European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genetic analysis, linkage, and association
tions or point mutations of the gene. More than 98% of deletions of the
DMD gene are readily detectable in affected males by using a PCR
reaction. We routenly use three multiplex PCR reactions to analyse
25 exons and the muscle and brain promoters. Recently, we started
to use the Multiplex Ligation-dependent Probe Amplification (MLPA)
technique, with the two commercial dystrophin probe mixes (P034/
P035 MRC Holland). This system allows the simultaneous hybridisation
and ligation of several probes, followed by PCR amplification and
analysis by capillary electrophoresis. We analysed with this technique
200 affected males and 60 female relatives for deletions and duplications
detection.
An sporadic DMD patient, presenting absence of dystrophin in the immunohystochemistry,
was analyzed firstable with by multiplex PCR
and no deletion was observed. Subsequently a deletion of exon 12
was observed with the P035 MLPA probe mix. The sequencing of
exon 12 showed the c.1438G>T, p.Gly480X mutation. This mutation
changes the penultimate nucleotide of the ligation site sequence and
consequently originates an apparent absence of exon 12.
In case of a single exon deletion pattern in MLPA reaction, it is mandatory
to perform the multiplex PCR and the sequencing of the exon in
order to check a point mutation.
P1029. High prevalence of GCK mutations and low prevalence of
HNF1A mutations in Italian MODY patients
N. Calza 1 , V. Mantovani 1,2 , P. Garagnani 1,3 , M. Cenci 2 , D. Bastia 1 , S. Salardi 4 , C.
Monciotti 5 , D. Luiselli 3 , G. Romeo 2 ;
1 Centro Ricerca Biomedica Applicata Policlinico S. Orsola-Malpighi, Bologna,
Italy, 2 U.O. Genetica Medica Policlinico S. Orsola-Malpighi, Bologna, Italy, 3 Antropologia
Molecolare Dip. Biologia Evoluzionistica Sperimentale Università di
Bologna, Bologna, Italy, 4 U.O. Endocrinologia Pediatrica Policlinico S. Orsola-
Malpighi, Bologna, Italy, 5 Dip. Pediatria Università di Padova, Padova, Italy.
Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous
group of disorders characterised by early onset non-insulindependent
diabetes mellitus, autosomal dominant inheritance and primary
defect in pancreatic beta cells function. Six genes have been associated
with different subtypes of the disease, but 15-20% of MODY
families do not exhibit any mutations in these genes. MODY2, caused
by glucokinase (GCK) mutations and MODY3, caused by hepatocytes
nuclear factor (HNF1A) mutations, are the most common forms; while
MODY1, 4, 5 and 6 are rare disorders. Aim of our study is to assess the
relative prevalence of MODY2 and MODY3 in Italian patients.
96 unrelated probands fitting MODY criteria were screened for GCK
mutations and, when negative, for HNF1A mutations. The analysis
was performed by DHPLC and direct sequencing. Mutations in the
GCK gene were detected in 33 of 96 (34.3%) families. 7 mutations
were previously undescribed: 3 missense ( E372D, C382X, H424Y),
2 deletions (Q106_M107 delinsL and G295fsdel CA), 1 splicing mutation
(IVS7+2T>C) and a stop codon suppression (X465insQ465+1_
X+145). Mutations in HNF1A gene were detected in 3 (3,1%) probands,
consisting in 2 new missense mutations (R159P and E508K)
and the known G31D. All mutations co-segregated with affected family
members, except for the de novo R159P mutation.
Our study indicates that defects in GCK/MODY2 gene are a very common
cause of MODY in Italian population, whereas HNF1A/MODY3
has a lower prevalence. Our data broadens our knowledge of the naturally
occurring GCK and HNF1A mutations repertoire.
P1030. Resolving a genetic paradox for Kostmann disease
through PGD
M. Malcov, D. Ben-Yosef, I. Roitberg, T. Cohen, T. Frumkin, A. Amit, Y. Yaron;
Tel-Aviv Sourasky medical center, Tel Aviv, Israel.
Introduction: Kostmann disease is a congenital immunodeficiency
syndrome amenable to bone marrow transplantation. The family described,
has a son affected with this disease. Genetic analysis identified
a known dominant mutation in the ELA2 gene in the affected child,
but also in the asymptomatic father. The parents requested preimplantation
genetic diagnosis (PGD), coupled with HLA matching, to obtain
a healthy suitable donor for the affected child.
Method: A PGD protocol was developed for the known mutation in
the ELA2 gene. The protocol was based on multiplex nested PCR for
direct analysis of the mutation, flanking polymorphic markers in the
ELA2 gene locus and HLA typing. The protocol was calibrated and
applied to single leukocytes isolated from the father, the mother and
2 2
the affected child.
Results: The amplification efficiency of the mutation was >90% in single
leukocytes from the affected child but only 67% in the father, suggesting
somatic mosaicism for the mutation. Analysis of single haploid
sperm cells from the father, using the same protocol, demonstrated 3
different sperm-cell populations: 1) sperm cells harboring the ELA2
mutation with the suspected allele, shared with the affected child, 2)
Sperm cells without the ELA2 mutation and the normal haplotype, and
3) sperm cells without the ELA2 mutation but with suspected allele,
shared with the affected child.
Conclusion: This was taken as evidence of somatic mosaicism for the
ELA2 mutation in the father, explaining why he is asymptomatic. These
data were also taken into consideration when deciding which embryos
to transfer.
P1031. A novel locus for Autosomal Dominant Distal Motor
Neuronopathy maps to chromosome 4q-ter
A. Magariello1 , L. Citrigno1 , L. Passamonti1 , A. Patitucci1 , F. L. Conforti1 , A. L.
Gabriele1 , R. Mazzei1 , T. Sprovieri1 , C. Ungaro1 , M. Bellesi2 , M. Muglia1 ;
1 2 Institute of Neurological Sciences, Mangone (CS), Italy, Institute of Neurological
Sciences, University Ancona, Ancona, Italy.
Distal hereditary motor neuronopathy (dHMN), also known as distal
spinal muscular atrophy, is a rare genetically and clinically heterogeneous
early-onset disorder, characterized by weakness and wasting of
distal limb muscles with possible pyramidal dysfunction, in absence of
overt sensory abnormalities. To date nine and three loci for autosomal
dominant dHMN and autosomal recessive dHMN have been described
respectively. We have previously reported a four generation kindred
characterized by atrophy and weakness of distal leg muscles associated
with pyramidal features without sensory abnormalities. Linkage
analysis excluded association to all the known loci for autosomal
dominant dHMN suggesting further locus heterogeneity for dHMN. A
genome wide search was performed by using 206 microsatellite markers
from the ABI PRISM Linkage Mapping Set LD 20. All genotyped
markers generated negative or nonsignificant LOD scores at all recombination
fractions tested, except for markers on chromosome 4. A
maximum LOD score of 3.19 at marker D4S408 was obtained, providing
evidence of linkage between the disease and this region. All affected
individuals in the family shared a commom haplotype between
D4S1552 and D4S426 ,which allowed the identification of a 20 cM
interval. The locus region contains many genes, including SNX25, a
member of the sortin nexin family. This gene was screened by using
DHPLC followed by sequencing of the variants. A nucleotide variation
was identified in the IVS13, but it was also present in the unaffected
members of the family suggesting that it is a polymorphism.
P1032. Association of mtDNA polymorphism with hypertension
and its complications
S. V. Buikin1 , M. V. Golubenko1 , V. V. Pogrebenkova1 , K. V. Puzyrev2 , I. V.
Tsymbalyuk3 , V. P. Puzyrev1,3 ;
1State Research Institute of Medical Genetics, Tomsk, Russian Federation,
2 3 State Research Institute of Cardiology, Tomsk, Russian Federation, Siberian
State Medical University, Tomsk, Russian Federation.
Hypertension is among most frequent cardiovascular diseases. It
is known that failing of energetic processes in cardiomyocyte mitochondira
plays substantial role in pathogenesis of cardiological diseases.
Mitochondrial DNA contains genes which encode subunits of
electron-transfer chain. Polymorphisms in these genes may influence
cardiovascular system function. To evaluate associations of mtDNA
polymorphisms with development of arterial hypertension, we have
studied sample of 147 patients (51 females, 96 males) with hypertension
and 137 healthy Russian individuals (81 females, 50 males).
Mean age in the samples was 48,3+5,5 and 47,6+10,2 years, respectively.
In the groups, ultrasound examination and 24-hours monitoring
of blood pressure was performed. Statistical analysis has shown that
in the hypertensive patients, prevalence of polymorphisms in positions
16292-16298 was revealed, so this locus might be designated as risk
factor for hypertension (OR=1,86; p=0,046). Frequencies of mitochondrial
polymorphisms were compared in the groups of patients with or
without left ventricular hypertrophy. It has been found that haplogroup
H was more frequent in hypertensive patients without hypertrophy
(OR=0,36; p=0,043) whereas haplogroup T was more frequent in the
patients with hypertrophy (OR=9,33; p=0,018). The findings suggest