Gian Marco Ghiggeri

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions (?Döhle-like? bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8?10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.The autosomal dominant, giant-platelet disorders1, May-Hegglin anomaly2,3 (MHA; MIM 155100), Fechtner syndrome4 (FTNS; MIM 153640) and Sebastian syndrome5 (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions (?Döhle-like? bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis4. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 810), which is expressed in platelets9 and upregulated during granulocyte differentiation10. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Genome-wide association study identifies susceptibility loci for IgA nephropathy

We carried out a genome-wide association study of IgA nephropathy, a major cause of kidney failure worldwide. We studied 1,194 cases and 902 controls of Chinese Han ancestry, with targeted follow up in Chinese and European cohorts comprising 1,950 cases and 1,920 controls. We identified three independent loci in the major histocompatibility complex, as well as a common deletion of CFHR1 and CFHR3 at chromosome 1q32 and a locus at chromosome 22q12 that each surpassed genome-wide significance (P values for association between 1.59 × 10−26 and 4.84 × 10−9 and minor allele odds ratios of 0.63–0.80). These five loci explain 4–7% of the disease variance and up to a tenfold variation in interindividual risk. Many of the alleles that protect against IgA nephropathy impart increased risk for other autoimmune or infectious diseases, and IgA nephropathy risk allele frequencies closely parallel the variation in disease prevalence among Asian, European and African populations, suggesting complex selective pressures.

IgA nephropathy, the most common cause of glomerulonephritis, is linked to 6q22–23

End-stage renal disease (ESRD) is a major public health problem, affecting 1 in 1,000 individuals and with an annual death rate of 20% despite dialysis treatment. IgA nephropathy (IgAN) is the most common form of glomerulonephritis, a principal cause of ESRD worldwide; it affects up to 1.3% of the population and its pathogenesis is unknown. Kidneys of people with IgAN show deposits of IgA-containing immune complexes with proliferation of the glomerular mesangium (Fig. 1). Typical clinical features include onset before age 40 with haematuria and proteinuria (blood and protein in the urine), and episodes of gross haematuria following mucosal infections are common; 30% of patients develop progressive renal failure. Although not generally considered a hereditary disease, striking ethnic variation in prevalence and familial clustering, along with subclinical renal abnormalities among relatives of IgAN cases, have suggested a heretofore undefined genetic component. By genome-wide analysis of linkage in 30 multiplex IgAN kindreds, we demonstrate linkage of IgAN to 6q22–23 under a dominant model of transmission with incomplete penetrance, with a lod score of 5.6 and 60% of kindreds linked. These findings for the first time indicate the existence of a locus with large effect on development of IgAN and identify the chromosomal location of this disease gene.

MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term “MHY9-related disease,” which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.

Prevalence of Mutations in Renal Developmental Genes in Children with Renal Hypodysplasia: Results of the ESCAPE Study

Renal hypodysplasia (RHD) is characterized by a reduced nephron number, small kidney size, and disorganized renal tissue. A hereditary basis has been established for a subset of affected patients, suggesting a major role of developmental genes that are involved in early kidney organogenesis. Gene mutations that have dominant inheritance and cause RHD, urinary tract anomalies, and defined extrarenal symptoms have been identified in TCF2 (renal cysts and diabetes syndrome), PAX2 (renal-coloboma syndrome), EYA1 and SIX1 (branchio-oto-renal syndrome), and SALL1 (Townes-Brocks syndrome). For estimation of the prevalence of these events, an unselected cohort of 99 unrelated patients with RHD that was associated with chronic renal insufficiency were screened for mutations in TCF2, PAX2, EYA1, SIX1, and SALL1. Mutations or variants in the genes of interest were detected in 17 (17%) unrelated families: One mutation, two variants, and four deletions of TCF2 in eight unrelated patients; four different PAX2 mutations in six families; one EYA1 mutation and one deletion in two patients with branchio-oto-renal syndrome; and one SALL1 mutation in a patient with isolated RHD. Of a total of 27 patients with renal cysts, six (22%) carried a mutation in TCF2. It is interesting that a SIX1 sequence variant was identified in two siblings with renal-coloboma syndrome as a result of a PAX2 mutation, suggesting an oligogenic inheritance. Careful clinical reevaluation that focused on discrete extrarenal symptoms and thorough family analysis revealed syndrome-specific features in nine of the 17 patients. In conclusion, 15% of patients with RHD show mutations in TCF2 or PAX2, whereas abnormalities in EYA1, SALL1, and SIX1 are less frequent.

COQ2 Nephropathy: A Newly Described Inherited Mitochondriopathy with Primary Renal Involvement

Primary coenzyme Q(10) (CoQ(10)) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Rarely, glomerular involvement has been reported. The COQ2 gene encodes the para-hydroxybenzoate-polyprenyl-transferase enzyme of the CoQ(10) synthesis pathway. We identified two patients with early-onset glomerular lesions that harbored mutations in the COQ2 gene. The first patient presented with steroid-resistant nephrotic syndrome at the age of 18 months as a result of collapsing glomerulopathy, with no extrarenal symptoms. The second patient presented at five days of life with oliguria, had severe extracapillary proliferation on renal biopsy, rapidly developed end-stage renal disease, and died at the age of 6 months after a course complicated by progressive epileptic encephalopathy. Ultrastructural examination of renal specimens from these cases, as well as from two previously reported patients, showed an increased number of dysmorphic mitochondria in glomerular cells. Biochemical analyses demonstrated decreased activities of respiratory chain complexes [II+III] and decreased CoQ(10) concentrations in skeletal muscle and renal cortex. In conclusion, we suggest that inherited COQ2 mutations cause a primary glomerular disease with renal lesions that vary in severity and are not necessarily associated with neurological signs. COQ2 nephropathy should be suspected when electron microscopy shows an increased number of abnormal mitochondria in podocytes and other glomerular cells.

Renal outcome in patients with congenital anomalies of the kidney and urinary tract

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a major cause of morbidity in children. We measured the risk of progression to end-stage renal disease in 312 patients with CAKUT preselected for the presence of anomalies in kidney number or size. A model of dialysis-free survival from birth was established as a function of the renal CAKUT categories of solitary kidney; unilateral and bilateral hypodysplasia; renal hypodysplasia associated with posterior urethral valves; and multicystic and horseshoe kidney. Cox regression analysis took into account the concomitant presence of vesicoureteral reflux, year of diagnosis, and time-varying values of serum creatinine, proteinuria, and hypertension. By 30 years of age, 58 patients had started dialysis, giving a yearly incidence of 0.023 over a combined 2474 patient risk years. The risk for dialysis was significantly higher for patients with a solitary kidney or with renal hypodysplasia associated with posterior urethral valves (hazard ratios of 2.43 and 5.1, respectively) compared to patients with unilateral or bilateral renal hypodysplasia, or multicystic or horseshoe kidney, and was independent of other prognostic factors. Our study shows that sub-clinical defects of the solitary kidney may be responsible for a poorer prognosis compared to more benign forms of CAKUT. Prospective studies are needed to validate these results.

Broadening the Spectrum of Diseases Related to Podocin Mutations

A total of 179 children with sporadic nephrotic syndrome were screened for podocin mutations: 120 with steroid resistance, and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistant patients presented homozygous mutations that were associated with early onset of proteinuria and variable renal lesions, including one case with mesangial C3 deposition. Single mutations of podocin were found in four steroid-resistant and in four steroid-dependent; five patients had the same mutation (P20L). Among these, two had steroid/cyclosporin resistance, two had steroid dependence, and one responded to cyclosporin. The common variant R229Q of podocin, recently associated with late-onset focal segmental glomerulosclerosis, had an overall allelic frequency of 4.2% versus 2.5% in controls. To further define the implication of R229Q, a familial case was characterized with two nephrotic siblings presenting the association of the R229Q with A297V mutation that were inherited from healthy mother and father, respectively. Immunohistochemistry with anti-podocin antibodies revealed markedly decreased expression of the protein in their kidneys. All carriers of heterozygous coding podocin mutation or R229Q were screened for nephrin mutation that was found in heterozygosity associated with R229Q in one patient. Finally, podocin loss of heterozygosity was excluded in one heterozygous child by characterizing cDNA from dissected glomeruli. These data outline the clinical features of sporadic nephrotic syndrome due to podocin mutations (homozygous and heterozygous) in a representative population with broad phenotype, including patients with good response to drugs. The pathogenetic implication of single podocin defects per se in proteinuria must be further investigated in view of the possibility that detection of a second mutation could have been missed. A suggested alternative is the involvement of other gene(s) or factor(s).

Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis.

Degeneration of photoreceptors is a common feature of ciliopathies, owing to the importance of the specialized ciliary structure of these cells. Mutations in AHI1, which encodes a cilium-localized protein, have been shown to cause a form of Joubert syndrome that is highly penetrant for retinal degeneration. We show that Ahi1-null mice fail to form retinal outer segments and have abnormal distribution of opsin throughout their photoreceptors. Apoptotic cell death of photoreceptors occurs rapidly between 2 and 4 weeks of age in these mice and is significantly (P = 0.00175 and 0.00613) delayed by a reduced dosage of opsin. This phenotype also shows dosage-sensitive genetic interactions with Nphp1, another ciliopathy-related gene. Although it is not a primary cause of retinal blindness in humans, we show that an allele of AHI1 is associated with a more than sevenfold increase in relative risk of retinal degeneration within a cohort of individuals with the hereditary kidney disease nephronophthisis. Our data support context-specific roles for AHI1 as a contributor to retinopathy and show that AHI1 may explain a proportion of the variability in retinal phenotypes observed in nephronophthisis.