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

Molecular and biochemical basis of disease
ataxic neuropathy always presented with associated muscular and/or
central neurological system features. The aim of our study was to test
whether POLG mutations can be responsible for isolated sensory ataxic
neuropathy. We screened 15 patients by direct sequencing. Seven
patients were men and the median age of the population was 57 years.
The presenting and only feature was ataxia caused by axonal sensory
neuropathy. A 50 year-old woman was found to be a compound
heterozygous carrying the c.1391T>C mutation (M464T) in combination
with the c.2302A>G substitution (K768E). No POLG mutation was
found in other patients. Nevertheless, a muscle biopsy was performed
in two cases. Ragged-red and COX negative fibers were found in one
patient, with multiple mtDNA deletions by both long range PCR and
Southern blot analysis. The patient was a 67 year-old man who developped
ataxic symptoms at the age of 47. ENMG revealed normal
motor potentials and absent sensory potentials in the four limbs. No
mutations were detected in ANT1 or Twinkle.
In conclusion, mitochondrial disease has to be considered as a cause
of isolated adult-onset sensory ataxic neuropathy.
P0862. Sepsis and mitochondrial DNA
A. Pyle 1 , S. V. Baudouin 2 , P. F. Chinnery 1 ;
1 Mitochondrial Research Group, Newcastle upon Tyne, United Kingdom, 2 Surgical
and Reproductive Sciences, Newcastle upon Tyne, United Kingdom.
Sepsis-induced multiple organ failure is the major cause of mortality
and morbidity in critically ill patients. However, the precise mechanisms
of this dysfunction are largely unknown. Genetic studies suggest
a strong genetic component to both the risk of developing sepsis
and the subsequent outcome in terms of survival.
The failure of cellular oxygen use appears to be important in the development
of multiple organ dysfunction in severe sepsis. Mitochondria
are small membrane-bound intracellular organelles. They are involved
in multiple cellular processes such as energy metabolism, apoptosis
and generation of reactive oxygen species (ROS).
Impaired mitochondrial function in septic patients is associated with
poor clinical outcome. Epidemiological studies indicate that premature
death from infection has a strong inherited component and natural
genetic variation in mitochondrial DNA (mtDNA) provides a potential
explanation.
Human mtDNA is maternally inherited. The population can be divided
into several mtDNA haplogroups on the basis of specific single nucleotide
polymorphisms (SNPs) scattered throughout the mitochondrial
genome, indicating that mutations accumulated by a discrete maternal
lineage during evolution. The SNPs that define the mtDNA haplogroups
are markers of a particular maternal lineage of mtDNA. The majority
of Europeans belong to one of nine haplogroups with haplogroup H
being the most common (44%). Our group found an improved survival
in septic patients with haplogroup H. MtDNA copy number was
measured in 59 haplogroup H patients and 34 control samples. Differences
in mtDNA copy number were observed between survivors and
non-survivors.
P0863. Histidine-Rich Calcium Binding Protein Interacts with
SERCA2 in a Ca-Dependent Manner
D. A. Arvanitis 1 , E. Vafiadaki 1 , B. A. Mitton 2 , K. N. Gregory 2 , F. Del Monte 3 , A.
Kontrogianni-Konstantopoulos 4 , E. G. Kranias 2,1 , D. Sanoudou 1 ;
1 Molecular Biology Division, Foundation for Biomedical Research, Academy of
Athens, Athens, Greece, 2 Department of Pharmacology and Cell Biophysics,
College of Medicine, University of Cincinnnati, Cincinnati, OH, United States,
3 Department of Cardiology, Harvard Medical School, Massachusetts General
Hospital, Boston, Boston, MA, United States, 4 Department of Physiology,
School of Medicine, University of Maryland Baltimore, Baltimore, Baltimore,
MD, United States.
Deregulation of Ca cycling by the sarcoplasmic reticulum (SR) in the
cardiomyocyte has been associated with depressed cardiac function
which may progress to heart failure. The histidine-rich calcium binding
protein (HRC) is a low affinity, high capacity Ca-handling SR protein
that binds to triadin. Through this interaction HRC may affect Ca release
by the ryanodine receptor. HRC overexpression in transgenic
mouse hearts was associated with decreased rates of SR Ca uptake
and delayed relaxation, which progressed to hypertrophy upon aging.
Using a combination of in vivo co-immunoprecipitations and pull-down
assays in human and mouse cardiac homogenates, and in vitro blot
overlay experiments with GST and MBP recombinant proteins, we
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identified the direct binding of HRC to SERCA2 in cardiac muscle. This
interaction involves the histidine and glutamic acid-rich domain of HRC
(320-460 aa) and part of the N-terminal cation transporter domain of
SERCA2 (74-90 aa), which projects into the SR lumen. The SERCA2
binding domain is upstream from the triadin binding region in human
HRC (609-699 aa). Ca-titration experiments indicated that the binding
of HRC to SERCA2 was reduced by 75%, when the Ca concentration
as raised from 0.1 to 100 μM. Increasing Ca-concentrations had opposite
effects on the HRC binding to triadin. Collectively, our data suggest
that HRC may be involved in the regulation of SR Ca-cycling by its
direct interaction with SERCA2 and triadin, which may be affected by
local Ca changes. Thus, HRC may mediate a fine cross-talk between
SR Ca uptake and release.
P0864. Mutation spectrum of IL2RG gene in Russian families
with X-linked combined immunodeficiency.
I. G. Sermyagina1 , S. M. Tverskaya1 , A. V. Polyakov1 , I. V. Kondratenko2 ;
1 2 Research Center for Medical Genetics, Moscow, Russian Federation, Russian
Children’s Clinical Hospital, Moscow, Russian Federation.
X chromosome-linked severe combined immunodeficiency (X-SCID)
is a rare disease of serious imbalance cellular and humoral immune
function. X-SCID results from a mutation in the gene encoding the
gamma subunit of the interleukin-2 receptor (IL2RG), a component
of several IL receptors. IL2RG gene contains 8 exons and has been
mapped to the Xq13.1 region.
Direct DNA sequencing of all exons and exon-intron junctions and
PCR-RFLP performed a search of IL2RG gene mutations.
We have detected 8 mutations in 9 unrelated families with X-SCID. In
this molecular investigation we identified 6 novel mutations, the others
have been reported.
In exon 2 and exon 3 of IL2RG gene we show three missence mutations:
reported - p.Glu68Lys, p.Tyr105Cys and novel - p.Cys72Trp. We
identified a new missense mutations (p.Cys182Ser) and (p.Cys182Tyr)
in exon 4 of IL2RG gene in a two affected probands with X-SCID. In a
two patients with X-linked combined immunodeficiency in our Center
we found two unicue splice mutations (IVS 5 as-2nt a->g) and (IVS 5
as-1nt g->a) in intron 5 of IL2RG gene. One insertion (c.837ins9bp) we
identified in a boy with X-SCID in exon 6 of IL2RG gene.
The mothers of all patients were heterozygous for the mutation. Also,
three prenatal diagnostics of X-SCID were made.
In this molecular investigation of nine familys with X-linked combined
immunodeficiency we have detected different mutations in exon 2, 3,
4, 6 and intron 5. We supposed two “hot spots” in exon 4 and in intron
5 of IL2RG gene.
P0865. Identification of small genomic deletions flanking the
human SHOX gene
S. Thomas, K. Baker, D. Bunyan, J. Harvey;
Wessex Regional Genetics Laboratory, Salisbury, United Kingdom.
In addition to point mutations and gene deletions, haploinsufficiency of
the human SHOX gene can also result from deletions 3’ of the gene.
These are generally 80 to 500 kb in size, with a 29kb minimal deleted
region, that exert a position effect. These classes of mutation account
for only 70% of Leri-Weill dyschondrosteosis (LWD) patients. Therefore
we have used the MRC-Holland SHOX MLPA kit supplemented
with additional probes to screen for novel types of SHOX mutation
in 73 cases in whom no mutation was previously identified (16 with
LWD, 12 with short stature and 45 with undefined skeletal dysplasia).
Upstream of SHOX we identified two deletions, of maximum size 73kb
and 160kb, each in a single patient. Neither deletion was present in 88
normal controls. Downstream (3’) of SHOX we identified two deletion
classes: 12 of the 73 patients (16%) carried an approximately 10kb
deletion and two LWD patients (3%) were compound heterozygotes for
the 10kb deletion and a larger overlapping deletion of up to 60kb. The
larger deletion was absent among the 88 controls, but there were 13
carriers of the 10kb deletion (15%). Among 12 LWD mutation positive
cases, there were two 10kb deletions (17%) and one larger deletion
(8%). In summary additional types of SHOX mutation are likely to exist,
although it will be difficult to assess their functional significance.
The deletions identified in this study do not appear to cause classical
SHOX haploinsufficiency, however it is possible they may act to modify
phenotypic expression.