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American Journal of Medical Genetics 44513-517 (1992) Phenotypic Evidence for a Common Pathogenesis in X-Linked Deafness Pedigrees and in Xq13-q21 Deletion Related Deafness W. Reardon, S. Roberts, P.D. Phelps, N.S. Thomas, L. Beck, R. Issac, and H.E. Hughes Institute of Medical Genetics W . R . , S.R., N.S.T., Ha.), Department of Ophthalmlogy (L.B.), Welsh Hearing Institute (R.I.), University Hospital of Wales, Heath Park, Cardiff, UX. and Department of Radiology, The Institute of Laryngology and Otology (PDPJ, 3301332 Gray’s Inn Rd., London, U.K. A structural cochlear abnormality has been observed by high resolution CT scanning in some families where X-linkeddeafnessis s e g regating. We now present evidence that the same abnormality is present in a deaf patient who has a deletion within Xq21. This observa- tion provides phenotypicevidencethat the ge- notypic basis of deafness is the same in both patient groups. It is also likely that the peri- lymphatic fluid “gusher” abnormality may be common to both. O 1992 Wiiey-Lisa, inc. KEY WORDS: deafness, CT scanning, X-linked INTRODUCTION Non syndromic X-linked deafness has been classified 1. Congenital sensorineural deafness (McKusick No 30450). 2. Progressive sensorineural deafness (McKusick No 30470). 3. High tone sensorineural deafness (McKusick No 30460). 4. Mixed conductiveand sensorineuraldeafness asso- ciated with perilymphatic gusher at stapes surgery (McKusick No 30440). In this condition stapedial sur- gery, aimed at improvinghearing by correctingthe con- ductive element of the deafness leads to a profuse flow of fluid from the perilymphatic space of the cochlea,the so called perilymphatic “gusher.” The noncongenital forms, types 2 and 3, are readily distinguishable from the others by their postlingual into 4 different types (McKusick, 1988): ~~~ Received for publication July 31,1991;revisionreceived May 19, 1992. Address reprint requests to Dr. W. Reardon, Department of Paediatric Genetics, Institute of Child Health, 30 Guilford St., London WC1 NlEH, U.K. O 1992 Wiley-Liss, Inc. onset. However, they account for only a minority of ped- igrees reported [Mohr and Majeroy, 1960; Livan 1961; Pelletier and Tanguay 19751. In the vast majority of pedigrees with nonsyndromic X-linked deafness, the deafness is of prelingual onset. Traditionally,these fam- ilies have been separated into types 1 and 4 on the basis of audiogram changes. Considerable doubt now sur- rounds the validity of this separation as there is evi- dence that the audiogram may be an unreliable delinea- tor of different forms of prelingual X-linked deafness [Reardon et al., 1991; Reardon et al., 1992; Glasscock, 19731. Moreover, linkage data on pedigrees with audi- ologically different forms of prelingual nonsyndromic X-linked deafness suggest a possible common locus [Reardon et al., 19911. In parallel with these developments, work has been progressing on the molecular investigation of deaf pa- tients with cytogenetically detectable X chromosome deletions. This form of inquiry stems from a report docu- menting 3 male patients, 2 brothers and a maternal uncle with deafness and choroideremia, an X-linked retina1 degenerative condition [Ayazi, 19811. Subse- quent molecular analysis showed a deletion in the Xq21 region and cytogenetic evaluation suggested a possible abnormality of Xq21 [Nussbaum et al., 1987; Merry et al., 19891. Other reports of male patients with cho- roideremia and a variety of clinical features, frequently includingdeafness,highlighted the associationbetween deafness and deletions of variable size in the Xq13-q21 region [Tabor et al., 1983; Rosenberg et al., 1986; Nussbaum et al., 1987;Hodgson et al., 19871.Molecular characterisation of these deletions has been invaluable in the cloning of the choroideremia gene and also sug- gests that the deafness gene in these patients is cen- tromeric to the choroideremia gene [Schwartz et al., 1988; Merry et al., 1989; Cremers et al., 1989, 19901. Most of these patients with deletions had sensorineural deafness [Cremers et al., 19891 and in one a perilympha- tic “gusher” was observed at stapes surgery [Merry et al., 19891. Linkage studies, performed in 2 separate cyto- genetically normal pedigrees with X-linked deafness and surgically confirmed “gusher,” but with no other

Phenotypic evidence for a common pathogenesis in X-linked deafness pedigrees and in Xq13–q21 deletion related deafness

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Page 1: Phenotypic evidence for a common pathogenesis in X-linked deafness pedigrees and in Xq13–q21 deletion related deafness

American Journal of Medical Genetics 44513-517 (1992)

Phenotypic Evidence for a Common Pathogenesis in X-Linked Deafness Pedigrees and in Xq13-q21 Deletion Related Deafness W. Reardon, S. Roberts, P.D. Phelps, N.S. Thomas, L. Beck, R. Issac, and H.E. Hughes Institute of Medical Genetics W . R . , S.R., N.S.T., H a . ) , Department of Ophthalmlogy (L.B.), Welsh Hearing Institute (R.I.), University Hospital of Wales, Heath Park, Cardiff, UX. and Department of Radiology, The Institute of Laryngology and Otology (PDPJ, 3301332 Gray’s Inn Rd., London, U.K.

A structural cochlear abnormality has been observed by high resolution CT scanning in some families where X-linked deafness is seg regating. We now present evidence that the same abnormality is present in a deaf patient who has a deletion within Xq21. This observa- tion provides phenotypic evidence that the ge- notypic basis of deafness is the same in both patient groups. It is also likely that the peri- lymphatic fluid “gusher” abnormality may be common to both. O 1992 Wiiey-Lisa, inc.

KEY WORDS: deafness, CT scanning, X-linked

INTRODUCTION Non syndromic X-linked deafness has been classified

1. Congenital sensorineural deafness (McKusick No 30450).

2. Progressive sensorineural deafness (McKusick No 30470).

3. High tone sensorineural deafness (McKusick No 30460).

4. Mixed conductive and sensorineural deafness asso- ciated with perilymphatic gusher at stapes surgery (McKusick No 30440). In this condition stapedial sur- gery, aimed at improving hearing by correcting the con- ductive element of the deafness leads to a profuse flow of fluid from the perilymphatic space of the cochlea, the so called perilymphatic “gusher.”

The noncongenital forms, types 2 and 3, are readily distinguishable from the others by their postlingual

into 4 different types (McKusick, 1988):

~~~

Received for publication July 31,1991; revision received May 19, 1992.

Address reprint requests to Dr. W. Reardon, Department of Paediatric Genetics, Institute of Child Health, 30 Guilford St., London WC1 NlEH, U.K.

O 1992 Wiley-Liss, Inc.

onset. However, they account for only a minority of ped- igrees reported [Mohr and Majeroy, 1960; Livan 1961; Pelletier and Tanguay 19751. In the vast majority of pedigrees with nonsyndromic X-linked deafness, the deafness is of prelingual onset. Traditionally, these fam- ilies have been separated into types 1 and 4 on the basis of audiogram changes. Considerable doubt now sur- rounds the validity of this separation as there is evi- dence that the audiogram may be an unreliable delinea- tor of different forms of prelingual X-linked deafness [Reardon et al., 1991; Reardon et al., 1992; Glasscock, 19731. Moreover, linkage data on pedigrees with audi- ologically different forms of prelingual nonsyndromic X-linked deafness suggest a possible common locus [Reardon et al., 19911.

In parallel with these developments, work has been progressing on the molecular investigation of deaf pa- tients with cytogenetically detectable X chromosome deletions. This form of inquiry stems from a report docu- menting 3 male patients, 2 brothers and a maternal uncle with deafness and choroideremia, an X-linked retina1 degenerative condition [Ayazi, 19811. Subse- quent molecular analysis showed a deletion in the Xq21 region and cytogenetic evaluation suggested a possible abnormality of Xq21 [Nussbaum et al., 1987; Merry et al., 19891. Other reports of male patients with cho- roideremia and a variety of clinical features, frequently including deafness, highlighted the association between deafness and deletions of variable size in the Xq13-q21 region [Tabor et al., 1983; Rosenberg et al., 1986; Nussbaum et al., 1987; Hodgson et al., 19871. Molecular characterisation of these deletions has been invaluable in the cloning of the choroideremia gene and also sug- gests that the deafness gene in these patients is cen- tromeric to the choroideremia gene [Schwartz et al., 1988; Merry et al., 1989; Cremers et al., 1989, 19901. Most of these patients with deletions had sensorineural deafness [Cremers et al., 19891 and in one a perilympha- tic “gusher” was observed at stapes surgery [Merry et al., 19891.

Linkage studies, performed in 2 separate cyto- genetically normal pedigrees with X-linked deafness and surgically confirmed “gusher,” but with no other

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514 Reardon et al.

clinical manifestations, independently mapped the gene for this condition to probes in the Xq13-q21 region with lod scores of 6.32 [Wallis et al., 19881 and 3.07 [Brunner et al., 19881. Brunner et al. [19881 have speculated that the gene for mixed deafness and perilymphatic “gusher” may be identical to the gene involved in deaf patients with Xq13-q21 deletions. However, the only supporting evidence for this to date is the observation of peri- lymphatic gusher in the patient with Xq13-q21 dele- tion related deafness [Merry et al., 19891.

More recently a multipedigree linkage study of fami- lies with X-linked deafness as the sole clinical feature, and in whom cytogenetic studies have been normal, has confirmed linkage to Xq13-q21 probes [Reardon et al., 19911. This study has demonstrated a readily identifia- ble cochlear abnormality by high resolution CT scan- ning in some affected patients [Phelps et al., 19913. This radiological abnormality is likely to hold the key to the anatomical basis of the perilymphatic “gusher” some- times seen in X-linked deafness and has been discussed in detail elsewhere [Phelps et al., 19911. In brief, it appears that the CT scan is detecting a fistulous connec- tion between the cerebrospinal fluid space and the peri- lymphatic space of the cochlea and this fistula gives rise to the gush of clear cerebrospinal fluid observed sur- gically. Moreover, the multipedigree study into X-linked deafness showed that this radiological abnormality is independent of audiogram type, as it is observed in fami- lies with both mixed deafness and sensorineural deaf- ness [Reardon et al., 1991, 19921.

Scanning abnormalities of the cochlea have not been reported previously in deaf patients with Xq deletions, although these would not be unexpected in view of the observation of “gusher” in one such patient [Merry et al., 19891. The purpose of this report is to provide radiologi- cal evidence that, in a patient with a cytogenetically visible Xq21 deletion, the anatomical basis of deafness is identical to that seen in cytogenetically normal patients from families in whom X-linked deafness is the sole clinical feature. In so doing, we present phenotypic evi- dence that the genetic basis of deafness is the same in both patient groups.

CLINICAL REPORT The pedigree is shown in Figure 1. The propositus

WJ, age 9 years, was referred with his first cousins IV3 ard IV5, al1 with a diagnosis of sensorineural deafness. Delivery was at 40 weeks gestation and he had a normal birth weight (3.2 kg). Prune belly syndrome was noted and thought to be related to bladder dilation and ureteric reflux, secondary to urethral folds. He was hy- potonic and early motor development was delayed with walking attained at 2 years. Clinical examination showed no dysmorphic features. He now walks indepen- dently, has poor social skills and attends a special school for children with deafness and psychomotor delay. He has a severe bilateral sensorineural deafness, of the order of 90 dB observed across most frequencies, and a disturbance of retinal pigment on ophthalmoscopy. This is characterised by a granular depigmented appearance with normal optic discs. Visual acuity is normal.

Patient IV, was born by forceps delivery of the after-

I I 1

3 ‘7’ 4 ’ & 5

/ Fig. 1. Family pedigree.

coming head following a breech presentation at term. Birth weight was 2.16 kg. Early motor milestones were delayed and he walked at 18 months. On examination, at age 9 years, he has no dysmorphic features and is a hyperactive child. He attends the same special educa- tional needs school as IV,. The deafness is bilateral, sensorineural, and of moderate to severe degree. He has similar ophthalmic pigmentary disturbances to the propositus.

Patient IV5 is now 15 months old. Birth weight was 3.26 kg at term and delivery was normal. Bilateral sensorineural deafness was detected early and motor milestones have been slow, sitting at 9 months and not walking at 15 months. Retina] examination does not show pigmentary abnormalities a t present.

As seen from Figure 1, the maternal granduncle (11,) has some features in common with our patients. This man, to whom access was not granted, is said to have profound intellectual impairment and severe deafness. His ophthalmic status is unknown.

Although unavailable for electrodiagnostic testing of the retinae, female patients 112, 1112, and 111, are clini- cally normal and have normal hearing. Ophthal- moscopic examination in 111, and 1115 showed normal visual acuity and optic discs. Despite some granularity of the fundi in 1112, no frank pigmentary disturbance was seen. III, has normal fundi. 112 was not examined oph- thalmoscopically or audiologically but claims to have normal hearing and vision. She had 7 first or early second trimester miscarriages.

CYTOGENETIC STUDIES Chromosome investigations had previously been un-

dertaken on patient IV, when it was considered that he might have fragile X syndrome and no abnormality was detected at that time. In view of the recent recognition of the association between deafness, retinal pigmentary disturbances, and Xq21 deletions these investigations were repeated. On this occasion chromosomes suitable for high resolution G band analysis were obtained from lymphocyte cultures using deoxycytidine release of a thymidine block [Wheater and Roberts, 19871 and GTG banded using a modification of the method of Seabright

Page 3: Phenotypic evidence for a common pathogenesis in X-linked deafness pedigrees and in Xq13–q21 deletion related deafness

X-Linked Deafness 515

a b C d

1123 l i 3 H -g 1 1 2

13 l 3 I 2 u 13 3

22 1 22 2 22 3

23

2 4 I I

27 I 27 2 ? 7 3

28

Fig. 2. GTG banded sex chromosomes showing deletion of Xq21.1 in (a) aunt of the propositus (III?), (b) mother of the propositus (III,), (c) the propositus (NI), and (d) an affeded cousin (IV3). “he deletion X chromosome is on the right in the heterozygotes and the Y chromosome is on the right in the boys. “he diagram of the X chromosome indicates the location of the deleted segment.

[19711. Replication banding was by an adaptation of the method of Perry and Wolff [19741, which included the addition of BrdU for the final 5 hr of culture to release a methotrexate block and exposure to UV light before treatment with 2 x SSC and staining with giemsa.

Analysis of both GTG and RBG banded preparations from the proband (IV,) and his 2 affected cousins (IV3 and IV,) showed a small deletion within Xq21 which resulted in this band being narrower than normal (Fig. 2). The banding patterns were consistent with a deletion of subband Xq21.1 with the proximal breakpoint located between q13.3 and q21.1 and the dista1 breakpoint within q21.2. The mother of the proband (111,) and her sister (1115) were heterozygous for the deletion. Replica- tion studies in the 2 female carriers indicated that the deleted X was preferentially inactivated with 73% (55 out of 75) of informative cells examined from the mother and 77% (58 out of 75) from her sister showing late replication of the deleted X.

Molecular deletion analysis of DNA from the 3 avail- able affected male patient studies have confirmed the cytogenetic findings. There is a complete deletion of the choroideremia gene sequences and al1 closely flanking DNA loci tested. The DNA deletion boundaries were defined proximally by DXS72 and distally by DXYS1.

RADIOLOGICAL STUDY High resolution cochlear CT scan was undertaken in

patient IV,. This showed a bulbous interna1 auditory meatus, incompletely separated from the basa1 bony coi1 of the cochlea and a dilated facial nerve canal (Fig. 3), exactly resembling the findings in cytogenetically nor- mal patients from pedigrees with X-linked deafness [Reardon et al., 1991; Phelps et al., 19911.

DISCUSSION The subject of this report had a cytogenetically visible

deletion at Xq21. The identification in him of the same cochlear abnormality as in some cytogenetically normal pedigrees who show linkage to Xq13-q21 probes sup- ports the thesis of Brunner et al. [19881 that the gene involved in “gusher” related X-linked deafness and the gene deleted in some Xq13-q21 deleted deaf patients may be identical or, a t least, may be identical in some instances.

As is almost invariably the case in heterozygotes with structural imbalances of the X chromosomes [Therman and Patau, 19741 the abnormal X chromosome was pref- erentially inactivated in the normal carriers in this family. Preferential inactivation of X chromosomes with

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516 Reardon et al.

Fig. 3. High resolution CT s a n of the cochlea, patient IV,. The arrow indicates the dilated facial neme canal. The normal distinction between the basa1 coi1 of the cochlea and the interna1 auditory meatus has been lost.

comparable small deletions in this region has been re- ported previously [Schwartz et al., 1988; Wells et al., 19911.

The retinal abnormalities in the affected boys in this family are consistent with the pigmentary changes found in the early stages of choroideremia, the onset of which usually occurs in the second or third decade of life. This is a condition that has been associated with dele- tions of the proximal part of Xq21 and with deafness [Cremers et al., 19901. The deletion of DNA sequences from the choroideremia gene supports this view. How- ever, a definitive clinical diagnosis could not be made in these boys. Absence of the characteristic funda1 abnor- malities, which have been reported in carriers of cho- roideremia [Hodgson et al., 19871, in the heterozygotes in this family could be accounted for by postulating that preferential inactivation of the abnormal X has occurred in the retinal cells.

It is unclear at present whether al1 patients with Xq13-q21 deletion related deafness have the cochlear abnormality. However, the observation of “gusher” in one deaf, deleted patient [Merry at al., 19891 together with the findings we report here emphasise that the association with “gusher” is not solely confined to pa- tients with nonsyndromic deafness of the mixed type, as previously thought. The inference for clinicians is that the detrimental surgical phenomenon of perilymphatic “gusher” may be observed in sensorineural as well as in mixed deafness, in patients whose sole clinical feature is deafness inherited as an X-linked trait, as well as in patients whose more extensive clinical signs reflect an

Xq13-q21 deletion. This observation further empha- sises the dangers inherent in relying exclusively on the audiogram to discriminate between different forms of X-linked deafness, and underlines the need for a new approach to classifying this condition.

ACKNOWLEDGMENTS We would like to thank Miss Elizabeth Little for her

assistance with the cytogenetic analysis, Dr. F. Cremers for making available the choroideremia gene probe, Ms. Rebecca Coffey and F’rofessor Marcus Pembrey for organ- ising the radiology studies, and Dr. Dafydd Stephens at the Welsh Hearing Institute for valuable discussion.

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X-Linked Deafness 517

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