Prevalence of retinitis pigmentosa in Slovenia

Prevalence of retinitis pigmentosa in Slovenia Peterlin B, Canki-Klain N, Morela V, Stirn B, Rainer S, Cerar V. Prevalence of retinitis pigmentosa in Slovenia. Clin Genet 1992: 42: 122-123. Two hundred and twenty-nine symptomatic patients with retinitis pigmentosa were ascertained in Slovenia between 1986 and 1990. Twentythree further patients were identified while data from 63 families (82 patients) were being collected. After correction for underascertainment, a prevalence of 1 in 6023 was estimated in the Slovene population (1 999477 in 1990). The highest prevalence of 1 in 1902 was found in the age group 65 years and older. Of 63 analysed families, 17 (27%) showed autosomal dominant, 13 (21%) autosomal recessive, and one family (1.5'!4) X-linked inheritance; in 30 families (47.5%) isolated cases were found; and in two families the mode of inheritance was impossible to determine.

6. Peterlin', N. Canki-Klainlf, V. #orelazf 6. SUrd, S. Roine9.' and V. Cera? 'Division of Medical Genetics, University Department of Obstetrics and Gynecology. University Medical Center, Ljubljana 2Univeuty Medical Faculty, Ljubljana, 'Department Of OphthdmOlOQy, Novo meSto Medical Center, 'Ophthalmology Clinic, University Medical Center, Ljubljana and 'University Department of Obstetrics and Gynecology, University Medical Centre. Ljubljana Slovenia

Key words: genetic types pigmentosa

- prevalence - retinitis

Dr. b r u t Peterlin. Univ. Dept. of Obstetrics and Gynecology, Univ. Medical Centre Ljubljana, Slajmerjeva 3, Y-61OOO L j u b l j a Slovenia Received 31 May 1991, revised 4 April, accepted for publication 23 April 1992

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disease and is one of the major causes of hereditary defective vision and blindness (Boughman et al. 1980, Kaplan et al. 1990). The prevalence of the disease and proportions of different genetic forms of R P vary in different populations (Francois 1961, Jay 1982, Hu 1982, Bundey & Crews 1984, Bunker et al. 1984). In the present study the population of RP patients in the state of Slovenia was analysed to estimate the prevalence and relative frequency of genetic types of R P in order to improve genetic counselling and prevention of the disease. Patients and methods

An attempt has been made to ascertain all symptomatic patients with RP living in Slovenia between 1986 and 1990. Two hundred and thirty-eight RP patients were referred from seven ophthalmological departments and Slovene associations for blind people. An effort was made to provide a complete diagnostic evaluation including detailed clinical examination, visual field measurement, dark adaptation testing and electroretinography of referred patients and their close relatives. If this was not possible, medical records were collected to confirm the diagnosis of RP. A pedigree was taken from 63 RP patients: from 122

eight patients while they were seeking genetic advice from the Department of Medical Genetics, Ljubljana, and from 55 patients by conducting a home visit to all accessible referred patients from four out of seven Slovene regional health authorities. Patients were classified according to the genetic type (autosomal dominant - AD, autosomal recessive - AR and X-linked recessive - XR), as described elsewhere (Kaplan et al. 1990). Isolated cases were defined as patients with no other family history or consanguinity recognised in the pedigree. Families where R P was a part of a syndrome, including five families with Usher syndrome, were not included in the study. The prevalence corrected for underascertainment was estimated as P =A/nN, where P =prevafence, A=number of affecteds in the population and N=size of the population at risk. K was estimated as Ca(a- l)/Ca(r- 1) where summation is over all families, and a = number of affecteds ascertained independently as probands and r = total number of affecteds (Emery 1986). Results

Of 238 symptomatic R P patients ascertained in Slovenia between 1986 and 1990, nine died before the year 1990. Therefore 229 patients (104 males

Retinitis pigmentosa in Slovenia Table 1. Age-specifi prevalence of RP patients in Slovenia Age group

No. of inhabitants in Slovenia in 1990

No. of RP patients'

Prevalence of RP

0-1 4 15-29 30-44 45-65 65 and over

413 569 450 260 466 894 453813 214 941

6 38 87 88 113

1 in 68928 1 in 11849 1 in 5367 1 in 5157 1 in 1902

*

Corrected for underascertainment.

and 125 females) were included in the prevalence study. While pedigrees were collected for 63 families (82 RP patients), a further 23 patients were identified. After correcting for underascertainment, the prevalence was estimated as 332 patients in the Slovene population of 1999477 in 1990, or 1 in 6023. The highest prevalence of 1 in 1902 was found in the age group 65 years or older (Table I). Sixty-three pedigrees were constructed and genetic types were determined (Table 2).

Acknowledyements The authors thank ophthalmologists Dr. B. Gracner, Dr. A. Kalan, Dr. Oblak, Dr. J. Srebrnic, Dr. L. Thalani-Pfeifer and Dr. J. Vrhovec for referring the patients.

Discussion

Two hundred and twenty-nine symptomatic patients were ascertained in the state of Slovenia between 1986and 1990.In 63 families with 82 previous RP patients, a further 23 affected individuals were ascertained through their relatives. The highest prevalence (1 in 1902) was found in the group aged 65 and older. Considering these two facts, it can therefore be argued that the study was somewhat biased towards finding patients with more severe disease or older patients with manifested disease. There is well-documented evidence that marked clinical heterogeneity regarding the seventy of the disease exists, even within different genetic forms of RP (Lyness et al. 1985,Kaplan et al. 1990). After correction for underascertainment, a prevalence of I in 6023 was estimated in Slovenia, an estimate similar to prevalences reported in Switzerland, 1 in 7000 (Amman et al. 1965),in the City of Birmingham, 1 in 4869 (Bundey & Crews 1984), Table 2 The proportions of genetic forms in the Slovene population Kindreds Mode of transmission

in the State of Maine, 1 in 4756 (Bunker et al. 1984), and in China, 1 in 4016 (Hu 1982). The proportions of different genetic forms (AD, AR and sporadic forms) in the Slovene population are comparable to the proportions reported by Fishman (1978) and Bunker et al. (1984) in the USA,Bird (1975)in England and Grsndahl(1986) in Norway. They differ from findings in the Swiss (Amman et al. 1965), Russian (Panteleeva 1969) and Chinese (Hu 1982) populations. The proportions of genetic forms in Slovenia are similar to those found in countries with a relatively low frequency of consanguineous marriages. The sex ratio of 0.83 (104males to 125 females) is also concordant with the small proportion of the XR form of inheritance (1.5%) in Slovenia. The results of the present study provide the basis for further genetic and clinical studies of RP in this region.

No.

_

AD AR XR Isolated cases Not determined

17 13 1 30 2

Total

63

-

% _

27 21 1.5 47.5 3 . . 100

Cases No.

%

~ __

43 26 2 30 4

41 25 2 28 4

105

100

References Amman F, Klein D, Franceschetti A. Genetic and epidemiological investigations of retinal and allied diseases in Switzerland. J Neurol Sci 1965: 2: 183-196. Bird AC. X-linked retinitis pigmentosa. Br J Ophthalmol 1975: 59: 1 17-197. Boughman A, Conneally M, Nance WE. Population genetic studies of retinitis pigmentosa. Am J Hum Genet 1980: 32: 223-235. Bunday S, Crews SJ. A study of retinitis pigmentosa in the City of Birmingham. I Prevalence. J Med Genet 1984: 21: 417420. Bunker CH. Berson EL, Bromley WC, Hayes RP, Roderick TH. Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol 1984: 9 7 357-365. Emery AEH. Methodology in medical genetics - an introduction to statistical methods, 2nd ed. Edinburgh: Churchill Livingstone, 1986: 39-40. Fishman GA. Retinitis pigmentosa: genetic percentages. Arch Ophthalmol 1978: 96: 822-826. Francois J. Hereditary tapetoretinal degenerations. In: Heredity in ophthalmology. St. Louis: C. V. Mosby, 1961: 441455. Grsndahl J. Tapeto-retinal degeneration in four Norwegian counties 11. Clin Genet 1986: 29: 1741. Hu DN. Genetic aspects of retinitis pigmentosa in China. Am J Med Genet 1982 12: 51-56. Jay M. On the heredity of retinitis pigmentosa. Br J Ophthalmol 1982: 6 6 405-416. Kaplan J, Bonneau D, Frezal J, Munnich A, Dufier JL. Clinical and genetic heterogeneity in retinitis pigmentosa. Hum Genet 1990 85: 635-642. Lyness AL, Ernest W. Quinlon MP, Clover GM, Arden GB. A clinical, psychophysical and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa. Br J Ophthalmol 1985: 69: 326-329. Panteleeva OA. On hereditary tapetoretinal degenerations. Vestn Oftalmol 1969 82: 53-56.

123

Reassessment of a chromosome 12s + marker bv fluorescent in situ hvbridization (FISH) J

J

Jeziorowska A, Houck GE Jr, Yao X-L Jr, Sklower-Brooks SL, Wisniewski KE, Jenkins EC,Wisniewski HM. Reassessment of a chromosome 12q + marker by fluorescent in situ hybridization (FISH). Clin Genet 1992: 42: 124-128.

A. Jaziorowska, 6. E. Horck, Jr., X.-L Yaa, S. L Sklowar-Braoks, K. E. Wisniewski, E. C. Jenkins and H. M. Wisniewski

We present a case previously described by Jenkins et al. (1983) as atypical Down syndrome (DS). The initial diagnosis was first made on the basis of phenotypic and cytogenetic data. This analysis was supported by studies of superoxide dismutase (SODI) activity that maps to band 21q22.1. Results from phenotypic, chromosome banding and SODl studies suggested a karyotype of 46,XX.- 12,+ t( 12pter to 12qter::21q21 to 21q22.?2). Using fluorescent in siru hybridization (FISH) for chromosome painting with D N A libraries derived from sorted human chromosomes to stain selectively the chromosomes No. 21 and No. 12. we demonstrate that the marker chromosome 12q + has no chromosome 21 content but it is derived from chromosome 12.

Department of Genetics, Institute for Basic Research in Developmental Disabilities, Staten Island, NY USA.

Key words: chromosome painting - fluorescent in Situ hybridization (FISH) marker chromosome 12q+ partial trisomy 12

-

-

Dr. Anna Jeziorowska. lnst for Basic Research in Developmental Disabilities Dept. of Genetics, 1050 Forest Hill Road, Staten Island, New York 10314, USA. Received 12 August 1991, revised and accepted for publication 24 April 1992

The increased definition offered by high resolution banding techniques has made it possible to characterize the chromosomal abnormalities in many patients. Some of the chromosomal abnormalities, however, cannot be accurately delineated by the available banding techniques, particufarly when they involve small chromosomal segments with ambiguous appearance in banding. These ambiguous banding patterns sometimes result in marker chromosomes that mimic the pattern of familiar chromosomes and syndromes (Dallapicola et al. 1980, Taviaux et al. 1989, Smit et al. 1990). In the case of our patient, a female with some DS stigmata phenotypically described as atypical DS, the cytogenetic results demonstrated a visible chromosomal abnormality presumed to involve chromosome 21 material as it was further supported by Cu-Zn superoxide dismutase (SODI) activity studies. The SODl gene mapped to 21q22.1 (Tan et al. 1973, Levanon et al. 1985) is closely associated with the gene complex believed to be responsible for DS (Rahmani et al. 1989, Korenberg et al. 1990). We recently had the opportunity to reevaluate the proposita who is now 21 years of age. Because of her phenotypic discrepancies from DS and the availability of molecular probes for chromosome 21 and 12, we applied molecular techniques to resolve this case. Analysis of chromosome 21 DNA sequences copy number had been applied initially to investi124

gate the patient’s possible partial trisomy 21 by Southern blot and slot blot techniques (results not shown). Those results showed two copies of all sequences examined with no suggestion of partial trisomy 21 as it was reported by McCormick et al. (1988). A possible explanation for this result was that the observed cytogenetic abnormality did not involve chromosome 21 material and that this patient represents a phenocopy of Down syndrome. The technique of in situ hybridization provides a valuable addition to classical cytogenetic procedures for the resolution of such abnormalities. Chromosomal fluorescent in situ hybridization (FISH) has made it possible to stain individual human chromosomes in metaphase spreads and in interphase nuclei, as the DNA of each chromosome occupies a discrete territory within the nucleus (Pinkel et al. 1986, 1988, Cremer et al. 1986, Lichter et al. 1988a, Trask 1991). Analysis of various chromosomal aberrations by in situ hybridization proved chromosome painting to be an appropriate technology to delineate such abnormalities (Lichter et al. 1988a, b, Fuscoe et al. 1989, Heppell-Parton & Waters 1991). The reliability of the FISH procedure, mainly depending on the specificity of the DNA probe used, combines the accuracy of an all-or-none response. In this report we demonstrate the further usefulness of the FISH technique in cfinical cytogenetics. We show that this approach can be used to detect

Reassessment of chromosome 12q + by FISH

de novo unbalanced abnormalities. We present molecular confirmation of the chromosome 12 origin of the additional material detected on the q terminus of the 12q+ end of the long arm of the 12q+ marker.

Case report When the proposita was initiiilly evaluated and reported at 11-112 years of age (Jenkins et al. 1983), she had several features suggestive of DS, including brachycephaly, low posterior hairline, Brushfield spots, dysplastic ears, flat profile, brachydactyly and wide space between the first and second toes. We had the opport.unity to reevaluate her at 21 years of age. Fig. 1 shows a recent photograph of the patient. She was a. well-proportioned moderately retarded young women. Her height was 166 cm (70%-ile) and her weight was 71 kg (85%ile). Her head circumference WiLS 55 cm (50Y0-ile). In addition to the previous findings, she was noted to have upwardly displaced pupils and an upward flare of the lateral aspect of the eyebrows. The palpebral fissures were narrow: The philtrum was flat and short. The uvula wa3 bifid. There was mandibular prognathism with marked underbite. Her feet were small and broad (andthere was fibu-

lar angulation of both great toes. Her skin was hyperkeratotic over the elbows and hands. She made poor eye contact and was shy. She had limitation of upward gaze. There was impairment of fine and gross coordination. An EEG was abnormal due to a slow background. An MRI revealed no significant intracranial abnormalities.

Material and methods pBS-21 and pBS-12 libraries were labeled with biotin-14-dATP by nick translation (BRL), and were separated on G-50 Sephadex by spin column. Both were hybridized at a concentration of 6-8 ng/pl. Repetitive sequences were blocked by the addition .of total genomic DNA at a concentration of 200 ng/pl. Pre-annealing conditions consisted of a 3-h incubation with biotin-labeled probe at 37°C in Hybrisol VI (Oncor), a hybridization buffer, followed by an additional aliquot of total genomic DNA and an additional 3-h incubation. The probe was denatured at 70°C for 5 min and placed in an ice bath until ready for use. Metaphase spreads were prepared according to routine protocol from a lymphoblastoid culture of the patient cell line GM 3997. Fresh metaphase spreads were treated with RNase at 37°C for 1 h. The slide preparations were then denatured for 2 min in 70% formamide/2X SSC at 70°C and dehydrated in a graded ethanol series. The probe mix was then applied to air-warmed slides (30 pl of mix sealed under a 22 x 50 mm glass coverslip) and hybridized for 16 h at 37°C in a moist chamber. After hybridization the slides were washed in 50% formamide/2X SSC for 20 rnin at 4 3 T , rinsed in two changes of 2X SSC at 37°C for 4 min each and placed in 1X PBS buffer (Oncor). Fluorescent detection was performed by alternating layers of FITC-avidin and anti-avidin antibody (Oncor) until two layers of avidin had been applied. Each layer was incubated for 20 min at 37°C under a plastic coverslip in a moist chamber. Slides were washed in 1 x PBD buffer at room temperature between applications. Preparations were counterstained in a propidium iodide (PI)/ antifade solution and analyzed using a Zeiss Axiophot microscope equipped with a FITC filter (450-490 nm). Results and Discussion

Fig. 1. Facies of the proposita at 21 years in age.

Our previous preliminary findings that the Southern blot analysis did not prove the GM 3997 cell line genomic DNA to contain any extra chromosome 21 material (data not shown) encouraged us to investigate further the origin of the additional genetic material found at the end of the long arm 125

Jeziorowska et al.

of the marker chromosome 12q+ by means of cytogenetic techniques as shown in Fig. 2. We applied FISH technique to paint chromosome 12 as well as chromosome 21 with the biotin labeled chromosome 12 and 21 libraries. Fig. 3a and Fig. 3b show the results of FISH with pBS-21 DNA and pBS-12 DNA respectively when hybridized to a metaphase spread of GM 3997 cell line. The pBS2 1 library showed preferential hybridization to chromosome 21 (Fig. 3a). Both chromosomes 21 were specifically and entirely painted. The 12q+ marker chromosome was not labeled by the chromosome 21 library. This indicated that the additional material on the 12q+ marker long arms originated from some other chromosome. Some minor binding sites were observed at or near the centromeric regions of other acrocentric chromosomes due to the repetitive sequence inserts present in the pBS 21 library (Pinkel et al. 1988). The hybridization of pBS 12 was more specific (Fig. 3b). Analysis of metaphase chromosomes indicated that it hybridized to both the normal and marker chromosome 12 uniformly along the full length of those target chromosomes. Thus the additional material on the marker 12q+ chromosome originated from this chromosome as a duplication. Many interphase nuclei showed distinct labeling of the two apparent chromosome 12 domains, as can be seen in the lower right comer of this photograph. Unlike the chromosome 21 DNA

'

* *

p !

0

12 Fig. 2. Two partial karyotype showing two examples of the 12q+ marker chromosome (arrows) and two normal No. 12 chromosomes, as well as an idiogram (ISCN 1985).The lines are drawn to connect all chromosomes at an apparent breakpoint of 12q24.31.

126

library, there were no minor nonspecific hybridization signals. These results demonstrate the high specificity of the pBS 12 probe in visualizing chromosome 12 sequences in metaphase spreads, and its value in monitoring the occurrence of chromosome 12 aberrations. The application of FISH with both chromosome 21 and 12 DNA libraries allowed the detection of chromosome 12 material on the 12q+ marker chromosome which was previously considered to

Fig. 3. Chromosome painting by FISH using biotin-labeled DNA specific for chromosome 21-pBS-21 (a) and chromosome 12-pBS-12 (b), respectively. Chromosomes 21 or 12 hybridized with either of these probes were visualized with the fluorochrome FITC, resulting in a yellow fluorescent staining of those chromosomes. Counterstaining of the chromosomes was performed with PI resulting in red staining. a; In sittr hybridization of pBS-21 DNA to a metaphase spread of the GM 3997 cell line showing complete fluorescent staining of both chromosomes 21 (arrows). b: In situ hybridization of pBS-12 DNA. The GM 3997 cell line metaphase spread displays two copies of chromosome 12 positively stained from pter to qter. The marker chromosome 12q+ is indicated by an arrow.

Reassessment of chromosome 12q+ by FISH

be of chromosome 21 origin. We believe that our results demonstrate a genuine de n o w chromosome 12 duplication. The question is what part of chromosome 12 is present in triplicate due to this rearrangement. In many instances, duplicated chromosomal material remains unidentified due to the overlapping of clinical features in partial trisomies and the incompleteness of the present human gene map. The clinical features of our patient described previously were consistent with, but not diagnostic for trisomy 21. It is well known that in cases of some de n o w chromoscmal imbalances involving only small segments of the human genome, quantitation of gene activity, such as SODl studies here, can be useful when considering various cytogenetic alternatives. The erythrocyte SODl activity was elevated, as in cases of confirmed trisomy 2 1. DNA analysis (Southern blot and slot blot) using the human SODl gene probe pS61-10, showed that the genotype of the proposita contained only two copies of the SODl gene (results not shown). It is possible that the elevated enzymatic activity of SODl may be influenced by some secondary factors such as generally disturbed protein synthesis (Baeteman et al. 1983, Jeziorowska et al. 1988). Chromosome 12 disorders mainly include 12p trisomies; 12p monosomy and 12q trisomies are rare syndromes (de Grouchy & Turleau 1984). Trisomy of the short arm of chromosome 12 represents a multiple congenital anomalies/mental retardation syndrome. The features of the syndrome are produced by gene sequences localized distally to band 1 2~1 2 ,which is the breakpoint in the reported partial trisomies (Stengel-Rutkowski et al. 1981, Pratt & Bulugahapitija et al. 1983). Guerrini et al. (1990) reviewed 26 cases from the literature. Trisomies involving the long arm of chromosome 12 are extremely rare with only a limited number of patients reported (Borgaonkar 1989). There are, however, several reports of partial unbalanced trisomy 12q (Harrod et al. 1980, de Muelenaere et al. 1980, Melnyk et a]. 1981, Zabel & Bauman 1981, Albert et al. 1986). The similarities in appearance, specific anomalies, and developmental pattern of those patients and those reported by Hobolth et al. (1974) and Hemming & Brown (1979) suggest that duplication of the 12q24 region results in a clinically identifiable syndrome. In general, trisomy 12q may be characterized by the following clinical features: psychomotor retardation, growth retardation, dyscephaly, hypertelorism, flat nasal bridge, down-turned mouth, micrognathia, low-set ears, poor Iobulation of the ears, short neck, loose skin of nape, wide-set nipples, sacral dimple, cryptorchidism and simian crease. These characteristic features are well observed in

infants or young cases, but gradually become obscure with age in adult cases. Skeletal abnormalities of the extremities may be a characteristic feature in trisomy 12q syndrome also. Chromosome break points are mostly distributed in the region of 12q(12q21.1 to q24.3) (Melnyk et al. 1981, Roberts et al. 1981). The features of our patient suggest that the duplicated segment is in the q arm. Her phenotype, however, differs from the reported cases in her lack of growth retardation, hypertelorism, micrognathia, or simian creases. To study this 12q+ marker chromosome in detail, a hybrid cell line containing only the above marker would be of invaluable help (Zhang et al. 1989, Warburton et al. 1990). For this purpose we will attempt to construct a human-hamster hybrid cell line derived from the fusion of the patient’s cell line. Microcells containing the 12q + marker can then be derived and, by further selection and cloning, cells carrying only the 12q + chromosome will be obtained. Further proof for the chromosome 12 origin of this marker will be possible by hybridization of 12p and 12q probes using Southem blotting and/or in situ analysis.

Acknowledgements The authors are grateful to Drs. Daniel Pinkel & Joe Gray, Lawrence Livermore National Laboratory, for providing us with the pBSI2 and pBS2I libraries used in this study. This work was funded by an N.I.H. grant PO-1-HD-22634-05 and by the New York State Office of Mental Retardation and Developmental Disabilities.

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