Anand N. Mhatre

APOL1 Variants Increase Risk for FSGS and HIVAN but Not IgA Nephropathy

A chromosome 22q13 locus strongly associates with increased risk for idiopathic focal segmental glomerulosclerosis (FSGS), HIV-1-associated nephropathy (HIVAN), and hypertensive ESRD among individuals of African descent. Although initial studies implicated MYH9, more recent analyses localized the strongest association within the neighboring APOL1 gene. In this replication study, we examined the six top-most associated variants in APOL1 and MYH9 in an independent cohort of African Americans with various nephropathies (44 with FSGS, 21 with HIVAN, 32 with IgA nephropathy, and 74 healthy controls). All six variants associated with FSGS and HIVAN (additive ORs, 1.8 to 3.0; P values 3 × 10(-2) to 5 × 10(-5)) but not with IgA nephropathy. In conditional and haplotype analyses, two APOL1 haplotypes accounted for virtually all of the association with FSGS and HIVAN on chromosome 22q13 (haplotype P value = 5.6 × 10(-8)). To assess the role of MYH9 deficiency in nephropathy, we crossbred Myh9-haploinsufficient mice (Myh9(+/-)) with HIV-1 transgenic mice. Myh9(+/-) mice were healthy and did not demonstrate overt proteinuria or nephropathy, irrespective of the presence of the HIV-1 transgene. These data further support the strong association of genetic variants in APOL1 with susceptibility to FSGS and HIVAN among African Americans.

Audiologic testing and molecular analysis of 12S rRNA in patients receiving aminoglycosides.

Background: Pathogenic mutations in the mitochondrial genome are associated with a wide variety of maternally inherited human diseases including sensorineural hearing loss (HL). A specific mutation, m.1555A>G in the mitochondrial 12S rRNA gene, is associated with predisposition to aminoglycoside ototoxicity and HL. Mutation screening in this gene has been recommended before use of aminoglycosides as a preventative strategy to reduce the risk of ototoxicity.

Two families with nonsyndromic low-frequency hearing loss harbor novel mutations in Wolfram syndrome gene 1

Although hereditary hearing loss is highly heterogeneous, only a few loci have been implicated with low-frequency hearing loss. Mutations in one single gene, Wolfram syndrome 1 (WFS1), have been reported to account for most familial cases with this type of hearing impairment. This study was conducted to determine the cause of nonsyndromic low-frequency hereditary hearing impairment in two large families. Two large families from Switzerland and United States with low-frequency hearing loss were identified. Genomewide linkage analysis was performed followed by mutation screening in the candidate gene WFS1 with direct DNA sequencing and restriction fragment analysis. Both families were linked to DFNA6/14/38 with lod scores>3. Two novel heterozygous missense mutations in WFS1 were identified: c.2311G>C leading to p.D771H in the Swiss family and c.2576G>C leading to p.R859P in the US family. The sequence alteration was absent in 100 control chromosomes. Nonsyndromic low-frequency hereditary hearing impairment seems to be predominantly a monogenic disorder due to WFS1. We confirm that most mutations in WFS1 associated with isolated low-frequency hearing loss are clustered in the C-terminal protein domain coded by exon 8.

Generation and characterization of mice with Myh9 deficiency

Mutant alleles of MYH9 encoding a class II non-muscle myosin heavy chain-A (NMMHC-IIA) have been linked to hereditary megathrombocytopenia with or without additional clinical features that include sensorineural deafness, cataracts, and nephritis. To assess its biological role in the affected targets, particularly the inner ear, we have generated and characterized mice with Myh9 deficiency. These mice were generated using the XA136 ES cell line (BayGenomics, http://baygenomics.ucsf.edu/) carrying gene trap insertion in Myh9, within the intron flanking exons 4 and 5. Mice heterozygous for the Myh9 null allele, Myh9+/− were expanded on C57BL/6J background. Intercross of the Myh9+/− mice did not yield Myh9−/− pups, indicating embryonic lethality, subsequently determined to occur at or before E7.5, thus precluding a post-natal analysis of the effects of complete Myh9 deficiency. The heterozygous mice were normal for their hearing, parameters of platelet integrity and renal function despite their Myh9 haplo-insufficiency. In addition, the age-dependent auditory threshold of the Myh9+/− mice and their wild type littermates, spanning from 3 to 12xa0months of age, were similar indicating that Myh9 haplo-insufficiency does not contribute towards accelerated age-related hearing loss (AHL). The embryonic lethality associated with the complete Myh9 deficiency establishes a critical role for this non-muscle myosin in fetal development. The results of these studies do not support the Myh9 haploinsufficiency as a pathogenic factor in the etiology of auditory dysfunction.

DFNA54, a third locus for low-frequency hearing loss

Nonsyndromic hereditary hearing impairment (NSHHI) is a highly heterogeneous disorder with more than 90 loci mapped, of which nearly one-half of the responsible genes are identified. In dominant NSSHI hearing loss is typically biased towards the high frequencies while low-frequency hearing loss is unusual. Only two NSHHI loci, DFNA1 and DFNA6/14/38, are associated with predominantly low- frequency loss. We mapped the loci harboring the gene responsible for autosomal dominant low-frequency hearing loss in a multigenerational family. The pedigree of a Swiss family with low-frequency hearing loss was established. Using genomic DNA, DFNA1 and DFNA6/14/38 were excluded by linkage analysis or by direct sequencing of the responsible gene. Genome-wide linkage analysis was performed using commercially available microsatellite markers. Two-point linkage analysis demonstrated linkage to chromosome 5q31, the locus for DFNA15, with a lod score of 6.32 at recombination fraction θ=0 for marker D5S436. Critical recombinations were seen at markers D5S1972 and D5S410. Sequencing of the corresponding gene POU4F3 yielded no pathogenic mutation segregating with the affected members. In addition to Wolfram syndrome gene 1 (DFNA6/14/38) and diaphanous (DFNA1) there is evidence for a third gene involved in low-frequency hearing loss located at DFNA15. Because of the differences in auditory phenotype and the absence of pathogenic mutation in the coding region of POU4F3 it is likely that there is a second gene in 5q31, designated DFNA54, associated with NSHHI.

Expression of Myh9 in the mammalian cochlea: Localization within the stereocilia

Mutations of non‐muscle myosin Type IIA or MYH9 are linked to syndromic or nonsyndromic hearing loss. The biologic function of MYH9 in the auditory organ and the pathophysiology of its dysfunction remain to be determined. The mouse represents an excellent model for investigating the biologic role of MYH9 in the cells and tissues affected by its dysfunction. A primary step toward the understanding of the role of MYH9 in hearing and its dysfunction is the documentation of its cellular and sub‐cellular localization within the cochlea, the auditory organ. We describe the localization of Myh9 within the mouse cochlea using a polyclonal anti‐Myh9‐antibody, generated against an 18 amino acid long peptide corresponding to the sequence at the C‐terminus of mouse Myh9. The anti‐Myh9 antibody identified a single, specific, immunoreactive band of 220 kDa in immunoblot analysis of homogenate from a variety of different mouse tissues. The Myh9 antibody cross‐reacts with the rat but not the human orthologue. Myh9 is expressed predominantly within the spiral ligament as well as in the sensory hair cells of the organ of Corti. Confocal microscopy of cochlear surface preparations, identified Myh9 within the inner and outer hair cells and their stereocilia. Localization of Myh9 within the stereocilia raises the possibility that mutations of MYH9 may effect hearing loss though disruption of the stereocilia structure.

Localization in stereocilia, plasma membrane, and mitochondria suggests diverse roles for NMHC-IIa within cochlear hair cells

NMHC-IIa, a nonmuscle myosin heavy chain isoform encoded by MYH9, is expressed in sensory hair cells and its dysfunction is associated with syndromic and nonsyndromic hearing loss. In this study, we investigate the ultrastructural distribution of NMHC-IIa within murine hair cells to elucidate its potential role in hair cell function. Using previously characterized anti-mouse NMHC-IIa antibody detected with immunogold labelling, NMHC-IIa was observed in the stereocilia, in the cytosol along the plasma membrane, and within mitochondria. Within stereocilia, presence of NMHC-IIa is observed throughout its length along the actin core, from the center to the periphery and at its base in the cuticular plate, suggesting a potential role in structural support. Within the sensory hair cells, NMHC-IIa was distributed throughout the cytoplasm and along the plasma membrane. A novel finding of this study is the localization of NMHC-IIa within the mitochondria, with the majority of the label along its inner membrane folds. The presence of NMHC-IIa within heterogeneous areas of the hair cell suggests that it may play different functional roles in these distinct regions. Thus, mutant NMHC-IIa may cause hearing loss by affecting hair cell dysfunction through structural and or functional disruption of its stereocilia, plasma membrane, and/or mitochondria.

MYH9-siRNA and MYH9 mutant alleles: expression in cultured cell lines and their effects upon cell structure and function.

MYH9 encodes a class II nonmuscle myosin heavy chain-A (NMHC-IIA), a widely expressed 1960 amino acid polypeptide, with translated molecular weight of 220 kDa. From studies of type II myosin in invertebrates and analogy with the skeletal and smooth muscle myosin II, NMHC-IIA is considered to be involved in diverse cellular functions, including cell shape, motility and division. The current study assessed the consequences of two separate, naturally occurring MYH9 dominant mutant alleles, MYH9(R702C) and MYH9(R705H) linked to syndromic and nonsyndromic hearing loss, respectively, upon diverse NMHC-IIA related functions in two separate cultured cell lines. MYH9-siRNA-induced inhibition of NMHC-IIA in HeLa cells or HEK293 cells resulted in alterations in their shape, actin cytoskeleton and adhesion properties. However, HeLa or HEK293 cells transfected with naturally occurring MYH9 mutant alleles, MYH9(R702C) or MYH9(R705H), as well as in vitro generated deletion derivatives, MYH9(DeltaN592) or MYH9(DeltaC570), were unaffected. The effects of MYH9-siRNA-induced suppression underline the critical role of NMHC-IIA in maintenance of cell shape and adhesion. However, the results also indicate that the NMHC-IIA mutants, R702C and R705H do not inactivate or suppress the endogenous wild type NMHC-IIA within the HeLa or HEK293 cell assay system.

Role of Cytomegalovirus, Epstein-Barr Virus, and Human Herpes Virus-8 in Benign Lymphoepithelial Cysts of the Parotid Gland

Objective: To provide background and evaluate the role of herpesviruses in benign lymphoepithelial cysts (BLC) of the parotid gland.

Aquaporin-2 and -4: Single nucleotide polymorphisms in Ménière's disease patients

Abstract The endolymphatic hydrops seen in Ménières disease (MD) results from an inner ear fluid disequilibrium that has a suspected inherited component. Aquaporin-2 and aquaporin-4 (AQP2 and AQP4) water transport proteins may contribute to abnormal fluid homeostasis seen in MD. Our objective was to screen for sequence alterations in AQP2 and AQP4 genes in a northern European population with MD. Amplification for AQP2 (n = 18) and AQP4 (n = 30) was performed for patients with MD. Sequences were screened with denaturing high powered liquid chromatography (DHPLC) and confirmed with sequencing. Allele frequencies were compared with previously reported normal populations. We found that DHPLC failed to identify sequence alterations in any sample. Sequencing identified three intronic and one 3’ untranslated region polymorphism in AQP2, and one polymorphism upstream from the start codon in AQP4. Two of the AQP2 intronic allele frequencies showed an A and C allele enrichment, respectively, compared with a reported mixed population (0.389 A vs. 0.00 A; 0.389 C vs. 0.00 C, p<0.001). The remaining polymorphism showed statistical difference from three non-Caucasian populations (0.611 A vs. 0.389 A, 0.375 A, and 0.280 A, p<0.05). The AQP4 allele frequency in the MD population was statistically different from a previously published Japanese population (0.800 G vs. 0.620 G, p = 0.0053) but not from a reported Caucasian population. We concluded that aquaporin polymorphisms may contribute to MD. Additional studies are needed to confirm these findings in well-defined population isolates and to determine if these polymorphisms lead to altered AQP protein function or levels.