Frequency distribution of hepatitis C virus genotypes in different geographical regions of Pakistan and their possible routes of transmission-1

Ectable HCV RNA by qualitative RT-PCR and viral load >500 IU/ml and belonged to any of the four provinces of Pakistan<p><b>Copyright information:</b></p><p>Taken from "Frequency distribution of hepatitis C virus genotypes in different geographical regions of Pakistan and their possible routes of transmission"</p><p>http://www.biomedcentral.com/1471-2334/8/69</p><p>BMC Infectious Diseases 2008;8():69-69.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2409346.</p><p></p

Additional file 2: of A novel LRAT mutation affecting splicing in a family with early onset retinitis pigmentosa

Table S2. Primer list (DOCX 12 kb)

Additional file 3: of A novel LRAT mutation affecting splicing in a family with early onset retinitis pigmentosa

Table S3. LINKAGE profile of Family 61,254 (XLSX 13 kb)

Additional file 1: of A novel LRAT mutation affecting splicing in a family with early onset retinitis pigmentosa

Table S1. Primer sequences and PCR conditions of linkage mapping panels, A. Marker specific primers, B. Primers with fluorescent tags, C. Amplification Protocol (DOCX 32 kb)

Point mutation in <i>PPIP5K2</i> is associated with DFNB100 hearing loss (HL).

<p>(A) Nucleotide sequence chromatograms of selected region of <i>PPIP5K2</i> exon 21 from homozygous WT (normal) and affected individuals harboring c.2510G>A [p.(Arg837His)] mutation segregating in DFNB100 families. (B) The human <i>PPIP5K2</i> gene has 33 exons. Alternate splicing of <i>PPIP5K2</i> gives rise to many different transcripts that are predicted to affect the composition of the non-catalytic intrinsically disordered region (IDR). The non-coding 5’ and 3’ UTRs are gray rectangles while sequences encoding the kinase, phosphatase (pase) and IDR domains, and other coding regions of exons are denoted by orange, light green, dark green, and black boxes, respectively. The nucleotide and amino acid variants of <i>PPIP5K2</i> are also shown. The blue bar under the phosphatase domain indicates the antigenic region of the commercially available polyclonal antibodies. (C) Schematic representation of PPIP5Ks biochemical function. Arrows indicate direction of the reaction. (D) PPIP5K2 amino acid conservation from eleven vertebrates and one non-vertebrate (Drosophila). Conserved amino acids are shaded in gray, while the p.Arg837 amino acid affected due to c.2510G>A variant, identified in our DFNB100 families, is noted with an arrow head. (E) The phosphatase activities of FLAG-tagged WT PPIP5K2 (21.5 ± 1.5 nmol/mg protein/min) and PPIP5K2^R837H (16.9 ± 2.1 nmol/mg protein/min) were determined as described in the Methods section (*p<0.05, n = 5; mean ± SE). (F) The kinase activities of FLAG-tagged WT PPIP5K2 (0.9 ± 0.21 nmol/mg protein/min) and PPIP5K2^R837H (1.44 ± 0.29 nmol/mg protein/min) were determined as described in the Methods Section (*p<0.03, n = 5; mean ± SE).</p

Mutations in Diphosphoinositol-Pentakisphosphate Kinase PPIP5K2 are associated with hearing loss in human and mouse

<div><p>Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, <i>DFNB100</i> on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in <i>PPIP5K2</i> that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the <i>Ppip5k2</i> phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.</p></div

<i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> mice exhibit late onset high-frequency decline in ABR wave I amplitudes.

<p>Average pure tone-evoked ABR wave I amplitudes and latencies in <i>Ppip5k2</i>^<i>+/+</i>, <i>Ppip5k2</i>^<i>+/K</i><i>^</i>, and <i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> at different time points (P60, P90, P120, P150) were assessed (n = 7; mean ± SEM). Significantly reduced wave I amplitudes at 24KHz were detected at P120 in <i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> mice (*p < 0.01). At P150 both the homozygous and heterozygous <i>Ppip5k2</i>^<i>K</i><i>^</i> mice had significantly reduced wave I amplitudes at 24KHz mice (**p < 0.05). These results suggest impaired late onset auditory neuronal function in PPIP5K2 mutant mice.</p

<i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> mice exhibit late onset high-frequency progressive HL.

<p>(A) Schematic of the <i>Ppip5k2</i>^<i>K</i><i>^</i> allele (left), and resulting truncated PPIP5K2 protein (right) with a truncated (only the first 82 residues) of the histidine acid phosphatase motif (HP). (B) The kinase activities of FLAG-tagged WT (0.9 ± 0.21) and PPIP5K2^1-466 (20.3 ± 1.0), which has kinase and HP motif but no phosphatase activity, and corresponds to the protein expressed by the mouse <i>Ppip5k2</i>^<i>K</i><i>^</i> allele (*p<0.001, n = 5; mean ± SE). (C) Levels of IP6, IP7 and IP8 were determined by HPLC in <i>PPIP5K1/2</i> double knockout (DKO) HEK293 cells transfected with various <i>PPIP5K2</i> constructs. Bar graphs display mean ± SE (n = 6, except PPIP5K2^1-466 n = 5). IP7/IP6 ratio in DKO cells (0.057 ± 0.009, n = 6), DKO cells transfected with FLAG-tagged WT (0.05 ± 0.006), PPIP5K2^R837H (0.048 ± 0.006) and PPIP5K2^1-466 (0.028 ± 0.0029; *p<0.011). Shown also IP8/IP6 ratio (*p<0.023) in DKO cells (0 ± 0), cells transfected with FLAG-tagged WT (0.0049 ± 0.0012), PPIP5K2^H837R (0.0051 ± 0.0014) and PPIP5K2^1-466 (0.028 ± 0.008) constructs. (D) Average click and pure tone-evoked ABR thresholds (dB SPL) in <i>Ppip5k2</i>^<i>+/+</i>, <i>Ppip5k2</i>^<i>+/K</i><i>^</i>, and <i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> at different time points (P60, P90, P120, P150) were assessed (n = 7; mean ± SEM). Raised mean ABR thresholds at 32 kHz were detected as early as P60 in <i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> mice and continued to increase with age. At P120 and P150, the <i>Ppip5k2</i>^<i>K</i><i>^</i>^<i>/K</i><i>^</i> mice had profound HL at higher frequencies [24 kHz (**p<0.001) and 32 kHz]. Also, <i>Ppip5k2</i>^<i>+/K</i><i>^</i> mice exhibited elevated hearing thresholds loss at higher frequencies [24 kHz (*p<0.05) and 32 kHz]. These results suggest impaired PPIP5K2 function in mouse is associated with late onset high-frequency progressive HL. However, as anticipated, due to age-related hearing loss in mice on a C57BL/6 genetic background, the ABR thresholds at 32 kHz were also elevated in WT mice at P120 and P150.</p

PPIP5K2 localized in sensory and non-sensory cells of mouse inner ear.

<p>(A) Expression of <i>Ppip5k2</i> in adult (P150) mice, normalized against <i>Gapdh</i> (ΔCT) and <i>Ppip5k1</i> expression (ΔΔCT). (B) Cross-section through one of the coils of inner ear showing diffuse cytoplasmic immunolabeling of PPIP5K2 throughout the cochlear duct, including spiral ganglion neurons (SG), organ of Corti (OC), and stria vascularis (SV). (C) Expression of PPIP5K2 persists at the ages tested in WT mice, from early postnatal day P16, up to 9 months of age. Scale bars: 100μm (panel A), and 20μm (panel C).</p

Pedigrees of families segregating DFNB100 deafness and representative pure-tone audiograms.

<p>(A, B) Squares and circles denote male and female family members, respectively. Filled symbols represent affected individuals. (C) Pure tone air conduction thresholds for family PKDF041 individuals VI:2 (9 yo male), and V:5 (42 yo male). Individual VI:2 hearing thresholds were within normal range, while individual V:5 had profound HL in both ears. Right ear air conduction: O; Left ear air conduction: X. (D) Pure tone air conduction thresholds for family PKDF751 individuals V:2 (23 yo female) revealed profound HL in both ears. In contrast, individual V:7 (20 yo male) had hearing threshold within normal range.</p