2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
P12.107
clinical and genetic heterogeneity of mal de meleda: exclusion
of genetic linkage to the ARS gene in a tunisian family
M. Bchetnia 1,2 , R. Chakroun 3 , A. Ben Brick 1 , C. Charfeddine 1 , S. Boubaker 4 , A.
Dhahri Ben Osman 3 , S. Abdelhak 1 , M. Mokni 2,3 ;
1 Molecular Investigation of Genetic Orphan Diseases Research Unit (MIGOD)
UR 26/04, Institut Pasteur de Tunis, Tunis, Tunisia, 2 Study of Hereditary Keratinization
Disorders Research Unit (THK) UR 24/04, La Rabta Hospital, Tunis,
Tunis, Tunisia, 3 Dermatology Department, La Rabta Hospital, Tunis, Tunis, Tunisia,
4 Anatomo-Pathology Department, Institut Pasteur de Tunis, Tunis, Tunisia.
Background. Mal de Meleda (MdM) is a rare form of palmoplantar
keratoderma (PPK) with autosomal recessive transmission. It is characterized
by diffuse erythema and hyperkeratosis of the palms and
soles. Recently, mutations in the ARS (component B) gene (ARS,
MIM: 606119) on chromosome 8q24.3 have been identified in families
with this disorder.
Objective. Clinical and genetic investigation of a consanguineous family
from Northern Tunisia with transgressive palmoplantar keratoderma
closely resembling the Mal de Meleda phenotype.
Methods. A family with six members, among them two were affected
individuals, was recruited for this study. Mutation screening of the ARS
gene was performed by direct sequencing and haplotype analysis
using 2 microsatellite markers (D8S1836 and D8S1751) flanking the
ARS gene.
Results. No mutation was found in the coding region and exon-intron
junctions of the ARS gene within the explored family. This genetic exclusion
of the ARS gene was confirmed by haplotype analysis and Lod
score calcul.
Conclusion. This is the second Tunisian Family with “MdM like” phenotype.
Our findings give further evidence for the molecular heterogeneity
of the MdM phenotype in North African population.
P12.108
Quantitative sequence analysis of FBN premature termination
codons provides evidence for incomplete NmD in leukocytes
I. Magyar 1 , D. Colman 1 , E. Arnold 1,2 , D. Baumgartner 3 , A. Bottani 4 , S. Fokstuen
4 , M. C. Addor 5 , W. Berger 1 , T. Carrel 6 , B. Steinmann 2 , G. Mátyás 1 ;
1 University of Zurich, Institute of Medical Genetics, Division of Medical Molecular
Genetics and Gene Diagnostics, Schwerzenbach, Switzerland, 2 University
Children’s Hospital, Division of Metabolism and Molecular Pediatrics, Zurich,
Switzerland, 3 Innsbruck Medical University, Department of Pediatric Cardiology,
Innsbruck, Austria, 4 Geneva University Hospitals, Division of Medical Genetics,
Geneva, Switzerland, 5 Centre Hospitalier Universitaire Vaudois, Service of
Medical Genetics, Lausanne, Switzerland, 6 University Hospital, Clinic for Cardiovascular
Surgery, Berne, Switzerland.
In order to assess the pathogenic effects of mutations, we improved,
evaluated, and used Sanger sequencing for quantification of SNP variants
in transcripts and gDNA samples. This improved assay resulted
in highly reproducible relative allele frequencies (e.g. for a heterozygous
gDNA 50.0±1.4%, P=0.05, and for a missense mutation-bearing
transcript 46.9±3.7%, P=0.05) with a lower detection limit of 3-9%. It
provided excellent accuracy (e.g. for a duplicated gDNA 66.6±2.2%,
P=0.05) and linear correlation between expected and observed relative
allele frequencies. This sequencing assay, which can also be used for
the quantification of CNVs, methylations, mosaicisms, and DNA pools,
enabled us to analyze transcripts of the FBN1 gene in fibroblasts and
blood samples of patients with suspected Marfan syndrome not only
qualitatively but also quantitatively. We present a total of 19 novel and
18 known FBN1 sequence variants leading to a premature termination
codon (PTC), 26 of which we analyzed by quantitative sequencing
both at gDNA and cDNA levels. The relative amounts of PTC-containing
FBN1 transcripts in fresh and PAXgene-stabilized blood samples
were significantly higher (33.0±3.9% to 80.0±7.2%, P=0.05) than those
detected in affected fibroblasts with inhibition of nonsense-mediated
mRNA decay (NMD) (11.0±2.1% to 25.0±1.8%, P=0.05), while in fibroblasts
without NMD inhibition no mutant alleles could be detected.
These results provide evidence for incomplete NMD in leukocytes and
have particular importance for RNA-based analyses not only in FBN1
but also in other genes.
P12.109
mutation analyses of MYH , ACTA , TAGLN, and TGFBR in
patients with suspected marfan syndrome
H. Burri 1 , E. Arnold 1,2 , C. Henggeler 1 , R. Perez 1 , S. Alonso 1 , S. Fokstuen 3 , A.
Croquelois 4 , M. Rohrbach 2 , T. Carrel 5 , B. Steinmann 2 , W. Berger 1 , G. Matyas 1 ;
1 Division of Medical Molecular Genetics and Gene Diagnostics, Institute of
Medical Genetics, University of Zurich, Zurich, Switzerland, 2 Division of Metabolism
and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland,
3 Geneva University Hospitals, Division of Medical Genetics, Geneva,
Switzerland, 4 Neuropsychology and Neurorehabilitation Department, Centre
Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 5 Clinic for Cardiovascular
Surgery, University Hospital, Berne, Switzerland.
Marfan syndrome (MFS) is an autosomal dominant disorder of connective
tissue, which displays variable manifestations in the skeletal, ocular,
and cardiovascular systems. Patients with suspected MFS referred
to us for molecular genetic testing carry the disease-causing mutation
in FBN1, TGFBR1, and TGFBR2 in the majority of cases. Negative
genetic testing results can be due to assay limitations or because the
disease-causing mutation is located in another gene. Indeed, mutations
in MYH11 and ACTA2 have recently been associated with familial
thoracic aortic aneurysms and dissections (TAAD), which overlap
with cardiovascular features of MFS, and TAGLN has been predicted
in silico as a candidate gene for TAAD. Furthermore, considering the
TGF-beta pathway, one could also expect mutations in the TGFBR3
gene. Here, we analysed MYH11, ACTA2, TAGLN, and TGFBR3 in
patients with suspected MFS in whom previous mutation analyses
of FBN1 and/or TGFBR1 and TGFBR2 detected no mutation. Exonby-exon
gDNA sequencing of 24 (MYH11), 89 (ACTA2), 46 (TAGLN),
and 12 (TGFBR3) unrelated patients revealed two putative pathogenic
mutations in MYH11 (p.Leu1752_Glu1755dup and p.R1794Q) as well
as one missense mutation in ACTA2 (p.I371T). Screening of TAGLN
and TGFBR3 revealed no putative pathogenic sequence changes.
Although the number of patients analysed in this study is still small,
our results indicate that screening for MYH11 and ACTA2 mutations
should be considered in patients with suspected MFS in whom mutation
analyses of FBN1, TGFBR1, and TGFBR2 revealed no diseasecausing
mutation, in particular in patients with cardiovascular complications
lacking skeletal features of MFS.
P12.110
Identification of genome wide targets of the UPF3B dependent
nonsense-mediated mRNA surveillance pathway in patients with
mutations in UPF3B using exon array
L. Nguyen, C. Shoubridge, A. Gardner, L. Vandeleur, M. Corbett, J. Gecz;
SA Pathology, North Adelaide, Australia.
Non-sense mediated mRNA decay (NMD) is a universal RNA surveillance
pathway that among other functions degrades mRNAs bearing
premature termination codons (PTC). We recently showed mutations
in UPF3B, an important member of this pathway, caused syndromic
and nonsyndromic mental retardation (MR). To assess the impact of
UPF3B null mutations and identify relevant genes regulated by NMD,
we performed expression profiling using RNA isolated from control and
patient lymphoblastoid cells using Affymetrix Human Exon 1.0 ST arrays.
Compared to controls, 633 genes were significantly de-regulated
in patients (30% up, 70% down, false discovery rate = 10%), including
down-regulation of UPF3B. Hence, NMD is only partially compromised
in the absence of UPF3B as its own PTC containing mRNA is degraded.
Comparison with previous microarray studies from UPF1, UPF2
or UPF3B knock down cell lines in human, fly and yeast generated
minimal overlap. Such low correspondence indicates these studies do
reflect the situation of a real knock out or reflects differences in the
tissue types and platforms used. Among the significantly de-regulated
genes identified in patients with mutations in UPF3B, ARHGAP24 was
highly up-regulated. ARHGAP24 is a negative regulator of Rho-GT-
Pase and has role in regulating cell polarity. We show that PC12 cells
over expressing ARHGAP24 failed to differentiate into neuronal-like
cells upon treatment with nerve growth factor. We have identified the
bona-fide targets of the UPF3B dependent NMD pathway and suggest
up-regulation of ARHGAP24 contributes to the phenotypes seen in patients
with MR due to mutations in UPF3B.