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

Molecular and biochemical basis of disease
P0815. Multiple Sclerosis: Defect on GD3 synthase as a factor
determining B-cell response
S. Husain1 , M. Schwalb1 , S. Cook1 , E. Vitale1,2 ;
1 2 UMDNJ-New Jersey Medical School, Newark, NJ, United States, CNR Institute
of Cybernetics, Pozzuoli (NA), Italy.
Cell surface gangliosides represent the major class of glycoconjugates
on neurons and bear the majority of sialic acid within the central nervous
system (CNS). In a previous study we found that a variant in
GD3 synthase (GD3S) was associated with a disruption of the ganglioside
pathway in a unique pedigree segregating Multiple Sclerosis. The
patients show a reduced expression of GD3 synthase enzyme which
leads to a different co-localization of GD3 ganglioside (GD3G) on peripheral
blood mononuclear cells (PBMC) and these could represent
a foundation of the disease in this family. Here we present evidence
that GD3S is mainly expressed on the B cell surface thus providing
evidence of a potential role of B cells in this autoimmune disease
The ability to recognize self from non self components it is crucial for
the immune system as this can lead to the disruption of the body’s own
tissue. MS is the result of a complex interaction of genes and environment
and the need to understand this complex mechanism is essential
to establish the correct therapies. Although MS is considered to be
mediated by T-helper 1 (Th1) cells, emerging evidence implicates B
lymphocytes and their products in the pathogenesis of this disorder.
Evidence from recent pathology studies has led to a renewed interest
in the role that chronically activated B cells may play in the pathogenesis
of MS, independent of antibody production.
P0816. Microsatellite Expansion in Myotonic Dystrophy Patients:
The Search for Underlying Pattern
B. Veytsman 1 , L. Akhmadeeva 2 , F. Morales 3,4 , G. Hogg 3 , T. Ashizawa 5 , P.
Cuenca 4 , G. del Valle 4 , R. Brian 4 , M. Sittenfeld 4 , A. Wilcox 3 , D. E. Wilcox 3 , D. G.
Monckton 3 ;
1 George Mason University, Fairfax, VA, United States, 2 Bashkir State Medical
University, Ufa, Russian Federation, 3 University of Glasgow, Glasgow, United
Kingdom, 4 Universidad de Costa Rica, San Jose, Costa Rica, 5 The University of
Texas Medical Branch, Galveston, TX, United States.
Microsatellite expansion is the cause of a number of severe diseases
including fragile X, Huntington disease and myotonic dystrophy. An interesting
common feature of these disorders is the instability of the mutation:
once expanded, the number of repeat units continues to change
in patient’s cells. An understanding of the mechanism and dynamics
of microsatellite expansion will provide more informative genotype to
phenotype relationships, which will in turn be of considerable utility
in allowing patients and families to make more informed life and reproductive
choices, and facilitate the clinical management of disease.
Recently (J. Theor. Biol., 242, 401-408 (2006)), a mathematical model
was proposed to describe the dynamics of microsatellite expansion
and the resulting distribution of repeat lengths in patient’s DNA. In this
work, we compare the theoretical predictions with observed repeat
length distributions in a large cohort of myotonic dystrophy type 1 patients.
We find that the theoretical predictions agree fairly well with the
clinical data with the observed distributions close to those predicted
by the mathematical model. We also used the clinical data to estimate
the theoretical parameters underlying the model: the rate of increase
of the number of repeats and the rate of widening the distribution. We
find that while these parameters have large individual variations, the
average values give reasonable predictions for the development of
mutations. These values can be used to estimate the initial mutation
(the number of repeats in the progenitor allele) and to predict the development
of the disease.
P0817. Molecular-genetic diagnostic of Juvenile
nephronophthisis.
A. Stepanova 1 , L. Prihodina 2 , A. Polyakov 1 ;
1 Research Centre for Medical Genetics, Moscow, Russian Federation, 2 The
Moscow scientific research institute of pediatrics and children’s surgery, Moscow,
Russian Federation.
Juvenile nephronophthisis (NPHP) is an autosomal recessive cystic
kidney disease, which leads to end-stage renal failure at an average
age of 13 years following the initial symptoms of polyuria, polydipsia,
renal salt loss, and secondary enuresis.
Juvenile nephronophtisis is most often caused by mutations in the
NPHP1 gene, encoding nephrocystin. About 70% of cases of Juvenile
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nephronophtisis are caused by large deletions in the 2q13 region.
Two previously described markers (NPHP804/6 and NPHPC11) localised
within the common NPHP1 deletion interval were amplified in
multiplex PCR with control markers - exon 13 of the PAH gene, mapping
outside the deleted region.
We have studied 6 unrelated patients. A homozygous deletion of the
NPHP1 gene was found in 3 of 6 probands.
All patients with deletion had polyuria, polydipsia, isosthenuria and diffusive
ostepenia.
This work was partly supported by Russian President’s grand NSh-
5736.2006.7.
P0818. Missense alterations in the NF1 gene: how to make sense
of them?
L. M. Messiaen, T. Callens, J. Williams, M. Herbst, B. R. Korf;
Medical Genomics Laboratory, University of Alabama at Birmingham, AL,
United States.
Neurofibromatosis type 1 (NF1), notorious for its phenotypic variability,
is characterized by neurofibromas, skinfold freckling and café-au-lait
spots. Recently, a relationship between a 3-bp inframe deletion in the
NF1 gene and a specific phenotype, i.e. absence of cutaneous neurofibromas,
was found. This finding holds promise that other missense
or 1-2 AA-deletions may exist that correlate a specific missense (or
1-2 AA-deletion) to the expression of a specific clinical phenotype. As
a first step, all putative missense alterations need to be classified correctly.
We developed a comprehensive NF1 assay using RNA- and DNAbased
techniques including long-range RT-PCR, cDNA sequencing,
FISH and MLPA and use this approach in a clinical setting. For all
patients, the phenotype is recorded using a standardized checklist.
So far, we identified a pathogenic mutation in 1400 unrelated patients.
All 172 different missense alterations, found in 299 patients, were further
analyzed for effect on splicing; presence/absence of another possible
deleterious NF1 mutation; presence/absence in >1000 control alleles;
evolutionary conservation; in silico predicted effect by PolyPhen,
SIFT and AGVGD; familial clinical and genetic assessment. Using this
approach, we classified 17 different “missense“ alterations, present in
46 patients, as splicing mutations, 19 novel alterations, residing in cis
or trans of a clearly deleterious mutation, as rare benign variants and
2 as variants of still unknown significance. The remaining 136 different
missense alterations were classified as pathogenic mutations. As
30 of these mutations are recurrent in unrelated families, collection of
phenotypic data of affected family members may provide additional
genotype-phenotype correlations.
P0819. Functional analysis of splicing mutations in exon 7 of
NF1 gene
I. Torrente 1 , I. Bottillo 1 , A. De Luca 1,2 , A. Schirinzi 1 , V. Guida 1 , S. Calvieri 3 , C.
Gervasini 4 , L. Larizza 4 , A. Pizzuti 1,2 , B. Dallapiccola 1,2 ;
1 CSS-Mendel Institute, Rome, Italy, 2 Department of Experimental Medicine
and Pathology, University of Rome “La Sapienza”, Rome, Italy, 3 Department of
Dermatology - Venereology and Plastic and Reconstructive Surgery, University
of Roma “La Sapienza”, Rome, Italy, 4 Division of Medical Genetics, San Paolo
School of Medicine, University of Milan, Milan, Italy.
Background. Neurofibromatosis type 1 is one of the most common autosomal
dominant disorders, affecting about 1:3,500 individuals. NF1
exon 7 displays weakly defined exon-intron boundaries, and is particularly
prone to missplicing.
Methods. In this study we investigated the expression of exon 7
transcripts using bioinformatic identification of splicing regulatory sequences,
and functional minigene analysis of four sequence changes
[c.910C>T (R304X), c.945G>A/c.946C>A (Q315Q/L316M), c.1005T>C
(N335N)] identified in exon 7 of three different NF1 patients.
Results. We detected three exonic splicing enhancers (ESEs) and one
putative exonic splicing silencer (ESS) element. The wild type minigene
assay resulted in three alternative isoforms, including a transcript
lacking NF1 exon 7 (NF1ΔE7). Both the wild type and the mutated
constructs shared NF1ΔE7 in addition to the complete messenger, but
displayed a different ratio between the two transcripts. In the presence
of R304X and Q315Q/L316M mutations, the relative proportion
between the different isoforms is shifted toward the expression of
NF1ΔE7, while in the presence of N335N variant, the NF1ΔE7 expression
is abolished.Conclusions. It appears mandatory to investigate the