2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
P05.131
A novel splicing mutation in the HNF1a gene in an italian family
with mODY3 disease
A. Cappelli1,2 , S. Silvestri3 , P. Staffolani1 , A. Consoli4 , L. Pianese1 ;
1 2 Laboratorio Medicina Molecolare, ASUR ZT13, Ascoli Piceno, Italy, School of
Advanced Studies: Environmental Sciences and Public Health, Camerino, Italy,
3 4 Scuola di Specializzazione in Biochimica Clinica, Camerino, Italy, Università
degli Studi “G. d’Annunzio”, Chieti, Italy.
Maturity-onset diabetes of the young (MODY) is a monogenic form
of diabetes mellitus characterized by autosomal dominant inheritance,
early age of onset and a primary insulin secretion defect . More than
200 different mutations in the hepatocyte nuclear factor 1a HNF1a
gene have been shown to cause a common type of MODY, called
MODY3 . Mutations span the entire gene and most types of mutations
have been described; e .g . insertion/deletion mutations, missense mutations,
nonsense mutations and splice site mutations . Recently, partial
and whole gene deletion mutations also were described .
In the present study we screened for mutation the HNF1a gene in a
proband which fulfilled the criteria for MODY3, using direct sequence.
The examination was extended to the proband’s family: an affected
father and unaffected mother and sister .
Here we report the identification of a novel HNF1a mutation at conserved
splice acceptor site of exon 5 (IVS4nt-1 G>T) that cosegregated
with diabetes in the family . Using a neural network based program,
this mutation might be expected to result in the skipping of the exon
immediately 3′ to the mutation and the utilization of the next available
AG site for exon 6 . Alternatively, a cryptic AG splice acceptor site in
intron 5, at -75 bp 5′ to the natural site, could be recruited resulting
in inclusion of some intronic sequence . However, truncation of the
protein results in both cases . Examination of mutant mRNA transcript
will be necessary in order to assess the precise consequence of this
mutation .
P05.132
contribution of the dHPLc to the diagnosis of VHL somatic
mosaicism
S. Lefebvre 1 , C. Gressier 1 , P. Delobre 1 , N. Burnichon 2,3 , S. Pinson 1 , A. Gimenez-Roqueplo
2,3 , R. Salomon 4 , S. Richard 5,6 , A. Calender 1 , S. Giraud 1 ;
1 Hospices civils de Lyon, Hôpital Edouard Herriot, Service de génétique, Lyon,
France, 2 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges
Pompidou , Département de génétique, Paris, France, 3 Université Paris Descartes,
Faculté de médecine, Paris, France, 4 Assistance Publique-Hôpitaux de
Paris, Hôpital Necker, Service de Néphrologie pédiatrique, Paris, France, 5 Faculté
de médecine Paris-Sud, Service de génétique oncologique EPHE, Paris,
France, 6 Institut Gustave Roussy, UMR8125, Villejuif, France.
Detection and characterization of somatic mosaicism are difficult because
of the low frequency of mutant alleles . We report here the diagnosis
of two cases of VHL somatic mosaicism identified by dHPLC.
The Von Hippel Lindau disease (VHL) is an autosomic dominant inherited
syndrome, predisposing to the development of various tumours,
notably retina hemangioblastomas and phaeochromocytomas . Analysis
for constitutional change of the VHL gene is realized by sequencing
and QMPSF . For two patients addressed for VHL suspicion, the
sequencing did not show mutation . However for each patient, on two
different frequent change sites, second peaks (respectively c .482G>A
and c .500G>C) were discriminated but their heights were small (1/5
of the normal peak) and difficult to differentiate from the usual background
noise .
We analyzed these two patients by dHPLC . For each patient, aberrant
profile was observed, similar but reduced relative to profile with known
mutation at the same site .
With dHPLC collector, we collected and sequenced separately the
various peaks . We could clearly observe a superposition of two nucleotides
in equivalent heights, which allowed us to confirm the VHL
somatic mosaicism .
These two patients need follow-up like other VHL patients even if they
have not presented all the characteristic attacks of this syndrome .
We began a retrospective work by dHPLC on the patients addressed
for a suspicion of VHL disease, with early or multiple attacks and without
identified mutation.
P05.133
interaction between mtHFR polymorphisms and development of
myopathyc process (hypothesis approbation)
V. C. Sacara1 , E. V. Scvortova2 ;
1Centre of reproductive health and medical genetics, Chisinau, Republic of
Moldova, 2Moldavian State University, Chisinau, Republic of Moldova.
Background:Polymorphisms of the MTHFR gene can influence the
methionine metabolic pathway and folate metabolism . The methylation
process is essential for the regulation of gene expression controlling
the development and function of the muscles . We noted that DMD
patients with the same deletion in the dystrophine gene had different
clinical features and severity of pathology process . The main conception
of our hypothesis is that exist an interaction between MTHFR polymorphisms
and development of myopathyc process .
Methods:We analyzed DNA from a case-control study in the RM of 50
DMD probands and 114controls . MTHFR variant alleles were determined
by a PCR-RFLP . Control group data is taken from publication
Skibola et al.,1999 . The genotyping protocol for the detection of the
MTHFR C677T and A1298C polymorphisms were adapted from MG-
Center, Moscow (prof . Polyakov A .) .Statistic analyses were performed
by using SISA .
Results:We found the MTHFR C677C allele present among 3(6%)DMD
and 61(53 .5%) controls (X2 =32,9,P=0,00), the C677T genotype among
27(54%)DMD, 39(34 .2%) controls (X2 =5,66,P=0,02), and the T677T
allele 20(40%)DMD, 14(12 .3%) controls (X2 =16,25,P=0,00) . For MTH-
FR 1298, the A1298A genotype was observed in 33(66%) of DMD,
49(43 .0%) of the controls (X2 =7,37,P=0,00), the A1298C allelic variant
was observed in 11(22%) DMD, 54(47 .4%) controls (X2 =9,35,P=0,00),
and the rarer C1298C variant was observed among 6(12%) case and
11(9 .6%) controls (X2 =0,21,P=0,65) .
Conclusions:According to our data were observed statistically significant
differences between DMD and control MTHFR genotypes, except
C1298C . The MTHFR T677T genotype was higher among DMD patients
as we know this mutation leads to reduced MTHFR activity and
influence on myopathyc process.
P05.134
mutational analysis of the mtHFR gene in four patients with
homocystinuria due to severe MTHFR deficiency
R. Urreizti 1,2 , U. Fanhoe 1 , A. Langkilde 1 , C. Esteves 1 , M. Cozar 1,2 , M. Vilaseca
3,2 , R. Artuch 3,2 , A. Baldellou 4 , L. Vilarinho 5 , B. Fowler 6 , A. Ribes 7,2 , D. R.
Grinberg 1,2 , S. Balcells 1,2 ;
1 Dpt de Genetica, IBUB, Universitat de Barcelona, Barcelona, Spain, 2 Centro
de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER),
ISCIII, Barcelona, Spain, 3 Department de Bioquimica Clinica, Hospital Sant
Joan de Deu, Barcelona, Spain, 4 Unidad de Enfermedades Metabolicas, Hospital
Infantil Miguel Servet, Zaragoza, Spain, 5 Instituto de Genetica Medica Jacinto
Magalhaes, Porto, Portugal, 6 Metabolic Unit, University Children’s Hospital,
Basel, Switzerland, 7 Divisio d’Errors Congenits del Metabolisme (IBC), Departament
de Bioquimica i Genetica Molecular, Hospital Clinic, Barcelona, Spain.
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction
of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate,
which acts as a methyl donor in the remethylation of homocysteine
to methionine . Disruption of MTHFR activity results in severe hyperhomocysteinemia
and causes vascular and neurological disorders
and developmental delay . Four patients with severe hyperhomocysteinemia
and hypomethioninemia were examined with respect to their
symptoms, their MTHFR enzyme activity and their genotypes at the
MTHFR gene . We found three novel mutations: two missense mutations
c .664G>T (p .V218L) and c .1316T>C (p .F435S), and a one bp
deletion c .1768delC (p .L590C fs X70) . We also found c .1420G>T
(p .E470X), a previously reported nonsense mutation . Four new genotypes
were identified: Patient 24 was homozygous for p.V218L and
the common polymorphism p .A222V (c .667C>T); patient 73 was homozygous
for p .E470X; patient 86 was homozygous for p .F435S and
the common polymorphism p .E429A (c .1298A>C); and patient 95 was
homozygous for c .1768delC . Patient 24 presented an MTHFR enzyme
activity below 7% of control level, while patients 73 and 95 had MTHFR
enzyme activities below 1% . All patients presented symptoms of severe
central nervous system disease and microcephaly . Two of them
suffered a fatal stroke (patient 73 at 18 months and patient 86 at age
14 years) . Patient 24 was the mildest, with a diagnosis at age 18 years .
Patient 95 could be diagnosed at age 11 months and is improving upon