Linkage Exclusion Between the Autosomal Dominant Polycystic Kidney Disease Locus and Chromosome 16...
Transcript of Linkage Exclusion Between the Autosomal Dominant Polycystic Kidney Disease Locus and Chromosome 16...
Journal of the American Society of Nephrology 913
Linkage Exclusion Between the Autosomal DominantPolycystic Kidney Disease Locus and Chromosome 16Markers in a New Family1
Jane E. Brissenden, Janet M. Roscoe,2 Nancy E. Simpson, and Melvin Silverman
J.E. Brissenden, Clinical Molecular Biology Unit, TorontoHospital, Toronto, Ontario, Canada
J.M. Roscoe, Division of Nephrology, The Wellesley
Hospital, Toronto, Ontario, Canada
N.E. Simpson, Departments of Biology and Paediatrics.
Queen’s University, Kingston, Ontario, Canada
M. Silverman, Division of Nephrology, Toronto Hospital,Toronto, Ontario, Canada
(J. Am. Soc. Nephrol. 1991; 2:913-919)
ABSTRACTA family segregating for autosomal dominant poly-cystic kidney disease (ADPKD) is reported. The clini-
cal picture was typical for ADPKD in some familymembers, although others showed mild involvement.DNA from family members was probed with sevenchromosome 16 sIngle-copy DNA sequences thatmapped to the telomere of the short arm of thechromosome. The most likely order of six of theprobes from the telomere is palpha3’HVR.64 at thedesignated locus D16S85, CRI-0327 at D16S63, CR1-090 at D16S45, CRI-0129 at D16S56, CRI-0133 atD16S58, and CRI-0 136 at D16S60, with the PKD I locusfor ADPKD between D16S85 and D16S63. The seventhprobe 24-1 at D16S80 had not been ordered in re-lation to the other sequences, but PKDI had beenmapped between it and D16S85. The three probesthat were informative in our family, palpha3’HVR.64,CRI-090, and CRI-0136 had been linked to the dis-ease locus at recombination frequencies of 4% andapproximately 6 and 12%, respectively. Linkage wasexcluded between the ADPKD locus in our familyand palpha3’HVR.64 at a recombination value of upto 6%. Linkage was also excluded between CRI-090and the disease locus at a recombination value ofup to 5%. The data for linkage between CRI-0136
Received April 9, 1991. Accepted June 18, 1991.
2Correspondence to Dr. J.M. Roscoe. Room 308. Jones BuildIng. 160 WetlesleySt. E., Toronto, Ontario, Canada M4V 1J3.
1046-6673/0204-0913$03.0O/OJournal of the American Society of NephrologyCopyright C 1991 by the American Society of Nephrology
and the ADPKD locus in our family were inconclusive.Multipoint analysis excluded the possibility that the
disease in this family lies between the flanking ge-netic markers that have previously been used todefine the genetic interval in which the most com-mon form of polycystic kidney disease, PKDI, lies.We have not made a positive assignment of theADPKD mutation in this family. This interesting familytherefore represents a new instance of ADPKD that isnot linked to chromosome 16 markers. Koryotypes ofkey family members were normal. The family origi-nated in the British Isles and raises the possibility of agreater worldwide presence of a second ADPKD lo-cus, not linked to chromosome 16 markers, than wasfirst realized.
Key Words: Restriction fragment length polymorphism, chro-
mosome 16, DNA probes, genetic, llnkage heterogeneity,
autosomal polycystic kidney disease
A utosomal dominant polycystic kidney disease(ADPKD) affects 1 in 400 to 1 in 1,000 individ-
uals or up to 600,000 people In the United Statesalone (1). It is inherited as an autosomab dominantcondition and has an apparent mutation rate of 6.5X b0� to 10 X i0� (2,3). Ontario, with a population
of about 3,900,000 people has about 3900 personssuffering from ADPKD. At least 50% (3,4) of affected
individuals progress to end-stage renal failure (ESRF)contributing about 9% of new ESRF cases per year.In 1989, about 48 of the approximately 829 new
cases of ESRF seen in Ontario were diagnosed asADPKD (5-7) as were 99 of 2.004 new end-stagerenal disease patients in Canada. Patients withADPKD suffer other difficulties during their lifespanin addition to end-stage renal disease. These difficul-ties Include abdominal masses or pain that requiresuch diagnostic intervention as computed tomo-graphic scans, renal colic with passage of blood clots
or stones, hypertension, urinary tract infections, he-maturia and proteinuria, and extrarenab cysts (in-cluding ovarian. hepatic, and pancreatic cysts). SomeADPKD patients suffer subarachnoid haemorrhage(4) or renal cell carcinomas (8).
Unfortunately. present clinical methods for early
diagnosis of ADPKD are imperfect. Clinical expres-sion of the disease is very variable, and ultrasonog-
ADPKD Not Linked to Chromosome 16 Probes
914 Volume 2 #{149}Number 4’ 1991
raphy, which is the most sensitive noninvasivemeans of detecting asymptomatic ADPKD, still
misses 5% of persons at risk in their thirties and14% of individuals at risk in their twenties (9-11).
Most people make their childbearing decisions duringthis time of diagnostic uncertainty, and some personswould wish to alter these decisions if they knew theyhad a 50% risk of passing on a deleterious gene.
The locus for ADPKD, known as PKD1, had beenmapped to the tebomere of the short arm of chromo-
some 16 at l6pl3.3 (12-14). Throughout the article.we shall distinguish this first mapped locus for the
disease as PKD 1 and refer to the disease as ADPKD.PKD1 was first linked to the D16S85 locus byusing a polymorphism detected by the probepalpha3’HVR.64 with a recombination frequency (0)
of 4% (13,14). The probe was formerly known as
3’HVR (the 3’ hypervarlabbe region) of the alpha 1chain of the hemoglobin gene (HBA1). Subsequently.
a series of five-chromosome 16 probes were mappedin the following order: CRI-0327 mapping at the DNAlocus D16S63, CRI-090 at D16S45, CRI-0129 atD16S56, CRI-0133 at D16S58, and CRI-0136 at
Dl 6S60 and were shown to be on the proximal sideof PKD1 (15,16) with D16S85 being distal to PKD1(15). A sixth proximal probe 24-1 (D16580) had been
liked to PKD1 with a recombination frequency of 4%(see reference 21), but its order in relation to theother five proximal probes was not established. To-
gether, the above chromosome 16 probes have thepotential to identify the disease by genetic linkageanalysis preclinicably, with an accuracy of 96% or
better (13-24). These probes are expected to be veryuseful in the preclinical diagnosis of PKD1-associ-ated ADPKD (25,26).
Early reports suggested that there was no geneticlinkage heterogeneity in ADPKD (27-29). Recently.however, reports have been published of families in
which apparently typical ADPKD is segregating and
the disease locus does not appear to be closely linkedto the chromosome i6p probes (30-33). Two of the
reported families originated from the Sicilian regionof Italy. and one originated from Copenhagen (30-32). Two additional families have been described inwhich the authors state that the disease phenotypes
and markers are segregating independently, al-though no supporting data were published (33).These latter two families are located in Newfound-land. The Copenhagen group has recently publishedsuggestive evidence for linkage of their unusual fam-ily’s ADPKD locus to a polymorphic locus from the
short arm of chromosome 2 (34). However, otherscientists have suggested that data from families inwhich the disease locus is apparently not linked tochromosome 16 markers may be a result of segrega-tion of familial chromosome rearrangements (35,36)or from statistical chance (37). An additional atypicalFrench family in which the disease was not linked to
the alpha gbobin probes has been reported (38), but,in this case, the diagnosis was not firm.
We describe a new family in which the ADPKDlocus does not appear to be linked to the chromosome16 markers. We have previously published brief re-ports of this family in abstract form (39-41). Thefamily is Canadian. of British descent. We have stud-ied the family with non-alpha globin flanking mark-ers as well as with the usual alpha gbobin markers to
detect chromosome rearrangement. These markers
all suggest a lack of linkage to the ADPKD locus.Additionally, the chromosomes of key family mem-bers were examined.
MATERIALS AND METHODS
We have studied 43 members of the Wellesley Hos-pital A family. Family members were assessed clini-cally by J.M.R. All participants gave informed con-sent.
DNA Probes
The probe palpha3’HVR.64 (Table 1) was gener-ously donated by Professor D.J. Weatherall of OxfordUniversity (Oxford, England) (13,14). The probes
(Table 1)CRI-090(D16S45), CRI-0129 (D16S56), CR1-0133 (D16S58), CRI-0136 (D16S60), and CRI-0327(D16S63) were purchased from Collaborative Re-
search Incorporated (CR1, Bedford, MA) (15,16). The
probe 24-1 (D16S80) (Table 1) was kindly suppliedby Dr. M.H. Breuning from the Rijksuniversiteit ofthe Netherlands in Leiden.
Polymorphism Detection
Blood samples were collected from all living mem-bers of the WHA family. Plasma, serum, and red cellswere stored at -70#{176}Cfor future use. DNA was ex-
tracted from the white cells by standard methods(42). DNA was analyzed after digestion to completionwith bacterial restriction enzymes (Table 1). DigestedDNA was separated by electrophoresis in 0.8% aga-rose gels when probed with the cosmid vectors and
1.0% gels when probed with palpha3’HVR.64 and24-1. After electrophoresis, the DNA was passively
transferred to nylon membranes (Hybond; Amer-sham Canada Limited, Oakville, Ontario) (43), hy-bridized to the 32P-labeled DNA probe, and radiola-
bebed by the obigonucleotide primer method of Fein-
berg and Vogebstein (44). Fragments hybridizing tothe probes were detected by autoradiography. andthe restriction fragments for each family memberwere defined by inspection.
The alpha globin polymorphism defined bypalpha3’HVR.64 derives from the variable repetitionof a tandem repeat unit resulting In eight size classesof DNA fragment (13). Unrelated individualsseldom share identIcal-appearing fragments. This
Brissenden et al
Journal of the American Society of Nephrology 915
TABLE I. Probes used for ADPKD family linkage study
Probe Enzyme LocusFragment SI:es
Mops To:
palpha3’HVR.64 Pvull D16S85 VNTRb 0.04 16p13.3CRI-090 EcoRI D16545 10.6, 9.4 (C)
20.0, 13.0, 6.8 (V)0.06 lopter to p13
CRI-0129 EcoRl D16S56 11.0, 8.8, 4.8 (C)Al 15.0, A2 8.0
0.09 16p13
CRI-0133 HindIlI D16S58 10.5, 5.6, 4.1, 2.92.5, 2.1 (C)7.0,6.4(V)
0.10 16p13
CRI-0136 H/nclI D16S60 10, 7.2, 4.6, 4.5, 3.2,2.4. 1.8 (C)
Al 3.8, A2 3.4, A32.7
BI 5.6, B2 5.2
0.12 16p13
CRI-0327 Hindlll Dl6563 16, 7, 5, 3, 2.5 (C)11.2, 9(V)
0.04 16p13
24-1 ToqI DI6S8O BI 3.8 (80%)B2 1.5,1.3(13%)B3 1.5(7%)B4 1.4 (rare)
0.04 16p13.3to p13.13
#{176}8, recombinotion frequency between the PKDI locus and the marker loci.
b VNTR, variable number tandem repeat eight sizes. C, constant fragment; V. variable fragment. Values in parentheses are the frequencies in
the population.
palpha3’HVR.64 probe was used along with othermarkers to check the paternity in this family because
non-paternity Is an occasional cause of apparentfailure to detect linkage.
Karyotyping from three family members was doneby standard methods (12) to rube out a possible fa-
milial chromosomal rearrangement as the cause of
failure to detect linkage in this family.
LINKAGE ANALYSIS
Pairwise lod scores (45) between the disease locusand the marker Id were determined by using the
computer program LIPED (46). The lod score is ameasure of the relative odds in favor of linkage ver-sus no linkage at different arbitrarily chosen recom-bination values (0) between 0 and 50%. The absenceof linkage corresponds to a recombination fraction of50%. The odds values are expressed as logarithms tothe base 10 and are called lods, an abbreviation forthe term logarithm of the odds. The recombinatlonvalue at which the lod score is maximal is taken as
the best estimate of the recombination frequencybetween two linked loci (maximum likelihoodmethod). Linkage is considered significant when the
lod score is �3 In cases where there is no prior reasonto believe that linkage should be present. Linkage of
an ADPKD locus to the studied chromosome 16 mark-ers has been demonstrated previously in a barge num-ber of families as described (12-29). Linkage is ex-
cluded when the lod score is �-2. and the data areInconclusive when the lod score is between -2 and+3 (45).
The probability that an individual at risk forADPKD will be diagnosed as having ADPKD increaseswith age. For an age of onset correction, we used theprobabilities for at-risk individuals actually carrying
the ADPKD gene after testing as negative, as calcu-lated by Bear et at. (10) for different age groups.These probabilities are: 0.34 for the age interval, 15to 20 yr; 0.14 for the interval, 20 to 30 yr; and 0.05for the interval, 30+ yr. A straight line age of onsetcorrection (47) was used in the linkage analysis.Penetrance (the probability that an individual whocarries the disease gene will be diagnosed as being
affected) was considered to increase linearly from avalue of 0.05 at birth to 0.99 at 40 yr of age or older.
The gene frequency of ADPKD was assumed to be0.008. The data were also analyzed by the LINKMAPprogram of the LINKAGE package of programs writ-
ten by M. Lathrop and J.M. Lalouel (48,49). Thisprogram is used for multipoint linkage studies. Themethod varies the position of one locus over a fixed
map of linked markers to detect the most likely mapposition of the variable marker. For LINKMAP, age
of onset was again corrected, this time as a stepfunction, based on the different liability classes de-scribed previously (Bear et at. (10)).
RESULTS
Clinical Assessment of Family Members
The index case in the family (Figure 1) (IV, 1)presented at age 21 with severe flank pain. Subse-quent surgery to reduce a parapelvic cyst thought to
Cl)UI
0C)(I)
a0-J
0.40.5
N
AFEE�TED ONLY
AU, DATA
ADPKD Not Linked to Chromosome 16 Probes
916 Volume 2 Number 4’ 1991
Figure 1. Pedigree of the family. The arrow points to theindex case. 0 and 0, male and female, respectively. Link-age data from top include palpha3’HVR.64, ADPKD normal(N) or affected (Af), CRI-090, and CRI-0136. 1,1-died ofrenal failure at age 90+-not assessed; 11.3-asympto-matic. Ultrasound (U/S)-ADPKD, moderate renal insuffi-ciency, creatinine of 271, 68 years old; 11,4-presenteduremic at age 65. U/S-typical ADPKD, end-stage renal dis-ease required peritoneal dialysis. Died of complications ofuremia; 11,8-U/S-typical ADPKD, creatinine probably nor-mal at 98 mmol/L; 11.9-U/S-multiple bilateral Cysts, ultrason-ically normal tissue between cysts; creatinine normal at 77mmol/L; 11,10-presented with anaemia at age 57. U/S-ADPKD, creatinine moderately increased at 150 mmol/L;lll,3-asymptomatic. U/S-ADPKD, creatinlne unknown, age40+; 111,4-asymptomatic. U/S-ADPKD, creatinine normal,age 40; III, 17-asymptomatic. U/S-mild ADPKD, renal func-tion normal, age 29; lV,1-flank pain age 21. U/S-two smallCysts (less than one centimeter in diameter); polycystickidney at laparotomy, normal renal function, hypertension;lV,3-asymptomatic, age 16. Three small renal cysts, nor-mal renal function, myotonic dystrophy inherited from fa-ther.
be obstructing the renal pelvis resulted in the diag-
nosis of bilateral extensive kidney cysts. Family his-tory revealed that the maternal grandmother of thepatient (II, 4), aged 65, had renal failure, and evalu-
ation revealed typical ADPKD. Ultrasound examina-tion of the grandmother’s two children (III, 3; III, 4)
showed that both had ADPKD. Both of these individ-
uals are aged in their forties and have well-preservedrenal function. Subsequent evaluation of the grand-mother’s eight living sibs (of nine) revealed four tohave evidence of mild ADPKD involvement (II, 3; II,
8; II, 9; II. 10). Two sibs (II, 9; II. 10), aged in theirearly sixties, have normal renal function and very
mild renal impairment, respectively. One of these
sibs (II, 9) has unusually well-preserved renal paren-chyma despite multiple, bilateral renal cysts. Two
sibs. one in the late fifties (II, 10) and one at age 68(II. 3), have moderate renal Impairment with typical
ADPKD. One sib (IV, 3) of the index case (IV, 1) fits
the criteria for mild, early ADPKD, and one sib (IV,4) has spbenic cysts only. These batter two sibs werecoded as “disease status unknown” for the LINKMAP
‘L’b.3 -0.2 -0.1 0.0 0.1 0.2 0.30 ro
> 0) 1’)
I2 2C,)
MAP DISTANCE (MALE)-MORGANS
Figure 2. ADPKD location likelihood relative to restrictionfragment length polymorphism locus map locations. Thelikelihood of a particular location of the ADPKD locus di-vided by the likelihood that the ADPKD locus is not linkedand expressed as log to the base 10 (lod) is plotted for anumber of positions of the ADPKD locus within a publishedmap (16) of the three informative markers studied. On the
x axis are plotted the map locations of the informativerestriction fragment length polymorphism markers relativeto an arbitrarilydefined zero. Included isthe region distalto CRI-0 136 with the telomere on the left.The distances(morgans) were derived by applying Haldane’s mappingfunction (48,49) to the published recombination data (16).The lod scores are plotted on the �‘ axis. The ADPKD locusIsset as the variable locus, and the age of onset correctiondescribed in the text was used. The solid line results fromanalysis of the entire family data. The broken line resultsfrom analysis of the affected individuals only. On the figure,the probe name paipha3’HVR.64 has been abbreviated3’HVR and probes CRI-090 and CRI-O 136 have been abbre-viated CR1090 and CR10136, respectively.
analysis. All other family members were clinically
normal.
DNA Results
Results of analysis of informative DNA markersfor the family members are summarized in Figure 1.Results are included for only 26 of the 43 familymembers whose DNA was studied without priorknowledge of their disease status. After clinical ex-amInation of a family member showed him/her to beADPKD negative beyond the age of 40, his/her chil-
dren were not studied further.Probe 24-1 and the CR1 probes, other than CRI-090
and CRI-0 136, were not informative in this family.There was no evidence for non-paternity in any of
the family units. The marker patterns of each childwere consistent with family relationships as statedas were each sib’s markers.
Linkage Analysis
Two-point lod scores between palpha3’HVR.64and ADPKD and between CRI-090 and ADPKD ex-
Brissenden et al
Journal of the American Society of Nephrology 917
TABLE 2. Lod scores for linkage between the ADPKD locus and three informative probes
ProbeRecombinat ion Value (8)
0.000 0.001 0.010 0.50 0.100 0.200 0.300 0.400
CRI-090 -3.97 -3.93 -3.29 -2.04 -1.44 -0.83 -0.46 -0.20palpha3’HVR.64 -9.54 -7.32 -4.71 -2.24 -1.09 -0.08 0.26 0.26CRI-0136 -1.62 -1.60 -1.42 -0.10 -0.65 -0.29 -0.11 -0.02
elude linkage in our family at recombination frac-tions of up to 0.06 and 0.05, respectively (Table 2).The lod score was inconclusive for linkage between
CRI-Ol 36 and the ADPKD locus. Data from the otherthree probes were noninformative and are not in-cluded in Figure 1 or in Table 2.
Figure 2 shows the results of multipoint analysisby using LINKMAP. Multipoint linkage analyses ex-
cluded the disease locus 0.20 morgans from the distalside of palpha3’HVR.64 to 0.43 morgans on the prox-
imal side when palpha3’HVR.64 was arbitrarily set
at 0.00. These results are still supported when thedata are analyzed by using affected family membersonly. Despite the reduced numbers of individuals
analyzed, the graph of lod equivalent scores (48,49)
is similar to that of the entire family (Figure 2). Thebed equivalent at -2.32 with 0 = 0 between theADPKD locus and pabpha3’HVR.64 excludes linkage.At 0=0 between CRI-090 and ADPKD, the lod equiv-alent is -1.67, and at 0 = 0 between CRI-0136 andADPCKD, It is -1.78, strongly suggesting no linkagein both cases. Even with data from affected individ-
uals only, the lod equivalent never rises above -1.58
over the whole region analyzed, supporting lack of
linkage with the ADPKD locus.
Results of Karyotypic Analysis
The chromosomes of individuals 11-6, 11-8. and 11-9
were normal with no evidence of rearrangement be-tween chromosome 16 and/or any other chromo-
somes at the 550-band stage (12). Individuals 11-8 and11-9 were affected with ADPKD, and 11-6 was unaf-
fected. Nevertheless, individuals 11-6 and 11-9 hadidentical pabpha3’HVR.64 restriction fragments, im-plying at least one crossover event between themarker and disease loci.
DISCUSSION
This interesting family in which the ADPKD locus
is not linked to the chromosome 16 DNA sequences.which are known to be linked to the locus for the
classic form of ADPKD (PKD 1), was first reported in
1988 (39).The family provides additional evidence for a sec-
ond locus for ADPKD. This family is of Interest be-
cause it was studied extensively with flanking mark-ers as well as with the alpha globin marker,
palpha3’HVR.64. In addition, the family originatedin the British Isles. The original two reported familiesin which ADPKD was not linked to chromosome 16markers were Italian and Italian-American in origin.raising the possibility that this form of ADPKD mightoriginate from a single geographical isolate. Our fam-
ily, plus others recently reported, increases the pos-
sibility of a greater worldwide presence of the secondlocus for ADPKD. It will therefore be necessary toconsider the likelihood of this second locus in all
ADPKD families studied with DNA markers.Grantham (35) and Hulten (36) have suggested the
possibility of a balanced/unbalanced familial chro-mosomab rearrangement to explain cases of apparent
exclusion of linkage of ADPKD to the chromosome
16 probes, in which linkage might In reality exist.
Because our data exclude linkage between ADPKDand two flanking markers on chromosome 16,palpha3’HVR.64. and CRI-090, and are highlysuggestive of exclusion between ADPKD and CR!-
0136. it would be necessary to postulate a deletion/insertion type of chromosome rearrangement to ex-
plain these data. Two chromosome 16 breaks very
close together, one above and one below the PKD1
locus, and insertion of the chromosome segment con-taining PKD 1 to another chromosomab location com-
patible with no abnormalities would be necessary toexplain our data by a balanced familial chromosomal
rearrangement. Familial balanced insertion re-arrangements have been associated with expressivitydifferences in retinoblastoma (50) as have familialdeletions (51), but these have so far been cytogenet-ically detectable. Familial deletions have also been
associated with such single gene conditions as mus-cular dystrophy (52) and X-binked lymphoprolifera-
tive disease (53,54). In these cases, the re-arrangement has also been detectable either cytoge-
netically or by the loss of closely linked DNA markersand, in some cases, by the presence of additionalclinical syndromes. Chromosomal studies of threeindividuals in this family, of whom one unaffected.64-yr-old woman was an obligate crossover (if theADPKD locus of this family is linked to chromosome
16 short arm markers), failed to detect a re-arrangement at the 550-band stage. Further studieswill be necessary to definitely rule out the possibility
of chromosome rearrangement in this family. Suchstudies include hIgh-resolution chromosome analy-
ADPKD Not Linked to Chromosome 16 Probes
918 Volume 2’ Number 4 #{149}1991
sis, cloning of the PKD1 gene, and/or mapping of the
second ADPKD locus and its cloning. In situ hybrid-ization of closely linked DNA markers in our family
would be helpful to rube out a subtle chromosomal
rearrangement.
ACKNOWLEDGMENTS
This study was supported by the Wellesley Hospital Renal Research
Fund and the Polycystic Kidney Disease Research Fund at the
University of Toronto. Part of this work was supported by the MRC
of Canada (to N.E.S.).We thank Dr. Andrew Pakstls from the laboratory of Dr. Ken Kidd
at Yale University for his generous and ongoing support in setting
up the linkage analysis programs, including LINKAGE and the VAX
version of LIPED In our laboratory. We also thank both Drs. KIdd
and Pakstls for their helpful discussions during the analysis of our
data and for performing critical analyses which were beyond the
capacity of our computer on their VAX. Without their help this paper
would not have become a reality. In addition, we appreciate the help
of Nancy Worth and the University of Toronto Faculty of Medicine
Division of Medical Computing in setting up the linkage analysis
programs at the University of Toronto. We thank Dr. E. Winsor.
Toronto Hospital. for performing the chromosomal analyses on fam-
ily members II. 6: II. 8; and II. 9. We also thank Mrs. S. Bandeau for
her excellent technical assistance and Doron Almegor for his tirelessand enthusiastic collection of family data and specimens and for
encouraging the family members to take part. We thank the family
members themselves for their unselfish participation in the study
and their referring family doctors for their helpful cooperation.
Lastly, we thank W.G. and J.w. Brissenden for their essential
support and encouragement.
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