Segregation of USH2A alleles and associated haplotypes within members of family 5014. Haplotypes associated with the USH2A mutation detected in patient 121-397 are boxed. Arrow indicates the index patient; squares, males; circles, females; filled circles, females with retinitis pigmentosa; slashes, deceased individuals; F, Cys759Phe (TGC>TTC); H, His752His (CAT>CAC); and +, wild-type USH2A sequence.
The DNA sequence of USH2A codons 752 to 759, in selected members of family 5014. ID numbers of patients and unaffected relatives are indicated on the left. The gray-shaded columns highlight the isocoding change in His752 on the left and the missense mutation in codon Cys759 on the right.
Allele distribution of microsatellite markers scattered along chromosome 1 in patient 121-397, her parents, and her sisters. The polymorphic markers and the USH2A gene were placed on the physical and cytogenetic maps of chromosome 1, according to the information contained in the NCBI database (http://www.ncbi.nlm.nih.gov/genome/guide/human). Allelotypes are given in columns below each family member's symbol and identification number in the schematic pedigree depicted at the top of the figure. Deduced regions of chromosome 1 isodisomy (ID) and heterodisomy(HD) in patient 121-397 are indicated to the left of her allelotypes. The 2 recombination events that likely occurred during paternal meiosis I are indicated by X's (see text and Figure 5). Square with slash indicates deceased male; circles, females; filled circle, female with retinitis pigmentosa; F, Cys759Phe (TGC>TTC); +, wild-type USH2A allele; and ND, not done.
Pure-tone air-conduction audiograms of patient 121-397 at age 54 years. The shaded areas indicate the hearing ranges (mean values ± 1 SD) for women with normal hearing aged 48 to 59 years11 and for patients with Usher syndrome type II (men and women) aged 50 to 59 years.12
Schematic diagram of the events leading to primary and secondary heterodisomy, compared with normal gametogenesis, for a given pair of autosomes. In regular meiosis, each pair of chromosomes is separated during the first meiotic division, whereas the sister chromatids of each chromosome detach during the second meiotic division. If nondisjunction occurs during the first meiotic division, both chromosomes of a pair (or none at all) can be transmitted to a gamete. In contrast, in a nondisjunction event occurring during the second meiotic division, a gamete can have 2 identical copies of the same chromosome formed from both of its sister chromatids. In this example, 2 homologous recombination events designated by X's (1 affecting each chromosome arm) occur in prophase of meiosis I (top row). If there is nondisjunction during meiosis I (second row, center column), 2 disomic gametes carrying heteroallelic regions near the centromere are generated, as well as 2 gametes that are nullisomic for that particular chromosome. Depending on the chromatid segregation during the second meiotic division, these disomic gametes can generate a condition of centromeric heterodisomy with bitelomeric isodisomy or complete heterodisomy (not depicted in the figure), both of which depend on subsequent gamete complementation or trisomy rescue. In contrast, a nondisjunction event during the second meiotic division (third row, right column) produces secondary heterodisomy with isodisomic centromeric regions and, possibly, heteroallelic sequences at the telomeres. The genotype detected in patient 121-397 (paternal heterodisomy with partial isodisomy for chromosome 1, with heteroallelic centromeric sequences and homoallelic regions at both telomeres) is likely to have originated from either of the 2 hyperhaploid spermatozoa (indicated with asterisks in the fourth row, middle column) produced by a nondisjunction event in meiosis I and subsequent gamete complementation or trisomy rescue in the zygote. Vertical hatched lines indicate how chromosomes or chromatids are assorted during cell division.
Rivolta C, Berson EL, Dryja TP. 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. Arch Ophthalmol. 2002;120(11):1566-1571. doi:10.1001/archopht.120.11.1566
EDWIN M.STONEMD, PhD
Copyright 2002 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2002
To evaluate a form of nonmendelian inheritance in a patient with retinitis pigmentosa (RP).
Direct DNA sequencing of the USH2A coding region and microsatellite analysis of polymorphic markers from chromosome 1 and other chromosomes.
A patient with RP without hearing loss caused by the homozygous mutation Cys759Phe in the USH2A gene on chromosome 1q was found to be the daughter of a noncarrier mother and a father who was heterozygous for this change. Further evaluation with microsatellite markers revealed that the patient had inherited 2 copies of chromosome 1 from her father and none from her mother. The paternally derived chromosome 1's were heteroallelic from the centromere of chromosome 1 to the proximal short and long arms. The distal regions of the short and long arms of chromosome 1 were homoallelic, including the region of 1q with the mutant USH2A allele. This genetic pattern is compatible with a phenomenon of uniparental primary heterodisomy with regions of homozygosity arising through a nondisjunction event during paternal meiosis I and subsequent trisomy rescue or gamete complementation. A paternal second cousin of the patient also had RP and also had an identical heterozygous mutation in the USH2A gene in the same codon. However, the analysis of an isocoding polymorphism 20 base pairs away and closely linked microsatellite markers in the patient and family members indicated that the 2 mutant alleles are unlikely to be identical by descent and that the 2 relatives fortuitously had RP and a mutation in the same codon of the USH2A gene.
This family illustrates that recessive RP without hearing loss can rarely be inherited from only 1 unaffected carrier parent in a nonmendelian manner.
The genetic counseling of families with recessively inherited eye diseases must take into consideration the possibility that an unaffected heterozygous carrier can have an affected offspring homozygous for the same mutation, even if the carrier's spouse has wild-type alleles at the disease locus.
SOME EXCEPTIONAL individuals inherit 2 copies of a chromosome from one parent and no copy from the other parent. This nonmendelian form of inheritance is called uniparental disomy and is a consequence of at least 2 independent errors occurring during meiosis or immediately after fertilization.1,2 If the chromosomes in the uniparentally inherited pair are identical, ie, if they originate from the same parental chromosome, the condition is termed isodisomy. Alternatively, if the members of the uniparentally inherited chromosome pair are different and originate from both chromosomes from a single parent, the condition is termed heterodisomy. Many examples of humans with isodisomy and heterodisomy have been reported, and almost all were ascertained because they had a genetic disease resulting either from a homozygous mutation on the affected chromosome pair or from unbalanced imprinting of genes on the affected chromosome pair.3 Very few cases of retinal disease have been reported with this aberrant mode of chromosomal transmission.4,5
The USH2A gene on chromosome 1q was first identified as a cause of Usher syndrome type II.6 Patients with this recessively inherited disease have both retinitis pigmentosa (RP) and incomplete hearing loss. We recently reported that certain mutations in the USH2A gene, such as the missense mutation Cys759Phe, can produce RP without hearing loss (ie, nonsyndromic RP).7 In this article, we describe our subsequent evaluation of one of the extended families from that study with 2 members affected with RP without hearing loss and with an USH2A mutation.
This study involved human subjects and conformed to the Declaration of Helsinki. The index patient in this study (003-281) was one of the subjects of a previous report from our group.7
In all cases, the diagnosis of RP was based on the results of an ophthalmological examination that included electroretinography.8
Blood samples were obtained from the relatives of the index patient, and leukocyte DNA was purified from those samples. An affected relative of the index patient (121-397) had a second blood sample drawn for chromosome analysis conducted using standard karyotyping methods in a clinical cytogenetics laboratory (Brigham and Women's Hospital, Boston, Mass). The DNA from one deceased family member (226-1742) was obtained 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 that twice the suggested amount of proteinase K was used.
To screen for mutations in the USH2A gene, genomic fragments were amplified from 20 to 100 ng of DNA. The primers used for the polymerase chain reaction (PCR) are provided at our laboratory's Web site (http://eyegene.meei.harvard.edu). Amplified DNA fragments were sequenced in both the sense and antisense directions using an ABI Prism 377 sequencer (Applied Biosystems, Foster City, Calif).
To amplify microsatellite markers from chromosome 1 from leukocyte DNA, we used commercially available primers (MapPairs; Research Genetics, Inc, Carlsbad, Calif). The position of 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; accessed October 10, 2001). For each primer pair, PCR cycling conditions were performed according to the manufacturer's protocol whereas the buffer composition and the annealing temperature were according to that reported in the Genome Database (http://www.gdb.org). The concentration of dATP, dTTP, and dGTP in the reaction buffer was 0.02 mM, and the concentration of dCTP was 0.002 mM supplemented with about 22.2 kBq of 33P-α-dCTP at 111 TBq/mmol. The PCR-amplified DNA fragments were diluted 1:1 (v:v) with a solution of 95% formamide, 20 mM of EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol before electrophoresis through 6% denaturing polyacrylamide gels. The PCR amplification of microsatellites used for testing parentage was performed as described by Alford et al.9 Detection of microsatellite alleles was performed by autoradiography.
The index patient (003-281) with RP was heterozygous for the missense mutation Cys759Phe (TGC to TTC).7 No other pathogenic mutation in the coding sequence or in the intron splice sites flanking the 21 exons of this gene was found by DNA sequence analysis. Analysis of DNA from this patient's parents showed that the Cys759Phe allele had been inherited from the patient's mother (see the schematic pedigree in Figure 1). Like every other index patient whom we have encountered with this missense change, this patient also had a syntenic isocoding change in codon His752 (CAT to CAC) only 20 base pairs(bp) away from the site of the missense mutation.7
The family was remarkable because there was a female second cousin on the father's side (patient 121-397) who also had nonsyndromic RP (Figure 1). On obtaining a blood sample from this relative, we found her to have the Cys759Phe mutation homozygously but without the usually associated His752 isocoding change. More remarkably, the mother of patient 121-397 did not carry this mutation (Figure 2).
To obtain more information about the origin of the Cys759Phe mutant alleles in patient 121-397, we extracted DNA from paraffin-embedded fragments of small intestine from the proband's deceased father; the fragments had been obtained at autopsy and stored in a hospital pathology department. Standard parentage testing with informative microsatellites on chromosomes 4q, 5q, 6p, 8p, 11p, 12p, 12q, 15q, and Xq9 was consistent with both parents of patient 121-397 being the biological parents (P>.997). Direct sequencing of the USH2A gene revealed that the proband's father was a heterozygous carrier of the Cys759Phe mutation without the His752 isocoding change (Figure 2).
We also analyzed several microsatellite markers scattered along both arms of chromosome 1. Patient 121-397 was heterozygous at marker loci near the centromere and the proximal short and long arms of chromosome 1, with both alleles identical to those in her father (Figure 3). The patient was homozygous at markers on the distal short and long arms, with the allele present at each locus identical to an allele present in the father. The homozygous (isodisomic) region on the long arm included the USH2A locus with the Cys759Phe mutation. The putative disease-causing haplotype defined by the closely linked microsatellite markers D1S229, D1S490, D1S237, and D1S47410 that was found in patient 121-397, her father, and his immediate relatives was absent from the affected second cousin who had the Cys759Phe mutation associated with the isocoding change at codon His752. These findings indicated that the Cys759Phe alleles in the affected second cousin were likely of independent origin (Figure 1).
To investigate the possibility that the mechanism responsible for the Cys759Phe homozygosity was uniparental disomy and, more specifically, paternal isodisomy rather than a situation of hemizygosity caused by an undetected deletion spanning the maternal USH2A gene, we obtained a karyotype of patient 121-397. This was normal, and, specifically, there was no observed abnormality of chromosome 1q where the USH2A gene lies (not shown). To search for a possible microdeletion not detected by cytogenetic analysis, we analyzed the closely linked microsatellite markers D1S229, D1S490, D1S237, and D1S474. As expected, patient 121-397 was homozygous for a paternal allele at each of these markers. Furthermore, evidence for the absence of a maternally inherited microdeletion came from the observation that the mother of patient 121-397 was heterozygous at 3 of these 4 marker loci, including marker D1S229 which is physically the closest marker to USH2A. We concluded that the patient had complete uniparental(paternal) heterodisomy for chromosome 1 with isodisomic segments at the distal ends of the short and long arms.
Ophthalmic evaluation of both the index patient (003-281) and the heterodisomic relative (121-397) documented the findings characteristic of RP, including fundi with attenuated retinal vessels and intraretinal bone-spicule pigment around the periphery of both eyes. At age 35 years, patient 003-281 had visual acuities of 20/25 OD and 20/30 OS and a mildly myopic refractive error (spherical equivalent, −0.25 averaged between the 2 eyes) There was a midperipheral scotoma extending from the 8° isopter to the 30° to 50° isopter in all meridians in both eyes with a V4e white test light in the Goldmann perimeter. Her electroretinograms (ERGs) were reduced but easily detected. The rod-plus-cone ERG amplitude in response to 0.5-Hz flashes of light was 43 µV, and the cone ERG amplitude in response to 30-Hz flashes of light was 7.2 µV (both amplitudes are means between the 2 eyes; normal amplitudes,≥350 µV and ≥50 µV, respectively). The cone ERG implicit time was abnormally prolonged (40 milliseconds in each eye; normal value, ≤32 milliseconds). At age 49 years, patient 121-397 had visual acuity of 20/40 in both eyes and a mean refractive error of +1.50 spherical equivalent. She had peripheral iridotomies from previous laser treatments for narrow-angle glaucoma. Her fields with the V4e test light were constricted to a central island extending to the 8° isopter with additional thin islands in the inferior field. The mean rod-plus-cone ERG amplitude was 3.2 µV, and the mean cone ERG amplitude was 0.67 µV. The mean cone ERG implicit time was delayed at 47 milliseconds. At age 54 years, pure-tone audiograms were normal (Figure 4).
The USH2A gene was first identified as a cause of Usher syndrome type II.6 Most of the mutations reported in this gene were found in patients with the syndromic form of RP. We have previously reported that a particular missense mutation in USH2A can also cause recessive, nonsyndromic RP.7 This mutation, Cys759Phe, is the one found homozygously in patient 121-397, who is the main subject of the current study. This patient clearly had no hearing loss. The reason that this particular mutation causes RP without hearing loss remains unexplained. It is also unknown whether there are other USH2A mutations that might cause nonsyndromic RP.
The molecular genetic findings in patient 121-397 indicate that she inherited no copy of chromosome 1 from her mother. The centromeric regions of her 2 copies of chromosome 1 are derived from her father's 2 copies of chromosome 1, whereas the 2 telomeric regions are derived from only 1 of the 2 paternal copies. The abnormal form of chromosome transmission for which the regions near the centromere are heteroallelic requires a nondisjunction event during meiosis I and is termed primary heterodisomy. In this case, the nondisjunction event must have occurred after 2 recombination events, one involving each arm of chromosome 1, so that the 2 chromosome 1 homologues in the aberrant sperm had regions of homozygosity on the distal short and long arms (Figure 5). It is possible that more than 2 recombinations might have occurred during meiosis I but were undetected because we did not analyze informative markers from the involved regions of chromosome 1. The aberrant sperm is likely to have fertilized an ovum that fortuitously had no chromosome 1, producing a balanced zygote through a mechanism referred to as gamete complementation. Alternatively, the aberrant sperm fertilized an ovum with a maternal chromosome 1 that was lost early in embryogenesis, a mechanism referred to as trisomy rescue. This latter mechanism is considered less likely since conceptuses trisomic for chromosome 1 are exceedingly rare and are thought to die before implantation.13 If trisomy rescue were the mechanism, it must have occurred during or soon after the first cell division of the zygote, and there was complete loss of the trisomic cells.
A few other examples of uniparental disomy of chromosome 1 have been reported, including examples of the disomic chromosome 1 being of either paternal5,14- 16 or maternal17- 19 origin. Figure 5 summarizes the errors that can occur in meiosis I and meiosis II to produce the abnormal gametes that would have 2 copies of chromosome 1. There are documented examples of uniparental disomy related to nondisjunction in meiosis I (resulting in both copies of chromosome 1 being transmitted from one parent) and meiosis II (resulting in 2 identical versions of one copy of chromosome 1 being transmitted from one parent). As in our case, almost all of the patients with uniparentally derived chromosome 1's were ascertained because of evaluations for recessive diseases that turned out to be caused by mutant alleles in the regions of chromosome 1 that were isodisomic. One case was ascertained fortuitously and had no genetic abnormalities associated with the chromosome 1 uniparental disomy.13 No case of uniparental disomy for chromosome 1 had any detected abnormality that might reflect genes on chromosome 1 that are differentially expressed through imprinting. Only one previously reported case had retinal degeneration; this patient had uniparental isodisomy leading to homozygosity for a splice-site mutation in the RPE65 gene.5
Cases with uniparental disomy involving chromosomes other than chromosome 1 have also been reported. Of particular relevance to ophthalmology is a case of isodisomy of chromosome 2 resulting in RP because of homozygosity for a mutation in the MERTK gene on that chromosome.4 Another report described a patient with rod monochromatism and chromosome 14 isodisomy.20 This case has been cited as evidence that a recessive gene causing rod monochromatism is on chromosome 14; this hypothetical gene remains unidentified. With the recent identification of the specific genes causing many hereditary ophthalmic diseases, it is likely that other examples of patients who have uniparental disomy will be discovered during the course of their molecular genetic analysis. This phenomenon is important for the genetic counseling of families with recessively inherited eye diseases because an unaffected heterozygous carrier can have an affected offspring homozygous for the same mutation even if the carrier's spouse has wild-type alleles at the disease locus.
Our case is unusual not only because of the uniparental disomy but also because the index patient had a relative who was also affected with RP and who also carried the same missense mutation in the USH2A gene. Although seemingly exceedingly improbable, our results clearly showed that this familial recurrence was fortuitous; the mutations in the 2 affected relatives were of independent origin. Other examples of close relatives with the same rare inherited ocular disease caused by independently arising mutations have been reported (eg, retinoblastoma).21- 23 The ascertainment of such improbable associations is likely due to the extremely large number of such potential associations that would be deemed worthy of note. Although each such potential association is improbable, the large number of possible associations makes it likely that some of them will actually occur.
Submitted for publication December 18, 2001; final revision received April 26, 2002; accepted June 13, 2002.
This study was supported by grants EY08683 and EY00169 from the National Institutes of Health, Bethesda, Md, and the Foundation Fighting Blindness, Owings Mills, Md.
We thank the patients for their participation in this study.
Corresponding author and reprints: Thaddeus P. Dryja, MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114 (e-mail: firstname.lastname@example.org).