Rearrangements within the facioscapulohumeral muscular dystrophy
locus: mechanism, timing and consequences.
Lemmers, R.
Citation
Lemmers, R. (2005, June 15). Rearrangements within the facioscapulohumeral muscular
dystrophy locus: mechanism, timing and consequences. Retrieved from
https://hdl.handle.net/1887/2699
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Institutional Repository of the University of Leiden
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171
Chapter 9.
Compl
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nformati
on i
n the di
agnosi
s of FSHD by tri
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e DNA anal
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Lemmers RJLF,de KievitP,van GeelM ,van der W ielen M JR,Bakker E,Padberg GW ,Frants RR,van der M aarelSM
Complete Allele Information
in the Diagnosis of
Facioscapu lohu meral
Mu scu lar Dystrophy by
T riple DN A Analysis
Richard J. L. F. Lemmers, B S c,1P eggy de K iev it,1Michel v an G eel, P hD,3Michiel J.R. v an der Wielen,1 E gbert B akker, P hD,1G eorge W. P adberg, MD, P hD,2
Ru ne R. Frants, P hD,1
and S ilv e`re M. v an der Maarel, P hD1
Facioscapulohumeral muscular dystrophy is caused by partial deletion of the D 4Z 4 repeat array on chromosome 4q 3 5 . G enetic diagnosis is based on siz ing of this repeat array, w hich is complicated by cross-hybridiz ation of a homologous polymorphic repeat array on chromosome 10 and by the freq uent ex changes betw een these chromo-somal regions. T he restriction enz yme XapI optimiz es the diagnosis of facioscapulohumeral muscular dystrophy by uniq uely digesting 4-deriv ed repeat units and leav ing 10-deriv ed repeat units undigested, thus complementing B ln I, w hich uniq uely digests 10-deriv ed repeat units. A triple analysis w ith E c o R I, E c o R I/B ln I, and XapI un-eq uiv ocally allow s characteriz ation of each of the four al-leles, w hether homogeneous or hybrid. T his is particu-larly useful in the case of identical siz ed 4-deriv ed and 10-deriv ed arrays, in situations of suspected facioscapu-lohumeral muscular dystrophy w ith nonstandard allele configurations, and for assignment of hybrid fragments to their original alleles.
Ann N eu rol 2001;50:816 – 819
Facioscapu lohu meral mu scu lar dystrophy (FS HD, MIM 158900) is characteriz ed by progressiv e weakness and atrophy of facial and shou lder girdle mu scles and a gradu al spread, v ariable in time, to abdominal and foot-extensor mu scles, followed by inv olv ement of u
p-per arm and pelv ic girdle mu scles. Recently, attention has been drawn to the absence of facial weakness and pelv ic girdle onset.1– 3 Clinical u ncertainties in some patients and the high freq u ency of new mu tations make reliable DN A diagnosis mandatory for genetic confirmation and cou nseling.
FS HD is associated with partial deletions of the polymorphic D4Z 4 repeat array on 4q 35. T his array is v isu aliz ed in EcoRI-digested DN A by hybridiz ation with probe p13E -11 (D4F104S 1), which recogniz es the region proximal to the D4Z 4 repeat (Fig 1). In normal people, the array v aries between 11 to 150 u nits; patients carry an allele of 1 to 10 u nits (10 to 38kb).4,5 DN A diagnosis is compromised by
cross-hybridiz ation of p13E -11 to a highly homologou s polymorphic array on 10q 26 .6 ,7 At least 10% of chro-mosome 10-deriv ed fragments in the popu lation are ,38kb and do not cau se disease. T he restriction en-z yme B ln I specifically recognien-z es chromosome 10-deriv ed u nits, bu t not chromosome 4-10-deriv ed u nits.8 T herefore, B ln I-insensitiv e chromosome 4 alleles can be discriminated from B ln I-sensitiv e chromosome 10 alleles by EcoRI/B ln I dou ble digestion (Fig 2B ; see Fig 1). P u lsed-field gel electrophoresis (P FG E ), allowing discrimination of fragments .50kb and separation of both 4 and 10 alleles, demonstrated the existence of 4-deriv ed arrays on chromosome 10 or 10-deriv ed ar-rays on chromosome 4.9 Hybrid repeat arrays consist-ing of mixtu res of 4- and 10-deriv ed u nits hav e also been identified, adding to the complexity of the DN A diagnosis. E specially in patients with a complex repeat array composition, in patients in whom the probe re-gion itself is also deleted, or in patients who display somatic mosaicism for the repeat array deletion, P FG E has prov ed v ery u sefu l.9 – 12
T hese complicated situ ations u nderscore the need for simple and reliable tests withou t u se of P FG E , particu larly when DN A testing is increasingly wanted not only as a confirmatory bu t also as an exclu sion test.
P atients and M ethods S u b jects
Control S u bjects 1 and 2 are from a random Du tch popu -lation. S u bject 3 is a de nov o FS HD patient. S u bject 4 is a 57 -year-old man who was seen by his local neu rologist for shou lder pain. When he claimed that his cou sin had FS HD, DN A testing was performed showing a shortened EcoRI frag-ment, indicating that he a gene carrier with a probability of greater than 98%. He was referred for a second opinion. Apart from pain, he reported progressiv e difficu lties raising his arms abov e his head for a period of 10 years. T he phys-ical examination showed mild m. infraspinatu s atrophy and weakness (MRC grade 4), as well as bilateral deltoid, biceps, and triceps weakness (grade 4). P ainfu l elev ation of the arms limited to 145 degrees was present bilaterally. E xorotation of From the 1Center for Hu man and Clinical G enetics, Leiden U
ni-v ersity Medical Center, Leiden;2Department of N eu rology, U niv
er-sity Medical Center N ijmegen, N ijmegen, T he N etherlands; and
3Department of Cancer G enetics, Roswell P ark Cancer Institu te,
B u ffalo, N Y .
Cu rrent address for Dr v an G eel: Department of Dermatology, U ni-v ersity Medical Center N ijmegen, P .O B ox 9101, 6 500 HB , T he N etherlands.
Receiv ed May 21, 2001, and in rev ised form S ep 10, 2001. Ac-cepted for pu blication S ep 10, 2001.
Address correspondence to Dr v an der Maarel, Leiden U niv ersity Medical Center, Department of Hu man and Clinical G enetics, Wassenaarseweg 7 2, 2333 AL Leiden, T he N etherlands.
E -mail: maarel@ lu mc.nl
the upper arms was restricted. He had no facial weakness or other signs of FSHD. Creatine kinase was normal and an extensive electromyelogram showed polyphasic potentials of normal duration and amplitude in the left biceps muscle only. Because the reduction of theEcoRI fragment after BlnI digestion was greater than 3kb, additional genetic studies were requested.
A cousin of Subject 4 was 65 years old and had a painful right shoulder for many years. A minor stroke 2 years before the examination had left the patient with a left-sided central facial paresis and a restricted endorotation of the right arm. The brother of this cousin was a 57-year-old left-handed man with shoulder pain of 25 years duration. He had an elevated left shoulder but no paresis or atrophy, and no signs compatible with FSHD. Ten years earlier, an orthopedic sur-geon had diagnosed his condition as bilateral rotator-cuff syndrome. Electromyelography at that time was done be-cause of bilateral supra- and infraspinatus atrophy, indicating mild neurogenic and myopathic features, which his neurolo-gist considered suggestive of FSHD.
D N A Isola tion
For linear gel electrophoresis, DNA was isolated from pe-ripheral blood lymphocytes essentially as described.13 For PFGE, peripheral blood lymphocytes were embedded in aga-rose plugs at a concentration of 7.5 3 105cells per plug. D N A D ia g nosis
DNA digestion, separation by linear gel electrophoresis or PFGE, Southern blotting, and hybridization were carried out as described previously.9,14 X a p I was purchased from
Fer-mentas. Blots were exposed for 16 to 24 hours to phospho-rimager screens and analyzed with the ImageQ uant software program (MBI Fermentas, St. Leon-Rot, Germany).
Results
Sequence comparison of eight (4-derived) D4Z4 units and seven 10-derived units from GenBank and se-quences generated in our laboratory showed the
pres-ence of a novel consistent differpres-ence between both units. A G-to-C transition (position 7515 in accession no. AF117653) results in a single X a p I restriction site in the 4-derived unit. The 10-derived unit is com-pletely resistant to X a p I. Although X a p I cuts the D4F104S1 locus, resulting in weaker hybridization sig-nals, probe p13E-11 is still applicable. A X a p I
restric-F ig 1 . R estriction m a p of th e EcoR I fra g m ents recog niz ed by p 1 3 E-1 1 (shaded) on ch rom osom es 4 (solid ) a nd 1 0 (op en) ba sed on one D 4 Z 4 unit in seq uence A F 1 1 7 6 5 3 . R estriction enz y m es a re EcoR I (E), BlnI (B) a nd X apI (X ) a nd restric-tion siz es a re in k iloba ses (k b). X apI cuts freq uently a round th e rep ea t a rra y , th erefore only relev a nt restriction fra g m ents a re ind ica ted .
F ig 2 . (A ) P ulsed -field g el electrop h oresis (P F G E) a na ly sis of th e rep ea t a rra y s on ch rom osom es 4 a nd 1 0 in th e D N A of control Subjects 1 a nd 2 a fter d ig estion w ith EcoR I/Hind III (E), EcoR I/BlnI (B) or X apI (X ) a nd h y brid iz a tion w ith p robe p 1 3 E-1 1 . In P F G E a na ly ses, Hind III is a d d ed to th e EcoR I restriction since th is enz y m e increa ses th e resolution w ith in th e 2 0 - to 5 0 -k b ra ng e a nd d oes not cut w ith in th e rep ea t a rra y itself. Ind iv id ua l 1 ca rries a sta nd a rd a llele con-fig ura tion of tw o 4 -d eriv ed (BlnI-resista nt) a lleles of 6 5 k b a nd 4 0 k b, a nd tw o 1 0 -d eriv ed (X apI-resista nt) a lleles of 1 0 0 k b a nd 5 0 k b. Ind iv id ua l 2 ca rries co-m ig ra ting 4 -d eriv ed a nd 1 0 -d eriv ed a lleles of 1 2 0 k b ba sed on th e resista nce to both enz y m es. (B) Sch em a tic p resenta tion of th e D 4 Z 4 locus (solid ) a nd its h om olog ue on ch rom osom e 1 0 (open). T h e rep ea t units a re rep resented by a n a rrow a nd m a y v a ry in num ber (n). T h e reg ion recog niz ed by p robe p 1 3 E-1 1 is shaded. T h e restriction sites for EcoR I (E), BlnI (B) a nd X apI (X ) a re ind ica ted . A bov e th e ch rom osom e, th e restriction fra g m ents a re v isua liz ed by solid ba rs, a fter d ig estion w ith th e p rop er en-z y m es for ch rom osom e 4 q ter. Below th e ch rom osom e, th e re-striction fra g m ents for 1 0 q ter a re v isua liz ed . T h e a llele siz es of Subject 1 a re ind ica ted on th e p rop er restriction fra g m ents.
tion site distal to the repeat array obviates a double digest (see Figs 1 and 2B).
We tested this difference in 50 healthy controls and 20 FSHD patients who had been examined previously for their repeat array constitution. No inconsistencies were identified; upon EcoRI/BlnI digestion, 4-derived arrays were visualized, while uponXapI digestion, only 10-derived repeat arrays were seen. As demonstrated in Figure 2A, XapI not only facilitates the proper assign-ment of each allele to its respective chromosome, as exemplified in control 1, but also assists in the identi-fication of equal-size 4-derived and 10-derived alleles, as shown in control 2. This individual carries co-migrating 4-derived and 10-derived alleles of 120kb, as inferred from its resistance to BlnI and XapI.
Patients 3 and 4 (Fig 3A) were referred for the agnosis of FSHD. Routine DNA diagnosis includes di-gestion of DNA withEcoRI and double digestion with EcoRI/BlnI prior to separation by linear gel electro-phoresis. As shown in Figure 3A, Patient 3 carries a standard FSHD 4-derived allele of 17kb, based on its BlnI resistance and XapI sensitivity, confirming the clinical diagnosis of FSHD.
Patient 4 carries a 30-kb fragment upon digestion withEcoRI, while EcoRI/BlnI double digestion shows a fragment of 17kb, possibly derived from the 30-kb EcoRI fragment. Based on these two enzymes, he was previously considered gene carrier. XapI demonstrates that the 30-kb EcoRI fragment is a homogeneous 10-derived array and that, therefore, the 17-kbEcoRI/BlnI fragment cannot be derived from the 30-kbEcoRI frag-ment. Further PFGE analysis (see Fig 3B) reveals the original allele from which the 17-kb EcoRI/BlnI frag-ment is derived. It shows a 4-derived (BlnI-resistant) allele of 90kb, two 10-derived (XapI-resistant) alleles of 80kb and 30kb, respectively, and a BlnI- and XapI-sensitive allele of 250kb. Since the 90-, 80-, and 30-kb fragments contain homogeneous arrays, the 17-kb EcoRI/BlnI fragment has to come from the hybrid 250-kb allele, ruling out FSHD. Subsequent DNA analysis of his cousins supported this interpretation be-cause it provided no evidence for repeat array frag-ments of less than 38kb (data not shown).
Discussion
In recent years, unusual manifestations of FSHD have gained considerable attention in the literature, empha-sizing the need for a reliable genetic test for FSHD.2,3,15–19 In a clinically highly selected
popula-tion, we reached a sensitivity and specificity of the test of more than 95%.14 This led to a liberal use of the test for exclusion purposes, and to a potential rise in the number of false-positive results.
The molecular diagnosis of FSHD is complicated by the co-hybridizing homologous repeat array on chro-mosome 10qter, which may vary between 1 to 150
units without pathological consequences, and by the presence of translocated or hybrid repeat arrays in some 21% of the Dutch population.9,11,20 In contrast to an earlier report,9 recent improvements in genetic
diagno-Fig 3. (A) L inear gel analysis of two potential facioscapulo-humeral muscular dystrophy (FSH D) patients for genetic diag-nosis of FSH D. Fragment sizes in the marker lane (M ) are indicated in the middle. The cross-hybridizing 9 .4-kb chromo-some Y fragment is indicated on the right. DNA of these indi-viduals was digested with EcoRI (E), EcoRI/BlnI (B) and with XapI (X) and separated in adjacent lanes. The new in-formative XapI fragment is indicated with an arrowhead. Patient 3 carries a standard FSH D allele of 17kb. The frag-ment is resistant to BlnI digestion and becomes reduced to 14kb due to the BlnI site 3kb distal to the prox imal EcoRI site; therefore, it contains chromosome 4-derived repeats and is sensitive to XapI digestion. In another hospital, Patient 4, clinically diagnosed as possibly having FSH D, was originally genetically diagnosed with FSH D with an hybrid allele of 30kb decreasing to 17kb after EcoRI/BlnI double digestion. Further analysis with XapI showed that the 30-kb fragment is a homogeneous 10-derived array, based on its XapI resistance. C onsequently, the 17-kb EcoRI/BlnI fragment cannot be de-rived from the 30-kb array. (B) Pulsed-field gel electrophoresis (PFGE) analysis of Patient 5 carrying a potential hybrid re-peat array of 30kb. C ombinatory analysis with EcoRI/ HindIII, BlnI and XapI shows that the 250-kb allele is the only allele that is sensitive to both discriminating enzymes. The other three alleles are homogeneous arrays, since they are resistant to BlnI or XapI. It is concluded that the 17-kb EcoRI/BlnI hybrid fragment (arrowhead) is not derived from the 30-kb allele, but rather from the 250-kb allele (arrow). FSH D is therefore ex cluded in this patient. (C ) Schematic representation of the hybrid allele of Patient 4. C hromosome 4-derived units are solid, 10-derived units are open. The restriction sites for EcoRI (E), BlnI (B) and XapI (X) are indicated. Fragment sizes of the hybrid allele in Patient 4 are indicated below the chromosome.
sis showed that only homogeneous or hybrid 4-derived arrays of less than 38kb on chromosome 4 have been identified in FSHD. Short homogeneous 10-derived arrays on chromosome 4 or, small repeat arrays on chromosome 10, irrespective of their composition, have never been associated with disease. Unfortunately, the current techniques do not permit determination of the originalEcoRI fragment from which hybrid EcoRI/BlnI fragments are derived.
Introduction of the restriction enzyme XapI refines FSHD diagnosis. XapI has opposite characteristics to BlnI: it recognizes a single restriction site within the 4-derived repeat unit, while the 10-derived repeat unit is XapI-resistant. Therefore, digestion of DNA with EcoRI, with EcoRI/BlnI, and with XapI, allows un-equivocal determination of chromosome 4-derived and 10-derived alleles. XapI is particularly useful when re-peat arrays are composed of clusters of 4-derived and 10-derived repeat units, in which it provides complete allele information. Patient 4 (see Fig 3A–C) exemplifies the power of this procedure, since a potential hybrid short repeat array of four 4-derived repeat units (17kb), followed by three 10-derived units (13kb) in a telo-meric direction, would have explained the 30-kbEcoRI fragment, in a clinically atypical FSHD patient. How-ever, the XapI digest indicates that the 30-kb EcoRI fragment is a homogeneous 10-derived repeat and that, therefore, the 17-kb EcoRI/BlnI hybrid fragment can-not have been derived from the 30-kbEcoRI fragment. Indeed, PFGE analysis demonstrates the presence of a 250-kbEcoRI fragment sensitive to BlnI and XapI from which the 17-kbEcoRI/BlnI fragment must be derived. This was supported by the finding that his supposedly affected cousins did not carry alleles ,38kb. Since we have not observed a small fragment with BlnI-sensitive repeats, residing on chromosome 4 and responsible for FSHD, there is no genetic support for the diagnosis of FSHD.
Although such complicated allele constitutions are probably rare, the increasing use of the DNA test as exclusion criterion necessitates the additional use of XapI to obtain complete allele information.
This work is funded by the Prinses Beatrix Fonds, the Dutch FSHD Foundation, the Muscular Dystrophy Association (USA), the FSH Society (USA), and the Association Franc¸aise Contre les Myopa-thies.
S.M.vd.M. is a Gisela Thier Fellow of the Leiden University Med-ical Center.
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