A 112 kb Deletion in Chromosome 19q13.42 Leads to Retinitis Pigmentosa

Abstract

Purpose.: This study sets out to identify novel mutations in PRPF31 in a cohort of autosomal dominant retinitis pigmentosa (adRP) patients with a history of nonpenetrance in the family.

Methods.: Twenty-one patients with history of nonpenetrant autosomal dominant retinitis pigmentosa were selected; all underwent full ophthalmic examination. Multiplex ligation-dependent probe analysis (MLPA) was performed and, where a deletion was found, further family members were recruited. An individual suspected to harbor a large deletion was used as a positive control. Analysis of single nucleotide polymorphisms in the upstream region was used to determine the extent of the deletion, and the breakpoint was then characterized by PCR and sequencing.

Results.: In one family, multiplex ligation-dependent probe analysis revealed a novel large deletion in 19q13.4 encompassing exons 1 to 13 of the PRPF31 gene. The mutation was characterized as a deletion of 112 kilobase (kb), encompassing over 90% of PRPF31 and five upstream genes: TFPT, OSCAR, NDUFA3, TARM-1, and VSTM-1. The breakpoint in the positive control family was also characterized. The mechanism of deletion in both families was Alu-mediated nonallelic homologous recombination.

Conclusions.: This study describes two large deletions, one in a previously reported family and one in a new family: the latter represents the largest deletion yet described on chromosome 19 and the first report of the involvement of VSTM-1. Remarkably, heterozygous deletion of this large region (encompassing six genes) produces little or no other clinical disease besides retinitis pigmentosa.

Heterozygous mutations in several ubiquitously expressed RNA splicing factors (PRPF3, PRPF6, PRPF8, PRPF31, and hBrr2) have been found to cause retinitis pigmentosa (RP), ^{1 –5} a clinically and genetically diverse group of disorders characterized by degeneration of the retinal photoreceptors. 

PRPF31 was identified as the causative gene at the RP11 locus, ⁴ with over 40 mutations identified to date, including nonsense, missense, insertion, and deletion mutations. A key feature of PRPF31 associated autosomal dominant retinitis pigmentosa (adRP) is the unique inheritance pattern. The affected families may show nonpenetrance: an individual carrying a mutant allele may not show disease symptoms; they may, however, have affected children. It had been suggested that the partial penetrance is due to the existence of two different wild type PRPF31 alleles, a high expressivity allele and a low expressivity allele. ⁶ If a patient carries a mutant allele and a high-expressivity wild type allele, then the residual level of PRPF31 protein (hPRP31) is sufficient for normal function. If, however, a patient has a mutant allele and a low expressivity allele, then hPRP31 level falls below the threshold for normal retinal function. It has been shown that asymptomatic mutation carriers have a two-fold higher expression level of PRPF31 compared with symptomatic individuals. ⁷  

hPRP31 is associated with the U4/U6 di-snRNP and interacts with hPRP6, to form the U4/U6-U5 tri-snRNP. ⁸ When hPRP31 expression is knocked down by RNA interference, U4/U6 di-snRNPs accumulate in the Cajal bodies and the U4/U6-U5 tri-snRNP cannot form. ⁹ It has been proposed that due to the high demands on splicing components in retinal photoreceptors, a slightly reduced level of splicing factor will have a cumulative detrimental effect on its proper functions. ¹⁰  

PRPF31 lies within a region that is rich in repeat elements, especially Alu repeats. Alu repeats are short-interspersed nuclear elements which account for 10% of the total genome content, although in chromosome 19 these repeats account for around 26.3%. ¹¹ Most of the sense and antisense Alu integration occurs also in the proximity of microRNAs and these together have been implicated in modulation of human genome architecture and in mediation of gene rearrangement in human disorders. ¹²  

In this study, we report a systematic analysis of insertions/deletions in PRPF31 in 21 RP patients with a family history of nonpenetrance. We have identified a 112 kb deletion that encompasses over 90% of PRPF31, together with 5 adjacent (upstream) genes. The mechanism of deletion was Alu-mediated nonallelic homologous recombination and is the largest reported so far in a patient with retinitis pigmentosa. 

Methods

Patient Selection

Twenty-one patients with a diagnosis of adRP were selected, all having had full ophthalmic examination including slit lamp examination, assessment of visual acuity, perimetry, color vision, and electrodiagnostic testing at Moorfields Eye Hospital. The patients' family history was examined for evidence of autosomal dominant inheritance with nonpenetrance. All patients were screened by direct sequencing for mutations in RHO, RDS, and PRPF31. Furthermore, they did not harbor known mutations in IMPDH1, NRL, PRPF8, PRPF3, NR2E3, RP9, and RP1. Informed consent was obtained from all patients before the research being conducted, which was performed according to the tenets of the Declaration of Helsinki. 

Multiplex Ligation-Dependent Probe Analysis (MLPA)

A kit which screens for large insertions or deletions in the four most commonly affected adRP genes: RHO, PRPF31, IMPDH1, and RP1, was used according to the protocol provided by the manufacturer (MRC-Holland P235 Retinitis Pigmentosa kit; MRC-Holland, Amsterdam, Netherlands). MLPA fragments were separated and sized (GeneScan 500 LIZ size standard on 3730 DNA Analyzer; both from Applied Biosystems, Inc., Cheshire, UK) and the results analyzed (GeneMarker v1.8; SoftGenetics, LLC, State College, PA). Each experiment was repeated at least three times to ensure reproducibility. 

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 on microsatellite markers in the 19q13.4 region. 

Single Nucleotide Polymorphism (SNP) Analysis

The NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) was used to select SNPs with high heterozygosity in the Caucasian population. Primer pairs were designed to amplify short fragments (150–400 base pairs) containing the SNP. PCR product was obtained (Taq polymerase; Bioline UK, Ltd., London, UK) following manufacturer's protocol using primers at 100 ng/μL PCR product was sequenced (BigDye Terminator v3.1; Life Technologies, Cheshire, UK), and the results were analyzed (SeqMan; DNAStar, Madison, WI). 

Breakpoint Characterization

Alu repeats within the region defined by SNP analysis were identified with a DNA sequence screening program (RepeatMasker browser; http://www.repeatmasker.org/). Primers were designed to unique upstream sequence to each Alu and PCR performed, as described previously. PCR product produced was then sequenced (as described) and aligned to reference genome sequence. 

Clinical Assessment of Patients Harboring Deletions

Detailed medical history was obtained, including a review of systems to identify any possible health problems in patients with PRPF31 deletion. A standard clinical examination was performed of all major systems, including cardiorespiratory, gastrointestinal, and nervous systems (including assessment of cranial nerves, peripheral motor nerves, and peripheral sensory nerves). Full blood count and metabolic bone profile biochemistry was also performed. 

Results

Twenty-one patients with RP recruited for this study showed autosomal dominant form of inheritance together with partial penetrance in their family pedigree. No mutations were identified in the entire PRPF31 gene by direct sequencing in any of the probands. No known mutations were identified in IMPDH1, NRL, PRPF8, PRPF3, NR2E3, RP9, RHO, RDS, and RP1. To investigate whether these patients had large deletions or insertions in PRPF31, their DNA samples were subjected to MLPA. As a positive control, we included individual IV:2 from AD2 family (hereafter referred to as individual AD2), as this family had previously been shown to have a large deletion in PRPF31 based on microsatellite data, although no specific deletion boundaries were defined in that study. 

Multiplex Ligation-Dependent Probe Analysis (MLPA)

Out of 21 patients tested, MLPA analysis identified a deletion encompassing multiple exons in PRPF31 in one patient. Patient RP15011 was identified as carrying a deletion encompassing exons 1 to 13 (Fig. 1A). Her parents were subsequently recruited for this study and their DNA samples were subjected to MLPA. The father of the proband carried the same deletion whereas the mother was normal (Figs. 1B and 1C). A deletion of exons 4–13 (inclusive) was detected in AD2 (Fig. 1D). 

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.

Deletion Breakpoint Characterization

To define the breakpoints of the deletion, 15 SNPs spanning a region of roughly 110 kb upstream of PRPF31 were analyzed in RP15011 and her parents. The 5′ border of the deletion was limited by rs54527245, as this SNP was found to be hemizygous. The apparent genotypes of the individuals were: RP15011 (AA), father (CC), and mother (AA). The parental genotypes are clearly incompatible with the daughter, and as such, the true genotypes were: RP15011 (A-), father (C-) and mother (AA). The maximal border of the SNP was limited by rs4806681, for which the patient and their father were heterozygous (AT). This region contains 22 Alu repeats lying in the same orientation as the PRPF31 intron 13 AluY (Fig. 2). 

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.

A PCR product was obtained using a forward primer lying in the intergenic region between CACNG6 and VSTM1 [primer coordinates (hg19): 54521019–54521047] and a reverse primer located within intron 13 of PRPF31 [primer coordinates (hg19): 54634595–54634572]. Sequencing of the product detected a deletion of 112 kb, encompassing the genes VSTM-1, OSCAR, TFPT, NDUFA3, TARM-1, and >90% of PRPF31 (Fig. 3A). Each of the two breakpoints resides within an AluY repeat, one located in the CACNG6 intergenic region and one within intron 13 of PRPF31 (Fig. 3B). The mechanism of deletion therefore appears to be unequal recombination between highly homologous AluY repeats. 

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.

For the characterization of breakpoint in AD2, PCR was performed with primers in intron 3 and intron 13 of PRPF31. Sequencing of the product identified an 11 kb deletion encompassing exons 4 to 13 of the gene, inclusive (Fig. 4). This breakpoint also occurs within highly homologous AluY repeats, again suggesting a mechanism of nonallelic homologous recombination. 

Figure 4.

 

Composite electropherogram showing the deletion breakpoint in AD2.

Clinical History and Examination

RP15011.

Individual RP15011 belongs to a large Caucasian family (Fig. 5A, individual IV:1) who, at age 33, had presented to Moorfields Eye Hospital with worsening peripheral vision and a 10-year history of night blindness. The most recent clinical examination showed Snellen visual acuity of 6/9 both eyes and visual field testing showed a midperipheral field defect. Bilateral optic disc pallor was present, with attenuated retinal vessels and intraretinal pigment migration to the midperiphery (Figs. 5B and 5C). Electroretinogram showed minimal photopic response to the single stimulus and grossly subnormal and delayed response to 30 Hz flicker. There was no rod cell response. Electro-oculogram was flat in both eyes (not shown). A diagnosis of retinitis pigmentosa was made. Family history showed both parents as unaffected. However, the paternal grandmother (II:2) and a great aunt (II:4) on the paternal side both had retinitis pigmentosa, and were registered blind in the fifth decade of life (Fig. 5A). The patient's sister (IV:2) reports significant night blindness, although this individual was unwilling to undergo formal ophthalmic examination. 

Figure 5.

 

Pedigree and fundus photograph of deletion patients. (A) Pedigree of RP15011; (B, C) fundi of RP15011 showing classic changes of RP; (D, E) fundi of III.2, the asymptomatic father of RP15011, showing no abnormality.

As the family history suggested a diagnosis of adRP, through the paternal side of the family, the father (III:2) underwent clinical examination at the age of 68. Ophthalmic examination was normal, with visual acuities of 6/6 both eyes and no visual field abnormality. The fundi were normal (Figs. 5D and 5E) and electroretinogram at the age of 58 revealed normal rod and cone cell function (not shown). This was highly suggestive of a family history of adRP with nonpenetrance. 

In light of the extent of the deletion, a full medical history and systems review was obtained from the proband, RP15011 and their asymptomatic father. There was no known medical history in either individual. Additionally, a basic clinical examination was performed of all major systems in RP15011. Particular attention was paid to the nervous system, with clinical assessment of the cranial nerves, peripheral sensory, and peripheral motor systems. There was no abnormality detected in any system. Full blood count and routine bone profile biochemistry was performed in RP15011. All parameters on the full blood count were within normal limits and no abnormality in calcium, phosphate, albumin, or alkaline phosphatase was observed. 

AD2 Family.

The clinical history AD2 family has been previously described fully by Abu-Safieh et al. ¹³  

Discussion

To date, only six large deletions have been identified in PRPF31, with the largest being 59 kb, found in a Swedish pedigree. ^{13 –15} The deletion identified in the Caucasian individual in this study is 112 kb and includes over 90% of PRPF31 and the five genes immediately upstream. The proband appears to be normal, except for the classical symptoms of RP, and her father appears to be entirely asymptomatic with good visual acuity, visual fields, normal retinal appearance, and no abnormality on electrophysiological testing. It is remarkable that a deletion of this magnitude has little (or no) effect on phenotype, but nevertheless these observations suggest that a single copy of the five genes (VSTM-1, OSCAR, TFPT, NDUFA3, TARM-1) is enough to carry out their normal functions. 

TFPT, also known as CF3 fusion partner or FB1, codes for a 253-amino acid protein, first identified as the fusion partner of the transcription factor E2A in some cases of pediatric pre-B-cell acute lymphoblastic leukemia. ¹⁶ A normal role for the human protein has not yet been described, but the rat homologue, Tfpt, has been shown to be proapoptotic and may modulate apoptosis in the brain. ¹⁷ There have been no reports of disease-causing mutations in this gene in humans, except as a fusion partner of E2A in leukemia. 

NDUFA3 encodes for the alpha subcomplex 3 of the NADH-ubiquinone oxidoreductase 1, the first enzyme in the electron transport chain of mitochondria. ¹⁸ Interestingly, homozygous or compound heterozygous mutations in subunits of complex I are a common cause of Leigh syndrome, an early onset, severe neurodegeneration characterized by multiple degenerative foci in the central nervous system. Mutations in many complex I components have been described, including NDUFS8, NDUFS7, and NDUFA2; although no mutations in NDUFA3 have been reported. ^{18 –20}  

OSCAR encodes for the protein osteoclast-associated receptor, an osteoclast-specific member of the leukocyte receptor complex family. Osteoclasts are a group of bone marrow-derived cells that play an essential role in bone resorption. Osteoclasts are differentiated from the same cell line that gives rise to monocytes and dendritic cells, osteoclastic differentiation being aided by osteoblasts. It is suggested that OSCAR is an important regulator of bone-specific osteoclast differentiation and formation. ²¹ TARM-1 is a poorly characterized gene that shares sequence homology to OSCAR, but has no defined function. VSTM1 encodes a recently described inhibitory immune receptor protein, thought to regulate the activity of phagocyte cells. ²² Despite the deletion of these genes, the patient did not show any abnormality in metabolic bone profile, or any evidence of immune system dysfunction. 

All four of the previously characterized large deletions have been mediated by homologous recombination between Alu repeats. ^14,15 This is not surprising as 26.3% of chromosome 19 is comprised of these repeats. ¹¹ The majority of the Alu repeats belong to old or intermediary classes (e.g., AluJ), whereas relatively few represent the young subfamilies (e.g., AluY). The young Alu repeats tend to reside within GC-rich regions and are most biologically active. ¹¹ The gene deletion breakpoints identified in the two samples in this study are both within AluY repeats, adding to the four previously reported. 

There may be an important role for repeat elements in chromosome 19 in disease causation, as well as in maintaining the high and low expression alleles of PRPF31. Gross gene rearrangements within this region, such as inversions and translocations, might be prevalent due to the high number of repeats. These rearrangements might cause adRP, but current techniques, like MLPA, are not be able to detect these abnormalities. Moreover, next generation sequencing of 19q13 is unlikely be successful due to the presence of these repeats. ²³  

The evolution of two wild type PRPF31 alleles could also be Alu dependent, as these elements have been shown to act as RNA polymerase III promoters, controlling the expression of microRNA on chromosome 19. ²⁴ Detailed analysis of this region is important to understand the regulation of PRPF31 and may account for the nonpenetrance of disease within families affected by PRPF31-associated adRP. 

Footnotes

 Supported by National Institute for Health Research UK (Moorfields Eye Hospital and Institute of Ophthalmology, London, United Kingdom).

Footnotes

 Disclosure: A.M. Rose, None; R. Mukhopadhyay, None; A.R. Webster, None; S.S. Bhattacharya, None; N.H. Waseem, None

The authors thank the patients for participating in this study and all members of the Bhattacharya laboratory for insightful discussions and suggestions.