contractions, a phenomenon that has been reported so far
only in healthy individuals, and observed also in our control
set, in terms of the presence of a rare polymorphism having
no effect on the phenotype.
The existence of a mutational hotspot in the region coding
for the polyalanine domain of PHOX2B needs to be postulated
since 92% of all mutations we detected so far in this gene
leads to variations in the length of the polyalanine tract.
There are at least nine genes in which alanine expansions
have been shown to cause human disease.
Moreover, 31 out
of 98 sequences coding for polyalanine domains in the
human genome have been found polymorphic, thus suggest-
ing the opportunity of screening similar sequences in
Although polyalanine tracts are
common in transcription factors, neither their normal
function nor the effects of their expansion are understood.
Polyalanine tracts have been found in repression motifs of
several proteins containing homeodomains and demon-
strated, in some cases, to be responsible for the repression
of target genes.
In other cases, polyalanine tracts act as
flexible spacer elements between functional domains.
polyalanine expansions observed in PHOX2B may either
directly interfere with protein-protein interactions, distort
an essential secondary structure, or act as dominant negative
factors. The correlation between phenotypic severity and
expansion size observed in CCHS suggests that tracts of 25 or
more polyalanines might confer a progressive gain of
function to PHOX2B, a hypothesis which will require further
During our study we have collected proof that polyalanine
expansion corresponds to a PHOX2B allele carried by
patients in their constitutive genomic DNA. Indeed, at first
in our sequencing experiments alanine expansions had the
appearance of an unclear background lying behind the
normal DNA sequence in a small proportion of patients.
Under the hypothesis that in individuals heterozygous for
defects involving length changes of the polyalanine stretch
of exon 3, the largest allele is always prone to unsuccessful
amplification by DNA polymerase, and to avoid incorrect
interpretation of the genotype of some patients and
controls, we have developed a proper PCR protocol. Results
reported in fig 4 have confirmed our hypothesis, showing
different amplification outcomes when using different
experimental protocols: when DNA amplification occurs
under standard procedures, the shortest product is always
observed while the largest amplimer can remain undetect-
able, as in the case of alanine expanded against wild-type
alanine stretches and of wild-type alanine stretches against
both the del38 allele and an alanine contracted tract in a
control (fig 4). Such a misleading methodological aspect can
cause mistakes in recording individuals’ genotypes and we
therefore suggest great care when approaching PHOX2B
mutational screening and especially for PHOX2B molecular
diagnosis in relatives of affected children. Indeed, following
observation of recurrence of PHOX2B defects and potential
transmission of the CCHS phenotype to progeny, an accurate
detection of PHOX2B mutations represents the ground for
genetic counselling, which has become indispensable for
couples with an affected child to determine the recurrence
risk to a fetus.
In conclusion, PHOX2B is confirmed as the major gene in
CCHS pathogenesis and autosomal dominant inheritance
with reduced penetrance is now demonstrated. Moreover, a
correlation between phenotypic severity and expansion
length of the polyalanine tracts can be assumed and used
to predict onset and progression of the disease in mutation
carriers. Finally, molecular testing and genetic counselling
has now become feasible, thus giving hopes to many families
We are extremely grateful to all the families who have participated in
this study and the clinicians who have reported details of patients
and provided blood samples. We want to thank Francesca Schena
and Giuseppe Santamaria for their technical help.
I Matera, T Bachetti, F Puppo, M Di Duca, R Ravazzolo, I Ceccherini,
Laboratorio di Genetica Molecolare, Istituto G Gaslini, Genova, Italy
M Di Duca, Laboratorio di Fisiopatologia dell’Uremia, Istituto G Gaslini,
F Morandi, UO Pediatria, Ospedale ‘‘Sacra Famiglia’’, Erba (CO), Italy
G M Casiraghi, UO Anestesia e Rianimazione, Ospedale Mandic,
Merate (Lecco), Italy
M R Cilio, Divisione di Neurologia, Ospedale Bambino Gesu’, Roma,
R Hennekam, Departments of Paediatrics and Clinical Genetics,
University of Amsterdam, The Netherlands
R Hofstra, Department of Medical Genetics, University of Groningen,
J G Scho¨ber, Kinderklinik Dritter Orden (vormals Lachnerklinik),
R Ravazzolo, Dipartimento di Pediatria e CEBR, Universita` di Genova,
G Ottonello, UO Anestesia e Rianimazione, Istituto G Gaslini, Genova,
This study was begun with the financial support of Telethon Italy (grant
no E791) which is gratefully acknowledged. The completion of our work
has been guaranteed by a grant obtained from Compagnia di San
Paolo. Finally, we gratefully acknowledge the financial support of the
European Community (contract no QLG1-CT-2001–01646) and of the
Ministry of University (FIRB project to RR).
Conflicts of interest: none declared.
Correspondence to: Isabella Ceccherini, Ph.D, Laboratorio Genetica
Molecolare, Istituto G Gaslini, L.go G Gaslini, 5, 16148 Genova, Italy;
Revised version received 14 November 2003
Accepted for publication 17 November 2003
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