Fig. 32.3 ClustalW alignment of RS proteins shows the tryptophan residue at position 92 (W92; boxed) has been highly conserved throughout evolution

ClustalW RS protein sequence alignment shows W92 is highly conserved, even in Danio rerio (Fig. 32.3).

Sequencing of 135 control X chromosomes confirmed this variant was not present in the normal population (data not shown). DNA sequencing of family members revealed the proband’s mother was heterozygous for this variant, the symptomatic younger brother did in fact posess the same novel 289T > G single base substitution, while the younger sister did not harbour any change (Fig. 32.2a, b).

32.4 Discussion

In this study we revealed the genetic diagnosis of individuals affected by XLRS in four Western Australian families. The exon five, frameshift 416delA mutation has previously been reported resulting in premature termination in the discoidin domain hence abrogating normal RS structure (The Retinoschisis Consortium 1998) making intra-cellular retention and degradation very likely. Clincal data was not presented for this family as the ophthalmic report indicated that in addition to Retinoschisis, an RP type syndrome was likley.

Also previously reported, the 52+1G > T missense mutation was found in two Western Australian families and is likely to result in a splicing defect predicted to cause a deleterious insertion or deletion towards the end of the leader sequence, a motif important in secretion of RS from the endoplasmic reticulum (Mashima et al. 1999). We showed that the full-field and mfERG results for an affected

Fig. 32.2 (continued) Solid square indicates affected male, in this case the proband (III:1; arrow). Central dot within a circle indicates carrier female (II:2). Question mark within a box indicates a possibly affected male (III:2). A circle containing the letter ‘N’ indicates a non-carrier female (III:4). (b) Electropherograms for part of the RS1 nucleotide sequence from exon four is shown in order from top to bottom for (1) an unrelated, normal control, (2) the proband (III:1) in which the 289T > G substitution was observed, (3) the proband’s symptomatic brother (III:2) in which this change was also observed, (4) the proband’s mother II:2, heterozygous for this change and marked by the letter K. The position of nucleotide 289T is marked by an arrow (top)

290 T. Lamey et al.

male with this mutation were typical for XLRS (Piao et al. 2003; Tantri et al. 2004; Tsang et al. 2007). The carrier, heterozygous for 52+1G > T recorded an abnormal mfERG trace array, sometimes observed in XLRS obligate carriers (Kim et al. 2006). Interestingly, the abnormal full-field ERG observed, is not usually seen in XLRS carriers. The likely explanation for this is unfavourably biased X-inactivation (Lyon 1962) or alternatively, the presence of another underlying condition.

To the best of our knowledge the 289T > G genetic variant has not before been reported. This single base substitution causes a W92G amino acid change. Importantly, the highly conserved W92 is one of three aromatic amino acids proposed to form the membrane lipid anchorage site (Fraternali et al. 2003) confirmed by site directed mutatengesis; the W92C mutant reduced the ability of RS to anchor to the preferred lipid moiety, phosphatidylserine, by 30–50% in vitro (Vijayasarathy et al. 2007). While the effect of a W92G transition in RS has not been confirmed experimentally, replacement of the large, hydrophobic, aromatic membrane binding tryptophan with the ambivalent and smallest of all amino acids, glycine, is likely to negatively affect anchorage as demonstrated for other membrane binding proteins (Ortega-Gutierrez and Lopez-Rodriguez 2005). Hence even if extracellular RS is present in this case, is is unlikely to be properly functional.

Given that (1) the 289T > G genetic variant was the the only change detected in RS1 DNA of the proband, (2) the proband’s mother is heterozygous for this single base substitution, (3) this change was absent from 135 control X chromosomes, and

(4) it causes a change in a crucial membrane binding residue in a protein that functions to bind photoreceptor and bi-polar cell membranes, there is strong evidence to suggest that 289T > G is pathogenic.

That RS1 is a small gene with retinal specific expression patterns makes this gene a suitable candidate for gene delivery gene therapies. Characterisation of disease causing mutations in patients affected by inherited retinal diease is an important preceeding step for such therapies, but also contributes towards a better understanding of the clinical manifestation of these diseases.

Acknowledgments The authors gratefully acknowledge the Western Australian Retinitis Pigmentosa Foundation for their generous funding, and the assistance of the Western Australian DNA Bank (NHMRC Enabling Facility) with DNA samples for this study.

References

Deutman AF (1971) Sex-linked retinoschisis. In: Deutman AF (ed) The hereditary dystrpohies of the posterior pole of the eye. Van Gorcum, Assen, Netherlands

Eksandh L, Andreasson S, Abrahamson M (2005) Juvenile x-linked retinoschisis with normal scotopic b-wave in the electroretinogram at an early stage of the disease. Ophthalmic Genet 26(3):111–117

Forsius H, Krause U, Helve J et al (1973) Visual acuity in 183 cases of x-chromosomal retinoschisis. Can J Ophthalmol 8(3):385–393

Fraternali F, Cavallo L, Musco G (2003) Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin. FEBS Lett 544(1–3):21–26

George ND, Yates JR, Moore AT (1995) X linked retinoschisis. Br J Ophthalmol 79(7):697–702 Hewitt AW, FitzGerald LM, Scotter LW et al (2005) Genotypic and phenotypic spectrum of x-

linked retinoschisis in australia. Clin Exp Ophthalmol 33(3):233–239

Kellner U, Brummer S, Foerster MH et al (1990) X-linked congenital retinoschisis. Graefes Arch Clin Exp Ophthalmol 228(5):432–437

Kim DY, Neely KA, Sassani JW et al (2006) X-linked retinoschisis: novel mutation in the initiation codon of the xlrs1 gene in a large family. Retina 26(8):940–946

Lyon MF (1962) Sex chromatin and gene action in the mammalian x-chromosome. Am J Hum Genet 14:135–148

Mashima Y, Shinoda K, Ishida S et al (1999) Identification of four novel mutations of the xlrs1 gene in japanese patients with x-linked juvenile retinoschisis. Mutation in brief no. 234. Online. Hum Mutat 13(4):338

Molday LL, Wu WW, Molday RS (2007) Retinoschisin (rs1), the protein encoded by the x-linked retinoschisis gene, is anchored to the surface of retinal photoreceptor and bipolar cells through its interactions with a na/k atpase-sarm1 complex. J Biol Chem 282(45):32792–32801

Ortega-Gutierrez S, Lopez-Rodriguez ML (2005) Cb1 and cb2 cannabinoid receptor binding studies based on modeling and mutagenesis approaches. Mini Rev Med Chem 5(7): 651–658

Piao CH, Kondo M, Nakamura M et al (2003) Multifocal electroretinograms in x-linked retinoschisis. Invest Ophthalmol Vis Sci 44(11):4920–4930

Pimenides D, George ND, Yates JR et al (2005) X-linked retinoschisis: clinical phenotype and rs1 genotype in 86 uk patients. JMedGenet 42(6):e35

Sauer CG, Gehrig A, Warneke-Wittstock R et al (1997) Positional cloning of the gene associated with x-linked juvenile retinoschisis. Nat Genet 17(2):164–170

Tantri A, Vrabec TR, Cu-Unjieng A et al (2004) X-linked retinoschisis: a clinical and molecular genetic review. Surv Ophthalmol 49(2):214–230

The Retinoschisis Consortium (1998) Functional implications of the spectrum of mutations found in 234 cases with x-linked juvenile retinoschisis. Hum Mol Genet 7(7):1185–1192

Tsang SH, Vaclavik V, Bird AC et al (2007) Novel phenotypic and genotypic findings in x-linked retinoschisis. Arch Ophthalmol 125(2):259–267

Vijayasarathy C, Takada Y, Zeng Y et al (2007) Retinoschisin is a peripheral membrane protein with affinity for anionic phospholipids and affected by divalent cations. Invest Ophthalmol Vis Sci 48(3):991–1000

Wang T, Waters CT, Rothman AM et al (2002) Intracellular retention of mutant retinoschisin is the pathological mechanism underlying x-linked retinoschisis. Hum Mol Genet 11(24):3097–3105 Wu WW, Molday RS (2003) Defective discoidin domain structure, subunit assembly, and endoplasmic reticulum processing of retinoschisin are primary mechanisms responsible for x-linked

retinoschisis. J Biol Chem 278(30):28139–28146

Wu WW, Wong JP, Kast J et al (2005) Rs1, a discoidin domain-containing retinal cell adhesion protein associated with x-linked retinoschisis, exists as a novel disulfide-linked octamer. J Biol Chem 280(11):10721–10730

Chapter 33

Mutation Frequency of IMPDH1 Gene of Han

Population in Ganzhou City

Li Shumei, Luo Xiaoting, Zeng Xiangyun, Hu Liqun, Xiong Liang, and Li Sisi

Abstract

Objective: Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) have recently been discovered that IMPDH1 gene plays a critical role in pathogenesis of autosomal dominant retinitis pigmentosa (adRP). Aiming towards an understanding of the molecular background of retinitis pigmentosa (RP), this paper investigates the mutation frequency of IMPDH1 genes in the Han patients with adRP in Ganzhou City.

Methods: The whole blood samples were collected randomly from 56 adRP patients and 62 unrelated normal controls who were residents of Han population in Ganzhou City, and then their genomic DNA samples were extracted respectively. Genic polymorphism was examined by the polymerase chain reaction and restriction- fragment-length polymorphisms (PCR-RFLP). The statistical significance of the data was further analyzed by SPSS 14.0 software.

Results: Mutation rate of IMPDH1 gene had no significance between in adRP patients and in the normal control by exact probabilities in 2 × 2 table (p = 0.232). The mutation frequency of IMPDH1gene in the Han samples was 3.6%.

Conclusion: The mutation frequency of IMPDH1 gene of the Han population in Ganzhou city was similar as approximately 2–5% of the adRP cases among Americans of European origin and Europeans.

33.1 Introduction

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative disorders characterized by progressive degeneration of the midperipheral retina, leading to night blindness, visual field constriction, and eventual loss of visual acuity (Gandra et al.

L. Shumei (B)

Department of Preventive Medicine, Gannan Medical College, Ganzhou, China e-mail: gnyxylsm@163.com

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_33,C Springer Science+Business Media, LLC 2010