal failure, and the anterior lenticonus, later. The demonstration of a dot-and-fleck retinopathy in any individual with a family history of Alport syndrome or with end-stage renal disease is diagnostic of Alport syndrome. The presence of anterior lenticonus or posterior polymorphous corneal dystrophy in any individual is highly suggestive of the diagnosis of Alport syndrome. Additional ocular features described in X-linked Alport sy...

Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies Quality Control Guidelines, Comments, and Recommendations for Testing in Other Autoimmune Diseases

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in Wegener granulomatosis, microscopic polyangiitis and its renal-limited variant (pauci-immune crescentic glomerulonephritis), and Churg-Strauss syndrome. The International Consensus Statement on testing and reporting of ANCA states that ANCA are demonstrated most readily in these conditions by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 or myeloperoxidase. The group that produced the International Consensus Statement has developed guidelines for the corresponding quality control activities, examples of comments for various IIF patterns and ELISA results, and recommendations for ANCA testing when inflammatory bowel disease and other nonvasculitic ANCA-associated autoimmune diseases are suspected.

Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in Wegener granulomatosis, microscopic polyangiitis and its renal-limited variant (pauciimmune crescentic glomerulonephritis), and Churg-Strauss syndrome. The International Consensus Statement on testing and reporting of ANCA states that ANCA are demonstrated most readily in these conditions by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 or myeloperoxidase. The group that produced the International Consensus Statement has developed guidelines for the corresponding quality control activities, examples of comments for various IIF patterns and ELISA results, and recommendations for ANCA testing when inflammatory bowel disease and other nonvasculitic ANCA-associated autoimmune diseases are suspected.

Expert Guidelines for the Management of Alport Syndrome and Thin Basement Membrane Nephropathy

Few prospective, randomized controlled clinical trials address the diagnosis and management of patients with Alport syndrome or thin basement membrane nephropathy. Adult and pediatric nephrologists and geneticists from four continents whose clinical practice focuses on these conditions have developed the following guidelines. The 18 recommendations are based on Level D (Expert opinion without explicit critical appraisal, or based on physiology, bench research, or first principles-National Health Service category) or Level III (Opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees-U.S. Preventive Services Task Force) evidence. The recommendations include the use of genetic testing as the gold standard for the diagnosis of Alport syndrome and the demonstration of its mode of inheritance; the need to identify and follow all affected members of a family with X-linked Alport syndrome, including most mothers of affected males; the treatment of males with X-linked Alport syndrome and individuals with autosomal recessive disease with renin-angiotensin system blockade, possibly even before the onset of proteinuria; discouraging the affected mothers of males with X-linked Alport syndrome from renal donation because of their own risk of kidney failure; and consideration of genetic testing to exclude X-linked Alport syndrome in some individuals with thin basement membrane nephropathy. The authors recognize that as evidence emerges, including data from patient registries, these guidelines will evolve further.

α1-Antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis

α1‐antitrypsin (α1‐AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti‐neutrophil cytoplasmic antibodies (ANCA). An increased incidence of α1‐AT phenotypes associated with dysfunctional α1‐AT or low serum levels has been reported in patients with anti‐PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of α1‐AT. Phenotypes usually associated with a moderate or severe reduction in α1‐AT serum levels or in dysfunctional activity were found more often in individuals with anti‐PR3 antibodies than in the general population: four of the 31 patients (13%) with anti‐PR3 antibodies had phenotypes MZ (n= 2), S (n= 1) or Z (n= 1) (P < 0·05). However, the corresponding α1‐AT serum levels were normal (n= 3) or elevated (n= 1). None of the 31 sera with anti‐PR3 antibodies had low levels of α1‐AT. No abnormal α1‐AT phenotype was demonstrated in seven patients with anti‐elastase antibodies, despite a low level of α1‐AT in one serum. Anti‐myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum oi‐AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti‐glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA‐associated diseases, but again none of 10 sera had an abnormal α1‐AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal α1‐AT phenotypes. These results confirm that patients with anti‐PR3 antibodies often have α1‐AT phenotypes that are usually associated with low serum levels of α1‐AT or with dysfunctional protein. Nevertheless, the incidence of anti‐PR3 antibodies in patients with abnormal α1‐AT phenotypes is very low. This probably reflects the rarity of Wegeners granulomatosis, the major disease associated with anti‐PR3 antibodies, and the relative frequency of abnormal α1‐AT phenotypes. The mechanism for the development of anti‐PR3 antibodies in patients with abnormal α1‐AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.

Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.

Collagen IV is the major protein found in basement membranes. It comprises three heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (“interactomes”) that indicated major structural landmarks, known and predicted ligand‐binding sites, and missense mutations, in order to identify functional and disease‐associated domains, potential interactions between ligands, and genotype–phenotype relationships. The maps documented more than 30 known ligand‐binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin, and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection, and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases, and heparin. The maps indicated that mutations affecting major ligand‐binding sites, for example, for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms, and muscle Cramps [HANAC] syndrome, and early‐onset Alport syndrome, respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. Hum Mutat 32:127–143, 2011.

Anti-neutrophil cytoplasm antibodies in rheumatoid arthritis.

Anti‐neutrophil cytoplasm antibodies (ANCA) occur occasionally in rheumatoid arthritis (RA), but their incidence and clinical significance have been unclear. In this study we have investigated 58 patients with RA. In 22 patients the disease was inactive and the remaining 36 with active disease were further subdivided into those without clinical evidence of vasculitis (26), those with cutaneous vasculitis (8) and those with systemic vasculitis (2). ANCA were demonstrated by indirect immunofluorescence in 10 of the 58 patients (17%), While both pcrinuclear (pANCA) and cytoplasmic (cANCA) staining were detected, pANCA were more common (70%). Neutrophil‐specific anti‐nuclear antibodies (ANNA) were demonstrated in a further eight sera (14%) and ANA were detected on Hep‐2 cells in 30 of the 58 sera (52%). ELISAs for the detection of anti‐myeloperoxidase and anti‐elastase antibodies were then established. Five sera with pANCA and five that contained ANNA were negative for both anti‐myeloperoxidase and anti‐elastase antibodies, suggesting other as yet unidentified cytoplasmic antigens as the target molecules. However, anti‐myeloperoxidase or anti‐elastase antibodies were found in four sera that had homogeneous or speckled ANA on both Hep‐2 cells and neutrophils. One serum contained both antibodies. The presence of ANCA detected by indirect immunofluorescence or of anti‐myeloperoxidase or anti‐elastase antibodies in these patients with RA was not associated with disease activity nor with the demonstration of cutaneous vasculitis or renal disease (P NS). A possible association with systemic vasculitis remains to be confirmed. There is an incomplete correlation between indirect immunofluorescence patterns and antibody specificity in ELISA systems.

Ocular manifestations of autosomal recessive Alport syndrome

Ocular abnormalities are common in X-linked Alport syndrome, but they have not been studied in patients with the rarer autosomal recessive disease. We have examined the eyes of a family with autosomal recessive Alport syndrome. Four of the eight offspring of a consanguineous marriage had renal failure and deafness by the age of 20 years. The diagnosis of Alport syndrome was confirmed on the ultrastructural demonstration of a lamellated glomerular basement membrane (GBM) in one affected family member. Autosomal recessive inheritance was suggested by the lack of linkage to the COL4A5/COL4A6 locus, and by linkage to the COL4A3/COL4A4 locus. All four affected family members had anterior lenticonus (or had had a lens replacement for this) and the three who were examined had a dot-and-fleck retinopathy. Neither of the two unaffected offspring who were examined nor the father had these abnormalities. The ocular manifestations of autosomal recessive Alport syndrome are probably identical to those for the X-linked form. Although the mutations in these diseases affect genes for different type IV collagen chains, these chains occur together in the basement membranes of the kidney, eye and ear, and abnormalities in any one may result in the same clinical phenotype.

COL4A3/COL4A4 Mutations and Features in Individuals with Autosomal Recessive Alport Syndrome

Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6-54 years). The median age at end stage renal failure was 22.5 years (range, 10-38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome.