Vision 2012; 18:1226-1237
Received 28 November 2011 | Accepted 7 May 2012 | Published 10 May 2012
Muhammad Ajmal,1,2,3 Muhammad Imran Khan,1,2 Shazia Micheal,1,2 Waqas Ahmed,1 Ashfa Shah,3 Hanka Venselaar,4,5 Habib Bokhari,1 Aisha Azam,6 Nadia Khalida Waheed,3,7 Rob W.J. Collin,2,5,8 Anneke I. den Hollander,2,5,8 Raheel Qamar,1,3 Frans P. M. Cremers1,2,5
The first two authors contributed equally to this work
1Department of Biosciences, Faculty of Science, COMSATS Institute of Information Technology, Islamabad, Pakistan; 2Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Shifa College of Medicine, Islamabad, Pakistan; 4Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 5Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 6Institute of Ophthalmology, Mayo Hospital, Lahore, Pakistan; 7Shifa International Hospital, Islamabad, Pakistan; 8Department of Ophthalmology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Correspondence to: Frans P.M. Cremers, Department of Human Genetics, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; Phone: +31-24-3613750; FAX: +31-24-3668752; email: email@example.com
Purpose: To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families.
Methods: Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements.
Results: The tubby-like protein 1 gene (TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation (c.1138A>G; p.Thr380Ala) in one family and a novel pathogenic variant (c.1445G>A; p.Arg482Gln) in the other family. Both variants were found to be present in a homozygous state in all affected individuals, were heterozygous present in the unaffected parents, and heterozygous present or absent in normal individuals. Affected individuals of both families showed an early-onset form of RP.
Conclusions: Homozygosity mapping, combined with candidate-gene analysis, successfully identified genetic defects in TULP1 in two large Pakistani families with early-onset retinitis pigmentosa.
The major cause of inherited blindness in humans is retinitis pigmentosa (RP; OMIM 268000). The clinical symptoms of RP are the loss of night vision in the early phase of disease, later followed by peripheral vision loss, tunnel vision, and sometimes complete blindness . Progression of the disease is mainly caused by the gradual loss of rod photoreceptor cells, which are mostly responsible for vision under low light conditions, and the subsequent loss of cone photoreceptor cells, which are involved in color vision under bright light conditions. The clinical diagnosis is based on fundus examination and electrophysiological analysis of rod and cone photoreceptor-cell function by measuring the scotopic and photopic responses, respectively, using electroretinography (ERG). The disease’s characteristics are the presence of pigmentary deposits (bone spicules) in the peripheral fundus, diminished or no ERG responses from rod and cone photoreceptor cells, and attenuation of the retinal blood vessels .
The disease is highly genetically heterogeneous, since 55 different genes and three loci have been identified as being associated with nonsyndromic RP (RetNet, Nov. 8, 2011). All Mendelian forms of inheritance have been observed for RP. Digenic forms and rare mitochondrial inheritance have also been reported [1-4].
Tubby-like protein 1 (TULP1; OMIM 602280) belongs to the tubby-like protein family. The TULP1 gene is located on the short arm of chromosome 6 . TULP1 is expressed specifically in the retina [5,6], and the encoded protein is thought to be involved in protein trafficking, such as the transport of rhodopsin from the inner segment to the outer segment via the connecting cilium .
The aim of the current study was to identify the underlying genetic causes of autosomal recessive RP (arRP) in Pakistani families by using genome-wide homozygosity mapping and Sanger sequencing of known retinal disease genes in the homozygous regions. We identified disease-causing mutations in TULP1 in two large families.
Before initiating this study, approval for this work was granted by the Ethics Committee/Institutional Review Board of the Shifa College of Medicine/Shifa International Hospital, Islamabad, Pakistan, and signed informed consent was obtained from all participating individuals in both families.
Two families from the Punjab province with individuals having night vision and daytime vision loss were included in the study. Their pedigrees were drawn (Figure 1A,B) using Haplopainter , and venous blood samples were collected in acid citrate dextrose-containing vacutainers (Becton Dickinson, Franklin Lakes, NJ). Fundus examinations were done, and ERG  measurements were made for probands in both families.
DNA was extracted by using a standard phenol chloroform extraction protocol  and stored at 4 °C. HumanOmniExpress (>700 K) single nucleotide polymorphism (SNP) microarrays from Illumina Inc. (San Diego, CA) were employed to search for homozygous regions in six affected individuals (Figure 1A) in family A and three affected individuals (Figure 1B) in family B. Genotype data were analyzed with Homozygosity Mapper , an online tool for homozygosity mapping using SNP genotyping data. Haplotype comparisons were also done for affected and normal individuals to identify the homozygous regions that were identical in all the affected individuals in each family.
Prior to Sanger sequencing, the 15 protein-coding exons of TULP1 and their flanking intronic sequences were amplified by PCR using standard conditions and reagents. PCR primers were designed with the online primer-designing tool Primer3  (available on request). Amplified PCR products were electrophoresed in 2% agarose gels containing ethidium bromide, and DNA bands were visualized on an ultraviolet transilluminator. PCR products were purified on PCR clean-up purification plates (NucleoFast™ 96 PCR, Cat. No. 743100.10; MACHEREY-NAGEL, Düren, Germany), according to the manufacturer’s protocol. Purified PCR products were subsequently used for Sanger sequencing in an automated DNA sequencer (Big Dye Terminator, version 3, on a 3730 DNA analyzer; Applied Biosystems, Foster City, CA).
Sequencing results were analyzed by using Vector NTI Advance™ 2011 software from Life Technologies/Invitrogen (Bleiswijk, Netherlands), by assembling the sequenced contigs and then visualizing the aligned sequences of the exons.
Restriction fragment length polymorphism analysis was performed to detect the presence or absence, of the identified mutations, in 100 ethnically matched control individuals. For the mutation identified in family A, restriction enzyme HpyCH4III was used, whereas in family B, restriction enzyme MspI was used. In both families, restriction enzyme recognition sites were abolished in the mutant sequences. Purified PCR products were used for restriction enzyme digestion, according to the manufacturer’s protocol (New England BioLabs, Ipswich, MA).
To check the evolutionary conservation of the mutated amino acids, the TULP1 orthologous protein sequences of the following species were aligned with the Vector NTI Advance™ 2011 software: humans (H. sapiens, ENSP00000229771); chimpanzees (P. troglodytes, ENSPTRP00000007764); mice (M. musculus, ENSMUSP00000049070); dogs (C. familiaris, ENSCAFP00000001922); chickens (G. gallus, ENSGALP00000009613); frogs (X. tropicalis, ENSXETP00000000899); tetraodons (T. nigroviridis, ENSTNIP00000004001); fruitflies (D. melanogaster, FBpp0088961); honeybees (A. mellifera, GB19892-PA); roundworms (C. elegans, F10B5.4), blood flukes (S. mansoni, Smp_058730__mRNA); and Arabidopsis (A. thaliana, AT1G76900.1).
In both families, the average age of disease onset was in the first decade of life. Ophthalmic examination of affected individuals from both families revealed the presence of attenuated retinal vessels and the optic disc to have a waxy, pale appearance (Figure 2). A yellow perifoveal annular ring, a characteristic feature of individuals carrying TULP1 mutations, was also clearly visible in family A (Figure 2A,B). In family B, the perifoveal ring was in the process of development (Figure 2C,D). Upon ERG, scotopic and photopic electrophysiological responses of rod and cone photoreceptors, respectively, were diminished in affected members of both families (Table 1). Neither nystagmus nor eye poking were present in either family.
Genome-wide SNP microarray data of six affected individuals of family A were analyzed with the help of homozygosity mapper, which revealed a single homozygous region (Figure 3A) of 3.9 Mb (from 32.9 Mb to 36.8 Mb; hg19) on chromosome 6, flanked by SNPs rs3132131 and rs236411. This homozygous region harbored TULP1, a gene known to be mutated in patients with Leber congenital amaurosis (LCA) and arRP. The sequence analysis identified a previously reported mutation, c.1138A>G (p.Thr380Ala) [14,15] in this family (Figure 1C).
Similarly, genome-wide SNP microarray data analysis of three affected individuals of family B, resulted in the identification of six homozygous regions (Figure 3B). After haplotype comparison, two regions, a 4.8 Mb region on chromosome 6 (from 33.8 Mb to 38.6 Mb, flanked by SNPs rs9296102 and rs7761629) and a 1.4 Mb region on chromosome 7 (from 132.3 Mb to 133.7 Mb, flanked by SNPs rs924368 and rs10249912), were found to be identical in all the affected individuals. TULP1 resides in the homozygous region on chromosome 6, and the sequence analysis revealed a novel mutation, c.1445G>A (p.Arg482Gln; Figure 1C). The homozygous chromosomal region on chromosome 7 did not contain a gene previously implicated in an inherited retinal disease such as arRP or LCA.
In families A and B, the variants c.1138A>G and c.1445G>A, respectively, were found to be present in a homozygous state in all affected individual, were heterozygous present in the unaffected parents, and absent or heterozygous present in normal individuals (Figure 1A,B). Both wild-type nucleotides were shown to be highly conserved, as evidenced by their phylogenetic p value  scores of 2.87 (c.1138A) and 6.10 (c.1445G) for family A and B, respectively. In addition, the encoded amino acids, p.Thr380 and p.Arg482, located in the C-terminal tubby domain, are highly conserved among different vertebrate and invertebrate species, while in a plant (Arabidopsis), isoleucine is present instead of threonine (Figure 4). These amino acids are completely conserved among the tubby, TULP1, TULP2, and TULP3 proteins . SIFT predicts that both TULP1 variants are “not tolerated” whereas Polyphen predicts that both are “probably damaging” with prediction scores of 0.827 and 1.000 for p.Thr380Ala and p.Arg482Gln, respectively.
A three-dimensional-structure prediction analysis by project HOPE predicts that the p.Thr380Ala mutation, due to the smaller size of the alanine residue, causes an empty space in the protein and possible rearrangements of surrounding residues (Figure 5A,B). Any hydrogen bonds made by threonine will also be lost, because alanine is a hydrophobic residue. Very close to Thr380 is a predicted inositol triphosphate binding site that might also be influenced by local conformational changes. The p.Arg482Gln variant changes a positively charged amino acid (arginine) to a neutral residue (glutamine), which results in the loss of interactions with negatively charged residues in its vicinity (Figure 5C). In view of its location in the three-dimensional structure, these changes may result in a loss of external interactions.
The p.Thr380Ala and p.Arg482Gln variants were not detected in 100 healthy ethnically matched control individuals.
In the current study, we report on two large families with several consanguineous marriages and multiple individuals with early-onset RP that were found to carry TULP1 mutations. Family A had 27 affected individuals who resided in the northern part of the Punjab province. This family had a strong custom of marrying inside the family. TULP1 mutations were previously reported to cause LCA, a congenital form of severe vision impairment or blindness and early-onset RP [14,15,18-31]. There is clinical and genetic overlap between (early-onset) arRP and LCA [32,33], and often it is very difficult to differentiate between these two conditions. Mutations in Crumbs homolog 1 (CRB1), Lecithin retinol acyltransferase (LRAT), Mer tyrosine kinase protooncogene (MERTK), Retinal pigment epithelium-specific protein, 65-KD (RPE65), Spermatogenesis-associated protein 7 (SPATA7), and TULP1 have been identified in individuals with LCA and arRP [18-21,34-47]. If a severe form of rod–cone dystrophy is observed beyond early childhood, and when there are no features reminiscent of LCA, such as nystagmus, or eye poking, the phenotype is classified as early-onset RP.
TULP family proteins (TUB, TULP1, TULP2, and TULP3) have crucial roles in embryonic development in vertebrates and take part in the proper functioning of the central nervous system [6,48]. TULP1 is expressed specifically in the rod and cone photoreceptor cells [5,6], and is involved in the transport of rhodopsin . In Tulp1−/− mice, photoreceptor degeneration precedes synaptic malfunction, and thus TULP1 may have a function in photoreceptor synapse development . A mutation of the same residue (p.Arg482Trp) was earlier found in combination with p.Leu504fsX140 in five affected individuals with severe early-onset RP .
The structural analyses of the TULP1 C-terminal domains of the mutant proteins suggest that the missense mutations identified in our study might have resulted in the destabilization of the mutant proteins, or might have influenced the putative interactions of the tubby domain. Among different species of animals and plants, p.Arg482 is located in the signature sequence (F-[KRHQ]-G-R-V-[ST]-x-A-S-V-K-N-F-Q) of the Tubby family of proteins, and this signature sequence contains 11 invariant amino acids that are highly conserved (Prosite) . Replacement of the wild-type residue with the mutant glutamine might affect the signature sequence; this might ultimately prevent the mutant TULP1 protein from functioning normally.
In family A, the presence of a typical yellow-colored perifoveal annular ring was also indicative of TULP1 involvement ; whereas in family B, the ring formation was incomplete (Figure 2C,D). The bone spicules absent from both families might still develop later in life. The age of both individuals who were clinically evaluated was 20 years.
The previously identified mutation p.Thr380Ala has only been reported in two unrelated Pakistani families [14,15]. Our findings concerning this mutation in yet another Pakistani family suggested that c.1138A>G might be a Pakistani founder mutation, although no link was established between any of these families. One family belonged to a northern area of Pakistan  while the other belonged to the southern part of Punjab . Our family belonged to the northern part of Punjab, which, however, does not exclude the possibility that this mutation represents a founder mutation in Pakistan.
Including our findings, 26 different TULP1 mutations have been identified in 33 families (Table 2). TULP1 mutations were found in 4.3% (10/231) of LCA families [14,18,24,25,27,29,30] and 2.4% (23/948) of arRP families [15,19-24,26,28,30,31,50]. TULP1 mutations causing arRP or LCA include two nonsense, two frame-shift, and seven splice-site mutations; a six-base-pair duplication; and 14 missense mutations (Table 2). Splice-site and protein-truncating mutations are distributed throughout the gene (Figure 6A), whereas the missense mutations are only present in the C-terminal tubby domain of TULP1 (Figure 6B).
TULP1 mutations are a frequent cause of LCA and arRP, and therefore represent an attractive therapeutic target. Thus far, TULP1 mutations have been found in a total of 136 individuals with LCA or arRP (Table 2). Through our studies, 33 additional patients with TULP1 mutations might benefit from genetic counseling and future gene-augmentation therapy.
In conclusion, we were able to identify one novel (c.1445G>A; p.Arg482Gln) and one previously identified (c.1138A>G; p.Thr380Ala) disease-causing mutation in TULP1 in Pakistani families with early-onset RP.
We thank both families for their participation. This work was supported by grant no. PAS/I-9/Project awarded (to R.Q.) by the Pakistan Academy of Sciences and a core grant from the Shifa College of Medicine. We also acknowledge the Higher Education Commission of Pakistan for awarding M.A. an IRSIP Scholarship, which allowed him to work at the Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. This work was also financially supported by the Foundation Fighting Blindness, United States (to F.P.M.C, A.I.d.H. and R.W.J.C.), the Netherlands Organisation for Scientific Research, TOP-grant 91209047 (to F.P.M.C. and A.I.d.H.), the Stichting Nederlands Oogheelkundig Onderzoek, the Nelly Reef Foundation, and the Stichting ter Verbetering van het Lot der Blinden (to F.P.M.C., R.W.J.C., and A.I.d.H.).