Note: Descriptions are shown in the official language in which they were submitted.
~'A21 16633
The present invention relates to a method for the
diagnosis of Familial Hemiplegic Migraine (FHM)
Background of the Invention
Migraine is an extremely frequent, and sometimes
incapacitating condition, affecting about 4% of children,
6% of men and 18% of women (Stewart, W.F. et al., Jama 267,
64-69, 1992, and Lance J.W., Mechanism and management of
headache, fourth ed. Butterworths Scientific, London,
1982). Its etiology is unknown and its pathophysiology
poorly understood. In the complete absence of laboratory
diagnostic tests, its diagnosis r~r~; n~ purely clinical,
based on the detailed description of symptoms by the
patient. Until recently, there has been a marked
interobserver diagnostic variability due to the lack of
explicit definition and precise diagnostic criteria. This
~A2 1 ~ 6~33
_
has led the experts of the International Headache Society
(I.H.S.) to propose a new classification for headache
disorders which provides, for the first time, operational
diagnostic criteria for all varieties of facial pain and
headache, including migraine (Headache Classification
Committee of the International Headache Society
Classification and diagnostic criteria for headache
disorders, cranial neuralgias and facial pain - Cephalalgia
8, Supplt 7, 19-28, 1988). The two most frequent varieties
are migraine without aura (formerly called "common"
migraine), and migraine with aura (formerly called
"classical" migraine) in which neurological symptoms
precede or accompany the headache.
Familial hemiplegic migraine (FHM) is considered
in the I.N.S. classification as a subtype of migraine with
aura, particular by its rarity, its autosomal dominant
pattern of transmission and by the presence of hemiplegia
as the salient feature of the aura (Blau, J.N. et al.,
Lancet 2, 1115-1116, 1955). Since its first description by
Clarke in 1910 (Clarke, J.M., Br. Med. J. 1, 1534-1538,
1910), it has been reported in around forty families. The
age of onset varies from 5 to 30 years with a predominance
during youth. Minor head trauma and cerebral angiography
are well established triggering factors (Blau, J.N. et al.
- see above - and Glista, G. et al., Mayo Clin. Proc. 50,
307-311, 1975). Attacks are characterized by the presence
-
~..
l,'A2~ r`6G33
of an hemiparesis or hemiplegia either isolated or
associated with other aura symptoms such as hemianopic
blurring of vision, unilateral paresthesiae or numbness,
and dysphasia (Whitty, C.W.M., J. Neurol. Neurosurg.
Psychiatry 16, 172-177, 1953). These symptoms usually last
30 to 60 minutes and are followed by a severe pulsatile
headache lasting a few hours or days. In severe attacks,
hemiplegia is often associated with fever, drowsiness,
confusion or coma which usually also resolve within a few
hours, days or sometimes weeks (Munte, T.F. et al., J.
Neurol. 237, 59-61, 1990, and Fitzimons, R.B. et al., Brain
108, 555-557, 1985).
Various combinations of neurological deficits
have been described in association with FHM: retinitis
pigmentosa, sensorineural deafness, (Young, G.F. et al.,
Arch. Neurol. 23, 201-209, 1970), essential tremor,
disordered ocular smooth poursuit (Zifkin, B. et al., Ann.
Neurol. 8, 329-332, 1980). The most frequent one is a
progressive cerebellar disturbance with dysarthria,
horizontal nystagmus and limb incoordination (Ohta, M. et
al., Neurology 17, 813-817, 1967, and Codina, A. et al.,
Rev. Neurol. (Paris) 124, 526-530, 1971). These deficits
are permanent and not part of the aura.
Summary of the invention
During the clinical investigation of patients
affected with another autosomal dominant neurological
.
- ~2~-6~33
condition, CADASIL, the Applicant noticed that some of the
patients also experienced recurrent attacks of severe
headache, with various aura symptoms including transient
hemiplegia (Tournier-Lasserve E. et al., Stroke 22, 1297-
1302, 1991). This led the Applicant to raise the hypothesis
that CADASIL and FHM, though clinically widely different,
might be due to the alteration of the same gene (Tournier-
Lasserve E. et al., Nature Genetics, vol. 3, 256-259, March
1993 and Joutel A. et al., Nature Genetics, vol. 5, 40-45,
o September 1993, which are both incorporated therein by
reference). The Applicant performed linkage analysis on 2
unrelated FHM families with a set of DNA markers spanning
the most probable location of CADASIL and establish the
most likely location of the FHM gene on chromosome 19,
within a 30 cM interval encompassing the most probable
location of the CADASIL gene. Locus assignment provides the
first biological marker for this disease.
Clinical heterogeneity of FHM was reported on the
basis of the presence of a cerebellar involvement occuring
in some families. In the reference herein enclosed (A.
Joutel et al, Nature Genetics, 1993, 5, 40-46) the
Applicant demonstrated linkage to chromosome 19 in 2
pedigrees of both types (with and without cerebellar
involvement).
The Applicant have now studied by linkage
analysis more than ten families affected with FHM and
~,d2 1 1 `~3
established genetic heterogeneity in the sense that
approximately fifty per cent only of the FHM families
tested map on chromosome 19 (A. Joutel, manuscript in
preparation). To our current knowledge, these is no
clinical difference between chromosome 19 linked and
unlinked families.
The invention has several objects:
- One object of the invention is to provide a
method for diagnosing a subgroup of FHM.
- Another object of the invention is to provide a
method to estimate the carrier risk for an at risk
individual within an affected family linked to chromosome
19 .
- Another object of the invention is to provide a
method to identify FHM families which are linked to
chro`mosome 19 allowing selection of genetically homogenous
groups of FHM families, this being potentially important in
terms of therapeutic trials.
- Another object of the invention is to provide a
method for screening families affected with other types of
migraine, with and without aura.
These objectives are attained by a method for the
genotypic diagnosis of FHM, by searching the presence of a
mutated gene responsible for the disease, comprising the
search of the presence of a mutation responsibie for FHM on
the chromosome 19, in the region of it comprised between
-
.
- ~A2~7~3~
the microsatellites D19S216 and D19S215, particularly of a
family or of an at risk individual, including foetus.
Preferably, this method is based on genetic
linkage analysis of human samples belonging to the members
of the affected families to be tested. The first step is to
establish linkage of the disease gene responsible for the
disease present in the family with the FHM locus. Linkage
analysis is conducted on families which structure is
suitable for such analysis, namely families comprising
multiple individuals which clinical status (healthy or
affected) has been unambiguously established (see Nature
Genetics, 1993, 5, 40-46).
Genetic linkage analysis is preferably conducted
with a set of highly polymorphic DNA markers
(microsatellites flanking highly polymorphic CA or GATA
repeats) spanning the most likely location intervals of
FHM, flanking markers beeing D19S216 and D19S215.
Markers are selected to give the best
informativity for a given family. Markers located on both
sides of the gene increase the accuracy of the diagnosis
and permits to reduce the risks of false diagnosis in case
of recombination. These markers include D19S216, D19S76,
D19S221, D19S179, D19S226, D19S252, D19S253, D19S244,
D19S415, D19S199, D19S215.
These microsatellites are described in the
microsatellites Genethon map (Weissenbach J. & al., Nature
~A2 1 ~ 6633
359, 794-801, 1992 which is incorporated therein by
reference) as well as in J. Weber et al, Am. J. Hum. Gen.
1993, 53, 1079-1095 . The oligonucleotide sequences serving
as primers, which are specific of each microsatellite, are
available in the Genome Data Bank (Accessing GDB and
OMIMTM at Johns Hopkins University, Baltimore, Maryland,
USA).
Preferably, the method comprises hybridization of
selected primers to the DNA to be tested, followed by DNA
amplification by Polymerase Chain Reaction amplification.
Preferably, the polymorphic amplified fragments so called
amplimers, are then separated according to their size by
electrophoresis on acrylamide denaturing gels, blotted on
nylon membranes and hybridized with CA12mer or GATA 5mer
radiolabelled probes. Data obtained from autoradiographies
are computed and Lod-score calculations are carried out
using the M-LINK program (Lathrop et al, P.N.A.S., 1984,
81, 3443-3446.). Based on MRI data, penetrance has been
established to be complete after 35 year old (see above).
The assertion of linkage or absence of linkage is
derived from statistical analysis, a lod-score above 3
establishing unambiguous linkage of the disease gene to the
tested marker. A lod-score value of 3 is in fact needed
only when the prior probability of linkage between a
disease gene and a marker is no more than random, which is
not anymore the case for FHM. The strictness of this
.
~21 1~633
criterion should be soon decreased according to the prior
probability values observed in on going epidemiological
studies.
Once genotypic analysis has established linkage
to chromosome 19 within a given FHM affected family,
genotypic analysis can be used in a second step for testing
of at risk individuals, members of an affected family for
example for predictive testing or help for differential
diagnosis when needed. Using linked polymorphic markers a
DNA based carrier risk can be calculated using the MLINK
program (Lathrop et al, P.N.A.S., 1984, 81, 3443-3446.).
Thus, the preferred general method with respect
to the kind of diagnosis comprises hybridyzing DNA
fragments from an individual with selected primer pairs
which hybridize selectively on DNA sequences flanking
allelic DNA polymorphisms situated in the said region
comprised between the microsatellites D19S216 and D19S21S,
including the latter, separately amplifying the DNA
polymorphisms flanked by the primers thus forming so-called
amplimers and analysing these polymorphisms in order to
assess the presence or absence of linkage or the DNA
polymorphism carrier risk by genetic linkage analysis.
The discovery by the inventors that the FHM gene
was linked to a specific region of chromosome 19 rendered
the principle of a genotypic diagnostic method available.
It is evident that from this information and the DNA
21 i b~33
polymorphisms disclosed therein, it is easy for the
specialist to test other markers which would be tested or
discovered later. The application of other markers in the
genotypic diagnosis of FHM r~m~i n~ within the scope of the
present invention.
Besides, on the basis of the therein-disclosed
microsatellites, it is easy to search other polymorphisms
which are closer to the gene responsible for FHM. The
method consists in the search of new polymorphisms which
are linked to at least one of the microsatellites disclosed
herein. The potential polymorphisms can be then submitted
to a linkage analysis in relation with the gene, in order
to determine the genetic distances and the Lod Scores.
Therefore, another object of the invention is to
propose a method for selecting polymorphisms which are
closely related to the gene responsible for FHM , wherein,
with the aid of DNA probes, one searches polymorphisms and
then one submit these polymorphisms to a linkage analysis
with the microsatellites D19S216, D19S76, D19S221, D19S179,
D19S226, D19S252, D19S253, D19S244, D19S415, D19S199,
D19S215. Thereafter, one can proceed with a linkage
analysis of the selected polymorphisms with respect to the
disease gene.
Brief description of the figures
Figure 1 : Pedigree of the first FHM family
analysed. The Roman numbers, on the left, represent the
633
generations, the numbers above the symbols identify the
individuals. Squares, males; circles, females; the affected
subjects are represented by a filled symbol and by a half-
filled symbol when the symptoms are assumed on the basis of
familial history; hatched symbols stand for subject of
unknown status ; At, cerebellar ataxia.
Figure 2 : analogous to Fig. 1 for the second FHM
family.
Figure 3 : Multipoint linkage analysis. Location
10scores for the FHM locus with respect to D19S216 (1),
D19S221 (2), D19S226 (3). The solid line indicates the 10:1
odds (1-lod-unit) interval for the placement of the locus.
Odds against alternatives are shown for the most likely
placements of FHM locus in each interval from the map.
15Figure 4 : analogous to Fig. 3, but with respect
to D19S221 (2), D19S226 (3) and D19S215 (4).
Figure 5 : Genetic regional map of chromosone 19.
Critical markers used for linkage analysis are indicated
with their respective genetic distances (~). Map is shown
to scale. Approximate location is shown for selected
markers from the CEPH database (version 5). The order of
the markers was determined as described, in Weissenbach, J
& al., Nature 359, 794-801 (1992).
Detailed specification
25For the purpose of the present genetic study, the
Applicant selected 2 large FHM pedigrees satisfying the
-
- ~A2 1 ~ ~33
I.H.S. criteria for FHM, one with cerebellar signs, the
other without. The Applicant performed linkage analysis
with a set of DNA markers spanning the best estimate
location interval for CADASIL, which was recently mapped on
chromosome 19 ~Tournier-Lasserve, E. et al., Nature
Genetics 3, 256-259, 1993). Linkage of FHM gene was
established to two markers, D19S221 and D19S226, with
respective maximum lod-scores of 8.24 and 8.07 at e = 0.03.
Multilocus analysis localizes the disease gene within the
30 cM (centiMorgans) region located between the D19S216 and
the D19S215 loci; this region encompasses the interval
spanned by D19S221 - D19S21S, which is estimated to be the
most likely location of CADASIL. Although at present it
cannot be excluded heterogeneity, these results suggest
that these two diseases may be due to the alteration of the
same gene.
Clinical evaluation
The first pedigree (Family 1, Fig. 1) is a three
generation Caucasian family originating from the center of
France. All three members from generation I were born most
likely of different fathers but of the same mother for whom
clinical history was not available. The grandmother on
their mother's side was said to have presented recurrent
attacks of transient hemiplegia with headache. Thirty eight
members of this family were examined and clinical data from
three other affected members were obtained by telephone.
.
_~ .
- ~ A i ;i l ~ 3
Twenty members have experienced attacks of
hemiplegic migraine satisfying I.H.S. diagnostic criteria
for FHM. Attacks started at a mean age of 17.5 years ~range
7-37) in generation II, and 12.1 years (5-26) in generation
III. All the patients presented typical hemiplegic attacks
and eight of them occasionally had severe attacks in which
hemiplegia was associated with confusion or coma and fever.
All symptoms always resolved without sequelae. Neurological
examination during non-affected periods disclosed
horizontal gaze nystagmus in 16 of these affected members
and cerebellar ataxia in one (I-2). Mental retardation was
present in another individual (III-21). Magnetic resonance
imaging (MRI) of the brain, performed on ten subjects (five
of them with severe attacks), did not show any abnormal
signal. There were no lesions suggestive of infarcts and
the brain white matter was normal. A cerebellar vermian
atrophy was present in two subjects (I-2, III-21). Cerebral
angiography, performed in four individuals, was normal in
three, however in one subject (II-7), who had suffered from
subarachnoid hemorrhage, a large middle cerebral artery
aneurysm was found. In one patient (II-6), two extremely
severe attacks were triggered, first, by a coronary
arteriography and second, by a cerebral angiography.
Attacks were triggered by minor head trauma in two other
patients.
Sixteen other members, with a mean age of 41.8
.
~'A2~ ~-663~
years (28-50) at generation II and 23.4 (18-35) at
generation III, born of an affected parent, were free of
attacks of hemiplegic migraine. Neurological examination
was normal in 14 of these subjects but disclosed
cerebellarataxia and mental retardation in one individual
(III-32) and horizontal gaze nystagmus in another (III-28).
The second pedigree (Family 2, Fig. 2),
originates from the south of France. It includes 22 living
members,belonging to four generations. Ten have presented
typical recurrent attacks of hemiplegic migraine which were
occasionally severe in three. Two others have experienced a
single episode of respectively transient hemiplegia without
headache (II-6) and prolonged hyperthermic confusion (III-
20). The mean age of onset was 10.2 years (6-15).
Neurological examination performed in eight affected
members and two healthy individuals was unremarkable. In
particular there was no nystagmus or cerebellar ataxia.
Brain MRI and cerebral angiography performed in one subject
(III-9) were normal.
Linkage analysis
The genetic linkage analysis was undertaken on
fifty-four members from the two families. Four highly
polymorphic markers spanning the region containing the
CADASIL locus on chromosome 19 were chosen for genotype
characterization. Allele frequencies were estimated
initlally from data on the CEPH panel (CEPH database
- ~A21 16633
14
version 6). The analysis was repeated with a frequency
estimated from the FHM families. Individuals were
considered affected for linkage analysis, based on the
occurrence of at least one hemiplegic migraine attack as
defined by the I.H.S. criteria. Initially, due to
uncertainty concerning penetrance, the disease status was
included only for affected members, all others being
considered of unkown phenotype. As a second step, healthy
individuals with a normal neurological examination were
considered as non-affected, and included in the analysis.
Family 1 included 20 affected members and 11 non-affected
members, and family 2 included 10 affected members and 2
non-affected members. Based on data published previously
from all families, the penetrance for FHM was estimated to
be approximately o.g.
Significant evidence of linkage was obtained when
either affected family members alone, or both affected and
non-affected family members were included in the analysis
of the selected chromosome 19 markers (Table 1). When
analyzing only affected members tTable 1, part a), combined
data showed a maximum lod score of 6.55 at e = o . 03 with
D19S226(AFM 256yc9). The lod score was 4.18 at e = o . os ( 1
recombinant : II-12) in family 1, and 2.54 at e = o in
family 2. The lod scores increased considerably when the
non-affected family members were included (Table 1, part
b). The maximum combined lod score was 8.24 at e = o . o 3
-
- ~' A 2 1 ~
with D19S221 (AFM 224ye9) even though the lod score for
the second family declined slightly because of an
unaffected individual ~ 11) who has inherited the
disease-associated allele. These results provide strong
evidence for a single disease locus in the two families.
Varying the penetrances or estimating the marker allele
frequencies for the FHM data did not affect these results
significantly.
Multipoint analysis
The order and sex-average recombination fractions
between the marker loci estimated from genotype data on a
subset of CEPH families are : D19S216 - 0.18 - D19S221 -
0.07 - D19S226 - 0.05 - D19S215 (Weissenbach, J. et al.,
Nature 359, 794-801, 1992). Due to computational
limitations, multilocus linkage analysis was undertaken
with subsets of three marker loci considered simultaneously
with the FHM or CADASIL locus (Lathrop, G.M. et al., Am. J.
Hum. Genet. 37, 482-498, 1985). The location scores are
reported with the subsets D19S216 - D19S221 - D19S226 and
D19S221 - D19S226 - D19S215. Penetrance 0.9 for the FHM
locus is assumed in the results presented here (Figures 3
and 4). Other subsets of the markers, and different
penetrances gave similar results, and led to no
modification in the conclusions regarding the intervals to
which the disease loci were assigned. As reported
previously, significantly greater recombination was found
16
in females compared to males in this region in the CEPH
families ~Tournier-Lasserve, E. et al., see above).
Location score analysis under the assumption of different
recombination fractions in males and females led to the
same conclusion regarding the locations of the FHM and
CADASIL loci (results not shown).
Multilocus linkage analysis provided conclusive
evidence that the FHM locus maps in the interior of the
region spanned by D19S216 - D19S221 - D19S226 - D19S215
(Fig. 5) ; the odds against placements outside of D19S216
or D19S215 were greater than 104:1 (Fig. 3 and 4). The most
likely location of the gene was between D19S216 and
D19S221, but its placement in the other intervals defined
by these markers can not be rejected. The odds against a
location within the intervals D19S221 - D19S226 and D19S226
- D19S215 were 48:1 and 330:1 respectively.
Examination of haplotypes suggested that the
evidence for a placement of the FHM locus distal to D19S221
is due largely to a recombination event observed for the
chromosome transmitted from individual I-l to II-12 in the
first FHM family. The individual II-12 is affected : her
affected father (I-l) is heterozygous for all four of the
marker loci. He has had three other affected offsprings who
have received the same paternal alleles at the four loci,
while individual II-12 has received the same allele of
D19S216 but different paternal alleles for the three other
17 l~A2~
loci.
Methodology
Markers. D19S216, D19S221, D19S226, D19S215 were
chosen from the microsatellites Genethon linkage map
(Weissenbach, J. et al., see above~. All oligonucleotides
sequences are available in the Genome Data Bank. The number
of alleles of the 4 critical markers used for pairwise and
multipoint linkage analysis, as well as their respective
frequencies were determined in the CEPH Caucasian reference
families (Tournier-Lasserve, E. et al., see above).
PCR. Polymorphic genomic sequences were amplified
by PCR using a PHC3 Techne apparatus. The reactions were
performed in a final volume of 50 ~l containing 200 ng of
genomic DNA, 125 ~M 4dNTP mix, lxPCR Boehringer Taq
Polymerase buffer, lU Boehringer Taq Polymerase, 1 ~M of
each primer. Samples were processed through 30 temperature
cycles (lst cycle, 94-C for 5 min ; 28 cycles including a
denaturation step at 92C for 1 min, an annealing step at
55 C for 1 min and an extension at 72C for 10 min).
After addition of 75 ~l of loading buffer the
samples were denatured for 10 min at 94C , then laid on a
6% acrylamide DNA sequencing gel. After blotting, nylon
membranes were fixed in 0.4M sodium hydroxyde and
hybridized with a (CA)12, P32 labelled probe for 14 hours.
Linkage analysis. Two point and multipoint
linkage analysis were performed using the LINKAGE package
~21 1~`633
18
assuming an autosomal dominant FHM gene with a O.OOOl
frequency. To test the effects of penetrance on the
results, the linkage analysis was performed with
penetrances of 0.8, O.9 and 1Ø Lod scores were calculated
at various recombination fractions for each marker.
Protocol of the diagnosis
The protocol which will be described hereinafter
is given by way of example and as such is not considered as
limitative. On the basis of the polymorphisms disclosed
therein, all known methods of genotypic diagnosis are
applicable.
Protocol :
- Obtaining DNA from the available family members whose
status with respect to the disease is known ; the nuclear
DNA can for example be isolated from peripheral blood
leucocytes, lymphoblastoid cell lines, cultured amniotic
fluid cells, or chorionic villi, by standard proteinase K
treatment and phenol-chlorophorm extraction techniques, and
amplified or digested with the appropriate restriction
enzymes if needed.
- Hybridyzing the selected set of primers to the DNA.
All oligonucleotide sequences serving as primers are
available in the Genome Data Bank.
- Amplifying the polymorphic alleles by the PCR technique,
using advantageously an automatic thermocycler apparatus
such as the PHC3 Techne apparatus. See for example
~,
(~A21 r~33
19
paragraph PCR in Methodology.
- Analysing the size of the amplification products
(amplimers), for example by electrophoresis on denaturating
acrylamid DNA sequencing gel, blotting on Nylon membranes,
hybridyzing with radiolabelled suitable repeats probes.
- Alternatively, restriction polymorphic fragments can be
analysed on agarose gels after Southern blotting.
- Data obtained from autoradio-graphies are computed and
Lod-score calculations are carried out using the M-LINK
program.
- The assertion of linkage or absence of linkage is derived
from statistical analysis, a Lod-score above 3 establishing
unambiguous linkage of the disease gene to the tested
marker. See above a lod-score <-2 excluding linkage.
~ Determination of the most informative polymorphims
(microsatellites) for the studied family.
When an at risk individual from an affected
family is to be tested for predictive diagnosis, the
protocol can be completed in the following manner:
~ ObtA;n;ng DNA from the individual in the same manner than
above.
- Hybridyzing the selected set of primers to the DNA.
- Amplifying as above.
- Analyzing the amplification products as above.
~ Using linked polymorphic markers, a DNA based carrier
risk can be calculated using the M-LINK program.
-
. .
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(~A 2 1 1 6633
- The same method can be used for prenatal carrier risk
diagnosis.
Table1 rai.~r~ise linkage data for FHM and chromosome 19 markers
a Affected subjects only
Locus Recombination fraction
0.00 0.05, 0.10 0.20 0.30 0.40 Z max
D19S216 Family 1-99.00 1.26 1.55 1.49 1.12 0.59 1.58 0.13
Family 2-99.00 -1.39 -0.91 -0.49 -0.25 -0.09 - -
Total-99.00 -0.13 0.64 1.00 0.87 0.50 1.00 0.21
D19S221 Family 11.34 3.6~ 3.49 2.86 2.00 0.99 3.63 0.05
Family 22.40 2.1 ~ 1.93 1.42 0.90 0.40 2.40 0.00
Total 3.74 5.8~ 5.42 4.28 2.90 1.39 5.83 0.03
D19S226 Family 11.94 4.18 3.99 3.24 2.26 1.13 4.18 0.05
Family 22.54 2.31 2.06 1.52 0.96 0.42 2.54 0.00
Total 4.48 6.49 6.05 4.76 3.22 1.55 6.55 0.03
Di9S215 Family 1-2.13 2.84 2.99 2.63 1.92 1.01 2.99 0.09 Family 2-99.00 -1.08 -0.56 -0.14 0.02 0.05
Total-99.00 1.76 2.43 2.49 1.94 1.06 2.58 0.15
b Affected and healthy subjects (penetrance=0.9)
0.00 0.050.10 0.20 0.30 0.40 Z max
D19S216 Family 1-6.47 1.251.68 1.72 1.35 0.74 1.78 0.15
Family 2-4.30 -1.40-0.91 -0.49 -0.25 -0.09 - -
Total-10.77 -0.150.77 -1.23 -1.10 0.65 1.30 0.22
D19S221 Family 10.75 6.335.96 4.81 3.38 1.70 6.36 0.03
Family 21.91 1.831.68 1.30 0.85 0.39 1.91 0.00
Total 2.66 8.167.64 6.11 4.23 2.09 8.24 0.03
D19S226 Family 10.35 6.065.78 4.73 3.35 1.70 6.07 0.04
Family 22.06 1.971.81 1.40 0.91 0.41 2.06 0.00
Total -2.41 8.037.59 6.13 4.26 2.11 8.07 0.03
- D19S215 Family 1-4.71 4.724.78 4.12 3.02 1.60 4.81 0.08
Family 2-7.86 -1.42-0.81 -0.26 -0.03 0.04 - -
Total-12.57 3.303.97 3.86 2.99 1.56 4.07 0.14
