Clin Genet 2013: 84: 290–293Printed in Singapore. All rights reserved

© 2012 John Wiley & Sons A/S.Published by John Wiley & Sons Ltd

CLINICAL GENETICSdoi: 10.1111/cge.12039

Letter to the Editor

Homozygosity mapping identifies geneticdefects in four consanguineous familieswith retinal dystrophy from Pakistan

To the Editor :Retinitis pigmentosa (RP) is a progressive retinal

degeneration, which primarily affects rod photoreceptorcells. Typical RP features include nyctalopia, bonespicule pigmentation, attenuation of retinal arterioles,and reduced electroretinogram (ERG) (1). Defects in36 genes are known for autosomal recessive RP (arRP)(http://www.sph.uth.tmc.edu/retnet), which explain thedisease in about 50% of the patients (2).

Homozygosity mapping has been very successful inidentifying genetic defects in the Pakistani populationas >80% of families show consanguinity. Homozy-gosity mapping was utilized to identify genetic defectsin four consanguineous Pakistani families. Patientswere informed about the aims and objectives of theproject before obtaining consent. Affected personsfrom families RP03, RP48, and RP55 had typicalfeatures of classical RP (Table 1). In family RP27,early-onset RP was observed along with maculardegeneration, which is indistinguishable from Lebercongenital amaurosis (LCA), as reported previously insome cases (Table 1) (3).

Genomic DNA samples of selected persons wereanalyzed using whole genome single nucleotidepolymorphism arrays (Fig. 1a). Family RP27 wasgenotyped using the Illumina 6K array, whilefamilies RP03, RP48, and RP55 were analyzed

Table 1. Clinical features of the probands with arRP or LCA

Family

Age atexamination

(years)Age of

onset (years) Fundus ERG VA (RE, LE) Diagnosis

RP03 30 10–15 ARV, peripheral BSP, prominentmacular degeneration

Rod-cone pattern 6/60, 6/60 RP

RP27 35 2–5 Prominent macular degeneration,widespread BSP

Extinguished LP Early onset RP/LCA

RP48 33 10–20 ARV, peripheral BSP, maculardegeneration

Rod-cone pattern CF RP

RP55 45 25–30 ARV, peripheral BSP, prominentmacular degeneration

Rod-cone pattern 6/60, 6/60 RP

ARV, attenuated retinal vessels; BSP, bone spicule pigmentation; CF, counting finger; ERG, electroretinogram; LCA, Leber congenitalamaurosis; LE, left eye; LP, light perception; RE, right eye; RP, Retinitis pigmentosa; VA, visual acuity.

on Human OmniExpress platforms. The genotypedata were analyzed with homozygosity mapper(http://www.homozygositymapper.org), the outputplots and details about the homozygous regions arepresented in Fig. 2 and Table 2, respectively. Sangersequencing was used to identify causative variants andto screen the identified mutations in probands from 50unsolved Pakistani retinal dystrophy (RD) families torule out founder effects.

RP03 : Of the eight homozygous regions shared bythe affected persons, the second largest region harboreda known arRP gene ABCA4 (MIM#601691). Sequenceanalysis revealed a homozygous nonsense mutation,c.6658C>T (p.Gln2220*), segregating with the disease(Fig. 1a). This null mutation was previously reported ina heterozygous state in a person with isolated cone-roddystrophy (4). The genotypic results are in agreementwith the genotype–phenotype model described forABCA4 mutations, where two severe mutations, such asin RP03, can cause a severe phenotype like RP (4, 5).

RP27 : The largest homozygous region contained twoknown arLCA genes, i.e. AIPL1 (MIM#604392) andGUCY2D (MIM#600179). Sequencing of all exons ofGUCY2D revealed no variation. Sequencing of AIPL1revealed a nonsense mutation, c.834G>A (p.Trp278*),which is the most common AIPL1 mutation describedworldwide, including Pakistan (3).

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(a)

(b)

Fig. 1. Segregation of genetic variants in families with autosomal recessive retinitis pigmentosa and protein modeling of PDE6B missense variant.(a) Pedigrees of retinal dystrophy families and segregation analysis of pathological variants. M1 (ABCA4; c.6658C>T, p.Gln2220*), M2 (AIPL1;c.834G>A, p.Trp278*), M3 (CERKL; c.847C>T, p.Arg283*), M4 (PDE6A; c.1630C>T, p.Arg544Trp). Individuals genotyped using whole genomesingle nucleotide polymorphism arrays are marked with asterisks. Arrows point to the probands. (b) Three-dimensional structure of heterotetramericcomplex of cyclic guanosine monophosphate phosphodiesterase (cGMP-PDE). (I) Structure of PDE6A (gray) and PDE6B (tan) protein complex inthe native conformation. Arg544 is colored green. (II) Part of PDE complex showing the location of the mutant residue, on the surface, close to themonomer interaction site. (III) Close-up of the mutation. Wild-type and mutant residues are shown and colored green and red, respectively. Yellowdots indicate the hydrogen bond formed by the wild-type residue, which will be lost due to the mutation.

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Fig. 2. Outputs of homozygosity mapper for four arRP families. The numbers on top indicate the chromosomes. Bars extending above 0.8 representsputative homozygous regions in all affected individuals.

Table 2. Homozygous regions found in arRP/LCA families

Family Chr.Flanking single

nucleotide polymorphismsRegion in

Mbp (hg19) Size in Mb RankingKnown arRP/LCA genes

RP03 1 rs743117;rs716581 94.4–102.3 7.9 2 ABCA42 rs20349;rs17722726 134.6–137.6 3.0 7 –4 rs730349;rs936232 21.2–25.8 4.6 5 –4 rs543337;rs1461605 82.7–92.0 9.3 1 –6 rs2894891;rs1475270 100.2–106.7 6.5 3 –7 rs4728251;rs1424376 131.8–137.7 5.9 4 –

14 rs1007813;rs9324014 96.0–100.2 4.2 6 –14 rs17172;rs872945 101.4–103.5 2.1 8 –

RP27 6 rs875590;rs9364286 166.1–169.2 3.1 4 –8 rs753012;rs115429695 8.9–11.8 2.9 5 –

17 rs7221818;rs1860300 5.8–11.1 5.3 1 AIPLI, GUCY2D17 rs2017167;rs34727469 32.5–36.8 4.3 3 –21 rs1892687;rs13625 36.3–40.6 4.3 2 –

RP48 2 rs6734093;rs12693396 182.2–185.7 3.5 1 CERKLRP55 5 rs7711546;rs10515714 143.6–154.9 11.3 1 PDE6A

RP48 : A single homozygous region on chromosome2 was identified, that encompassed a known arRP geneCERKL (MIM#608381). Sequence analysis of CERKLrevealed a nonsense mutation, c.847C>T (p.Arg283*),which was first identified in two Spanish families andseven other probands with arRP (6).

RP55 : A single homozygous region on chromosome5 was observed, which contained PDE6A (MIM#180071). Sequence analysis revealed a novel

homozygous missense variant, c.1630C>T(p.Arg544Trp), segregating with the disease (Fig. 1a).Eighteen different mutations have been reported inPDE6A, the majority (56%; 10/18) are missense (7).The variant was considered to be likely pathogenic as itwas not detected in 80 ethnically matched controls, andonline prediction tools (PolyPhen2 and SIFT) predictit to be damaging. Homology based protein modelinganalysis of mutant and wild-type proteins indicate that

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the mutation likely disrupts the hydrogen and ionicinteractions between the monomers and will destabilizethe structure and affect the function of protein (Fig. 1b).

In conclusion, through homozygosity mapping andsequence analysis, we were able to identify the geneticdefects in 55% Pakistani RD families. The identificationof genetic defects will not only be helpful in geneticcounseling but will also be beneficial in selectingpatients that are eligible for gene therapy in near future.

MI Khana,b

M Ajmala,b,c

S Micheala,b,d

M Azama,b

A Hussaina

A Shahzadc

H Venselaare

H Bokharia

IJ de Wijsb

LH Hoefslootb

NK Waheedf

RWJ Collinb,g

AI den Hollanderb,d,g

R Qamara,c

FPM Cremersa,b,g

aDepartment of Biosciences, COMSATS Institute ofInformation Technology, Islamabad, Pakistan,

bDepartment of Human Genetics, Radboud UniversityNijmegen Medical Centre, Nijmegen, The Netherlands,

cPCR Laboratories, Shifa College of Medicine, Islamabad,Pakistan,

dDepartment of Ophthalmology,eRadboud University Nijmegen Medical Centre, Centre for

Molecular and Biomolecular Informatics, Nijmegen,The Netherlands,

fDepartment of Ophthalmology, Shifa InternationalHospital, Islamabad, Pakistan, and

gNijmegen Centre for Molecular Life Sciences, RadboudUniversity Nijmegen Medical Centre, Nijmegen,

The Netherlands

References1. Heckenlively JR. Retinitis pigmentosa. Philadelphia: Lippincott, 1988.2. den Hollander AI, Black A, Bennett J, Cremers FPM. Lighting a candle

in the dark: advances in genetics and gene therapy of recessive retinaldystrophies. J Clin Invest 2010: 120: 3042–3053.

3. Dharmaraj S, Leroy BP, Sohocki MM et al. The phenotype ofLeber congenital amaurosis in patients with AIPL1 mutations. ArchOphthalmol 2004: 122: 1029–1037.

4. Maugeri A, Klevering BJ, Rohrschneider K et al. Mutations in theABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am J Hum Genet 2000: 67: 960–966.

5. Cremers FPM, van de Pol TJR, van Driel M et al. Autosomal recessiveretinitis pigmentosa and cone-rod dystrophy caused by splice sitemutations in the Stargardt’s disease gene ABCR. Human Mol Genet1998: 7: 355–362.

6. Avila-Fernandez A, Riveiro-Alvarez R, Vallespin E et al. CERKLmutations and associated phenotypes in seven Spanish families withautosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci2008: 49: 2709–2713.

7. Dryja TP, Rucinski DE, Huang Chen S, Berson EL. Frequencyof mutations in the gene encoding the a subunit of rod cGMP-phosphodiesterase in autosomal recessive retinitis pigmentosa. InvestOphthalmol Vis Sci 1999: 40: 1859–1865.

Correspondence:Muhammad Imran KhanDepartment of Biosciences COMSATS Institute of InformationTechnology Islamabad PakistanTel.: +92 5190405036Fax: +92 519247008e-mail: mimranmani@gmail.com

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