Paternal Uniparental Heterodisomy with Partial Isodisomy Of

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 heterozygous 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 ofS 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

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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 without 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-

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 10 His Gly Ser Val Asn Lys Phe Cys D1S237, and D1S474 that was found in patient 121- 397, her father, and his immediate relatives was absent His Phe from the affected second cousin who had the Cys759Phe CAT GGC T CA G T G AAC AAATTC T G C mutation associated with the isocoding change at codon His752. These findings indicated that the Cys759Phe al- C T leles in the affected second cousin were likely of independent origin (Figure 1). To investigate the possibility that the mechanism re- sponsible 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

226-1644 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. Fur- 226-1742 thermore, 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 physi- cally the closest marker to USH2A. We concluded that the patient had complete uniparental (paternal) hetero- 121-397 disomy for chromosome 1 with isodisomic segments at the distal ends of the short and long arms.

CLINICAL FINDINGS

Ophthalmic evaluation of both the index patient (003- 003-281 281) and the heterodisomic relative (121-397) documented the findings characteristic of RP, including fundi with attenuated retinal vessels and intraretinal bone- Figure 2. The DNA sequence of USH2A codons 752 to 759, in selected spicule pigment around the periphery of both eyes. At members of family 5014. ID numbers of patients and unaffected relatives are age 35 years, patient 003-281 had visual acuities of 20/25 indicated on the left. The gray-shaded columns highlight the isocoding OD and 20/30 OS and a mildly myopic refractive error change in His752 on the left and the missense mutation in codon Cys759 on the right. (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 ments had been obtained at autopsy and stored in a hos- eyes with a V4e white test light in the Goldmann perim- pital pathology department. Standard parentage testing eter. Her electroretinograms (ERGs) were reduced but with informative microsatellites on chromosomes 4q, 5q, easily detected. The rod-plus-cone ERG amplitude in re- 6p, 8p, 11p, 12p, 12q, 15q, and Xq9 was consistent with sponse to 0.5-Hz flashes of light was 43 µV, and the cone both parents of patient 121-397 being the biological par- ERG amplitude in response to 30-Hz flashes of light was ents (P Ͼ .997). Direct sequencing of the USH2A gene 7.2 µV (both amplitudes are means between the 2 eyes; revealed that the proband’s father was a heterozygous car- normal amplitudes, Ն350 µV and Ն50 µV, respec- rier of the Cys759Phe mutation without the His752 iso- tively). The cone ERG implicit time was abnormally coding change (Figure 2). prolonged (40 milliseconds in each eye; normal value, We also analyzed several microsatellite markers Յ 32 milliseconds). At age 49 years, patient 121-397 scattered along both arms of chromosome 1. Patient had visual acuity of 20/40 in both eyes and a mean re- 121-397 was heterozygous at marker loci near the cen- fractive error of +1.50 spherical equivalent. She had pe- tromere and the proximal short and long arms of chro- ripheral iridotomies from previous laser treatments for mosome 1, with both alleles identical to those in her fa- narrow-angle glaucoma. Her fields with the V4e test ther (Figure 3). The patient was homozygous at markers light were constricted to a central island extending to on the distal short and long arms, with the allele present the 8° isopter with additional thin islands in the inferior at each locus identical to an allele present in the father. field. The mean rod-plus-cone ERG amplitude was 3.2 The homozygous (isodisomic) region on the long arm µV, and the mean cone ERG amplitude was 0.67 µV. included the USH2A locus with the Cys759Phe muta- The mean cone ERG implicit time was delayed at 47 tion. The putative disease-causing haplotype defined by milliseconds. At age 54 years, pure-tone audiograms the closely linked microsatellite markers D1S229, D1S490, were normal (Figure 4).

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Chromosome 1 (Mb) 0 36.33 36.32 36.31 36.23 10 36.22 36.21 36.13 20 36.12 36.11 35.3 35.2 30 35.1 226-1644 226-1742 121-397 226-1642 226-1643 34.3 40 34.2 ID 34.1 D1S1615 2,2 1,2 1,1 2,1 2,2 33 50 X 32.3 32.2 D1S1161 3,2 1,2 1,2 3,2 ND,ND 60 p 32.1 31.3 MYCL1 1,3 2,2 2,2 1,2 3,2 31.2 70

31.1 D1S1613 1,1 ND,ND 1,1 1,1 1,1 80

22.3 D1S1162 1,2 2,3 2,3 1,3 1,3 22.2 90 22.1 21.3 D1S1611 1,3 ND,ND 2,2 1,2 1,2 21.2 100 21.1 13.3 13.2 110 D1S1159 1,1 2,3 2,3 1,3 1,3 13.1 12 11.2 120 D1S1166 2,3 1,3 HD 1,3 2,3 3,3 11.1 11 130 D1S534 2,3 1,2 1,2 3,2 2,2 12 140 D1S400 2,2 1,1 1,1 2,1 2,1 21.1 21.2 150 21.3 D1S1651 4,3 1,2 1,2 4,2 3,1 22 160 23.1 D1S518 2,3 1,1 1,1 2,1 ND,ND 23.2 170 23.3 D1S1593 2,4 1,3 1,3 2,3 4,1 24.1 24.2 24.3 180 X 25.1 D1S1660 1,2 1,1 1,1 1,1 2,1 q 25.2 25.3 190 USH2A +,+ +,F F,F +,F +,+ 31.1 31.2 200 31.3 D1S229 2,1 3,3 3,3 2,3 2,3 32.1 210 32.2 D1S490 4,1 2,3 3,3 4,3 4,2 32.3 ID 41 220 42.11 42.12 D1S237 2,2 1,3 1,1 2,1 2,3 42.13 230 42.2 D1S474 1,2 1,1 1,1 1,1 1,1 42.3 240 43 D1S2811 1,1 1,2 1,1 1,1 1,1 44 250

Figure 3. 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 (TGCϾTTC); +, wild-type USH2A allele; and ND, not done.

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Frequency, Hz x Homologous 250 5001000 2000 4000 8000 Recombination –10 Prophase 0 (Meiosis I) x

20 Normal 30

50 (Meiosis I)

60 First Meiotic Division

Hearing Level, dB 70

80 Usher II 90

100

110 Right Ear (Meiosis II) Left Ear

120 Second Meiotic Division

Figure 4. 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

COMMENT Normal Gametogenesis Nondisjunction Nondisjunction in Meiosis I in Meiosis II The USH2A gene was first identified as a cause of Usher (Primary Heterodisomy) (Secondary Heterodisomy) 6 syndrome type II. Most of the mutations reported in this Figure 5. Schematic diagram of the events leading to primary and secondary gene were found in patients with the syndromic form of heterodisomy, compared with normal gametogenesis, for a given pair of autosomes. In regular meiosis, each pair of chromosomes is separated during RP. We have previously reported that a particular mis- the first meiotic division, whereas the sister chromatids of each chromosome sense mutation in USH2A can also cause recessive, non- detach during the second meiotic division. If nondisjunction occurs during the syndromic RP.7 This mutation, Cys759Phe, is the one found first meiotic division, both chromosomes of a pair (or none at all) can be homozygously in patient 121-397, who is the main sub- 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 ject of the current study. This patient clearly had no hear- same chromosome formed from both of its sister chromatids. In this example, ing loss. The reason that this particular mutation causes 2 homologous recombination events designated by X’s (1 affecting each RP without hearing loss remains unexplained. It is also chromosome arm) occur in prophase of meiosis I (top row). If there is nondisjunction during meiosis I (second row, center column), 2 disomic unknown whether there are other USH2A mutations that gametes carrying heteroallelic regions near the centromere are generated, as might cause nonsyndromic RP. well as 2 gametes that are nullisomic for that particular chromosome. The molecular genetic findings in patient 121-397 in- Depending on the chromatid segregation during the second meiotic division, dicate that she inherited no copy of chromosome 1 from these disomic gametes can generate a condition of centromeric heterodisomy with bitelomeric isodisomy or complete heterodisomy (not depicted in the her mother. The centromeric regions of her 2 copies of figure), both of which depend on subsequent gamete complementation or chromosome 1 are derived from her father’s 2 copies of trisomy rescue. In contrast, a nondisjunction event during the second meiotic chromosome 1, whereas the 2 telomeric regions are de- division (third row, right column) produces secondary heterodisomy with isodisomic centromeric regions and, possibly, heteroallelic sequences at the rived from only 1 of the 2 paternal copies. The abnormal telomeres. The genotype detected in patient 121-397 (paternal heterodisomy form of chromosome transmission for which the regions with partial isodisomy for chromosome 1, with heteroallelic centromeric near the centromere are heteroallelic requires a nondis- sequences and homoallelic regions at both telomeres) is likely to have originated from either of the 2 hyperhaploid spermatozoa (indicated with junction event during meiosis I and is termed primary het- asterisks in the fourth row, middle column) produced by a nondisjunction erodisomy. In this case, the nondisjunction event must have event in meiosis I and subsequent gamete complementation or trisomy rescue occurred after 2 recombination events, one involving each in the zygote. Vertical hatched lines indicate how chromosomes or chromatids arm of chromosome 1, so that the 2 chromosome 1 ho- are assorted during cell division. mologues 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 oc- chromosome 1 that was lost early in embryogenesis, a curred during meiosis I but were undetected because we mechanism referred to as trisomy rescue. This latter mecha- did not analyze informative markers from the involved re- nism is considered less likely since conceptuses trisomic gions of chromosome 1. The aberrant sperm is likely to for chromosome 1 are exceedingly rare and are thought have fertilized an ovum that fortuitously had no chromo- to die before implantation.13 If trisomy rescue were the some 1, producing a balanced zygote through a mecha- mechanism, it must have occurred during or soon after nism referred to as gamete complementation. Alterna- the first cell division of the zygote, and there was com- tively, the aberrant sperm fertilized an ovum with a maternal plete loss of the trisomic cells.

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 A few other examples of uniparental disomy of chro- This study was supported by grants EY08683 and mosome 1 have been reported, including examples of the EY00169 from the National Institutes of Health, Bethesda, Md, disomic chromosome 1 being of either paternal5,14-16 or ma- and the Foundation Fighting Blindness, Owings Mills, Md. ternal17-19 origin. Figure 5 summarizes the errors that can We thank the patients for their participation in this occur in meiosis I and meiosis II to produce the abnor- study. mal gametes that would have 2 copies of chromosome 1. Corresponding author and reprints: Thaddeus P. Dryja, There are documented examples of uniparental disomy re- MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, lated to nondisjunction in meiosis I (resulting in both cop- Boston, MA 02114 (e-mail: thaddeus_dryja@meei ies of chromosome 1 being transmitted from one parent) .harvard.edu). and meiosis II (resulting in 2 identical versions of one copy of chromosome 1 being transmitted from one parent). As REFERENCES in our case, almost all of the patients with uniparentally derived chromosome 1’s were ascertained because of evalu- 1. Engel E. A new genetic concept: uniparental disomy and its potential effect, iso- ations for recessive diseases that turned out to be caused disomy. Am J Med Genet. 1980;6:137-143. by mutant alleles in the regions of chromosome 1 that were 2. Kotzot D. Abnormal phenotypes in uniparental disomy (UPD): fundamental as- pects and a critical review with bibliography of UPD other than 15. Am J Med isodisomic. 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Am J Hum Genet. 1994;55: monochromatism and chromosome 14 isodisomy.20 This 190-195. 10. Sumegi J, Wang JY, Zhen DK, et al. The construction of a yeast artificial chro- case has been cited as evidence that a recessive gene caus- mosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) ing rod monochromatism is on chromosome 14; this hy- gene in 1q41. Genomics. 1996;35:79-86. 11. Cruickshanks KJ, Wiley TL, Tweed TS, et al. Prevalence of hearing loss in older pothetical gene remains unidentified. With the recent iden- adults in Beaver Dam, Wisconsin. The Epidemiology of Hearing Loss Study. Am tification of the specific genes causing many hereditary J Epidemiol. 1998;148:879-886. ophthalmic diseases, it is likely that other examples of pa- 12. Wagenaar M, van Aarem A, Huygen P, et al. Hearing impairment related to age in Usher syndrome types 1B and 2A. 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Complete paternal uniparental isodisomy for rier’s spouse has wild-type alleles at the disease locus. chromosome 1 revealed by mutation analyses of the TRKA (NTRK1) gene en- Our case is unusual not only because of the unipa- coding a receptor tyrosine kinase for nerve growth factor in a patient with con- genital insensitivity to pain with anhidrosis. Hum Genet. 2000;107:205-209. rental disomy but also because the index patient had a rela- 16. Takizawa Y, Pulkkinen L, Chao SC, et al. Mutation report: complete paternal uni- tive who was also affected with RP and who also carried parental isodisomy of chromosome 1: a novel mechanism for Herlitz junctional the same missense mutation in the USH2A gene. Al- epidermolysis bullosa. J Invest Dermatol. 2000;115:307-311. 17. Pulkkinen L, Bullrich F, Czarnecki P, Weiss L, Uitto J. Maternal uniparental di- though seemingly exceedingly improbable, our results somy of chromosome 1 with reduction to homozygosity of the LAMB3 locus in clearly showed that this familial recurrence was fortu- a patient with Herlitz junctional epidermolysis bullosa. Am J Hum Genet. 1997; 61:611-619. itous; the mutations in the 2 affected relatives were of in- 18. Dufourcq-Lagelouse R, Lambert N, Duval M, et al. Chediak-Higashi syndrome dependent origin. Other examples of close relatives with associated with maternal uniparental isodisomy of chromosome 1. Eur J Hum the same rare inherited ocular disease caused by indepen- Genet. 1999;7:633-637. 19. Takizawa Y, Pulkkinen L, Shimizu H, et al. Maternal uniparental meroisodisomy dently arising mutations have been reported (eg, in the LAMB3 region of chromosome 1 results in lethal junctional epidermolysis retinoblastoma).21-23 The ascertainment of such improb- bullosa. 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