Genetics A 112 kb Deletion in Chromosome 19q13.42 Leads to Retinitis Pigmentosa Anna M. Rose,1 Rajarshi Mukhopadhyay,2 Andrew R. Webster,2 Shomi S. Bhattacharya,1 and Naushin H. Waseem1 PURPOSE. This study sets out to identify novel mutations in nonsense, missense, insertion, and deletion mutations. A key PRPF31 in a cohort of autosomal dominant retinitis pigmen- feature of PRPF31 associated autosomal dominant retinitis pig- tosa (adRP) patients with a history of nonpenetrance in the mentosa (adRP) is the unique inheritance pattern. The affected family. families may show nonpenetrance: an individual carrying a METHODS. Twenty-one patients with history of nonpenetrant mutant allele may not show disease symptoms; they may, autosomal dominant retinitis pigmentosa were selected; all however, have affected children. It had been suggested that underwent full ophthalmic examination. Multiplex ligation- the partial penetrance is due to the existence of two different wild type PRPF31 alleles, a high expressivity allele and a low dependent probe analysis (MLPA) was performed and, where a 6 deletion was found, further family members were recruited. An expressivity allele. If a patient carries a mutant allele and a individual suspected to harbor a large deletion was used as a high-expressivity wild type allele, then the residual level of positive control. Analysis of single nucleotide polymorphisms PRPF31 protein (hPRP31) is sufficient for normal function. If, in the upstream region was used to determine the extent of the however, a patient has a mutant allele and a low expressivity deletion, and the breakpoint was then characterized by PCR allele, then hPRP31 level falls below the threshold for normal and sequencing. retinal function. It has been shown that asymptomatic muta- tion carriers have a two-fold higher expression level of PRPF31 RESULTS. In one family, multiplex ligation-dependent probe compared with symptomatic individuals.7 analysis revealed a novel large deletion in 19q13.4 encompass- hPRP31 is associated with the U4/U6 di-snRNP and interacts ing exons 1 to 13 of the PRPF31 gene. The mutation was with hPRP6, to form the U4/U6-U5 tri-snRNP.8 When hPRP31 characterized as a deletion of 112 kilobase (kb), encompassing expression is knocked down by RNA interference, U4/U6 over 90% of PRPF31 and five upstream genes: TFPT, OSCAR, di-snRNPs accumulate in the Cajal bodies and the U4/U6-U5 NDUFA3, TARM-1, and VSTM-1. The breakpoint in the positive tri-snRNP cannot form.9 It has been proposed that due to the control family was also characterized. The mechanism of dele- high demands on splicing components in retinal photorecep- tion in both families was Alu-mediated nonallelic homologous tors, a slightly reduced level of splicing factor will have a recombination. cumulative detrimental effect on its proper functions.10 CONCLUSIONS. This study describes two large deletions, one in a PRPF31 lies within a region that is rich in repeat elements, previously reported family and one in a new family: the latter especially Alu repeats. Alu repeats are short-interspersed nu- represents the largest deletion yet described on chromosome clear elements which account for 10% of the total genome 19 and the first report of the involvement of VSTM-1. Remark- content, although in chromosome 19 these repeats account for ably, heterozygous deletion of this large region (encompassing around 26.3%.11 Most of the sense and antisense Alu integra- six genes) produces little or no other clinical disease besides tion occurs also in the proximity of microRNAs and these retinitis pigmentosa. (Invest Ophthalmol Vis Sci. 2011;52: together have been implicated in modulation of human ge- 6597–6603) DOI:10.1167/iovs.11-7861 nome architecture and in mediation of gene rearrangement in human disorders.12 eterozygous mutations in several ubiquitously expressed In this study, we report a systematic analysis of insertions/ HRNA splicing factors (PRPF3, PRPF6, PRPF8, PRPF31, and deletions in PRPF31 in 21 RP patients with a family history of hBrr2) have been found to cause retinitis pigmentosa (RP),1–5 nonpenetrance. We have identified a 112 kb deletion that a clinically and genetically diverse group of disorders charac- encompasses over 90% of PRPF31, together with 5 adjacent terized by degeneration of the retinal photoreceptors. (upstream) genes. The mechanism of deletion was Alu-medi- PRPF31 was identified as the causative gene at the RP11 ated nonallelic homologous recombination and is the largest locus,4 with over 40 mutations identified to date, including reported so far in a patient with retinitis pigmentosa. METHODS From the 1Department of Genetics, UCL Institute of Ophthalmol- 2 ogy, London, United Kingdom; and Moorfields Eye Hospital, London, Patient Selection United Kingdom. Supported by National Institute for Health Research UK (Moor- Twenty-one patients with a diagnosis of adRP were selected, all having fields Eye Hospital and Institute of Ophthalmology, London, United had full ophthalmic examination including slit lamp examination, as- Kingdom). sessment of visual acuity, perimetry, color vision, and electrodiagnostic Submitted for publication May 11, 2011; revised June 8, 2011; testing at Moorfields Eye Hospital. The patients’ family history was accepted June 14, 2011. examined for evidence of autosomal dominant inheritance with non- Disclosure: A.M. Rose, None; R. Mukhopadhyay, None; A.R. Webster, None; S.S. Bhattacharya, None; N.H. Waseem, None penetrance. All patients were screened by direct sequencing for mu- Corresponding author: Anna M. Rose, Department of Genetics, tations in RHO, RDS, and PRPF31. Furthermore, they did not harbor UCL Institute of Ophthalmology, Bath Street, London EC1V 9EL, UK; known mutations in IMPDH1, NRL, PRPF8, PRPF3, NR2E3, RP9, and [email protected]. RP1. Informed consent was obtained from all patients before the Investigative Ophthalmology & Visual Science, August 2011, Vol. 52, No. 9 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 6597 Downloaded from jov.arvojournals.org on 09/27/2021 6598 Rose et al. IOVS, August 2011, Vol. 52, No. 9 research being conducted, which was performed according to the http://www.repeatmasker.org/). Primers were designed to unique up- tenets of the Declaration of Helsinki. stream sequence to each Alu and PCR performed, as described previ- ously. PCR product produced was then sequenced (as described) and Multiplex Ligation-Dependent Probe aligned to reference genome sequence. Analysis (MLPA) A kit which screens for large insertions or deletions in the four most Clinical Assessment of Patients commonly affected adRP genes: RHO, PRPF31, IMPDH1, and RP1, was Harboring Deletions used according to the protocol provided by the manufacturer (MRC- Holland P235 Retinitis Pigmentosa kit; MRC-Holland, Amsterdam, Detailed medical history was obtained, including a review of sys- Netherlands). MLPA fragments were separated and sized (GeneScan tems to identify any possible health problems in patients with 500 LIZ size standard on 3730 DNA Analyzer; both from Applied PRPF31 deletion. A standard clinical examination was performed of Biosystems, Inc., Cheshire, UK) and the results analyzed all major systems, including cardiorespiratory, gastrointestinal, and (GeneMarker v1.8; SoftGenetics, LLC, State College, PA). Each ex- nervous systems (including assessment of cranial nerves, peripheral periment was repeated at least three times to ensure reproducibil- motor nerves, and peripheral sensory nerves). Full blood count and ity. metabolic bone profile biochemistry was also performed. Where a deletion was found, other family members were recruited and MLPA was performed on their DNA sample. An affected individual from AD2 family (individual IV:2) was used as a positive control.13 This family had been suspected to harbor a large deletion in PRPF31 based RESULTS on microsatellite markers in the 19q13.4 region. Twenty-one patients with RP recruited for this study Single Nucleotide Polymorphism (SNP) Analysis showed autosomal dominant form of inheritance together The NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) with partial penetrance in their family pedigree. No muta- was used to select SNPs with high heterozygosity in the Caucasian tions were identified in the entire PRPF31 gene by direct population. Primer pairs were designed to amplify short fragments sequencing in any of the probands. No known mutations (150–400 base pairs) containing the SNP. PCR product was obtained were identified in IMPDH1, NRL, PRPF8, PRPF3, NR2E3, (Taq polymerase; Bioline UK, Ltd., London, UK) following manufac- RP9, RHO, RDS, and RP1. To investigate whether these turer’s protocol using primers at 100 ng/␮L PCR product was se- patients had large deletions or insertions in PRPF31, their quenced (BigDye Terminator v3.1; Life Technologies, Cheshire, UK), DNA samples were subjected to MLPA. As a positive control, and the results were analyzed (SeqMan; DNAStar, Madison, WI). we included individual IV:2 from AD2 family (hereafter referred to as individual AD2), as this family had previously Breakpoint Characterization been shown to have a large deletion in PRPF31 based on Alu repeats within the region defined by SNP analysis were identified microsatellite data, although no specific deletion boundaries with a DNA sequence screening program (RepeatMasker browser; were defined in that study. FIGURE 1. MLPA profile of individual RP15011 (A), her asymptomatic father (B), healthy mother (C), and individual IV2 from AD2 family (D). The horizontal lines denote the normal ratio of exons of PRPF31. The dark squares below the horizontal line indicate a single copy exon. Downloaded from jov.arvojournals.org on 09/27/2021 IOVS, August 2011, Vol. 52, No. 9 A 112 kb Deletion in Chromosome 19q13 Leads to adRP 6599 Multiplex Ligation-Dependent Probe the product detected a deletion of 112 kb, encompassing Analysis (MLPA) the genes VSTM-1, OSCAR, TFPT, NDUFA3, TARM-1, and Ͼ90% of PRPF31 (Fig. 3A). Each of the two breakpoints Out of 21 patients tested, MLPA analysis identified a deletion resides within an AluY repeat, one located in the CACNG6 encompassing multiple exons in PRPF31 in one patient.