Genetics A 112 kb Deletion in 19q13.42 Leads to

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 (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 . 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 : 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 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

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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 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.

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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. intergenic region and one within 13 of PRPF31 (Fig. Patient RP15011 was identified as carrying a deletion encom- 3B). The mechanism of deletion therefore appears to be passing exons 1 to 13 (Fig. 1A). Her parents were subse- unequal recombination between highly homologous AluY quently recruited for this study and their DNA samples were repeats. subjected to MLPA. The father of the proband carried the For the characterization of breakpoint in AD2, PCR was same deletion whereas the mother was normal (Figs. 1B and performed with primers in intron 3 and intron 13 of 1C). A deletion of exons 4–13 (inclusive) was detected in PRPF31. Sequencing of the product identified an 11 kb AD2 (Fig. 1D). deletion encompassing exons 4 to 13 of the gene, inclusive (Fig. 4). This breakpoint also occurs within highly homolo- Deletion Breakpoint Characterization gous AluY repeats, again suggesting a mechanism of nonal- lelic homologous recombination. To define the breakpoints of the deletion, 15 SNPs spanning a region of roughly 110 kb upstream of PRPF31 were ana- lyzed in RP15011 and her parents. The 5Ј border of the Clinical History and Examination deletion was limited by rs54527245, as this SNP was found to be hemizygous. The apparent genotypes of the individu- RP15011. Individual RP15011 belongs to a large Cauca- als were: RP15011 (AA), father (CC), and mother (AA). The sian family (Fig. 5A, individual IV:1) who, at age 33, had parental genotypes are clearly incompatible with the daugh- presented to Moorfields Eye Hospital with worsening pe- ter, and as such, the true genotypes were: RP15011 (A-), ripheral vision and a 10-year history of night blindness. The father (C-) and mother (AA). The maximal border of the SNP most recent clinical examination showed Snellen visual acu- was limited by rs4806681, for which the patient and their ity of 6/9 both eyes and visual field testing showed a mid- father were heterozygous (AT). This region contains 22 Alu peripheral field defect. Bilateral optic disc pallor was pres- repeats lying in the same orientation as the PRPF31 intron ent, with attenuated retinal vessels and intraretinal pigment 13 AluY (Fig. 2). migration to the midperiphery (Figs. 5B and 5C). Electro- A PCR product was obtained using a forward primer lying retinogram showed minimal photopic response to the single in the intergenic region between CACNG6 and VSTM1 stimulus and grossly subnormal and delayed response to 30 [primer coordinates (hg19): 54521019–54521047] and a Hz flicker. There was no rod cell response. Electro-oculo- reverse primer located within intron 13 of PRPF31 [primer gram was flat in both eyes (not shown). A diagnosis of coordinates (hg19): 54634595–54634572]. Sequencing of retinitis pigmentosa was made. Family history showed both

FIGURE 2. Alu repeats in the region of potential deletion between SNPs rs4806681 and rs54527245. Those in the same orientation as PRPF31 intron 13 Alu, and thus potential mediators of the deletion, are seen in dark gray.

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FIGURE 3. Characterization of breakpoint in RP15011. (A) Graphical representation of deletion identified in RP15011. (B) Composite electropherogram showing the breakpoint of the deletion. The sequence in AluY2 is not entirely clear (marked with an asterisk) due to enzymatic slippage, caused by presence of Alu poly-A tracts. Unique sequence (sufficient to identify the AluY2 repeat) is, however, visible on either side.

parents as unaffected. However, the paternal grandmother In light of the extent of the deletion, a full medical (II:2) and a great aunt (II:4) on the paternal side both had history and systems review was obtained from the proband, retinitis pigmentosa, and were registered blind in the fifth RP15011 and their asymptomatic father. There was no decade of life (Fig. 5A). The patient’s sister (IV:2) reports known medical history in either individual. Additionally, a significant night blindness, although this individual was un- basic clinical examination was performed of all major sys- willing to undergo formal ophthalmic examination. tems in RP15011. Particular attention was paid to the ner- As the family history suggested a diagnosis of adRP, vous system, with clinical assessment of the cranial nerves, through the paternal side of the family, the father (III:2) peripheral sensory, and peripheral motor systems. There underwent clinical examination at the age of 68. Ophthal- was no abnormality detected in any system. Full blood count mic examination was normal, with visual acuities of 6/6 and routine bone profile biochemistry was performed in both eyes and no visual field abnormality. The fundi were RP15011. All parameters on the full blood count were normal (Figs. 5D and 5E) and electroretinogram at the age of within normal limits and no abnormality in calcium, phos- 58 revealed normal rod and cone cell function (not shown). phate, albumin, or alkaline phosphatase was observed. This was highly suggestive of a family history of adRP with AD2 Family. The clinical history AD2 family has been nonpenetrance. previously described fully by Abu-Safieh et al.13

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FIGURE 4. Composite electropherogram showing the deletion breakpoint in AD2.

DISCUSSION resorption. Osteoclasts are differentiated from the same cell line that gives rise to monocytes and dendritic cells, oste- To date, only six large deletions have been identified in oclastic differentiation being aided by osteoblasts. It is sug- PRPF31, with the largest being 59 kb, found in a Swedish gested that OSCAR is an important regulator of bone-specific 13–15 pedigree. The deletion identified in the Caucasian indi- osteoclast differentiation and formation.21 TARM-1 is a vidual in this study is 112 kb and includes over 90% of poorly characterized gene that shares to PRPF31 and the five genes immediately upstream. The pro- OSCAR, but has no defined function. VSTM1 encodes a band appears to be normal, except for the classical symp- recently described inhibitory immune receptor protein, toms of RP, and her father appears to be entirely asymptom- thought to regulate the activity of phagocyte cells.22 Despite atic with good visual acuity, visual fields, normal retinal the deletion of these genes, the patient did not show any appearance, and no abnormality on electrophysiological abnormality in metabolic bone profile, or any evidence of testing. It is remarkable that a deletion of this magnitude has immune system dysfunction. little (or no) effect on phenotype, but nevertheless these All four of the previously characterized large deletions observations suggest that a single copy of the five genes have been mediated by homologous recombination between (VSTM-1, OSCAR, TFPT, NDUFA3, TARM-1) is enough to 14,15 Alu repeats. This is not surprising as 26.3% of chromo- carry out their normal functions. some 19 is comprised of these repeats.11 The majority of the TFPT, also known as CF3 fusion partner or FB1, codes for Alu repeats belong to old or intermediary classes (e.g., AluJ), a 253- protein, first identified as the fusion part- whereas relatively few represent the young subfamilies ner of the transcription factor E2A in some cases of pediatric 16 (e.g., AluY). The young Alu repeats tend to reside within pre-B-cell acute lymphoblastic leukemia. A normal role for 11 the human protein has not yet been described, but the rat GC-rich regions and are most biologically active. The gene homologue, Tfpt, has been shown to be proapoptotic and deletion breakpoints identified in the two samples in this may modulate apoptosis in the brain.17 There have been no study are both within AluY repeats, adding to the four reports of disease-causing mutations in this gene in humans, previously reported. except as a fusion partner of E2A in leukemia. There may be an important role for repeat elements in NDUFA3 encodes for the alpha subcomplex 3 of the chromosome 19 in disease causation, as well as in maintain- NADH-ubiquinone 1, the first in the ing the high and low expression alleles of PRPF31. Gross of mitochondria.18 Interestingly, gene rearrangements within this region, such as inversions homozygous or compound heterozygous mutations in sub- and translocations, might be prevalent due to the high units of complex I are a common cause of , number of repeats. These rearrangements might cause adRP, an early onset, severe neurodegeneration characterized by but current techniques, like MLPA, are not be able to detect multiple degenerative foci in the central nervous system. these abnormalities. Moreover, next generation sequencing Mutations in many complex I components have been de- of 19q13 is unlikely be successful due to the presence of scribed, including NDUFS8, NDUFS7, and NDUFA2; al- these repeats.23 though no mutations in NDUFA3 have been reported.18–20 The evolution of two wild type PRPF31 alleles could also be OSCAR encodes for the protein osteoclast-associated re- Alu dependent, as these elements have been shown to act as ceptor, an osteoclast-specific member of the leukocyte re- RNA polymerase III promoters, controlling the expression of ceptor complex family. Osteoclasts are a group of bone microRNA on chromosome 19.24 Detailed analysis of this re- marrow-derived cells that play an essential role in bone gion is important to understand the regulation of PRPF31 and

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FIGURE 5. Pedigree and fundus pho- tograph of deletion patients. (A) Ped- igree of RP15011; (B, C) fundi of RP15011 showing classic changes of RP; (D, E) fundi of III.2, the asymp- tomatic father of RP15011, showing no abnormality.

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