OPHTHALMIC MOLECULAR GENETICS SECTION EDITOR: EDWIN M. STONE, MD, PhD Paternal Uniparental Heterodisomy With Partial Isodisomy of Chromosome 1 in a Patient With Retinitis Pigmentosa Without Hearing Loss and a Missense Mutation in the Usher Syndrome Type II Gene USH2A Carlo Rivolta, PhD; Eliot L. Berson, MD; Thaddeus P. Dryja, MD Objective: To evaluate a form of nonmendelian inher- cue or gamete complementation. A paternal second cousin itance in a patient with retinitis pigmentosa (RP). of the patient also had RP and also had an identical het- erozygous mutation in the USH2A gene in the same codon. Methods: Direct DNA sequencing of the USH2A cod- However, the analysis of an isocoding polymorphism 20 ing region and microsatellite analysis of polymorphic base pairs away and closely linked microsatellite mark- markers from chromosome 1 and other chromosomes. ers in the patient and family members indicated that the 2 mutant alleles are unlikely to be identical by descent Results: A patient with RP without hearing loss caused and that the 2 relatives fortuitously had RP and a muta- by the homozygous mutation Cys759Phe in the USH2A tion in the same codon of the USH2A gene. gene on chromosome 1q was found to be the daughter of a noncarrier mother and a father who was heterozy- Conclusion: This family illustrates that recessive RP with- gous for this change. Further evaluation with microsat- out hearing loss can rarely be inherited from only 1 un- ellite markers revealed that the patient had inherited 2 affected carrier parent in a nonmendelian manner. copies of chromosome 1 from her father and none from her mother. The paternally derived chromosome 1’s were Clinical Relevance: The genetic counseling of fami- heteroallelic from the centromere of chromosome 1 to lies with recessively inherited eye diseases must take into the proximal short and long arms. The distal regions of consideration the possibility that an unaffected hetero- the short and long arms of chromosome 1 were homoal- zygous carrier can have an affected offspring homozy- lelic, including the region of 1q with the mutant USH2A gous for the same mutation, even if the carrier’s spouse allele. This genetic pattern is compatible with a phenom- has wild-type alleles at the disease locus. enon of uniparental primary heterodisomy with regions of homozygosity arising through a nondisjunction event during paternal meiosis I and subsequent trisomy res- Arch Ophthalmol. 2002;120:1566-1571 OME EXCEPTIONAL individu- most all were ascertained because they had als inherit 2 copies of a chro- a genetic disease resulting either from a ho- mosome from one parent and mozygous mutation on the affected chro- no copy from the other par- mosome pair or from unbalanced imprint- ent. This nonmendelian form ing of genes on the affected chromosome Sof inheritance is called uniparental di- pair.3 Very few cases of retinal disease have somy and is a consequence of at least 2 in- been reported with this aberrant mode of dependent errors occurring during meio- chromosomal transmission.4,5 sis or immediately after fertilization.1,2 If The USH2A gene on chromosome 1q the chromosomes in the uniparentally in- was first identified as a cause of Usher syn- herited pair are identical, ie, if they origi- drome type II.6 Patients with this reces- nate from the same parental chromo- sively inherited disease have both retini- From the Ocular Molecular some, the condition is termed isodisomy. tis pigmentosa (RP) and incomplete Genetics Institute (Drs Rivolta Alternatively, if the members of the uni- hearing loss. We recently reported that cer- and Dryja) and the parentally inherited chromosome pair are tain mutations in the USH2A gene, such Berman-Gund Laboratory for the Study of Retinal different and originate from both chro- as the missense mutation Cys759Phe, can Degenerations (Dr Berson), mosomes from a single parent, the con- produce RP without hearing loss (ie, non- 7 Harvard Medical School, dition is termed heterodisomy. Many ex- syndromic RP). In this article, we de- Massachusetts Eye and Ear amples of humans with isodisomy and scribe our subsequent evaluation of one Infirmary, Boston, Mass. heterodisomy have been reported, and al- of the extended families from that study (REPRINTED) ARCH OPHTHALMOL / VOL 120, NOV 2002 WWW.ARCHOPHTHALMOL.COM 1566 ©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 with 2 members affected with RP without hearing loss and with an USH2A mutation. METHODS SUBJECTS This study involved human subjects and conformed to the Dec- laration of Helsinki. The index patient in this study (003-281) was one of the subjects of a previous report from our group.7 226-1683 226-1641 226-1644 226-1742 226-1649 CLINICAL AND FUNCTIONAL INVESTIGATIONS F + + + codon 759 + + + F + F H + + + codon 752 + + + + + + 5 3 2 3 D1S229 2 1 3 3 3 3 In all cases, the diagnosis of RP was based on the results of 4 3 3 2 D1S490 4 1 2 3 2 3 an ophthalmological examination that included electroreti- 5 2 2 3 D1S237 2 2 3 1 3 1 nography.8 1 1 1 1 D1S474 1 2 1 1 1 1 MOLECULAR ANALYSIS Blood samples were obtained from the relatives of the index patient, and leukocyte DNA was purified from those samples. 003-281 121-397 226-1642 226-1643 F + codon 759 F F + F + + An affected relative of the index patient (121-397) had a sec- H + codon 752 + + + + + + ond blood sample drawn for chromosome analysis conducted 5 2 D1S229 3 3 2 3 2 3 using standard karyotyping methods in a clinical cytogenetics 4 3 D1S490 3 3 4 3 4 2 laboratory (Brigham and Women’s Hospital, Boston, Mass). The 5 2 D1S237 1 1 2 1 2 3 DNA from one deceased family member (226-1742) was ob- 1 1 D1S474 1 1 1 1 1 1 tained from the paraffin blocks of intestinal tissue stored in the pathology department of a local hospital. The method of DNA purification from the paraffin blocks was according to the DNeasy Tissue Kit from Qiagen Inc (Valencia, Calif), except 226-1650 226-1651 that twice the suggested amount of proteinase K was used. codon 759 + F + F To screen for mutations in the USH2A gene, genomic frag- codon 752 + + + + ments were amplified from 20 to 100 ng of DNA. The primers D1S229 2 3 2 3 used for the polymerase chain reaction (PCR) are provided at D1S490 3 3 3 3 D1S237 2 1 2 1 our laboratory’s Web site (http://eyegene.meei.harvard.edu). Am- D1S474 1 1 1 1 plified DNA fragments were sequenced in both the sense and antisense directions using an ABI Prism 377 sequencer (Ap- Figure 1. Segregation of USH2A alleles and associated haplotypes within plied Biosystems, Foster City, Calif). members of family 5014. Haplotypes associated with the USH2A mutation To amplify microsatellite markers from chromosome 1 from detected in patient 121-397 are boxed. Arrow indicates the index patient; leukocyte DNA, we used commercially available primers (Map- squares, males; circles, females; filled circles, females with retinitis pigmentosa; slashes, deceased individuals; F, Cys759Phe (TGCϾTTC); H, Pairs; Research Genetics, Inc, Carlsbad, Calif). The position of His752His (CATϾCAC); and +, wild-type USH2A sequence. these markers on chromosome 1 was determined according to the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/genome/guide/human; ac- other pathogenic mutation in the coding sequence or in cessed October 10, 2001). For each primer pair, PCR cycling the intron splice sites flanking the 21 exons of this gene conditions were performed according to the manufacturer’s pro- was found by DNA sequence analysis. Analysis of DNA tocol whereas the buffer composition and the annealing tem- perature were according to that reported in the Genome Da- from this patient’s parents showed that the Cys759Phe al- tabase (http://www.gdb.org). The concentration of dATP, dTTP, lele had been inherited from the patient’s mother (see the and dGTP in the reaction buffer was 0.02 mM, and the con- schematic pedigree in Figure 1). Like every other index centration of dCTP was 0.002 mM supplemented with about patient whom we have encountered with this missense 22.2 kBq of 33P-␣-dCTP at 111 TBq/mmol. The PCR-amplified change, this patient also had a syntenic isocoding change DNA fragments were diluted 1:1 (v:v) with a solution of 95% in codon His752 (CAT to CAC) only 20 base pairs (bp) formamide, 20 mM of EDTA, 0.05% bromophenol blue, and away from the site of the missense mutation.7 0.05% xylene cyanol before electrophoresis through 6% dena- The family was remarkable because there was a fe- turing polyacrylamide gels. The PCR amplification of micro- male second cousin on the father’s side (patient 121- satellites used for testing parentage was performed as de- 397) who also had nonsyndromic RP (Figure 1). On ob- scribed by Alford et al.9 Detection of microsatellite alleles was performed by autoradiography. taining a blood sample from this relative, we found her to have the Cys759Phe mutation homozygously but with- out the usually associated His752 isocoding change. More RESULTS remarkably, the mother of patient 121-397 did not carry this mutation (Figure 2). MOLECULAR FINDINGS To obtain more information about the origin of the Cys759Phe mutant alleles in patient 121-397, we ex- The index patient (003-281) with RP was heterozygous tracted DNA from paraffin-embedded fragments of small for the missense mutation Cys759Phe (TGC to TTC).7 No intestine from the proband’s deceased father; the frag- (REPRINTED) ARCH OPHTHALMOL / VOL 120, NOV 2002 WWW.ARCHOPHTHALMOL.COM 1567 ©2002 American Medical Association.